A weak post-acid fermented milk containing a sleep-regulating functional ingredient and a preparation method, starter and application thereof based on temperature-regulated fermentation

Abstract

The application belongs to the technical field of fermented dairy products. A weak post-acid fermented milk containing sleep-regulating functional components and a preparation method, starter and application thereof based on temperature-adjusted fermentation are disclosed. The weak post-acid starter is prepared by using Streptococcus thermophilus complex, and the fermented milk with GABA content of 34 mg / 100 g or more is prepared by two-stage temperature-adjusted fermentation, which can effectively improve the GABA synthesis efficiency. In the application, Streptococcus thermophilus for producing GABA is used to replace Lactobacillus bulgaricus in the traditional starter, so that the post-acid problem is avoided from the source of the strain, and the efficient accumulation of GABA is realized in a short time through the temperature-adjusted fermentation process. The preparation process is simple, the obtained fermented milk has weak post-acid and good flavor, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of fermented dairy product technology, specifically a weakly acidic fermented milk containing sleep-regulating functional components, its preparation method based on temperature-controlled fermentation, the starter culture, and its application. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a functional substance with sedative and sleep-inducing activities. With increasing consumer focus on sleep health, GABA-containing fermented dairy products, which combine sleep-aiding properties with dairy nutrition, have a promising market prospect. However, existing technologies for preparing such products mainly face the following three problems:

[0003] Firstly, traditional fermented milk typically uses a combination of Streptococcus thermophilus and Lactobacillus bulgaricus as a starter culture. Lactobacillus bulgaricus retains a strong ability to produce acid even after fermentation and during refrigerated storage, leading to post-fermentation. Post-fermentation not only makes the product excessively sour and degrades its flavor and quality, but also necessitates a low-temperature cold chain to inhibit the activity of residual bacteria, increasing storage and distribution costs and shortening the product's quality stability period within its shelf life.

[0004] Secondly, regarding the methods of adding GABA, directly adding GABA exogenously has problems such as high cost, potential off-flavors, or impact on product texture. While in-situ production of GABA using microbial fermentation often faces challenges, such as poor strain growth in the milk environment, excessive acid production leading to overly acidic products unsuitable for direct consumption, and the need for prolonged fermentation, which reduces production efficiency.

[0005] Third, while existing technologies include methods for producing GABA through lactic acid bacteria fermentation, most focus on preparing fermentation broths with high concentrations of GABA rather than its direct application in fermented dairy products. For example, Chinese patent CN102108370B discloses a method for producing γ-aminobutyric acid (GABA), which uses lactic acid bacteria NBRC 12005 in MRSG medium for fermentation and achieves a GABA yield of over 13.1 g / L through segmented addition of the substrate monosodium glutamate and segmented oxygen control. However, the technical solution has the following limitations: (1) Its culture medium is a nitrogen-rich MRSG system without milk components, which is not a fermented milk culture medium based on cow's milk, and cannot directly prepare drinkable fermented milk products; (2) The fermentation cycle is as long as 90~106h, and the production efficiency is extremely low, which is difficult to meet the requirements of cycle and cost for industrial production of dairy products; (3) Its segmented temperature and oxygen control is the core of promoting the growth of strains and the induction of GABA synthase, but it does not involve key issues such as flavor quality, post-acid control and product palatability in fermented milk; (4) Its process requires the addition of substrate and the creation of an anaerobic environment multiple times, which is complicated and not conducive to the standardization and large-scale production of dairy products.

[0006] In summary, there is currently a lack of a method for preparing fermented milk that can efficiently produce GABA in a milk matrix without relying on Lactobacillus bulgaricus and avoiding post-acidity issues, and that optimizes the fermentation process to balance production efficiency and product flavor. Summary of the Invention

[0007] One objective of this invention is to provide a weakly acidic fermented milk containing sleep-regulating functional components, and its preparation method, starter culture, and application based on temperature-controlled fermentation, in order to solve the problem of post-acidity caused by the use of Lactobacillus bulgaricus in existing GABA-containing fermented dairy products, as well as the problems of poor strain compatibility, complex processes, and poor product flavor when existing GABA fermentation technology is applied to dairy systems.

[0008] This invention discovers that not all Streptococcus thermophilus strains have the ability to produce GABA. However, multiple Streptococcus thermophilus strains with GABA-producing ability obtained through screening, when combined, significantly increase the GABA production compared to single-strain fermentation. Moreover, the fermented product has a good flavor and does not require the addition of Lactobacillus bulgaricus, thus avoiding the post-acidity problem from the source of the strain.

