CN115624065A

CN115624065A - Preparation method of fermented yogurt rich in conjugated linoleic acid and omega-3 fatty acid

Info

Publication number: CN115624065A
Application number: CN202211331317.4A
Authority: CN
Inventors: 陈振华; 陈玮南
Original assignee: Hunan Deyuan Zhongchuang Technology Co ltd
Current assignee: Hunan Deyuan Zhongchuang Technology Co ltd
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-01-20

Abstract

The invention discloses a preparation method of fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid, belonging to the technical field of preparation of fermented milk. The invention strictly controls the feeding and yoghourt fermentation links of the dairy cows and improves the contents of conjugated linoleic acid and omega-3 fatty acid in the raw milk. The fermented yoghourt is rich in nutrition, has high contents of conjugated linoleic acid and omega-3 fatty acid, enhances the health care effect of the yoghourt, is constantly drunk every day, can obviously improve three highs after 3-6 months, improves the sleep quality, relieves constipation, enhances the immunity and improves the health condition of drinkers.

Description

Preparation method of fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid

Technical Field

The invention relates to the technical field of fermented milk preparation, in particular to a preparation method of fermented yogurt rich in conjugated linoleic acid and omega-3 fatty acid.

Background

The yogurt is a fermented product of milk, has a long history and is rich in nutrition. The yoghourt contains rich high-quality protein, vitamin B1, vitamin B2 and rich calcium. The protein content of the yoghourt is generally 3%, the proportion of the essential amino acid meets the requirement of a human body, and the yoghourt belongs to high-quality protein. Compared with milk, the yogurt can improve symptoms of lactose intolerance and constipation, and help to maintain normal gastrointestinal functions. However, during the production, storage and transportation of yogurt, whey protein precipitation, dehydration, etc. may occur in yogurt, resulting in loss of nutrients and a decreased sensory experience.

The stabilizer is added into the yoghourt product to keep the stable state of the yoghourt and reduce the whey separation, which is a universal and feasible method. Chinese patent CN103081995A discloses a compound stabilizer, fermented yoghurt containing the same and a preparation method of the fermented yoghurt, wherein the compound stabilizer of the invention contains esterified modified starch, pectin, sodium alginate, agar, xanthan gum and gelatin; the viscosity of the sodium alginate is 500-1000 mPas. The preparation method comprises the following steps: (1) uniformly mixing the compound stabilizer, the sweetening agent, water and pre-sterilized non-antibiotic milk; (2) homogenizing; (3) pasteurization; (4) cooling, adding leaven for fermentation; (5) turning over the cylinder; (6) pasteurization; (7) rapidly cooling, and storing under sterile condition. The fermented yoghourt produced by the secondary pasteurization technology and the aseptic filling technology can still keep the stability of physicochemical and sanitary indexes after being stored for 3-6 months at the normal temperature of 20-40 ℃. The normal-temperature sale of the fermented yoghourt with the protein being more than or equal to 2.3 percent and the long-distance sale of the fermented yoghourt due to the limitation of a cold chain are realized. However, in practical applications, the chemical composition of the compound stabilizer contains an aldehyde acid structure, wherein the aldehyde structure is unstable and is easily oxidized, so that the effect of the stabilizer is reduced, and the quality of the product is affected.

The active substances with the functions of scavenging free radicals and resisting oxidation are added into the yoghourt to effectively inhibit the oxidation process of the yoghourt. Chinese patent CN114831181A provides an antialcoholic and hepatoprotective yoghourt and a preparation method thereof, wherein raw milk is separated to obtain milk fat and skim milk, and the milk fat and the skim milk are respectively sterilized; extracting dihydromyricetin from caulis et folium Tenebrianae to obtain powdered dihydromyricetin; inoculating the composite zymophyte powder into skim milk for passage to obtain activated bacterial liquid, adding dihydromyricetin powder, mixing, and fermenting to obtain dihydromyricetin fermented milk; mixing the butter fat and the concentrated whey, hydrating, homogenizing and sterilizing, cooling, inoculating flavor fermentation bacteria, adding an enzyme preparation, and fermenting to obtain the flavor butter fat, so that the flavor and the mouthfeel of the yogurt are improved; and finally, sterilizing the dihydromyricetin fermented milk and the flavored milk fat, mixing, refrigerating and aging to obtain the hangover-alleviating and liver-protecting yogurt. The yogurt has good effects of nourishing and protecting liver, expelling toxin, clearing intestine and relieving alcoholism, and has rich flavor. Dihydromyricetin has good effects of scavenging free radicals and resisting oxidation, but has poor water solubility, reduces the processability and has the technical problem of poor compatibility with an aqueous emulsion system of the yoghourt.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the present invention provides a method for preparing fermented yogurt with rich nutrition and good stability.

Conjugated linoleic acid is a newly discovered nutrient and has certain health care effect. It is mainly present in milk, and can improve the content of conjugated linoleic acid in the milk of herbivore by changing the structure of feed, and is beneficial to cardiovascular health, but the omega-3 fatty acid is thermolabile and has poor taste, so that it is the best choice to combine with yoghurt.

