EP2209886A1 - Growth medium for lactic acid bacteria - Google Patents
Growth medium for lactic acid bacteriaInfo
- Publication number
- EP2209886A1 EP2209886A1 EP08844567A EP08844567A EP2209886A1 EP 2209886 A1 EP2209886 A1 EP 2209886A1 EP 08844567 A EP08844567 A EP 08844567A EP 08844567 A EP08844567 A EP 08844567A EP 2209886 A1 EP2209886 A1 EP 2209886A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- source
- lactic acid
- formate
- lactase
- purine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- 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
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- 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
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
- A23C9/1275—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss using only lactobacteriaceae for fermentation in combination with enzyme treatment of the milk product; using enzyme treated milk products for fermentation with lactobacteriaceae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01108—Lactase (3.2.1.108)
Definitions
- the present invention provides lactic acid bacterial cultures.
- the present invention provides cultures comprising lactic acid bacteria, a dairy substrate (e.g., milk or cream), at least one formate source, and at least one purine source.
- the cultures comprise exogenous lactase enzyme.
- the present invention also provides cultures of cells comprising the culture medium and lactic acid bacteria.
- the present invention further provides starter compositions (i.e., starter cultures) for starting the culture.
- the compositions of the present invention find use in the production of fermented milk products (e.g., yogurt, cheese and/or fermented milk beverages).
- Fermented milk products include dairy foods that have been fermented using lactic acid bacteria such as Lactobacillus and Lactococcus.
- the fermentation process converts carbohydrate into lactic acid, which increases the shelf-life of the product, as well as enhancing the taste and improves the digestibility of milk.
- Cheese, yogurt, sour cream and creme fraiche are examples of such products.
- the present invention provides lactic acid bacterial cultures.
- the present invention provides cultures comprising lactic acid bacteria, a dairy substrate (e.g., milk or cream), at least one formate source, and at least one purine source.
- the cultures comprise exogenous lactase enzyme.
- the present invention also provides cultures of cells comprising the culture medium and lactic acid bacteria.
- the present invention further provides starter compositions (i.e., starter cultures).
- the compositions of the present invention find use in the production of fermented milk products (e.g., yogurt, cheese and/or fermented milk beverages).
- the invention provides a bacterial culture comprising a dairy substrate, a population of lactic acid bacteria, lactase, a source of formate, and a source of purine.
- the cultures further comprise at least one exogenous lactase enzyme.
- the bacterial culture comprises a dairy substrate, a population of lactic acid bacteria, an exogenous lactase, a source of formate, and a source of purine.
- the present invention also provides cultures of cells comprising the culture medium and lactic acid bacteria.
- the present invention further provides starter compositions (Le., starter cultures), hi some embodiments, the compositions of the present invention find use in the production of fermented milk products (e.g., yogurt, cheese and/or fermented milk beverages).
- the present invention provides compositions for starting a culture (i.e., a "starter culture”).
- the composition is frozen and comprises a population of lactic acid bacteria and exogenous lactase enzyme.
- the composition is used in methods that include combining the above-described starter composition with a dairy substrate (e.g., milk or cream) to make a bacterial culture.
- a dairy substrate e.g., milk or cream
- the bacterial culture is maintained under culture conditions to make a fermented milk product (e.g., yogurt, cheese or fermented milk beverages).
- the present invention also provides bacterial cultures.
- the bacterial culture comprises: a dairy substrate; a population of lactic acid bacteria; and exogenous lactase enzyme.
- the dairy substrate is milk or cream. It is contemplated that milk from any suitable animal will find use in the present invention, including but not limited to cow, goat, sheep, yak, buffalo, bison, etc. Indeed it is intended that the present invention encompass milk obtained from any mammal.
- the bacteria are members of the genera Lactococcus, Lactobacillus or Streptococcus.
- the dairy substrate is not a lactose-hydrolyzed dairy substrate.
- the lactase enzyme is present at a concentration of about 0.1 ⁇ g/ml to about 10 mg/ml.
- the lactic acid bacteria population is in lag phase in this culture.
- the culture is prepared by inoculating milk with a starter composition comprising the lactase enzyme and lactic acid bacteria.
- the bacterial culture is prepared using a method that includes combining a dairy substrate with a population of lactic acid bacteria and isolated lactase enzyme.
- the population of lactic acid bacteria and the isolated lactase enzyme are combined simultaneously, while in other embodiments the lactic acid bacteria and isolated lactase enzyme are sequentially combined.
- the bacterial cultures further comprise at least one source of formate.
- the bacterial cultures are frozen and/or freeze-dried.
- the present invention also provides further compositions for starting cultures.
- the composition is frozen and/or freeze-dried, and comprises: a population of lactic acid bacteria; at least one formate source; and at least one purine source.