[0009] Based on this, the present invention further discovered that when using the above-mentioned compound fermenting agent for milk fermentation, fermentation temperature and fermentation time have a significant impact on the synthesis efficiency of GABA. Therefore, the present invention employs a two-stage temperature-controlled fermentation process: by setting appropriate temperature conditions at different fermentation stages, while ensuring good bacterial growth and acid production, the accumulation rate of GABA is significantly improved, and fermented milk products with adequate GABA content and suitable acidity can be obtained in a shorter time.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a weakly acidic fermented milk containing sleep-regulating functional components, wherein the raw materials for making its effective components, by weight, include: 80-90 parts of liquid milk, 0.05-0.18 parts of monosodium glutamate and 1.4-3 parts of milk protein;

[0012] The mildly acidic fermented milk containing sleep-regulating components is obtained by fermentation with a mildly acidic starter.

[0013] The strains in the weak post-acid fermentation agent are Streptococcus thermophilus subsp. JMCC16, Streptococcus thermophilus subsp. JMCC0019 and Streptococcus thermophilus subsp. JMCC0022.

[0014] The weakly acidic fermented milk containing sleep-regulating components has a GABA content ≥34mg / 100g.

[0015] As a limitation of the present invention, the raw materials also include 6 to 10 parts by weight of sucrose;

[0016] The milk protein is whey protein powder, whey protein concentrate, whey protein isolate, caseinate, or a combination thereof.

[0017] Secondly, the present invention provides a weak post-acid fermenting agent for preparing the above-mentioned weak post-acid fermented milk containing sleep-regulating functional components, wherein the ratio of viable bacteria of Streptococcus salivarius subsp. thermophile JMCC16, Streptococcus salivarius subsp. thermophile JMCC0019 and Streptococcus salivarius subsp. thermophile JMCC0022 in the weak post-acid fermenting agent is 16:35:5~10.

[0018] Among them, Streptococcus thermophilus subspecies JMCC16, also known as Streptococcus thermophilus JMCC16, was deposited on November 16, 2015, at the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 11672. It was published in Chinese Invention Patent No. CN109536406B on May 13, 2022.

[0019] Streptococcus thermophilus subspecies JMCC0019, also known as Streptococcus thermophilus JMCC0019, was deposited on July 14, 2017, at the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.14426. It was published in Chinese Invention Patent No. CN108004167B on November 3, 2020.

[0020] Streptococcus thermophilus subspecies JMCC0022, also known as Streptococcus thermophilus JMCC16, was deposited on May 28, 2018, at the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 15822. It was published in Chinese Invention Patent No. CN109576157B on August 28, 2020.

[0021] As a further limitation of the present invention, the weakly acidic fermentation agent also includes Lactobacillus delbrueckii subsp. bulgaricus JMCC0018.

[0022] The viable count ratio of *Lactobacillus delbrueckii* subsp. bulgaricus JMCC0018 to *Streptococcus salivarius* subsp. thermophilicus JMCC16 was 0.01:16.

[0023] Lactobacillus delbrueckii subspecies bulgaricus JMCC0018, also known as Streptococcus thermophilus JMCC0018, was deposited in 2017 at the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.14425. It was published in Chinese invention patent application with publication number CN109601620A on April 12, 2019.

[0024] Thirdly, the present invention provides a method for preparing a weakly acidic fermented milk containing sleep-regulating components, characterized by comprising the following steps performed sequentially:

[0025] S1. Take the raw material and a stabilizer accounting for 0.1%~2.2% of the total weight of the raw material, mix them evenly, homogenize, sterilize, and obtain liquid A;

[0026] S2. Take liquid A, cool it to the inoculation temperature, inoculate it with a weak post-acid fermentation agent, ferment until the pH is 4.42~4.5, stop fermentation, and allow it to mature to obtain a weak post-acid fermented milk containing sleep-regulating components.

[0027] Fourthly, the present invention provides a method for preparing a weakly acidic fermented milk containing sleep-regulating functional components based on temperature-controlled fermentation. After inoculating the weakly acidic starter culture agent into the material solution A in step S2 above, the mixture is first fermented at a first fermentation temperature for 2.5 to 3.5 hours, and then the fermentation temperature is adjusted to a second fermentation temperature and fermentation continues until the total fermentation time is 6.5 to 7 hours.

[0028] The first fermentation temperature is 42℃, and the second fermentation temperature is 36℃;

[0029] Post-ripening involves standing at 3.5~5.5℃ for 8~16 hours.

[0030] Preferably, after fermentation, the milk is broken by stirring at 800 rpm for 2 minutes and then concentrated to obtain the fermented milk product.

[0031] Furthermore, the stabilizer is at least one selected from polydextrose, acetylated distarch phosphate, agar, pectin, diacetyl tartaric acid mono- and diglycerides, xanthan gum, and gelatin.

