A compound starter culture, fermented milk and a method for preparing the same

Abstract

The present application relates to the technical field of fermented milk, in particular to a compound starter culture, fermented milk and a preparation method thereof. The compound starter culture provided by the present application can significantly improve the content of functional substances with the effect of improving sub-health and the function of relieving sub-health in the fermented milk prepared therefrom, while improving the taste and flavor and stability of the fermented milk. The fermented milk provided by the present application has excellent taste and flavor, the taste is delicate and smooth, and the taste experience is melting in the mouth, the flavor is rich and full, the overall product preference is excellent, and the fermented milk has the characteristics of long shelf life, convenient storage and consumption, etc. In addition, the fermented milk can significantly regulate intestinal flora, affect neurotransmitter levels, improve sleep quality, relieve anxiety, and effectively relieve sub-health symptoms.

Description

Technical Field

[0001] This invention relates to the field of fermented milk technology, and in particular to a compound fermenting agent, fermented milk and its preparation method. Background Technology

[0002] With the improvement of living standards and the development of the social economy, people's lifestyles have changed. Various unhealthy eating habits, as well as work and life pressures, can all harm human health. In recent years, the sub-health state has gradually attracted social attention, and how to prevent and alleviate sub-health has become a research hotspot. Sub-health status (SHS) refers to a state between health and disease, specifically manifested as symptoms such as lethargy, fatigue, sleep disorders, anxiety, depression, and memory loss, even without diseases or defects in organs, tissues, or functions. A WHO predictive survey shows that 75% of the global population is in a sub-health state, with only 5% being healthy. The sub-health status of young and middle-aged people aged 20-45 is particularly severe. Therefore, sub-health is a potential health threat that cannot be ignored and urgently requires attention and timely intervention.

[0003] In recent years, functional fermented milk has gradually attracted attention. However, existing functional fermented milks mostly acquire their functional properties by adding plant extracts and other functional ingredients. Currently, there are no reports of probiotic fermented milk that can alleviate sub-health conditions without adding plant extracts or other functional ingredients and utilizing fermentation strains. Summary of the Invention

[0004] This invention provides a compound fermentation agent, the fermented milk obtained therefrom, and a method for preparing fermented milk.

[0005] To address the problems existing in current technologies, this invention aims to develop functional probiotic fermented milk that alleviates sub-health conditions. Functional probiotic fermented milk needs to simultaneously achieve functionality, good taste and flavor, and stability. During the research and development process, this invention discovered that the fermentation strain has a significant impact on the functionality, stability, and taste of probiotic fermented milk in alleviating sub-health conditions. By comparing the above-mentioned properties of fermented milk obtained from different probiotic blends, this invention identified a blended fermentation agent that can simultaneously achieve functionality, good taste and flavor, and stability. The fermented milk produced using this agent not only has high taste and flavor and stability but also significantly improved functionality in alleviating sub-health conditions.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a compound fermentation agent, wherein the compound fermentation agent comprises a basic fermentation agent and synergistic fermentation probiotics;

[0008] The co-fermenting probiotics include Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8.

[0009] The preservation number of the thermophilic subspecies of Streptococcus salivarius MN-ZLW-002 is CGMCC No.3817;

[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] This invention has discovered that five probiotic strains—Streptococcus salivarius thermophilus subsp. MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8—can synergistically enhance the functionality of the fermented probiotic milk in alleviating sub-health conditions during fermentation through metabolism and enzyme production. The absence or replacement of any one of these probiotic strains will lead to a significant decrease in the functionality of the fermented milk in alleviating sub-health conditions and / or in terms of taste, flavor, and stability.

[0015] Among the above strains, *Streptococcus thermophilus* subspecies MN-ZLW-002 has been disclosed in patent application CN102465108A. It was deposited on May 7, 2010, 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 *Streptococcus thermophilus*, with accession number CGMCC No. 3817.

[0016] Lactobacillus helveticus H9 has been disclosed in patent application CN102329763 A and 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), classified and named Lactobacillus helveticus, with accession number CGMCC No. 4811.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In the compound fermentation agent described above, the ratio of the effective live bacteria count of the basic fermentation agent to the co-fermenting probiotics is 1:(1-20).