- the formate source is a formate salt (e.g., sodium formate).
- the purine source is inositol 5' monophosphate, inosine, hypoxantine, guanine, or disodium-5'-inosinate.
- the starter composition comprises a population of lactic acid bacteria at a concentration in the range of about 10 6 to about 10 13 CFU/g, a source of formate at a concentration of about 10% to about 50% (w/w); and a source of purine at a concentration of about 10% to about 50% (w/w).
- the methods include combining the above-described starter composition with a dairy substrate (e.g., milk or cream) to make a bacterial culture.
- the bacterial culture is maintained under culture conditions to make a fermented milk product (e.g., yogurt, cheese, or fermented milk beverages).
- the present invention also provides further bacterial cultures.
- the bacterial culture comprises: a dairy substrate; a population of lactic acid bacteria; a source of formate; and a source of purine.
- the dairy substrate is milk or cream. It is contemplated that milk from any suitable animal will find use in the present invention, including but not limited to cow, goat, sheep, yak, buffalo, bison, etc. Indeed it is intended that the present invention encompass milk obtained from any mammal.
- the bacteria are members of the genera Lactococcus, Lactobacillus or Streptococcus.
- the formate source is present at a concentration of about 0.25 ppm to about 50 ppm, and the source of purine is present at a concentration of about 0.5 ppm to about 100 ppm.
- the lactic acid bacteria population is in lag phase in this culture.
- the culture is prepared by inoculating milk with a starter composition comprising sources of formate and purine, and lactic acid bacteria.
- the composition for starting a culture comprises: a population of lactic acid bacteria, exogenous lactase enzyme, a source of formate; and a source of purine.
- the bacterial culture comprises: a dairy substrate, a population of lactic acid bacteria, exogenous lactase enzyme, a source of formate; and a source of purine.
- cultures are made by combining a dairy substrate with a starter composition containing all of the added components.
- the dairy substrate is simultaneously combined with the added components, while in other embodiments, the dairy substrate is sequentially combined with the added components.
- any of the above-described bacterial cultures will find use in methods comprising: maintaining the bacterial culture under conditions suitable for fermentation, to make a fermented milk product.
- the methods provide for the production of lactic acid by the population of lactic acid bacteria, and lowering of the pH of the culture.
- the fermented milk product is yogurt, cheese or a fermented milk beverages.
- the methods further include adding a flavoring and/or a stabilizer to the fermented milk product.
- the methods further include packaging the fermented milk product.
- the present invention also provides fermented products made using any of the methods provided herein.
- Figure 1 provides a graph of pH relative to time, for four cultures of lactic acid bacteria.
- the present invention provides lactic acid bacterial cultures.
- the present invention provides cultures comprising lactic acid bacteria, a dairy substrate (e.g., milk or cream), at least one formate source, and at least one purine source.
- the cultures comprise exogenous lactase enzyme.
- the cultures comprise exogenous lactase enzyme combined with at least one formate source, and / or at least one purine source.
- the present invention also provides cultures of cells comprising the culture medium and lactic acid bacteria.
- the present invention further provides starter compositions (Ie., starter cultures) for starting the culture.
- the starter compositions comprise exogenous lactase enzyme which can be combined with at least one formate source, and / or at least one purine source.
- the compositions of the present invention find use in the production of fermented milk products (e.g., yogurt, cheese and/or fermented milk beverages).
- the present invention provides bacterial cultures comprising a dairy substrate (e.g., milk), a population of lactic acid bacteria, and exogenous lactase enzyme.
- a dairy substrate e.g., milk
- Other embodiments provide a bacterial culture containing a dairy substrate, a population of lactic acid bacteria, at least one formate source, and at least one purine source.
- these bacterial cultures are starter compositions comprising a population of lactic acid bacteria, at least one source of formate, and at least one source of purine.
- the starter composition is used to inoculate a dairy substrate, in order to produce a culture.
- the present invention provides a variety of compositions, including bacterial cultures and compositions for starting such cultures. The present invention also provide various methods for using the present compositions.
- the lactase-related embodiments and the format/purine- related embodiments are combined (e.g., providing a bacterial culture containing a dairy substrate, a population of lactic acid bacteria, exogenous lactase enzyme, at least one formate source, and at least one purine source). In some further embodiments at least one formate source or at least one purine source find use.
- lactase enzyme and/or sources of formate and purine, facilitate the growth of the lactic acid bacteria in the culture, particularly during the early growth stages (i.e., when the bacteria are in lag phase).
- the added components allow the culture to reach a steady state pH in the range of about 3.6 to about 6.0, earlier than an equivalent culture that does not contain the added lactase enzyme, and/or formate and/or purine sources.
