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WO2025045422A1 - New strains and blends for texture and flavor in plant-based cheese-analogues - Google Patents

New strains and blends for texture and flavor in plant-based cheese-analogues Download PDF

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Publication number
WO2025045422A1
WO2025045422A1 PCT/EP2024/069310 EP2024069310W WO2025045422A1 WO 2025045422 A1 WO2025045422 A1 WO 2025045422A1 EP 2024069310 W EP2024069310 W EP 2024069310W WO 2025045422 A1 WO2025045422 A1 WO 2025045422A1
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Prior art keywords
pea
dsm
streptococcus thermophilus
fermented
culture
Prior art date
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PCT/EP2024/069310
Other languages
French (fr)
Inventor
Carmen MASIÁ CALABUIG
Raquel FERNANDEZ
Vera Kuzina POULSEN
Kim Ib Soerensen
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Chr. Hansen A/S
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Publication of WO2025045422A1 publication Critical patent/WO2025045422A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C20/00—Cheese substitutes
    • A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
    • A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/155—Kefiri
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/175—Rhamnosus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/249—Thermophilus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/24—Lactobacillus brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to a method for improving the organoleptic experience of a plant-based cheese-analogue, and the resulting products as such.
  • bacterial blends that through fermentation of a pea-base yield a gel or matrix with improved texture and flavor that resemble that of dairy cheese.
  • dairy cheese is an ancient practice that started as a way of preserving milk and turned into the development of fermented products with a broad range of flavors and interesting textures that are nowadays widely consumed on a regular basis.
  • the characteristic organoleptic properties of cheese are directly linked to the production processes, the performance of different microorganisms, and most importantly, the nature of dairy milk.
  • This colloidal dispersion of fat globules stabilized by casein micelles presents extremely particular behavior upon heat treatment or acidification, and its replication with plant raw materials a difficult challenge to overcome.
  • milk is heat-treated to ensure safety and inactivate endogenous microorganisms that could be present in the raw product.
  • proteins denature and unfold.
  • Microorganisms e.g. lactic acid bacteria
  • lactic acid bacteria are then inoculated and start fermenting the milk, namely transforming lactose into lactic acid, and thus acidifying the media.
  • This pH drop complements the action of rennet, encouraging proteins to interact with each other and start forming a protein network known as curd, that entraps fat globules in its pores and conforms the firm texture of cheese.
  • Milk casein is responsible for the formation of this three-dimensional network, and its characteristic molecular structure is responsible for its versatility to confer liquid but also gel-like textures. Accordingly, bacteria play several important roles, including dairy milk preservation through acidification to avoid spoilage, texture development through lactic acid production to encourage protein aggregation and curd formation, and flavor and aroma development through proteolysis and lipolysis.
  • current plant-based cheeseanalogues comprise additives, including oils, such as coconut oil, hydrocolloids, such as agar agar or carrageenans, or gums, such as guar, xanthan or Arabic gum.
  • oils such as coconut oil
  • hydrocolloids such as agar agar or carrageenans
  • gums such as guar, xanthan or Arabic gum.
  • the flavor profile of the currently commercialized plant-based cheese-analogues is achieved by cheese flavoring agents or by the characteristic flavor of the plant raw material.
  • the texture of currently available plant-based cheese-analogues does not meet the expectation of the consumers and the beany, nutty, or earthy aftertaste from plant raw material has low acceptance levels among consumers.
  • Fermentation of the plant-base has the potential to improve both texture and flavor of plant-based cheese-analogues.
  • Plant protein gels are three-dimensional protein networks that can be induced by fermentation. In this process, the liquid matrix is converted into a gel structure through protein aggregation that is characterized as soft matter, since it is composed of protein molecules that are dispersed in a liquid matrix, giving them a semi-solid consistency.
  • the textural and rheological properties of these gels can vary with pH, ionic strength, reducing agents, or protein concentration, among other factors.
  • the gelation dynamics of plant protein gels are dependent on the molecular structure of the proteins present in the gel.
  • the protein-protein interaction conditions gel strength, but also other factors such as the size of the protein aggregates, if the protein network is fine or coarse and how well-structured it is, and the degree of cross-linking between the proteins.
  • the potential of fermentation in flavor development is dual: it can reduce the off-flavor from the raw material and it can boost the intensity of acid and dairy like notes reminiscent of animal milk products.
  • the flavor profile of dairy cheese is formed by a complex mixture of very diverse volatile organic compounds (VOC) and it depends on factors such as VOC and precursors already present in milk and the metabolism of the bacteria that are fermenting it, among others.
  • VOC volatile organic compounds
  • VOC products of the carbohydrate metabolism are easier to obtain through plant protein matrices. Supplementation of those with simple sugars enables bacteria to convert them into diacetyl or acetoin as they do with lactose. VOC products of protein metabolism are produced after protein hydrolysis occurring during cheese maturation, and, as well as VOC products of fat metabolism, they are directly linked to the nature of those proteins and lipids that are present in animal milk.
  • Pea protein is an exceptional plant-base as a dairy alternative because it is easily digestible, rich in iron, and largely hypoallergenic. Fermentation of the pea-base has great potential to modify and improve the physicochemical and sensorial profile of proteins.
  • bacterial cultures comprising a blend of lactic acid bacteria which together facilitate fermentation of a plant base raw material, such as pea protein, into a plant-based product which provides the consumer with an organoleptic experience similar to that gained from dairy cheese.
  • the present invention relates to a method for fermentation of a pea-base raw material into a gel or matrix with a texture and flavor similar to that of a dairy cheese. Fermentation is performed by a carefully selected bacterial blend that comprises a combination of lactic acid bacteria which together in consortium provide a self- supporting three-dimensional gel with a firm texture and improved flavor.
  • the bacterial blends promote development of a flavor profile with volatile organic compounds (VOCs) of enhanced dairy-like notes and diminished plant raw material aftertaste.
  • VOCs volatile organic compounds
  • the present invention also provides pea-based cheese-analogues with improved firmness and flavor.
  • an object of the present invention relates to the provision of a method for preparing a plant-based cheese-analogue with an organoleptic experience resembling that of a dairy-based cheese.
  • Another object of the present invention relates to the provision of bacterial blends that promote development of dairy-like cheese texture and flavor of a pea-base.
  • an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • Another aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a still further aspect of the present invention relates to use of a bacterial blend in the production of a fermented pea-based cheese-analogue, wherein the bacterial blend comprises:
  • Figure 1 shows gel firmness of pea-base compositions fermented with single lactic acid bacteria strains at 30 °C and 40 °C.
  • Figure 3 shows acidification curves of the different 64 bacterial blends upon fermentation.
  • Figure 4 shows gel firmness quantified as positive penetration area of gels based on pea-base compositions fermented with each of the 64 bacterial blends.
  • Figure 5 shows principal component analysis (PCA) bi-plots of the first two principal components in pea-base compositions fermented with each of the 64 different bacterial blends.
  • PCA principal component analysis
  • Figure 7 shows levels of dairy-associated VOCs detected in pea-base compositions fermented with each of the 64 bacterial blends.
  • A diacetyl
  • B acetoin
  • C 2,3- pentanedione
  • D 3-methyl butanal
  • E 2-pentanone
  • F dimethyl-disulfide
  • G 2-heptanone
  • the term "dairy” refers to the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels.
  • plant-based cheese-analogue refers to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more milk constituents have been replaced with other ingredients and the resulting food resembles the original product.
  • the milk constituents are replaced completely or substantially with plant material, such as a pea-base comprising pea protein.
  • pea-base composition refers to the plant-based material used as a base for fermentation.
  • the pea-base composition comprises a peaprotein component, which can be, but is not limited to, a pea protein isolate, a pea protein concentrate, pea protein powder, or a pea protein flour.
  • the pea-base composition may comprise additional ingredients beneficial for the fermentation, such as sugars.
  • lactic acid bacteria refers to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, catalase negative, acid tolerant, non-motile, non- sporulating, microaerophilic or anaerobic bacteria.
  • lactic acid bacteria During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus generally responsible for the acidification of milk and for the texture of various dairy products. Beyond production of lactic acid, also acetic acid, formic acid and propionic acid are generated by lactic acid bacteria.
  • the industrially most useful lactic acid bacteria include, but are not limited to, Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Enterococcus spp.
  • Lactic acid bacteria may also be referred to by the abbreviation LAB.
  • the term “fermentation” or “fermenting” refers to a process wherein carbohydrates are transformed into a range of metabolites through chemical reactions carried out by microorganisms, such as a bacterial culture.
  • the carbohydrates are provided in a pea-base composition and the bacterial culture comprises one or more lactic acid bacteria.
  • lactic acid metabolites produced during fermentation include also volatile organic compound that can affect the flavour of the fermentation product.
  • VOCs Volatile organic compounds
  • volatile organic compound refers to low-molecular weight organic compounds with high vapor pressure, i.e. they evaporate easily at room temperature.
  • VOCs are metabolites produced by the bacterial cells and can contribute to all phases of tasting; odour, flavor and aftertaste.
  • Volatile organic compounds may also be referred to by the abbreviation VOC.
  • Pea protein is an exceptional plant-base as a dairy alternative because it is easily digestible, rich in iron, and largely hypoallergenic. Fermentation of the pea base has great potential to modify and improve the physicochemical and sensory properties of proteins, thus creating pleasant textures and flavors that might improve the quality of plant-based cheese-analogues.
  • Tailoring bacteria cultures comprising a blend of lactic acid bacteria which together facilitate fermentation of a plant base raw material into a final product with a texture and VOC profile that provides the consumer with an organoleptic experience similar to that gained from dairy cheese is a complex task. The interplay between multiple species of bacteria is hard to predict and careful selection is needed to realize the qualities of the individual species in a final balanced product.
  • bacterial blends which may be used advantageously in the fermentation of a pea-based composition to produce a fermented pea-base product with excellent texture and flavor.
  • the bacterial blends include species of the Lactobacillus genus.
  • Lactobacillus genus taxonomy was updated in 2020.
  • the new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if nothing else is noticed.
  • table 1 presents a list of new and old names of some Lactobacillus species relevant to the present invention.
  • the inventors have identified bacterial species with suitable acidification profiles that can contribute to gel firmness and improvement of flavor profile in plant-based protein gels.
  • the best performing strains were selected for a subsequent compounding to further design versatile combinations of strains that can improve texture and flavor in plant-based cheese analogues.
  • the lactic acid bacteria of the blends are selected so that none of the bacteria inadvertently suppress the proliferation and/or beneficial properties of the other bacteria in the blends.
  • some species was found to be favorable for development of a firm texture, while others contributed mainly to improvement of flavor through a desired VOC profile, including removal of off-flavor compounds and production of dairy-like compounds.
  • Texture developing species included Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei. Flavor developing species included Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:
  • the pea-base composition comprises pea proteins which during fermentation are crosslinked to provide a matrix- or gel-like structure.
  • the pea protein is typically extracted from yellow split peas and may be provided in several different forms.
  • One preferred form is pea protein isolate in which the outer shell of the peas is removed and the remainder is milled into a flour.
  • the flour then undergoes separation of fibers and starch in a filtration process and is finally distilled into a white precipitate known as pea protein isolate or analogously as pea protein powder.
  • the proteins in the pea protein component are mostly denatured during the purification process.
  • the amount of pea protein may be adjusted in accordance with the desired properties of the final fermented pea-based product.
  • the auxiliary culture comprises a Lactobacillus helveticus and a Pediococcus acidilactici.
  • the auxiliary culture comprises a Lactobacillus helveticus and a Lacticaseibacillus casei.
  • the auxiliary culture comprises a Pediococcus acidilactici and a Lactobacillus helveticus.
  • the auxiliary culture comprises a Pediococcus acidilactici and a Lacticaseibacillus casei.
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Pediococcus acidilactici.
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Lacticaseibacillus casei.
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lacticaseibacillus casei, and a Pediococcus acidilactici.
  • the auxiliary culture comprises a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei.
  • the first culture comprises a Lactiplantibacillus plantarum
  • the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei.
  • the S. thermophilus is S. thermophilus DSM 34725.
  • the Lacticaseibacillus rhamnosus is Lacticaseibacillus rhamnosus DSM 33870.
  • the Lactobacillus bulgaricus is Lactobacillus bulgaricus DSM 28910.
  • the Lactobacillus helveticus is Lactobacillus helveticus DSM 19499.
  • the Pediococcus acidilactici is Pediococcus acidilactici DSM 28307.
  • the Lacticaseibacillus casei is Lacticaseibacillus casei ATCC55544.
  • the first culture comprises a Lactiplantibacillus plantarum
  • the second culture comprises S. thermophilus DSM 34725 and Lacticaseibacillus rhamnosus DSM 33870
  • the auxiliary culture comprises Lactobacillus bulgaricus DSM 28910, Lactobacillus helveticus DSM 19499, Pediococcus acidilactici DSM 28307, and Lacticaseibacillus casei ATCC55544.
  • thermophilus and Lacticaseibacillus rhamnosus DSM 33870 thermophilus and Lacticaseibacillus rhamnosus DSM 33870
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei.
  • the first culture comprises a Lactiplantibacillus plantarum
  • the second culture comprises a S.
  • thermophilus and a Lacticaseibacillus rhamnosus thermophilus and a Lacticaseibacillus rhamnosus
  • the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, Pediococcus acidilactici DSM 28307, and a Lacticaseibacillus casei.
  • the first culture comprises a Lactiplantibacillus plantarum
  • the second culture comprises a S.
  • Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lentilactobacillus kefiri, the second culture comprises Streptococcus thermophilus, and the third culture comprises Lactococcus lactis.
