A starter culture for fermented milk, fermented milk and its preparation method

Abstract

This invention relates to the field of fermented milk technology, specifically to a starter culture for fermented milk, fermented milk, and a method for preparing the same. The starter culture provided by this invention, through the synergistic effect of various bacterial strains, can significantly increase the content of functional substances that improve sub-health conditions and alleviate sub-health symptoms in fermented milk during preparation. It promotes the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the intestines, significantly regulating the intestinal flora, increasing the content of the neurotransmitter GABA, and decreasing the content of 5-HT and 5-HIAA, which is beneficial for improving sleep quality and relieving anxiety. Simultaneously, it can better ensure the taste, flavor, and stability of the fermented milk. Fermented milk prepared using the above-mentioned starter culture has a good taste and flavor, long shelf life, and functionality, effectively alleviating sub-health symptoms, with no toxic side effects, a sweet, smooth, and delicate taste, a rich and full flavor, and overall good appeal.

Description

Technical Field

[0001] This invention relates to the field of fermented milk technology, and in particular to a starter culture for fermented milk, fermented milk, and a method for preparing the same. Background Technology

[0002] Fermented milk is loved by consumers for its rich nutritional value. Currently, influenced by the trend of health and wellness, consumers are increasingly inclined to buy fermented milk with functional attributes.

[0003] In the 1980s, Professor Buchmann first proposed the concept of "sub-health." Sub-health (SHS) refers to a state between health and disease, characterized by the absence of organ, tissue, or functional diseases or defects, yet exhibiting symptoms such as lethargy, fatigue, sleep disorders, anxiety, depression, and memory loss. With improved living standards, socioeconomic development, and changes in lifestyle, various unhealthy dietary habits and work / life stress can all harm human health. A WHO predictive survey shows that 75% of the global population is in a sub-healthy state, with only 5% being healthy, and the sub-health status of young and middle-aged adults aged 20-45 is particularly severe. Therefore, sub-health is a significant potential health threat that urgently requires attention and timely intervention. However, currently, there are no probiotic fermented milk products that can alleviate sub-health.

[0004] Fermentation strains are the core of fermented milk preparation. During fermentation, they participate in metabolism through their own processes or enzymes, utilizing some carbohydrates, proteins, and fats in the fermentation raw materials to produce metabolic products such as organic acids, amino acids, extracellular polysaccharides, and some aromatic compounds, thereby giving fermented milk its unique texture and flavor. Currently, there are no reports on fermentation strains for probiotic fermented milk used to alleviate sub-health conditions. Summary of the Invention

[0005] This invention provides a starter culture for fermented milk, the fermented milk obtained therefrom, and a method for preparing fermented milk.

[0006] To address the problems existing in current technologies, this invention aims to develop functional probiotic fermented milk that alleviates sub-health symptoms. Functional fermented milk must not only guarantee good functionality but also maintain the typical properties of fermented milk, such as taste, flavor, and stability. During the research and development process, this invention discovered that the fermentation strain has a significant impact on the functionality, stability, taste, and flavor of probiotic fermented milk. By comparing the aforementioned properties of fermented milk obtained from different probiotic blends, this invention found that four probiotic strains—Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8—can synergistically act in terms of metabolism and enzyme production during fermentation. Combined with a basic starter culture, the resulting fermented milk not only possesses superior taste, flavor, and stability but also exhibits significantly enhanced functionality in alleviating sub-health conditions.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a starter culture for fermented milk, the starter culture for fermented milk comprising a basic starter culture and co-fermenting probiotics;

[0009] The co-fermenting probiotics include Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8.

[0010] The preservation number of the Lactobacillus helveticus H9 is CGMCC No. 4811;

[0011] The preservation number of the Lactobacillus paracasei PC-01 is CGMCC No. 17537;

[0012] The preservation number of the Lactobacillus plantarum LP-6 is CGMCC No. 16661;

[0013] The preservation number of the Bifidobacterium lactis Probio-M8 is CGMCC No.18610.

[0014] Among the above strains, Lactobacillus helveticus H9 has been disclosed in patent application CN102329763 A. It was deposited on April 28, 2011, 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, postcode 100101), and classified as Lactobacillus helveticus, with accession number CGMCC No. 4811.

