CN117179054A - Selenium-enriched lactobacillus beverage and preparation method thereof - Google Patents
Selenium-enriched lactobacillus beverage and preparation method thereof Download PDFInfo
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- CN117179054A CN117179054A CN202311119836.9A CN202311119836A CN117179054A CN 117179054 A CN117179054 A CN 117179054A CN 202311119836 A CN202311119836 A CN 202311119836A CN 117179054 A CN117179054 A CN 117179054A
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- 239000011669 selenium Substances 0.000 title claims abstract description 181
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- Dairy Products (AREA)
- Non-Alcoholic Beverages (AREA)
Abstract
The invention relates to the technical field of fermented dairy products, in particular to a selenium-enriched lactobacillus beverage and a preparation method thereof. The invention provides a selenium-enriched lactobacillus beverage which comprises the following raw materials in parts by weight: 25-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is selenium-enriched lactobacillus helveticus. The invention uses the lactobacillus helveticus as the carrier to bring the selenium into the selenium-rich lactic acid beverage, improves the selenium content in the selenium-rich lactic acid beverage, and has the selenium content more than or equal to 8.0 mug/100 g, which is far higher than the selenium content of the traditional dairy products, and simultaneously, the mouthfeel, the flavor, the texture stability and the living bacteria content of the beverage are obviously improved, and the influence of the selenium addition in the traditional lactic acid beverage on the fermentation performance is overcome.
Description
Technical Field
The invention relates to the technical field of fermented dairy products, in particular to a selenium-enriched lactobacillus beverage and a preparation method thereof.
Background
Lactic acid bacteria beverage (lactobacilli) is a fermented acidic milk-containing beverage, usually taking milk or dairy products as raw materials, sterilizing, cooling, inoculating emulsion prepared by lactic acid bacteria fermentation, and then adding ingredients to prepare the beverage, wherein the protein content of the product is not less than 0.7g/100g or 100ml.
Selenium is one of trace elements necessary for human body, and has various functions of resisting oxidation, resisting bacteria, enhancing immunity, etc. But the human body cannot synthesize by itself and must acquire the substance through an exogenous substance. The selenium content of the existing dairy products in the market is low and is far lower than the reference intake of dietary nutrients of Chinese residents (60 mug/day). However, the selenium content in natural foods is relatively low, and most of the selenium-enriched foods on the market reach a certain selenium level by additional addition or biochemical transformation. There is a certain risk of bioconversion, for example, incomplete conversion of inorganic selenium to organic selenium, resulting in residual inorganic selenium and higher toxicity.
Chinese patent CN 104255916A discloses a method for producing selenium-enriched yoghurt in family, which comprises the following steps: (1) preparing sodium selenite aqueous solution; (2) Sterilizing fresh milk, and adding sodium selenite water solution into the sterilized fresh milk to obtain mixed raw milk, wherein the adding amount of the sodium selenite water solution is 10-2500 mug of selenium in 1L of fresh milk, and the fresh milk is fresh milk or fresh goat milk; (3) Adding 5-15g of probiotic powder into the mixed raw milk to obtain a raw milk fermentation material; (4) Fermenting the fermented milk material at 38-43deg.C for 4-10 hr to obtain the final product. The method has the advantages of simple process, low cost and short time consumption in the preparation process, but the inorganic sodium selenite is added, so that the method has low bioavailability, high toxicity and food safety risk.
Chinese patent CN 107873839A discloses a method for preparing selenium-enriched tea yogurt, which comprises the steps of extracting selenium element from tea rich in organic selenium element to obtain selenium-enriched tea refined extract component, mixing the selenium-enriched tea refined extract component with pretreated fresh milk, and fermenting to obtain selenium-enriched tea yogurt. The addition amount of the selenium-enriched tea refined extract component in the invention is 0.3 percent of the weight of fresh milk. According to the invention, the tea leaves are soaked in hot water and then freeze-dried to obtain a refined extract, and milk is added to prepare the selenium-enriched yoghurt, but the content of selenium element which can be dissolved from the tea leaves in a hot water leaching mode is low.
In addition, according to the health and/or taste requirements of people, new lactobacillus beverages prepared by adding different flavor nutrients into lactobacillus beverages are becoming a development trend, and some lactobacillus beverages are added with vitamins, minerals, fruit juice or substances such as amino acids, peptides or functional factors with health care effect.
The walnut protein is prepared into peptide products with different fragment sizes and biological activities through biological enzymolysis technology, and the peptide products have a plurality of biological activities and are attracting great attention. Researches show that the walnut peptide has the effects of scavenging free radicals, relieving fatigue, improving memory and the like. The typical representative of the dairy products related to the walnut in the market is that walnut kernel is ground into milk, lactic acid bacteria are inoculated and fermented, and the direct ground walnut kernel is obtained by adding high grease to the milk, so that the stability of a final dairy system is affected, meanwhile, the walnut protein in the walnut kernel is not hydrolyzed, the solubility is poor, the biological activity does not have a certain influence on the uniformity of the product, the molecular weight of the walnut protein is high, and the walnut protein is not easy to digest and absorb by a human body.
Disclosure of Invention
The invention aims to solve the technical problem of providing a selenium-enriched lactobacillus beverage and a preparation method thereof, and in order to achieve the purposes, the invention provides the following technical scheme:
in a first aspect, the invention provides a selenium-enriched lactobacillus beverage, which comprises the following raw materials in parts by weight: 25-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
In one embodiment of the invention, the selenium-enriched lactobacillus beverage comprises the following raw materials in parts by weight: 35-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
In one embodiment of the invention, the selenium-enriched lactobacillus beverage comprises the following raw materials in parts by weight: 35-40 parts of cow milk, 0.1-1 part of walnut peptide, 0.08-0.1 part of first fermentation inoculant and 0.08-0.15 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
In one embodiment of the invention, the selenium-enriched lactobacillus helveticus is prepared by inoculating lactobacillus helveticus into a selenium-containing culture medium for fermentation culture; preferably, the lactobacillus helveticus is lactobacillus helveticus Zhegu LBH-VI (Lactobacillus helveticus Zhegu LBH-VI), wherein the lactobacillus helveticus Zhegu LBH-VI is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 7 days in 2023, and the preservation number is CCTCC NO: m2023095.
In one embodiment of the invention, the content of organic selenium in the lactobacillus helveticus is 100-125 mug/g.
In one embodiment of the invention, the selenium-enriched lactic acid bacteria beverage comprises, based on the weight of the selenium-enriched lactic acid bacteria beverageThe selenium content in the selenium-enriched lactobacillus beverage is not less than 8.0 mug/100 g; preferably, the viable count of the selenium-enriched lactobacillus beverage in the shelf life is more than or equal to 10 9 CFU/ml。
In one embodiment of the present invention, the cow milk is one or more selected from the group consisting of whole cow milk, low fat cow milk and defatted cow milk.
In one embodiment of the invention, the molecular weight of the walnut peptide is 800-1000Da; preferably, the walnut peptide is prepared by enzymolysis of walnut through protease.
In one embodiment of the present invention, the first fermentation starter is selected from one or more of streptococcus salivarius thermophilus subspecies, lactobacillus delbrueckii subspecies, bifidobacterium lactis, lactococcus lactis subspecies and lactobacillus paracasei, preferably streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies, more preferably the mass ratio of streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies is 2-3:1-1.5 on a dry matter basis.
In one embodiment of the present invention, the streptococcus salivarius thermophilus subspecies are streptococcus salivarius subsp thermophilus 932 (Straptococcus salivarias subsp. Thermophilus 932), which was deposited with the China Center for Type Culture Collection (CCTCC) at 2023, 6 and 5 days, with a deposit number of CCTCC NO: m2023902;
And/or, the Lactobacillus delbrueckii subspecies are Lactobacillus delbrueckii subspecies Dangxiong LB V III (Lactobacillus delbrueckii subsp. Lactis Dangxiong LB V III), and are preserved in China Center for Type Culture Collection (CCTCC) for 3 months and 23 days in 2023, wherein the preservation number is CCTCC NO: M2023396;
and/or, the lactococcus lactis subspecies are lactococcus lactis subspecies 954 (Lactococcus lactis subsp. Lactis 954) which are preserved in China Center for Type Culture Collection (CCTCC) in 6 and 5 days of 2023, and the preservation number is CCTCC NO: m2023904;
and/or the lactobacillus paracasei is lactobacillus paracasei ALI Plateau LPA-i (lactobacillus paracasei ALI Plateau LPA-i).
In one embodiment of the invention, the feedstock further comprises 5.0-8.5 parts of sweetener;
preferably, the sweetener is selected from one or more than two of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycoside, preferably sucrose.
In one embodiment of the invention, the raw materials further comprise 0.3-0.7 parts of stabilizer;
preferably, the stabilizer is selected from one or more of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate and sodium citrate;
More preferably, the mass ratio of the sodium carboxymethyl cellulose, the pectin, the sodium tripolyphosphate and the sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.7.
In one embodiment of the invention, the raw materials further comprise 0.11-0.6 parts of an acidulant;
preferably, the acidulant is selected from citric acid, lactic acid and sodium citrate, preferably the mass ratio of citric acid, lactic acid and sodium citrate is 5-25:5-15:1-20.
In one embodiment of the invention, the selenium-enriched lactobacillus beverage is prepared by fermenting a fermentation base material comprising cow milk and walnut peptide at a variable temperature.
In a second aspect, the invention also provides a preparation method of the selenium-enriched lactobacillus beverage, which comprises the following steps:
(1) Mixing raw materials comprising cow milk and walnut peptide to prepare a fermentation base material;
(2) Sequentially inoculating a first fermentation microbial inoculum and a second fermentation microbial inoculum into the fermentation base material prepared in the step (1) to perform variable-temperature fermentation to prepare fermented milk;
(3) And (3) blending the fermented milk prepared in the step (2) to obtain the selenium-enriched lactobacillus beverage.
In one embodiment of the present invention, in the preparation method, the blending in the step (3) includes adding a stabilizer and/or an acidulant into the selenium-enriched lactobacillus beverage prepared in the step (2) to perform mixing and homogenization treatment.