  • the first culture comprises Lentilactobacillus kefiri
  • the second culture comprises Streptococcus thermophilus
  • the third culture comprises Lactococcus lactis.
  • the first culture comprises a Lentilactobacillus kefiri
  • the second culture comprises Streptococcus thermophilus DSM 34727, and a Lacticaseibacillus rhamnosus
  • the third culture comprises a Lactococcus lactis.
  • the first culture comprises Lentilactobacillus kefiri
  • the second culture comprises Streptococcus thermophilus DSM 19242, and a Lacticaseibacillus rhamnosus
  • the third culture comprises a Lactococcus lactis.
  • the first culture comprises a Lentilactobacillus kefiri
  • the second culture comprises a Streptococcus thermophilus and Lacticaseibacillus rhamnosus DSM 33870
  • the third culture comprises a Lactococcus lactis.
  • the first culture comprises a Lentilactobacillus kefiri
  • the second culture comprises a Streptococcus thermophilus and a Lacticaseibacillus rhamnosus
  • the third culture comprises Lactococcus lactis DSM 34729.
  • a further embodiment of the present invention relates to one of the following strains and composition comprising the strains, as well as and uses thereof for plant-based fermentation:
  • Bacterial metabolism and its enzymes are responsible for the breakdown of chemical compounds in the pea-base composition and their transformation into new products that contribute to the flavor profile of a fermented product.
  • Preferred were bacterial blends that produced higher levels of VOCs found in dairy cheese, such as diacetyl, 2,3- pentanone, acetoin and 3-methylbutanal.
  • Ketones diacetyl, acetoin and 2,3- pentandione
  • 3-methylbutanal may confer unripe, apple-like, sweet and fruity notes.
  • ketones which are commonly found in cheese, the content of which typically increases during ripening, and bring fruity-floral notes to the cheese as well as green, blue cheese (2-heptanone), and hot milk and musty (2-nonanone) aromas.
  • an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented peabased product prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3- pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2-heptanone, 2-nonanone, and 3-methyl butanal.
  • VOCs dairy-like volatile organic compounds
  • aldehydes 2,4-decadienal, hexanal, heptanal, 2-hexenal, 2-heptenal, octanal, 2-octenal, and pentanal are contemplated as major contributors to the characteristic beany flavor of pea.
  • higher levels of hexanal are considered to be one of the major compounds responsible for off-flavor in pea protein.
  • bacterial blends that can reduce these off-flavor VOCs. It has been found that the strains producing acetoins in the removal of off-flavor compounds.
  • One of the bacterial species inducing the lowest rates of beany VOCs was L. rhamnosus.
  • an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4-decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
  • VOCs volatile organic compounds
  • the method is not limited to a particular amount of bacterial blend for fermenting the pea-base composition.
  • the amount may be adjusted to vary the texture and flavor of the desired end product, e.g. some fermented pea-based cheese-analogues may benefit from higher amount of bacterial blend to build a stronger texture and flavor, whereas other products may be better suited with less of the bacterial blend to generate a softer texture and more mild flavor.
  • a preferred content of bacterial blend has been set at which texture and flavor is developed without using unnecessary high loads of bacteria.
  • an embodiment of the present invention relates to the method as described herein, wherein the content of bacterial blend is in the range of about 0.01 % (w/w) to about 0.1 % (w/w), with respect to the total weight of the fermented pea-based composition.
  • the pea-base composition is acidified during fermentation as the lactic acid bacteria converts carbohydrates into lactic acid and other metabolites. Lactic acid bacteria which induce fast acidification are preferred for food safety reasons. Fermentation of the peabase composition is continued until a predetermined pH is reached. This target pH value can be set, amongst others, based on the desired texture of the fermented pea-based product. Typically, the predetermined pH value will be less than pH 5 to realise the full benefits of the fermentation process where texture as well as flavour is developed. However, for preparation of less acidic products, a slightly higher pH, such as pH 5.5 may be preferred.
  • an embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.
  • Another embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is in the range of about pH 4 to about pH 5, such as about pH 4.3 to about pH 4.7.
  • a further embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is in the range of about pH 5 to about pH 5.5.
  • the pea-base composition has been transformed into a matrix or gel with mechanical textural attributes similar to a dairy cheese. This include a certain gel strength and firmness that keeps the product from collapsing under pressure, but also some elasticity as would be expected from a dairy cheese.
  • an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is in the form of a matrix or gel.
  • Another embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.
  • the form and texture of the fermented pea-based product is sustained by the pea protein network built during fermentation.
  • the gel formation is achieved without additives such as texturizers or thickening agents.
  • Producers of plant-based products, such as cheese-analogues, would like to avoid use of these additives because it would reduce cost and give a cleaner label.
  • an embodiment of the present invention relates to the method as described herein, wherein fermented pea-based product does not contain any texturizers.
  • Another embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, hydrocolloids and gums.
  • a further embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, agar agar, carrageenans, guar gum, xanthan gum and Arabic gum.
  • the fermented pea-based product obtained from the present method has firm texture and improved development of flavour with reduced off-taste caused by VOCs with beany notes.
  • a fermented pea-based product such as a cheeseanalogue
  • the fermented pea-based product, such as a cheese-analogue is achieved by use of a bacterial blend comprising a consortium of lactic acid bacteria with complementary properties yielding both firm texture and balanced flavor.
  • an aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.
  • Another aspect of the present invention relates to a bacterial blend comprising:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • Yet another aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • a further aspect of the present invention relates to use of a bacterial blend in the production of a fermented pea-based cheese-analogue, wherein the bacterial blend comprises:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • a method for producing a fermented pea-based product comprising the following steps:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus, and
  • the second culture comprises at least two different Streptococcus thermophilus strains, such as three different Streptococcus thermophilus strains, preferably two different Streptococcus thermophilus strains.
  • the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
  • the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lactis DSM 34729, Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
  • lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lact
  • the first culture comprises at least one lactic acid bacteria selected from the group consisting of: Lentilactobacillus kefiri DSM 34723, and Lactiplantibacillus plantarum DSM 34728.
  • the second culture comprises at least one lactic acid bacteria selected from the group consisting of: Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Streptococcus thermophilus DSM 34745 and Lacticaseibacillus rhamnosus DSM 33870.
  • Streptococcus thermophilus DSM 34745 Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
  • the third culture comprises at least one lactic acid bacteria selected from the group consisting of: Lactococcus lactis DSM 34729, and Levilactobacillus brevis DSM 34744.
  • the bacterial blend comprises the following lactic acid bacteria:
  • Streptococcus thermophilus DSM 19242 Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
  • Streptococcus thermophilus DSM 34726 Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
  • the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.
  • the pea protein component comprises at least about 50% (w/w) protein, such as at least about 60% (w/w) protein, such as at least about 70% (w/w) protein, preferably at least about 80% (w/w) protein, with respect to the total weight of the pea protein component.
  • X24 The method according to any one of items X22 or X23, wherein the pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the pea-base composition.
  • X25 The method according to any one of items X22-X24, wherein the pea-base composition has a content of pea protein component of about 5 % (w/w), with respect to the total weight of the pea-base composition.
  • X35 The method according to any one of items X32-X34, wherein the one or more sugars are sucrose and/or glucose. X35. The method according to any one of the preceding items, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial blend.
  • the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.
  • X44 The method according to item X43, wherein the texturizers are selected from the group consisting of coconut oil, hydrocolloids and gums.
  • X45 The method according to any one of items X43 or X44, wherein the texturizers are selected from the group consisting of coconut oil, agar agar, carrageenans, guar gum, xanthan gum and Arabic gum.
  • the fermented pea-based product comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
  • VOCs dairy-like volatile organic compounds
  • the fermented pea-based product comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4- decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
  • VOCs volatile organic compounds
  • a fermented pea-based product obtainable by the method according to any one of the preceding items.
  • a bacterial blend comprising:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • a fermented pea-based cheese-analogue comprising:
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • the fermented pea-based cheese-analogue according to item Zl wherein the fermented pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the fermented pea-base composition.
  • Streptococcus thermophilus DSM 34745 Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
  • Streptococcus thermophilus DSM 19242 Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
  • Streptococcus thermophilus DSM 34726 Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
  • the fermented pea-based cheese-analogue according to any one of items Z1-Z7, wherein the fermented pea-based cheese-analogue comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented peabased cheese-analogue prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
  • VOCs dairy-like volatile organic compounds
  • the fermented pea-based cheese-analogue according to any one of items Z1-Z8, wherein the fermented pea-based cheese-analogue comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based cheese-analogue prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4- decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
  • VOCs volatile organic compounds
  • the fermented pea-based cheese-analogue according to any one of items Z1-Z9, wherein the fermented pea-based cheese-analogue has a gel firmness in the range of about 200 g*ms to about 300 g*ms.
  • a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
  • a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
  • the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
  • the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lactis DSM 34729, Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
  • the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870
  • Lacticaseibacillus rhamnosus DSM 33870 Lacticaseibacillus rhamnosus DSM 33870
  • any one of items W1-W5 wherein the fermented pea-based cheese-analogue comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented pea-based cheese-analogue prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
  • VOCs dairy-like volatile organic compounds
  • VOCs volatile organic compounds
  • a bacterial strain in selected from the group consisting of:
  • Streptococcus thermophilus DSM 19242 Streptococcus thermophilus DSM 19242.
  • a pea-based cheese analogue comprising a bacterial strain selected from the group consisting of:
  • Streptococcus thermophilus DSM 34727 Streptococcus thermophilus DSM 19242.
  • This example describes how the fermented pea-based product can be produced by fermentation of a pea-base composition with lactic acid bacteria. The procedure was used both for test of single bacterial strains and for bacterial blends.
  • Pea protein isolate (ProFam®580, ADM, Chicago, IL, USA) with a composition of 81.3% protein, 7% fat, and 9% fiber was suspended at 5% w/w in a water solution of 1% w/w glucose- and 1% w/w sucrose (Sigma Aldrich, Soborg, Denmark) and mixed at 8,100 rpm with a mixer (L5M Laboratory Mixer, Silverson, Chesham, United Kingdom) for 2 min.
  • the suspension was emulsified with 5% sunflower oil (Ollineo, Budapest, Hungary) with the same mixer at 8,100 rpm for 2 min and was subjected to high pressure homogenization in an homogenizer (GEA Lab Homogenizer PandaPLUS 2000, GEA, Parma, Italy) at two stages (150; 50 bars) in one pass. Homogenized emulsions were then pasteurized at 90°C for 20 min while stirring in a water bath, stored at room temperature over night, pasteurized again under the same conditions and cooled down to 4°C , prior to microbial inoculation.
  • an homogenizer GEA Lab Homogenizer PandaPLUS 2000, GEA, Parma, Italy
  • lactic acid bacteria strains were sourced from Chr. Hansen 's Culture Collection (Chr. Hansen A/S, Horsholm, Denmark), comprising different Lactobacilli, Lactococci, Leuconostoc and Streptococcus thermophilus strains. All strains were cultured from a frozen glycerol stock in MRS pH 6.3-6.7, MRS pH 5.4 and M17 for 24 to 48 hours at 30°C, 37°C, and 40°C according to their suitable growth media and temperature.
  • the pea-base composition was stained with 5% pH colour indicator (1 : 1 wt% bromocresol purple salt and bromocresol green salt, Sigma Aldrich, St. Louis, US) and 990 pl stained pea-base composition was inoculated with 10 pl bacterial culture overnight in 96-well plates and incubated at 30°C and 40°C for 22 hours on top of flatbed scanners (HP ScanJet G4010). Colour changes were recorded every 6 min through Hue values with pH Multiscan software v.5.1 (HNH Consult Aps, 9530 Stovring, Denmark). The pH of the samples at end of fermentation was approx. 4.5. Samples were then stored overnight under refrigeration.
  • Fermentation with single strains (1% inoculum) and with strain blends was carried out in 2 different formats: 1 ml samples in 1 ml sterile 96-well microtiter plates (MTP) (Saveen Werner ApS, Limhamn, Sweden) for pH and texture measurements and 3 ml samples in headspace vials for analysis of targeted volatile compounds. Samples for acidification and texture measurements were incubated at 30 °C and 40 °C and samples for VOC analysis were incubated at 37 °C after analyzing the results from the previous two tests. After 20 hours of incubation, the samples were stored overnight under refrigeration. The acidification of the strain blends was measured using the same setup but at an incubation temperature of 37 °C.
  • the method produced 90 fermented pea-based gels that were stored and put forward for further evaluation with the aim of identifying lactic acid bacteria which individually contribute advantageously to the two main parameters; texture and flavor.
  • Example 2 First screening round - capacity of individual strains for promoting texture and flavor
  • Fermented pea-based gels through fermentation with 90 individual lactic acid bacteria strains were prepared as described in Example 1. Each of the gels were evaluated as described below.
  • the gel firmness of the gels in the 96-deepwell MTPs was analyzed after overnight storage under refrigeration using a penetration test with a Hamilton Star robot and custom-made metal micro-tools and a precision balance (Mettler Toledo, Columbus, Ohio, United States).
  • the MTPs were placed on the precision, and the micro-tools of 4 mm of plunger diameter penetrated 22 mm of each sample at a time.
  • the precision balance where the plates were located recorded the force resistance every 10 ms.
  • the obtained values time (ms) on the x-axis and force (g) on the y-axis) were plotted and the positive area under the curve was calculated for each replicate and used as a measurement for gel firmness.
  • VOCs volatile organic compounds
  • the GC oven program was as follows: 60 °C/2 min, Ramp 1 : 45 °C /min to 230 °C, hold 0.5 min. Identification of VOCs was based on retention time in comparison with that of the standards. Data were processed using Chromeleon software (Version 7.2.7, Thermo Engineer Inc., Denmark). Results were calculated as peak height divided by baseline noise (signal-to-noise, S/N).
