Preparation method and application of leavening agent and fermented milk thereof

Abstract

The invention provides a leavening agent and a preparation method and application of fermented milk of the leavening agent. The invention provides a leavening agent. The leavening agent comprises bifidobacterium lactis and a neutral protease isolated from bacillus polymyxa. The invention also provides a method for simultaneously promoting protein digestion and absorption and calcium absorption and utilization of the fermented milk or preparing the fermented milk. The invention also provides related fermented milk, food and application.

Description

Technical Field

[0001] This invention belongs to the field of fermented dairy products, and more specifically, this invention relates to a starter culture, a method for preparing fermented milk, and its application. Background Technology

[0002] Fermented dairy products are widely popular among consumers due to their health and delicious properties. Currently, probiotics are commonly used in fermented dairy products to perform specific functions, such as improving immunity, regulating gut microbiota, and improving constipation. However, the application of probiotics in improving the nutrient absorption of fermented milk is relatively lacking. Compared with milk before fermentation, fermented milk has certain advantages in nutrient digestion and absorption, but problems such as low calcium bioavailability and slow protein absorption rate still exist. At the same time, my country has a large population with lactose intolerance, and fermented milk still contains a lot of lactose, which can easily cause symptoms such as diarrhea and abdominal pain, thus hindering nutrient absorption.

[0003] Therefore, there is an urgent need to explore a method that can simultaneously improve the bioavailability of protein and calcium in fermented milk, thereby enhancing the nutritional value of fermented milk and better meeting consumers' demand for healthy and delicious yogurt. Summary of the Invention

[0004] The purpose of this invention is to provide a fermentation agent and its preparation method and application in fermented milk.

[0005] In a first aspect, the present invention provides a fermentation agent comprising Bifidobacterium lactis and a neutral protease isolated from Bacillus polymyxa.

[0006] In one or more embodiments, the Bifidobacterium lactis includes Bifidobacterium lactis BB-12, Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8; wherein, Bifidobacterium lactis Ca360 is Bifidobacterium lactis Ca360 with accession number CGMCC No. 32403, Bifidobacterium lactis MN-Gup is Bifidobacterium lactis MN-Gup with accession number CGMCC No. 15578, and Bifidobacterium lactis M8 is Bifidobacterium lactis M8 with accession number CGMCC No. 18610.

[0007] In one or more embodiments, the amount of Bifidobacterium lactis added to the fermenting agent is 1 × 10⁻⁶. 6 cfu / g ~1×10 10 cfu / g.

[0008] In one or more embodiments, the neutral protease isolated from Bacillus polymyxa is ADMIL.

[0009] In one or more embodiments, the fermenting agent further includes fermenting bacteria or cultures thereof other than Bifidobacterium lactis.

[0010] In one or more embodiments, the fermenting bacteria is any one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. milk fat, Lactococcus lactis subsp. diacetyl, and Lactobacillus rhamnosus.

[0011] In one or more embodiments, the fermenting bacteria are selected from any group of the following: (A) Streptococcus thermophilus and Lactobacillus bulgaricus; (B) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis subsp. milk fat; (C) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum; (D) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus helveticus; (E) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis diacetyl subsp. (F) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus rhamnosus.

[0012] In one or more embodiments, the total inoculum amount of fermenting bacteria other than Bifidobacterium lactis is 50U~180U / ton.

[0013] A second aspect of the present invention provides a method for simultaneously improving the digestibility and absorption of protein and the absorption and utilization of calcium in fermented milk, or for preparing fermented milk, the method comprising: fermenting a milk raw material using a fermenting agent as described in any embodiment of the present invention.

[0014] In one or more embodiments, the fermenting agent includes: The amount of Bifidobacterium lactis added is 1×10⁻⁶. 6 cfu / g ~1×10 10 cfu / g; The amount of neutral protease isolated from Bacillus polymyxa added is 0.02~0.05g / 100g milk raw material; The total inoculum amount of fermentation bacteria other than Bifidobacterium lactis is 50U~180U / ton.

[0015] In one or more embodiments, the dairy raw materials include: fresh milk, reconstituted milk, and dairy products.

[0016] In one or more embodiments, the fresh milk includes raw cow's milk and raw sheep's milk.

[0017] In one or more embodiments, the dairy products include milk powder, condensed milk, cream, and milk powder.

[0018] In one or more embodiments, the milk raw material also includes lactase.

[0019] In one or more embodiments, the lactase is a transGOS type lactase.

[0020] In one or more embodiments, the transGOS lactase includes Nurica and Aspergillus oryzae β-galactosidase.

[0021] In one or more embodiments, the amount of lactase added is 0.1~0.6 g / 100g milk raw material.

[0022] In one or more embodiments, the dairy raw material also includes a sweetener.

[0023] In one or more embodiments, the sweetener includes sucrose, white sugar, fructose syrup, maltitol, mogroside, xylitol, sucralose, neotame, and aspartame.

[0024] In one or more embodiments, the amount of sweetener added is 5-8g of sweetener per 100g of milk raw material.

[0025] In one or more embodiments, the fermentation further includes one or more of the following conditions: (1) The fermentation temperature is 30~45℃; (2) The end point of fermentation is when the pH of the fermented milk is less than or equal to 4.5; (3) The fermentation time is 4 to 12 hours.

[0026] In one or more embodiments, the method further includes the steps of chemicaling, homogenizing, sterilizing, cooling, demulsifying, and / or filling.

[0027] A third aspect of the present invention provides a fermented milk prepared using the method described in any embodiment of the present invention.

[0028] In a fourth aspect, the present invention provides a food product comprising the fermented milk described in any embodiment of the present invention.

[0029] A fifth aspect of the invention provides applications selected from the following: (1) The application of the starter culture described in any embodiment of the present invention in the preparation of fermented milk; (2) The application of the fermented milk described in any embodiment of the present invention in the preparation of food; (3) The application of the combination of Bifidobacterium lactis as defined in any embodiment of the present invention and neutral protease isolated from Bacillus polymyxa as defined in any embodiment of the present invention in the preparation of starter culture; (4) The application of the fermenting agent described in any embodiment of the present invention in improving the digestion and absorption of protein in fermented milk and / or improving the absorption and utilization of calcium in fermented milk; (5) The application of the fermented milk described in any embodiment of the present invention in improving the digestion and absorption of protein in food and / or improving the absorption and utilization of calcium in food; (6) The application of the combination of Bifidobacterium lactis as defined in any embodiment of the present invention and neutral protease isolated from Bacillus polymyxa as defined in any embodiment of the present invention in improving the digestion and absorption of protein in food and / or improving the absorption and utilization of calcium in food.