A preparation method of fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid comprises the following steps:

s1, preparing raw materials according to a proportion, grinding 15-30 parts of full-fat soybean, 40-60 parts of corn and 5-15 parts of linseed by weight, adding 10-15 parts of wheat bran and 1-2 parts of active yeast, and placing the mixture in a fermentation tank for fermentation for 10-15 hours at a constant temperature of 35-43 ℃; after fermentation is finished, continuously adding the baking soda, the calcium bicarbonate, the mineral substances, the vitamins and the salt, and uniformly mixing to obtain concentrated feed;

s2, proportioning the concentrated feed and the coarse feed in proportion, adding compound vegetable oil into the feed additionally, stirring the feed by a stirrer, feeding the feed by a cow in a lactation period freely, and collecting raw milk;

s3, sampling and extracting fat from the raw milk, and measuring the content of conjugated linoleic acid and omega-3 fatty acid;

s4, standardizing the raw milk according to the result in the step S3; the conjugated linoleic acid content of the raw milk is more than or equal to 40mg/g calculated by milk fat; the content of omega-3 fatty acid is more than or equal to 5 percent according to the milk content; for those which do not meet the standard, the product standard is met by adding natural extracts;

s5, sterilizing the raw milk subjected to the standardized treatment, wherein the sterilization temperature is 75-95 ℃, and the duration time is 5-30 minutes; cooling the sterilized raw milk to 40-43 ℃, inoculating a fermentation strain, and fermenting at the temperature for 5-6 h to obtain primary fermented yoghourt;

s6, adding conjugated linoleic acid and omega-3 fatty acid into the primary fermented yoghourt, uniformly mixing, and then performing secondary fermentation, wherein the temperature of the secondary fermentation is 35-39 ℃, and the fermentation time is 3-4 hours; and immediately cooling the fermented yoghourt to 2-6 ℃, adding mogroside and agaricus blazei murill chitosan type stabilizer, uniformly mixing, and canning to obtain the fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid.

Preferably, the formula of the roughage in the step S2 is as follows, and the roughage is prepared from the following components in parts by weight: 65-85 parts of green pasture and 35-25 parts of hay; the green pasture comprises at least two of broussonetia papyrifera leaves, mulberry leaves, sweet grassiness, corn stalks and ryegrass; the hay comprises at least one of alfalfa and oats.

Preferably, the mass ratio of the concentrated feed to the coarse feed in the step S2 is 35:65.

preferably, the components of the composite vegetable oil in step S2 and the addition amount of each lactating cow per day are as follows: 100-200 g of soybean oil, 150-300 g of sunflower seed oil and 50-250 g of linseed oil or safflower oil.

Preferably, the natural extract in step S4 is at least one of conjugated linoleic acid and omega-3 fatty acid; the conjugated linoleic acid is a safflower cold-pressed extract; the omega-3 fatty acid is a cold pressed extract of linseed oil.

Preferably, the fermentation strains in step S5 are lactobacillus bulgaricus and streptococcus thermophilus in a mass ratio of 1: 1-2 to obtain mixed strains; the mass ratio of the fermentation strain to the raw milk is 1:20000 to 30000.

Preferably, the addition amount of the conjugated linoleic acid in the step S6 is 0.8-3.6 g/kg; the addition amount of omega-3 fatty acid is 1-2 wt%.

Preferably, the addition amount of the mogrosides in the step S6 is 0.02-0.08 wt%; the additive amount of the agaricus blazei murill chitosan type stabilizer is 0.6 to 1.5 weight percent.

In order to improve the stability of the yoghourt product, the invention prepares and uses an agaricus blazei murill chitosan type stabilizer. The stabilizer is prepared by taking agaricus blazei murill as a raw material, and firstly extracting and preparing agaricus blazei murill chitosan; and then reacting D-leucine tert-butyl ester hydrochloride with dihydromyricetin, connecting the two through carbonyl provided by bis (p-nitrophenyl) carbonate, and performing substitution reaction on the product with amino in the agaricus blazei murrill chitosan to finally obtain the agaricus blazei murrill chitosan type stabilizer.

The agaricus blazei murill chitosan type stabilizer is irregular and flaky, has smooth and porous surface and filamentous branches, and shows good water holding performance. Due to the presence of pores and branched structures, the stabilizer can be combined with water in the yoghourt and facilitates cross-linking. The stabilizer wraps the spherical milk protein in the filamentous branches to form a denser protein gel net, and the water holding capacity of the yoghourt is increased through the stretched conformation of the cavity and the gel net, so that the water loss and protein separation in the processes of yoghourt production, storage and transportation are reduced.

The chitosan structure of the agaricus blazei murill chitosan type stabilizer contains hydroxyl and negative charge groups, and the chitosan structure is easy to combine with water to form hydrogen bonds, so that the yogurt stability is improved. The combination of dihydromyricetin and agaricus blazei murill chitosan is not realized by traditional oxidative free radical grafting, so that the possibility that free radicals are formed on phenolic hydroxyl groups at a plurality of different positions is avoided, and the reaction process is stable and controllable; the dihydromyricetin structure is introduced into the agaricus blazei murill chitosan, so that the water solubility of the agaricus blazei murill chitosan is improved, the product has amphipathy, and the stabilizing agent has an antioxidant effect while exerting a stabilizing effect.