In one embodiment of the present invention, in the above preparation method, the fermentation base further comprises a sweetener, preferably, the sweetener is one or more selected from sucrose, erythritol, xylitol, maltitol, sucralose, or steviol glycoside, preferably sucrose.
In one embodiment of the present invention, in the above preparation method, the step (2) of sequentially inoculating the first fermentation starter and the second fermentation starter to perform the temperature swing fermentation includes inoculating the first fermentation starter to ferment at 40-45 ℃, and then inoculating the second fermentation starter to ferment at 35-39 ℃;
preferably, the first fermentation inoculant is inoculated for fermentation at 40-45 ℃ for 10-12 hours until the final acidity is 90-100 DEG T, and then the second fermentation inoculant is inoculated for fermentation at 35-39 ℃ for 8-10 hours until the final acidity is 160-180 DEG T.
In one embodiment of the present invention, in the above preparation method, the preparation method further comprises a step of homogenizing the fermentation base material before inoculation, preferably at a homogenizing temperature of 50-65 ℃, more preferably at a homogenizing pressure of 10-50MPa;
preferably, the homogenizing treatment further comprises a step of sterilizing the fermentation base material, preferably at a sterilization temperature of 90-100 ℃, more preferably for a sterilization time of 200-400s.
In one embodiment of the present invention, the above preparation method further comprises a step of stirring and demulsification to obtain fermented milk after the first fermentation and after the second fermentation, preferably at a stirring speed of 20-40r/min, and more preferably for a stirring time of 10-20min.
The invention has the beneficial effects that:
1. according to the invention, the lactobacillus helveticus is used as a carrier to bring selenium into the fermentation process of the selenium-enriched lactic acid beverage, so that the selenium content in the selenium-enriched lactic acid beverage is increased, the selenium content in the prepared lactic acid beverage is more than or equal to 8.0 mug/100 g and is far higher than the selenium content (less than or equal to 0.6 mug/100 g) of the traditional dairy product, and meanwhile, the taste, the flavor, the texture stability and the living bacteria number content of the selenium-enriched lactic acid beverage are all obviously improved, and the influence of the selenium addition in the lactic acid beverage in the prior art on the fermentation performance is overcome;
2. the invention uses cow milk and walnut peptide with fatigue relieving effect as fermentation base material to prepare lactobacillus beverage, and animal experiments prove that the lactobacillus beverage prepared by the invention has the effect of obviously relieving fatigue.
Description of strains
Lactobacillus helveticus Zhegu LBH-VI (Lactobacillus helveticus Zhegu LBH-VI) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 7 days in 2023, and the preservation number is CCTCC NO: m2023095, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
The streptococcus salivarius thermophilus subspecies 932 (Straptococcus salivarias subsp. Thermophilus 932) used in the invention is preserved in China center for type culture collection (CCT CC) in 2023, 6 and 5 days, and the preservation number is CCTCC NO: m2023902, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
The Lactobacillus delbrueckii subspecies Dangxiong LB V III (Lactobacillus delb rueckii subsp. Lactis Dangxiong LB V III) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) in 3.month 23 of 2023, the preservation number is CCTCC NO: M2023396, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052 Lactobacillus delbrueckii subspecies lactis.
Lactococcus lactis subspecies 954 (Lactococcus lactis subsp. Lactis 954) used in the invention are preserved in China Center for Type Culture Collection (CCTCC) in 6 and 5 days of 2023, and the preservation number is CCTCC NO: m2023904, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
Lactobacillus paracasei AL1 Plateau LPA-1 (lactobacillus paracasei AL1 Plateau LPA-1) used in the present invention was preserved in the China Center for Type Culture Collection (CCTCC) for 10 months and 25 days in 2021, and the preservation number is CCTCC NO: m20211312, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052. This strain is described in chinese application CN202210217216.8 (publication No. CN114480214 a).
Detailed Description
In order to make the purposes, technical schemes and technical effects of the embodiments of the present invention more clear, the technical schemes in the embodiments of the present invention are clearly and completely described. The embodiments described below are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art without the benefit of the teachings of this invention, are intended to be within the scope of the invention.
At present, the existing technical route of the selenium-enriched lactic acid beverage is to directly add inorganic selenium or selenium-enriched raw materials into cow milk for fermentation, however, the inorganic selenium has lower bioavailability and higher toxicity, the added selenium-enriched raw materials are added additionally, the addition amount is less, and the selenium content of the final product is lower. In addition, the existing walnut dairy products are prepared by directly squeezing walnut or adding walnut protein into cow milk, but the walnut pulp has higher fat content, higher molecular weight, poorer solubility and difficult digestion and absorption. Therefore, the walnut products with better solubility and easy absorption are required to be searched for as ingredients, so that the prepared fermented dairy product has good texture and ensures the original taste and functional characteristics of the lactobacillus beverage.
In a first aspect, in a specific embodiment of the present invention, the present invention provides a selenium-enriched lactobacillus beverage, which comprises the following raw materials in parts by weight: 25-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
The lactobacillus helveticus is prepared by inoculating lactobacillus helveticus into a selenium-containing culture medium for fermentation culture, the lactobacillus helveticus can be prepared into liquid, powder or granule, and the lactobacillus helveticus can be prepared by the following steps:
the lactobacillus helveticus strain preserved in the glycerol tube is activated, the first activation is inoculated into MRS broth culture medium according to 1-5% of inoculum size, and is cultured for 12-36h at 35-40 ℃, and the second activation is cultured for 6-24h according to 1-3% of inoculum size at 35-40 ℃. Inoculating the activated bacterial liquid into MRS broth culture medium according to the inoculum size of 5-15%, culturing and fermenting at 35-40 ℃, adding sterile sodium selenite solution to a final concentration of 30 mug/mL for selenium enrichment when fermenting for 3-5h, and continuing fermenting for 19-21h. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the supernatant, and washing the bacterial body twice with sterile physiological saline to obtain the selenium-enriched Lactobacillus helveticus bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by weight: 5-15% of skim milk powder, 4-8% of maltodextrin, 2-8% of sucrose and the balance of pure water, and the components of the protective agent are fully stirred and uniformly mixed, and are preserved in a constant-temperature water bath kettle at 90 ℃ for 30min for sterilization and disinfection for standby.
Preparing the cleaned selenium-enriched lactobacillus helveticus bacterium mud and a freeze-drying protective agent according to the weight ratio of 1:1-5, adding the bacterium mud into the sterilized freeze-drying protective agent, fully and uniformly mixing in a sterile freeze-drying bottle, pre-freezing for 12 hours in a refrigerator at the temperature of minus 30 ℃, and freeze-drying the pre-frozen bacterium milk in a vacuum freeze dryer for 48 hours to obtain the selenium-enriched lactobacillus helveticus freeze-drying bacterial powder. Through detection, the selenium content of the fungus powder is 100-125 mug/g.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 25-65 parts by weight, 30-65 parts by weight, 35-65 parts by weight, 40-65 parts by weight, 45-65 parts by weight, 50-65 parts by weight, 25-60 parts by weight, 25-55 parts by weight, 25-50 parts by weight, 25-45 parts by weight or 25-40 parts by weight of cow milk. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 25 parts by weight, 30 parts by weight, 35 parts by weight, 40 parts by weight, 45 parts by weight, 50 parts by weight, 55 parts by weight, 60 parts by weight or 65 parts by weight of cow's milk, or cow's milk within a range of values defined by any 2 of the specific values as the endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.1-2 parts by weight, 0.5-2 parts by weight, 1-2 parts by weight, 0.1-1.5 parts by weight or 0.1-1 parts by weight of walnut peptide. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.1 weight part, 0.5 weight part, 1 weight part, 1.5 weight part or 2 weight parts of walnut peptide, or cow milk in a numerical range of any 2 specific numerical values as defined by the endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.01-0.1 part by weight, 0.01-0.09 part by weight, 0.01-0.08 part by weight, 0.01-0.07 part by weight, 0.01-0.06 part by weight, 0.01-0.05 part by weight, 0.02-0.1 part by weight, 0.03-1 part by weight, 0.04-0.1 part by weight, or 0.05-1 part by weight of the first fermentation starter. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.01 part by weight, 0.02 part by weight, 0.03 part by weight, 0.04 part by weight, 0.05 part by weight, 0.06 part by weight, 0.07 part by weight, 0.08 part by weight, 0.09 part by weight, or 0.1 part by weight of the first fermentation starter, or cow milk in a numerical range of any 2 specific numerical values as defined above. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.08-0.16 parts by weight, 0.08-0.15 parts by weight, 0.08-0.14 parts by weight, 0.08-0.13 parts by weight, 0.08-0.12 parts by weight, 0.09-0.16 parts by weight, 0.10-0.16 parts by weight, 0.11-0.16 parts by weight or 0.12-0.1 parts by weight of the second fermentation starter. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.08 parts by weight, 0.09 parts by weight, 0.10 parts by weight, 0.11 parts by weight, 0.12 parts by weight, 0.13 parts by weight, 0.14 parts by weight, 0.15 parts by weight or 0.16 parts by weight of the second fermentation starter, or cow milk within a range of values between any 2 of the specific values as defined above. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the invention, the lactobacillus helveticus is lactobacillus helveticus Zhegu LBH-VI (Lactobacillus helveticus Zhegu LBH-VI), wherein the lactobacillus helveticus Zhegu LBH-VI is preserved in the chinese typical culture collection (CCTCC) at 2023, month 2 and 7, with a preservation number of cctccc NO: m2023095.
In some embodiments of the invention, the selenium-enriched lactic acid bacteria beverage has a selenium content of ∈8.0 μg/100g; preferably, the viable count of the selenium-enriched lactobacillus beverage in the shelf life is more than or equal to 10 9 CFU/ml。
The shelf life of the selenium-enriched lactobacillus beverage refers to a period of time in which the selenium-enriched lactobacillus beverage can keep stable in texture under the storage condition of 2-6 ℃, and meanwhile, the mouthfeel, the sensory and the microbial content of the selenium-enriched lactobacillus beverage are ensured to be in ideal states, and any nutritional value stated by a label is reserved. The quality guarantee period of the selenium-enriched lactobacillus beverage is 30 days.
In some embodiments of the present invention, cow milk used in the present invention refers primarily to fresh milk or reconstituted milk that should meet the domestic raw fresh cow milk acquisition criteria, including but not limited to whole cow milk, low fat cow milk, or a combination of two or more of skimmed cow milk.