  • lactic acid bacteria single strains were evaluated for their ability to ferment a peabase composition. The evaluation was based on acidification speed, contribution to texture, and volatile organic compound (VOC) profile, including removal of off-flavor ("beany" flavor) and production of dairy-like compounds.
  • VOC volatile organic compound
  • Non-inoculated control samples within the 96-well MTP showed no change of color from the initial blue color and therefore no capacity to lower the sample pH.
  • Samples inoculated with a bacterial strain turned light green, indicating a change of pH as a result of acidification.
  • Fast acidification correlates to the ability of the strains to produce lactic acid, and therefore to their capacity to metabolize carbon sources in the pea-base composition.
  • Fast acidification was prioritized as main criteria for the selection of the lead candidate strains. Because a fast pH drop could hinder any potential background growth, such as growth of endogenous flora. Therefore, fast deep acidifiers able to acidify to Hue values of 180 within 8 hours were selected. A few strains with lower acidification capacity, including L kefiri, were still included in the selection due to their good texturizing properties.
  • the gel firmness of the fermented gels was positively correlated to the acidification capacity of the strains.
  • the fast acidifying candidates contributed the most to the gel firmness with values mainly above 400 g*ms, whereas at 40 °C the vast majority of the samples presented values above 600 g*ms ( Figure 1) .
  • strains that contributed the most to gel firmness were of the species L. kefiri, L. paracasei, and L. plantarum (strains 19, 22 and 38, respectively). These strains were selected for the second screening stage as texturizing strains.
  • VOCs Volatile organic compounds
  • Diacetyl production was significantly increased in a set of S. thermophilus strains, as well in a few L. rhamnosus strains ( Figure 2A). Encouragingly, the L. kefiri strain that promoted texture also contributed to production of diacetyl. Samples fermented with other lactobacilli such as L. fermentum, L. bulgaricus, L. paracasei, or L. plantarum did not show significant levels of diacetyl after fermentation.
  • 3-methylbutanal was only detected in samples fermented with L. brevis, L. sanfranciscensis, or L. lactis. This aldehyde has been found in Gouda, Cheddar, and Camembert cheeses and described as a powerful malty and cheese odorant. Although the detected signal-to-noise (S/N) values for 3-methylbutanal were close to the limit of quantification (data not shown), they were included in the strain selection for their potential in the production of this compound when combined with other strains but also to embrace the diversity of species within the blend design.
  • S/N signal-to-noise
  • strains comprised one L. kefiri, one L. plantarum, one L. paracasei, six S. thermophilus, one L. rhamnosus, one L. brevis, one L. sanfranciscensis and one L. lactis.
  • the present application thus identifies the strains as well as its use in fermentation of plant-based matrices.
  • Example 3 Identification of advantageous blends of lactic acid bacteria strains
  • Fermented pea-based gels through fermentation with 64 different bacterial blends and 2 alternative bacterial blends were prepared (see Table 3 and Table 4) as described in Example 1, and a D-optimal model studied the effect of the factors, based on 192 runs including triplicates of each combination.
  • the pea-base composition was fermented in the same 96-well MTPs and headspace vials set-up as in the first screening step, but all samples were incubated at 37 °C for 20 h.
  • Table 3 Overview of the compounding of lactic acid bacteria strains in the 64 different bacterial blends for screening.
  • Table 4 Overview of the compounding of lactic acid bacteria strains in the 2 alternative bacterial blends for screening.
  • the volatile organic compounds in the samples fermented with the designed blends were analyzed after fermenting 3 ml of pea-base composition directly in a 20 ml headspace vial together with the strain blends.
  • Samples were prepared in duplicates and then analyzed by headspace solid phase microextraction gas chromatography coupled to mass spectrometry (HS-SPME-GC-MS) after one week of storage under refrigeration.
  • the instrument was a Multi Purpose Sampler (Gerstel, MSCI, Skovlunde, Denmark), with a 7890B GO (Agilent Technologies, Denmark) and a 5977A MS (Agilent Technologies, Denmark).
  • VOCs were extracted by SPME using a DVB/Car/PDMS-fiber (Supelco 57299, VWR, Denmark) for 20 min at 60 °C, desorbed splitless at 270 °C on a TenaxTA-filled liner (Gerstel 012438, MSCI, Skovlunde, Denmark) kept at -30 °C. After fiber desorption, the TenaxTA-filled liner was heated to 300 °C and the trapped VOCs were transferred splitless and separated on a DB-5MS UI column 30m x 0.25mm x 1 pm (Agilent 122- 5533UI, Agilent Technologies, Denmark) at 170 kPa constant pressure using helium as carrier gas.
  • Oven temperature program was as follows: starting at 32 °C/2min - increased to 102°C at 10 °C/min - further increased to 145°C at 5 °C/min - further increased to 200 °C at 15 °C/min - further increased to 200 °C at 15 °C/min - further increased to 280 °C at 20 °C/min - hold at 280 °C for 5 min.
  • the mass spectrometer operated in electron impact mode at -70eV and the analyzer was scanning from 29-209 amu.
  • PCA Principal Component Analysis
  • VOC volatile organic compound
  • Example 2 In the first round of the screening (Example 2), the majority of slow-acidifying strains were discarded to obtain a fast pH drop and avoid background growth. Cultures, namely mixes of different single strains, are more robust and beneficial for fast acidification than single strains, and therefore their acidification capacity is more stable. This assumption was confirmed by the acidification curves of the 64 bacterial blends ( Figure 3), which all showed relatively fast acidification and within the acceptable spectrum where background growth is minimised.
  • the acidification curves show the blends being separated into two overall groupings, although all of them show similar acidification patterns with a pH drop after around 6 hours of incubation.
  • the upper grouping includes curves with a value above 170 hue after 10 hours of incubation, whereas the lower grouping (highlighted by circle) contained the samples that presented values below 170 hue after 10 hours of incubation.
  • strain STH89 was only present in the lower grouping samples, suggesting that this strain significantly improves acidification speed. Consequently, the pH drop occurs 2-3 hours earlier in the lower grouping than in the higher grouping, presenting a marked advantage in terms of food safety and process optimization.
  • the positive area of the curve measured by the HTP penetration test was taken as a measurement of gel hardness or firmness - the higher the value, the harder the consistency of the sample.
  • the hardness was defined as the resistance of the sample to penetration.
  • VOCs Volatile organic compounds
  • PCA principal component analysis
  • dairy-associated VOCs such as diacetyl, acetoin, or 2,3-pentanedione, among other metabolites, were found in the upper-right quadrant, where also many of the blends can be found in the scores plot, while the pea-base composition and the chemically-acidified sample were located in the opposite side of the Y axis. This observation emphasizes the need of fermentation for producing dairy notes from the pea-base composition.
  • Blend 6 comprising L. kefiri performed well in the removal of all beany VOCs ( Figure 6A-H).
  • Blends 62, 63 and 64 performed significantly good at producing compounds such as diacetyl, 2-pentanone, acetoin, and 2-heptanone.
  • Dimethyl disulfide, an important fraction of the Cheddar cheese flavor was also produced in high levels by blend 63 and 64. All three blends contain L. rhamnosus, suggesting its importance when combined with S. thermophilus.
  • Blends B6 and A2 along the positive side of SC2 was attributed to higher contents of acetic acid, while Al and B64 blends, located on the negative side of SC2, had higher concentrations of 2,3- pentanedione, acetoin, diacetyl and benzaldehyde.
  • Acetoin, diacetyl and acetoin are normally associated with fermented dairy aromas (Reyes-Diaz et al., 2020), which confirms that blend B64 produces cheese-related VOCs, as previously observed (Masia, Fernandez-Varela, Poulsen, et al., 2023).
  • alcohols exhibit higher odor thresholds than aldehydes e.g., 1-hexanol has a detection threshold of 500 ppb in the water (Guadagni et al., 1963) while that of hexanal is 4.5 ppb in water (Flath et al., 1967), therefore their overall contribution to product odor is lower. Compared to the other blends, this higher degradation of aldehydes could result in a lower intensity of undesirable beany- related odors in B6 samples.
  • Samples fermented using A2 were located in the opposite quadrant to samples fermented with Al in the scores plot.
  • Samples fermented with Al had the highest concentrations of 2,3-pentanedione, a desirable VOC associated with dairy notes (Cheng 2010), while samples fermented by A2 had higher levels of acetic acid, a compound that can impart vinegary notes but that at low levels has been associated with the pleasant aroma of Cheddar cheese (Murtaza et al., 2014).
  • Beany flavor in pea protein Recent advances in formation mechanism, analytical techniques and microbial fermentation mitigation strategies. Food Bioscience, 56, 103166. https://doi.Org/10.1016/j.fbio.2023.103166.
  • fermented pea-based products with great texture and excellent flavor profile could be achieved through fermentation with compounded bacterial blends with complementing attributes.
  • Results also showed that the chosen bacterial blends produced desirable dairy-related VOCs, such as diacetyl and acetoin, especially by B64 and A2.
  • the deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany.
  • the strain Lactobacillus paracasei subsp. paracasei (also referred to as Lacticaseibacillus paracasei, Lacticaseibacillus casei, CRL431 and L. casei 431®) was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Tissue type Collection Center, 10801 University Boulevard, Manassas, VA 20110, USA on 24 January 1994 under accession number ATCC 55544.
  • the well-known probiotic bacterium is commercially available from Chr. Hansen A/S, 10-12 Boege Alle, DK-2970 Hoersholm, Denmark, under the product name Probio-Tec® F-DVS L. casei-431®, Item number 501749, and under the product name Probio-Tec® C-Powder-30, Item number 687018.

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Abstract

The present invention relates to a method for improving the organoleptic experience of a plant-based cheese-analogue, and the resulting products as such. In particular, bacterial blends that through fermentation of a plant base yield a gel or matrix with improved texture and flavor that resemble that of dairy cheese. The present application also provides bacterial strains which contribute to gel firmness or improve of flavor profile in plant-based

Description

NEW STRAINS AND BLENDS FOR TEXTURE AND FLAVOR IN PLANT-BASED CHEESE-ANALOGUES
Technical field of the invention
The present invention relates to a method for improving the organoleptic experience of a plant-based cheese-analogue, and the resulting products as such. In particular, bacterial blends that through fermentation of a pea-base yield a gel or matrix with improved texture and flavor that resemble that of dairy cheese.
Background of the invention
The production of dairy cheese is an ancient practice that started as a way of preserving milk and turned into the development of fermented products with a broad range of flavors and interesting textures that are nowadays widely consumed on a regular basis. The characteristic organoleptic properties of cheese are directly linked to the production processes, the performance of different microorganisms, and most importantly, the nature of dairy milk. This colloidal dispersion of fat globules stabilized by casein micelles presents extremely particular behavior upon heat treatment or acidification, and its replication with plant raw materials a difficult challenge to overcome.
In the production of dairy cheese, milk is heat-treated to ensure safety and inactivate endogenous microorganisms that could be present in the raw product. During this process, proteins denature and unfold. Microorganisms, e.g. lactic acid bacteria, are then inoculated and start fermenting the milk, namely transforming lactose into lactic acid, and thus acidifying the media. This pH drop complements the action of rennet, encouraging proteins to interact with each other and start forming a protein network known as curd, that entraps fat globules in its pores and conforms the firm texture of cheese. Milk casein is responsible for the formation of this three-dimensional network, and its characteristic molecular structure is responsible for its versatility to confer liquid but also gel-like textures. Accordingly, bacteria play several important roles, including dairy milk preservation through acidification to avoid spoilage, texture development through lactic acid production to encourage protein aggregation and curd formation, and flavor and aroma development through proteolysis and lipolysis.
The demand for plant-based alternatives to dairy cheese is exponentially growing, mostly due to an increasing awareness of sustainability and the large amount of resources that animal protein production requires, and consumers are looking for products that can provide a similar experience to eating dairy cheese but are based on plant-based raw materials. However, the structure of plant proteins differs from those of dairy proteins and so does their behavior under fermentation conditions. Moreover, the available macronutrients in plant matrices are also different from those in dairy milk, and consequently, the flavor compounds produced by bacteria will also be different.
Accordingly, two of the most important challenges in plant-based cheese-analogue production are texture and flavor development. Producers trying to replicate dairy cheese mostly rely on functional ingredients such as texturizers and flavoring agents for the development of these attributes.
To obtain a texture characteristic for dairy cheese, current plant-based cheeseanalogues comprise additives, including oils, such as coconut oil, hydrocolloids, such as agar agar or carrageenans, or gums, such as guar, xanthan or Arabic gum. The flavor profile of the currently commercialized plant-based cheese-analogues is achieved by cheese flavoring agents or by the characteristic flavor of the plant raw material. However, the texture of currently available plant-based cheese-analogues does not meet the expectation of the consumers and the beany, nutty, or earthy aftertaste from plant raw material has low acceptance levels among consumers.
Fermentation of the plant-base has the potential to improve both texture and flavor of plant-based cheese-analogues.