[0030] In one or more embodiments, the promotion of protein digestion and absorption includes: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids.

[0031] In one or more embodiments, the promotion of calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent absorption rate of calcium, and / or improving exercise capacity.

[0032] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0033] Figure 1 The technical roadmap of the present invention for simultaneously improving the bioavailability of protein and calcium in fermented milk.

[0034] Figure 2 Results of mouse motor function assessment. Fermented milk prepared using the methods described in Example 2 and Comparative Example 1 was administered to mice by gavage for 30 days, while the blank control group was administered 0.9% physiological saline by gavage. Detailed Implementation

[0035] Through in-depth research, the inventors discovered that adding Bifidobacterium lactis and protease to the starter culture significantly promotes protein digestion and absorption in vivo, enhances calcium absorption, and improves the apparent bioavailability of calcium. Therefore, this invention provides a method for simultaneously improving the bioavailability of protein and calcium in fermented milk, the method comprising adding Bifidobacterium lactis and a neutral protease isolated from Bacillus polymyxa to the starter culture.

[0036] Fermentation agent

[0037] This invention provides a starter culture comprising Bifidobacterium lactis and a neutral protease isolated from Bacillus polymyxa. The term "starter culture" refers to a reagent that can be used for substrate fermentation. In this document, the starter culture generally refers to a reagent used for dairy product fermentation. The starter culture typically comprises one or more microorganisms that can be used for fermentation, or a culture of one or more microorganisms that can be used for fermentation.

[0038] The term "Bifidobacterium lactis" refers to a species of bacteria within the genus Bifidobacterium. This invention has found that using Bifidobacterium lactis in a starter culture significantly enhances the digestion and absorption of protein and improves calcium bioavailability compared to using other Bifidobacteria (e.g., Bifidobacterium longum). In some embodiments, the Bifidobacterium lactis includes Bifidobacterium lactis BB-12, Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8. In some embodiments, the Bifidobacterium lactis is selected from any one or more of Bifidobacterium lactis BB-12, Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8. In some specific embodiments, the Bifidobacterium lactis is selected from any one or more of Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8. It should be understood that Bifidobacterium lactis BB-12, Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8 are all commercially available.

[0039] In this invention, the *Bifidobacterium lactis* BB-12 refers to *Bifidobacterium animalis* subsp. *bifidobacterium lactis* (Bifidobacterium animalis). B. animalis subsp. lactis The anaerobic probiotic strain of , whose whole genome sequence can be found in NCBI GenBank accession number CP001853.2.

[0040] In this invention, the *Bifidobacterium lactis* Ca360 is deposited at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) and classified as *Bifidobacterium animalis* subsp. *lactamella* (…). Bifidobacterium animalis subsp. lactis (The accession number is CGMCC No. 32403).

[0041] In this invention, the *Bifidobacterium lactis* MN-Gup is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and belongs to the subspecies *Bifidobacterium animalis* (Lactobacillus). Bifidobacterium animalis subsp.lactis The accession number is CGMCC No. 15578.

[0042] In this invention, the *Bifidobacterium lactis* M8 is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and is classified and named *Bifidobacterium lactis*. Bifidobacterium lactis The accession number is CGMCCNo.18610.

[0043] It should be understood that the *Bifidobacterium lactis* can be added to the starter culture in any suitable form, such as, but not limited to, live *Bifidobacterium lactis*, fermentation broth, concentrate, dilution, precipitate, or lyophilized powder (bacterial powder), all of which are within the scope of protection of this invention. In some embodiments, when added to the starter culture, the amount of *Bifidobacterium lactis* added may be 1 × 10⁻⁶. 6 CFU / g or higher, for example, 1×10 7 cfu / g or higher, 1×10 8 CFU / g or higher, more specifically, 1×10⁻⁶ 6 cfu / g ~1×10 10 cfu / g, 1×10 6 cfu / g ~1×10 9 cfu / g, 1×10 6 cfu / g ~1×10 8 cfu / g or 1×10 7 cfu / g ~1×10 8 cfu / g.

[0044] The term "protease" refers to a class of enzymes that hydrolyze proteins into peptides or amino acids, including acidic proteases, neutral proteases, and basic proteases. Neutral proteases typically refer to proteases with an optimal pH of 6-8. Neutral proteases include neutral proteases isolated from Fusarium (e.g., Galaya Smooth) and neutral proteases isolated from Bacillus polymyxa (e.g., ADMIL). In some embodiments, the neutral protease isolated from Bacillus polymyxa is ADMIL. ADMIL is commercially available. In some embodiments, ADMIL loses or substantially loses its enzymatic activity at a pH less than or equal to 4.6. In some embodiments, when added to the starter culture, the amount of neutral protease isolated from Bacillus polymyxa added is 1-2.5 g / 100 g of raw material. In some embodiments, when using the starter culture to prepare fermented milk, the amount of neutral protease isolated from Bacillus polymyxa added is 0.02-0.05 g / 100 g of milk raw material.

[0045] The starter culture may also include fermenting bacteria or cultures thereof other than Bifidobacterium lactis. In some embodiments, the fermenting bacteria may be any one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. milk fat, Lactococcus lactis subsp. diacetyl, and Lactobacillus rhamnosus. In some embodiments, the fermenting bacteria include Streptococcus thermophilus and Lactobacillus bulgaricus, and optionally any one or more of Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. milk fat, Lactococcus lactis subsp. diacetyl, and Lactobacillus rhamnosus. In some specific embodiments, the fermenting bacteria are selected from any group of the following: (A) Streptococcus thermophilus and Lactobacillus bulgaricus; (B) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactococcus lactis subsp. milk fat; (C) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus plantarum; (D) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus helveticus; (E) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactococcus lactis subsp. diacetyl; (F) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus rhamnosus. In some implementations, when added to the starter culture, the total inoculum of fermenting bacteria other than Bifidobacterium lactis can be 50 U to 180 U / ton, for example, 50 U / ton, 60 U / ton, 70 U / ton, 80 U / ton, 90 U / ton, 100 U / ton, 110 U / ton, 120 U / ton, 130 U / ton, 140 U / ton, 150 U / ton, 160 U / ton, 170 U / ton, 180 U / ton, and any combination thereof, more specifically, for example, 150 ± 30 U / ton, 150 ± 25 U / ton, 150 ± 20 U / ton, 150 ± 15 U / ton, 150 ± 10 U / ton, 150 ± 5 U / ton, 150 ± 3 U / ton, 150 ± 2 U / ton, or 150 ± 1 U / ton.