[0015] Lactobacillus paracasei PC-01 has been disclosed in patent application CN111575207A and was deposited on April 9, 2019, 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, postcode 100101). It is classified and named Lactobacillus paracasei, with accession number CGMCC No. 17537.

[0016] Lactobacillus plantarum LP-6 has been disclosed in patent application CN111548972A and was deposited on October 31, 2018 at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with the classification name Lactobacillus plantarum and accession number CGMCC No. 16661.

[0017] Bifidobacterium lactis (Probio-M8) has been disclosed in patent application CN111172074A and was deposited on September 20, 2019, 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, postcode 100101). It is classified and named Bifidobacterium lactis, with accession number CGMCC No. 18610.

[0018] Experimental verification has shown that Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 work together during fermentation. The absence or replacement of any one of these probiotic strains will lead to a significant decrease in the fermented milk's ability to alleviate sub-health symptoms and / or its taste, flavor, and stability.

[0019] In the fermentation agents for fermented milk described above, the ratio of the effective live bacteria count of the basic fermentation agent to the co-fermenting probiotics is 1:(0.1-5).

[0020] In this invention, the basic starter culture is the same starter culture used to prepare conventional, non-functional fermented milk. Controlling the inoculation amounts of the basic starter culture and the co-fermenting probiotics within the aforementioned ratio range is more conducive to the synergistic effect of the co-fermenting probiotics and the basic starter culture, thereby enhancing the function of the fermented milk in alleviating sub-health symptoms.

[0021] In the co-fermented probiotics mentioned above, the effective live bacteria ratio of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 is (0.5-5):100:200:200.

[0022] This invention has found that Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 can better exert a synergistic effect within the above-mentioned inoculation ratio range, thereby improving the function of fermented milk in alleviating sub-health symptoms, while better ensuring taste, flavor, and stability.

[0023] The basic starter cultures mentioned above include Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0024] Preferably, the basic fermentation agent includes Streptococcus salivarius thermophilus subsp. MN-ZLW-001, Streptococcus salivarius thermophilus subsp. MN-ZLW-002 and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003;

[0025] The preservation number of the thermophilic subspecies of Streptococcus salivarius MN-ZLW-001 is CGMCC No.3816;

[0026] The preservation number of the thermophilic subspecies of Streptococcus salivarius MN-ZLW-002 is CGMCC No. 3817;

[0027] The Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 has the accession number CGMCC No. 3818.

[0028] The aforementioned *Streptococcus salivarius* subsp. *thermophilus* MN-ZLW-001, *Streptococcus salivarius* subsp. *thermophilus* MN-ZLW-002, and *Lactobacillus delbrueckii* subsp. *bulgaricus* MN-ZLW-003 have all been disclosed in patent application CN116410880A.

[0029] Preferably, in the basic fermentation agent, the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-001, Streptococcus salivarius subsp. thermophilus MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is (1-50):(1-50):1.

[0030] Using the basic starter culture described above can better synergize with the aforementioned co-fermenting probiotics to jointly improve the taste, flavor, stability, and functionality of fermented milk.

[0031] As a preferred embodiment of the present invention, the basic fermenting agent is MN01, which was previously developed by the applicant.

[0032] Secondly, the present invention provides the application of the above-described starter culture for fermented milk in the preparation of fermented milk.

[0033] Specifically, the application includes: using the fermenting agent for fermented milk as a fermenting agent to ferment milk raw materials to obtain fermented milk.

[0034] Preferably, the fermented milk has the function of relieving sub-health symptoms, improving sleep quality and / or relieving anxiety.

[0035] Thirdly, the present invention provides a fermented milk, wherein the raw materials of the fermented milk include milk raw materials and the fermenting agent for fermented milk described above.

[0036] In addition to milk raw materials and starter culture, the raw materials for the fermented milk also include sweeteners, starch and seaweed powder.

[0037] Preferably, the raw materials comprise the following components in parts by weight: 76-98 parts of milk raw material, 1.0-10.5 parts of sweetener, 1.20-2.40 parts of starch, 0.15-0.25 parts of seaweed powder, and 0.04-0.2 parts of fermenting agent for fermented milk.

[0038] This invention has discovered that by using the above-mentioned optimized starter culture, it is possible to ensure that the fermented milk has a suitable viscosity and excellent stability without adding edible colloids, but only adding a small amount of food ingredient seaweed powder. It will not exhibit water separation or stratification within a 6-month shelf life, and the fermented milk has a refreshing and smooth taste.