[0032] Furthermore, the stabilizer is prepared by mixing 1.6 parts by weight of polydextrose, 0.3 parts by weight of acetylated distarch phosphate, 0.06 parts by weight of agar, 0.2 parts by weight of pectin and 0.06 parts by weight of diacetyl tartaric acid mono- and diglycerides.

[0033] Fifthly, the present invention provides the application of a weakly acidic fermentation agent, specifically for use in the preparation of any dosage form among powders, granules, emulsions, tablets, blocks, sticks, oral liquids, and capsules.

[0034] Furthermore, weak post-acid starter cultures are used to prepare fermented milk, fermented milk beverages, fermented buttermilk, fermented cream, or frozen fermented dairy products.

[0035] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0036] This invention solves the problem of post-acidity in fermented milk from the source of the microbial strain. In traditional fermented milk, the continuous acid production of Lactobacillus bulgaricus during refrigeration is the main cause of post-acidity. However, this invention uses a combination of three thermophilic streptococci with GABA-producing ability, JMCC16, JMCC0019, and JMCC0022, obtained through screening, as a starter culture. There is no need to add Lactobacillus bulgaricus, and the resulting fermented milk has weak post-acidity, avoiding flavor deterioration caused by post-acidity and dependence on strict low-temperature cold chain.

[0037] This invention achieves highly efficient GABA synthesis through strain combination. Single GABA-producing Streptococcus thermophilus strains exhibit low GABA yield during milk fermentation. However, this invention, by combining three strains in a specific ratio of 16:35:5~10, significantly increases GABA yield to over 34 mg / 100g, demonstrating the synergistic effect of the combination.

[0038] This invention employs a two-stage temperature-controlled fermentation process, which further increases GABA yield while ensuring production efficiency. By optimizing GABA synthase activity at 42℃ in the early stage of fermentation, the pH is rapidly reduced to approximately 4.5, the optimal level for GABA production. Simultaneously, a second stage of fermentation at 36℃ extends the period of high GABA production. Products meeting GABA content standards can be obtained within a total fermentation time of 6.5–7 hours. Compared to isothermal fermentation, GABA yield is significantly increased within the same fermentation time. Detailed Implementation

[0039] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. MRS liquid culture medium is a finished product from Beijing Modern Reagent Co., Ltd.; the Nagqu 4580 probiotic is from Hebei Yiran Biotechnology Co., Ltd., and has been disclosed in Chinese Patent ZL202010705870.4. *Streptococcus salivarius* subsp. *thermophilicus* JMCC16, JMCC0019, JMCC0022, JMCC0024, JMCC0031, JMCC0032, *Lactobacillus deuterans* subsp. *bulgaricus* JMCC0018, and Nagqu 4580 are all publicly available strains, deposited at Junlebao R&D Center.

[0041] Example 1

[0042] This embodiment describes a mildly acidic fermented milk containing sleep-regulating components and its preparation method, as detailed below:

[0043] S1. Ingredients and Pretreatment

[0044] Take 88 kg of raw milk after acceptance, 0.1 kg of monosodium glutamate, 2.2 kg of whey protein powder, 7 kg of white sugar, and a stabilizer made by mixing 1.6 kg of polydextrose, 0.3 kg of acetylated distarch phosphate, 0.06 kg of agar, 0.2 kg of pectin, and 0.06 kg of diacetyl tartaric acid mono- and diglycerides. The total amount of stabilizer is 2.22 kg.

[0045] Monosodium glutamate (MSG) is added as a byproduct of monosodium glutamate (MSG).

[0046] Preheat raw milk (i.e., liquid milk) to 50°C, add milk protein (i.e., whey protein powder), granulated sugar (i.e., sucrose), monosodium glutamate, and stabilizer, stir until dissolved, and add water to a total weight of 100 kg. Homogenize all the liquid, sterilize at 98°C for 15 minutes to induce browning, and cool to the fermentation temperature to obtain liquid A.

[0047] S2. Inoculation and Fermentation

[0048] Preparation of weakly acidic fermentation spawn: *Streptococcus salivarius* subsp. *thermophilus* JMCC16, JMCC0019, and JMCC0022 were cultured separately in MRS liquid medium at 37°C for 24 hours. After three subcultures at a 5% inoculum size, activation culture was completed until the viable count reached 1.5 × 10⁻⁶ cells / mL. 9 CFU / mL, mixed at a weight ratio of 16:35:7, to obtain a weakly acidic fermentation agent.

[0049] The weak post-acid fermenting agent was inoculated into liquid A at an inoculation rate of 60 g / t. Fermentation was first carried out at 42℃ (i.e., the first fermentation temperature) for 3 hours, and then the fermentation temperature was adjusted to 36℃ (i.e., the first fermentation temperature). The second fermentation temperature was maintained and fermentation continued for 4 hours until the pH reached 4.42. The total fermentation time was 7 hours. Fermentation was then terminated and the mixture was allowed to stand at 4℃ for 12 hours for post-maturation to obtain a weak post-acid fermented milk containing sleep-regulating functional components.

