A composite probiotic starter and use thereof

Abstract

The application discloses a kind of composite probiotic ferment and its application, belong to the technical field of ferment.The application provides a kind of ferment, by gastrointestinal tract tolerance good, intestinal colonization ability strong, with a variety of known probiotic function, in the list of food usable strain directory Commercial probiotic composition;The fermented milk prepared using the provided ferment can be comparable with commercial ferment in fermentation rate, storage quality, but the viable count during storage, sensory evaluation result is significantly higher than that of the fermented milk prepared by commercial ferment;Compared with the fermented milk prepared by using commercial ferment, it contains more oligopeptide and amino acid, sugar and its derivative, fatty acid and its derivative and other small molecule metabolites;Nutritional value is higher, has better probiotic effect.

Description

Technical Field

[0001] This invention relates to the field of fermentation agent technology, and in particular to a compound probiotic fermentation agent and its application. Background Technology

[0002] Probiotics have important physiological functions for the body, such as regulating the balance of intestinal flora, lowering cholesterol, anti-oxidation, anti-infection, and enhancing human immunity. They have broad application prospects in the food and medical fields.

[0003] Bifidobacteria, as dominant microorganisms in the gastrointestinal tract, play an irreplaceable role in maintaining intestinal health. In recent years, various clinical, in vivo, and in vitro studies have demonstrated that Bifidobacteria play a crucial role in protecting the integrity of the intestinal barrier, contributing to diseases such as inflammatory bowel disease, irritable bowel syndrome, cancer, diarrhea, and lactose intolerance, thereby maintaining a healthy and stable state of the body. Lactobacillus rhamnosus is widely present in the gastrointestinal tracts of humans and animals, and can tolerate the acidic environment of gastric acid and bile, thus effectively colonizing the intestines and exerting its probiotic effects. LGG (ATCC53103), isolated from the intestines of healthy humans, possesses probiotic effects such as regulating intestinal flora structure, improving intestinal physiological function, preventing and treating diarrhea, enhancing the body's immune function, protecting the gastric mucosa, reducing the activity of hydrolytic enzymes, reducing toxins, and preventing tooth decay.

[0004] Fermented milk is rich in protein, calcium, and various nutrients. After fermentation by lactic acid bacteria, proteins are broken down into peptides and amino acids, and lactose is hydrolyzed into galactose and glucose, which are then converted into lactic acid. The resulting fine curds are easier for the gastrointestinal tract to digest and absorb, making it suitable for people with lactose intolerance. Furthermore, fermented milk performs well in regulating gut microbiota and preventing and treating diabetes, cardiovascular diseases, and liver diseases, making it a popular functional food.

[0005] Currently, fermented milk production mostly uses commercial starter cultures, primarily composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. These starter cultures mainly function to produce acid and enhance flavor in the fermentation system, their core value being to ensure the flavor and texture of fermented dairy products, while their probiotic effects are relatively limited. Currently, the probiotics in commercially available probiotic fermented milk are mostly added as supplementary ingredients. During fermentation and storage, they compete with these commercial starter cultures for nutrients or spatial sites. This competition significantly affects the survival rate of the probiotics, resulting in a substantial reduction in their probiotic effects. Summary of the Invention

[0006] The purpose of this invention is to provide a compound probiotic fermentation agent and its application, in order to solve the problem of poor probiotic effect of commercial fermentation agents, and to provide a fermentation agent with good gastrointestinal tolerance, strong intestinal colonization ability, good fermentation quality, higher nutritional value and better sensory quality of the fermented milk prepared.

[0007] To achieve the above objectives, the present invention provides a compound probiotic starter, wherein the compound probiotic starter is composed of Lactobacillus rhamnosus. Lacticaseibacillus rhamnosus HN001, Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. Lactis HN019 and Lactobacillus rhamnosus Lacticaseibacillus rhamnosus LGG composition.

[0008] Preferably, the fermenting agent contains *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus HN001: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. Lactis HN019: Lactobacillus rhamnosus Lacticaseibacillus rhamnosus The live bacteria count ratio of LGG is 5:1:1.

[0009] The application of the compound probiotic starter as described above in the preparation of dairy products, including fermented milk.

[0010] Dairy products prepared from the above-mentioned compound probiotic starter.

[0011] The application of the compound probiotic fermentation agent described above in the preparation of oligopeptides and amino acids, wherein the oligopeptides and amino acids are obtained by extraction / separation / purification of the product after fermentation with the compound probiotic fermentation agent; the oligopeptides and amino acids include isoleucine, phenylalanine, prolylproline, phenylalanine, tryptophan-tyrosine, methionine-tyrosine and γ-aminobutyric acid.

[0012] The application of the compound probiotic fermentation agent as described above in the preparation of sugars and their derivatives, wherein the sugars and their derivatives are obtained by extraction / separation / purification of the product after fermentation with the compound probiotic fermentation agent; the sugars and their derivatives include melibiose, galactooligosaccharides, sedoheptulose, and N-acetylgalactosamine.

[0013] The application of the compound probiotic fermentation agent described above in the preparation of fatty acids and their derivatives, wherein the fatty acids and their derivatives are obtained by extraction / separation / purification of the product after fermentation with the compound probiotic fermentation agent; the fatty acids and their derivatives include palmitamide, mevalonic acid and phosphatidic acid.

[0014] The above-mentioned compound probiotic fermentation agent is used in the preparation of bioactive small molecules. The bioactive small molecules are obtained by extraction / separation / purification of the product after fermentation with the compound probiotic fermentation agent. The bioactive small molecules include nicotinyl glycine, thioctinamide, and dephosphorylated coenzyme A.