[0021] In this invention, the basic starter culture is the starter culture used to prepare conventional, non-functional fermented milk, and its main function is to promote the fermentation process of the milk raw materials. Controlling the inoculation amounts of the basic starter culture and the co-fermenting probiotics within the above-mentioned ratio range is more conducive to the synergistic effect of the co-fermenting probiotics and the basic starter culture, thereby improving the function of fermented milk in alleviating sub-health conditions, as well as its taste, flavor, and stability.

[0022] Among the co-fermented probiotics mentioned above, the ratio of effective viable bacteria counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 is 300:(0.5-5):100:200:(100-300).

[0023] This invention has found that the amount of *Lactobacillus helveticus* H9 added to the synergistic fermentation probiotics needs to be controlled at the aforementioned low level. Increasing its addition will affect the flavor of the fermented milk, presumably because the increased addition may enhance the protein hydrolysis capacity of *Lactobacillus helveticus*, resulting in a poor flavor. *Streptococcus salivarius* subsp. *thermophilus* MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 can better exert a synergistic effect within the aforementioned inoculum ratio range, improving the function of fermented milk in alleviating sub-health conditions, while better ensuring taste, flavor, and stability.

[0024] The basic starter cultures mentioned above include Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0025] Preferably, in the basic fermentation agent, the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is (1-100):1.

[0026] As a preferred embodiment of the present invention, the base starter is Danisco YO-MIX T12. This starter is commercially available and features rapid fermentation with a prominent sweet cream and milky flavor.

[0027] This invention has found that using the above-mentioned basic starter culture can better synergize with the co-fermenting probiotics to jointly promote the taste, flavor, stability, and functionality of fermented milk.

[0028] Secondly, the present invention provides the application of the above-described compound fermentation agent in the preparation of fermented milk.

[0029] Specifically, the application includes: fermenting raw materials for fermented milk preparation, including milk raw materials, with the compound fermenting agent to obtain fermented milk.

[0030] Preferably, the fermented milk has the function of relieving sub-health symptoms, improving sleep quality and / or relieving anxiety.

[0031] Thirdly, the present invention provides a fermented milk, wherein the raw materials of the fermented milk include milk raw materials, prebiotics and the compound fermentation agent described above.

[0032] In addition to milk raw materials, prebiotics, and compound fermentation agents, the raw materials of the fermented milk also include sweeteners, stabilizers, etc.

[0033] Preferably, the raw materials comprise the following components in parts by weight: 76-92 parts milk raw material, 0.5-8.5 parts sweetener, 1.5-2.6 parts stabilizer, 2.0-3.0 parts prebiotic, and 0.04-0.2 parts compound fermentation agent;

[0034] The inoculum size of the basic fermentation agent was 5.0 × 10⁻⁶. 4 -5.0×10 5 The inoculum density of CFU / g fermentation substrate and the co-fermentation probiotic inoculum density is 1.0 × 10⁻⁶. 5 -2.0×10 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.

[0035] In some specific embodiments of the present invention, the compound fermentation agent includes 0.02-0.12 parts of basic fermentation agent and 0.02-0.08 parts of synergistic fermentation probiotics.

[0036] The addition of prebiotics to the fermented milk can further significantly improve its taste and stability.

[0037] The prebiotic is selected from one or more of polydextrose, resistant dextrin, inulin, soybean oligosaccharide, cottonseed oligosaccharide, spirulina, and chitosan oligosaccharide.

[0038] Preferably, the prebiotic is inulin. The appropriate addition of inulin can significantly reduce the particle size of fermented milk and improve the smoothness of the product. This is presumably because inulin contains both short-chain and long-chain fructans, which are more conducive to the uniform dispersion of fat globules in the fermented milk system.

[0039] The stabilizer mentioned above is one or more selected from physical starch, citrus fiber, and seaweed powder.

[0040] Preferably, the raw materials of the fermented milk do not contain edible colloids. This invention has found that by using the above-mentioned optimized compound fermenting agent, a smooth texture and system stability can be achieved without adding edible colloids, relying solely on the effects of physical starch, citrus fiber, seaweed powder, and other food ingredient stabilizers.