[0050] The fermented milk obtained in this example was found to contain 69.8 mg / 100g of GABA. The product had a weak acidity and, after being stored at 15°C for 21 days, the acidity was ≤120°T. Sensory evaluation showed that the flavor was good.

[0051] Effect Experiment Example 1

[0052] This experimental example compares the GABA production of different thermophilic streptococcal strains and the control starter culture to compare the ability of each strain to produce GABA independently.

[0053] The test strains were *Streptococcus salivarius* subsp. *thermophilus* JMCC16, JMCC0019, JMCC0022, JMCC0024, JMCC0031, JMCC0032, and *Lactobacillus deuterans* subsp. *bulgaricus* JMCC0018, as well as the control starter culture Nagqu 4580 (Chinese Patent ZL202010705870.4). Each strain was inoculated into fermentation substrate (solution A) prepared according to the raw material ratio in Example 1, and fermented statically at 40°C for 48 h, followed by post-ripening at 4°C for 24 h to obtain fermentation samples. The GABA content in the fermentation samples was detected by liquid chromatography. This experiment was commissioned to Hebei Yiran Biotechnology Co., Ltd. for detection, and the results are shown in Table 1.

[0054] Table 1. Results of GABA content in each fermentation sample

[0055]

[0056] The results showed that not all Streptococcus thermophilus strains had the ability to produce GABA. The three strains JMCC16, JMCC0019, and JMCC0022 used in this invention had the ability to produce GABA, but the yield of a single strain was significantly lower than that of the control fermentation agent Naqu 4580 containing Lactobacillus. This indicates that a single Streptococcus thermophilus strain does not have an advantage in GABA production compared with a compound fermentation agent containing Lactobacillus.

[0057] Effect Experiment Example 2

[0058] This experimental example was used to analyze the effect of different compound ratios on GABA production. Seven compound schemes were set up, and strains were mixed in corresponding proportions to prepare corresponding starter cultures. These starter cultures were inoculated into the same fermentation substrate as in Experiment 2, and fermentation was carried out according to the process in Experiment 2. The GABA content in the resulting fermentation samples was measured, and the results are shown in Table 2.

[0059] Table 2. GABA yield results for different fermentation agents

[0060]

[0061] The results showed that the highest GABA yield was achieved when the ratio of JMCC16, JMCC0019, and JMCC0022 was 16:35:7. As the proportion of JMCC0022 increased, the GABA yield first decreased and then slightly increased. After being made into a weak post-acid fermenting agent, the yield was significantly higher than that of a single strain. Meanwhile, the addition of Lactobacillus de Germany subsp. Bulgaria did not significantly increase the GABA yield.

[0062] This indicates that consuming 300g of fermented milk prepared with compound formula 1 (i.e., the weak post-acid fermenting agent of Example 1 of this invention) daily can achieve the minimum effective dose of 100mg for adults.

[0063] Effect Experiment Example 3

[0064] This experimental example was used to analyze the effect of fermentation time on GABA yield and product acidity. The weak post-acid fermentation agent of Example 1 of this invention was used to ferment the feed solution A prepared in Example 1, and the GABA yield and pH of the fermentation product at different fermentation times were recorded. The results are shown in Table 3.

[0065] Table 3. GABA yield results for different starter cultures

[0066]

[0067] The results showed that as the fermentation time was extended, the GABA yield increased significantly, but the pH at the end of the fermentation continued to decrease, and the acidity of the product increased. After the fermentation time exceeded 24 hours, although the GABA yield was high, the pH had dropped to about 4.0, and the fermented lactic acid was too high, making it unsuitable for drinking.

[0068] Effect Experiment Example 4

[0069] In this experimental example, the protein content in the liquid A prepared in Example 1 was adjusted to 3% and 4% by adjusting the amount of whey protein powder added. The liquids were fermented at 40℃ for 24h and 48h respectively. The GABA content of the fermentation products was detected to analyze the effect of protein content in the substrate on GABA yield. The results are shown in Table 4.

[0070] Table 4. GABA production results at different milk protein contents.

[0071]

[0072] The results showed that increasing the milk protein content in feed solution A did not increase GABA production. In fact, when the protein content increased from 3% to 4%, GABA production decreased by about 50% under the same fermentation time. Excessively high protein content is actually detrimental to GABA synthesis.