[0039] The aforementioned starter culture for fermented milk includes a basic starter culture and co-fermenting probiotics, wherein the preferred inoculum amount of the basic starter culture is 1.0 × 10⁻⁶. 5 -1.0×10 7 CFU / g fermentation substrate, preferably 5.0 × 10⁻⁶. 5 -5.0×10 6 The optimal inoculum size for co-fermenting probiotics is 1.0 × 10⁻⁶ CFU / g fermentation substrate. 5 -2.0×10 7 CFU / g fermentation substrate, preferably 5.0 × 10⁻⁶. 5 -5.0×10 6 CFU / g fermentation base. The fermentation base consists of raw materials other than the starter culture. The resulting fermented milk exhibits superior functionality in alleviating sub-health symptoms, a smoother and more delicate texture, and a richer, fuller flavor.

[0040] In some specific embodiments of the present invention, the starter culture for fermented milk includes 0.02-0.12 parts of basic starter culture and 0.02-0.08 parts of co-fermenting probiotics.

[0041] Preferably, the raw materials comprise the following components in parts by weight: 88-92 parts milk raw material, 6-9 parts sweetener, 1.20-1.80 parts starch, 0.20-0.25 parts seaweed powder, and 0.04-0.2 parts fermenting agent for fermented milk.

[0042] The sweeteners mentioned above are selected from one or more of the following: white sugar, glucose, fructose, aspartame, cyclamate, acesulfame potassium, erythritol, xylitol, and sucralose.

[0043] The milk raw materials mentioned above are raw milk and / or reconstituted milk.

[0044] Preferably, the sweetener is white sugar. The milk raw material is raw milk. The starch is corn starch.

[0045] The raw materials for the fermented milk may also include, or not include, protein powder, dietary fiber, water, etc., as needed.

[0046] This invention was validated through human trials. By combining metagenomics, metabolomics, and questionnaires, the changes in gut microbiota and intestinal metabolites in subjects before and after fermented milk intervention were analyzed, along with the effect of fermented milk on improving symptoms such as sleep disorders and low mood in sub-healthy individuals. The results showed that the fermented milk of this invention can effectively improve symptoms such as sleep disorders and low mood in sub-healthy individuals by regulating the composition and structure of gut microbiota, thereby affecting the production of short-chain fatty acids, neurotransmitters, and other microbial metabolites, or regulating the levels of immune factors.

[0047] Fourthly, the present invention provides a method for preparing the fermented milk described above, wherein the raw materials are mixed and fermented, and after fermentation, demulsification and cooling are performed.

[0048] Preferably, the milk raw materials, sweetener, starch and seaweed powder are mixed to obtain a mixture, which is then sterilized and inoculated with a fermenting agent for fermentation.

[0049] Preferably, the fermentation temperature is 33-43℃, more preferably 35-38℃, and even more preferably 36-37℃.

[0050] After fermentation, the milk is quickly broken down and cooled to 2-10°C, and then subjected to two pasteurization processes to obtain the fermented milk.

[0051] The beneficial effects of this invention include at least the following: the fermenting agent for fermented milk provided by this invention, through the synergistic effect of various bacterial strains, can significantly increase the content of functional substances that improve sub-health and alleviate sub-health symptoms in fermented milk when used in fermented milk preparation. It promotes the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the intestine, which can significantly regulate the intestinal flora, improve intestinal health, regulate human metabolism and immune function, promote the increase of neurotransmitter GABA content, and reduce the content of 5-HT and 5-HIAA, which is beneficial to improve sleep quality and relieve anxiety. At the same time, it can also better ensure the taste, flavor, and stability of fermented milk.

[0052] Fermented milk prepared using the above-mentioned fermenting agent has excellent taste and flavor, long shelf life and functionality. It can effectively relieve sub-health symptoms, has no toxic side effects, tastes sweet, delicate and smooth, has a rich and full flavor, and is generally well-liked. Moreover, the product does not separate or separate when stored at room temperature for 6 months, and is convenient to store and consume. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0054] Example 1

[0055] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 91.0 parts raw milk, 7.0 parts white sugar, 1.8 parts starch, 0.2 parts seaweed powder, and a basic starter culture MN01 (the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-001, Streptococcus salivarius subsp. thermophilus MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is 1:1:1) with an inoculation amount of 1.0 × 10⁻⁶. 6 The cfu / g inoculum of the co-fermenting probiotics (with a live bacteria count ratio of 3:100:200:200 of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 2.0 × 10⁻⁶. 6 cfu / g.