[0041] Preferably, the stabilizer is physical starch and citrus fiber. The preferred mass ratio of physical starch to citrus fiber is 6:1-10:1. Physical starch and citrus fiber work synergistically in the fermented milk system of this invention to form a stable suspension structure, preventing water separation and stratification. Citrus fiber is a dietary fiber extracted from citrus fruits. A possible preparation method is: citrus peel / pulp is crushed and washed, then extracted and separated, followed by water / alcohol washing, drying, and ultra-fine grinding with a 500-800 mesh mesh. Both physical starch and citrus fiber are food-grade raw materials, resulting in a stable fermented milk system with a natural, non-sticky taste, while also achieving label cleanliness.

[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 above-mentioned milk raw materials are raw milk and / or reconstituted milk.

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

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

[0046] This invention was validated through human trials. Using 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 significantly regulate gut microbiota. By adjusting the composition and structure of gut microbiota, it can influence the production of short-chain fatty acids, neurotransmitters, and other microbial metabolites, or regulate the levels of immune factors, effectively improving symptoms such as sleep disorders and low mood in sub-healthy individuals and reducing the severity of sub-health.

[0047] Fourthly, the present invention provides a method for preparing the fermented milk described above, the method comprising: mixing the raw materials and fermenting them, and after fermentation, breaking the emulsion and cooling the mixture.

[0048] Preferably, the compound fermentation agent is activated by dual-frequency ultrasound before being added.

[0049] This invention has found that dual-frequency ultrasonic activation is highly effective for the microorganisms in the compound fermentation agent. By first activating the compound fermentation agent with dual-frequency ultrasonic before inoculating it into the fermentation substrate for fermentation, the taste, flavor, and stability of the fermented milk can be significantly improved, as well as its functionality in alleviating sub-health conditions.

[0050] Preferably, the ultrasonic frequencies of the dual-frequency ultrasonic activation are 20kHz and 40kHz, the ultrasonic power is 400-600W, and the time is 10-30min.

[0051] Preferably, the fermentation ends when the pH is 4.40-4.50 and the acidity is 70-80°T.

[0052] Preferably, the fermentation temperature is 38-43°C.

[0053] Preferably, the method includes: mixing milk raw materials, prebiotics, sweeteners, and stabilizers to obtain a mixture; sterilizing the mixture; cooling it to 38-43°C; adding a compound fermentation agent activated by dual-frequency ultrasonic treatment for fermentation; when the pH reaches 4.40-4.50 and the acidity reaches 70-80°T, rapidly breaking the emulsion and cooling it to 2-8°C; post-ripening for 6-12 hours; performing a second pasteurization treatment; and then processing it through a smoothing filter to obtain fermented milk.

[0054] The beneficial effects of the present invention include at least the synergistic effect of the various strains in the compound fermentation agent provided by the present invention, which can significantly increase the content of functional substances that improve sub-health and alleviate sub-health status in the fermented milk produced therefrom, while improving the taste, flavor and stability of the fermented milk.

[0055] The fermented milk provided by this invention has excellent taste and flavor, with a delicate and smooth texture that melts in the mouth. It boasts a rich and full-bodied flavor, exhibiting excellent overall appeal. Furthermore, it demonstrates high stability, showing no water separation or stratification even after 6 months of storage at room temperature. It features a long shelf life and convenient storage and consumption. In addition, the fermented milk promotes the production of acetic acid, propionic acid, and butyric acid in the intestines, significantly regulating intestinal flora, neurotransmitter levels, and anti-inflammatory factor levels. This improves sleep quality, alleviates anxiety, and effectively relieves sub-health symptoms. Detailed Implementation

[0056] 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.

[0057] Example 1

[0058] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 88.0 parts raw milk, 7.0 parts white sugar, 2.0 parts stabilizer, 3.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 1.0 × 10⁻⁶. 5 The effective viable count ratio of the co-fermented probiotics (Streptococcus thermophilus subsp. MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 300:3:100:200:200, and the inoculum size was 1.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0059] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0060] Raw milk, sweetener, stabilizer, and inulin were mixed and sterilized. The mixture was then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 500W, ultrasonic time: 15min), and co-fermenting probiotics were added for fermentation. The fermentation temperature was 40°C. The fermentation endpoint was reached when the pH reached 4.40 and the acidity reached 70°T. The mixture was then rapidly demulsified and cooled to 4°C. After 12 hours of post-ripening, the mixture underwent a second pasteurization process and was then processed through a smoothing filter to obtain fermented milk.