[0073] Effect Experiment Example 5

[0074] In this experimental example, the amount of monosodium glutamate (MSG) added to the liquid A prepared in Example 1 was set into groups of 0.1% and 0.2%, respectively. The mixture was fermented at 40°C for 24 h and 48 h, respectively, with other conditions being the same. The GABA content of the fermentation product was detected, and the preference for the fermentation product was evaluated using a sensory evaluation method with a maximum score of 10. The results are shown in Table 5.

[0075] Table 5. GABA production results with different monosodium glutamate (MSG) addition levels.

[0076]

[0077] The results showed that increasing the amount of monosodium glutamate (MSG) added from 0.1% to 0.2% improved GABA production, but significantly reduced product preference, indicating that excessive MSG addition would significantly affect product flavor.

[0078] Effect Experiment Example 6

[0079] This experimental example sets the addition amount of the weak post-acid fermenting agent in Example 1 to 60 g / t and 600 g / t, and the fermentation time to 6 h, 12 h and 18 h, respectively. The feed liquid A obtained in Example 1 is fermented at 40 °C, and the GABA content of the fermentation product is detected to analyze the effect of the addition amount of weak post-acid fermenting agent on GABA yield. The results are shown in Table 6.

[0080] Table 6. GABA yield results with different amounts of weakly acidified fermenting agent.

[0081]

[0082] The results showed that increasing the amount of weak post-acid fermentation agent could increase GABA production. With the same fermentation time, increasing the amount of agent by 10 times increased the GABA content by about 25%.

[0083] Effect Experiment Example 7

[0084] In this experimental example, the fermentation temperature of the liquid A prepared in Example 1 was set to a fixed 36℃, 39℃ or 42℃. The fermentation was stopped when the pH reached 4.42. The pH value of the fermented milk was measured every half hour. After the fermentation was completed, the GABA content was measured to investigate the effect of different fermentation temperatures on GABA yield and pH changes during the fermentation process. The results are shown in Tables 7 and 8.

[0085] Table 7. GABA yield results at different fixed fermentation temperatures.

[0086]

[0087] The results in Table 7 show that different fixed fermentation temperatures have no significant effect on GABA yield.

[0088] Table 8. Monitoring results of pH changes at different fixed fermentation temperatures

[0089]

[0090] Table 8 shows that within the optimal pH range of 4.5–4.0 for GABA synthase, the fermentation duration at 36°C and 42°C was 3 h or 4 h, significantly longer than the 2 h at 39°C. GABA yield may be closely related to the duration of the low pH stage.

[0091] Effect Experiment Example 8

[0092] This experimental example verifies the effect of the two-stage temperature-controlled fermentation method in Example 1 on GABA yield. Specifically, it measures the pH change and final GABA yield during fermentation and compares them with those in Experiment 8. The results are shown in Table 9.

[0093] Table 9. Monitoring results of pH changes in the thermoregulatory fermentation process

[0094]

[0095] The results showed that adjusting the fermentation temperature shortened the total fermentation time to 7 hours and extended the fermentation time for high GABA production below pH 4.5 to 4 hours, resulting in a GABA yield of 69.8 ± 3.17 mg / 100g, which was significantly better than the fixed fermentation temperature process in Experiment Example 7.

[0096] Effect Experiment Example 9

[0097] In this experimental example, the amount of monosodium glutamate (MSG) added to the feed solution A prepared in Example 1 was set to be 2%, 6%, and 10%, respectively. The weakly acidic starter culture from Example 1 was inoculated, and the fermentation temperature was fixed at 37°C for 36 h, 68 h, and 96 h, respectively. The GABA content in the fermentation product was measured after each fermentation. Alternatively, MRS medium with 10% MSG was used. During fermentation, the pH was measured every 6 h and adjusted to 4.5 with NaHCO3. Fermentation continued for 96 h, and the GABA content was measured again. This was to analyze the extreme GABA yield of the weakly acidic starter culture of the present invention under conditions of increased MSG addition and extended fermentation time, verifying its potential in the preparation of high GABA content products. The results are shown in Table 10.

[0098] Table 10 Results of GABA content in fermentation products of each group

[0099]

[0100] The results showed that by adding 10% monosodium glutamate to MRS medium and adjusting the pH to 4.5, the GABA content reached 46.75 g / L after 96 hours. Even without considering flavor enhancement, the GABA yield increased significantly after increasing the amount of monosodium glutamate added and extending the fermentation time. If a suitable acidity environment is maintained through pH adjustment during fermentation, the GABA content can still be significantly increased. This indicates that the compound fermentation agent screened in this invention has excellent potential for high GABA production, and fermentation conditions can be flexibly adjusted according to product positioning.