One of the issues for development of texture is that the gel strength is generally weaker in plant protein systems than in dairy curds, and therefore the textural and rheological properties of these plant protein gels remain far from those of dairy curds and cheeses. Plant protein gels are three-dimensional protein networks that can be induced by fermentation. In this process, the liquid matrix is converted into a gel structure through protein aggregation that is characterized as soft matter, since it is composed of protein molecules that are dispersed in a liquid matrix, giving them a semi-solid consistency. The textural and rheological properties of these gels can vary with pH, ionic strength, reducing agents, or protein concentration, among other factors. Moreover, the gelation dynamics of plant protein gels are dependent on the molecular structure of the proteins present in the gel. The protein-protein interaction conditions gel strength, but also other factors such as the size of the protein aggregates, if the protein network is fine or coarse and how well-structured it is, and the degree of cross-linking between the proteins. The potential of fermentation in flavor development is dual: it can reduce the off-flavor from the raw material and it can boost the intensity of acid and dairy like notes reminiscent of animal milk products. The flavor profile of dairy cheese is formed by a complex mixture of very diverse volatile organic compounds (VOC) and it depends on factors such as VOC and precursors already present in milk and the metabolism of the bacteria that are fermenting it, among others. Therefore, replicating such a complex VOC mixture with different starting raw materials is a challenge since the precursors of the typical VOC of dairy cheese might not be found in plant protein matrices. VOC products of the carbohydrate metabolism are easier to obtain through plant protein matrices. Supplementation of those with simple sugars enables bacteria to convert them into diacetyl or acetoin as they do with lactose. VOC products of protein metabolism are produced after protein hydrolysis occurring during cheese maturation, and, as well as VOC products of fat metabolism, they are directly linked to the nature of those proteins and lipids that are present in animal milk.
While starter cultures for plant materials are currently available in the market, these bacterial ingredients target plant proteins in general and are not specifically designed for either product type or plant source. However, the bacterial strains required for a plant-based cheese-analogue might not be same as for that of a plant-based yoghurtanalogue. Moreover, bacterial strains might perform differently depending on the plantbase substrate it grows in and is fermenting.
One plant source which is gaining much interest for development of new sustainable food products is pea. Pea protein is an exceptional plant-base as a dairy alternative because it is easily digestible, rich in iron, and largely hypoallergenic. Fermentation of the pea-base has great potential to modify and improve the physicochemical and sensorial profile of proteins.
However, as outlined above, it remains difficult to predict the combination of bacterial strains needed to provide a balanced product within a specific food category, such as cheeses, when based on a particular plant source, such as pea. For instance, the contribution of lactic acid bacteria to flavor development depends largely on strain metabolism and the compounds that are available in the starting plant raw material.
For these reasons there is a clear need for identifying favourable combinations or blends of lactic acid bacteria capable of transforming plant-bases, such as pea protein, into plant-based food products, such as plant-based cheese-analogues, with improved texture and flavor.
Thus, it would be advantageous to provide bacterial cultures comprising a blend of lactic acid bacteria which together facilitate fermentation of a plant base raw material, such as pea protein, into a plant-based product which provides the consumer with an organoleptic experience similar to that gained from dairy cheese.
Moreover, it would be advantageous to provide a method which yields fermented peabased products with excellent texture and flavor mimicking that of a dairy cheese, and with the resulting products being devoid of texturizers and flavor additives.
Summary of the invention
The development of plant-based alternatives to dairy-based cheeses is driven mainly by the sustainability agenda and the awareness of reducing the resource burden required by animal protein production. However, the plant-based cheese-analogue options available on the market today fail to meet the organoleptic expectations of the consumers. In particular, presently available plant-based cheese-analogues lack firmness and have beany, nutty, or earthy aftertaste.
The present invention relates to a method for fermentation of a pea-base raw material into a gel or matrix with a texture and flavor similar to that of a dairy cheese. Fermentation is performed by a carefully selected bacterial blend that comprises a combination of lactic acid bacteria which together in consortium provide a self- supporting three-dimensional gel with a firm texture and improved flavor. In particular, the bacterial blends promote development of a flavor profile with volatile organic compounds (VOCs) of enhanced dairy-like notes and diminished plant raw material aftertaste. The present invention also provides pea-based cheese-analogues with improved firmness and flavor.
Therefore, an object of the present invention relates to the provision of a method for preparing a plant-based cheese-analogue with an organoleptic experience resembling that of a dairy-based cheese.
Another object of the present invention relates to the provision of bacterial blends that promote development of dairy-like cheese texture and flavor of a pea-base. Thus, an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:
(i) providing a pea-base composition,
(ii) adding a bacterial blend to the pea-base composition, said bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus, and
(iii) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.
Another aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.
Yet another aspect of the present invention relates to a bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
A further aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:
- a fermented pea-base composition, and bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and
Lacticaseibacillus rhamnosus. A still further aspect of the present invention relates to use of a bacterial blend in the production of a fermented pea-based cheese-analogue, wherein the bacterial blend comprises:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
An even further aspect of the present invention relates to a bacterial strain selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
Brief Description of the Figures
Figure 1 shows gel firmness of pea-base compositions fermented with single lactic acid bacteria strains at 30 °C and 40 °C.
Figure 2 shows levels of (A) diacetyl, (B) 2,3-pentanedione, and (C) acetoin present in pea-base compositions fermented with single lactic acid bacteria strains. Results are reported as signal-to-noise (S/N) values. The "B" sample corresponds to an unfermented pea-base composition. The "C" sample corresponds to an unfermented pea-base composition which has been chemically acidified.
Figure 3 shows acidification curves of the different 64 bacterial blends upon fermentation. Figure 4 shows gel firmness quantified as positive penetration area of gels based on pea-base compositions fermented with each of the 64 bacterial blends.
Figure 5 shows principal component analysis (PCA) bi-plots of the first two principal components in pea-base compositions fermented with each of the 64 different bacterial blends. (A) Scores plot labelled by the different bacterial blends including the unfermented base and a chemically acidified sample. (B) Loadings plot labelled by the different detected VOCs in all samples.
Figure 6 shows levels of beany VOCs detected in pea-base compositions fermented with each of the 64 bacterial blends. (A) 2,4-decadienal, (B) 2-octenal, (C) pentenal, (D) 2- hexenal, (E) hexanal, (F) heptanal, (G) 2-heptenal, and (H) octanal.
Figure 7 shows levels of dairy-associated VOCs detected in pea-base compositions fermented with each of the 64 bacterial blends. (A) diacetyl, (B) acetoin, (C) 2,3- pentanedione, (D) 3-methyl butanal, (E) 2-pentanone, (F) dimethyl-disulfide, and (G) 2-heptanone.
Detailed description of the invention
Definitions
Prior to outlining the present invention in more details, a set of terms and conventions is first defined:
Dairy
In the present context, the term "dairy" refers to the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels.
The terms "dairy" and "milk" may be used interchangeably herein. Thus, for most practical purposes, the term "dairy-based" products (or cheese) refers to products (or cheese) obtained from cow's milk.
Plant-based cheese-analogues
In the present context, the term "plant-based cheese-analogue" refers to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more milk constituents have been replaced with other ingredients and the resulting food resembles the original product. The milk constituents are replaced completely or substantially with plant material, such as a pea-base comprising pea protein.
Pea -base composition
In the present context, the term "pea-base composition" refers to the plant-based material used as a base for fermentation. The pea-base composition comprises a peaprotein component, which can be, but is not limited to, a pea protein isolate, a pea protein concentrate, pea protein powder, or a pea protein flour. The pea-base composition may comprise additional ingredients beneficial for the fermentation, such as sugars.
Lactic acid bacteria
In the present context, the term "lactic acid bacteria" refers to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, catalase negative, acid tolerant, non-motile, non- sporulating, microaerophilic or anaerobic bacteria.
During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus generally responsible for the acidification of milk and for the texture of various dairy products. Beyond production of lactic acid, also acetic acid, formic acid and propionic acid are generated by lactic acid bacteria.
The industrially most useful lactic acid bacteria include, but are not limited to, Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Enterococcus spp.
Lactic acid bacteria may also be referred to by the abbreviation LAB.
Fermentation
In the present context, the term "fermentation" or "fermenting" refers to a process wherein carbohydrates are transformed into a range of metabolites through chemical reactions carried out by microorganisms, such as a bacterial culture. Herein, the carbohydrates are provided in a pea-base composition and the bacterial culture comprises one or more lactic acid bacteria. Besides lactic acid, metabolites produced during fermentation include also volatile organic compound that can affect the flavour of the fermentation product.
Volatile organic compounds (VOCs)
In the present context, the term "volatile organic compound" refers to low-molecular weight organic compounds with high vapor pressure, i.e. they evaporate easily at room temperature. VOCs are metabolites produced by the bacterial cells and can contribute to all phases of tasting; odour, flavor and aftertaste.
Volatile organic compounds may also be referred to by the abbreviation VOC.
About
Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ± 10% about the number 10, which is anything between 9% and 11%).
New bacterial strain blends
Two of the main hurdles in manufacture of plant-based cheese-analogues are to produce a product with texture and flavor resembling that of a corresponding dairy cheese. Current efforts typically include addition of texturizers and flavoring agents, but the resulting mechanical textural attributes and taste do not meet the expectations of the consumers. Thus, a sustainable and improved organoleptic offering to the consumers is needed to attain a more sizable commitment to plant-based cheese-analogues.
Therefore, there is an increasing interest in new ways of producing gels where the plant proteins are the main structural units of the gel structure. Pea protein is an exceptional plant-base as a dairy alternative because it is easily digestible, rich in iron, and largely hypoallergenic. Fermentation of the pea base has great potential to modify and improve the physicochemical and sensory properties of proteins, thus creating pleasant textures and flavors that might improve the quality of plant-based cheese-analogues. Tailoring bacteria cultures comprising a blend of lactic acid bacteria which together facilitate fermentation of a plant base raw material into a final product with a texture and VOC profile that provides the consumer with an organoleptic experience similar to that gained from dairy cheese is a complex task. The interplay between multiple species of bacteria is hard to predict and careful selection is needed to realize the qualities of the individual species in a final balanced product.
Herein are identified bacterial blends which may be used advantageously in the fermentation of a pea-based composition to produce a fermented pea-base product with excellent texture and flavor. The bacterial blends include species of the Lactobacillus genus.
It will be appreciated that the Lactobacillus genus taxonomy was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if nothing else is noticed. For the purpose of the present invention, table 1 presents a list of new and old names of some Lactobacillus species relevant to the present invention.
Figure imgf000012_0001
Table 1. New and old names of some Lactobacillus species relevant to the present invention.
The inventors have identified bacterial species with suitable acidification profiles that can contribute to gel firmness and improvement of flavor profile in plant-based protein gels. The best performing strains were selected for a subsequent compounding to further design versatile combinations of strains that can improve texture and flavor in plant-based cheese analogues. The lactic acid bacteria of the blends are selected so that none of the bacteria inadvertently suppress the proliferation and/or beneficial properties of the other bacteria in the blends. In particular, some species was found to be favorable for development of a firm texture, while others contributed mainly to improvement of flavor through a desired VOC profile, including removal of off-flavor compounds and production of dairy-like compounds. Texture developing species included Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei. Flavor developing species included Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
Thus, an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:
(i) providing a pea-base composition,
(ii) adding a bacterial blend to the pea-base composition, said bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus, and
(iii) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.
The pea-base composition comprises pea proteins which during fermentation are crosslinked to provide a matrix- or gel-like structure. The pea protein is typically extracted from yellow split peas and may be provided in several different forms. One preferred form is pea protein isolate in which the outer shell of the peas is removed and the remainder is milled into a flour. The flour then undergoes separation of fibers and starch in a filtration process and is finally distilled into a white precipitate known as pea protein isolate or analogously as pea protein powder. The proteins in the pea protein component are mostly denatured during the purification process. The amount of pea protein may be adjusted in accordance with the desired properties of the final fermented pea-based product.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.
Another embodiment of the present invention relates to the method as described herein, wherein the pea protein component comprises at least about 50% (w/w) protein, such as at least about 60% (w/w) protein, such as at least about 70% (w/w) protein, preferably at least about 80% (w/w) protein, with respect to the total weight of the pea protein component.
A further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the pea-base composition.
A still further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition has a content of pea protein component of about 5 % (w/w), with respect to the total weight of the pea-base composition.
The pea-base composition is a viscoelastic material, preferably in liquid form, and may comprise additional ingredients in addition to the pea protein component. Oil may be included in the pea-base composition to mimic the fat contained in dairy cheese, thereby improving the mouthfeel of the final product. The oil may be in liquid form at room temperature, i.e. have a melting temperature below room temperature, such as below 0 °C. Examples of such oils include, but are not limited to, sunflower oil and rapeseed oil.
Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is a viscoelastic material.
Another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is in liquid form.
Yet another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is in the form of a colloidal suspension.
Still another embodiment of the present invention relates to the method as described herein, wherein the colloidal suspension is an emulsion or sol, preferably an emulsion.
A further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises one or more oils or fats. An even further embodiment of the present invention relates to the method as described herein, wherein the one or more oils or fats are liquid at room temperature and/or have a melting temperature of less than 0°C.
Sugars can be added to the pea-base composition to induce fermentation. They act as carbohydrate substrates in the fermentation reaction and as nutrient for increased microbial proliferation. The method is not limited to any specific sugars or amount thereof.
Another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises one or more sugars.
Another embodiment of the present invention relates to the method as described herein, wherein the content of sugar is in the range of about 0.5 % (w/w) to about 5 % (w/w), such as about 1 % (w/w) to about 3 % (w/w), with respect to the total weight of the pea-base composition.
A further embodiment of the present invention relates to the method as described herein, wherein the one or more sugars are monosaccharides and/or disaccharides.
A still further embodiment of the present invention relates to the method as described herein, wherein the one or more sugars are sucrose and/or glucose.
Heat treatment may be applied prior to fermentation to ensure further denaturation in case some proteins of the pea protein component as supplied would only be partially denatured. Heat treatment induces protein unfolding which exposes the hydrophobic domains that are normally hidden inside the protein structure. With a gradual acidification occurring during fermentation, the electrostatic repulsion between proteins is reduced, and these start interacting with each other forming a protein network.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial blend.