In some embodiments of the invention, the molecular weight of the walnut peptide is 800-1000Da; preferably, the walnut peptide is prepared by degreasing walnut and carrying out enzymolysis on protease. The preparation method of the walnut peptide comprises the following steps: (1) Degreasing walnut to obtain defatted walnut cake (fat content is less than or equal to 1%), adding water to prepare feed liquid, adjusting pH to 8-10, stirring for 10-30min, separating and removing precipitate, adjusting pH to 4-5, stirring for 10-30min, and separating and collecting precipitate to obtain walnut protein; (2) Preparing walnut protein into dispersion liquid, regulating pH to 8-10, adding 1-3% protease, preferably alkaline protease and papain in the mass ratio of 1-2:0.5-1.5, performing enzymolysis at 45-60deg.C for 4-6 hr, inactivating enzyme, separating and drying to obtain walnut peptide.
In some embodiments of the present invention, the first fermentation starter is selected from one or more of streptococcus salivarius thermophilus subspecies, lactobacillus delbrueckii subspecies, lactobacillus paracasei, bifidobacterium lactis, and lactococcus lactis subspecies, preferably streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies, and more preferably streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies, in a mass ratio of 2-3:1-1.5.
In some embodiments of the invention, the feedstock further comprises 5.0-8.5 parts of sweetener; preferably, the sweetener is selected from one or more than two of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycoside, preferably sucrose.
In some embodiments of the invention, the feedstock further comprises 0.3 to 0.7 parts of a stabilizer; the stability and quality of the selenium-enriched lactobacillus beverage can be improved by adding the stabilizer, preferably, the stabilizer is one or more than two of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate and sodium citrate; more preferably, the mass ratio of the sodium carboxymethyl cellulose, the pectin, the sodium tripolyphosphate and the sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.7.
In some embodiments of the invention, the feedstock further comprises 0.11-0.6 parts of an acidulant; preferably, the acidulant is selected from citric acid and/or lactic acid, preferably the mass ratio of citric acid to lactic acid is 5-25:5-15.
In some embodiments of the invention, the selenium-enriched lactic acid bacteria beverage is prepared by subjecting a fermentation base comprising cow's milk and walnut peptide to temperature swing fermentation.
In some embodiments of the invention, the invention provides a selenium-enriched lactobacillus beverage, which comprises the following raw materials in percentage by weight: 25-65% of cow milk, 0.1-2% of walnut peptide, 0.01-0.1% of a first fermentation microbial agent, 0.08-0.16% of a second fermentation microbial agent and 33-73% of water, wherein the second fermentation microbial agent is selenium-enriched lactobacillus helveticus.
In some embodiments of the invention, the invention provides a selenium-enriched lactobacillus beverage, which comprises the following raw materials in percentage by weight: 25-40% of cow milk, 0.1-2% of walnut peptide, 0.01-0.1% of a first fermentation microbial agent, 0.08-0.16% of a second fermentation microbial agent and 50-69% of water, wherein the second fermentation microbial agent is selenium-enriched lactobacillus helveticus.
In some embodiments of the invention, the feedstock further comprises 5-8.5% sweetener.
In some embodiments of the invention, the feedstock further comprises 0.3-0.7% of a stabilizer.
In some embodiments of the invention, the feedstock further comprises 0.11-0.6% of an acidulant.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 25-65%, 30-65%, 35-65%, 40-65%, 45-65%, 50-65%, 25-60%, 25-55%, 25-50%, 25-45% or 25-40% by weight of cow milk. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% by weight of cow's milk, or cow's milk within a range of values defined by any 2 of the specific values as endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.1-2%, 0.5-2%, 1-2%, 0.1-1.5% or 0.1-1% by weight of the walnut peptide. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.1%, 0.5%, 1%, 1.5% or 2% by weight of the walnut peptide, or cow milk within a range of values defined by any 2 of the specific values as endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.01-0.1%, 0.01-0.09%, 0.01-0.08%, 0.01-0.07%, 0.01-0.06%, 0.01-0.05%, 0.02-0.1%, 0.03-1%, 0.04-0.1% or 0.05-1% by weight of the first fermentation agent. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% by weight of the first fermentation starter, or cow's milk within a range of values defined by any 2 of the specific values as endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage material may comprise 0.08-0.16%, 0.08-0.15%, 0.08-0.14%, 0.08-0.13%, 0.08-0.12%, 0.09-0.16%, 0.10-0.16%, 0.11-0.16%, or 0.12-0.16% by weight of the second fermentation starter. In some embodiments, the selenium-enriched lactic acid bacteria beverage material may comprise 0.08%, 0.09%, 0.10%, 0.12%, 0.13%, 0.14%, 0.15%, or 0.16% by weight of the second fermentation starter, or cow's milk within a range of values defined by any 2 of the specific values as endpoints. It should be understood that any of the above ranges may be combined with any other ranges in the specific embodiments, as long as the selenium-enriched lactic acid bacteria beverage of the desired properties of the present invention is obtained.
In a second aspect, the invention also provides a preparation method of the selenium-enriched lactobacillus beverage, which comprises the following steps:
(1) Mixing raw materials comprising cow milk and walnut peptide to prepare a fermentation base material;
(2) Sequentially inoculating a first fermentation microbial inoculum and a second fermentation microbial inoculum into the fermentation base material prepared in the step (1) to perform variable-temperature fermentation to prepare fermented milk;
(3) And (3) blending the fermented milk prepared in the step (2) to obtain the selenium-enriched lactobacillus beverage.
In some embodiments of the present invention, the preparation in step (3) may be performed by adding a stabilizer to the fermented milk for stabilization, adding an acidulant for acidification, metering in water, homogenizing, and the like, as needed.
In some embodiments of the invention, the fermentation base further comprises a sweetener.
The invention adopts two-stage inoculation and variable temperature fermentation processes, which remarkably improves the flavor, taste and texture stability of the selenium-enriched lactobacillus beverage and improves the number of viable bacteria in shelf life.
In some embodiments of the present invention, the step (2) of sequentially inoculating the first fermentation starter and the second fermentation starter to perform the temperature-variable fermentation refers to inoculating the first fermentation starter, fermenting at 40-45 ℃, inoculating the second fermentation starter, and fermenting at 35-39 ℃; preferably, the first fermentation inoculant is inoculated for fermentation at 40-45 ℃ for 10-12 hours until the final acidity is 90-100 DEG T, and then the second fermentation inoculant is inoculated for fermentation at 35-39 ℃ for 8-10 hours until the final acidity is 160-180 DEG T.
The first fermentation in the invention refers to the fermentation of inoculating the first fermentation inoculant at 40-45 ℃; the second fermentation refers to the fermentation of inoculating the second fermentation starter at 35-39 ℃.
The method adopts the two-stage inoculation and variable temperature fermentation process to prepare the viable bacteria type lactobacillus beverage, so that the lactobacillus beverage has rich taste and good tissue state, and the viable bacteria quantity of the lactobacillus beverage in the shelf life is improved.
In some embodiments of the invention, the preparation method further comprises a step of homogenizing the fermentation base prior to inoculation, preferably at a homogenization temperature of 50-65 ℃, more preferably at a homogenization pressure of 10-50MPa; preferably, the homogenizing treatment further comprises a step of sterilizing the fermentation base material, preferably at a sterilization temperature of 90-100 ℃, more preferably for a sterilization time of 200-400s.
In some embodiments of the invention, the fermentation broth is stirred to break the emulsion after each fermentation is completed, the breaking is to stir the curd block possibly existing, so that the ingredients added subsequently are uniformly mixed in the feed liquid, preferably at a stirring speed of 20-40r/min, and more preferably for a stirring time of 10-20min.
The advantageous effects of the present invention are further illustrated by the following specific examples.
The raw materials or reagents used in the present invention are all purchased from market mainstream factories, and are analytically pure grade raw materials or reagents which can be conventionally obtained without any particular limitation as long as they can function as intended.
No particular technique or condition is identified in this example, which is performed according to techniques or conditions described in the literature in this field or according to product specifications.
Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but the technical scope of the present invention is not limited to these examples. Unless otherwise specified, all percentages, parts and ratios used in the present invention are based on mass.
The reagents and instrument sources used in the examples below are shown in Table 1.
Table 1 Table of raw Material information used in examples
| Raw materials | Model/purity | Vendor' s |
| Citric acid | The purity is more than or equal to 99.5 percent | Wuhan Cheng Ze food ingredients Co.Ltd |
| Lactic acid | The purity is more than or equal to 95 percent | HENAN JINDAN LACTIC ACID TECHNOLOGY Co.,Ltd. |
| Sodium citrate | Purity is more than or equal to 99 percent | The food ingredients of the Wuhan Cheng Ze areLimited company |
| Sodium carboxymethyl cellulose | Low viscosity | XIAMEN OUKAI TECHNOLOGY Co.,Ltd. |
| Alkaline protease | AP-200A | Angel Yeast Co.,Ltd. |
| Papain | 2021070301 | Angel Yeast Co.,Ltd. |
| Pectin | Low ester | XIAMEN OUKAI TECHNOLOGY Co.,Ltd. |
| Sodium tripolyphosphate | Purity is more than or equal to 85 percent | Wuhan Cheng Ze food ingredients Co.Ltd |
| Sucrose | First level | Guangxi Phoenix sugar Co., ltd |
| Xylitol | The purity is more than or equal to 98.5 percent | Shandong Futaste Co.,Ltd. |
| Erythritol | The purity is more than or equal to 99.5 percent | Shandong Futian technology groupLimited Co. |
| Steviol glycoside | RA96, 400 times | Wuhan Cheng Ze food ingredients Co.Ltd |
| Maltitol | Purity is more than or equal to 98 percent | Shandong Futaste Co.,Ltd. |
Example 1
1. Preparation of walnut peptide
Selecting dry and mildew-free walnut, placing in 0.5% NaOH solution, soaking in water bath at 65deg.C for 30min, and peeling. Drying peeled semen Juglandis in oven at 40deg.C, pulverizing with pulverizer, wrapping with gauze, squeezing oil with small sesame oil hydraulic squeezer, maintaining at 35Mpa for 30min to obtain semen Juglandis cake, and pulverizing to 20 mesh. Removing oil from the walnut pulp by supercritical extraction, wherein the extraction pressure is 35MPa, the extraction temperature is 40 ℃, the extraction time is 3h, the pressure of the separation kettle 1 is 10-12MPa, the temperature is 40 ℃, the pressure of the separation kettle 2 is 5-7MPa, and the temperature is 40 ℃. The deoiling walnut cake (fat content is less than or equal to 1%) is obtained.