[0101] Example 2

[0102] This embodiment describes a method for preparing a weakly acidic fermented milk containing sleep-regulating components, as detailed below:

[0103] S1. Ingredients and Pretreatment

[0104] Take 80 kg of raw milk after acceptance, 0.05 kg of monosodium glutamate, 1.4 kg of whey protein powder, 6 kg of white sugar, and a stabilizer made by mixing 1.6 kg of polydextrose, 0.3 kg of acetylated distarch phosphate, 0.06 kg of agar, 0.2 kg of pectin, and 0.06 kg of diacetyl tartaric acid mono- and diglycerides. The total amount of stabilizer is 2.22 kg.

[0105] Preheat raw milk to 50°C, add whey protein powder, white sugar, monosodium glutamate, and stabilizer, stir until dissolved, and add water to a total weight of 100 kg. Homogenize all the liquids, sterilize at 98°C for 15 minutes to induce browning, and cool to fermentation temperature to obtain liquid A.

[0106] S2. Inoculation and Fermentation

[0107] Preparation of weakly acidic fermentation spawn: *Streptococcus salivarius* subsp. *thermophilus* JMCC16, JMCC0019, and JMCC0022 were cultured separately in MRS liquid medium at 37°C for 24 hours. After three subcultures at a 5% inoculum size, activation culture was completed until the viable count reached 1.5 × 10⁻⁶ cells / mL. 9 CFU / mL, mixed at a live bacteria ratio of 16:35:5, to obtain a weakly acidic fermentation agent.

[0108] The weak post-acid fermenting agent was inoculated into the feed liquid A at an inoculation rate of 50 g / t. Fermentation was first carried out at 42℃ for 2.5 h, then the fermentation temperature was adjusted to 36℃ and maintained at the second fermentation temperature for another 4 h. Fermentation continued until the pH reached 4.42. The total fermentation time was 6.5 h. Fermentation was then terminated and the mixture was allowed to stand at 3.5℃ for 8 h for post-maturation to obtain a weak post-acid fermented milk containing sleep-regulating components.

[0109] The fermented milk obtained in this example was tested and found to contain 68.2 mg / 100g of GABA, which meets the requirement of ≥34 mg / 100g. The product also showed weak acidity, with an acidity of ≤120°T after 21 days of storage at 15°C. Sensory evaluation showed that the flavor was good.

[0110] Example 3

[0111] This embodiment describes a method for preparing a weakly acidic fermented milk containing sleep-regulating components, as detailed below:

[0112] S1. Ingredients and Pretreatment

[0113] Take 85 kg of raw milk after acceptance, 0.115 kg of monosodium glutamate, 2.2 kg of whey protein powder, 8 kg of white sugar, and a stabilizer made by mixing 0.8 kg of polydextrose, 0.2 kg of acetylated distarch phosphate, 0.04 kg of agar, 0.1 kg of pectin, 0.04 kg of diacetyl tartaric acid mono- and diglycerides, 0.2 kg of xanthan gum, and 0.3 kg of gelatin. The total amount of stabilizer is 1.68 kg.

[0114] Preheat raw milk to 50°C, add whey protein powder, white sugar, monosodium glutamate, and stabilizer, stir until dissolved, and add water to a total weight of 100 kg. Homogenize all the liquids, sterilize at 98°C for 15 minutes to induce browning, and cool to fermentation temperature to obtain liquid A.

[0115] S2. Inoculation and Fermentation

[0116] Preparation of weakly acidic fermentation spawn: *Streptococcus salivarius* subsp. *thermophilus* JMCC16, JMCC0019, and JMCC0022 were cultured separately in MRS liquid medium at 37°C for 24 hours. After three subcultures at a 5% inoculum size, activation culture was completed until the viable count reached 1.5 × 10⁻⁶ cells / mL. 9 CFU / mL, mixed at a live bacteria ratio of 16:35:8, to obtain a weakly acidic fermentation agent.

[0117] The weak post-acid fermenting agent was inoculated into the feed liquid A at an inoculation rate of 65 g / t. Fermentation was first carried out at 42℃ for 3 hours, and then the fermentation temperature was adjusted to 36℃. The fermentation was continued at the second fermentation temperature for 3.8 hours until the pH reached 4.46. The total fermentation time was 6.8 hours. Fermentation was then terminated, and the mixture was allowed to stand at 5.5℃ for 16 hours for post-maturation to obtain a weak post-acid fermented milk containing sleep-regulating components.

[0118] The fermented milk obtained in this example was tested and found to contain 68.1 mg / 100g of GABA, which meets the requirement of ≥34 mg / 100g. The product also showed weak acidity, with an acidity of ≤120°T after 21 days of storage at 15°C. Sensory evaluation showed that the flavor was good.