[0046] Methods for preparing fermented milk

[0047] The present invention also provides a method for preparing fermented milk, the method comprising the step of fermenting milk raw materials using the fermenting agent described in any embodiment of the present invention.

[0048] The term "milk raw material" refers to the raw material used in the preparation of fermented milk, typically as a substrate for a starter culture. The milk raw material can be fresh milk (e.g., raw cow's milk, raw goat's milk, etc.), reconstituted milk, or dairy products (e.g., milk powder, condensed milk, cream, milk powder, etc.). In some embodiments, the milk raw material is raw cow's milk.

[0049] In some embodiments, lactase may also be added to the milk raw material. The term "lactase" refers to an enzyme capable of breaking down lactose into glucose and galactose. In some embodiments, the lactase may be a transGOS lactase. The term "transGOS lactase" is a lactase with transglycosidic activity, capable of hydrolyzing lactose (breaking it down into glucose and galactose) while simultaneously recombinating some lactose molecules to generate galacto-oligosaccharides. In some embodiments, the transGOS lactase described in this invention includes *Aspergillus oryzae* β-galactosidase. It should be understood that both *Aspergillus oryzae* β-galactosidase are commercially available. *Aspergillus oryzae* β-galactosidase can be referenced in CN120005857B (application number 202311474445.9), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the amount of lactase added to the milk raw material may be 0.1~0.6 g / 100g milk raw material.

[0050] In some embodiments, sweeteners such as sucrose, white sugar, high-fructose corn syrup, maltitol, mogroside, xylitol, sucralose, neotame, and aspartame may be added to the milk raw material. In some specific embodiments, the sweetener is sucrose. The amount of sweetener added can be adjusted as needed and in accordance with the requirements of laws and regulations in the field; for example, 5-8g of sweetener (e.g., sucrose) may be added per 100g of milk raw material. The milk raw material described in this invention may also contain food additives. The term "food additive" refers to artificially synthesized or natural substances added to food to improve its quality, color, aroma, and flavor, as well as for preservation, freshness, and processing needs. Food additives include, but are not limited to: food flavorings, food fragrances, base substances in chewing gum, and processing aids for the food industry.

[0051] In this invention, during fermentation, the starter culture comprises *Bifidobacterium lactis*, a neutral protease isolated from *Bacillus polymyxa*, and fermenting bacteria, wherein the fermenting bacteria include *Streptococcus thermophilus* and *Lactobacillus bulgaricus*, and optionally one or more of *Lactobacillus plantarum*, *Lactobacillus helveticus*, *Lactococcus lactis* subsp. *milk fat*, *Lactococcus lactis* subsp. *diacetyl*, and *Lactobacillus rhamnosus*. In some embodiments, the amount of *Bifidobacterium lactis* added during fermentation may be 1 × 10⁻⁶. 6 CFU / g or higher, for example, 1×10 7 cfu / g or higher, 1×10 8 CFU / g or higher, more specifically, 1×10⁻⁶ 6 cfu / g ~1×10 10 cfu / g, 1×10 6 cfu / g ~1×109 cfu / g, 1×10 6 cfu / g ~1×10 8 cfu / g or 1×10 7 cfu / g ~1×10 8 cfu / g. In some implementations, when used for fermentation, the amount of neutral protease isolated from Bacillus polymyxa added is 0.02~0.05g / 100g of milk raw material. In some embodiments, when used for fermentation, the total inoculum amount of the fermenting bacteria can be 50U to 180U / ton, for example, 50U / ton, 60U / ton, 70U / ton, 80U / ton, 90U / ton, 100U / ton, 110U / ton, 120U / ton, 130U / ton, 140U / ton, 150U / ton, 160U / ton, 170U / ton, 180U / ton, and any combination thereof, more specifically, for example, 150±30U / ton, 150±25U / ton, 150±20U / ton, 150±15U / ton, 150±10U / ton, 150±5U / ton, 150±3U / ton, 150±2U / ton, or 150±1U / ton.

[0052] In some embodiments, the fermentation temperature is 30–45°C, for example 35–45°C, 37–44°C, 36–43°C, 38–42°C, or 39–41°C. In some embodiments, fermentation is stopped when the pH of the fermented milk is less than or equal to 4.5. Therefore, the fermentation time is the time from the start of fermentation to the end point when the pH of the fermented milk is less than or equal to 4.5, for example, it can be 4–12 hours.

[0053] In some specific embodiments, the fermentation step includes: using Bifidobacterium lactis, a neutral protease isolated from Bacillus polymyxa, and fermentation bacteria (preferably including Streptococcus thermophilus and Lactobacillus bulgaricus, and optionally any one or more of Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. milk fat, Lactococcus lactis subsp. diacetyl and Lactobacillus rhamnosus) as starter cultures, inoculating them into milk raw material supplemented with lactase (preferably transGOS lactase) for fermentation, and stopping fermentation when the pH is less than or equal to 4.5.

[0054] In some embodiments, the preparation method may further include steps of chemicaling, homogenization, sterilization, cooling (e.g., quenching), demulsification and / or filling, which may be performed using methods known in the art.

[0055] Fermented milk

[0056] The present invention also provides fermented milk prepared using the preparation method described in any embodiment of the present invention. The term "fermented milk" refers to a dairy product obtained by inoculating a milk raw material with a starter culture and fermenting it, resulting in increased acidity and viscosity.

[0057] The fermented milk described in this invention can be used directly as a food (including health food), or it can be added to one or more food-grade acceptable materials, or it can be used to prepare an intermediate composition (e.g., a food premix) suitable for subsequent addition to food-grade acceptable materials. This invention also provides a food comprising fermented milk prepared using the preparation method described in any embodiment of this invention.