Another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is subjected to heat treatment at a temperature sufficient for denaturing the pea proteins. Still another embodiment of the present invention relates to the method as described herein, wherein said heat treatment is performed at a temperature of at least about 80°C, such as at least about 85°C, preferably about 90°C.
An even further embodiment of the present invention relates to the method as described herein, wherein the heat treatment is performed for at least about 5 min, such as at least about 10 min, such as at least about 15 min, preferably at least about 20 min.
All bacterial blends comprising one of the identified texturizing species (first culture) performed well and produced fermented products with a firmness resembling that of dairy-based cheese. Thus, it appears sufficient to include a single of the texturizing species. Lentilactobacillus kefiri was the texturizing species encompassed in the most preferred bacterial blends and contributed also to flavor by production of VOCs such as diacetyl and acetoin.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the first culture comprises only a single lactic acid bacteria strain.
Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises a Lentilactobacillus kefiri strain.
A further embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product has a gel firmness in the range of about 200 g*ms to about 300 g*ms.
The fermentation with the bacterial blend promotes development of an ensemble of volatile organic compounds (VOCs) resulting in a fermented product with a flavor resembling that of a corresponding dairy-based cheese. In particular, this is achieved by detecting a broad spectrum of VOCs and selecting the bacterial blends which best removed off-flavor, especially green and beany notes, and induced formation of dairy notes compounds such as cheesy and buttery. These selected compounds comprised aldehydes, ketones, esters, furans, and sulphur derivative compounds. The beany flavor may result from a combination of different sensorial attributes, as combination of mold, earthy, green, and fresh pea. The beany, green and/or grassy flavor of pea protein is mostly characterized by the aldehydes hexanal, 2-hexenal, heptanal, 2-heptenal, octanal, 2-octenal, pentanal, and 2,4-decadienal. Therefore, the degradation of these VOCs through fermentation is of great interest for plant-based cheese-analogues production.
The bacterial blends presented herein comprise combinations of lactic acid bacteria which together upon fermentation of the pea-base composition produce a flavor with excellent cheesy notes and minimal aftertaste. These "flavoring" lactic acid bacteria are included in the bacterial as a second culture to supplement the first culture comprising the texturizing strains. It has been found that the combination of Streptococcus thermophilus and Lacticaseibacillus rhamnosus is particularly good for flavor development.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least one Streptococcus thermophilus strain.
Another embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least two different Streptococcus thermophilus strains, such as three different Streptococcus thermophilus strains, preferably two different Streptococcus thermophilus strains.
Yet another embodiment of the present invention relates to the method as described herein, wherein the second culture comprises a Lacticaseibacillus rhamnosus strain.
A further embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least two different lactic acid bacteria strains, preferably three different lactic acid bacteria strains.
A still further embodiment of the present invention relates to the method as described herein, wherein the second culture comprises two different Streptococcus thermophilus strains and a Lacticaseibacillus rhamnosus strain.
The bacterial blend may further comprise one or more lactic acid bacteria that produce 3-methylbutanal, which is an aldehyde derived from isoleucine and leucine providing malty and nutty notes. This VOC is known to be present in dairy cheese, at it is therefore contemplated that bacteria promoting 3-methylbutanal may be favourable for the flavor perception of the fermented pea-based product. Herein are identified bacterial species which induce production of 3-methylbutanal while at the same time work well in consortium with the texturizing and flavoring bacterial cultures.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
Another embodiment of the present invention relates to the method as described herein, wherein the third culture comprises a Lactococcus lactis strain.
A further embodiment of the present invention relates to the method as described herein, wherein the third culture comprises only a single lactic acid bacteria strain.
The present invention has also identified specific lactic acid bacteria strains that have been demonstrated as particularly beneficial for producing a dairy-like cheese product based on a pea-base composition. The strains improve the key parameters, including firmer texture, less off-taste VOCs and more VOCs with cheesy and buttery notes.
Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
The most preferred texturizing strains were capable of producing a firm fermented peabased product irrespective of the other strains included in the bacterial blends. A single Lentilactobacillus kefiri strain was encompassed in the most preferred bacterial blends, and thus it is contemplated that this particular strain may also contribute to flavor development when used in combination with some flavoring strains. Thus, an embodiment of the present invention relates to the method as described herein, wherein the first culture comprises at least one lactic acid bacteria selected from the group consisting of:
Lentilactobacillus kefiri DSM 34723, and Lactiplantibacillus plantarum DSM 34728.
A preferred embodiment of the present invention relates to the method as described herein, wherein the first culture comprises the Lentilactobacillus kefiri strain deposited as DSM 34723.
The best flavoring strains were found among the species Streptococcus thermophilus and Lacticaseibacillus rhamnosus. Several S. thermophilus strains were found to produce a favorable VOC profile with contribution to increased levels of diacetyl, 2,3- pentadione and/or acetoin. The identified L. rhamnosus strain worked together with different S. thermophilus strains to contribute amongst others with induction of acetoin levels.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least one lactic acid bacteria selected from the group consisting of:
Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Streptococcus thermophilus DSM 34745 and Lacticaseibacillus rhamnosus DSM 33870.
Another embodiment of the present invention relates to the method as described herein, wherein the second culture comprises the Lacticaseibacillus rhamnosus strain deposited as DSM 33870.
A further embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least one Streptococcus thermophilus strain selected from the group consisting of:
Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
A still further embodiment of the present invention relates to the method as described herein, wherein the second culture comprises at least one Streptococcus thermophilus strain selected from the group consisting of:
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
A Lactococcus lactis strain was found to be a good contributor of 3-methylbutanal and was included in several of the most preferred bacterial blends. It is contemplated that this particular strain is beneficial in particular in combination with the identified S. thermophilus and L. rhamnosus strains.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the third culture comprises at least one lactic acid bacteria selected from the group consisting of:
Lactococcus lactis DSM 34729, and
Levilactobacillus brevis DSM 34744.
Another embodiment of the present invention relates to the method as described herein, wherein the third culture comprises the Lactococcus lactis strain deposited as DSM 34729.
Some particularly favourable bacterial blends has been identified as part of the screening process. These blends comprise a mix of texturizing strains and flavoring strains that contribute to the production of VOCs providing cheesy notes and reduction of VOCs with off-flavors.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 19242,
Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729. Another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 34726, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
Other bacterial blends which provided fermented pea-based products with good characteristics were also identified. The resulting products all had great texture resembling dairy cheese and cheesy flavours.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Levilactobacillus brevis DSM 34744,
- Lentilactobacillus kefiri DSM 34723,
- Streptococcus thermophilus DSM 34727, and
- Streptococcus thermophilus DSM 19242.
Another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Levilactobacillus brevis DSM 34744,
- Lactiplantibacillus plantarum DSM 34728,
- Streptococcus thermophilus DSM 34745, and
- Streptococcus thermophilus DSM 19242.
Still another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Levilactobacillus brevis DSM 34744,
- Lentilactobacillus kefiri DSM 34723,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 19242.
A further embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Levilactobacillus brevis DSM 34744,
- Lentilactobacillus kefiri DSM 34723,
- Lacticaseibacillus rhamnosus DSM 33870, - Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 19242.
A still further embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Levilactobacillus brevis DSM 34744,
- Lactiplantibacillus plantarum DSM 34728,
- Lacticaseibacillus rhamnosus DSM 33870,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 34727.
An even further embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lactiplantibacillus plantarum DSM 34728,
- Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34745,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 34727.
Another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lactiplantibacillus plantarum DSM 34728,
- Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34726,
- Streptococcus thermophilus DSM 34727, and
- Streptococcus thermophilus DSM 19242.
Yet another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lentilactobacillus kefiri DSM 34723,
- Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 34727.
Still another embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lentilactobacillus kefiri DSM 34723, - Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34745,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 19242.
A further embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lentilactobacillus kefiri DSM 34723,
- Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34726, and
- Streptococcus thermophilus DSM 19242.
A still further embodiment of the present invention relates to the method as described herein, wherein the bacterial blend comprises the following lactic acid bacteria:
- Lactiplantibacillus plantarum DSM 34728,
- Lactococcus lactis DSM 34729,
- Streptococcus thermophilus DSM 34726,
- Streptococcus thermophilus DSM 34727, and
- Streptococcus thermophilus DSM 19242.
Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lactiplantibacillus plantarum, and the second culture comprises Streptococcus thermophilus.
Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lactiplantibacillus plantarum, and the second culture comprises Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lactiplantibacillus plantarum, and the second culture comprises Lacticaseibacillus rhamnosus.
Yet another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lactiplantibacillus plantarum, the second culture comprises S. thermophilus and/or Lacticaseibacillus rhamnosus, and the bacterial blend further comprises an auxiliary culture, the auxiliary culture comprising one or more lactic acid bacteria selected from the group consisting of Lactobacillus bulgaricus, Lactobacillus helveticus, Pediococcus acidilactici, and Lacticaseibacillus casei. In one version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus. In another version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus. In another version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici. In one version of this embodiment, the auxiliary culture comprises a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Lactobacillus helveticus. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Pediococcus acidilactici. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus and a Pediococcus acidilactici. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus and a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici and a Lactobacillus helveticus. In a preferred version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici and a Lacticaseibacillus casei. In a preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Pediococcus acidilactici. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Lacticaseibacillus casei. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lacticaseibacillus casei, and a Pediococcus acidilactici. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei.
In a preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In a version of this embodiment, the S. thermophilus is S. thermophilus DSM 34725. In another version of this embodiment, the Lacticaseibacillus rhamnosus is Lacticaseibacillus rhamnosus DSM 33870.
In an embodiment, the Lactobacillus bulgaricus is Lactobacillus bulgaricus DSM 28910. In an embodiment, the Lactobacillus helveticus is Lactobacillus helveticus DSM 19499. In an embodiment, the Pediococcus acidilactici is Pediococcus acidilactici DSM 28307. In an embodiment the Lacticaseibacillus casei is Lacticaseibacillus casei ATCC55544.
In a preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises S. thermophilus DSM 34725 and Lacticaseibacillus rhamnosus DSM 33870, and the auxiliary culture comprises Lactobacillus bulgaricus DSM 28910, Lactobacillus helveticus DSM 19499, Pediococcus acidilactici DSM 28307, and Lacticaseibacillus casei ATCC55544.
In an embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises S. thermophilus DSM 34725 and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and Lacticaseibacillus rhamnosus DSM 33870, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises Lactobacillus bulgaricus DSM 28910, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, Lactobacillus helveticus DSM 19499, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, Pediococcus acidilactici DSM 28307, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the first culture comprises a Lactiplantibacillus plantarum, the second culture comprises a S. thermophilus and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and Lacticaseibacillus casei ATCC55544.
Another embodiment of the present invention relates to the method as described herein, wherein the first culture comprises Lentilactobacillus kefiri, the second culture comprises Streptococcus thermophilus, and the third culture comprises Lactococcus lactis. In a preferred version of this embodiment, the first culture comprises Lentilactobacillus kefiri, the second culture comprises Streptococcus thermophilus, and the third culture comprises Lactococcus lactis. In a specific version of this embodiment, the first culture comprises Lentilactobacillus kefiri DSM 34723, the second culture comprises Streptococcus thermophilus DSM 34727, Streptococcus thermophilus DSM 19242, and Lacticaseibacillus rhamnosus DSM 33870, and the third culture comprises Lactococcus lactis DSM 34729. In another version of this embodiment, the first culture comprises Lentilactobacillus kefiri DSM 34723, the second culture comprises a Streptococcus thermophilus, and a Lacticaseibacillus rhamnosus, and the third culture comprises a Lactococcus lactis. In another version of this embodiment, the first culture comprises a Lentilactobacillus kefiri, the second culture comprises Streptococcus thermophilus DSM 34727, and a Lacticaseibacillus rhamnosus, and the third culture comprises a Lactococcus lactis. In another version of this embodiment, the first culture comprises Lentilactobacillus kefiri, the second culture comprises Streptococcus thermophilus DSM 19242, and a Lacticaseibacillus rhamnosus, and the third culture comprises a Lactococcus lactis. In another version of this embodiment, the first culture comprises a Lentilactobacillus kefiri, the second culture comprises a Streptococcus thermophilus and Lacticaseibacillus rhamnosus DSM 33870, and the third culture comprises a Lactococcus lactis. In yet another version of this embodiment, the first culture comprises a Lentilactobacillus kefiri, the second culture comprises a Streptococcus thermophilus and a Lacticaseibacillus rhamnosus, and the third culture comprises Lactococcus lactis DSM 34729.
A further embodiment of the present invention relates to one of the following strains and composition comprising the strains, as well as and uses thereof for plant-based fermentation:
Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lactis DSM 34729, Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242. The strains have been isolated for due to very good performance and contribution to gel firmness and their production of volatile organic compounds. It is therefore envisioned that the strains can be used by itself or in blends.
Bacterial metabolism and its enzymes are responsible for the breakdown of chemical compounds in the pea-base composition and their transformation into new products that contribute to the flavor profile of a fermented product. Preferred were bacterial blends that produced higher levels of VOCs found in dairy cheese, such as diacetyl, 2,3- pentanone, acetoin and 3-methylbutanal. Ketones (diacetyl, acetoin and 2,3- pentandione) are common constituents of a wide range of dairy products and have a major influence on cheese aroma and low perception threshold, and 3-methylbutanal may confer unripe, apple-like, sweet and fruity notes. Also of interest is the production of ketones which are commonly found in cheese, the content of which typically increases during ripening, and bring fruity-floral notes to the cheese as well as green, blue cheese (2-heptanone), and hot milk and musty (2-nonanone) aromas.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented peabased product prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3- pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2-heptanone, 2-nonanone, and 3-methyl butanal.