[0119] Example 4

[0120] This embodiment describes a method for preparing a weakly acidic fermented milk containing sleep-regulating components, as detailed below:

[0121] S1. Ingredients and Pretreatment

[0122] Take 90 kg of raw milk after acceptance, 0.18 kg of monosodium glutamate, 3.0 kg of whey protein powder, 10 kg of white sugar, and a stabilizer made by mixing 1.6 kg of polydextrose, 0.3 kg of acetylated distarch phosphate, 0.06 kg of agar, 0.2 kg of pectin, and 0.06 kg of diacetyl tartaric acid mono- and diglycerides. The total amount of stabilizer is 2.22 kg.

[0123] Preheat raw milk to 50°C, add whey protein powder, white sugar, monosodium glutamate, and stabilizer, stir until dissolved, and add water to a total weight of 100 kg. Homogenize all the liquids, sterilize at 98°C for 15 minutes to induce browning, and cool to fermentation temperature to obtain liquid A.

[0124] S2. Inoculation and Fermentation

[0125] Preparation of weakly acidic fermentation spawn: *Streptococcus salivarius* subsp. *thermophilus* JMCC16, JMCC0019, and JMCC0022 were cultured separately in MRS liquid medium at 37°C for 24 hours. After three subcultures at a 5% inoculum size, activation culture was completed until the viable count reached 1.5 × 10⁻⁶ cells / mL. 9CFU / mL, mixed at a live bacteria ratio of 16:35:10, to obtain a weakly acidic fermentation agent.

[0126] The weak post-acid fermenting agent was inoculated into the feed liquid A at an inoculation rate of 80g / t. Fermentation was first carried out at 42℃ for 3.5h, then the fermentation temperature was adjusted to 36℃ and maintained at the second fermentation temperature for another 3.5h. Fermentation continued until the pH reached 4.50. The total fermentation time was 7h. Fermentation was then terminated and the mixture was allowed to stand at 5℃ for 9h for post-maturation to obtain a weak post-acid fermented milk containing sleep-regulating components.

[0127] The fermented milk obtained in this example was tested and found to contain 72.3 mg / 100g of GABA, which meets the requirement of ≥34 mg / 100g. The product also showed weak acidity, with an acidity of ≤120°T after 21 days of storage at 15°C. Sensory evaluation showed that the flavor was good.

[0128] In other embodiments, the other conditions are the same as in Example 1, except that: the amount of liquid milk is adjusted to be in the range of 80-90 kg, such as 82 kg, 86 kg, or 89 kg; the amount of monosodium glutamate is adjusted to be in the range of 0.05-0.18 kg, such as 0.08 kg, 0.12 kg, or 0.15 kg; the amount of milk protein is adjusted to be in the range of 1.4-3.0 kg, such as 1.8 kg, 2.5 kg, or 2.8 kg; the amount of sucrose is adjusted to be in the range of 6-10 kg, such as 7 kg, 8 kg, or 9 kg; and the total amount of stabilizer is adjusted to be in the range of 0.1%-2.2% of the total weight of raw materials, such as 0.1%, 1%, or 1%. The inoculum concentrations were adjusted to 0.5%, 2.0%, and 2.2%; the inoculum amount was adjusted to be within the range of 50-80 g / t, such as 55 g / t, 70 g / t, and 75 g / t; the final pH of fermentation was adjusted to be within the range of 4.42-4.50, such as 4.44, 4.48, and 4.50; the first fermentation stage time in the temperature-controlled fermentation process was adjusted to 2.5-3.5 h, such as 2.8 h and 3.2 h, and the total fermentation time was 6.5-7 h, such as 6.6 h and 6.9 h. The resulting fermented milk GABA content all met the requirement of ≥34 mg / 100 g, and the product had weak post-acidity. When whey protein powder was replaced by concentrated whey protein, whey protein isolate, sodium caseinate, or a combination thereof, the GABA content and post-acidity control of the resulting fermented milk were not significantly different from those in the above examples. When the viable count ratio of JMCC16, JMCC0019, and JMCC0022 in the weak post-acid fermentation agent is in other ratios within the range of 16:35:5 to 10, such as 16:35:6, 16:35:7, or 16:35:9, or when Lactobacillus delbrueckii subsp. bulgaricus JMCC0018 is added to the fermentation agent so that the viable count ratio of Lactobacillus delbrueckii to JMCC16 is 0.01:16, the resulting fermented milk has GABA content that meets the requirements and post-acidity control is good.

[0129] When the stabilizer is any one or more combinations of polydextrose, acetylated distarch phosphate, agar, pectin, diacetyl tartaric acid mono- and diglycerides, xanthan gum, and gelatin, such as using 0.5 kg of xanthan gum alone, 0.5 kg of gelatin alone, or a combination of 1.0 kg of polydextrose and 0.3 kg of pectin, the GABA content and post-acidity control of the resulting fermented milk meet the requirements.