Preferably, the preparation method of the agaricus blazei murill chitosan type stabilizer comprises the following steps of:

m1, crushing dried agaricus blazei murill into powder, and dispersing the powder in water through ultrasonic treatment to obtain an agaricus blazei murill powder suspension; heating the agaricus blazei murill powder suspension, centrifugally separating to collect supernatant, adding a sodium hydroxide aqueous solution into the supernatant, and adjusting the pH value of the supernatant to be neutral to obtain neutral supernatant; adding 4-6 times of absolute ethyl alcohol into the neutral supernatant for precipitation, and filtering to obtain a filter cake; mixing aqueous hydrogen peroxide with absolute ethyl alcohol to obtain a treatment solution, and treating the filter cake at normal temperature by using the treatment solution to obtain a filter cake mixed solution; adding 5-10 times of absolute ethyl alcohol into the filter cake mixed liquor after the step is finished, precipitating again, filtering and collecting precipitates, washing the precipitates by the absolute ethyl alcohol, and drying to obtain agaricus blazei murrill chitosan for later use;

m2, under the anaerobic condition, uniformly mixing 3.4-5.8 parts of D-leucine tert-butyl ester hydrochloride, 3.9-6.7 parts of N, N-diisopropylethylamine and 90-160 parts of tetrahydrofuran to obtain a first reaction solution for later use; uniformly mixing 4.7-7.8 parts of bis (p-nitrophenyl) carbonate and 35-60 parts of tetrahydrofuran to obtain a second reaction solution for later use; uniformly mixing 2.3-3.9 parts of dihydromyricetin and 15-25 parts of tetrahydrofuran to obtain a third reaction solution for later use;

m3, uniformly mixing the first reaction solution and the second reaction solution under an anaerobic condition to obtain a reaction mixture, and adding the third reaction solution into the reaction mixture for reaction; after the reaction is finished, adding absolute ethyl alcohol with the same volume as the reaction product into the reaction product, filtering to obtain filtrate, and freeze-drying the filtrate to obtain myricetin derivatives for later use;

m4, dissolving 1.4 to 1.9 parts of agaricus blazei murill chitosan into 75 to 100 parts of acetic acid aqueous solution with the concentration of 1 weight percent to obtain agaricus blazei murill chitosan solution; dissolving 2.1-2.9 parts of myricetin derivative in 10-20 parts of acetone to obtain myricetin derivative solution; under the anaerobic condition, uniformly mixing the agaricus blazei murill chitosan solution and the myricetin derivative solution, and carrying out substitution reaction under the catalysis of 0.8-1.0 part of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride; cooling to normal temperature after the substitution reaction is finished, adding 300-450 parts of acetone with the temperature of 0-4 ℃ into the reaction product for precipitation, then filtering and collecting a filter cake, washing the filter cake with acetone, washing with water, and drying to obtain the agaricus blazei murrill chitosan type stabilizer.

More preferably, in the agaricus blazei murill powder suspension in the step M1, the mass fraction of the agaricus blazei murill powder is 3 to 6.5%.

More preferably, the temperature of the heating treatment in the step M1 is 85 to 100 ℃, and the treatment time is 2 to 6 hours.

More preferably, the concentration of the aqueous sodium hydroxide solution in the step M1 is 0.1 to 0.2mol/L.

Further preferably, the volume ratio of the aqueous hydrogen peroxide solution to the absolute ethyl alcohol in the step M1 is 1:1 to 3, and the concentration of the aqueous hydrogen peroxide solution is 28 to 36 weight percent.

More preferably, in the step M1, in the process of treating the filter cake with the treatment solution, the mass ratio of the treatment solution to the filter cake is 15 to 40:1, the treatment time is 6 to 12 hours.

More preferably, the reaction temperature in the step M3 is 25-35 ℃, and the reaction time is 12-36 h.

More preferably, the reaction temperature of the substitution reaction in the step M4 is 65-85 ℃, and the reaction time is 18-48 h.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The introduction and the function of part of raw materials in the formula of the invention are as follows:

conjugated linoleic acid: the conjugated linoleic acid is one of essential fatty acids for human and animals, but is a substance which cannot be synthesized into the conjugated linoleic acid with remarkable pharmacological action and nutritional value, and is beneficial to human health. The conjugated linoleic acid has certain physiological functions of resisting tumor, oxidation, mutation and bacteria, reducing cholesterol of human bodies, resisting atherosclerosis, improving immunity, improving bone density, preventing and treating diabetes, promoting growth and the like. Conjugated linoleic acid can increase physical consumption after entering into the body, so that the body fat deposition can be effectively reduced in the aspect of weight control.

Omega-3 fatty acids: the omega-3 fatty acid is eicosapentaenoic acid from fish and animals, contains five unsaturated bonds, docosahexaenoic acid and six unsaturated bonds, and has stronger function of regulating blood fat because the two unsaturated bonds contain more unsaturated bonds. Omega-3 fatty acids derived from fish oil can lower blood pressure, regulate blood lipid, and especially reduce triglycerides. Omega-3 fatty acids can reduce the risk of death from cardiovascular disease and reduce joint stiffness and pain, can increase bone density after supplementation, and also have vasodilating and antithrombotic effects.