Among the aldehydes, 2,4-decadienal, hexanal, heptanal, 2-hexenal, 2-heptenal, octanal, 2-octenal, and pentanal are contemplated as major contributors to the characteristic beany flavor of pea. In particular, higher levels of hexanal are considered to be one of the major compounds responsible for off-flavor in pea protein. Favored are therefore bacterial blends that can reduce these off-flavor VOCs. It has been found that the strains producing acetoins in the removal of off-flavor compounds. One of the bacterial species inducing the lowest rates of beany VOCs was L. rhamnosus.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4-decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
The method is not limited to a particular amount of bacterial blend for fermenting the pea-base composition. The amount may be adjusted to vary the texture and flavor of the desired end product, e.g. some fermented pea-based cheese-analogues may benefit from higher amount of bacterial blend to build a stronger texture and flavor, whereas other products may be better suited with less of the bacterial blend to generate a softer texture and more mild flavor. However, herein a preferred content of bacterial blend has been set at which texture and flavor is developed without using unnecessary high loads of bacteria.
Thus, an embodiment of the present invention relates to the method as described herein, wherein the content of bacterial blend is in the range of about 0.01 % (w/w) to about 0.1 % (w/w), with respect to the total weight of the fermented pea-based composition.
The pea-base composition is acidified during fermentation as the lactic acid bacteria converts carbohydrates into lactic acid and other metabolites. Lactic acid bacteria which induce fast acidification are preferred for food safety reasons. Fermentation of the peabase composition is continued until a predetermined pH is reached. This target pH value can be set, amongst others, based on the desired texture of the fermented pea-based product. Typically, the predetermined pH value will be less than pH 5 to realise the full benefits of the fermentation process where texture as well as flavour is developed. However, for preparation of less acidic products, a slightly higher pH, such as pH 5.5 may be preferred.
Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.
Another embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is in the range of about pH 4 to about pH 5, such as about pH 4.3 to about pH 4.7. A further embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is in the range of about pH 5 to about pH 5.5.
At the end of the fermentation the pea-base composition has been transformed into a matrix or gel with mechanical textural attributes similar to a dairy cheese. This include a certain gel strength and firmness that keeps the product from collapsing under pressure, but also some elasticity as would be expected from a dairy cheese.
Therefore, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is in the form of a matrix or gel.
Another embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.
The form and texture of the fermented pea-based product is sustained by the pea protein network built during fermentation. The gel formation is achieved without additives such as texturizers or thickening agents. Producers of plant-based products, such as cheese-analogues, would like to avoid use of these additives because it would reduce cost and give a cleaner label.
Thus, an embodiment of the present invention relates to the method as described herein, wherein fermented pea-based product does not contain any texturizers.
Another embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, hydrocolloids and gums.
A further embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, agar agar, carrageenans, guar gum, xanthan gum and Arabic gum.
The fermented pea-based product obtained from the present method has firm texture and improved development of flavour with reduced off-taste caused by VOCs with beany notes. Provided herein is therefore a fermented pea-based product, such as a cheeseanalogue, which have mechanical properties and a flavor profile that resemble those of a dairy cheese. The fermented pea-based product, such as a cheese-analogue, is achieved by use of a bacterial blend comprising a consortium of lactic acid bacteria with complementary properties yielding both firm texture and balanced flavor.
Accordingly, an aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.
Another aspect of the present invention relates to a bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
Yet another aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:
- a fermented pea-base composition, and bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
A further aspect of the present invention relates to use of a bacterial blend in the production of a fermented pea-based cheese-analogue, wherein the bacterial blend comprises:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus. The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for producing the fermented pea-based product and all its features, which may readily be part of the resulting fermented pea-based product, such as a fermented pea-based cheese-analogue. Moreover, said features may also be transferred to the bacterial blend as such or use of the same for production of a fermented pea-based cheese-analogue. Embodiments and features of the present invention are also outlined in the following items.
Items
XI. A method for producing a fermented pea-based product, the method comprising the following steps:
(i) providing a pea-base composition,
(ii) adding a bacterial blend to the pea-base composition, said bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus, and
(iii) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.
X2. The method according to item XI, wherein the first culture comprises a Lentilactobacillus kefiri strain.
X3. The method according to any one of items XI or X2, wherein the first culture comprises only a single lactic acid bacteria strain. X4. The method according to any one of the preceding items, wherein the second culture comprises at least one Streptococcus thermophilus strain.
X5. The method according to any one of the preceding items, wherein the second culture comprises at least two different Streptococcus thermophilus strains, such as three different Streptococcus thermophilus strains, preferably two different Streptococcus thermophilus strains.
X6. The method according to any one of the preceding items, wherein the second culture comprises a Lacticaseibacillus rhamnosus strain.
X7. The method according to any one of the preceding items, wherein the second culture comprises at least two different lactic acid bacteria strains, preferably three different lactic acid bacteria strains.
X8. The method according to any one of the preceding items, wherein the second culture comprises two different Streptococcus thermophilus strains and a Lacticaseibacillus rhamnosus strain.
X9. The method according to any one of the preceding items, wherein the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
X10. The method according to item X9, wherein the third culture comprises a Lactococcus lactis strain.
XI 1. The method according to any one of items X9 or X10, wherein the third culture comprises only a single lactic acid bacteria strain.
X12. The method according to any one of the preceding items, wherein the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lactis DSM 34729, Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
X13. The method according to any one of the preceding items, wherein the first culture comprises at least one lactic acid bacteria selected from the group consisting of: Lentilactobacillus kefiri DSM 34723, and Lactiplantibacillus plantarum DSM 34728.
X14. The method according to any one of the preceding items, wherein the first culture comprises the Lentilactobacillus kefiri strain deposited as DSM 34723.
X15. The method according to any one of the preceding items, wherein the second culture comprises at least one lactic acid bacteria selected from the group consisting of: Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Streptococcus thermophilus DSM 34745 and Lacticaseibacillus rhamnosus DSM 33870.
X16. The method according to any one of the preceding items, wherein the second culture comprises the Lacticaseibacillus rhamnosus strain deposited as DSM 33870.
X17. The method according to any one of the preceding items, wherein the second culture comprises at least one Streptococcus thermophilus strain selected from the group consisting of:
Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
X18. The method according to any one of the preceding items, wherein the third culture comprises at least one lactic acid bacteria selected from the group consisting of: Lactococcus lactis DSM 34729, and Levilactobacillus brevis DSM 34744. X19. The method according to any one of the preceding items, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
X20. The method according to any one of items X1-X18, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 34726, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
X21. The method according to any one of the preceding items, wherein the content of bacterial blend is in the range of about 0.01 % (w/w) to about 0.1 % (w/w), with respect to the total weight of the fermented pea-based composition.
X22. The method according to any one of the preceding items, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.
X23. The method according to item X22, wherein the pea protein component comprises at least about 50% (w/w) protein, such as at least about 60% (w/w) protein, such as at least about 70% (w/w) protein, preferably at least about 80% (w/w) protein, with respect to the total weight of the pea protein component.
X24. The method according to any one of items X22 or X23, wherein the pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the pea-base composition. X25. The method according to any one of items X22-X24, wherein the pea-base composition has a content of pea protein component of about 5 % (w/w), with respect to the total weight of the pea-base composition.
X26. The method according to any one of the preceding items, wherein the pea-base composition is a viscoelastic material.
X27. The method according to any one of the preceding items, wherein the pea-base composition is in liquid form.
X28. The method according to any one of the preceding items, wherein the pea-base composition is in the form of a colloidal suspension.
X29. The method according to item X28, wherein the colloidal suspension is an emulsion or sol, preferably an emulsion.
X30. The method according to any one of the preceding items, wherein the pea-base composition comprises one or more oils or fats.
X31. The method according to any one of the preceding items, wherein the one or more oils or fats are liquid at room temperature and/or have a melting temperature of less than 0°C.
X32. The method according to any one of the preceding items, wherein the pea-base composition comprises one or more sugars.
X33. The method according to item X32, wherein the content of sugar is in the range of about 0.5 % (w/w) to about 5 % (w/w), such as about 1 % (w/w) to about 3 % (w/w), with respect to the total weight of the pea-base composition.
X34. The method according to any one of items X32 or X33, wherein the one or more sugars are monosaccharides and/or disaccharides.
X35. The method according to any one of items X32-X34, wherein the one or more sugars are sucrose and/or glucose. X35. The method according to any one of the preceding items, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial blend.
X36. The method according to any one of the preceding items, wherein the pea-base composition is subjected to heat treatment at a temperature sufficient for denaturing the pea proteins.
X37. The method according to any one of items X35 or X36, wherein said heat treatment is performed at a temperature of at least about 80°C, such as at least about 85°C, preferably about 90°C.
X38. The method according to any one of items X35-X37, wherein the heat treatment is performed for at least about 5 min, such as at least about 10 min, such as at least about 15 min, preferably at least about 20 min.
X39. The method according to any one of the preceding items, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.
X40. The method according to any one of the preceding items, wherein the predetermined pH is in the range of about pH 4 to about pH 5, such as about pH 4.3 to about pH 4.7.
X41. The method according to any one of the preceding items, wherein the fermented pea-based product is in the form of a matrix or gel.
X42. The method according to any one of the preceding items, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.
X43. The method according to any one of the preceding items, wherein fermented peabased product does not contain any texturizers.
X44. The method according to item X43, wherein the texturizers are selected from the group consisting of coconut oil, hydrocolloids and gums. X45. The method according to any one of items X43 or X44, wherein the texturizers are selected from the group consisting of coconut oil, agar agar, carrageenans, guar gum, xanthan gum and Arabic gum.
X46. The method according to any one of the preceding items, wherein the fermented pea-based product comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
X47. The method according to any one of the preceding items, wherein the fermented pea-based product comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based product prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4- decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
X48. The method according to any one of the preceding items, wherein the fermented pea-based product has a gel firmness in the range of about 200 g*ms to about 300 g*ms.
Yl. A fermented pea-based product obtainable by the method according to any one of the preceding items.
Ul. A bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
U2. A bacterial blend according to item Ul, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723, Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
Zl. A fermented pea-based cheese-analogue comprising:
- a fermented pea-base composition, and bacterial blend comprising:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
Z2. The fermented pea-based cheese-analogue according to item Zl, wherein the fermented pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the fermented pea-base composition.
Z3. The fermented pea-based cheese-analogue according to any one of items Zl or Z2, wherein the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
Z4. The fermented pea-based cheese-analogue according to any one of items Z1-Z3, wherein the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
Z5. The fermented pea-based cheese-analogue according to any one of items Z1-Z4, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 19242, Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
Z6. The fermented pea-based cheese-analogue according to any one of items Z1-Z4, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 34726, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729.
Z7. The fermented pea-based cheese-analogue according to any one of items Z1-Z6, wherein the content of bacterial blend is in the range of about 0.01 % (w/w) to about 0.1 % (w/w), with respect to the total weight of the fermented pea-based composition.
Z8. The fermented pea-based cheese-analogue according to any one of items Z1-Z7, wherein the fermented pea-based cheese-analogue comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented peabased cheese-analogue prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
Z9. The fermented pea-based cheese-analogue according to any one of items Z1-Z8, wherein the fermented pea-based cheese-analogue comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based cheese-analogue prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4- decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal. Z10. The fermented pea-based cheese-analogue according to any one of items Z1-Z9, wherein the fermented pea-based cheese-analogue has a gel firmness in the range of about 200 g*ms to about 300 g*ms.
Wl. Use of a bacterial blend in the production of a fermented pea-based cheeseanalogue, wherein the bacterial blend comprises:
- a first culture comprising one or more lactic acid bacteria selected from the group consisting of Lentilactobacillus kefiri, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
\N2. The use according to item Wl, wherein the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
W3. The use according to any one of items Wl or W2, wherein the bacterial blend comprises one or more lactic acid bacteria selected from the group consisting of: Levilactobacillus brevis DSM 34744, Lentilactobacillus kefiri DSM 34723, Lactiplantibacillus plantarum DSM 34728, Lacticaseibacillus rhamnosus DSM 33870, Lactococcus lactis DSM 34729, Streptococcus thermophilus DSM 34745, Streptococcus thermophilus DSM 34726, Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
W4. The use according to any one of items W1-W3, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 19242,
Streptococcus thermophilus DSM 34727, Lacticaseibacillus rhamnosus DSM 33870, and Lactococcus lactis DSM 34729. W5. The use according to any one of items W1-W3, wherein the bacterial blend comprises the following lactic acid bacteria:
Lentilactobacillus kefiri DSM 34723,
Streptococcus thermophilus DSM 34726,
Lacticaseibacillus rhamnosus DSM 33870, and
Lactococcus lactis DSM 34729.
W6. The use according to any one of items W1-W5, wherein the fermented pea-based cheese-analogue comprises increased amounts of one or more dairy-like volatile organic compounds (VOCs) compared to a fermented pea-based cheese-analogue prepared without the bacterial blend, wherein the one or more dairy-like VOCs are selected from the group consisting of diacetyl, 2-pentanone, 2,3-pentanedione, acetoin, dimethyl-sulfide, 2-hexanone, 2- heptanone, 2-nonanone, and 3-methyl butanal.
W7. The use according to any one of items W1-W6, wherein the fermented pea-based cheese-analogue comprises reduced amounts of one or more beany volatile organic compounds (VOCs) compared to a fermented pea-based cheese-analogue prepared without the bacterial blend, wherein the one or more beany VOCs are selected from the group consisting of 2,4- decadienal, 2-octenal, pentanal, 2-hexenal, hexanal, heptanal, 2-heptenal, and octanal.