Distilled water is added into deoiling walnut pulp according to the mass ratio of 1:20 to obtain feed liquid, the pH value of the feed liquid is regulated to 9.0 by using 0.1% NaOH solution, the rotating speed is set to be 100r/min, stirring is carried out for 10min, the treated feed liquid is transferred into a centrifugal bottle for centrifugation at 4000r/min for 10min, the supernatant solution is collected, the pH value of the feed liquid is regulated to 4.5 by using 0.1% hydrochloric acid solution, the rotating speed is set to be 100r/min, stirring is carried out for 10min, the treated feed liquid is transferred into the centrifugal bottle for centrifugation at 4000r/min for 10min, and sediment is collected.
Adding distilled water into the precipitate according to the mass ratio of 1:10, regulating the pH value of the feed liquid to 9.0 by using a 0.1% NaOH solution, adding 2% protease (alkaline protease: papain=1.5:1), heating the feed liquid to 50 ℃, carrying out enzymolysis for 5 hours, carrying out enzyme deactivation treatment (90 ℃ for 10 minutes) on the feed liquid after the enzymolysis is finished, transferring the feed liquid 2 into a centrifugal bottle for 4000r/min for centrifuging for 10 minutes, collecting supernatant, carrying out rotary concentration on the supernatant at 50 ℃ after film passing, pre-freezing the concentrated solution in a refrigerator at-30 ℃ for 12 hours, and freeze-drying the pre-frozen concentrated solution in a vacuum freeze dryer for 48 hours to obtain the walnut polypeptide with the molecular weight of 952.433Da.
2. Screening and identification method and identification result of lactobacillus helveticus Zhegu LBH-VI (Latin name Lactobacillus helveticus Zhegu LBH-VI)
1) Taking 25g of Tibetan hucho plain herdsman homemade yak milk residues and 225mL of sterile physiological saline, uniformly mixing to obtain uniform sample liquid, carrying out gradient dilution on the sample liquid, and respectively taking 10 -3 、10 -4 、10 -5 、10 -6 The diluted solution was spread on an MRS medium plate, and after culturing at 37℃for 48 hours, colonies were grown in the MRS medium.
2) Bacterial primary screening
According to the standard colony characteristics of lactobacillus helveticus, single colony is selected for separation and purification, continuous culture is carried out, and continuous separation and purification is carried out for at least three times, so as to obtain purified colony.
Culturing characteristics: the optimal growth temperature is 37 ℃, and the culture medium is facultative anaerobic and grows in MRS culture medium.
3) Acid production experiment
Single colony obtained by primary screening of strain is treated with strain containing 0.2% CaCO 3 Culturing on MRS culture medium plate, culturing at 37deg.C for 48 hr, observing transparent ring-free condition around colony, and selecting single colony with strong acid-producing ability and large transparent ring for further separation and purification.
Morphological features: the growth state in MRS agar medium is: the growth forms of the bacteria are milky white colonies, round, smooth in edge, protruding thalli and rough in surface in MRS agar culture medium.
4) Gram staining
And (3) carrying out gram staining on the strain obtained by the re-screening, wherein the gram staining is typical positive, and the obtained strain is the target strain. The cells are observed under a microscope to be in a short rod shape, without flagellum, without producing spores and without moving.
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and not producing gas is subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis with NCBI GenBank database, so that the result shows that the strain is lactobacillus helveticus. The 16SrDNA gene sequence of the strain is shown in SEQ ID NO. 1:
CAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGATTTACTTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGCAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCCATCCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTC
The strain is named as Lactobacillus helveticus (Lactobacillus helveticus) Zhegu LBH-VI strain, and is preserved in China Center for Type Culture Collection (CCTCC) in 2 nd month 7 of 2023, and the preservation number is CCTCC NO: M2023095.
3. Preparation of selenium-enriched lactobacillus helveticus
The Lactobacillus helveticus strain Zhegu LBH-VI (Latin name Lactobacillus helveticus Zhegu LBH-VI) deposited in the glycerol tube was activated, the first activation was inoculated in MRS broth medium at an inoculum size of 3%, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours at an inoculum size of 2%. Inoculating the activated bacterial liquid into 2L of MRS broth culture medium according to 10% of inoculum size, culturing and fermenting at 37 ℃, adding sterile sodium selenite solution to a final concentration of 30 mug/mL for selenium enrichment when fermenting for 4h, and continuing fermenting for 20h. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the supernatant, washing the bacterial cells twice with sterile physiological saline to obtain selenium-enriched lactobacillus helveticus bacterial sludge, and measuring the bacterial sludge water content.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned selenium-enriched lactobacillus helveticus bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing the bacterial mud with the sterilized freeze-drying protective agent in a sterile freeze-drying bottle, pre-freezing the bacterial mud in a refrigerator at the temperature of minus 30 ℃ for 12 hours, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the selenium-enriched lactobacillus helveticus freeze-drying bacterial powder. Through detection, the selenium content of the fungus powder is 100-125 mug/g.
4. Screening and identification method and identification result of streptococcus salivarius thermophilus 932 (Straptococcus salivarias subsp. Thermophilus 932)
1) Taking 25g of Tibetan hucho plain herdsman homemade yak milk residues and 225mL of sterile physiological saline, uniformly mixing to obtain uniform sample liquid, carrying out gradient dilution on the sample liquid, and respectively taking 10 -3 、10 -4 、10 -5 、10 -6 The diluted solution was spread on an MRS medium plate, and after culturing at 37℃for 48 hours, colonies were grown in the MRS medium.
2) Bacterial primary screening
According to the standard colony characteristics of streptococcus salivarius thermophilus subspecies, single colony is selected for separation and purification, continuous culture is carried out, and the separation and purification are carried out for at least three times continuously, so as to obtain purified colony.
Culturing characteristics: the optimal growth temperature is 42 ℃, and the culture medium is facultative anaerobic and grows in MRS culture medium.
3) Acid production experiment
Single colony obtained by primary screening of strain is treated with strain containing 0.2% CaCO 3 Culturing on MRS culture medium plate, culturing at 42deg.C for 48 hr, observing the condition of no transparent ring around colony, and selecting single colony with strong acid production capacity and large transparent ring for further separation and purification.
Morphological features: the growth state in MRS agar medium is: the growth form in MRS agar culture medium is slightly yellowish at the bottom, and the surface is milky colony, irregular edge, raised thallus and smoother surface.
4) Gram staining
And (3) carrying out gram staining on the strain obtained by the re-screening, wherein the gram staining is typical positive, and the obtained strain is the target strain. The cells are observed under a microscope to be in a sphere rod shape, have no flagella, do not produce spores and do not move.
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and not producing gas is subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis with NCBI GenBank database, so that the result shows that the strain is streptococcus salivarius thermophilus subspecies. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 2:
TTAGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCA
the strain is named as streptococcus salivarius thermophilus 932 (Latin name Straptococcus salivari as subsp. Thermophilus 932) and is preserved in China Center for Type Culture Collection (CCTCC) in the 6 th month and 5 th year of 2023, and the preservation number is CCTCC NO: m2023902.
5. Preparation of streptococcus salivarius thermophilus subspecies 932 microbial inoculum
The Streptococcus salivarius thermophilus 932 (Straptococcus salivarias subsp. Thermophilus 932) strain deposited in the glycerol tube was activated, the first activation was inoculated in an inoculum size of 3% in MRS broth medium, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours in an inoculum size of 2%. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the centrifugal supernatant, and washing the bacterial body twice with sterile physiological saline to obtain the streptococcus salivarius subspecies thermophilus 932 bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned streptococcus salivarius thermophilus subspecies 932 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing the bacterial mud with the sterilized freeze-drying protective agent in a sterile freeze-drying bottle, pre-freezing the bacterial milk in a refrigerator at the temperature of minus 30 ℃ for 12 hours, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the streptococcus salivarius thermophilus subspecies 932 freeze-drying bacterial powder.
6. Screening and identifying method and identifying result of Lactobacillus delbrueckii subspecies Dangxiong LB V III (Lactobacillus delbrueckii subsp. Lactis Dangxiong LB V III)
1) Enrichment of strains: pulverizing milk powder sample of 5g Tibetan Dandelion (altitude 4350 m) at hercynanchum, adding 45ml sterile physiological saline solution, dispersing uniformly, adding 2ml above dispersion into 20ml sterilized litmus milk culture medium, standing in 37 deg.C incubator, and acidifying the culture medium
And taken out when solidified and appeared pink.
2) Culturing single colonies: diluting litmus milk culture medium bacterial liquid to 10 -5 100 μl of the mixture is sucked and coated on a BL solid culture medium plate, and the mixture is placed in a 37 ℃ incubator for culture until colonies are formed. On the BL plate, the colony is smooth and slightly convex, moist, and has a non-smooth edge, colorless to slightly white, a diameter of 1-3mm, and the back of the colony is yellowish.
3) Screening acid-producing strains: single colonies obtained from the primary screening were isolated in the presence of 0.2% CaCO 3 The BL solid culture medium plate is streaked and cultured for 36 hours in a 37 ℃ incubator, after the culture is finished, whether transparent rings are generated around the colony is observed, and single colony with the transparent ring diameter of 3-3.5cm is selected for further separation and purification.