Agaricus blazei murill: the name learning: agaric blazei Murill is a fungus of the family Agaricaceae, genus Agaricus. Agaricus blazei is a precious fungus used both as food and medicine. The mushroom meat crisp has delicious taste and almond flavor, and is rich in nutrition. Has high anticancer activity, and has effects in enhancing cellular immunity, lowering blood sugar, regulating blood pressure, resisting arteriosclerosis, improving hyperosteogeny, and tranquilizing mind.

Dihydromyricetin: has effects in scavenging free radicals, resisting oxidation, resisting thrombosis, resisting tumor, and relieving inflammation; also has effects in relieving alcoholism, preventing alcoholic liver and fatty liver, inhibiting liver cell deterioration, and reducing incidence of hepatocarcinoma.

The invention has the beneficial effects that:

compared with the prior art, the fermented yoghourt has rich nutrition, high content of conjugated linoleic acid and omega-3 fatty acid, and enhanced health care effect, is constantly drunk every day, and can obviously improve three highs after 3-6 months, improve sleep quality, relieve constipation, enhance immunity and improve the health condition of drinkers.

Compared with the prior art, the agaricus blazei murill chitosan is prepared by extracting the agaricus blazei murill as a raw material; and then reacting D-leucine tert-butyl ester hydrochloride with dihydromyricetin, connecting the two through carbonyl provided by bis (p-nitrophenyl) carbonate, and performing substitution reaction on the product with amino in the agaricus blazei murrill chitosan to finally prepare the agaricus blazei murrill chitosan type stabilizer.

Compared with the prior art, the invention uses the agaricus blazei murill chitosan type stabilizer to improve the stability of the yoghourt and solves the technical problem that the existing stabilizer is easy to oxidize. The agaricus blazei murill chitosan type stabilizer increases the water holding capacity of the yoghourt through the stretching conformation of the cavity and the gel net, reduces water loss and protein separation in the production, storage and transportation processes of the yoghourt, reserves the nutrient substances of the yoghourt, and improves the sensory experience of drinkers.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Some raw material parameters in the comparative examples and examples of the invention are as follows:

active yeast, ATCC9080, cat no: XY-WSW-1545, available from Shanghai Xuan Biotechnology Ltd;

minerals and vitamins, premix, cat # s: n855, brand: beijing Longyue pasture, available from Beijing Longyue pasture Biotechnology Ltd;

conjugated linoleic acid, cold-pressed extract of safflower, supplied by wakawa walce biotechnology limited;

omega-3 fatty acids, food grade, cold-pressed extract of linseed oil, provided by Shandong Teng Wang chemical Co Ltd;

mogroside, cat number: 20201213, shenzhen Lefu Biotech, inc.;

lactobacillus bulgaricus, ATCC11842, cat No.: XK-SH-4829, available from Shanghai 261810, biotech, inc.;

streptococcus thermophilus, ATCC19258, cat no: ZIKER0211, available from Shenzhen Zinzaceae Biotech Limited;

dried Agaricus blazei Murill, supplied by Yunnan Yunrei agricultural science and technology Co., ltd;

dihydromyricetin, food grade, CAS No.: 27200-12-0;

d-leucine tert-butyl ester hydrochloride, CAS No.: 13081-32-8.

Example 1

A fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid is prepared by the following method:

s1, preparing raw materials in proportion, grinding 15kg of full-fat soybeans, 40kg of corns and 5kg of flaxseeds into powder, adding 10kg of wheat bran and 1kg of active yeast, and fermenting for 12 hours in a fermentation tank at a constant temperature of 43 ℃; after the fermentation is finished, continuously adding 1kg of baking soda, 1kg of calcium bicarbonate, 2kg of mineral matter, vitamin and 0.5kg of salt, and uniformly mixing to obtain concentrated feed;

s2, mixing the concentrated feed and the coarse feed according to a mass ratio of 35:65, proportioning, adding compound vegetable oil in the feed, stirring by a stirrer, feeding by cows in lactation period freely, and collecting to obtain raw milk;

s3, sampling raw milk to extract fat, and obtaining the contents of conjugated linoleic acid and omega-3 fatty acid respectively by referring to a method in GB 5009.168-2016 (determination of fatty acid in food safety national standard food);

s4, standardizing the raw milk according to the result in the step S3; calculated by milk fat, the conjugated linoleic acid content of the raw milk is more than or equal to 40mg/g; the content of omega-3 fatty acid is more than or equal to 5 percent according to the milk content; for the part which does not meet the standard, the part reaches the product standard by adding the cold pressed safflower extract and the cold pressed linseed oil extract;

s5, sterilizing the raw milk subjected to the standardized treatment, wherein the sterilization temperature is 95 ℃, and the duration time is 30 minutes; cooling the sterilized raw milk to 43 ℃, inoculating a fermentation strain, and fermenting at the temperature for 6 hours to obtain primary fermented yoghourt;

s6, adding conjugated linoleic acid and omega-3 fatty acid into the primary fermented yoghourt, uniformly mixing, and then performing secondary fermentation at the temperature of 39 ℃ for 4 hours; and immediately cooling the fermented yoghourt to 4 ℃, adding mogroside, uniformly mixing, and canning to obtain the fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid.