[0061] Example 2

[0062] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 88.0 parts raw milk, 7.0 parts white sugar, 2.0 parts stabilizer, 3.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 5.0 × 10⁻⁶. 4 The effective viable count ratio of the co-fermented probiotics (Streptococcus thermophilus subsp. MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 300:3:100:200:200, and the inoculum size was 1.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0063] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0064] Raw milk, sweetener, stabilizer, and inulin are mixed and sterilized. The mixture is then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 400W, ultrasonic time: 30min), and co-fermenting probiotics are added for fermentation. The fermentation temperature is 40°C. When the pH reaches 4.50 and the acidity reaches 80°T, the milk is rapidly broken and cooled to 4°C. After 12 hours of post-ripening, the mixture undergoes a second pasteurization process and is then processed through a smoothing filter to obtain fermented milk.

[0065] Example 3

[0066] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 89.0 parts raw milk, 7.0 parts white sugar, 2.0 parts stabilizer, 2.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 5.0 × 10⁻⁶. 5The effective viable count ratio of the co-fermented probiotics (Streptococcus thermophilus subsp. MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8) was 300:3:100:200:200, and the inoculum size was 2.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0067] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0068] Raw milk, sweetener, stabilizer, and inulin were mixed and sterilized. The mixture was then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 600W, ultrasonic time: 10min), and co-fermenting probiotics were added for fermentation. The fermentation temperature was 40°C. When the pH reached 4.46 and the acidity reached 75°T, the mixture was rapidly demulsified and cooled to 4°C. After 12 hours of post-ripening, the mixture underwent a second pasteurization process and was then processed through a smoothing filter to obtain fermented milk.

[0069] Example 4

[0070] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 89.0 parts raw milk, 7.0 parts white sugar, 2.0 parts stabilizer, 2.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 1.0 × 10⁻⁶. 5 The cfu / g of the co-fermenting probiotics (the effective viable count ratio of *Streptococcus thermophilus* subsp. salivarius MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 300:3:100:200:200) was 1.0 × 10⁻⁶. 5 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0071] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0072] Raw milk, sweetener, stabilizer, and inulin were mixed and sterilized. The mixture was then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 500W, ultrasonic time: 15min), and co-fermenting probiotics were added for fermentation. The fermentation temperature was 40°C. When the pH reached 4.46 and the acidity reached 75°T, the milk was rapidly broken and cooled to 4°C. After 12 hours of post-ripening, the mixture underwent a second pasteurization process and was then processed through a smoothing filter to obtain fermented milk.

[0073] Example 5

[0074] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 88.0 parts raw milk, 6.9 parts white sugar, 2.6 parts stabilizer, 2.5 parts inulin, and the inoculum amount of the basic starter YO-MIX T12 is 1.0 × 10⁻⁶. 5 The cfu / g of the co-fermenting probiotics (the ratio of effective viable counts of *Streptococcus thermophilus* subsp. salivarius MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 300:5:100:200:100) was 1.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0075] This embodiment also provides a method for preparing the above-mentioned fermented milk, which is the same as that in Example 4.

[0076] Example 6

[0077] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 87.0 parts raw milk, 8.5 parts white sugar, 2.0 parts stabilizer, 2.5 parts inulin, and the inoculum amount of the basic starter YO-MIX T12 is 1.0 × 10⁻⁶. 5 The cfu / g of the co-fermenting probiotics (the ratio of effective viable counts of *Streptococcus thermophilus* subsp. salivarius MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 300:5:100:200:300) was 1.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 6:1.

[0078] This embodiment also provides a method for preparing the above-mentioned fermented milk, which is the same as that in Example 4.

[0079] Example 7

[0080] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 88.0 parts raw milk, 8.0 parts white sugar, 2.0 parts stabilizer, 2.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 1.0 × 10⁻⁶. 5 The cfu / g of the co-fermenting probiotics (the ratio of effective viable counts of *Streptococcus thermophilus* subsp. salivarius MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 300:5:100:200:300) was 1.0 × 10⁻⁶.6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 10:1.