4) Gram stain preliminary identification: gram staining the strain in the step (3), if the bacterial stain is purple, the strain is gram positive bacteria, and the strain is directly eliminated from the gram negative bacteria if the stain is red. The identification result shows that: the obtained strain is gram positive bacteria, does not move, has no spore, no flagellum and no capsule, the thallus is in a thin rod shape, presents a single rod or a chain, the strain is the target strain,
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and not producing gas is subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis in NCBI GenBank database, so that the result shows that the strain is Lactobacillus delbrueckii subspecies lactis. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 3:
GATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACACCATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAAACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAAAACCTTACCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGTGCAGTACAACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGCTCTCAGATCGGACTGCGGGCTGCAACTCGCCTGCACGAAACTGGAATCGCTAGTAATCTCGGATCACCACGCCGCGGTGAATACGTTCCCGCGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGTCGGTGAGATAACCTTTATAGGAGTCAGCCGCCTAA
the strain is named as Lactobacillus delbrueckii subspecies Dangxiong LB V III (Latin name Lactobacillus delbrueckii subsp. Lactis Dangxiong LB V III) and is preserved in China Center for Type Culture Collection (CCTCC) for 3 months and 23 days in 2023, and the preservation number is CCTCC NO: M2023396.
7. Preparation of Lactobacillus delbrueckii subspecies Dangxiong LB V III microbial inoculum
The Lactobacillus delbrueckii subspecies Dangxiong LB V III strain preserved in the glycerol tube was activated, the first activation was inoculated in MRS broth medium at an inoculum size of 3%, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours at an inoculum size of 2%. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the centrifugal supernatant, washing the bacterial cells twice with sterile physiological saline to obtain Lactobacillus delbrueckii subspecies Dangxiong LB V III bacterial mud, and measuring the water content of the bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned Lactobacillus delbrueckii subspecies Dangxiong LB V III bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing in a sterile freeze-drying bottle, pre-freezing for 12 hours in a refrigerator at the temperature of minus 30 ℃, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain Lactobacillus delbrueckii subspecies Dangxiong LB V III freeze-dried bacterial powder.
8. Preparation of selenium-enriched lactobacillus beverage
1) Preparation of fermentation base material
Checking raw cow milk according to the standard requirement of GB 19301, and carrying out standardized treatment on the cow milk which is checked to be qualified, so that the milk protein is 2.9g/100g; taking 650g of standardized cow milk, heating to 60 ℃, uniformly mixing 20g of walnut peptide and 65g of sucrose, adding the mixture into the standardized cow milk, shearing for 15min by using a shearing emulsifying machine 3000r, homogenizing under the conditions of 60 ℃ and 25MPa (wherein the low pressure is 5 MPa), heating to 95 ℃ after homogenization, preserving heat for 300s, sterilizing, and then pumping into a fermentation tank;
2) First fermentation
Cooling the fermentation base material prepared in the step 1) to 42 ℃, adding streptococcus salivarius thermophilus subspecies 932 freeze-dried bacterial powder and lactobacillus delbrueckii lactobacillus subspecies Dangxiong LB V III freeze-dried bacterial powder (wherein the addition amount of streptococcus salivarius thermophilus subspecies 932 and lactobacillus delbrueckii subspecies Dangxiong LB V III is 0.1g based on the dry bacterial matter, the mass ratio of streptococcus salivarius thermophilus subspecies 932 to lactobacillus delbrueckii subspecies Dangxiong LB V III is 2:1), stirring for 5min under the condition of 30r/min to ensure that the streptococcus salivarius thermophilus subspecies 932 freeze-dried bacterial powder and the lactobacillus delbrueckii subspecies Dangxiong LB V III freeze-dried bacterial powder are uniformly dispersed, and fermenting under the condition of 42 ℃ until the acidity is 100 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
3) Second fermentation
Cooling the fermented milk after the first fermentation to 37 ℃, adding selenium-enriched lactobacillus helveticus freeze-dried bacterial powder (wherein the adding amount of the selenium-enriched lactobacillus helveticus is 1g based on the dry bacterial matter), stirring for 5min at 30r/min to uniformly disperse the lactobacillus helveticus freeze-dried bacterial powder, fermenting at 37 ℃, and stopping the acidity at 160 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
4) Secondary mixing material
Adding 6g of stabilizer into 75 ℃ hot water for dissolution, wherein the stabilizer comprises sodium carboxymethylcellulose, pectin, sodium tripolyphosphate and sodium citrate, the mass ratio of the sodium carboxymethylcellulose to the pectin to the sodium tripolyphosphate is sodium citrate=1:2:0.3:0.5, shearing for 15min by using a shearing emulsifying machine 3000r, sterilizing the feed liquid at the sterilizing temperature of 75 ℃ for 15s, cooling the feed liquid to 25 ℃ after sterilization, and stirring and mixing the feed liquid with the fermented milk obtained after the second fermentation in the step 3), wherein the stirring speed is 30r/min and the stirring time is 20min;
5) Adding sour agent
Preparing 1.8g of citric acid, 1g of lactic acid and 0.3g of sodium citrate into an acid liquor with the solid content of 10%, sterilizing the acid liquor at the sterilization temperature of 75 ℃ for 15 seconds, and cooling to 25 ℃ after sterilization; the liquid in the step 4) is subjected to constant volume by using sterile distilled water (the addition amount of the sterile distilled water is 1000g for the liquid with the acidity adjusted), and the acidity of the liquid is adjusted to 65 DEG T by using the prepared sterile acid liquid;
finally, homogenizing the feed liquid under the conditions of 25 ℃ and 20MPa; then quantitatively filling and sealing are carried out in a sterile environment, and the filling temperature is 15 ℃; and (5) transferring the mixture to a refrigeration house after filling, and refrigerating at 6 ℃.
Example 2
Preparation of selenium-enriched lactobacillus beverage
1) Preparation of fermentation base material
Checking raw cow milk according to the standard requirement of GB 19301, and carrying out standardized treatment on the cow milk which is checked to be qualified, so that the milk protein is 2.9g/100g; taking 350g of standardized cow milk, heating to 60 ℃, uniformly mixing 1g of walnut peptide prepared in the step 1 of the embodiment 1 with 50g of sucrose, adding the mixture into the standardized cow milk, shearing for 15min by using a shearing emulsifying machine 3000r, homogenizing under the conditions of 60 ℃ and 25MPa (wherein the low pressure is 5 MPa), heating to 95 ℃ for 300s for sterilization after homogenization, and then pumping the mixture into a fermentation tank;
2) First fermentation
Cooling the fermentation base material prepared in the step 1) to 45 ℃, adding the lyophilized powder of the streptococcus salivarius thermophilus subspecies 932 and the lyophilized powder of the Lactobacillus delbrueckii subspecies Dangxiong LB V III prepared in the example 1 (wherein the addition amount of the streptococcus salivarius thermophilus subspecies 932 and the Lactobacillus delbrueckii subspecies Dangxiong LB V III is 1g based on the dry matter of the thallus), stirring for 5min under the condition of 30r/min to ensure that the lyophilized powder is uniformly dispersed, and fermenting under the condition of 45 ℃ to ensure that the termination acidity is 90 DEG T; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
3) Second fermentation
Cooling the fermented milk after the first fermentation to 39 ℃, adding the selenium-enriched lactobacillus helveticus freeze-dried powder prepared in the example 1 (wherein the adding amount of the selenium-enriched lactobacillus helveticus is 0.8g based on the dry matter of the thallus), stirring for 5min at 30r/min to uniformly disperse the selenium-enriched lactobacillus helveticus freeze-dried powder, fermenting at 39 ℃, and stopping the acidity at 180 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
4) Secondary mixing material
Adding 3g of stabilizer into 75 ℃ hot water for dissolution, wherein the stabilizer comprises sodium carboxymethylcellulose, pectin, sodium tripolyphosphate and sodium citrate, the mass ratio of the sodium carboxymethylcellulose to the pectin to the sodium tripolyphosphate is sodium citrate=0.5:1.5:0.2:0.3, shearing the solution for 15min by using a shearing emulsifying machine 3000r, sterilizing the solution at the sterilizing temperature of 75 ℃ for 15s, cooling the solution to 25 ℃ after sterilization, and stirring and mixing the solution with the fermented milk obtained after the second fermentation in the step 3), wherein the stirring speed is i 30r/min, and the stirring time is 20min;
5) Adding sour agent
Preparing 0.5g of citric acid, 0.5g of lactic acid and 0.1g of sodium citrate into an acid liquor with the solid content of 10%, sterilizing the acid liquor at the sterilization temperature of 75 ℃ for 15 seconds, and cooling to 25 ℃ after sterilization; the liquid in the step 4) is subjected to constant volume by using sterile distilled water (the addition amount of the sterile distilled water is 1000g for the liquid with the acidity adjusted), and the acidity of the liquid is adjusted to 65 DEG T by using the prepared sterile acid liquid;
finally, homogenizing the feed liquid under the conditions of 25 ℃ and 20MPa; then quantitatively filling and sealing are carried out in a sterile environment, and the filling temperature is 15 ℃; and (5) transferring the mixture to a refrigeration house after filling, and refrigerating at 6 ℃.
Example 3
1. Screening and identifying method and identifying result of lactococcus lactis subspecies 954 (Lactococcus lactis subsp. Lactis)
1) Mixing 25g of Tibetan hucho taimen domestic yak milk residues with 225mL of sterile physiological saline to obtain uniform sample liquid, carrying out gradient dilution on the sample liquid, respectively taking 10-3, 10-4, 10-5 and 10-6 concentration diluted liquid, coating and culturing on an MRS culture medium flat plate, and culturing at 30 ℃ for 48 hours to grow colonies on the MRS culture medium.
2) Bacterial primary screening
According to the standard colony characteristics of streptococcus salivarius thermophilus subspecies, single colony is selected for separation and purification, continuous culture is carried out, and the separation and purification are carried out for at least three times continuously, so as to obtain purified colony.
Culturing characteristics: the optimal growth temperature is 30 ℃, and the plant is facultative anaerobic and grows in MRS culture medium.
3) Acid production experiment
Single colony obtained by primary screening of strain is treated with strain containing 0.2% CaCO 3 Culturing on MRS culture medium plate, culturing at 30deg.C for 48 hr, observing the condition of no transparent ring around colony, and selecting single colony with strong acid production capacity and large transparent ring for further separation and purification.
Morphological features: the growth state in MRS agar medium is: the growth form in MRS agar culture medium is yellowish at the bottom, and the surface is yellowish opaque colony, regular edge, low protrusion of thallus and smooth surface.