[0056] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0057] (1) After mixing raw milk, sweetener, starch and seaweed powder, sterilize them, then inoculate with basic starter culture and co-fermenting probiotics for fermentation at a temperature of 36°C.

[0058] (2) When the pH value reaches 4.55, the milk is quickly broken and cooled to 2-10℃. After two pasteurization treatments, fermented milk is obtained.

[0059] Example 2

[0060] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 90.0 parts raw milk, 8.0 parts white sugar, 1.8 parts starch, 0.2 parts seaweed powder, and a basic starter culture MN01 (the ratio of the effective viable counts of Streptococcus salivarius subsp. MN-ZLW-001, Streptococcus salivarius subsp. MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is 50:1:1) with an inoculum amount of 5.0 × 10⁻⁶. 5 The cfu / g inoculum of the co-fermenting probiotics (with a live bacteria count ratio of 3:100:200:200 of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 2.0 × 10⁻⁶. 6 cfu / g.

[0061] This embodiment also provides a method for preparing the above-mentioned fermented milk, which is the same as that in Example 1.

[0062] Example 3

[0063] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 90.0 parts raw milk, 8.0 parts white sugar, 1.75 parts starch, 0.25 parts seaweed powder, and a basic starter culture MN01 (the ratio of the effective viable counts of Streptococcus salivarius subsp. MN-ZLW-001, Streptococcus salivarius subsp. MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is 1:50:1) with an inoculum amount of 5.0 × 10⁻⁶. 6 The cfu / g concentration of the co-fermenting probiotics (the effective viable count ratio of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 was 5:100:200:200) was 5.0 × 10⁻⁶. 5 cfu / g.

[0064] This embodiment also provides a method for preparing the above-mentioned fermented milk, which is the same as that in Example 1.

[0065] Example 4

[0066] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 90.6 parts raw milk, 8.0 parts white sugar, 1.2 parts starch, 0.2 parts seaweed powder, and a basic starter culture MN01 (the ratio of the effective viable counts of Streptococcus salivarius subsp. MN-ZLW-001, Streptococcus salivarius subsp. MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is 50:50:1) with an inoculum amount of 1.0 × 10⁻⁶. 6 The cfu / g concentration of the co-fermenting probiotics (the ratio of effective viable bacteria to effective live bacteria of *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 0.5:100:200:200) was 5.0 × 10⁻⁶. 6 cfu / g.

[0067] This embodiment also provides a method for preparing the above-mentioned fermented milk, which differs from the preparation method in Example 1 only in that the fermentation temperature is 37°C.

[0068] Example 5

[0069] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 90.6 parts raw milk, 8.0 parts white sugar, 1.2 parts starch, 0.2 parts seaweed powder, and a basic starter culture MN01 (the ratio of the effective viable counts of Streptococcus salivarius subsp. MN-ZLW-001, Streptococcus salivarius subsp. MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is 1:1:1) with an inoculum amount of 1.0 × 10⁻⁶. 6 The cfu / g inoculum of the co-fermenting probiotics (with a live bacteria count ratio of 3:100:200:200 of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 1.0 × 10⁻⁶. 6 cfu / g.

[0070] This embodiment also provides a method for preparing the above-mentioned fermented milk, which differs from the preparation method in Example 1 only in that the fermentation temperature is 37°C.

[0071] Comparative Example 1

[0072] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that Lactobacillus helveticus H9 is removed from the co-fermenting probiotics, while the proportions of the remaining strains remain unchanged.

[0073] The preparation method of the fermented milk described above is the same as in Example 4.

[0074] Comparative Example 2

[0075] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that Lactobacillus paracasei PC-01 is removed from the co-fermenting probiotics, while the proportions of the remaining strains remain unchanged.

[0076] The preparation method of the fermented milk described above is the same as in Example 4.

[0077] Comparative Example 3

[0078] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that Lactobacillus plantarum LP-6 is removed from the co-fermenting probiotics, while the proportions of other strains remain unchanged.