[0081] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0082] Raw milk, white sugar, stabilizer, and inulin were mixed and sterilized. The mixture was then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 500W, ultrasonic time: 15min), and co-fermenting probiotics were added for fermentation. The fermentation temperature was 43°C. When the pH reached 4.46 and the acidity reached 75°T, the mixture was rapidly demulsified and cooled to 4°C. After 12 hours of post-ripening, the mixture underwent a second pasteurization process and was then processed through a smoothing filter to obtain fermented milk.

[0083] Example 8

[0084] This embodiment provides a fermented milk, the raw materials of which include the following components in parts by weight: 87.0 parts raw milk, 7.5 parts white sugar, 2.5 parts stabilizer, 3.0 parts inulin, and the basic starter culture YO-MIX T12 inoculum amount is 1.0 × 10⁻⁶. 5 The cfu / g of the co-fermenting probiotics (the ratio of effective viable bacteria counts of *Streptococcus thermophilus* subsp. salivarius MN-ZLW-002, *Lactobacillus helveticus* H9, *Lactobacillus paracasei* PC-01, *Lactobacillus plantarum* LP-6, and *Bifidobacterium lactis* Probio-M8 was 300:0.5:100:200:100) was 1.0 × 10⁻⁶. 6 cfu / g; wherein the stabilizer is physical starch and citrus fiber in a mass ratio of 8:1.

[0085] This embodiment also provides a method for preparing the above-mentioned fermented milk, the steps of which are as follows:

[0086] Raw milk, sweetener, stabilizer, and inulin were mixed and sterilized. The mixture was then cooled to 40°C. YO-MIX T12, a basic starter culture activated by dual-frequency ultrasonic treatment (simultaneous action of 20kHz and 40kHz, ultrasonic power: 400W, ultrasonic time: 15min), and co-fermenting probiotics were added for fermentation. The fermentation temperature was 38°C. When the pH reached 4.45 and the acidity reached 77°T, the milk was rapidly broken and cooled to 4°C. After 12 hours of post-ripening, the mixture underwent a second pasteurization process and was then processed through a smoothing filter to obtain fermented milk.

[0087] Comparative Example 1

[0088] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the inoculum amount of the basic starter culture YO-MIX T12 is 4.0 × 10⁻⁶.4 cfu / g.

[0089] The preparation method of the fermented milk described above is the same as in Example 1.

[0090] Comparative Example 2

[0091] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the inoculum size of the co-fermenting probiotics is 1.0 × 10⁻⁶. 4 cfu / g.

[0092] The preparation method of the fermented milk described above is the same as in Example 1.

[0093] Comparative Example 3

[0094] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the inoculum size of the co-fermenting probiotics is 2.0 × 10⁻⁶. 7 cfu / g.

[0095] The preparation method of the fermented milk described above is the same as in Example 1.

[0096] Comparative Example 4

[0097] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermenting probiotics is 300:0.1:100:200:200.

[0098] The preparation method of the fermented milk described above is the same as in Example 1.

[0099] Comparative Example 5

[0100] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermenting probiotics is 300:10:100:200:200.

[0101] The preparation method of the fermented milk described above is the same as in Example 1.

[0102] Comparative Example 6

[0103] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the ratio of the effective live bacteria counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermentation probiotics is 300:3:100:200:50.

[0104] The preparation method of the fermented milk described above is the same as in Example 1.

[0105] Comparative Example 7

[0106] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that the ratio of the effective viable counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8 in the co-fermenting probiotics is 300:0.5:100:200:400.

[0107] The preparation method of the fermented milk described above is the same as in Example 1.

[0108] Comparative Example 8

[0109] This comparative example provides a fermented milk with the same raw material composition as Example 1. The only difference between the preparation method and Example 1 is that the ultrasonic power for activating the fermentation strain is 300W and the time is 15min.

[0110] Comparative Example 9

[0111] This comparative example provides a fermented milk with the same raw material composition as Example 1. The only difference between the preparation method and Example 1 is that the ultrasonic power for activating the fermentation strain is 700w and the time is 15min.

[0112] Comparative Example 10

[0113] This comparative example provides a fermented milk with the same raw material composition as Example 1. The only difference between the preparation method and Example 1 is that the ultrasonic power for activating the fermentation strain is 500W and the time is 40min.

[0114] Comparative Example 11

[0115] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that: in the co-fermenting probiotics, Lactobacillus helveticus H9 is replaced with Lactobacillus helveticus R0052 (which can be purchased commercially).