[0058] The term "food-acceptable material" refers to food ingredients or their processed products (e.g., raw milk, whey protein powder, whey powder, etc.), nutritional additives used in food (e.g., sugars such as lactose, fructooligosaccharides, galactooligosaccharides, etc.; dietary fiber; prebiotics; proteins such as α-lactalbumin, β-lactalbumin, lactoferrin, milk fat globule membrane protein, etc.; lipids; minerals such as calcium, iron, phosphorus, zinc, etc.; vitamins; nervonic acid; nucleotides; choline; taurine), excipients or adjuvants (e.g., calcium carbonate, calcium phosphate, various sugars, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, etc.). The food of this invention can be in any form suitable for oral administration, such as powder, tablets, capsules, granules, solutions, suspensions, etc. In some embodiments, the food is a general food or a special food. In some embodiments, the special food is infant food, such as infant formula. "Infant" as used in this invention includes infants and toddlers, generally referring to people aged 0-36 months. The term "infant" refers to people aged 0-12 months. The term "infant" refers to a person aged 12 to 36 months.

[0059] application

[0060] This invention provides applications selected from the following: (1) The use of the starter culture described in any embodiment of the present invention in the preparation of fermented milk (including the preparation of fermented milk that promotes protein digestion and absorption and / or promotes calcium absorption and utilization); (2) The application of the fermented milk described in any embodiment of the present invention in the preparation of food (including the preparation of health food that promotes protein digestion and absorption and / or promotes calcium absorption and utilization); (3) The application of the combination of Bifidobacterium lactis described in any embodiment of the present invention and the neutral protease isolated from Bacillus polymyxa described in any embodiment of the present invention in the preparation of a starter culture; (4) The application of the fermenting agent described in any embodiment of the present invention in improving the digestion and absorption of protein in fermented milk and / or improving the absorption and utilization of calcium in fermented milk; (5) The application of the fermented milk described in any embodiment of the present invention in improving the digestion and absorption of protein in food and / or improving the absorption and utilization of calcium in food; (6) The application of the combination of Bifidobacterium lactis as described in any embodiment of the present invention and the neutral protease isolated from Bacillus polymyxa as described in any embodiment of the present invention in improving the digestion and absorption of protein in food and / or improving the absorption and utilization of calcium in food.

[0061] In this invention, the promotion of protein digestion and absorption includes, but is not limited to: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids. In some embodiments, the proteins (e.g., total amino acids, essential amino acids, branched-chain amino acids) include proteins (e.g., total amino acids, essential amino acids, branched-chain amino acids) found in plasma, ileum, etc.

[0062] In this invention, promoting calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent digestibility and absorption rate of calcium, and / or improving athletic performance (e.g., but not limited to increasing the time to exhaustion on the treadmill, increasing grip strength, etc.).

[0063] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0064] Experimental materials

[0065] Bifidobacterium lactis BB-12 was purchased from Chr. Hansen.

[0066] Bifidobacterium lactis Ca360, deposited at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), is classified as Bifidobacterium animalis subsp. lactis. Bifidobacterium animalis subsp. lactis The artifact, with accession number CGMCC No. 32403, was provided by Mengniu High-Tech Dairy Products (Beijing) Co., Ltd.

[0067] Bifidobacterium lactis MN-Gup, deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), belongs to the subspecies of Bifidobacterium animalis (Bifidobacterium lactis). Bifidobacterium animalis subsp.lactis The artifact, with accession number CGMCCNo. 15578, was provided by Mengniu High-Tech Dairy Products (Beijing) Co., Ltd.

[0068] Bifidobacterium lactis M8, deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), is classified and named Bifidobacterium lactis. Bifidobacterium lactis The document, with accession number CGMCC No.18610, was provided by Mengniu High-Tech Dairy Products (Beijing) Co., Ltd.

[0069] Preparation Example

[0070] This invention provides a method for preparing fermented milk, as illustrated in the schematic diagram below. Figure 1 As shown, the formulations of Examples 1-15 and Comparative Examples 1-8 are shown in Table 1.

[0071] 1. Preparation method 1

[0072] The fermented milk in Examples 1, 4, Comparative Example 1, and 5 was prepared using Preparation Method 1, with the following steps: (1) Preparation of fermentation base: After mixing raw milk and ingredients, dissolve the mixture at 55-65℃ while stirring for 10 minutes. After homogenization and standardization, sterilize at 85-95℃ for 5-10 minutes.

[0073] (2) Inoculation and fermentation: When the substrate cools to about 40°C, inoculate the starter culture and Bifidobacterium lactis culture, stir and add neutral protease, mix evenly and ferment at 42°C for 4-6 hours. When the pH of the yogurt drops below 4.5, break the emulsion at 20 rpm for 2 minutes. The added neutral protease will be inactivated when the pH drops below 4.6.

[0074] (3) Cooling and filling: Cool to 10-25℃ and then fill to prepare fermented milk.

[0075] 2. Preparation method 2

[0076] The remaining examples and comparative examples were prepared using preparation method 2, and the steps are as follows: (1) Preparation of fermentation base: After mixing raw milk and ingredients, dissolve the mixture at 55-65℃ while stirring for 10 minutes. Add lactase and react while stirring for 60-90 minutes. After homogenization and standardization, sterilize at 85-95℃ for 5-10 minutes. The lactase will be completely inactivated after heat sterilization.

[0077] (2) Inoculation and fermentation: When the substrate cools to about 40°C, inoculate the starter culture and Bifidobacterium lactis culture, stir and add neutral protease, mix evenly and ferment at 42°C for 4-6 hours. When the pH of the yogurt drops below 4.5, break the emulsion at 20 rpm for 2 minutes. The added neutral protease will be inactivated when the pH drops below 4.6.

[0078] (3) Cooling and filling: Cool to 10-25℃ and then fill to prepare fermented milk.

[0079] Table 1. Ingredient Table of Examples and Comparative Examples

[0080] Experimental methods: 1. Acidity value and pH detection The pH of the fermented milk samples was determined using a pH meter. Acidity values ​​were determined according to the method described in AKTAR T. Physicochemical and sensory characterisation of different yogurt production methods [J]. International Dairy Journal, 2022, 125: 105245. 10.00 g of accurately weighed fermented milk sample was mixed with 20 mL of distilled water, titrated with 0.1 mol / L sodium hydroxide standard solution, and the acidity value was calculated. The measurement results were obtained from two replicate measurements of three replicate samples.