[0015] Therefore, the specific technical effects of the compound probiotic starter and its application provided by the present invention are as follows: (1) This invention provides a starter culture consisting of a commercially available probiotic, Lactobacillus rhamnosus, which has good gastrointestinal tolerance, strong intestinal colonization ability, and various known probiotic functions and is listed in the list of microbial strains that can be used in food. Lacticaseibacillus rhamnosus HN001, Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. Lactis HN019 and Lactobacillus rhamnosus Lacticaseibacillus rhamnosus LGG composition; (2) Fermented milk prepared using the starter culture provided by the present invention can be comparable to commercial starter culture in terms of fermentation rate and storage quality, but the number of viable bacteria and sensory evaluation results during storage are significantly higher than those of fermented milk prepared using commercial starter culture. The starter culture provided by the present invention can be used to prepare fermented milk and improve the sensory quality of fermented milk. (3) Fermented milk prepared using the starter culture provided by the present invention contains more oligopeptides and amino acids, sugars and their derivatives, fatty acids and their derivatives, and other small molecule metabolites, including isoleucine, phenylalanine, proline, phenylalanine, tryptophan-tyrosine, methionine, γ-aminobutyric acid, melibiose, galactooligosaccharides, sedoheptulose, N-acetylgalactosamine, palmitamide, mevalonic acid, phosphatidic acid, nicotinylglycine, thioctinamide, and dephosphorylated coenzyme A, etc., compared with fermented milk prepared using commercial starter culture. It has higher nutritional value and better probiotic effects.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results of microrheological measurements during the fermentation of each group of fermented milk are shown in the efficacy test section of this invention; where I and V represent the EI trend; II and VI represent the FI value trend; III and VII represent the SLB value trend; and IV and VIII represent the MVI trend. Figure 2 The graph shows the pH and TA changes during the storage of fermented milk in groups A, B, C, and S during the effectiveness test of this invention; where I represents pH and II represents TA. Figure 3 The graph shows the pH and TA changes during the storage of fermented milk in groups A, E, D, L, and S during the effectiveness test of this invention; where I represents pH and II represents TA. Figure 4 The graph shows the changes in water-holding capacity of fermented milk in each group during storage, which is part of the efficacy test of this invention; where I represents groups A, B, C, and S; and II represents groups A, E, D, L, and S. Figure 5 The graph shows the viscosity changes of fermented milk in each group during storage, which is part of the efficacy test of this invention; where I represents groups A, B, C, and S; and II represents groups A, E, D, L, and S. Figure 6 The graph shows the changes in viable bacteria count during storage of fermented milk in each group during the efficacy test of this invention; where I represents groups A, B, C, and S; and II represents groups A, E, D, L, and S. Figure 7 The sensory evaluation results of each group of fermented milk compound probiotic fermented milk during storage are shown in the efficacy test section of this invention; where I represents groups A, B, C, and S; and II represents groups A, E, D, L, and S. Figure 8 The above is a PCA graph of the metabolic activity of fermented milk in each group on day 14 of storage, which is part of the efficacy test of this invention. Figure 9 The graphs show the metabolic PLS-DA of each group of fermented milk on day 14 of storage, which are part of the efficacy test of this invention. Figure 10 This is a heatmap of the differences in metabolites among the fermented milk samples stored on day 14 of the invention's efficacy testing. Figure 11 This is a KEGG pathway enrichment diagram of differential metabolites in fermented milk from different groups on day 14 of storage, which is part of the efficacy test of this invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The instruments, reagents, materials, and strains used in the examples were all obtained commercially. The method for detecting the number of viable bacteria involved in the examples was based on the national standard GB 4789.35-2016, "National Food Safety Standard - Microbiological Detection of Lactic Acid Bacteria in Food". The methods and steps not described in detail in the examples are all conventional techniques in the field.

[0022] Example 1 Fermented milk is prepared using the starter culture provided by this invention. The starter culture provided by this invention is composed of Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus HN001, Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp lactis HN019 and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The composition of LGG, HN001, HN019 and LGG had viable counts of 1.07 × 10⁻⁶. 7 CFU / mL, 2.14×10 6 CFU / mL and 2.14×10 6 CFU / mL (viable bacteria ratio of 5:1:1), the specific steps are as follows: After preheating whole milk to 60-65℃, add 6.5% granulated sugar to the milk and stir to promote sugar dissolution. Then, homogenize the fully dissolved milk in a high-pressure homogenizer at 20MPa. After homogenization, maintain at 95℃ for 30 minutes, then rapidly cool in ice water to the inoculation temperature of 37℃. The final inoculation concentration is then 1.07 × 10⁻⁶. 7 HN001 CFU / mL, 2.14×10 6 HN019 CFU / mL and 2.14×10 6 CFU / mL LGG (viable cell ratio 5:1:1). After thorough mixing, ferment at the optimal growth temperature of 37℃ until the pH drops to 4.5±0.1, which is the fermentation endpoint. Immediately stop fermentation by placing the mixture in an ice-water bath. Then, store at 4℃. This is designated as group E.

[0023] Comparative Example 1 Fermented milk was prepared using the commercial starter culture PYS-010, following the same method as in Example 1, except that the starter culture provided in this invention was replaced with PYS-010. The amount of PYS-010 added was 0.03‰, and the total viable count was 1.5 × 10⁻⁶. 7 CFU / mL. After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as S group.

[0024] Comparative Example 2 The method for preparing fermented milk is exactly the same as in Example 1, except that it uses Bifidobacterium animalis subsp. lactis (… Bifidobacterium animalis subsp lactis HN019, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus HN001 and LGG are the starter cultures; the viable cell counts of HN001, HN019, and LGG are all 5 × 10⁻⁶. 6 CFU / mL (viable cell ratio 1:1:1). After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as Group A.