4) Gram staining
And (3) carrying out gram staining on the strain obtained by the re-screening, wherein the gram staining is typical positive, and the obtained strain is the target strain. The cells are observed under a microscope to be in a sphere rod shape, have no flagella, do not produce spores and do not move.
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and not producing gas is subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis with NCBI GenBank database, so that the result shows that the strain is lactococcus lactis subspecies. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 4:
GGAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTGTGTTTAAAGTTTTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCATCCAGTCGGTACAAGTACCAACCTTCAGCGCTCAACTTGCATGTATTAGGCACGCCGCCAGCGTTC
The strain is named as a lactococcus lactis subspecies 954 (Lactococcus lactis subsp.lactis) strain, and is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, 6 and 5, and the preservation number is CCTCC NO: m2023904.
2. Preparation of lactobacillus lactis subspecies 954 microbial inoculum
The lactococcus lactis subspecies 954 strain preserved in the glycerol tube was activated, the first activation was inoculated into MRS broth medium at an inoculum size of 3%, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours at an inoculum size of 2%. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the supernatant, and washing the bacterial body twice with sterile physiological saline to obtain lactococcus lactis subspecies 954 bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned lactococcus lactis subspecies 954 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing in a sterile freeze-drying bottle, pre-freezing for 12 hours in a refrigerator at the temperature of minus 30 ℃, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the lactococcus lactis subspecies 954 freeze-dried bacterial powder.
3. Preparation of selenium-enriched lactobacillus beverage
1) Preparation of fermentation base material
Checking raw cow milk according to the standard requirement of GB 19301, and carrying out standardized treatment on the cow milk which is checked to be qualified, so that the milk protein is 2.9g/100g; taking 250g of standardized cow milk, heating to 60 ℃, taking 5g of walnut peptide prepared in the step 1 of the example 1, 20g of maltitol, 20g of xylitol, 40g of erythritol and 5g of stevioside, uniformly mixing, adding the mixture into the standardized cow milk, shearing for 15min by using a shearing emulsifying machine 3000r, homogenizing under the conditions of 60 ℃ and 25MPa (wherein the low pressure is 5 MPa), heating to 95 ℃ for 300s for sterilization after homogenization, and then pumping the mixture into a fermentation tank;
2) First fermentation
Cooling the fermentation base material prepared in the step 1) to 40 ℃, adding lactococcus lactis subspecies 954 freeze-dried bacterial powder (wherein the adding amount of the lactococcus lactis subspecies 954 is 0.5g based on the dry bacterial matter), stirring for 5min at 30r/min to uniformly disperse the lactococcus lactis subspecies 954, fermenting at 40 ℃, and stopping acidity at 100 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
3) Second fermentation
Cooling the fermented milk after the first fermentation to 35 ℃, adding the selenium-enriched lactobacillus helveticus freeze-dried bacterial powder (wherein the adding amount of the selenium-enriched lactobacillus helveticus is 1.2g based on the dry bacterial matter) prepared in the example 1, stirring for 5min at 30r/min to uniformly disperse the selenium-enriched lactobacillus helveticus freeze-dried bacterial powder, fermenting at 35 ℃, and stopping the acidity at 160 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
4) Secondary mixing material
Adding 5g of stabilizer into 75 ℃ hot water for dissolution, wherein the stabilizer comprises sodium carboxymethylcellulose, pectin, sodium tripolyphosphate and sodium citrate, the mass ratio of the sodium carboxymethylcellulose to the pectin to the sodium tripolyphosphate is sodium citrate=1.3:2.5:0.5:0.7, shearing the solution for 15min by using a shearing emulsifying machine 3000r, sterilizing the solution at the sterilizing temperature of 75 ℃ for 15s, cooling the solution to 25 ℃ after sterilization, and stirring and mixing the solution with the fermented milk obtained after the second fermentation in the step 3), wherein the stirring speed is 30r/min and the stirring time is 20min;
5) Adding sour agent
Preparing 2.5g of citric acid, 1.5g of lactic acid and 2g of sodium citrate into an acid liquor with the solid content of 10%, sterilizing the acid liquor at the sterilization temperature of 75 ℃ for 15 seconds, and cooling to 25 ℃ after sterilization; the liquid in the step 4) is subjected to constant volume by using sterile distilled water (the addition amount of the sterile distilled water is 1000g for the liquid with the acidity adjusted), and the acidity of the liquid is adjusted to 65 DEG T by using the prepared sterile acid liquid;
finally, homogenizing the feed liquid under the conditions of 25 ℃ and 20MPa; then quantitatively filling and sealing are carried out in a sterile environment, and the filling temperature is 15 ℃; and (5) transferring the mixture to a refrigeration house after filling, and refrigerating at 6 ℃.
Example 4
1. Lactobacillus paracasei AL1 Plateau LPA-1 freeze-dried powder
The Lactobacillus paracasei AL1 Plateau LPA-1 strain stored in the glycerol tube was activated, the first activation was inoculated in an inoculum size of 3% in MRS broth medium, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours in an inoculum size of 2%. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the centrifugal supernatant, and washing the bacterial body twice with sterile physiological saline to obtain lactobacillus paracasei AL1 Plateau LPA-1 bacterial sludge.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned lactobacillus paracasei AL1 Plateau LPA-1 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing the bacterial mud in a sterile freeze-drying bottle, pre-freezing the bacterial mud in a refrigerator at the temperature of minus 30 ℃ for 12 hours, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain lactobacillus paracasei AL1 Plateau LPA-1 freeze-drying bacterial powder.
2. Preparation of selenium-enriched lactobacillus beverage
1) Preparation of fermentation base material
Checking raw cow milk according to the standard requirement of GB 19301, and carrying out standardized treatment on the cow milk which is checked to be qualified, so that the milk protein is 2.9g/100g; taking 400g of standardized cow milk, heating to 60 ℃, taking 10g of walnut peptide prepared in the step 1 of the embodiment 1, 30g of sucrose, 20g of xylitol and 20g of sucrose, uniformly mixing, adding the mixture into the standardized cow milk, shearing for 15min by using a shearing emulsifying machine 3000r, homogenizing under the conditions of 60 ℃ and 25MPa (wherein the low pressure is 5 MPa), heating to 95 ℃ for 300s for sterilization after homogenization, and then pumping the mixture into a fermentation tank;
2) First fermentation
Cooling the fermentation base material prepared in the step 1) to 42 ℃, adding lactobacillus paracasei AL1 Plateau LPA-1 freeze-dried bacterial powder (wherein the adding amount of the lactobacillus paracasei AL1 Plateau LPA-1 is 0.8g based on the dry bacterial matter), stirring for 5min at 30r/min to uniformly disperse, fermenting at 40 ℃, and stopping the acidity to be 100 DEG T; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
3) Second fermentation
Cooling the fermented milk after the first fermentation to 37 ℃, adding the selenium-enriched lactobacillus helveticus freeze-dried bacterial powder (wherein the adding amount of the selenium-enriched lactobacillus helveticus is 1.5g based on the dry bacterial matter) prepared in the example 1, stirring for 5min at 30r/min to uniformly disperse the selenium-enriched lactobacillus helveticus freeze-dried bacterial powder, fermenting at 37 ℃, and stopping the acidity at 160 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
4) Secondary mixing material
Adding 7g of stabilizer into 75 ℃ hot water for dissolution, wherein the stabilizer comprises sodium carboxymethylcellulose, pectin, sodium tripolyphosphate and sodium citrate, the mass ratio of the sodium carboxymethylcellulose to the pectin to the sodium tripolyphosphate is sodium citrate=1:2:0.3:0.5, shearing for 15min by using a shearing emulsifying machine 3000r, sterilizing the feed liquid at the sterilizing temperature of 75 ℃ for 15s, cooling the feed liquid to 25 ℃ after sterilization, and stirring and mixing the feed liquid with the fermented milk obtained after the second fermentation in the step 3), wherein the stirring speed is 30r/min, and the stirring time is 20min;
5) Adding sour agent
Preparing 2g of citric acid, 1g of lactic acid and 1g of sodium citrate into an acid solution with the solid content of 10%, sterilizing the acid solution at the sterilization temperature of 75 ℃ for 15 seconds, and cooling to 25 ℃ after sterilization; the liquid in the step 4) is subjected to constant volume by using sterile distilled water (the addition amount of the sterile distilled water is 1000g for the liquid with the acidity adjusted), and the acidity of the liquid is adjusted to 65 DEG T by using the prepared sterile acid liquid;
finally, homogenizing the feed liquid under the conditions of 25 ℃ and 20MPa; then quantitatively filling and sealing are carried out in a sterile environment, and the filling temperature is 15 ℃; and (5) transferring the mixture to a refrigeration house after filling, and refrigerating at 6 ℃.
Comparative example 1
The difference from example 1 is that comparative example 1 was not added with walnut peptide, and the other is the same as example 1.
Comparative example 2
The difference from example 1 is that the second fermentation of comparative example 2 was not supplemented with lactobacillus helveticus enriched with selenium, otherwise the same as in example 1. The preparation method comprises the following steps:
1) Preparation of fermentation base material
Same as in example 1
2) First fermentation
Cooling the fermentation base material prepared in the step 1) to 42 ℃, adding the lyophilized powder of the streptococcus salivarius thermophilus subspecies 932 and the lyophilized powder of the Lactobacillus delbrueckii subspecies Dangxiong LB V III prepared in the example 1 (wherein the addition amount of the streptococcus salivarius thermophilus subspecies 932 and the Lactobacillus delbrueckii subspecies Dangxiong LB V III is 0.1g based on the dry matter of the thallus, the mass ratio of the streptococcus salivarius thermophilus subspecies 932 to the Lactobacillus delbrueckii subspecies Dangxiong LB V III is 2:1), stirring for 5min under the condition of 30r/min to ensure that the streptococcus salivarius thermophilus subspecies 932 and the Lactobacillus delbrueckii subspecies Dangxiong LB V III are uniformly dispersed, and fermenting under the condition of 42 ℃ to stop acidity of 100 DEG T; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
2) Second fermentation
Cooling the fermented milk after the first fermentation to 37 ℃, stirring for 5min at 30r/min to uniformly disperse the fermented milk, fermenting at 37 ℃ to stop the acidity at 160 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
The other steps are the same as in example 1.