[0079] The preparation method of the fermented milk described above is the same as in Example 4.

[0080] Comparative Example 4

[0081] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that Bifidobacterium lactis Probio-M8 is removed from the co-fermenting probiotics, while the proportions of other strains remain unchanged.

[0082] The preparation method of the fermented milk described above is the same as in Example 4.

[0083] Comparative Example 5

[0084] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that: in the co-fermenting probiotics, Bifidobacterium lactis Probio-M8 is replaced with Bifidobacterium BB12.

[0085] The preparation method of the fermented milk described above is the same as in Example 4.

[0086] Comparative Example 6

[0087] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that the ratio of effective live bacteria counts of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermentation probiotics is 10:100:200:200.

[0088] The preparation method of the fermented milk described above is the same as in Example 4.

[0089] Comparative Example 7

[0090] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that the ratio of the effective live bacteria counts of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermentation probiotics is 0.3:100:200:200.

[0091] The preparation method of the fermented milk described above is the same as in Example 4.

[0092] Comparative Example 8

[0093] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that: in the co-fermenting probiotics, Lactobacillus helveticus H9 is replaced with Lactobacillus helveticus R0052 (which can be purchased commercially).

[0094] The preparation method of the fermented milk described above is the same as in Example 4.

[0095] Comparative Example 9

[0096] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that: in the co-fermenting probiotics, *Lactobacillus paracasei* PC-01 is replaced with *Lactobacillus paracasei* L.CASEI 431. TM (Available through retail channels).

[0097] The preparation method of the fermented milk described above is the same as in Example 4.

[0098] Comparative Example 10

[0099] This comparative example provides a fermented milk whose raw material composition differs from that of Example 4 only in that: in the co-fermenting probiotics, Lactobacillus plantarum LP-6 is replaced with Lactobacillus plantarum R1012 (which can be purchased commercially).

[0100] The preparation method of the fermented milk described above is the same as in Example 4.

[0101] Example 1: Evaluation of the physicochemical parameters, stability, and taste / flavor of fermented milk

[0102] The fermented milk in the above examples and comparative examples was evaluated for its physicochemical parameters (particle size, viscosity), stability (clarification index, water separation and stratification during shelf life), and taste and flavor (taste, flavor description, preference rating).

[0103] The sensory evaluation method is as follows: A suitable amount of test sample is placed in a 50mL tasting cup for sensory evaluation by 50 participants. The color and texture are observed under natural light, the aroma is smelled, the participants rinse their mouths with warm water, and the taste is evaluated. The sensory evaluation includes a preference test for the test sample (overall preference score of 1-9, percentage of participants with a score of 6 or higher), with evaluation criteria shown in Table 1, and a descriptive test (15 sensory attribute descriptive words, 1-5 points), with evaluation criteria shown in Table 2.

[0104] Table 1 Overall Preference Evaluation Criteria

[0105]

[0106] Table 2 Evaluation Criteria for Sensory Descriptive Tests

[0107]

[0108] The evaluation results are shown in Table 3.

[0109] Table 3. Physicochemical parameters, stability, and taste evaluation results of fermented milk.

[0110]

[0111]

[0112] Experiment Example 2: Detection of Functional Substance Content in Fermented Milk

[0113] The content of functional substances in the probiotic fermented milk of Examples 1-5 and the fermented milk of each comparative example were detected. The experimental method and results are described in detail below.

[0114] Reagents: LC-MS grade methanol (MeOH) and LC-MS grade acetonitrile (ACN) were purchased from Fisher Scientific (Loughborough, UK); 2-amino-3-(2-chlorophenyl)-propionic acid was produced by Aladdin, Shanghai, China; formic acid was obtained from TCI; ammonium formate was obtained from Sigma; ultrapure water was generated using the Milli Q system (Millipore, Bedford, USA).

[0115] Instruments: High-speed centrifuges were provided by Hunan Xiangyi Experimental Equipment Co., Ltd., centrifugal vacuum evaporators were from Ependorf China Co., Ltd., vortex mixers were provided by Haimen Qilin Bell Laboratory Instruments Co., Ltd., and microporous membrane filters (0.22μm) were purchased from Tianjin Jienteng Experimental Equipment Co., Ltd.