[0081] 2. Bifidobacterium viable count determination

[0082] The selective plate pour anaerobic culture counting method specified in the National Food Safety Standard for Microbiological Examination of Food (GB 4789.35-2023) and the National Food Safety Standard for Microbiological Examination of Food (GB 4789.34-2016) was adopted. 25 g (mL) of fermented milk sample was weighed and homogenized with 225 mL of sterile diluent preheated to 36±1℃ to prepare a 1:10 homogenate. After serial dilution by 10 times, 1 mL of the homogenate with an appropriate dilution was injected into a sterile Petri dish. Modified MRS agar containing mupirocin lithium salt and L-cysteine ​​hydrochloride was poured in and mixed well. After the agar solidified, it was anaerobically incubated at 36±1℃ for 48-72 h. Plates with colony counts in the range of 30-300 CFU were selected for counting. The viable number of Bifidobacteria was calculated using the formula, with the unit being CFU / g (mL).

[0083] 3. In vitro digestion experiment of fermented milk

[0084] Before in vitro digestion, the protein concentration of the sample was adjusted to be consistent (3.00 g / 100 mL) with deionized water. First, 5 mL of sample was mixed with 3 mL of simulated gastric juice, then 6 M hydrochloric acid was added to adjust the pH to 3.0, and water was added to bring the volume to 8.9975 mL. The sample was incubated in a shaking water bath at 37°C for 5 min, then 2.5 μL of 0.3 M CaCl2 and 1 mL of pepsin stock solution were added to bring the final pepsin concentration in the system to 2000 U / mL. The sample and enzyme mixture was incubated in a water bath at 37°C for 120 min for gastric digestion. After 0, 5, 15, 30, 60, 90, and 120 min, the centrifuge tubes were removed at the corresponding time points, and a predetermined amount of 1 M NaOH was added to the digested sample to raise the pH to 7.0. The sample was then placed in a boiling water bath for 5 min, cooled on ice, heated with water to a total volume of 20 mL, aliquoted into microcentrifuge tubes, and stored at -80°C until analysis. After 120 min of gastric digestion, the remaining sample was adjusted to pH 7.0 using a predetermined amount of 1M NaOH. Then, 6 mL of simulated intestinal fluid and 1 mL of bile salt stock solution were added, and the volume was increased to 18.980 mL with a predetermined amount of water. The sample was annealed at 37°C for 5 min, then mixed with 20 μL of 0.3 M CaCl2, and 1 mL of stock trypsin solution was added to bring the final trypsin concentration to 100 U / mL. The mixture was then incubated at 37°C for 120 min. At 0, 5, 15, 30, 60, 90, and 120 min, the centrifuge tubes were removed, and the samples were immersed in a boiling water bath for 5 min and immediately cooled on ice to terminate the enzymatic digestion reaction. The digested samples were aliquoted into microcentrifuge tubes and stored at -80°C until further analysis.

[0085] 4. Animal experiments

[0086] Following the methods described in the literature SONG R, LI G, ZHAO L, et al. Decreased Amino Acid Transporter LAT2 Is the Main Determinant of Impaired Protein Utilization During Aging[J]. Engineering, 2024, 42: 88-98, we used C57BL / 6J mice to study the in vivo digestion and absorption of yogurt samples. This experimental protocol has been approved by Beijing Gushi Testing Technology Co., Ltd. for animal welfare and animal experiment ethics (GSCS-2025-025). Six-week-old male C57BL / 6J mice (Huafukang) were housed in an SPF environment with a 12-hour light / 12-hour dark circadian rhythm. After one week of acclimatization, all mice were fasted for 16 hours until day 8, but were allowed free access to water. At 8:00 AM on day 8, 3-5 drops of orbital venous blood were collected from the mice. After standing for 1 hour, the collected blood samples were centrifuged (3500 g, 15 min), and the plasma was collected and stored at -80°C. Blood collection caused no other harm, and all mice remained active after the procedure. Subsequently, the mice underwent a 7-day acclimatization and equilibration period, during which they were allowed free access to water and standard mouse food. Mice were fasted overnight (16 h) until day 16 and were gavaged with fermented milk samples prepared according to the methods of Examples 1-15 and Comparative Examples 1-8 (0.01389 mL / kg). Two hours after gavage, the mice were euthanized, and blood samples and ileal chyme were collected. The ileum was perfused twice with 0.1 mL of phosphate buffer containing a mixture of protease inhibitors, and the perfusion fluid was collected in test tubes. Finally, all samples were stored at -80°C until analysis.

[0087] 5. Determination of free amino acids

[0088] The free amino acid content in the samples was determined according to the research method of Qin et al. (QIN X, YU C, GENG X, et al. Heat treatment affects gastric digestion kinetics but not amino acid bioavailability of milk proteins: a pig study [J]. Food Chemistry, 2025, 491: 145198.). For in vitro digested samples, after freeze-drying and thorough grinding and mixing, 20 mg of the sample was mixed with 200 μL of pure water containing 1 mM PMSF by vortexing, and 500 μL of deionized water was added. After mixing, the mixture was homogenized at 8500 rpm for 30 s, repeated 6 times with an interval of 10 s each time. Then the sample was centrifuged at 4℃ and 12000 rpm for 15 min, and 400 μL of the supernatant was collected, freeze-dried, and used for later use. The lyophilized sample was reconstituted with 300 μL of acetonitrile aqueous solution (85:15, v / v), centrifuged at 4℃ and 13000 rpm for 20 min, and the supernatant was collected, diluted 50 times, and transferred to an LC vial for detection.

[0089] For animal samples, 20 μL of plasma or ileal perfusion fluid was added to 80 μL of cold methanol to precipitate non-amino acid proteins. The sample was then centrifuged at 4°C and 13,000 rpm for 10 min. 160 μL of the supernatant was collected and freeze-dried for later use. The freeze-dried sample was reconstituted with 160 μL of acetonitrile aqueous solution (85:15, v / v), centrifuged at 4°C and 12,000 rpm for 15 min, and 70 μL of the supernatant was transferred to an LC vial for analysis.

[0090] Accurately weigh the appropriate amount of amino acid standard into a 5 mL volumetric flask to prepare a standard stock solution. Mix the standard stock solution in a certain proportion to prepare a 200 ppm mixed standard solution, and then dilute it serially to obtain a series of standard solutions. Use these solutions together with the sample to generate a standard curve.