- cultures reach a steady state pH in less than about 70%, less than about 80%, less than about 90% or less than about 95% of the time of an equivalent culture that does not contain the added components.
- the resultant time saving is in the region of about 0.5 to about 10 hours per fermentation run. In some further embodiments, the time saving is from about 1 to about 2 hours per fermentation run.
- the sources of formate and purine in the culture medium negate the inhibitory effects of oxygen in the culture medium.
- the resultant fermented milk product tastes sweeter, and contain less lactose than fermented milk products produced without added lactase.
- the present invention be limited to any particular mechanism of action, nor theory.
- the term "food” refers to any nutritional item that provides nourishment to a plant and/or animal. It is not intended that the term be limited to any particular item, as it is used in reference to any substance taken into and assimilated by a plant or animal to keep it alive. It is also not intended that the term be limited to "solid" food, as liquid nourishment is encompassed by the definition. Indeed in some embodiments, liquid nourishment is preferred over solid food items. In some preferred embodiments, the term is specifically used in reference to food for human consumption.
- the term "feed” refers to any nutritional item that provides nourishment to non-human animals. It is not intended that the term be limited to any particular item, as it is used in reference to any substance taken into and assimilated by a plant or animal to keep it alive. It is also not intended that the term be limited to "solid” food, as liquid nourishment is encompassed by the definition. Indeed in some embodiments, liquid nourishment is preferred over solid food items.
- the term "isolated” with respect to bacteria refers to bacteria that have been isolated from their natural environment and cultured in vitro prior to use.
- the term “isolated” refers to a protein or other molecule that is removed from at least one component with which it is naturally associated.
- the term “purified” refers to a molecule that it is at least about 50%, at least about 90%, or at least about 99% free of components with which it is naturally associated.
- the term “dairy substrate” refers to milk of an animal, as well as components of the milk (e.g., cream), and combinations of milk and cream (e.g., half and half).
- milk refers to whole milk produced by an animal, as well as processed forms thereof.
- any suitable form of milk finds use in the present invention, including milk that includes or does not include whey, as well as whole milk, raw milk, skim milk, evaporated milk, reconstituted milk, condensed milk, pasteurized milk, unpasteurized, homogenized, non-homogenized, or re-hydrated milk powder.
- milk in any form will find use in the present invention.
- cream obtained from any suitable milk finds use.
- milk obtained from any suitable mammal finds use in the present invention, including but not limited to milk obtained from sources such as bovines, ovines, caprines, porcines, yaks, buffaloes, water buffaloes, bison, canines, felines, primates, equities, etc.
- cream refers to the higher-butterfat layer that is skimmed from the top of milk before homogenization.
- the fat contained in the milk is lighter than the other milk components and rises to the top of the milk.
- any form of cream be encompassed, including but not limited to whole cream, light cream, coffee cream, table cream, medium cream, whipping or light whipping cream, extra heavy or manufacturer's cream, clotted cream, double cream, etc.
- the cream is rehydrated cream powder. Indeed, it is not intended that the present invention be limited to any particular form of cream. It is also intended that as with milk, any suitable source of cream finds use in the present invention.
- lactose-hydrolyzed dairy substrate refers to a dairy substrate that has been pre-treated with lactase enzyme to reduce lactose levels. Thus, these dairy substrates have lower lactose levels than a non-lactose-hydrolyzed dairy substrate.
- lactic acid bacteria refers to any of a clade of Gram-positive, acid tolerant, non-sporulating, non-respiring rod or cocci that produce lactic acid as the major metabolic end-product of carbohydrate fermentation.
- bacteria include, but are not limited to, bacteria of the genus Lactococcus, Lactobacillus and Streptococcus.
- culture refers to any sample or item that contains one or more microorganisms.
- Purure cultures are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to “mixed cultures,” which are cultures in which more than one genus and/or species of microorganism are present. In some embodiments of the present invention, pure cultures find use.
- Lactobacillus refers to members of the genus Lactobacillus, in the family Lactobac ⁇ llaceae. These bacteria are Gram-positive facultatively anaerobic bacteria that represent a major part of the bacterial group often referred to as "lactic acid bacteria.” Various species of Lactobacillus have been identified, including but not limited to L. acidophilus, L. bulgaricus, L. casei, L. delbrueckii, L. fermentum, L. plantarum, L. reuteri, etc. It is intended that the genus include species that have been reclassified ⁇ e.g., due to changes in the speciation of organisms as the result of genetic and other investigations) or renamed for marketing and/or other purposes.
- microbiological media As used herein, the terms “microbiological media,” “culture media,” and “media” refer to any suitable substrate for the growth and reproduction of microorganisms. The term encompasses solid plated media, as well as semi-solid and liquid microbial growth systems.