The formula of the coarse feed in the step S2 is as follows: 65kg of green pasture and 35kg of hay; the green pasture is sweet grassiness and cornstalks, and the mass ratio of the sweet grassiness to the cornstalks is 1:1; the hay is alfalfa.

The components of the composite vegetable oil in the step S2 and the addition amount of each lactating cow per day are as follows: 150g of soybean oil, 225g of sunflower seed oil and 150g of linseed oil.

In the step S5, the fermentation strains are Lactobacillus bulgaricus and Streptococcus thermophilus according to the mass ratio of 1:1.5 obtaining mixed strains; the mass ratio of the fermentation strain to the raw milk is 1:25000.

in the step S6, the addition amount of the conjugated linoleic acid is 2.4g/kg, and the addition amount of the omega-3 fatty acid is 1.5wt%.

The addition amount of the mogroside in the step S6 is 0.02wt%.

Example 2

s1, preparing raw materials in proportion, grinding 15kg of full-fat soybeans, 40kg of corns and 5kg of flaxseeds into powder, adding 10kg of wheat bran and 1kg of active yeast, and fermenting for 12 hours in a fermentation tank at a constant temperature of 43 ℃; after the fermentation is finished, continuously adding 1kg of baking soda, 1kg of calcium bicarbonate, 2kg of mineral substances, vitamins and 0.5kg of salt, and uniformly mixing to obtain concentrated feed;

s2, mixing the concentrated feed and the coarse feed according to a mass ratio of 35:65, proportioning, adding compound vegetable oil into the feed, stirring by a stirrer, feeding by cows in lactation period freely, and collecting to obtain raw milk;

s4, standardizing the raw milk according to the result in the step S3; the conjugated linoleic acid content of the raw milk is more than or equal to 40mg/g calculated by milk fat; the content of omega-3 fatty acid is more than or equal to 5 percent according to the milk content; for the product which does not meet the standard, the product standard is met by adding the safflower cold-pressed extract and the linseed oil cold-pressed extract;

s5, sterilizing the raw milk subjected to standardized treatment, wherein the sterilization temperature is 95 ℃, and the sterilization duration is 30 minutes; cooling the sterilized raw milk to 43 ℃, inoculating a fermentation strain, and fermenting at the temperature for 6 hours to obtain primary fermented yogurt;

s6, adding conjugated linoleic acid and omega-3 fatty acid into the primary fermented yoghourt, uniformly mixing, and then performing secondary fermentation at the temperature of 39 ℃ for 4 hours; and immediately cooling the fermented yoghourt to 4 ℃, adding mogroside and agaricus blazei murill chitosan type stabilizer, uniformly mixing, and canning to obtain the fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid.

In the step S5, the fermentation strains are Lactobacillus bulgaricus and Streptococcus thermophilus according to the mass ratio of 1:1.5 obtaining mixed strains; the mass ratio of the fermentation strain to the raw milk is 1:25000。

In the step S6, the addition amount of the mogrosides is 0.02wt%, and the addition amount of the agaricus blazei murill chitosan type stabilizer is 0.8wt%.

The preparation method of the agaricus blazei murill chitosan type stabilizer comprises the following steps:

pulverizing dried Agaricus blazei Murill into powder, and dispersing in water by ultrasonic treatment to obtain 4.5 wt% Agaricus blazei Murill powder suspension; heating the agaricus blazei murill powder suspension at 100 ℃ for 4 hours, centrifuging, collecting supernatant, adding a 0.1mol/L sodium hydroxide aqueous solution into the supernatant, and adjusting the pH of the aqueous solution to be neutral to obtain neutral supernatant; adding 5 times of absolute ethyl alcohol into the neutral supernatant for precipitation, and filtering to obtain a filter cake; mixing a 32wt% aqueous hydrogen peroxide solution and absolute ethyl alcohol in a volume ratio of 1:2 to obtain a treatment fluid, and treating the filter cake at normal temperature by using the treatment fluid, wherein the mass ratio of the treatment fluid to the filter cake is 25:1, treating for 8 hours to obtain filter cake mixed liquor; after the step is finished, adding 10 times of absolute ethyl alcohol into the filter cake mixed liquor for precipitation again, filtering and collecting precipitates, washing the precipitates by the absolute ethyl alcohol, and drying to obtain the agaricus blazei murill chitosan type stabilizer.