Comparative example 3
Preparation of selenium-enriched lactobacillus beverage
3) Preparation of fermentation base material
Same as in example 1
4) First fermentation
Cooling the fermentation base material prepared in the step 1) to 42 ℃, adding the lyophilized powder of the streptococcus salivarius thermophilus subspecies 932 and the lyophilized powder of the lactobacillus delbrueckii lactobacillus Dangxiong LB V III prepared in the example 1 (wherein the addition amount of the streptococcus salivarius thermophilus subspecies 932 and the lactobacillus delbrueckii lactobacillus Dangxiong LB V III is 0.1g based on dry matter of the thallus, the mass ratio of the streptococcus salivarius thermophilus subspecies 932 and the lactobacillus delbrueckii lactobacillus Dangxiong LB V III is 2:1), and the lyophilized powder of the lactobacillus helveticus rich in the example 1 (wherein the addition amount of the lactobacillus helveticus is 1g based on dry matter of the thallus), stirring for 5min under the condition of 30r/min to ensure that the lyophilized powder is uniformly dispersed, and fermenting under the condition of 42 ℃, and stopping the fermentation under the condition of 100 DEG T; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying;
2) Second fermentation
Cooling the fermented milk after the first fermentation to 37 ℃, stirring for 5min at 30r/min to uniformly disperse the fermented milk, fermenting at 37 ℃ to stop the acidity at 160 ℃; stirring for 15min at the rotating speed of 30r/min after fermentation is finished, and demulsifying; cooling to 25 ℃;
The other steps are the same as in example 1.
Comparative example 4
The difference from example 1 is that the second fermentation temperature in the preparation of the selenium-enriched lactic acid bacteria beverage is 42 ℃, otherwise the same as in example 1.
Comparative example 5
The difference from example 1 is that the temperature of the first fermentation in the preparation of the selenium-enriched lactic acid bacteria beverage is 37 ℃, otherwise the same as in example 1.
Comparative example 6
The difference from example 1 is that the amount of the walnut peptide used in the preparation of the selenium-enriched lactic acid bacteria beverage is 25g, otherwise the same as in example 1.
Examples 1-4 and comparative examples 1-6 of the present invention lactic acid bacteria beverages were tested using the following method:
1. determination of selenium content
The selenium content (. Mu.g/100 g) of the lactic acid bacteria beverages prepared in examples 1 to 4 and comparative examples 1 to 6 was measured by the following method, and the results are shown in Table 2.
The method for measuring the selenium content comprises the following steps: selenium content in selenium-enriched lactobacillus beverages was detected according to GB 5009.93-2017 (first method) hydride atomic fluorescence spectrometry.
TABLE 2 selenium content in lactic acid bacteria beverages prepared in examples 1 to 4 and comparative examples 1 to 6
2. Sensory evaluation
10 dairy professionals with sensory training were randomly selected as sensory panelists, with the scoring criteria as shown in table 3 below.
TABLE 3 sensory evaluation criteria for lactic acid bacteria beverages
The scores of the sensory panelists were averaged and the index scores were added to give a total score, with higher scores representing higher product satisfaction and results shown in table 4.
TABLE 4 results of sensory testing of lactic acid bacteria beverages
3. Determination of viable count in shelf life
The lactic acid bacteria beverages prepared in examples 1 to 4 and comparative examples 1 to 6 were stored at a low temperature of 2 to 6 ℃ for a shelf life of 30 days, and the viable count content (CTU/g) in the lactic acid bacteria beverages was measured by sampling on days 1,10,20 and 30, respectively, as follows, and the results are shown in Table 5.
The method for detecting the number of living bacteria comprises the following steps: the total number of lactic acid bacteria colonies was determined by the dilution plate method.
TABLE 5 viable count content during shelf life
| Days of preservation | For 1 day | For 10 days | For 20 days | For 30 days |
| Example 1 | 5.9×10 10 | 5.8×10 10 | 5.6×10 10 | 5.4×10 10 |
| Example 2 | 7.7×10 9 | 7.5×10 9 | 7.3×10 9 | 7.1×10 9 |
| Example 3 | 5.8×10 10 | 5.8×10 10 | 5.7×10 10 | 5.6×10 10 |
| Example 4 | 9.3×10 10 | 9.2×10 10 | 9.2×10 10 | 9.0×10 10 |
| Comparative example 1 | 2.4×10 9 | 2.3×10 9 | 2.0×10 9 | 1.9×10 9 |
| Comparative example 2 | 7.4×10 7 | 7.1×10 7 | 6.9×10 7 | 6.7×10 7 |
| Comparative example 3 | 3.7×10 8 | 3.6×10 8 | 3.4×10 8 | 3.2×10 8 |
| Comparative example 4 | 4.4×10 8 | 4.2×10 8 | 4.1×10 8 | 3.8×10 8 |
| Comparative example 5 | 3.5×10 8 | 3.3×10 8 | 3.1×10 8 | 2.9×10 8 |
| Comparative example 6 | 5.3×10 10 | 5.2×10 10 | 5.0×10 10 | 4.8×10 10 |
4. Precipitation rate of lactobacillus beverage
The lactic acid bacteria beverages prepared in examples 1 to 4 and comparative examples 1 to 6 were tested for precipitation (%) as follows:
taking out lactobacillus beverage from a refrigerator with the temperature of 2-6 ℃, uniformly mixing, weighing 10g of the sample into a 10mL centrifuge tube, putting the sample into a centrifuge at 3000rpm for 10min, weighing the precipitation weight, and calculating the precipitation rate of the lactobacillus beverage according to the following formula;
Precipitation rate of lactic acid bacteria beverage = m 1 /m×100％
m 1 -mass of the precipitate in grams (g);
m—weighing the mass of the sample in grams (g);
the process was repeated three times and an average value was obtained.
The results are shown in Table 6.
TABLE 6 precipitation Rate of lactic acid bacteria beverages of examples 1-4 and comparative examples 1-6
| Product(s) | Precipitation rate of lactic acid bacteria beverage (%) |
| Example 1 | 0.51％ |
| Example 2 | 0.70％ |
| Example 3 | 0.59％ |
| Example 4 | 0.62％ |
| Comparative example 1 | 1.89％ |
| Comparative example 2 | 2.23％ |
| Comparative example 3 | 4.30％ |
| Comparative example 4 | 3.20％ |
| Comparative example 5 | 3.71％ |
| Comparative example 6 | 4.96％ |
As is clear from Table 2, the content of selenium in the lactic acid bacteria beverages prepared in examples 1 to 4 of the present invention was 8.5 to 9.7. Mu.g/100 g, which exceeded 8. Mu.g/100 g. Comparative example 2 no added selenium-enriched lactobacillus helveticus was added, and the selenium content in the lactobacillus beverage was only 0.5 μg/100g. As can be seen from Table 4, the lactic acid bacteria beverages prepared in examples 1 to 4 of the present invention have good fineness, no granular or powdery feel, moderate sour and sweet taste, and smooth mouthfeel. Comparative example 1 was free of added walnut peptide, had poor fineness, slightly grainy or powdery feel, and had a thin mouthfeel; comparative example 2 was free of added selenium-enriched lactobacillus helveticus, and was poor in fineness and taste except for low selenium content; comparative example 3 the first and second fermentation broths were added simultaneously to the fermentation base for fermentation, and the produced lactic acid bacteria beverage was poor in fineness, grainy or powdery, and thinner in taste; the comparative example 4 and the comparative example 5 both adopt constant temperature fermentation, and the fineness and the taste of the lactobacillus beverage prepared by the constant temperature fermentation are not the same as those of the lactobacillus beverage prepared by the embodiment 1; the comparative example 6 has a large amount of the walnut peptide added, which also results in a decrease in the fineness and taste of the lactic acid beverage.
As can be seen from Table 5, the viable count of the lactic acid bacteria of examples 1 to 4 of the present invention is not less than 10 in the shelf life 9 (CTU/g), demonstrating the relatively high content of lactic acid bacteria in the lactic acid beverage produced using the formulation and process of the present invention.
As can be seen from Table 6, the precipitation rate of the lactic acid bacteria beverages prepared in examples 1-4 of the present invention is less than 1%, and the precipitation rate of comparative example 1 is 1.89% without adding walnut peptide; comparative example 2 without addition of lactobacillus helveticus enriched with selenium, precipitation rate was 2.23%; comparative example 3 the first and second fermentation broths were added simultaneously to the fermentation base for fermentation to produce a lactic acid bacteria beverage having a precipitation rate of 4.3%; the comparative example 4 and the comparative example 5 both adopt constant temperature fermentation, and the precipitation rate of the prepared lactobacillus beverage is 3.2 percent and 3.71 percent no matter the fermentation is high temperature fermentation or low temperature fermentation; in comparative example 6, the amount of the walnut peptide added is large, and the precipitation rate of the prepared lactobacillus beverage is 4.96%.
Experimental example 1
Animal experiment for resisting exercise-induced fatigue
The exercise-induced fatigue resistance of the lactic acid bacteria beverages of example 1 and comparative example 1 was evaluated in this experiment. 48 SD rats, SPF grade, male, were purchased from the university of three gorges laboratory animal center at 5 weeks of age. The animals were fed adaptively for 7 days before the test, 16 animals per cage. Experimental grouping: blank control group (gavage saline), experimental group 1 (gavage lactic acid bacteria beverage of example 1), experimental group 2 (gavage lactic acid bacteria beverage of comparative example 1).
All rats were run on a treadmill daily for four weeks of progressive load exercises, running at 20m/min for 10min for the first week, 25m/min for 20min for the second week, 30m/min for 20min for the third week, and 35m/min for the fourth week for 30min. During running, all rats were gastric lavaged and the four-week trained rats were forced to run at 40m/min until exhausted, and their running time was recorded to determine their running endurance. Rats were determined to be exhausted when they remained on the panel for more than 10 seconds, and the results are shown in table 7,
TABLE 7 running time of rats
| Blank control group | Experiment group 1 | Experiment group 2 | |
| Running time of Qi exhaustion, min | 43.1±0.1 | 55.0±0.7 | 45.2±0.6 |
Compared with the blank control group, the running time of the two experimental groups is increased by 27.9 percent and 4.8 percent respectively, which shows that the lactic acid bacteria beverage added with the walnut peptide can prolong the running time of the rats, strengthen exercise endurance and have the anti-fatigue effect.