[0116] Methods and Algorithms: Global Untargeted Metabolomics examines the qualitative and quantitative changes of all small molecule metabolites in the system, providing high-throughput and full annotation of specific substances; based on the Thermo Q-Exactive high-resolution mass spectrometry platform, it integrates MoNA, METLIN, HMDB, LipidSearch, as well as a self-built database system and proprietary core algorithms.

[0117] The results are shown in Tables 4 and 5. Compared with Comparative Examples 1 and 5, the contents of the five substances related to sub-health improvement in the fermented milk of Examples 1-5 were significantly increased (P<0.05). The results indicate that the fermented milk of the present invention has the potential to improve sub-health.

[0118] Table 4 shows the relative content multiples of each differentially functional substance in the fermented milk of Example 4 compared to Examples 1-3, 5 and Comparative Examples 1 and 5.

[0119]

[0120] Note: The values ​​in Table 4 are the ratios of the content of each functional substance in the fermented milk of Example 4 to the content of each functional substance in the fermented milk of Examples 1-3, 5 and Comparative Examples 1 and 5. The higher the corresponding ratio, the lower the content of the functional substance.

[0121] Table 5. Differential functional substances and functional characteristics in fermented milk

[0122]

[0123] Furthermore, testing revealed that the content of the five functional substances shown in Table 5 in the fermented milk of the other comparative examples was also significantly lower than that in Examples 1-5.

[0124] Experiment Example 3: Human Experiment with Fermented Milk

[0125] While ensuring product stability and flavor, the probiotic fermented milk from Example 4 was selected for a human trial. The experimental setup method and the effect of the fermented milk in alleviating sub-health symptoms are described in detail below.

[0126] 1. Establishment of experimental study on the relief of sub-health conditions by probiotic fermented milk in people.

[0127] 1.1 Volunteer Inclusion Criteria

[0128] A. No gender restriction, age 30-60;

[0129] B. Sub-health assessment scale (SHMS) diagnosis of sub-health state (physiological conversion score <68 points, psychological conversion score <67 points, or social conversion score <67 points);

[0130] C. Voluntarily sign the informed consent form.

[0131] 1.2 Exclusion Criteria

[0132] A. Those who do not meet the above inclusion criteria;

[0133] B. Subjects who have been diagnosed with other diseases;

[0134] C. Subjects whose blood routine and biochemical indicators are outside the normal range may be in a disease state;

[0135] D. Has used antibiotics, probiotics, or synbiotics within the past month;

[0136] E. Individuals allergic to the sample or its components;

[0137] F. Has taken anti-anxiety, antidepressant or other psychotropic medications in the past month.

[0138] A human trial investigating the use of probiotic fermented milk to alleviate sub-health conditions recruited 49 volunteers. Participants consumed 200g / day of probiotic fermented milk for four consecutive weeks. Blood and stool samples were collected at the start of the trial (T0), on day 28 (T1), and 14 days after the trial (T2). Questionnaires were completed, including the Sub-health Measurement Scale Version (SHMS) and the Pittsburgh Sleep Quality Index (PSQI). The SHMS included three subscales: physical sub-health (PS), mental sub-health (MS), and social sub-health (SS). Lower total scores indicated more severe sub-health conditions, while lower PSQI scores indicated better sleep quality. The Wilcoxon Test was used to examine the differences in questionnaire results before and after probiotic consumption.

[0139] The results of the population experiment on the sub-health rating scale are shown in Table 6.

[0140] Table 6 Questionnaire survey on the intervention of fermented milk in sub-health symptoms

[0141]

[0142]

[0143] As shown in Table 6, the SHMS scores of subjects who consumed probiotic fermented milk were significantly higher at the end of the trial period (T1) than at the beginning of the trial (P = 0.02), and the effect persisted at the end of the follow-up period (T2), indicating that probiotic fermented milk significantly alleviated the sub-health state of the subjects. Furthermore, consuming probiotic fermented milk significantly reduced the subjects' PSQI, and the effect persisted at the end of the follow-up period (T2), indicating that drinking probiotic fermented milk can effectively improve the sleep quality of sub-healthy individuals (P = 0.0015).