- additive refers to any compound, composition, element and/or other material that is added to the culture media to enhance microbial growth and/or production of a desired protein.
- lactase refers to a glycoside hydrolase that catalyzes the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers. Lactases have an activity described as EC 3.2.1.108, according to IUBMB enzyme nomenclature. The systematic name for a lactase is ⁇ -D-galactopyranosyl ⁇ (l ⁇ 4)- ⁇ -D-glucopyranose. [038] As used herein, the term “lactase unit” refers to the measurement of lactase activity.
- one lactase unit hydrolyzes 1 uM of o-nitrophenyl-beta-D-galactoside to o-nitrophenol and beta-galactosidase per minute at pH 4.5, and 30°C.
- the unit is the same, but the pH is 6.5 and the temperature is 37 0 C.
- exogenous lactase enzyme when used with reference to a bacterial culture that contains such an enzyme, refers to an enzyme that is not secreted by the bacteria in the culture (Le., the lactase is added to the medium).
- the term “endogenous lactase enzyme” refers to lactase that is produced by the lactic acid bacteria in a culture. In some embodiments, the endogenous lactase enzyme is over- expressed by the lactic acid bacteria, such that the concentration of lactase in the composition is greater than it would be if the lactic acid bacteria did not overexpress the lactase enzyme.
- the term “frozen” refers to a solid state composition that is at a low temperature (e.g., less than about O 0 C). In some preferred embodiments, the term refers to a composition that is at a temperature of about -2O 0 C or less.
- the terms “freeze dried” and “lyophilized” refer to a solid state composition that is dried by freezing the composition in a high vacuum. As with freezing, these methods are well known in the art and any suitable method finds use in the present invention.
- the terms “source of formate” and “formate source” refer to a compound that when added to a culture of cells, provides formate. In some embodiments, the source of formate releases formate into a growth medium, while in other embodiments, the formate source is metabolized to produce formate. In some preferred embodiments, the formate source is exogenous. In some particularly preferred embodiments, formate is not provided by the dairy substrate.
- the terms “source of purine” and “purine source” refer to a compound that when added to a culture of cells, provides purine nucleotides. In some embodiments, the source of purine releases purine into a growth medium, while in other embodiments, the purine source is metabolized to produce purine. In some preferred embodiments, the purine source is exogenous. In some particularly preferred embodiments, purine is not provided by the dairy substrate.
- the term “exogenously added,” with reference to a growth medium refers to a compound that is added in addition to other components of the growth medium. For example, if a growth medium contains a dairy substrate and a source of formate that is exogenously added, the source of formate is not present in the dairy substrate. Rather, the source of formate is added in addition to the dairy substrate.
- the term "fermented milk product” refers to a food product made by fermentation of dairy substrate using at least one species of lactic acid bacteria.
- the products have a low pH (e.g., a pH of about 3.6 to about 6.0).
- the products contain lactic acid.
- Fermented milk products are provided in any form, including frozen, liquid or solid form.
- the products contain living bacteria, while in some alternative embodiments, no living bacteria are present. Fermented milk products include, but are not limited to cheese, yogurt, sour cream, creme fraiche, and fermented milk beverages.
- the present invention provides bacterial cultures.
- the bacterial culture comprises: dairy substrate (e.g., milk or cream); a population of lactic acid bacteria; at least one source of formate; and at least one source of purine.
- dairy substrate e.g., milk or cream
- a population of lactic acid bacteria at least one source of formate
- at least one source of purine at least one source of purine.
- the present invention also provides growth media comprising all of the above components with the exception of the population of lactic acid bacteria.
- the bacterial culture comprises: dairy substrate; a population of lactic acid bacteria; and exogenous lactase enzyme.
- the present invention also provides growth media comprising all of the above components with, the exception of the population of lactic acid bacteria.
- the bacterial culture comprises a dairy substrate, a population of lactic acid bacteria, an exogenous lactase eirzyme, at least one source of formate, and/or at least one source of purine.
- the present invention also provides growth media comprising all of the above components with the exception of the population of lactic acid bacteria.
- the major component of the bacterial culture is the dairy substrate (e.g., milk).
- the dairy substrate component of the culture is milk from any suitable animal species.
- the dairy substrate comprises at about least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the volume of the culture.
- Any suitable form of milk finds use in the present invention, including milk that includes or does not include whey, as well as whole milk, raw milk, skim milk, evaporated milk, reconstituted milk, condensed milk, or re- hydrated milk powder. Indeed, it is contemplated that milk in any form will find use in the present invention.
- the dairy substrate is not lactose-hydrolyzed milk (i.e., it has not been pre-treated to reduce the lactose content of the milk (e.g., by either enzyme treatment or filtration).