[0025] Comparative Example 3 The method for preparing fermented milk is exactly the same as in Example 1, except that it uses Bifidobacterium animalis subsp. lactis (… Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus HN001 and Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis HN019 is the starter culture, and the viable cell counts of BB-12, HN001, and LGG are all 5 × 10⁻⁶. 6 CFU / mL (viable cell ratio 1:1:1). After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as Group B.

[0026] Comparative Example 4 The method for preparing fermented milk was exactly the same as in Example 1, except that the viable counts of BB12, HN001, HN019, and LGG were all 3.75 × 10⁻⁶. 6 CFU / mL (viable cell ratio 1:1:1:1). After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as Group C.

[0027] Comparative Example 5 The method for preparing fermented milk is exactly the same as in Example 1, except that the viable counts of HN001, HN019, and LGG are 1.25 × 10⁻⁶. 7 CFU / mL, 1.25×10 6 CFU / mL and 1.25×10 6 CFU / mL (viable cell ratio 10:1:1). After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as Group D.

[0028] Comparative Example 6 The method for preparing fermented milk is exactly the same as in Example 1, except that the viable counts of HN001, HN019, and LGG are 6.82 × 10⁻⁶. 6 CFU / mL, 1.36×10 6 CFU / mL and 6.82×10 6 CFU / mL (viable cell ratio 5:1:5). After fermentation was terminated, the product was refrigerated at 4°C. This group is designated as group L.

[0029] Effect test The fermentation agent composition information of Examples 1, Comparative Examples 1-6 is shown in Table 1.

[0030] Table 1 Inoculation amount for different strains

[0031] (1) Microrheological measurements during fermentation.

[0032] Fermented milk was prepared using the method described in Example 1. After inoculating with the corresponding starter culture and mixing, 20 mL of each sample was placed in a sample bottle of a Rheolaser Master optical microrheometer for fermentation. The microrheometer temperature was set to 37°C. After setting the parameters in the software, the Rheolaser Master optical microrheometer was used for continuous measurement. The measurement was stopped after the fermentation endpoint was reached, and the software and optical microrheometer were stopped. Based on the root mean square displacement (MSD) curve, four different indices reflecting the structural characteristics of the sample were calculated: Elastic Index (EI), Solid Liquid Balance (SLB), Macroscopic Viscosity Index (MVI), and Fluidity Index (FI).

[0033] ① As a function of time, the elastic modulus (EI) directly reflects the change in elastic modulus, i.e., storage modulus, over a certain period of time. It can quickly and easily characterize the elastic modulus of a sample. The gel structure strength and stability of fermented milk samples are positively correlated with the magnitude of the EI value. Changes in elasticity factor during fermentation are as follows: Figure 1 As shown in Figure I, the EI values ​​of the fermented milk samples remained stable within 5 hours. This may be because in the early stage of fermentation, casein has not yet formed a gel network structure, resulting in lower viscosity and elasticity, higher fluidity, and thus exhibiting a lower EI value.

[0034] Around 5 hours, group S, using a commercial starter culture, reached the inflection point first, rapidly rising vertically to the gel point. Group B also began to rise at 5 hours and reached its gel point around 7.5 hours. Groups A and C reached their gel points successively after 10 hours. As fermentation progressed, the casein in the samples dissociated and then re-aggregated to form a gel network structure. The EI values ​​of each group successively rose to their maximum values ​​and then stabilized. Group S reached the gel point the fastest and remained higher than the other three groups during fermentation, indicating that Group S had the shortest fermentation time, fastest coagulation, and a stronger gel structure. Groups A-C showed similar trends, with relatively similar EI values ​​in the later stages of fermentation. However, Group B formed a gel structure faster, indicating that this compound fermented milk had a relatively faster coagulation rate. Figure 1 As shown in Figure V, the EI values ​​of each group successively increased to their maximum values ​​and then tended to stabilize. Around 5 hours, the commercially viscoelastic fermented milk group S was the first to show an inflection point, with its EI value rising vertically to its maximum. The other four compound groups began to rise after 10 hours and subsequently reached their gel points. Among the four compound groups, the EI value of group E was relatively higher than the other groups, indicating that the fermented milk sample with this compound ratio exhibited better viscoelasticity.

[0035] ② The change in FI reflects the speed of microscopic particle movement in the fermented milk sample. When the FI value is about 10 Hz, it is a high flow factor, indicating that the fermented milk sample is in a liquid state. -2 Hz represents the low flow factor, indicating that the fermented milk sample is solid. The speed of particle movement and the degree of flowability are directly proportional to the FI value. The trend of FI value change is shown below. Figure 1 As shown in Figure II, from the start of fermentation to 4 hours, the FI values ​​of all groups were higher than 10. 2 Hz indicates that all four groups of fermented milk were in a liquid state at this time, while group S showed an inflection point around 4 hours and rapidly decreased, dropping to 10 Hz around 5 hours. -1 ~10 -2 The curdling rate gradually leveled off after 5 hours, eventually forming a solidified state. Groups B and A also showed the same trend after 5 hours. The comparison results were similar to the EI values. Group B reached its inflection point the fastest and then decreased rapidly, followed by Group A, and finally Group C. This indicates that Groups B and S had a faster curdling rate than Groups A and C. Figure 1 As shown in VI, the FI value of group S reached an inflection point around 5 hours and then dropped vertically, reaching 10 around 5 hours later. -1 ~10 -2 The Hz trend began to slow down and approached a horizontal state, indicating that its fluidity decreased and it began to gradually solidify. The other four compound groups all showed the same downward trend after 10 hours. At the end of fermentation, the FI values ​​of all five samples were at 10. -1 ~10 -2 The range of Hz indicates that all five groups of fermented milk samples have high viscous liquid properties.