[0144] 2. The effect of probiotic fermented milk on intestinal flora

[0145] DNA was extracted from fecal samples. The DNA that met sequencing requirements was then randomly fragmented, end-repaired, ligated with adapters, and used to construct libraries. The samples were then sequenced using Illumina GAIIx and HiSeq2000 to analyze the effects of probiotic fermented milk on gut microbiota functional genes and metabolic pathways.

[0146] The results showed that there was no obvious clustering trend after drinking fermented milk, meaning that drinking fermented milk did not affect the overall structure of the gut microbiota in sub-healthy individuals. After drinking fermented milk, the diversity of gut microbiota in sub-healthy individuals decreased, but the difference was not significant. Comparing the gut microbiota with those that showed significant differences in relative abundance between 0 days and 4 weeks after drinking fermented milk, it was found that the abundance of Firmicutes and Bacteroidetes decreased, while the abundance of Proteobacteria and Actinobacteria increased (Table 7).

[0147] Table 7. Effects of drinking fermented milk on the relative abundance (%) of dominant bacterial phyla in sub-healthy individuals.

[0148]

[0149]

[0150] The results of the study on the effects of probiotic fermented milk on the characteristic gut microbiota of sub-healthy individuals (Table 8) revealed changes in 10 bacterial genera associated with sub-health: the abundance of 4 beneficial bacteria increased, while the abundance of 6 harmful bacteria decreased. These results indicate that fermented milk has the effect of improving the gut microbiota of sub-healthy individuals.

[0151] Table 8. Effects of fermented milk on gut microbiota in sub-healthy individuals

[0152]

[0153] In conclusion, fermented milk can fine-tune the intestinal flora, increasing beneficial bacteria and reducing harmful bacteria, which plays a positive role in maintaining intestinal flora homeostasis.

[0154] 3. The effect of probiotic fermented milk on fecal metabolites in sub-healthy individuals

[0155] Fecal samples were pretreated, and the full spectrum of fecal samples was determined non-targeted using LC-MS technology.

[0156] After consuming fermented milk, volunteers showed significant differences in their fecal metabolites: the levels of creatinine, L-carnitine, and lithocholic acid changed markedly. L-carnitine promotes increased fatty acid metabolism and facilitates the conversion of fat into energy, thus contributing to weight loss and anti-fatigue effects. Lithocholic acid regulates Treg cell differentiation and inhibits pro-inflammatory cytokines, playing a role in regulating human metabolism and immune function; therefore, it is speculated that fermented milk can improve gut health.

[0157] In summary, the mechanism by which the fermented milk of the present invention alleviates sub-health is through fine-tuning the intestinal flora, regulating inflammatory factors and secondary bile acid metabolites, and producing metabolites that are beneficial to the host.

[0158] 4. The effect of probiotic fermented milk on short-chain fatty acids in the feces of sub-healthy individuals

[0159] Short-chain fatty acids (SCFAs) are indirect nutrients produced by gut microbiota and typically have significant physiological regulatory effects, such as providing some of the energy needed by the human body, regulating electrolyte balance, protecting the intestinal mucosal barrier, promoting nutrient absorption, regulating lipid metabolism, inhibiting intestinal inflammation, and exhibiting anti-tumor effects and modulating immune responses. Table 9 shows that compared with baseline, the acetic acid content in the intestines of subjects who consumed probiotic fermented milk significantly increased (P<0.05), while the contents of propionic acid and butyric acid both increased, but the differences were not significant.

[0160] Table 9. Short-chain fatty acid production in fecal microbiota of sub-healthy volunteers.

[0161]

[0162] 5. Effects of probiotic fermented milk on serum neurotransmitters in sub-healthy individuals

[0163] Neurotransmitters are the mediators of communication between cells and play a wide and important regulatory role. The gut microbiota also produces some neuroactive metabolites, such as neurotransmitters or their precursors, which may directly or indirectly affect neuronal activity and cognitive function in the brain. Major neurotransmitters, such as dopamine, acetylcholine, and gamma-aminobutyric acid (GABA), are well-known bioactive metabolites that regulate synaptic transmission in neurons of the central nervous system and have properties that influence sleep, appetite, mood, and cognition.