- the concentration of lactose in a dairy substrate is at least about 4% (e.g., about 4% to about 5% or about 4.5% to about 5%) lactose.
- the dairy substrate is cow's milk having a lactose concentration of about 4.7% (w/v), goat's milk having a lactose concentration of about 4.1%, buffalo's milk having a lactose concentration of about 4.86%, yak milk having a lactose concentration of about 4.93%, or sheep's milk having a lactose concentration of about 4.6%.
- the population of lactic acid bacteria is a population of lactic acid bacteria that has GRAS status. In some preferred embodiments, the population of lactic acid bacteria is in the Order Lactobacillales.
- the bacteria in the culture are a single species (i.e., a "pure culture"), while in other embodiments, the bacteria comprise a mixture of species within the following genera: Abiotrophia, Aerococcus, Bifidobacterium, Carnobacterium, Enterococcus, Lactobacillus (including, but not limited to: L. acidophilus, L. brevis, L. delbrueckii ssp. bulgaricus, L. casei, L. delbruecMi, L. fermentum, L. helveticus, L. plantarum, L. rhamnosus, L. reuteri, and/or L.
- Abiotrophia Aerococcus
- Bifidobacterium Carnobacterium
- Enterococcus Enterococcus
- Lactobacillus including, but not limited to: L. acidophilus, L. brevis, L. delbrueckii ssp. bulgaricus, L
- Lactococcus including, but not limited to: L. garvieae, L. lactis, L. piscium, L. plantarum, and/or L. raffinolactis
- Leuconostoc Oenococcus
- Pediococcus Streptococcus (including, but not limited to S. thermophilus)
- Tetragenococcus Vagococcus and Weissella.
- the population of bacteria in the culture may be at any phase of growth, including lag phase, logarithmic phase, and stationary phase, or may be dormant.
- the exogenous lactase enzyme is any suitable lactase that is active in milk. Such enzymes are known in the art and are commonly employed in treatments for lactose intolerance, and for other applications (e.g., for pre-treatment of milk to lower lactose levels; See e.g., Reprod. Nutr. Dev., 42:127-32 [2002]).
- the lactase is a commercially available lactase, (e.g., the lactase of Kluyveromyces fragilis [K m ⁇ xianus var. marxianus]; Artolozaga et al.
- the lactase is an acid lactase having an optimal lactase activity at a pH ranging from approximately 4 to approximately 6.
- the lactase is a neutral lactase having an optimal lactase activity at a pH ranging from approximately 5.5 to approximately 7.
- acid lactases are used.
- acid and neutral lactases are used.
- Non-limiting examples of acid lactase is Lactase DS (Amano Enzyme, Nagoya, Japan).
- Non-limiting examples of neutral lactase include Maxilact® (DSM, Herleen, The Netherlands) and Lactase Godo (Godo Shusei Co., Tokyo, Japan).
- the enzyme is purified, while in other embodiments it is obtained from a natural isolate, or is produced using recombinant means.
- the lactase enzyme is present at a concentration that provides the desired effect. In some preferred embodiments, the lactase enzyme is present at a concentration in the range of about 0.1 ⁇ g/ml to about 10 mg/ml, although it is contemplated that concentrations outside of this range will find use in the present invention. In some embodiments, the lactase is present at a concentration of about 0.2 ⁇ g/ml to about 100 ⁇ g/ml. In still further embodiments, the concentration of lactase ranges from 0.5 to 20 units of lactase per milliliter of milk.
- the lactase present in the culture is exogenous (i.e., it is not secreted by the cells in the culture).
- the lactase is at a concentration that is significantly higher then the level of any lactase secreted by the bacteria in the culture.
- the formate source(s) comprise(s) any compound that releases formate into solution, or that can be metabolized by a lactic acid bacterium to produce formate.
- a formate salt is used (e.g., sodium formate, potassium formate, calcium formate, and ammonium formate).
- the formate source is present in the bacterial culture at a concentration of about 0.5 ppm to about 50 ppm, (e.g., about 1 ppm to about 20 ppm, or about 2 ppm to about 10 ppm).
- the source of formate is exogenous in that it is not supplied by the dairy substrate.
- any suitable source of purine that releases purine into solution, or that can be metabolized by a lactic acid bacterium to produce a purine nucleotide finds use in the present invention.
- the purine source is metabolizable into any purine required for cell growth (e.g., AMP, ADP, ATP, cAMP, NADH and GTP).
- Suitable purines include, but are not limited to inositol 5' monophosphate, inosine, hypoxanthine, and guanine.
- the source of purine is present in the bacterial culture at a concentration of about 1 ppm to about 100 ppm (e.g., about 2 ppm to about 50 ppm, or about 5 ppm to about 20 ppm). In some particularly preferred embodiments, the source of purine is exogenous in that it is not supplied by the dairy substrate.