[0091] The instrument used for detection was a liquid chromatography-mass spectrometry (LC-MS) system. The LC column was a BEN Admine column (2.1 mm × 100 mm, 1.7 µL) equipped with an Administrator guard column (2.1 mm × 10 mm, 1.7 µL). Chromatographic separation was performed at 45 °C. Mobile phase A was an aqueous solution containing 20 mM ammonium acetate and 0.5% formic acid, and mobile phase B was an aqueous solution of acetonitrile containing 20 mM ammonium acetate and 0.5% formic acid (85:15, v / v). Mobile phase A + mobile phase B = 100%.

[0092] Operating conditions: Gradient elution was performed at a mobile phase rate of 0.3 mL / min. The gradient elution program was as follows: 0–10.0 min, mobile phase B was kept constant at 15%; 10.0–14.0 min, mobile phase B was kept constant at 100%; 14.0–14.1 min, mobile phase B decreased from 100% to 15%; and 14.1–17 min, mobile phase B was kept constant at 15%.

[0093] Data acquisition employed multi-reaction monitoring (MRM), including ion scanning parameters for 20 amino acids. All data acquisition and analysis were performed on a SCIEX OS instrument. Operating parameters were set as follows: source temperature 550℃; ion source gas 1 55 psi; ion source gas 2 55 psi; curtain gas 35 psi; declustering voltage 50 V; ion spray voltage 5500 V. During automated MS / MS acquisition, instrument scanning parameters were set to a mass / charge ratio range of 60–1000 Da and an accumulation time of 100 ms.

[0094] 6. Calcium content determination

[0095] First, the samples were microwave-digested, with thorough agitation. 10 mL of each sample was weighed into a clean inner digestion vessel, and 5 mL of nitric acid and 2 mL of ultrapure water were added sequentially to each vessel. The inner digestion vessel containing the sample mixture was heated on an open thermostatic electric heating plate at 150°C until no white smoke was emitted. The vessel was then removed and allowed to cool naturally to room temperature. The inner digestion vessel was then inserted into the outer digestion vessel, the outer cap was tightened, and the vessel was placed in the microwave digestion system. Digestion was performed in four stages. After digestion, the sample was a colorless and transparent liquid. It was transferred without loss to a 50 mL volumetric flask and diluted to the mark with ultrapure water. The total calcium content in the processed sample was measured using inductively coupled plasma mass spectrometry (ICP-MS).

[0096] 7. Measurement of calcium-related indicators

[0097] Four-week-old male C57BL / 6J mice (Huafukang) were housed in an SPF environment with a 12-hour light / 12-hour dark circadian rhythm. After one week of acclimatization feeding, gavage was initiated and continued for 30 days.

[0098] After the experiment, urine and feces of mice were collected for 24 hours using metabolic cages. After anesthetizing the mice, blood samples were collected by taking blood from the orbital cavity. The mice were then dissected, and bone tissues such as the femur were removed, and soft tissues were removed. The calcium concentration of blood and urine was measured by colorimetry (referring to the instructions of the calcium test kit (C004-1-1, Nanjing Jiancheng Bioengineering Institute)). After pretreatment of bones and feces, the calcium concentration in fecal samples was measured by inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS detection method and biological sample pretreatment method in Wang Xuesheng et al., "Determination of Calcium in Rat Poisoned with Nano-Lead Sulfide by ICP-MS", Industrial Hygiene and Occupational Diseases, 2014, 40(2) were used for detection. The apparent calcium absorption rate was calculated as follows: Apparent calcium absorption rate (%) = [(calcium ingested - calcium excreted) / calcium ingested] × 100%.

[0099] 8. Measurement of mouse motor indicators

[0100] Four-week-old male C57BL / 6J mice (Huafukang) were housed in an SPF environment with a 12-hour light / 12-hour dark circadian rhythm. After one week of acclimatization, gavage was initiated and continued for 30 days. Motor parameters of the mice were measured after 30 days.

[0101] First, the initial weight of the mice was recorded before the experiment began. The weight of the mice was measured weekly using an electronic balance and recorded continuously until the end of the experiment. After the experiment, the exercise endurance of the mice was assessed using a treadmill testing system. Before the test, the mice underwent acclimatization training for 3 days. The treadmill speed was set to a constant 5 m / min, the incline to 0°, and the current to 0.3 mA. Each mouse exercised on the treadmill for approximately 10 minutes.

[0102] To avoid initial maladaptation affecting the results, the treadmill parameters were set as follows: initial speed 5 m / min, increasing by 2 m / min every 3 minutes, stimulation current 0.3 mA, and constant incline 5°. A single mouse was placed on the treadmill after startup, and its movement was observed. The time it took to reach exhaustion was recorded as the mouse's treadmill exhaustion time. During testing, the treadmill incline and speed were gradually increased, and the longest continuous movement time of the mouse was recorded as an indicator of its treadmill ability. A grip strength tester was used to measure the mice's grip strength. The mice were suspended by gripping the grid or crossbar of the device, and their grip strength was tested. Each mouse underwent three repeated tests, and the maximum grip strength value was recorded. The measurement results reflect the mouse's muscle strength and neuromuscular function. After the experiment, the mice were euthanized, dissected, and the femur and other bones were removed, weighed, and recorded. Changes in bone weight will be used to assess the mouse's skeletal development.

[0103] 9. Data Processing Methods

[0104] Experimental data were analyzed using SPSS 31 (IBM, USA) through one-way ANOVA, with P < 0.05 considered statistically significant. Tukey's post-hoc test was used for comparisons between groups. Significant differences were expressed using different letters (same letter indicates no significant difference, different letters indicate significant difference, P < 0.05). Data were plotted using GraphPad Prism 10, and all data are expressed as mean ± standard deviation.

[0105] Test Example 1: Detection of acidity, pH, and viable Bifidobacterium count

[0106] In this test case, the acidity, pH and viable number of Bifidobacteria in the fermented milk prepared by the methods of Examples 1-15 and Comparative Examples 1-8 were tested, and the results are shown in Table 2.

[0107] Table 2. Results of acidity value, pH and viable count tests

[0108] Note: In Table 2, D represents the date of fermentation, D+1 represents the second day after fermentation is completed, and / indicates no testing.

[0109] As shown in Table 2, the fermented milk prepared using the methods of Examples 1-15 and Comparative Examples 1-8 showed no significant difference in acidity and pH on day D+1, indicating that the formulation and preparation method of the present invention have little impact on the basic physicochemical properties of the product. In terms of the number of viable Bifidobacteria, Example 6 was significantly higher than the other groups, mainly due to its higher initial number of added viable bacteria. The number of viable bacteria in the other Example 1 groups showed no significant difference. Comparative Example 4 had a lower initial number of viable bacteria, so the number of viable bacteria in the fermented milk was significantly different from the other groups.