[0130] The weak post-acid fermenting agents from the various embodiments of the present invention were prepared into any one of the following dosage forms: powder, granules, emulsion, tablets, blocks, sticks, oral liquids, and capsules. Fermented milk was prepared using the same process as in Examples 1, 2, or 3. The resulting products showed little difference in GABA content and post-acid control compared to those prepared with fresh fermenting agents. When the fermenting agents were used to prepare fermented milk beverages, fermented buttermilk, fermented cream, or frozen fermented dairy products, the resulting products were found to have GABA content that met the requirements of the corresponding products, and the post-acidity was weak.

[0131] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A mildly acidic fermented milk containing sleep-regulating components, characterized in that, The raw materials for making its active ingredients, by weight, include: 80-90 parts liquid milk, 0.05-0.18 parts monosodium glutamate, and 1.4-3 parts milk protein; The mildly acidic fermented milk containing sleep-regulating components is obtained by fermentation with a mildly acidic starter. The strains in the weak post-acid fermentation agent are Streptococcus thermophilus subsp. JMCC16, Streptococcus thermophilus subsp. JMCC0019 and Streptococcus thermophilus subsp. JMCC0022. The weakly acidic fermented milk containing sleep-regulating components has a GABA content ≥34mg / 100g and an acidity ≤120°T when stored at 15℃ for 21 days.

2. The weakly acidic fermented milk containing sleep-regulating components according to claim 1, characterized in that, The raw materials also include 6-10 parts by weight of sucrose; The milk protein is whey protein powder, whey protein concentrate, whey protein isolate, caseinate, or a combination thereof.

3. A weakly acidified fermenting agent for preparing the weakly acidified fermented milk containing sleep-regulating functional components as described in claim 1 or 2, characterized in that, The viable cell ratio of Streptococcus thermophilus subsp. JMCC16, Streptococcus thermophilus subsp. JMCC0019, and Streptococcus thermophilus subsp. JMCC0022 in the weak post-acid fermentation agent is 16:35:5~10.

4. The weakly acidic fermentation agent according to claim 3, characterized in that, It also includes Lactobacillus delbrueckii subsp. bulgaricus JMCC0018; The viable count ratio of *Lactobacillus delbrueckii* subsp. bulgaricus JMCC0018 to *Streptococcus salivarius* subsp. thermophilicus JMCC16 was 0.01:

16.

5. A method for preparing a weakly acidic fermented milk containing sleep-regulating functional components as described in claim 1 or 2, characterized in that, This includes the following steps performed sequentially: S1. Take the raw material and a stabilizer accounting for 0.1%~2.2% of the total weight of the raw material, mix them evenly, homogenize, sterilize, and obtain liquid A; S2. Take liquid A, cool it to the inoculation temperature, and inoculate it at 50~80g / t with a viable count of not less than 1×10⁻⁶. 9 A weakly acidic starter culture (CFU / mL) is used to ferment the milk until the pH reaches 4.42-4.

5. Fermentation is then terminated, followed by ripening to obtain a weakly acidic fermented milk containing components that regulate sleep.

6. A method for preparing a weakly acidic fermented milk containing sleep-regulating functional components based on temperature-controlled fermentation, characterized in that, After the feed liquid A described in claim 5 is inoculated with a weak post-acid fermentation agent, it is first fermented at the first fermentation temperature for 2.5 to 3.5 hours, and then the fermentation temperature is adjusted to the second fermentation temperature and fermentation continues until the total fermentation time is 6.5 to 7 hours. The first fermentation temperature is 42℃, and the second fermentation temperature is 36℃; Post-ripening involves standing at 3.5~5.5℃ for 8~16 hours.

7. The method for preparing a weakly acidic fermented milk containing sleep-regulating functional components according to claim 6, characterized in that, The stabilizer is at least one selected from polydextrose, acetylated distarch phosphate, agar, pectin, diacetyl tartaric acid mono- and diglycerides, xanthan gum, and gelatin.

8. The method for preparing a weakly acidic fermented milk containing sleep-regulating functional components according to claim 7, characterized in that, The stabilizer is prepared by mixing 1.6 parts by weight of polydextrose, 0.3 parts by weight of acetylated distarch phosphate, 0.06 parts by weight of agar, 0.2 parts by weight of pectin and 0.06 parts by weight of diacetyl tartaric acid mono- and diglycerides.

9. The application of the weakly acidic fermenting agent according to claim 3 or 4, characterized in that, The weak post-acid fermentation agent is used to prepare any one of the following dosage forms: powder, granules, emulsion, tablets, blocks, sticks, oral liquids, and capsules.

10. The application of the weakly acidic fermenting agent according to claim 9, characterized in that, The weak post-acid fermenting agent is used to prepare fermented milk, fermented milk beverages, fermented buttermilk, fermented cream, or frozen fermented dairy products.

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