[0036] ③ The size of the SLB (strain modulus) is directly proportional to the viscoelasticity of the fermented milk. When the SLB value is 0~0.5, it indicates that the fermented milk sample exhibits an elastic modulus and is in a solid state; when the SLB value is 0.5~1, it indicates that the fermented milk sample exhibits a viscous modulus and is in a liquid state. Figure 1 As shown in section III, from the start of fermentation to approximately 4 hours, the SLB values ​​of all samples fluctuated and remained liquid because the casein in the fermented milk had not yet formed a gel structure. However, as fermentation progressed, the net negative charge of the casein micelles decreased, leading to a reduction in electrostatic repulsion between micelles. The casein micelles began to dissociate, and the dissociated casein re-aggregated to form a gel structure. The SLB value of group S showed an inflection point around 4 hours and then slowly increased, indicating that the casein in the fermented milk re-aggregated to form particles, increasing viscosity, and reaching the gel point earlier than other groups. Among groups A-C, the SLB value of group B started to rise first and remained higher than other groups, indicating that group B had a faster curdling rate, greater viscoelasticity, and stronger sample stability. Figure 1As shown in Figure VII, the SLB value of group S first showed an inflection point around 5 hours and gradually increased, indicating that the casein in the fermented milk increased viscosity due to the re-aggregation of casein into colloidal particles, reaching the gel point earlier. Meanwhile, the four compound groups also began to slowly increase after 10 hours of fermentation, reaching 0.5-0.6 at the fermentation endpoint.

[0037] ④MVI reflects the viscosity characteristics of a sample at the micrometer scale and can represent the viscosity change trend of fermented milk samples. Figure 1 As shown in section IV, the MVI values ​​of each group of fermented milk were low in the initial stage of fermentation, fluctuating to varying degrees. The MVI value of group S showed an inflection point around 4 hours and then rose vertically. The variation patterns of groups A-C were very similar to those of the EI value. The MVI values ​​of groups A, B, and C also showed inflection points and reached their maximum values ​​after 5 hours. This indicates that as fermentation time increased, the strain continuously produced acid, causing the pH value to continuously decrease, leading to the continuous dissociation of casein. At the end of fermentation, the casein micelles in each group of fermented milk samples gradually aggregated to form a relatively stable gel network structure, resulting in decreased solubility, increased viscosity, and a continuous increase in the MVI value until it reached its maximum value and then leveled off. Figure 1 As shown in Figure VIII, the MVI value reached an inflection point around 5 hours and then rose rapidly. Similarly, the four compound groups also began to rise vertically after 10 hours and reached their maximum value. This indicates that as fermentation progresses, the casein micelles in each group of fermented milk samples gradually aggregate to form a relatively stable gel network structure. Among them, the MVI value of group E at the end of fermentation was slightly higher than that of the other groups, which suggests that the viscosity of this compound ratio may be higher than that of the other groups.

[0038] (2) Samples of fermented milk from each group were taken on days 1, 7, 14, and 21 after storage, and the following indicators were measured for the fermented milk prepared from each group: ①pH value: After the fermented milk samples were brought to room temperature, the pH values ​​of the experimental group and the control group were measured using a Leici PHS-3 C pH meter. The average value was taken after measuring each sample three times.

[0039] ② Titration of acidity: Weigh 10.0g of fermented milk, add 20mL of distilled water and mix. Then add 2-3 drops of phenolphthalein indicator and shake well. Titrate with 0.1mol / L NaOH solution until the color turns slightly red or light pink and does not fade within 30s. Record the amount of NaOH used. According to GB 5009.239—2016 "Determination of acidity of food", calculate the titratable acidity (TA) of fermented milk on storage days 1, 7, 14 and 21 using the following formula I. Perform three parallel determinations and take the average value.

[0040] TA (°T)=V / M×100 (Formula I); In the formula: V represents the volume of NaOH standard solution consumed (mL); M represents the sample mass of the fermented milk sample (g).

[0041] pH and titratable acidity (TA), as quantitative parameters characterizing lactose conversion efficiency and direct indicators for determining the fermentation endpoint, are key parameters for assessing post-acidification and storage stability of fermented milk during storage. The trends of pH and acidity changes in the four groups of fermented milk during storage are shown below. Figure 2 As shown, throughout the entire storage period, the pH values ​​of fermented milk in groups A-C and the control group all showed a continuous decreasing trend with the extension of storage time, while the trend of titratable acidity (TA) was negatively correlated with this. The mechanism is that although the main fermentation stage ends after fermentation, lactic acid bacteria can still slowly ferment under 4°C refrigeration. They use the nutrients in the fermented milk as growth and metabolic substrates to slowly metabolize lactose, producing lactic acid through homofermentation via glycolysis and heterofermentation via phosphoketolase, causing the acidity to continuously increase. This series of dynamic changes is the post-acidification phenomenon of fermented milk.

[0042] Throughout the storage period, the pH values ​​of groups A and C were significantly higher than those of groups B and S. P <0.05). For example... Figure 2 As shown in Figure I, the pH values ​​of groups A and C changed relatively slowly during storage, while group B had the lowest initial pH. The decrease in pH value of the control group S was similar to that of group B.

[0043] Similarly, as Figure 2 As shown in Figure II, the titratable acidity of each group of fermented milk samples showed a significant upward trend with the extension of storage time. Group A and Group C reached 108.50 and 106.67°T respectively at the end of storage. Group B and Group S were significantly higher than Group A and Group C, reaching 115.30 and 116.43°T respectively. This reflects that the metabolic activity of Group A and Group C was relatively stable throughout the storage period, with better storage stability and relatively weak post-acidification.