W8. The use according to any one of items W1-W7, wherein the fermented pea-based cheese-analogue has a gel firmness in the range of about 200 g*ms to about 300 g*ms.
QI. A bacterial strain in selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and
Streptococcus thermophilus DSM 19242. Q2. A composition for fermenting plant-based, preferably pea-based cheese analogue, comprising a bacterial strain selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and
Streptococcus thermophilus DSM 19242.
Q3. A pea-based cheese analogue comprising a bacterial strain selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and Streptococcus thermophilus DSM 19242.
The invention will now be described in further details in the following non-limiting examples.
Examples
Example 1: Preparation of fermented pea-based product
This example describes how the fermented pea-based product can be produced by fermentation of a pea-base composition with lactic acid bacteria. The procedure was used both for test of single bacterial strains and for bacterial blends.
Method
Preparation of pea-base composition
Pea protein isolate (ProFam®580, ADM, Chicago, IL, USA) with a composition of 81.3% protein, 7% fat, and 9% fiber was suspended at 5% w/w in a water solution of 1% w/w glucose- and 1% w/w sucrose (Sigma Aldrich, Soborg, Denmark) and mixed at 8,100 rpm with a mixer (L5M Laboratory Mixer, Silverson, Chesham, United Kingdom) for 2 min. The suspension was emulsified with 5% sunflower oil (Ollineo, Budapest, Hungary) with the same mixer at 8,100 rpm for 2 min and was subjected to high pressure homogenization in an homogenizer (GEA Lab Homogenizer PandaPLUS 2000, GEA, Parma, Italy) at two stages (150; 50 bars) in one pass. Homogenized emulsions were then pasteurized at 90°C for 20 min while stirring in a water bath, stored at room temperature over night, pasteurized again under the same conditions and cooled down to 4°C , prior to microbial inoculation.
Fermentation
A diverse selection of 90 lactic acid bacteria strains was sourced from Chr. Hansen 's Culture Collection (Chr. Hansen A/S, Horsholm, Denmark), comprising different Lactobacilli, Lactococci, Leuconostoc and Streptococcus thermophilus strains. All strains were cultured from a frozen glycerol stock in MRS pH 6.3-6.7, MRS pH 5.4 and M17 for 24 to 48 hours at 30°C, 37°C, and 40°C according to their suitable growth media and temperature.
The pea-base composition was stained with 5% pH colour indicator (1 : 1 wt% bromocresol purple salt and bromocresol green salt, Sigma Aldrich, St. Louis, US) and 990 pl stained pea-base composition was inoculated with 10 pl bacterial culture overnight in 96-well plates and incubated at 30°C and 40°C for 22 hours on top of flatbed scanners (HP ScanJet G4010). Colour changes were recorded every 6 min through Hue values with pH Multiscan software v.5.1 (HNH Consult Aps, 9530 Stovring, Denmark). The pH of the samples at end of fermentation was approx. 4.5. Samples were then stored overnight under refrigeration.
Fermentation with single strains (1% inoculum) and with strain blends was carried out in 2 different formats: 1 ml samples in 1 ml sterile 96-well microtiter plates (MTP) (Saveen Werner ApS, Limhamn, Sweden) for pH and texture measurements and 3 ml samples in headspace vials for analysis of targeted volatile compounds. Samples for acidification and texture measurements were incubated at 30 °C and 40 °C and samples for VOC analysis were incubated at 37 °C after analyzing the results from the previous two tests. After 20 hours of incubation, the samples were stored overnight under refrigeration. The acidification of the strain blends was measured using the same setup but at an incubation temperature of 37 °C.
Results and conclusion
The method produced 90 fermented pea-based gels that were stored and put forward for further evaluation with the aim of identifying lactic acid bacteria which individually contribute advantageously to the two main parameters; texture and flavor.
Example 2: First screening round - capacity of individual strains for promoting texture and flavor
The selection of 90 lactic acid bacteria were screened for their acidification performance, contribution to gel firmness, and their ability to remove of off-flavors and develop cheese-like flavors. The main leads for each of the previously mentioned parameters were selected for a subsequent compounding to further design versatile combinations that can improve texture and flavor in plant-based cheese-analogue products.
Method
Fermented pea-based gels through fermentation with 90 individual lactic acid bacteria strains were prepared as described in Example 1. Each of the gels were evaluated as described below.
Firmness evaluation
The gel firmness of the gels in the 96-deepwell MTPs was analyzed after overnight storage under refrigeration using a penetration test with a Hamilton Star robot and custom-made metal micro-tools and a precision balance (Mettler Toledo, Columbus, Ohio, United States). The MTPs were placed on the precision, and the micro-tools of 4 mm of plunger diameter penetrated 22 mm of each sample at a time. The precision balance where the plates were located recorded the force resistance every 10 ms. The obtained values (time (ms) on the x-axis and force (g) on the y-axis) were plotted and the positive area under the curve was calculated for each replicate and used as a measurement for gel firmness.
Detection of volatile organic
Figure imgf000044_0001
The analysis and quantification of volatile organic compounds (VOCs) in samples fermented in headspace vials by single strains was performed with the Perkin Elmer TurboMatrix HS-110 static headspace sampler connected to a Perkin Elmer Clarus 690 GC Series coupled to a Flame Ionization Detector (GC-FID) (Perkin Elmer, MA, USA), equipped with an HP-FFAP column, 25 m x 0.2 mm x 0.33 m (Agilent Technologies, Glostrup, Denmark), using helium as carrier gas. Before injection of an aliquot of headspace above the sample, the vial was incubated for 37 min at 70 °C. The GC oven program was as follows: 60 °C/2 min, Ramp 1 : 45 °C /min to 230 °C, hold 0.5 min. Identification of VOCs was based on retention time in comparison with that of the standards. Data were processed using Chromeleon software (Version 7.2.7, Thermo Scientist Inc., Denmark). Results were calculated as peak height divided by baseline noise (signal-to-noise, S/N).
Statistical analysis
In the first round of screening individual lactic acid bacteria strains, ANOVA followed by Dunnett's multiple comparisons test was performed to compare the presence of VOCs in each fermented sample to that in the non-fermented sample. All statistical analyses were performed with JMP Pro 16 (SAS Institute, Cary, North Carolina, United States), and p values of less than 0.05 were interpreted as significant differences.
Results
90 lactic acid bacteria single strains were evaluated for their ability to ferment a peabase composition. The evaluation was based on acidification speed, contribution to texture, and volatile organic compound (VOC) profile, including removal of off-flavor ("beany" flavor) and production of dairy-like compounds.
Acidification
Non-inoculated control samples within the 96-well MTP showed no change of color from the initial blue color and therefore no capacity to lower the sample pH. Samples inoculated with a bacterial strain turned light green, indicating a change of pH as a result of acidification.
Fast acidification correlates to the ability of the strains to produce lactic acid, and therefore to their capacity to metabolize carbon sources in the pea-base composition. Fast acidification was prioritized as main criteria for the selection of the lead candidate strains. Because a fast pH drop could hinder any potential background growth, such as growth of endogenous flora. Therefore, fast deep acidifiers able to acidify to Hue values of 180 within 8 hours were selected. A few strains with lower acidification capacity, including L kefiri, were still included in the selection due to their good texturizing properties. Gel firmness
The gel firmness of the fermented gels was positively correlated to the acidification capacity of the strains. At 30 °C, the fast acidifying candidates contributed the most to the gel firmness with values mainly above 400 g*ms, whereas at 40 °C the vast majority of the samples presented values above 600 g*ms (Figure 1) . With these results in mind, the top acidifying and texturizing strains of each species were chosen to keep diversity in the selection.
The strains that contributed the most to gel firmness were of the species L. kefiri, L. paracasei, and L. plantarum (strains 19, 22 and 38, respectively). These strains were selected for the second screening stage as texturizing strains.
Volatile organic compounds (VOCs)
Each fermented sample was compared to the non-fermented sample ("base") and a study of the statistically significant differences in the levels of each VOC was carried out to define a desired performance of each strain. A chemically-acidified sample ("chem") was also included to evaluate if the presence of certain VOCs is attributed to acidic conditions or to the metabolic activity of the strains.
The result from the VOC analysis of fermentation by single lactic acid bacteria strains is displayed in Figure 2.
All samples showed significantly lower levels of hexanal in comparison to the nonfermented sample. Furthermore, there was no significant differences between the chemically acidified sample and the non-fermented sample, which reflects the positive effect of fermentation on the removal of beany flavor-related VOCs.
Diacetyl production was significantly increased in a set of S. thermophilus strains, as well in a few L. rhamnosus strains (Figure 2A). Encouragingly, the L. kefiri strain that promoted texture also contributed to production of diacetyl. Samples fermented with other lactobacilli such as L. fermentum, L. bulgaricus, L. paracasei, or L. plantarum did not show significant levels of diacetyl after fermentation.
The presence of 2,3-pentanedione was only detected in the sample fermented by the L. kefiri strain and in samples fermented by S. thermophilus (Figure 2B). Acetoin production was promoted in many strains, hereunder again the L. kefiri strain which promoted also texture building (Figure 2C). The L. rhamnosus strains were also effective in producing acetoin, and so was several S. thermophilus strains.
3-methylbutanal was only detected in samples fermented with L. brevis, L. sanfranciscensis, or L. lactis. This aldehyde has been found in Gouda, Cheddar, and Camembert cheeses and described as a powerful malty and cheese odorant. Although the detected signal-to-noise (S/N) values for 3-methylbutanal were close to the limit of quantification (data not shown), they were included in the strain selection for their potential in the production of this compound when combined with other strains but also to embrace the diversity of species within the blend design.
Conclusion
Out of the 90 strains a subset of 13 strains of the highest performance were selected for their contribution to gel firmness and their production of diacetyl, 2,3-pentanedione, acetoin, and 3-methylbutanal (Table 2). These strains comprised one L. kefiri, one L. plantarum, one L. paracasei, six S. thermophilus, one L. rhamnosus, one L. brevis, one L. sanfranciscensis and one L. lactis. The present application thus identifies the strains as well as its use in fermentation of plant-based matrices.
Figure imgf000047_0001
Table 2. Individual candidate strains selected after the first screening. Example 3: Identification of advantageous blends of lactic acid bacteria strains
The best strains identified during initial screening of individual strains were compounded to find strain combinations that could potentially turn into new starter cultures with great competences for pea-based cheese-analogue applications.
Method
Fermented pea-based gels through fermentation with 64 different bacterial blends and 2 alternative bacterial blends were prepared (see Table 3 and Table 4) as described in Example 1, and a D-optimal model studied the effect of the factors, based on 192 runs including triplicates of each combination. The pea-base composition was fermented in the same 96-well MTPs and headspace vials set-up as in the first screening step, but all samples were incubated at 37 °C for 20 h.
Figure imgf000048_0001
Figure imgf000049_0001
Table 3. Overview of the compounding of lactic acid bacteria strains in the 64 different bacterial blends for screening.
Figure imgf000049_0002
Figure imgf000050_0001
Table 4. Overview of the compounding of lactic acid bacteria strains in the 2 alternative bacterial blends for screening.
Each of the gels were evaluated as described below.
Firmness evaluation
Gel firmness was measured as described in Example 2.
Extended VOC metabolic fingerprinting analysis
The volatile organic compounds in the samples fermented with the designed blends were analyzed after fermenting 3 ml of pea-base composition directly in a 20 ml headspace vial together with the strain blends. Samples were prepared in duplicates and then analyzed by headspace solid phase microextraction gas chromatography coupled to mass spectrometry (HS-SPME-GC-MS) after one week of storage under refrigeration. The instrument was a Multi Purpose Sampler (Gerstel, MSCI, Skovlunde, Denmark), with a 7890B GO (Agilent Technologies, Denmark) and a 5977A MS (Agilent Technologies, Denmark).
VOCs were extracted by SPME using a DVB/Car/PDMS-fiber (Supelco 57299, VWR, Denmark) for 20 min at 60 °C, desorbed splitless at 270 °C on a TenaxTA-filled liner (Gerstel 012438, MSCI, Skovlunde, Denmark) kept at -30 °C. After fiber desorption, the TenaxTA-filled liner was heated to 300 °C and the trapped VOCs were transferred splitless and separated on a DB-5MS UI column 30m x 0.25mm x 1 pm (Agilent 122- 5533UI, Agilent Technologies, Denmark) at 170 kPa constant pressure using helium as carrier gas. Oven temperature program was as follows: starting at 32 °C/2min - increased to 102°C at 10 °C/min - further increased to 145°C at 5 °C/min - further increased to 200 °C at 15 °C/min - further increased to 200 °C at 15 °C/min - further increased to 280 °C at 20 °C/min - hold at 280 °C for 5 min. The mass spectrometer operated in electron impact mode at -70eV and the analyzer was scanning from 29-209 amu.
NIST 17 library search and Retention Indexes were used for identification of VOCs. Feature extraction was done using MassHunter Quantitative Analysis (Version 10.2, Build 10.2.733.8, Agilent Technologies, Denmark) and results calculated as peak height divided by baseline noise (signal-to-noise, S/N). Statistical analysis
For the design of bacterial blends, ANOVA followed by Tukey test was performed to observe differences between the different blends and also the effect of the single strains belonging to different categories on acidification, gel firmness and VOC present in the fermented matrix. All statistical analyses were performed with JMP Pro 16 (SAS Institute, Cary, North Carolina, United States), and p values of less than 0.05 were interpreted as significant differences.