Example 3

The procedure for preparing fermented yogurt rich in conjugated linoleic acid and omega-3 fatty acids in this example was substantially the same as in example 2, except that the preparation method of the agaricus blazei murill chitosan-type stabilizer in this example was different.

m1, crushing dried agaricus blazei into powder, and dispersing the powder in water through ultrasonic treatment to obtain an agaricus blazei powder suspension liquid with the mass fraction of 4.5% of agaricus blazei powder; heating the agaricus blazei murill powder suspension at 100 ℃ for 4 hours, centrifuging, collecting supernatant, adding a 0.1mol/L sodium hydroxide aqueous solution into the supernatant, and adjusting the pH of the aqueous solution to be neutral to obtain neutral supernatant; adding 5 times of anhydrous ethanol into the neutral supernatant for precipitation, and filtering to obtain a filter cake; mixing a 32wt% aqueous hydrogen peroxide solution and absolute ethyl alcohol in a volume ratio of 1:2 to obtain a treatment fluid, and treating the filter cake at normal temperature by using the treatment fluid, wherein the mass ratio of the treatment fluid to the filter cake is 25:1, treating for 8 hours to obtain filter cake mixed liquor; adding 10 times of anhydrous ethanol into the filter cake mixed liquor after the step is finished, precipitating again, filtering and collecting precipitate, washing the precipitate with the anhydrous ethanol, and drying to obtain agaricus blazei murrill chitosan for later use;

under the protection of M2 and nitrogen, 3.4kg of D-leucine tert-butyl ester hydrochloride, 3.9kg of N, N-diisopropylethylamine and 90kg of tetrahydrofuran are uniformly mixed to obtain a first reaction solution for later use; uniformly mixing 4.7kg of bis (p-nitrophenyl) carbonate and 35kg of tetrahydrofuran to obtain a second reaction solution for later use; uniformly mixing 2.3kg of dihydromyricetin and 15kg of tetrahydrofuran to obtain a third reaction solution for later use;

m3, under the protection of nitrogen, uniformly mixing the first reaction solution and the second reaction solution to obtain a reaction mixture, adding the third reaction solution into the reaction mixture, and reacting at 30 ℃ for 24 hours; after the reaction is finished, adding absolute ethyl alcohol with the same volume into the reaction product, filtering to obtain a filtrate, and freeze-drying the filtrate to obtain the myricetin derivative for later use;

m4, dissolving 1.4kg of agaricus blazei murill chitosan in 75kg of acetic acid aqueous solution with the concentration of 1wt% to obtain an agaricus blazei murill chitosan solution; dissolving 2.1kg of myricetin derivative in 10kg of acetone to obtain myricetin derivative solution; under the protection of nitrogen, uniformly mixing an agaricus blazei murill chitosan solution and a myricetin derivative solution, and carrying out substitution reaction under the catalysis of 0.8kg of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, wherein the reaction temperature is 80 ℃, and the reaction time is 24 hours; cooling to normal temperature after the substitution reaction is finished, adding 300kg of acetone with the temperature of 4 ℃ into the reaction product for precipitation, then filtering and collecting a filter cake, washing the filter cake with acetone, washing with water and drying to obtain the agaricus blazei murrill chitosan type stabilizer.

Comparative example 1

s1, preparing raw materials according to a proportion, grinding 15kg of full-fat soybeans, 40kg of corns and 5kg of flaxseeds into powder, adding 10kg of wheat bran and 1kg of active yeast, and fermenting for 12 hours in a fermentation tank at a constant temperature of 43 ℃; after the fermentation is finished, continuously adding 1kg of baking soda, 1kg of calcium bicarbonate, 2kg of mineral substances, vitamins and 0.5kg of salt, and uniformly mixing to obtain concentrated feed;

s2, mixing the concentrated feed and the coarse feed according to a mass ratio of 35:65, proportioning, stirring by a stirrer, feeding by dairy cows in lactation period freely, and collecting to obtain raw milk;

s6, adding conjugated linoleic acid and omega-3 fatty acid into the primary fermented yoghourt, uniformly mixing, and then performing secondary fermentation at the temperature of 39 ℃ for 4 hours; and immediately cooling the fermented yoghourt to 4 ℃, adding mogroside, uniformly mixing, and canning to obtain the fermented yoghourt.

the addition amount of the conjugated linoleic acid in the step S6 is 2.4g/kg,addition amount of omega-3 fatty acidIs 1.5wt%。

The addition amount of the mogroside in the step S6 is 0.02wt%.

Test example 1

In the preparation process of the fermented yoghourt rich in the conjugated linoleic acid and the omega-3 fatty acid, the dairy cattle collects raw milk after ingesting different feeds; the content test of conjugated linoleic acid and omega-3 fatty acid in the raw milk is carried out by referring to an internal standard method and steps thereof in GB 5009.168-2016 (determination of fatty acid in national standard food for food safety), and test results keep three significant digits. The results of the tests for the content of conjugated linoleic acid and omega-3 fatty acids in the raw milk are shown in table 1.

Table 1:

as can be seen from the test results in table 1, the addition of the complex vegetable oil to the cow feed can increase the content of conjugated linoleic acid and omega-3 fatty acid in raw milk. The conjugated linoleic acid and the omega-3 fatty acid respectively have the effects of reducing cholesterol of a human body, resisting atherosclerosis, reducing blood pressure and regulating blood fat; the content of conjugated linoleic acid and omega-3 fatty acid in the raw milk is higher, and the content of the nutrient substances in the prepared yoghourt is correspondingly higher. The fermented yoghourt is rich in nutrition, and the health-care effect of the yoghourt is enhanced.