[0110] Test Example 2: Total amino acid, essential amino acid, and branched-chain amino acid content in mouse plasma 2 hours postprandial

[0111] In this test case, fermented milk was prepared using the preparation methods of Examples 1-15 and Comparative Examples 1-8. The total amino acid, essential amino acid and branched-chain amino acid content of mouse plasma was measured 2 hours after gavage. The results are shown in Table 3.

[0112] Table 3. Detection of free amino acid content in mouse plasma

[0113] As shown in Table 3, there was no significant difference in the total amino acid content in mouse plasma before the test. Two hours after the start of the test, the total amino acid content in plasma of Examples 1-15 significantly increased, with Example 6 showing better results than the other examples, indicating that the addition of Bifidobacterium longum significantly promotes protein absorption. Example 2 showed better results than Example 4, indicating that the addition of lactase further enhances the protein absorption-promoting effect. The total amino acid content in Comparative Examples 1-8 was significantly lower than that in Examples 1-15. Comparative Example 2 showed significantly better results than Comparative Examples 1, 3, and 5, indicating that the use of protease alone can improve protein absorption, but the effect is still limited compared to Examples 1-15. Compared with Examples 1-3 and 5, Comparative Example 6 shows that replacing Bifidobacterium lactis with Bifidobacterium longum significantly reduced the protein absorption-promoting effect.

[0114] The results for essential amino acids showed that Examples 1-15 all significantly promoted the absorption of essential amino acids, with Example 6 showing the most significant effect. Comparative Examples 1-8 did not show a significant improvement compared to Examples 1-15. Branched-chain amino acids are more beneficial for muscle synthesis. The results showed that Examples 1-15 all significantly improved the absorption of branched-chain amino acids, with Example 6 showing the most significant effect.

[0115] Test Example 3: Total amino acid, essential amino acid, and branched-chain amino acid content in mouse ileal chyme

[0116] In this test case, fermented milk was prepared using the preparation methods of Examples 1-15 and Comparative Examples 1-8. After being administered to mice by gavage for 2 hours, the total amino acid, essential amino acid, and branched-chain amino acid content in the ileal chyme of the mice was detected. The results are shown in Table 4.

[0117] Table 4. Detection of free amino acid content in mouse digesta

[0118] As shown in Table 4, the amino acid content in mouse chyme reflects the digestion and absorption of food. Lower amino acid content indicates greater absorption by the body. The total amino acid results for Comparative Examples 1-8 are generally higher than those for Examples 1-15, indicating that the fermented milk prepared by gavage using the methods of Comparative Examples 1-8 is less conducive to amino acid absorption compared to the fermented milk prepared by gavage using the methods of Examples 1-15. The total amino acid content of Comparative Examples 2 and 4 is significantly lower than other comparative groups, indicating that the use of protease can improve amino acid absorption. The amino acid content of chyme in Example 6 is lower than other example groups, indicating that Example 6 has better amino acid absorption. The results for essential amino acids and branched-chain amino acids also show that the residual essential amino acid content in Examples 1-15 is significantly lower than that in Comparative Examples 1-8, indicating that the fermented milk prepared by gavage using the methods of Examples 1-15 can promote the absorption of essential amino acids and branched-chain amino acids, with Example 6 showing the best technical effect.

[0119] Test Example 4: Calcium Absorption and Utilization in Mice

[0120] In this test case, fermented milk was prepared using the preparation methods of Examples 1-15 and Comparative Examples 1-8. After being administered to mice by gavage for 30 days, the calcium absorption and utilization of the mice were tested, and the results are shown in Table 5.

[0121] Table 5. Dietary calcium utilization in mice

[0122] As shown in Table 5, the total calcium intake from food remained constant. Blood calcium reflects the absorption of calcium in the intestine. Example 2 showed significantly better results than other examples, indicating that Bifidobacterium lactis Ca360 was more effective in promoting calcium absorption. Compared with Comparative Example 5, Example 2 had a stronger effect on promoting calcium absorption, indicating that with the same dosage of Bifidobacterium lactis Ca360, the further use of protease and lactase could significantly increase calcium absorption. Example 3 showed better calcium absorption than Comparative Example 4, indicating that insufficient Bifidobacterium lactis addition would affect the effect on calcium absorption. Examples 2 and 3 showed better results than Comparative Example 2, indicating that the addition of Bifidobacterium lactis was necessary. Examples 2 and 3 showed better results than Comparative Example 3, indicating that the addition of Bifidobacterium lactis was more effective in promoting calcium absorption than other types of Bifidobacterium, such as Bifidobacterium longum in Comparative Example 3. Bone calcium level indicates the amount of calcium ultimately converted by the body. The bone calcium content of Examples 1-15 was significantly higher than that of Comparative Examples 1-8, with Example 2 showing significantly better results than other examples. Urinary and fecal calcium levels reflect the body's calcium excretion. The results showed that Examples 1-15 significantly reduced calcium excretion through urine and feces, and there was no significant difference in effect between Examples 1-15. This indicates that the fermented milk prepared using the methods described in Examples 1-15 improves calcium utilization and thus enhances bone calcium deposition by simultaneously increasing calcium absorption and reducing the body's calcium excretion. Regarding the final calcium digestibility and absorption rate, Examples 1-15 showed a higher rate than Comparative Examples 1-8, and Example 2 achieved a calcium absorption and utilization rate of 47.55%.

[0123] Test Example 5: Assessment of Mouse Motor Ability

[0124] In this test case, using the fermented milk prepared according to the methods of Example 2 and Comparative Example 1, mice were administered the milk by gavage for 30 days, and their motor abilities were then assessed to further verify the effect of improved calcium utilization on the motor abilities of mice. The results are as follows: Figure 2 As shown.

[0125] like Figure 2 As shown, compared with Comparative Example 1, the fermented milk prepared by the preparation method of Example 2 significantly increased the time to exhaustion in mice running and significantly improved their grip strength, indicating that the fermented milk prepared by the preparation method of Example 2 can ultimately improve the athletic ability of mice by enhancing the absorption and utilization of protein and calcium.

[0126] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A leavening agent, characterized in that, The fermentation agent includes Bifidobacterium lactis and a neutral protease isolated from Bacillus polymyxa.