And immediately after the fourth week endurance test is finished, the rats are sacrificed, and corresponding tissues are taken to detect corresponding biochemical indexes.
(1) After anesthesia, the heart was bled, left at room temperature for 2h, and the serum was isolated by centrifugation for 20 min. The skeletal muscles of the liver and leg were taken and washed with physiological saline. All samples were stored in a-80 ℃ refrigerator;
(2) Serum: detecting the nitrogen content of lactic acid and urea;
(3) Muscle: taking skeletal muscle of the leg, homogenizing, adding 5mL of 100mmol/L potassium phosphate buffer into 0.1g, centrifuging the homogenate at 4 ℃ for 15min, taking supernatant, and detecting the content of lactic acid and glycogen;
(4) Liver: 0.1g of frozen liver homogenate was taken and soaked in phosphate buffer (pH 6.8). The homogenate was then centrifuged at 4℃for 10min, and the supernatant was taken to determine glycogen content.
The results of the above detection are shown in Table 8
TABLE 8 results of fatigue-related biochemical marker detection
Reserves of hepatic glycogen and myoglycogen are decisive factors for the generation of fatigue, and generally the more energy reserved in the body, the later the time the body has to develop fatigue. A large amount of ATP is consumed in the long-time movement process of the organism, so that the aerobic oxidation process can not provide enough energy in time, the organism can perform anaerobic glycolysis, so that lactic acid accumulation can reduce the pH of body fluid, and the lactic acid content is an important biochemical index for accurately reflecting the fatigue degree of the organism. Urea is an end product of protein metabolism, and the content range of urea can be used as an important index for evaluating exercise quantity, and is an evaluation index of protein metabolism in human body. The nitrogen balance in extremely severe exercise bodies can be broken, and the nitrogen content is increased, so that the urea nitrogen content in the body and the exercise tolerance of the body are obviously inversely related.
As can be seen from table 8, the lactic acid content in serum and skeletal muscle of the rat subjected to the lactic acid bacteria beverage intragastric administration of example 1 and the urea nitrogen content in serum were significantly lower than those of the blank group. The consumption of the glycogen in the blank group leads to the participation of hepatic glycogen in decomposition energy supply, while the consumption of the hepatic glycogen in the experimental group 1 is less, and the consumption of the glycogen is mainly used, so that the supply of the glycogen in the experimental group 1 is sufficient, and the hepatic glycogen is used as a reserve energy supply material, thereby effectively improving the exercise endurance of rats. Comparing the indexes of the experimental group 1 and the experimental group 2, the data of the experimental group 1 are found to be far better than the data of the experimental group 2, and the data of the experimental group 2 are not greatly different from the blank group, which indicates that the lactobacillus beverage of the experimental group 1 has stronger anti-fatigue activity.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
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Claims (20)
1. The selenium-enriched lactobacillus beverage is characterized by comprising the following raw materials in parts by weight: 25-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
2. The selenium-enriched lactic acid bacteria beverage according to claim 1, wherein the selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 part of first fermentation inoculant and 0.08-0.16 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
3. The selenium-enriched lactic acid bacteria beverage according to claim 1 or 2, characterized in that the selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-40 parts of cow milk, 0.1-1 part of walnut peptide, 0.08-0.1 part of first fermentation inoculant and 0.08-0.15 part of second fermentation inoculant, wherein the second fermentation inoculant is lactobacillus helveticus rich in selenium.
4. A selenium-enriched lactic acid bacteria beverage according to any of claims 1-3, characterized in that the selenium-enriched lactobacillus helveticus is prepared by inoculating lactobacillus helveticus in a selenium-containing medium for fermentation culture; preferably, the lactobacillus helveticus is lactobacillus helveticus Zhegu LBH-VI (Lactobacillus helveticus Zhegu LBH-VI), wherein the lactobacillus helveticus Zhegu LBH-VI is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 7 days in 2023, and the preservation number is CCTCCNO: m2023095.
5. The selenium-enriched lactobacillus beverage according to claim 4, wherein the organic selenium content in the lactobacillus helveticus is 100-125 μg/g.
6. The selenium-enriched lactic acid bacteria beverage of any of claims 1-5, wherein the selenium content of the selenium-enriched lactic acid bacteria beverage is ∈ 8.0 μg/100g by weight of the selenium-enriched lactic acid bacteria beverage; preferably, the viable count of the selenium-enriched lactobacillus beverage in the shelf life is more than or equal to 10 9 CFU/ml。
7. The selenium-enriched lactic acid bacteria beverage of any of claims 1-6, wherein the cow's milk is selected from one or more of whole cow's milk, low-fat cow's milk, or skim cow's milk.
8. The selenium-enriched lactic acid bacteria beverage of any of claims 1-7, wherein the molecular weight of the walnut peptide is 800-1000Da; preferably, the walnut peptide is prepared by enzymolysis of walnut through protease.
9. Selenium-enriched lactic acid bacteria beverage according to any of claims 1-8, characterized in that the first fermentation starter is selected from one or more of streptococcus salivarius thermophilus, lactobacillus delbrueckii subspecies lactis, bifidobacterium lactis, lactococcus lactis subspecies lactis and lactobacillus paracasei, preferably streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies lactis, more preferably the mass ratio of streptococcus salivarius thermophilus subspecies and lactobacillus delbrueckii subspecies lactis is 2-3:1-1.5 on a dry matter basis.
10. The selenium-enriched lactic acid bacteria beverage of claim 9, wherein the streptococcus salivarius thermophilus subspecies are streptococcus salivarius thermophilus 932 (Straptococcus salivarias subsp. Thermophilus 932) which was preserved in China Center for Type Culture Collection (CCTCC) at 2023, 6 and 5 days with a preservation number of CCTCC NO: m2023902;
and/or, the Lactobacillus delbrueckii subspecies are Lactobacillus delbrueckii subspecies Dangxiong LB V III (Lactobacillus delbrueckii isub. Lactis Dangxiong LB V III), and are preserved in China Center for Type Culture Collection (CCTCC) at 3 months of 2023, with the preservation number of CCTCC NO: M2023396;
and/or, the lactococcus lactis subspecies are lactococcus lactis subspecies 954 (Lactococcus lactis subsp. Lactis 954) which are preserved in China Center for Type Culture Collection (CCTCC) in 6 and 5 days of 2023, and the preservation number is CCTCC NO: m2023904;
and/or the lactobacillus paracasei is lactobacillus paracasei AL1 Plateau LPA-1 (lactobacillus paracaseiAL Plateau LPA-1).
11. The selenium-enriched lactic acid bacteria beverage of any of claims 1-10, wherein the raw material further comprises 5.0-8.5 parts of a sweetener;
preferably, the sweetener is selected from one or more than two of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycoside, preferably sucrose.
12. The selenium-enriched lactic acid bacteria beverage of any of claims 1-11, wherein the raw material further comprises 0.3-0.7 parts of a stabilizer;
preferably, the stabilizer is selected from one or more of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate and sodium citrate;
more preferably, the mass ratio of the sodium carboxymethyl cellulose, the pectin, the sodium tripolyphosphate and the sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.7.
13. The selenium-enriched lactic acid bacteria beverage of any of claims 1-12, wherein the raw material further comprises 0.11-0.6 parts of a sour agent;
preferably, the acidulant is selected from citric acid, lactic acid and sodium citrate, preferably the mass ratio of citric acid, lactic acid and sodium citrate is 5-25:5-15:1-20.
14. The selenium-enriched lactic acid bacteria beverage of any of claims 1-13, wherein the selenium-enriched lactic acid bacteria beverage is prepared by subjecting a fermentation base comprising cow's milk and walnut peptide to temperature swing fermentation.
15. The method for preparing a selenium-enriched lactic acid bacteria beverage according to any of claims 1 to 14, characterized in that the method comprises the following steps:
(1) Mixing raw materials comprising cow milk and walnut peptide to prepare a fermentation base material;
(2) Sequentially inoculating a first fermentation microbial inoculum and a second fermentation microbial inoculum into the fermentation base material prepared in the step (1) to perform variable-temperature fermentation to prepare fermented milk;
(3) And (3) blending the fermented milk prepared in the step (2) to obtain the selenium-enriched lactobacillus beverage.
16. The method according to claim 15, wherein the preparing in the step (3) comprises adding a stabilizer and/or an acidulant to the selenium-enriched lactobacillus beverage prepared in the step (2) to perform mixing and homogenizing treatment.
17. The method of preparation according to claim 15 or 16, wherein the fermentation base further comprises a sweetener, preferably one or more of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycosides, preferably sucrose.
18. The method according to any one of claims 15 to 17, wherein the sequential addition of the first and second fermenting agents in step (2) for the temperature swing fermentation comprises inoculating the first fermenting agent for fermentation at 40 to 45 ℃ and then inoculating the second fermenting agent for fermentation at 35 to 39 ℃;
preferably, the first fermentation inoculant is inoculated for fermentation at 40-45 ℃ for 10-12 hours until the final acidity is 90-100 DEG T, and then the second fermentation inoculant is inoculated for fermentation at 35-39 ℃ for 8-10 hours until the final acidity is 160-180 DEG T.
19. The method according to any one of claims 15 to 18, further comprising a step of homogenizing the fermentation base material prior to inoculation, preferably at a homogenization temperature of 50-65 ℃, more preferably at a homogenization pressure of 10-50MPa;
preferably, the homogenizing treatment further comprises a step of sterilizing the fermentation base material, preferably at a sterilization temperature of 90-100 ℃, more preferably for a sterilization time of 200-400s.
20. The method according to any one of claims 15 to 19, further comprising the step of stirring to break the emulsion both after the first fermentation and after the second fermentation, preferably at a stirring speed of 20 to 40r/min, more preferably for a stirring time of 10 to 20min.
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| CN202311119836.9A CN117179054A (en) | 2023-08-31 | 2023-08-31 | Selenium-enriched lactobacillus beverage and preparation method thereof |
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| CN202311119836.9A CN117179054A (en) | 2023-08-31 | 2023-08-31 | Selenium-enriched lactobacillus beverage and preparation method thereof |
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