[0116] The preparation method of the fermented milk described above is the same as in Example 1.

[0117] Comparative Example 12

[0118] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 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).

[0119] The preparation method of the fermented milk described above is the same as in Example 1.

[0120] Comparative Example 13

[0121] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that: in the co-fermenting probiotics, Lactobacillus plantarum LP-6 is replaced with Lactobacillus plantarum R1012 (which can be purchased commercially).

[0122] The preparation method of the fermented milk described above is the same as in Example 1.

[0123] Comparative Example 14

[0124] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that: in the co-fermenting probiotics, Bifidobacterium lactis Probio-M8 is replaced with Bifidobacterium BB12 (which can be purchased commercially).

[0125] The preparation method of the fermented milk described above is the same as in Example 1.

[0126] Comparative Example 15

[0127] This comparative example provides a fermented milk whose raw material composition differs from that of Example 1 only in that: in the co-fermenting probiotics, Streptococcus thermophilus subsp. MN-ZLW-002 is replaced with Streptococcus thermophilus subsp. T-101004 (purchased from Chr. Hansen).

[0128] The preparation method of the fermented milk described above is the same as in Example 1.

[0129] Example 1: Evaluation of the physicochemical parameters, stability, and taste / flavor of fermented milk

[0130] 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).

[0131] 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.

[0132] Table 1 Overall Preference Evaluation Criteria

[0133]

[0134] Table 2 Evaluation Criteria for Sensory Descriptive Tests

[0135]

[0136] The evaluation results are shown in Table 3.

[0137] Table 3. Physicochemical parameters, stability, and taste evaluation results of fermented milk.

[0138]

[0139]

[0140]

[0141] Experiment Example 2: Detection of Functional Substance Content in Fermented Milk

[0142] While ensuring product stability and flavor, the functional substance content of the probiotic fermented milk from each embodiment and the fermented milk from each comparative example were tested. The experimental method and results are described in detail below.

[0143] 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).

[0144] 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.

[0145] 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.

[0146] The results are shown in Tables 4 and 5. Compared with Comparative Examples 1-4 and Comparative Example 8, the contents of 14 substances related to sub-health improvement in the probiotic fermented milk of Examples 1-8 were significantly increased (P<0.05). The results indicate that the fermented milk of the present invention has the potential to improve sub-health.

[0147] Table 4. Relative content multiples of each differentially functional substance in the fermented milk of Example 1 compared with Examples 2-8 and Comparative Examples 1-4 and 8.

[0148]

[0149]

[0150] Note: The values ​​in Table 4 are the ratios of the content of each functional substance in the fermented milk of Example 1 to the content of each functional substance in the fermented milk of Examples 2-8 and Comparative Examples 1-4 and 8, respectively. The higher the corresponding ratio, the lower the content of the functional substance.

[0151] Table 5. Differential functional substances and functional characteristics in fermented milk

[0152]

[0153]

[0154] Furthermore, testing revealed that the content of the 14 functional substances shown in Table 5 in the fermented milk of the other comparative examples was also significantly lower than that in Examples 1-8.

[0155] Experiment Example 3: Human Experiment with Fermented Milk

[0156] While ensuring product stability and flavor, the probiotic fermented milk from Example 1 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.

[0157] 1. Establishment of experimental study on the relief of sub-health conditions by Biostime fermented milk in people.

[0158] 1.1 Volunteer Inclusion Criteria

[0159] A. No gender restriction, age 30-60;

[0160] 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);

[0161] C. Voluntarily sign the informed consent form.

[0162] 1.2 Exclusion Criteria

[0163] A. Those who do not meet the above inclusion criteria;

[0164] B. Subjects who have been diagnosed with other diseases;

[0165] C. Subjects whose blood routine and biochemical indicators are outside the normal range may be in a disease state;

[0166] D. Has used antibiotics, probiotics, or synbiotics within the past month;

[0167] E. Individuals allergic to the sample or its components;

[0168] F. Has taken anti-anxiety, antidepressant or other psychotropic medications in the past month.