[0044] During storage periods of 1-14 days, the TA value increased relatively rapidly, while the upward trend slowed significantly during 14-21 days. This phenomenon may be due to the gradual decrease in pH value within the fermented milk system as storage time increases, and the higher acidity environment inhibits the activity of the bacterial strains, thereby suppressing the continued acid production process. Previous studies have confirmed that fermented milk products with a pH value maintained in the range of 4.0-4.6 and a titratable acidity maintained in the range of 70°T-110°T have a good taste, meeting both production needs and consumer preferences. When the lactic acid content is at an ideal level, it imparts a unique sour taste to the product, enhances the viscosity of the system, and provides preservative effects. Therefore, the acidity of all four groups of fermented milk products met this requirement during storage.

[0045] like Figure 3 As shown, the pH values ​​of groups E and D were significantly lower than the other three groups during storage, while their titratable acidity (TA) values ​​were significantly higher. This may be because, based on a total inoculum of 1.5 × 10⁷ CFU / mL for all four groups of fermented milk, groups E and D had a higher proportion of *Lactobacillus rhamnosus* HN001, resulting in stronger acid-producing capacity and a faster acid production rate, thus maintaining a consistently low pH and a high TA value. Furthermore, from... Figure 3 The pH trend in group I shows that towards the end of storage, the decrease in pH value in group E slowed down and remained between 4.1 and 4.2. Figure 3 As shown in section II, during storage days 7-21, the increase in titration acidity (TA) value in group E was more gradual than in other groups, and was very similar to that of the control group S. Furthermore, it remained between 100 and 110°T at the end of storage. This indicates that the compound ratio can maintain good shelf-life stability during storage, and both of the above indicators were within the ideal range at the end of storage. The fermented milk has moderate acidity, which can give the product a unique sour taste and enhance the viscosity of the system. This can both meet the taste requirements of consumers and have a certain preservation effect.

[0046] ③ Water-holding capacity: Weigh 20g of fermented milk sample and place it in a funnel with qualitative filter paper. Place the funnel on a 50mL Erlenmeyer flask and let it stand at room temperature for 2 hours. Immediately weigh the sample and record the mass of the filtrate. Set up three parallel sets for each sample. Calculate the water-holding capacity using Formula II.

[0047] (Formula II); In the formula: m1 represents the mass of the filtrate (g); m2 represents the mass of the sample (g).

[0048] Water-holding capacity reflects the ability of fermented milk to retain moisture and is positively correlated with its tissue state, making it an important indicator for evaluating the structural stability of fermented milk. Higher water-holding capacity indicates a stronger binding force of the gel structure to free water, less whey separation, and greater system stability. Good water-holding capacity not only gives fermented milk a smooth and delicate texture but also effectively reduces moisture loss during storage, thereby maintaining product stability and extending shelf life. Figure 4 It can be seen that the water-holding capacity of the four groups of fermented milk samples showed similar trends during the 21-day storage period, all exhibiting an upward trend in the early stage of storage and a downward trend in the later stage. In the early stage of storage, the whey in the fermented milk is more stable in the protein network structure and usually has a higher water-holding capacity. However, in the later stage of storage, as the acidity increases, the whey precipitation also increases, which destroys the original dense gel structure, resulting in a decrease in water-holding capacity, and consequently, a decline in the flavor, taste, and texture of the product.

[0049] Depend on Figure 4As shown in section I, the water-holding capacity of groups A and S is significantly higher than that of groups B and C. P <0.05), indicating that this compound formulation can effectively improve the water-holding capacity of fermented milk and stabilize it during storage, thus contributing to improved sensory quality and storage stability. Figure 4 As shown in Figure II, during storage days 1-7, the water-holding capacity of the control group S was higher than that of the compound group, while in the later stage of storage, the water-holding capacity of groups A and E was significantly improved. P <0.05), especially Group E, whose water-holding capacity was significantly higher than other groups and exceeded that of Group S during storage from day 14 to day 21. It can be inferred that this compound ratio can effectively improve the water-holding capacity of fermented milk, thereby improving the sensory quality and storage stability of fermented milk.

[0050] ④ Viscosity: After the fermented milk has returned to room temperature, weigh an appropriate amount of fermented milk sample into a weighing cup and measure it using the Brookfield viscometer with its own No. 4 rotor. The rotor speed is 100 r / min, the measurement time is 30 s, and the torque is 10%~100%. To reduce error, perform three parallel measurements and record the results.

[0051] Viscosity is a key parameter indicating the degree of viscosity of a fluid. The viscosity of fermented milk reflects the stability of its protein gel structure and assesses the quality of its texture. It is also a crucial indicator for evaluating the quality and shelf-life stability of fermented milk. Higher viscosity indicates a more uniform and dense texture, and lower fluidity. The increase in viscosity of fermented milk is mainly due to two factors: the viscosity-producing characteristics of the bacterial strains and the precipitation of casein into a gel state, resulting in larger particles. Investigating the changes in viscosity during storage and its regulatory mechanisms can help improve product quality and extend shelf-life stability.

[0052] The viscosity changes of fermented milk during storage, as follows: Figure 5 As shown, the overall trend is consistent with the results for water retention. Figure 5 In group I, the viscosity of fermented milk samples A, B, C, and S all showed a trend of first increasing and then decreasing. With prolonged storage, the viscosity significantly increased, reaching its maximum on day 14, and then began to decrease. In the early stages of storage, this may be due to the production of extracellular polysaccharides and other substances by lactic acid bacteria during growth, along with the significant production of viscosity; or it may be due to the increased acidity causing casein in the four groups of fermented milk samples to precipitate. The increased casein precipitation transformed into a gel-like structure, forming larger curd-like particles, which manifested as a gradual increase in viscosity.