- the bacterial culture is prepared using any suitable method. Indeed, it is not intended that the present invention be limited to any particular method for producing the bacterial culture. However, in some preferred embodiments and as described in greater detail below, the culture is made by combining a dairy substrate (e.g., milk or cream), with a starter composition that contains lactic acid bacteria and the sources of formate and/or purine. In some further particularly preferred embodiments, the culture further comprises at least one lactase enzyme. These starter compositions are described in greater detail below. In some alternate embodiments, the culture is made by independently adding the various components to the dairy substrate simultaneously, while in other embodiments, the various components are sequentially added to the dairy substrate.
- a dairy substrate e.g., milk or cream
- a starter composition that contains lactic acid bacteria and the sources of formate and/or purine.
- the culture further comprises at least one lactase enzyme. These starter compositions are described in greater detail below.
- the culture is made by independently adding the various components to the dairy substrate simultaneously, while in other embodiment
- the culture is prepared by mixing a frozen starter composition comprising the cells and sources of purine and formate, and a powdered or liquid form of the enzyme with the dairy substrate.
- the starter composition and the enzyme are added simultaneously, while in some alternative embodiments they are added in rapid succession (i.e., within 5 minutes of the first addition).
- the above-described bacterial culture finds use in methods that include maintaining the culture under conditions suitable for fermentation.
- the bacterial culture is used to produce a fermented milk product.
- the conditions are suitable for the production of lactic acid by the population of lactic acid bacteria, and/or for lowering the pH of the culture (e.g., to a pH of about 3.6 to about 6.0).
- the fermented milk product is cheese, while in other embodiments, it is yogurt, cultured sour cream, kefir, buttermilk, acidophilus milk, creme fra ⁇ che, kumis, shubat, quark, filmj ⁇ lk, Smetana, skyr, villik, or koumiss.
- the yogurt is thermophilic fermented milk made using Lactobacillus bulgaricus or Streptococcus thermophilus).
- the cultured sour cream is a mesophilic fermented pasteurized cream with an acidity of at least about 0.5%, made using Lactococcus lactis subsp. lactis).
- the cultured buttermilk is a mesophilic fermented pasteurized milk made using Lactococcus lactis (e.g., Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis biovar. diacetylactis) and/or Leuconostoc (e.g., Leuconostoc mesenteroides subsp. cremoris).
- Lactococcus lactis e.g., Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis biovar. diacetylactis
- Leuconostoc e.g., Leuconostoc mesenteroides subsp. cremoris
- the acidophilus milk is a thermophilic fermented milk beverage, often low-fat (i.e., about 2%, or about 1.5%) or nonfat (i.e., about 0.5%), made using, Lactobacillus acidophilus.
- the creme fraiche is a mesophilic fermented cream made using lactic acid bacteria that are naturally presenting in the cream.
- the product is kumis made using mare milk, shubat made using camel milk, quark, filmjolk, smetana, skyr or viili, or koumiss. Methods for making such fermented milk products are well known, and are readily adapted by the addition of sources of purine and formate, as described above.
- a flavoring composition is added to the culture prior to use.
- the flavoring composition is savory and in some embodiments contains salt, spices, herbs, flavorings, colorants, purees or pieces of vegetables and/or cereals.
- the flavoring composition is sweet and in some embodiments, contains sugar, honey, syrup, flavorings, colorants, compote, jam, marmalade, preserves, pulps, and/or pieces of fruit (e.g., dried, preserved, candied, and/or fresh).
- the flavoring composition is stabilized with a stabilizer, such that is given the necessary consistency for extrusion.
- Suitable stabilizers include but are not limited to food gums (e.g., locust bean gum, guar gum, xanthan gum, starches, short texture native or modified starch, and pectin).
- the food gums are used individually, while in other embodiments, they are used as an admixture.
- the methods of the present invention further include packaging the fermented milk product (e.g., to make a product that can be shipped and commercially sold). In other embodiments, the fermented milk product is shipped to a remote location prior to packaging.
- the fermented milk product is old as a drink or food (e.g., a cheese, fermented milk drink, lactic acid bacteria beverage, yogurt, cultured milk, kefir, or the like).
- the product is plain, while in other embodiments, it is flavored, sweetened or contains additional components (e.g., fruit).
- the product is provided in any suitable form (e.g., soft, hard, liquid, or a frozen product).
- the present invention also provides fermented products made using the methods provided herein.
- the fermented milk product tastes sweeter than equivalent products made without the additional components.
- the relative sweetness of lactose is approximately 20% of sucrose whereas glucose and galactose are 60% as sweet as sucrose (Fennema, 1996 ; Iversen, 1983).