Test example 2

The stability of the fermented yogurt is characterized by water retention and taste evaluation; the method and the steps for measuring the water binding capacity in a water binding capacity test reference document (Li Hui, wu Weidu, zhu Hui, etc.. Research on measurement of viscosity and water binding capacity of stirred yoghurt [ J ]. Grain and food industry, 2018, 25 (2): 56-59.DOI; mouthfeel evaluation test references (li hua, wang hua, chen cai, etc.. Stability study of unsaturated fatty acid DHA in yogurt [ J ] china dairy industry, 2017, 45 (6): 52-54.) were performed.

In the water retention test, the yogurt is packaged and stored at normal temperature, and the water retention test is performed after packaging and on day 21. Weigh 15g of sample (weight m, g) into a 50mL centrifuge tube (weight m) ₁ And g) setting the centrifugal force to be 300g and the centrifugal time to be 10min, pouring out precipitated water after centrifugation, and weighing the weight (m) of the sample and the centrifugal tube again ₂ G), calculating the water analysis rate according to the following formula:

in the taste evaluation test, the yogurt is packaged separately and stored at normal temperature, and the taste evaluation is carried out on the 21 st day. The taste evaluation is performed according to a literature method by using a 10-point strength scale method, the larger the fishy smell is, the higher the score is, and the result is averaged.

The results of the stability testing of the fermented yoghurts are shown in table 2.

Table 2:

in Table 2, "/" indicates that there is no bleeding according to the evaluation criteria in the above-mentioned documents.

As can be seen from the test results of Table 2, example 3 achieved good stability with the agaricus blazei chitosan type stabilizer, did not show water separation and delamination after 21 days of storage, and had the best taste. This result may be caused by the fact that the chitosan-type stabilizer of Agaricus blazei Murill has an irregular sheet shape, a smooth and porous surface, and filamentous branches, and exhibits good water-holding properties. Due to the pores and the branched structure, the stabilizer can be combined with water in the yoghourt and is favorable for cross-linking. The stabilizer wraps the spherical milk protein in the filamentous branches to form a denser protein gel net, and the water holding capacity of the yoghourt is increased through the stretched conformation of the cavity and the gel net, so that the water loss and protein separation in the processes of yoghourt production, storage and transportation are reduced.

Test example 3

Sensory evaluation of fermented yogurts the specific methods and procedures in the references (snow, liufei, liujing, etc.. Agaricus blazei murill yogurt development and storage stability research [ J ]. Food industry science and technology, 2014, 35 (11): 206-209. Doi. When the fermented yoghourt is evaluated, 12 professional technicians with sensory evaluation experience form an evaluation group, and the evaluation standard of the evaluation group is shown in table 3. The sensory evaluation composite score results of the fermented yoghurts are shown in table 4.

Table 3:

table 4:

as can be seen from the sensory evaluation test results of the fermented yogurt, the product has better comprehensive score after the agaricus blazei murill chitosan type stabilizer is used. The agaricus blazei murill chitosan type stabilizer can improve the water holding capacity of the yoghourt, prevent water loss and protein separation, reduce the whey separation of products and improve the tissue state.

Claims

1. A preparation method of fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid is characterized by comprising the following steps:

s1, preparing raw materials in proportion, grinding 15-30 parts of full-fat soybean, 40-60 parts of corn and 5-15 parts of linseed by weight, adding 10-15 parts of wheat bran and 1-2 parts of active yeast, and fermenting in a fermentation tank at constant temperature of 35-43 ℃ for 10-15 hours; after fermentation, continuously adding 1-3 parts of baking soda, 1-2 parts of calcium bicarbonate, 2-5 parts of mineral substances and vitamins and 0.5-2.5 parts of salt, and uniformly mixing to obtain concentrated feed;

s3, sampling and extracting fat from raw milk, and determining the content of conjugated linoleic acid and omega-3 fatty acid;

s5, sterilizing the raw milk after standardized treatment, wherein the sterilization temperature is 75-95 ℃, and the duration is 5-30 minutes; cooling the sterilized raw milk to 40-43 ℃, inoculating a fermentation strain, and fermenting at the temperature for 5-6 hours to obtain primary fermented yoghourt;

s6, adding conjugated linoleic acid and omega-3 fatty acid into the primarily fermented yoghourt, uniformly mixing, and then performing secondary fermentation at the temperature of 35-39 ℃ for 3-4 hours; and immediately cooling the fermented yoghourt to 2-6 ℃, adding mogroside and agaricus blazei murill chitosan type stabilizer, uniformly mixing, and canning to obtain the fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid.

2. The method for preparing fermented yoghurt rich in conjugated linoleic acid and omega-3 fatty acid according to claim 1, which is characterized in that: in the step S2, the mass ratio of the concentrated feed to the coarse feed is 35:65; the formula of the coarse feed comprises the following components in parts by weight: 65-85 parts of green pasture and 35-25 parts of hay; the green pasture comprises at least two of broussonetia papyrifera leaves, mulberry leaves, sweet grassiness, corn stalks and ryegrass; the hay comprises at least one of alfalfa and oats; the components of the composite vegetable oil and the addition amount of each lactating cow per day are as follows: 100-200 g of soybean oil, 150-300 g of sunflower seed oil and 50-250 g of linseed oil or safflower oil.