[0164] Fasting venous blood was collected from the subjects upon waking in the morning, centrifuged (3000 rpm, 10 min) after standing, and the serum levels of 21 neurotransmitters were measured. These neurotransmitters were 4-aminobutyric acid (GABA), histamine (HIsA), pyridine carboxylic acid (PA), tyramine (TyrA), acetylcholine chloride (Ach), glutamine (Gln), glutamate (Glu), dopamine hydrochloride (DA), histidine (His), tryptophan (TrpA), norepinephrine hydrochloride (NE), serotonin hydrochloride (5-HT), tyrosine (Tyr), adrenaline hydrochloride (E), kynurenic acid (KynA), 5-hydroxyindoleacetic acid (5-HIAA), levodopa (DOPA), tryptophan (Trp), xanthuric acid (XA), kynurenine (Kyn), and vanillylmandelic acid (VMA). The results of some neurotransmitter levels are shown in Table 10.

[0165] Table 10 Results of serum neurotransmitter content detection in sub-healthy volunteers

[0166]

[0167] The results showed that after consuming probiotic fermented milk for 4 weeks, the serum GABA levels significantly increased, while the levels of 5-HT and 5-HIAA significantly decreased (P<0.05). Studies have shown that a deficiency of GABA in the body can lead to anxiety, restlessness, fatigue, and worry. Therefore, consuming probiotic fermented milk can improve anxiety in sub-healthy individuals.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A starter culture for room-temperature fermented milk, characterized in that, The fermenting agent for room-temperature fermented milk consists of a basic fermenting agent and co-fermenting probiotics; The co-fermenting probiotics are Lactobacillus helveticus (Lactobacillus tumefaciens). Lactobacillus helveticus H9, Lactobacillus paracasei ( Lactobacillus paracasei PC-01, Lactobacillus plantarum ( Lactobacillus plantarum LP-6 and Bifidobacterium lactis ( Bifidobacterium lactis Probio-M8 consists of; The preservation number of the Lactobacillus helveticus H9 is CGMCC No. 4811; The preservation number of the Lactobacillus paracasei PC-01 is CGMCC No. 17537; The preservation number of the Lactobacillus plantarum LP-6 is CGMCC No. 16661; The preservation number of the Bifidobacterium lactis Probio-M8 is CGMCC No. 18610; The basic starter culture consists of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. In the fermentation agent for room temperature fermented milk, the ratio of the effective live bacteria count of the basic fermentation agent to the co-fermenting probiotics is 1:(0.1-5). In the co-fermented probiotics, the ratio of effective live bacteria counts of Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 is (0.5-5):100:200:

200.

2. The starter culture for room-temperature fermented milk according to claim 1, characterized in that, The basic fermentation agent consists of Streptococcus salivarius thermophilus subsp. MN-ZLW-001, Streptococcus salivarius thermophilus subsp. MN-ZLW-002 and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003; The preservation number of the *Streptococcus salivarius* subsp. *thermophilus* MN-ZLW-001 is CGMCC No. 3816; The preservation number of the thermophilic subspecies of Streptococcus salivarius MN-ZLW-002 is CGMCC No.3817; The Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 has the accession number CGMCC No. 3818.

3. The starter culture for room-temperature fermented milk according to claim 2, characterized in that, In the basic fermentation agent, the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-001, Streptococcus salivarius subsp. thermophilus MN-ZLW-002, and Lactobacillus delbrueckii subsp. bulgaricus MN-ZLW-003 is (1-50):(1-50):

1.

4. The use of the starter culture for room temperature fermented milk according to any one of claims 1 to 3 in the preparation of room temperature fermented milk.

5. A room-temperature fermented milk, characterized in that, The raw materials for the room-temperature fermented milk include milk raw materials and the fermenting agent for room-temperature fermented milk as described in any one of claims 1 to 3.

6. The room-temperature fermented milk according to claim 5, characterized in that, The raw materials comprise the following components in parts by weight: 76-98 parts milk raw material, 1.0-10.5 parts sweetener, 1.20-2.40 parts starch, 0.15-0.25 parts seaweed powder, and 0.04-0.2 parts starter for room temperature fermented milk.

7. The room-temperature fermented milk according to claim 6, characterized in that, The sweetener is selected from one or more of white sugar, glucose, fructose, erythritol, and xylitol; And / or, the milk raw material is raw milk and / or reconstituted milk.

8. The method for preparing room-temperature fermented milk according to any one of claims 5 to 7, characterized in that, The raw materials are mixed and fermented. After fermentation, demulsification and cooling are carried out.

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