[0053] The viscosity began to decrease after 14 days, possibly because colloidal calcium phosphate in the fermented milk sample was continuously dissolved as the pH value decreased during the later stages of storage. This not only disrupted the tight cross-linking structure between protein molecules but also led to increased whey precipitation, resulting in a decrease in the viscosity of the fermented milk system. It is also possible that the prolonged storage time inhibited the growth of lactic acid bacteria, reducing their viscosity-producing ability and further contributing to the decrease in viscosity. The viscosity of group A was significantly higher than the other three groups. P <0.05), this change is the same as the change in water retention. It can be inferred that using this compound combination for fermentation allows the fermented milk to construct a denser gel network structure within 21 days of storage by regulating the symbiotic metabolism of the strains, promoting the secretion of extracellular polysaccharides and cross-linking of casein micelles, effectively binding water and reducing whey separation, maintaining a relatively high viscosity, thereby obtaining a better texture and taste.

[0054] like Figure 5 As shown in Figure II, the overall viscosity trend of the five groups of fermented milk during storage was similar to that of the water-holding capacity results, showing an initial increase followed by a decrease. With prolonged storage, the viscosity significantly increased, reaching a peak on day 14, and then began to decrease. The viscosity of group E was significantly higher than that of the other three groups within 21 days. P <0.05), this change is consistent with the change in water-holding capacity, which suggests that the ratio of this strain can enable fermented milk to build a denser gel network structure during storage, maintain a relatively high viscosity, reduce whey separation, and thus obtain better sensory quality and storage stability.

[0055] ⑤ Viable Bacterial Count Determination: Weigh 25g of fermented milk into a sterile blue-capped bottle containing a certain number of glass beads, add sterile physiological saline to a final volume of 250mL, and shake for 15min to prepare a 1:10 initial dilution. Using a sterile pipette, pipette 1mL of the above dilution into a test tube containing 9mL of sterile physiological saline, vortex to mix thoroughly, and then perform serial dilutions. Select an appropriate dilution gradient, draw 1mL into a sterile petri dish, then pour in an appropriate amount of culture medium, mix using a figure-eight motion, set up three replicates for each gradient, and incubate under strict anaerobic conditions at 37℃ for 48h. Count the final colonies.

[0056] Changes in viable cell count during storage are important parameters for assessing the growth status and probiotic effects of lactic acid bacteria in fermented milk, and are also key indicators for characterizing starter culture activity and inoculum suitability. The trends in viable cell count during storage for each group are shown below. Figure 6 As shown, by Figure 6 As shown in Figure I, all four groups of fermented milk exhibited a trend of first increasing and then decreasing. The viable bacteria counts of groups A, B, and C all reached 10 in the early stage of storage. 9CFU / mL, significantly higher than group S throughout the entire storage period ( P <0.05). Meanwhile, the viable bacteria counts in all four groups of fermented milk increased from day 1 to day 7, reaching their highest value on day 7, and then showed a downward trend. Figure 6 As shown in section II, the viable bacterial counts of all five groups of fermented milk exhibited a trend of first increasing and then decreasing, increasing from day 1 to day 7 and reaching a maximum on day 7, after which they began to gradually decrease, and all could reach 10 in the early stage of storage. 9 CFU / mL. Throughout the entire storage period, the viable cell counts in all four compound groups were significantly higher than those in the commercially available group S (CFU / mL). P <0.05. The viable bacterial counts in groups A, E, and D were significantly higher than those in groups L and S from day 1 to day 21. P <0.05), and has remained at 10 9 CFU / mL, indicating a high level of viable bacteria, especially group E, which was significantly higher than the other four groups after day 14. P The concentration of live bacteria in fermented milk is less than 0.05%, indicating that this compound ratio allows the fermented milk to maintain a higher level of live bacteria throughout its shelf life, demonstrating a significant advantage and enabling it to more effectively exert the beneficial effects of probiotics. According to the National Food Safety Standard of the People's Republic of China, "Fermented Milk" (GB19302-2025), the minimum requirement for the content of live lactic acid bacteria in fermented milk is not less than 1×10⁻⁶. 6 CFU / mL, the number of viable bacteria in all four groups of fermented milk samples met this standard during storage.

[0057] ⑥ Determination of texture.

[0058] A suitable amount of fermented milk sample was weighed into a special sample bottle, and the textural properties of the fermented milk sample were determined using a TA.XT plus texture analyzer (equipped with an A / BE reverse extrusion probe). The method for testing yogurt consistency was set as follows: initial test speed 1.5 mm / s, mid-test speed 1.0 mm / s, and final test speed 1.5 mm / s; initial stress 2.0 g; compression degree 20%; compression time 5 s; and test distance 20 mm. Each sample was measured in triplicate. The results are shown in Table 2. The trends of the four groups of fermented milk samples during 21 days of storage are shown in Table 9. The textural characteristics of the fermented milk samples generally increased gradually from day 1 to day 14, reaching a maximum at day 14 and then showing varying degrees of decline, similar to the trends of water-holding capacity and viscosity. During the first 14 days of storage, the increased acidity continuously strengthened the gel network structure of the fermented milk, leading to a continuous increase in its hardness, consistency, cohesion, and viscosity index. The decline in textural properties towards the end of storage is likely due to the lower pH and excessive acidification in the fermented milk system at that stage, resulting in post-acidification and casein hydration, which worsened the texture of the fermented milk. At this point, the gel network structure of the fermented milk was damaged, changing from dense to loose, exhibiting significant whey separation, thus causing a decrease in various textural parameters. Throughout the entire storage period, the textural properties of compound group B were generally significantly higher than those of the other three groups. P <0.05), therefore, it is preliminarily inferred that the combination of HN001, HN019 and LGG strains for compound fermentation has certain advantages, which can improve texture characteristics, reduce whey precipitation, and further improve the sensory quality of fermented milk. This can provide a reference for the commercial application of compound fermentation agents and the development and utilization of fermentation agents.