Principal Component Analysis (PCA) was used to explore the effect of fermentation and visualize the fermented samples compared to the unfermented base and the chemically acidified sample. The PCA was performed with Matlab software (MATLAB® R2022b 9.13.0.2105380, MathWorks) and using PLS Toolbox 8.9 (Eigenvector Research Inc., USA).
Results
64 bacterial blends and the 2 alternative blends were evaluated for their ability to ferment a pea-base composition. The evaluation was based on acidification speed, contribution to texture, and volatile organic compound (VOC) profile, including removal of off-flavor ("beany" flavor) and production of dairy-like compounds.
Acidification
In the first round of the screening (Example 2), the majority of slow-acidifying strains were discarded to obtain a fast pH drop and avoid background growth. Cultures, namely mixes of different single strains, are more robust and beneficial for fast acidification than single strains, and therefore their acidification capacity is more stable. This assumption was confirmed by the acidification curves of the 64 bacterial blends (Figure 3), which all showed relatively fast acidification and within the acceptable spectrum where background growth is minimised.
The acidification curves show the blends being separated into two overall groupings, although all of them show similar acidification patterns with a pH drop after around 6 hours of incubation. The upper grouping includes curves with a value above 170 hue after 10 hours of incubation, whereas the lower grouping (highlighted by circle) contained the samples that presented values below 170 hue after 10 hours of incubation. Upon analysis it was found that strain STH89 was only present in the lower grouping samples, suggesting that this strain significantly improves acidification speed. Consequently, the pH drop occurs 2-3 hours earlier in the lower grouping than in the higher grouping, presenting a marked advantage in terms of food safety and process optimization.
Gel firmness
Based on the high-throughput penetration test results from the screening (Example 2), all the designed blends purposefully comprised one of the 3 strains that contributed the most to gel firmness, namely L. kefiri, L. plantarum, or L. paracasei. It was to investigate if the presence of any other strains in the blend would attenuate or enhance their texturizing ability.
The positive area of the curve measured by the HTP penetration test was taken as a measurement of gel hardness or firmness - the higher the value, the harder the consistency of the sample. The hardness was defined as the resistance of the sample to penetration. There was no significant difference between gels fermented with the 64 different blends (Figure 4). All positive area values varied between 284,6 and 186,5 g*ms, which can be considered satisfactory and supports the notion that the three texturizing strains perform well when combined with other strains.
Volatile organic compounds (VOCs)
In the design of bacterial blends, an extended VOC analysis was performed to identify a broader range of compounds in the samples fermented with the strain blends. 79 different compounds from different compound groups such as alcohols, acetaldehydes, aromatic, esters, furans, ketones and sulfur compounds were detected in the fermented pea-base composition samples.
A principal component analysis (PCA) revealed that all the fermented samples were located far away from the unfermented base and from the chemically-acidified sample (Figure 5A). The non-fermented samples were located in the lower right corner of the plot, in the same area as the majority of the aldehydes found in the pea-base composition raw material, whereas all the fermented samples were located on the opposite side (Figure 5B). This highlights the significant effect of fermentation on the reduction of beany off-notes. Moreover, dairy-associated VOCs such as diacetyl, acetoin, or 2,3-pentanedione, among other metabolites, were found in the upper-right quadrant, where also many of the blends can be found in the scores plot, while the pea-base composition and the chemically-acidified sample were located in the opposite side of the Y axis. This observation emphasizes the need of fermentation for producing dairy notes from the pea-base composition.
Out of the total detected VOCs, 15 were selected for further statistical analysis due to their contribution to the removal of green and beany flavor or the production of cheesy and dairy-associated flavor.
2,4-decadienal, hexanal, heptanal, 2-hexenal, 2-heptenal, octanal, 2-octenal, and pentanal were evaluated as major contributors to the characteristic beany flavor of pea (Figure 6A-H). There were no significant differences in the removal of pentanal, octanal,
2-heptenal, heptanal, and hexanal between the 64 different blends. In contrast, there were significant differences between blends on the removal of 2-octenal, 2,4- decadienal, and 2-hexenal. Blend 6 comprising L. kefiri performed well in the removal of all beany VOCs (Figure 6A-H).
Significant differences were found among the 64 blends in the formation of dairy- associated VOCs, where some blends did produce insignificant levels and others produced significantly higher levels (Figure 7A-G). Diacetyl, acetoin, 2,3-pentanedione,
3-methylbutanal, 2-pentanone, 2-heptanone, and dimethyl disulfide were considered relevant dairy-associated compounds. Blends 62, 63 and 64 performed significantly good at producing compounds such as diacetyl, 2-pentanone, acetoin, and 2-heptanone. Dimethyl disulfide, an important fraction of the Cheddar cheese flavor was also produced in high levels by blend 63 and 64. All three blends contain L. rhamnosus, suggesting its importance when combined with S. thermophilus.
An additional scores plot was generated for four performant bacterial blends, Al, A2, B6, and B64. A clear separation of the four different bacterial blends can be observed in the scores plot (Fig. 8), where each blend is located in a different quadrant: samples fermented by the bacterial blend B64 and A2 were located on the positive side of SCI, while those fermented with B6 and Al were located on the negative side of SCI. This separation was mainly driven by the higher concentration of 2,3-butanedione (diacetyl) in A2 and B64, which was previously reported to produce an aroma profile of butter and traditional Prato cheese (Domingos et al., 2019). Separation of blends B6 and A2 along the positive side of SC2 was attributed to higher contents of acetic acid, while Al and B64 blends, located on the negative side of SC2, had higher concentrations of 2,3- pentanedione, acetoin, diacetyl and benzaldehyde. Acetoin, diacetyl and acetoin are normally associated with fermented dairy aromas (Reyes-Diaz et al., 2020), which confirms that blend B64 produces cheese-related VOCs, as previously observed (Masia, Fernandez-Varela, Poulsen, et al., 2023).
Further examination of the loadings plot revealed that B6 samples were separated from others due to generally higher values of alcohols, such as 1-hexanol, 2-octen-l-ol, 2- heptanol, and 2-hepten-l-ol. Alcohols are commonly formed through the degradation of corresponding C6, C7 and C8 aldehydes (e.g., hexanal to 1-hexanol) mediated by alcohol dehydrogenase or via oxidative breakdown of their corresponding fatty acids (Marilley & Casey, 2004). Typically, alcohols exhibit higher odor thresholds than aldehydes e.g., 1-hexanol has a detection threshold of 500 ppb in the water (Guadagni et al., 1963) while that of hexanal is 4.5 ppb in water (Flath et al., 1967), therefore their overall contribution to product odor is lower. Compared to the other blends, this higher degradation of aldehydes could result in a lower intensity of undesirable beany- related odors in B6 samples.
Samples fermented using A2 were located in the opposite quadrant to samples fermented with Al in the scores plot. Samples fermented with Al had the highest concentrations of 2,3-pentanedione, a desirable VOC associated with dairy notes (Cheng 2010), while samples fermented by A2 had higher levels of acetic acid, a compound that can impart vinegary notes but that at low levels has been associated with the pleasant aroma of Cheddar cheese (Murtaza et al., 2014). Other compounds typically related to fermented products, such as acetic acid and hexanoic acid (Alemayehu et al., 2014; Diaz-Muniz et al., 2006), as well compounds related to beany aromas such as 1- pentanol, and 2-octenal, 2-heptanone, and 2-pentylfuran (Xiang et al., 2023), were all also more prominent in A2 samples.
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Domingos, L. D., Souza, H. A. L. de, Mariutti, L. R. B., Benassi, M. de T., Bragagnolo, N., & Viotto, W. H. (2019). Fat reduction and whey protein concentrate addition alter the concentration of volatile compounds during Prato cheese ripening. Food Research International, 119, 793-804. https://doi.Org/10.1016/j.foodres.2018.10.062
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Guadagni, D. G., Buttery, R. G., & Okano, S. (1963). Odour thresholds of some organic compounds associated with food flavours. Journal of the Science of Food and Agriculture, 14(10), 761-765. https://doi.org/10.1002/jsfa.2740141014
Marilley, L., & Casey, M. G. (2004). Flavours of cheese products: Metabolic pathways, analytical tools and identification of producing strains. International Journal of Food Microbiology, 90(2), 139-159. https://doi.org/10.1016/50168-1605(03)00304-0
Masia, C., Fernandez-Varela, R., Poulsen, V. K., Jensen, P. E., & Sorensen, K. I. (2023). Composition of bacterial blends for fermentation-induced pea protein emulsion gels using multi-property screening of lactic acid bacteria. Food Bioscience, 56, 103333. https://doi.Org/10.1016/j.fbio.2023.103333 Murtaza, M. A., Ur-Rehman, S., Anjum, F. M., Huma, N., & Hafiz, I. (2014). Cheddar Cheese Ripening and Flavor Characterization: A Review. Critical Reviews in Food Science and Nutrition, 54(10), 1309-1321. https://doi.org/10.1080/10408398.2011.634531
Xiang, L., Jiang, B., Xiong, Y. L., Zhou, L., Zhong, F., Zhang, R., Bin Tahir, A., 8<. Xiao, Z. (2023). Beany flavor in pea protein: Recent advances in formation mechanism, analytical techniques and microbial fermentation mitigation strategies. Food Bioscience, 56, 103166. https://doi.Org/10.1016/j.fbio.2023.103166.
Conclusion
The Example demonstrates that advantageous bacterial blends were produced and key lactic acid bacteria species were identified. In particular, it was found that fermented pea-based products with great texture and excellent flavor profile could be achieved through fermentation with compounded bacterial blends with complementing attributes. Results also showed that the chosen bacterial blends produced desirable dairy-related VOCs, such as diacetyl and acetoin, especially by B64 and A2.
Deposits and Expert Solution
The applicant requests that a sample of the deposited microorganism stated in table 5 below may only be made available to an expert, until the date on which the patent is granted.
The applicant requests that the availability of the deposited microorganism referred to in Rule 33 EPC shall be effected only by the issue of a sample to an independent expert nominated by the requester (Rule 32(1) EPC). If an expert solution has been requested, restrictions concerning the furnishing of samples apply.
The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany.
The Budapest Treaty provides that any restriction of public access to samples of deposited biological material must be irrevocably removed as of the date of grant of the relevant patent.
Figure imgf000057_0001
Table 5. Deposited strain made at the depositary institution DSMZ.
The strain Lactobacillus paracasei subsp. paracasei (also referred to as Lacticaseibacillus paracasei, Lacticaseibacillus casei, CRL431 and L. casei 431®) was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Tissue type Collection Center, 10801 University Blvd, Manassas, VA 20110, USA on 24 January 1994 under accession number ATCC 55544. The well-known probiotic bacterium is commercially available from Chr. Hansen A/S, 10-12 Boege Alle, DK-2970 Hoersholm, Denmark, under the product name Probio-Tec® F-DVS L. casei-431®, Item number 501749, and under the product name Probio-Tec® C-Powder-30, Item number 687018.
References
Zheng et al. (2020), Int. J. Syst. Evol. Microbiol., 70, 2782-2858

Claims

Claims
1. A method for producing a fermented pea-based product, the method comprising the following steps:
(i) providing a pea-base composition,
(ii) adding a bacterial blend to the pea-base composition, said bacterial blend comprising:
- a first culture comprising a Lentilactobacillus kefiri strain, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus, and
(iii) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.
2. The method according to claim 1, wherein the bacterial blend further comprises a third culture comprising one or more lactic acid bacteria selected from the group consisting of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, and Lactococcus lactis.
3. The method according to any one of claims 1 or 2, wherein the first culture comprises Lentilactobacillus kefiri DSM 34723.
4. The method according to any one of the preceding claims, wherein the second culture comprises at least one lactic acid bacteria selected from the group consisting of:
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 19242,
Streptococcus thermophilus DSM 34727,
Streptococcus thermophilus DSM 34745 and
Lacticaseibacillus rhamnosus DSM 33870.
5. The method according to any one of the preceding claims, wherein the third culture comprises at least one lactic acid bacteria selected from the group consisting of:
Lactococcus lactis DSM 34729, and
Levilactobacillus brevis DSM 34744.
6. The method according to any one of the preceding claims, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.
7. The method according to claim 6, wherein the pea-base composition has a content of pea protein component in the range of about 1 % (w/w) to about 20% (w/w), such as about 2 % (w/w) to about 15 % (w/w), such as about 3 % (w/w) to about 10 % (w/w), with respect to the total weight of the pea-base composition.
8. The method according to any one of the preceding claims, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial blend.
9. The method according to any one of the preceding claims, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.
10. The method according to any one of the preceding claims, wherein the fermented pea-based product is in the form of a matrix or gel.
11. The method according to any one of the preceding claims, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.
12. A fermented pea-based product obtainable by the method according to any one of the preceding claims.
13. A bacterial blend comprising:
- a first culture comprising a Lentilactobacillus kefiri strain, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
14. A fermented pea-based cheese-analogue comprising:
- a fermented pea-base composition, and bacterial blend comprising:
- a first culture comprising a Lentilactobacillus kefiri strain, and - a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
15. Use of a bacterial blend in the production of a fermented pea-based cheeseanalogue, wherein the bacterial blend comprises:
- a first culture comprising a Lentilactobacillus kefiri strain, and
- a second culture comprising one or more lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lacticaseibacillus rhamnosus.
16. A bacterial strain selected from the group consisting of:
Levilactobacillus brevis DSM 34744,
Lentilactobacillus kefiri DSM 34723,
Lactiplantibacillus plantarum DSM 34728,
Lacticaseibacillus rhamnosus DSM 33870,
Lactococcus lactis DSM 34729,
Streptococcus thermophilus DSM 34745,
Streptococcus thermophilus DSM 34726,
Streptococcus thermophilus DSM 34727, and
Streptococcus thermophilus DSM 19242.
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