[0169] A human trial investigating the use of probiotic fermented milk to alleviate sub-health conditions recruited 51 volunteers. Participants consumed 200g / day of the fermented milk for four consecutive weeks. Blood and stool samples were collected at the start of the trial (T0), 28 days after consumption (T1), and 14 days after the follow-up period (T2). Questionnaires were completed, including the Sub-health Measurement Scale Version (SHMS) and the Pittsburgh Sleep Quality Index (PSQI). The SHMS scale comprises 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. The specific results of the sub-health measurement scale survey in the target population are shown in Table 6.

[0170] Table 6 Questionnaire survey on the intervention of fermented milk in sub-health symptoms

[0171]

[0172]

[0173] The results showed that the SHMS scores of subjects who consumed 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. Consuming probiotic fermented milk significantly reduced the subjects' PSQI (P < 0.01), 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.

[0174] 2. The effect of Biostime fermented milk on intestinal flora

[0175] DNA was extracted from fecal samples. DNA samples meeting sequencing requirements were sequentially subjected to random fragmentation, end repair, adapter ligation, and library construction. The samples were then sequenced using Illumina GAIIx and HiSeq2000 to analyze the effects of fermented milk on bacterial functional genes and metabolic pathways.

[0176] The results are shown in Table 7. No obvious clustering trend was observed after consuming fermented milk, indicating that it did not affect the overall structure of the gut microbiota in sub-healthy individuals. While the diversity of gut microbiota decreased after consuming fermented milk, the difference was not significant. Comparing the gut microbiota with those showing significant differences in relative abundance between days 0 and 4 weeks of fermented milk consumption, it was found that the abundance of Firmicutes, Proteobacteria, and Bacteroidetes decreased, while the abundance of Actinobacteria increased.

[0177] Table 7. Effects of drinking fermented milk on the relative abundance (%) of dominant bacterial phyla in sub-healthy individuals.

[0178]

[0179] The results of the study on the effects of probiotic fermented milk on the characteristic flora of sub-healthy individuals (Table 8) showed that nine bacterial genera associated with sub-health changed: the abundance of four beneficial bacteria increased and the abundance of five harmful bacteria decreased; the results indicate that fermented milk products have the effect of improving the gut microbiota of sub-healthy individuals.

[0180] Table 8. Effects of Synbiotic Fermented Milk on Intestinal Characteristic Microbiota in Sub-healthy Individuals

[0181]

[0182] In summary, 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.

[0183] 3. Effects of Biostime fermented milk on fecal metabolites in sub-healthy individuals

[0184] Fecal samples were pretreated, and the full spectrum of fecal samples was determined non-targeted using LC-MS. After consuming fermented milk, the metabolites in the volunteers' feces showed significant differentiation: the levels of creatinine, L-2,4-diaminobutyric acid (2,4-diaminobutyric acid), and docosahexaenoyl serotonin changed significantly after consuming the fermented milk. The level of Kyotorphin (L-tyrosine-L-arginine) increased significantly after the follow-up period. 2,4-Diaminobutyric acid is a non-linear, non-competitive inhibitor of γ-aminobutyric acid (GABA) transaminase activity, capable of inducing an increase in GABA levels. Docosahexaenoyl serotonin exerts an anti-inflammatory effect by attenuating IL-13 and IL-17 signaling in macrophages. L-tyrosine-L-arginine is a neuropeptide with neuromodulatory effects, playing a role in pain regulation in the brain. The above results confirm that probiotic fermented milk can alleviate the sub-health status of subjects, and its potential mechanism may be through increasing the levels of anti-inflammatory substances and other regulatory factors related to sub-health. In summary, the mechanism by which probiotic fermented milk alleviates sub-health is through fine-tuning the gut microbiota, regulating inflammatory factors and other regulatory factors related to sub-health, and producing metabolites beneficial to the host.

[0185] 4. The effect of probiotic fermented milk on short-chain fatty acids in the feces of sub-healthy individuals

[0186] 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 the baseline period, the levels of acetic acid, propionic acid, and lactic acid in the intestines of subjects increased after consuming probiotic fermented milk (P>0.05), but the differences were not statistically significant.

[0187] Table 9. Short-chain fatty acid production in fecal microbiota of sub-healthy volunteers.

[0188]

[0189] 5. Effects of probiotic fermented milk on serum neurotransmitters in sub-healthy individuals

[0190] 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.