[0059] Table 2 Changes in textural properties of fermented milk during storage

[0060] Note: Uppercase letters indicate differences between groups. P <0.05), lowercase letters indicate within-group differences ( P< 0.05).

[0061] Table 3. Changes in the textural properties of compound fermented milk during storage.

[0062] Note: Uppercase letters indicate differences between groups. P <0.05), lowercase letters indicate within-group differences ( P< 0.05).

[0063] The textural properties of the five groups of fermented milk during 21 days of storage are shown in Table 3. The textural characteristic values ​​generally increased gradually from day 1 to day 14, reaching their maximum value at day 14 before decreasing to varying degrees, similar to the trends in water-holding capacity and viscosity. During the first 14 days of storage, acidity gradually increased, and the gel network structure of the fermented milk continuously strengthened, leading to a gradual increase in hardness, consistency, cohesion, and viscosity index. The decrease in textural characteristic values ​​at the end of storage may be due to the continued decrease in pH leading to excessive acidification. Post-acidification and casein hydration deteriorated the texture of the fermented milk, damaging the gel network structure and causing whey precipitation, thus reducing the various textural characteristic parameters. During storage, the textural characteristic values ​​of group E were significantly higher than those of the other four groups at all four time points. P (<0.05), therefore, it can be preliminarily determined that the compound ratio of this strain has a significant advantage in improving the textural properties of fermented milk, and can further improve the quality of fermented milk, providing theoretical basis and data support for the subsequent product development and process optimization of compound fermentation agents.

[0064] ⑦ Sensory evaluation of fermented milk.

[0065] A sensory evaluation table (as shown in Table 4) was developed based on the Chinese dairy industry standard RHB104-2020. A sensory evaluation team composed of 20 trained food science graduate students evaluated the fermented milk of each group on days 1, 7, 14, and 21 after storage, according to the sensory evaluation table (Table 6). After each tasting, the sensory evaluators rinsed their mouths three times with warm water before proceeding to the next group to prevent confusion between the tastes of different groups. During the evaluation process, the evaluators were not informed whether they were tasting the experimental or control group of fermented milk.

[0066] Table 4 Sensory Evaluation Form

[0067] Note: Taste and aroma do not involve sweetness; only sourness is evaluated.

[0068] Sensory evaluation can reflect the quality differences of fermented milk and directly reflect its popularity from the consumer's perspective, making it an important standard for assessing the quality of fermented milk. In this experiment, the evaluation was mainly based on four aspects: color, aroma, taste, and texture. The sensory evaluation scores of each group of fermented milk samples during storage are as follows: Figure 7 As shown in Figure I, the fermented milk in group A had the highest score during storage, followed by group B, while groups C and S had the lowest scores. During storage from day 1 to 14, the scores of groups A and B were significantly higher than those of groups C and S. P(<0.05) On day 7 of storage, the sensory scores of all groups showed an upward trend and reached their highest values, indicating that after a short period of post-ripening, the fermented milk achieved an optimal balance in flavor, color, and texture. Subsequently, with prolonged storage, the fermented milk in the later stages of storage exhibited problems such as increased acidity, decreased viscosity, and decreased water-holding capacity, resulting in varying degrees of decline in sensory scores. In summary, the four groups of fermented milk samples performed well in terms of color, aroma, taste, and texture during the initial storage period, possessing the characteristic color and aroma of fermented milk, a moderate sweet-sour ratio, and a delicate texture. Throughout the entire storage period, group A had a higher sensory score than the other groups, suggesting that the starter culture of group A could provide better sensory quality, thus indicating higher market acceptance.

[0069] Further investigation was conducted into the sensory quality of fermented milk under different compounding ratios, such as... Figure 7 As shown in Figure II, the sensory scores of fermented milk in groups E and L were relatively high, and the changes in sensory scores among the groups were not significant throughout the storage period, remaining above 80 points from day 7 onwards. This indicates that after a brief period of post-ripening, the fermented milk reached its optimal level in terms of flavor, color, and texture, which remained until the end of storage. This further demonstrates the good sensory quality stability of this compound combination during storage. In summary, the fermented milk samples of this compound combination exhibited good performance in terms of color, aroma, taste, and texture throughout the storage period, and maintained a consistently high score, reflecting good shelf-life stability. Furthermore, from day 7 onwards, the sensory score of group E was slightly higher than that of the other groups. Based on this comprehensive analysis, it can be inferred that fermented milk prepared using the strain ratio of group E has certain advantages in sensory quality and storage stability, thus leading to its higher popularity and market acceptance.

[0070] (3) Analysis of beneficial metabolites in fermented milk of each group.

[0071] 1) Comparative analysis of metabolites in each group.

[0072] The results of various index measurements of five groups of fermented milk (A, D, E, L, and S) showed that, at the four measurement time points throughout the storage period, the indicators of each group of fermented milk at 14 days of storage were generally better than those at the other three storage time points. Therefore, this invention first uses PCA to compare the differences in metabolites among the five groups of fermented milk samples stored for 14 days, analyzing the differences between and within groups. By observing the principal component analysis model score plots of the five groups of fermented milk samples at 14 days of storage, it can be seen that each scatter point represents a sample, and the color of the scatter points indicates different groups. The closer the sample points are, the more similar the types and contents of metabolites in the samples; conversely, the farther apart the sample points are, the greater the difference in their overall metabolic levels, reflecting the overall distribution trend of the samples.