2. The fermenting agent according to claim 1, characterized in that, The Bifidobacterium lactis includes Bifidobacterium lactis BB-12, Bifidobacterium lactis Ca360, Bifidobacterium lactis MN-Gup, and Bifidobacterium lactis M8; wherein, Bifidobacterium lactis Ca360 is Bifidobacterium lactis Ca360 with accession number CGMCC No. 32403, Bifidobacterium lactis MN-Gup is Bifidobacterium lactis MN-Gup with accession number CGMCC No. 15578, and Bifidobacterium lactis M8 is Bifidobacterium lactis M8 with accession number CGMCC No. 18610.

3. The fermenting agent according to claim 2, characterized in that, In the said leaven, the added amount of the Bifidobacterium lactis is 1 x 10 6 cfu / g ~ 1 x 10 10 cfu / g.

4. The fermenting agent according to claim 1, characterized in that, The neutral protease isolated from Bacillus polymyxa is ADMIL.

5. The fermenting agent as described in claim 1, characterized in that, The fermentation agent also includes fermenting bacteria or their cultures other than Bifidobacterium lactis.

6. The fermenting agent as described in claim 5, characterized in that, The fermenting bacteria are any one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. milk fat, Lactococcus lactis subsp. diacetyl, and Lactobacillus rhamnosus.

7. The fermenting agent as described in claim 6, characterized in that, The fermentation bacteria are selected from any one of the following groups: (A) Streptococcus thermophilus and Lactobacillus bulgaricus; (B) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis subsp. milk fat; (C) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum; (D) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus helveticus; (E) Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis diacetyl subsp. (F) Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus rhamnosus.

8. The fermenting agent as described in claim 6 or 7, characterized in that, The total inoculum amount of fermentation bacteria other than Bifidobacterium lactis is 50U~180U / ton.

9. A method for simultaneously improving the digestibility and absorption of protein and the absorption and utilization of calcium in fermented milk, or for preparing fermented milk, characterized in that, The method includes the step of fermenting dairy raw materials using the fermenting agent according to any one of claims 1-8.

10. The method as described in claim 9, characterized in that, In the fermentation agent: The added amount of the B. lactis is 1 x 10 6 cfu / g ~ 1 x 10 10 cfu / g; The amount of neutral protease isolated from Bacillus polymyxa added is 0.02~0.05g / 100g milk raw material.

11. A method for simultaneously improving the digestibility and absorption of protein and the absorption and utilization of calcium in fermented milk, or for preparing fermented milk, characterized in that, The method includes the step of fermenting milk raw materials using the fermenting agent according to claim 5 or 8, wherein the total inoculation amount of fermenting bacteria other than Bifidobacterium lactis is 50U~180U / ton.

12. The method as described in claim 9 or 11, characterized in that, The dairy raw materials include: fresh milk, reconstituted milk, and dairy products.

13. The method as described in claim 12, characterized in that: The fresh milk includes raw cow's milk and raw goat's milk; The dairy products include milk powder, condensed milk, cream, and milk powder.

14. The method as described in claim 9 or 11, characterized in that, The milk raw material also includes lactase.

15. The method as described in claim 14, characterized in that, The lactase mentioned is a transGOS type lactase.

16. The method as described in claim 15, characterized in that, The transGOS lactases include Nurica and Aspergillus oryzae β-galactosidase.

17. The method as described in claim 14, characterized in that, The amount of lactase added is 0.1~0.6 g / 100g of milk raw material.

18. The method as described in claim 9 or 11, characterized in that, The dairy raw materials also include sweeteners.

19. The method as described in claim 18, characterized in that, The sweeteners include sucrose, white sugar, fructose syrup, maltitol, mogroside, xylitol, sucralose, neotame, and aspartame.

20. The method as described in claim 18, characterized in that, The amount of sweetener added is 5-8g per 100g of milk raw material.

21. The method as described in claim 9 or 11, characterized in that, The fermentation process also includes one or more of the following conditions: (1) The fermentation temperature is 30~45℃; (2) The end point of fermentation is when the pH of the fermented milk is less than or equal to 4.5; (3) The fermentation time is 4 to 12 hours.

22. The method as described in claim 9 or 11, characterized in that, The method further includes steps of material preparation, homogenization, sterilization, cooling, demulsification and / or filling.

23. The method as described in claim 9 or 11, characterized in that, The promotion of protein digestion and absorption includes: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids.

24. The method as described in claim 9 or 11, characterized in that, The promotion of calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent absorption rate of calcium, and / or improving exercise capacity.

25. A fermented milk, characterized in that, The fermented milk is prepared using the method described in any one of claims 9-24.

26. A food product, characterized in that, The food product includes the fermented milk as described in claim 25.

27. The use of the starter culture agent according to any one of claims 1-8 in the preparation of fermented milk, improving the digestion and absorption of protein in fermented milk, and / or improving the absorption and utilization of calcium in fermented milk.

28. The application as described in claim 27, characterized in that, The promotion of protein digestion and absorption includes: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids.

29. The application as described in claim 27, characterized in that, The promotion of calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent absorption rate of calcium, and / or improving exercise capacity.

30. The use of the fermented milk of claim 25 in the preparation of food, in improving the digestion and absorption of protein in food, and / or in improving the absorption and utilization of calcium in food.

31. The application as described in claim 30, characterized in that, The promotion of protein digestion and absorption includes: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids.

32. The application as described in claim 30, characterized in that, The promotion of calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent absorption rate of calcium, and / or improving exercise capacity.

33. The use of the combination of Bifidobacterium lactis as defined in any one of claims 1-8 and the neutral protease isolated from Bacillus polymyxa as defined in any one of claims 1-8 in the preparation of a starter culture, in improving the digestion and absorption of protein in food, and / or in improving the absorption and utilization of calcium in food.

34. The application as described in claim 33, characterized in that, The promotion of protein digestion and absorption includes: promoting the digestion and absorption of total amino acids, promoting the digestion and absorption of essential amino acids, and / or promoting the digestion and absorption of branched-chain amino acids.

35. The application as described in claim 33, characterized in that, The promotion of calcium absorption and utilization includes promoting the absorption of calcium ions in the blood, promoting the utilization of calcium ions in bones, reducing the excretion of calcium ions in urine, reducing the excretion of calcium ions in feces, increasing the apparent absorption rate of calcium, and / or improving exercise capacity.

Images