3. The method for preparing fermented yogurt with enriched conjugated linoleic acid and omega-3 fatty acids as claimed in claim 1, wherein the method comprises the following steps: the natural extract in the step S4 is conjugated linoleic acid and omega-3 fatty acid; the conjugated linoleic acid is a safflower cold-pressed extract; the omega-3 fatty acid is a cold pressed extract of linseed oil.

4. The method for preparing fermented yoghurt rich in conjugated linoleic acid and omega-3 fatty acid according to claim 1, which is characterized in that: in the step S5, the fermentation strains are Lactobacillus bulgaricus and Streptococcus thermophilus according to the mass ratio of 1: 1-2 to obtain mixed strains; the mass ratio of the fermentation strain to the raw milk is 1:20000 to 30000.

5. The method for preparing fermented yoghurt rich in conjugated linoleic acid and omega-3 fatty acid according to claim 1, which is characterized in that: in the step S6, the addition amount of the conjugated linoleic acid is 0.8-3.6 g/kg, and the addition amount of the omega-3 fatty acid is 1-2 wt%; the addition amount of the mogroside is 0.02-0.08 wt%, and the addition amount of the agaricus blazei murill chitosan type stabilizer is 0.6-1.5 wt%.

6. The method for preparing fermented yogurt rich in conjugated linoleic acid and omega-3 fatty acids as claimed in claim 1 or 5, wherein the agaricus blazei murill chitosan type stabilizer is prepared by the following steps in parts by weight:

m1, crushing the dried agaricus blazei murill into powder, and dispersing the powder in water through ultrasonic treatment to obtain an agaricus blazei murill powder suspension; heating the agaricus blazei murill powder suspension, centrifugally separating to collect supernatant, adding a sodium hydroxide aqueous solution into the supernatant, and adjusting the pH value of the supernatant to be neutral to obtain neutral supernatant; adding 4-6 times of absolute ethyl alcohol into the neutral supernatant for precipitation, and filtering to obtain a filter cake; mixing aqueous hydrogen peroxide with absolute ethyl alcohol to obtain a treatment solution, and treating the filter cake at normal temperature by using the treatment solution to obtain a filter cake mixed solution; adding 5-10 times of absolute ethyl alcohol into the filter cake mixed liquor after the step is finished, precipitating again, filtering and collecting precipitates, washing the precipitates by the absolute ethyl alcohol, and drying to obtain agaricus blazei murrill chitosan for later use;

m3, uniformly mixing the first reaction liquid and the second reaction liquid under an anaerobic condition to obtain a reaction mixture, and adding the third reaction liquid into the reaction mixture for reaction; after the reaction is finished, adding absolute ethyl alcohol with the same volume into the reaction product, filtering to obtain a filtrate, and freeze-drying the filtrate to obtain the myricetin derivative for later use;

m4, dissolving 1.4-1.9 parts of agaricus blazei murill chitosan into 75-100 parts of acetic acid aqueous solution with the concentration of 1wt% to obtain agaricus blazei murill chitosan solution; dissolving 2.1-2.9 parts of myricetin derivative in 10-20 parts of acetone to obtain myricetin derivative solution; under the anaerobic condition, uniformly mixing the agaricus blazei murill chitosan solution and the myricetin derivative solution, and carrying out substitution reaction under the catalysis of 0.8-1.0 part of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride; cooling to normal temperature after the substitution reaction is finished, adding 300-450 parts of acetone with the temperature of 0-4 ℃ into the reaction product for precipitation, then filtering and collecting a filter cake, washing the filter cake with acetone, washing with water, and drying to obtain the agaricus blazei murrill chitosan type stabilizer.

7. The method for preparing fermented yoghurt rich in conjugated linoleic acid and omega-3 fatty acid according to claim 6, wherein the method comprises the following steps: in the step M1, the mass fraction of the agaricus blazei murill powder in the agaricus blazei murill powder suspension is 3-6.5%; the temperature of the heating treatment is 85-100 ℃, and the treatment time is 2-6 h; the concentration of the sodium hydroxide aqueous solution is 0.1-0.2 mol/L; the volume ratio of the mixed hydrogen peroxide aqueous solution to the absolute ethyl alcohol is 1:1 to 3, the concentration of the aqueous hydrogen peroxide solution is 28 to 36 weight percent; in the process of treating the filter cake by the treating fluid, the mass ratio of the treating fluid to the filter cake is 15-40: 1, the treatment time is 6 to 12 hours.

8. The method for preparing fermented yoghurt rich in conjugated linoleic acid and omega-3 fatty acid according to claim 6, wherein the method comprises the following steps: the reaction temperature in the step M3 is 25-35 ℃, and the reaction time is 12-36 h.

9. The method for preparing fermented yogurt with enriched conjugated linoleic acid and omega-3 fatty acids as claimed in claim 6, wherein: the reaction temperature of the substitution reaction in the step M4 is 65-85 ℃, and the reaction time is 18-48 h.

10. A fermented yoghourt rich in conjugated linoleic acid and omega-3 fatty acid is characterized in that: prepared by the method of any one of claims 1 to 9.