[0191] Fasting venous blood was collected from the subjects upon waking in the morning. After standing, the blood was centrifuged (3000 rpm, 10 min) to detect the levels of 21 neurotransmitters in the subjects' serum, namely 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 detection results of some neurotransmitters are shown in Table 10.

[0192] Table 10. Neurotransmitter production in the serum of sub-healthy volunteers.

[0193]

[0194] Analysis revealed a significant decrease in serum VMA, 5-HT, and NE levels in subjects after consuming probiotic fermented milk for 4 weeks (P<0.05). Studies indicate that VMA is a major metabolite of adrenaline and noradrenaline, and extreme anxiety and essential hypertension can both lead to elevated VMA levels. Furthermore, NE levels were higher than normal in patients with depression. These results suggest that consuming probiotic fermented milk may improve anxiety in sub-healthy individuals by regulating the levels of neurotransmitters such as NE and VMA, thereby influencing the nervous system.

[0195] 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 compound starter culture for room temperature fermented milk, characterized in that, The compound starter culture for room temperature fermented milk consists of a basic starter culture and synergistic fermentation probiotics; The co-fermenting probiotics consist of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6, and Bifidobacterium lactis Probio-M8. The preservation number of the thermophilic subspecies of Streptococcus salivarius MN-ZLW-002 is CGMCC No.3817; 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; In the compound starter culture for room temperature fermented milk, the ratio of the effective live bacteria count of the basic starter culture and the co-fermenting probiotics is 1:(1-20). The ratio of effective viable bacteria counts of Streptococcus salivarius subsp. thermophilus MN-ZLW-002, Lactobacillus helveticus H9, Lactobacillus paracasei PC-01, Lactobacillus plantarum LP-6 and Bifidobacterium lactis Probio-M8 in the co-fermented probiotics is 300:(0.5-5):100:200:(100-300).

2. The compound starter culture for room temperature fermented milk according to claim 1, characterized in that, The basic fermentation agent includes Streptococcus salivarius thermophilus subsp. and Lactobacillus delbrueckii bulgaricus subsp.

3. The compound starter culture for room temperature fermented milk according to claim 1, characterized in that, In the basic fermentation agent, the effective viable count ratio of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is (1-100):

1.

4. The application of the compound 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, prebiotics, and the compound 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: 76-92 parts milk raw material, 0.5-8.5 parts sweetener, 1.5-2.6 parts stabilizer, 2.0-3.0 parts prebiotic, and 0.04-0.2 parts compound fermentation agent for room temperature fermented milk; The inoculum size of the basic fermentation agent was 5.0 × 10⁻⁶. 4 -5.0×10 5 The inoculum size for co-fermenting probiotics is 1.0 × 10⁻⁶ CFU / g fermentation substrate. 5 -2.0×10 6 CFU / g fermentation substrate.

7. The room-temperature fermented milk according to claim 5 or 6, characterized in that, The prebiotic is selected from one or more of polydextrose, resistant dextrin, inulin, soybean oligosaccharide, cottonseed oligosaccharide, spirulina, and chitosan oligosaccharide.

8. The room-temperature fermented milk according to claim 6, characterized in that, The stabilizer is one or more selected from physical starch, citrus fiber, and seaweed powder.

9. The room-temperature fermented milk according to claim 6, characterized in that, The sweetener is selected from one or more of the following: white sugar, glucose, fructose, aspartame, cyclamate, acesulfame potassium, erythritol, xylitol, and sucralose.

10. The room-temperature fermented milk according to claim 5 or 6, characterized in that, The milk raw material is raw milk and / or reconstituted milk.

11. The method for preparing room-temperature fermented milk according to any one of claims 5 to 10, characterized in that, The method includes: mixing the raw materials and fermenting them, followed by demulsification and cooling after fermentation.

12. The method according to claim 11, characterized in that, Before adding the compound fermentation agent, the compound fermentation agent is first activated by dual-frequency ultrasound; The dual-frequency ultrasonic activation uses ultrasonic frequencies of 20kHz and 40kHz, ultrasonic power of 400-600W, and a duration of 10-30min.

13. The method according to claim 11 or 12, characterized in that, The fermentation was completed when the pH was 4.40-4.50 and the acidity was 70-80°T. And / or, the fermentation temperature is 38-43°C.

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