- Hydrolysis of 70% of lactose present in milk increases the sweetness by an amount comparable to the addition of approximately 2% of sucrose (Lindamood et al. 1989).
- the sweeter taste enables dairies and/or dairy processors to decrease the level of added natural or artificial sugars to the fermented products. This results in healthier fermented products since these products contain a reduced amount of sweetener(s).
- the present invention will provide cost savings for the dairy industry, as a decreased amount of added sweetener in fermented products is facilitated by the present invention. It is also contemplated that the present invention will provide cost savings to the agro food industries (e,g, the bakery industry), as fermented products having a sweeter taste to make food product (e.g., cakes, pancakes, biscuits) with a reduced amount of added sweetener are provided. In some embodiments, this additional sweetness is at least partially due to the production of galactose and glucose (i.e., from lactose metabolism). In some further embodiments, the fermented products have a more appealing texture when frozen due to reduced crystallization than similar products made using standard methods known in the art.
- agro food industries e,g, the bakery industry
- fermented products having a sweeter taste to make food product e.g., cakes, pancakes, biscuits
- this additional sweetness is at least partially due to the production of galactose and glucose (i.e., from lac
- the fermented product is a "low lactose" product, which contains less lactose (e.g., at least about 10% less, at least about 20% less, at least about 30% less, at least about 50% less, or at least about 70% less) than products made without the additional components.
- the final lactose concentration of a product made using the methods of the present invention is about 0.1% to about 3%, while in some preferred embodiments the concentration is from about 0.5% to about 2%.
- the lactose level is reduced to a level below lOOppm in the final fermented product, or to a level that permits the fermented product to be considered or labeled as "lactose free.”
- the present invention provides starter culture compositions.
- the starter composition is combined with a dairy substrate (e.g., milk or cream), to produce a cell culture, as described in greater detail herein.
- the starter composition is a frozen composition.
- the starter culture is packaged in a way that facilitates easy addition of the composition to a dairy substrate, in order to produce a culture.
- the present invention provides starter compositions comprising frozen pellets of cell culture, at least one source of formate and/or at least one source of purine.
- the starter composition comprises lactic acid bacteria, and exogenous lactase enzyme at a concentration higher than its concentration in a cell culture without the addition of exogenous lactase.
- the starter composition contains lactic acid bacteria, a source of formate, and/or a source of purine, wherein the concentrations of formate and/or purine are higher than their concentrations in the culture of cells without the added sources of formate and/or purine.
- the starter composition contains frozen pellets of cell culture, a source of formate, and a source of purine.
- a starter composition comprises the cells, the sources of purine and formate, and exogenous lactase.
- Starter compositions containing lactic acid bacteria are well known in the art (See e.g., U.S. Patent Appln. Publns. 20060204484, 20050053582 and 20050032196, as well as Speckman et at. LDairy Sci., 57:165-173 [1974]; Jakubowska eta!., Acta Microbiol. PoL, 1980 29:135-44 [1980]); Gibson et al, Appl. Microbiol, 14:665-9 [1966]); and Keogh et al. , Appl. Microbiol., 19:928-31 [1970]).
- the starter culture is modified to contain the sources of formate and purine, as discussed above.
- the lactic acid bacteria are present at a concentration in the range of about 10 6 CFU/g to about 10 13 CFU/g, for example.
- the sources of formate and purine are each independently present at a concentration of about 5% to about 80% (e.g., about 10% to about 50% (w/w)).
- the enzyme is present at a concentration of about 10 mg/ml to about 200 mg/ml.
- the concentration of lactase blended within the starter composition is set to provide a lactase concentration in the process milk from about 0.5 to about 20 units of lactase per milliliter of milk.
- a starter composition is made by adding appropriate amounts of the powdered forms of the sources of formate and purine to a lyophilized (i.e., freeze dried) cell culture.
- the starter composition is made by blending a frozen bacterial pellet with a frozen solution containing the sources of formate and purine.
- the lactase is added at any time during the preparation of the starter composition.
- the starter culture is made by inoculating hydrolyzed 1-5% milk supplemented with carbohydrates (e.g., glucose, lactose, and/or sucrose), yeast extract, peptones and/or minerals.
- carbohydrates e.g., glucose, lactose, and/or sucrose
- yeast extract e.g., yeast extract
- peptones e.g., peptones and/or minerals.
- the pH is controlled, such that it is maintained at about pH 6 (i.e., using NH 4 OH).
- the fermentate is cooled and the cells concentrated in a centrifuge. Ih some embodiments, the concentrate is then canned or placed in suitable containers for freezing in liquid nitrogen or dripped as drops into liquid nitrogen.
- the pellets containing the sources of formate and purine are also made by dripping solutions containing those compounds (either separately or mixed) into liquid nitrogen.
- the culture pellets and formate/purine pellets are then mixed to provide starter compositions that contain those components suitable concentrations.
- the starter composition is combined with a dairy substrate to produce the bacterial cultures described above. Kits
- kits for inoculating a dairy substrate with a starter culture comprise at least one culture of lactic acid bacteria (frozen or liquid), isolated lactase enzyme (powdered or a liquid stabilized in glycerol), a source of formate and a source of purine (powdered or a dissolved in a liquid, such as water).
- the various components of the kit are present in separate containers, while in other embodiments, compatible components are precombined into a single container, as desired.
- the kits further comprise a starter composition.
- the present invention provides kits for inoculating a dairy substrate.
- kits comprise a frozen or liquid starter culture of lactic acid bacteria, at least one source of formate and/or at least one source of purine.
- the purine and/or formate sources are in powder form, while in other embodiments the purine and/or formate sources are in liquid (e.g., dissolved in water).
- the various components of the kit are provided in separate containers, while in some further embodiments, compatible components are precombined into a single container, as desired.
- the kits contain at least one starter composition.
- kits further comprise instructions for using the components of the kit to practice the methods of the present invention.
- the instructions for practicing the subject methods are generally recorded on a suitable recording medium, and may contain information on the amount of dairy substrate that is to be inoculated.
- the instructions are printed on a substrate, such as paper or plastic, etc.
- the instructions are provided in the kits as a package insert and/or on the labeling of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
- the fermented milk product is packaged (e.g., to make a product that can be shipped and sold). In other embodiments, the fermented milk product is shipped to a remote location prior to packaging.
- the fermented milk product is sold as a drink, while in other embodiments, it is sold as a food (e.g., cheese, fermented milk drink, lactic acid bacteria beverage, yogurt, cultured milk, kefir, or the like).
- the product is plain, while in other embodiments, it is flavored, sweetened, and/or contain fruit or vegetables. In some embodiments, it is a soft product, while in other embodiments, it is hard, liquid, or frozen.
- Example 2 The same experiment as described in Example 2 was performed w/ a freeze dried yogurt culture ⁇ Streptococcus thermophilus and Lactobacillus bulgaricus). In this example the milk was at a temperature of 108 0 F. Inoculation rate was 0.003%. Again, lactase was added in accordance with the following table, and the pH of the culture was measured 2.5 hrs later.
- the combination booster i.e., Na-formate, IMP, and lactase
- the milk was at a temperature of 86 0 F (30 0 C).
- the inoculation rate of the culture was 0.025%, and the pH was measured at 4 hours. The results are provided below.
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Abstract
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US98390907P | 2007-10-30 | 2007-10-30 | |
US2020508P | 2008-01-10 | 2008-01-10 | |
PCT/IB2008/003302 WO2009056979A1 (en) | 2007-10-30 | 2008-10-30 | Growth medium for lactic acid bacteria |
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DE102010009582A1 (en) | 2010-02-05 | 2011-08-11 | Vitacare GmbH & Co. KG, 60318 | Agent for use in lactase deficiency and lactose intolerance |
CN103249830A (en) * | 2010-12-10 | 2013-08-14 | 帝斯曼知识产权资产管理有限公司 | Starter culture compositions |
AU2015201040B2 (en) * | 2011-04-20 | 2015-11-12 | Dupont Nutrition Biosciences Aps | Production of cheese with s. thermophilus |
US20140134292A1 (en) * | 2011-04-20 | 2014-05-15 | Dupont Nutrition Biosciences Aps | Production of cheese with s. thermophilus |
US20200205431A1 (en) * | 2017-06-15 | 2020-07-02 | Dsm Ip Assets B.V. | Frozen enzyme pellets |
WO2024115523A1 (en) | 2022-11-29 | 2024-06-06 | Dsm Ip Assets B.V. | Process for preparing a frozen or freeze-dried solid additive composition |
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US4956186A (en) * | 1989-10-25 | 1990-09-11 | Borden, Inc. | Process for the production of low calorie yogurt |
CA2594378C (en) * | 2005-01-05 | 2015-02-24 | Chr. Hansen A/S | Use of compounds involved in biosynthesis of nucleic acids to increase yield of bacterial cultures |
KR101530055B1 (en) * | 2005-10-11 | 2015-06-29 | 프로바이오티컬 에스.피.에이. | Method and related uses for non-allergic probiotic bacterial cultures |
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Non-Patent Citations (2)
Title |
---|
' LEARY ET AL: "Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme", APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMERICAN SOCIETY FOR MICROBIOLOGY, 1 July 1976 (1976-07-01), pages 89 - 94, XP055280416, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC170011/pdf/aem00006-0101.pdf> [retrieved on 20160614] * |
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