[0073] like Figure 8As shown, PCA extracts key variance information from the data, mapping high-dimensional metabolite data to a two-dimensional principal component space. Principal component 1 (PC1) contributes 56.7% of the variance, principal component 2 (PC2) contributes 9.9%, and the total variance is 66.6%, effectively reflecting the core metabolic differences among the five groups of fermented milk samples. The score plot clearly shows that samples in groups A, D, E, L, and S exhibit high intra-group clustering with no significant dispersion, indicating that the metabolite composition of these five groups of fermented milk samples has high consistency and stability after 14 days of storage, demonstrating good experimental repeatability and minimal outlier interference between samples.

[0074] Meanwhile, it can be seen that the five groups of samples formed three completely independent clusters without overlap in the principal component space, with clear boundaries between groups. Among them, the samples of groups D and A highly overlapped, both clustering in the negative half-axis of PC1 and the region close to the 0 axis of PC2, indicating that the metabolite composition and characteristics of the two groups of fermented milk were highly similar after 14 days of storage, and the differences in metabolic phenotypes were small. Similarly, the samples of groups E and L clustered closely, and the positions of the two groups of samples highly overlapped, which also indicated that their metabolic characteristics were highly consistent, forming a significant separation from groups D and A. Group S, as the control group, formed a completely independent cluster with the other four groups of samples, without any overlap. This shows that compared with the control group, the metabolite profiles of fermented milk in groups A, D, E, and L underwent significant specific changes after 14 days of storage. The introduction of compound probiotics played a decisive regulatory role in the metabolic characteristics of fermented milk during storage, and there were essential differences in the metabolic phenotypes between the two groups.

[0075] Furthermore, groups A, D, E, and L, as different proportions of the same compound, exhibited two clear clustering models in the principal component space. This reflects that different compound ratios have significant gradient and proportional dependence differences in the precise regulation of the metabolic characteristics of fermented milk during storage, providing key visualization evidence for subsequent screening of the optimal compound ratio and analysis of the synergistic metabolic mechanism among strains.

[0076] PLS-DA reduces the dimensionality of high-dimensional metabolic data by maximizing inter-group differences, effectively enhancing the distinguishability of metabolic characteristics between different groups. In the PLS-DA model constructed in this invention, Principal Component 1 contributes 56.5% of the variance, and Principal Component 2 contributes 9.3%. The two principal components collectively explain 65.8% of the total variability, indicating a good model fit that fully reflects the differences in metabolic characteristics among the five groups of fermented milk samples after 14 days of storage.

[0077] From the score Figure 9The inter-group clustering pattern was clearly observed. The replicates from groups A, D, E, L, and S all showed high clustering with no obvious dispersion or outliers, indicating that the metabolite composition of each group of fermented milk was stable after 14 days of storage. The experiment showed good reproducibility and high data reliability, providing a solid foundation for subsequent screening of differential metabolites. The inter-group differences among the five fermented milk samples were largely consistent with the PCA results. Group S formed a completely independent cluster with no overlap with groups A, D, E, and L. This result further validated the PCA conclusion: compared to the control group, the probiotic compound treatment significantly affected the metabolite profile of fermented milk during storage, showing significant differences in metabolic phenotype compared to the control group. The introduction of compound probiotics was the core factor driving the significant changes in metabolic characteristics.

[0078] Furthermore, groups A, D, E, and L are four compound combinations of the same formula but with different ratios. In the PLS-DA model, they exhibit clear gradient separation characteristics: the samples of groups D and A, and groups E and L, are highly overlapping. This also indicates that the metabolite composition of the two clustered samples is more similar, and there is no significant difference in metabolic phenotype. However, they are all significantly different from the other three fermented milk samples. This also intuitively reflects that adjusting the compound ratio can play a significant regulatory role in the metabolic characteristics of fermented milk during storage. At the same time, the synergistic metabolic effects of the strains under different ratios are significantly different, thus forming three completely independent cluster regions.

[0079] 2) Screening for differential metabolites.

[0080] To identify significantly different metabolites among different groups of fermented milk, a P-value < 0.05 and VIP > 1.2 were used as screening criteria. MetaboAnalyst 6.0 online platform was used to screen for differentially expressed metabolites among different samples after 14 days of storage. The screening results are as follows: Figure 10 As shown, a total of 62 differential metabolites were identified, mainly including oligopeptides and amino acids, carbohydrates and their derivatives, fatty acids and their derivatives, and other small molecule metabolites.

[0081] ①Oligopeptides and amino acid derivatives.

[0082] During fermentation, casein in fermented milk is broken down into amino acids and short peptides. A higher content of free amino acids in the fermented milk indicates stronger transport and hydrolysis capabilities of the lactic acid bacteria, enabling the release of large amounts of free amino acids. Groups A and E, after 14 days of storage, showed higher levels of certain oligopeptides and amino acid derivatives compared to other groups, including isoleucine, phenylalanine, proline, phenylalanine, tryptophan-tyrosine, methionine, and γ-aminobutyric acid (GABA).

[0083] Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter widely distributed in the central nervous system. Studies have found that it has multiple physiological functions, including promoting sleep, reducing anxiety, lowering blood pressure, and regulating glucose metabolism. GABA can also regulate pancreatic hormone secretion and promote glycogen synthesis, providing new ideas for the adjunctive treatment of diabetes. Based on its significant physiological activity, functional foods enriched with GABA through lactic acid bacteria fermentation have become a research hotspot.

[0084] Furthermore, studies have shown that the main form of protein absorption by the human body also includes oligopeptides, especially dipeptides and tripeptides. The absorption rate of proteins in oligopeptide form is 2 to 2.5 times higher than that of amino acids. Isoleucyltyrosine, phenylalanylproline, prolylproline, phenylalanylphenylalanine, tryptophan-tyrosine, and methionyltyrosine are all dipeptides composed of two amino acids linked by peptide bonds. The portion of the dipeptide absorbed by cells can be hydrolyzed into free amino acids, thereby reducing the antagonistic effects between free amino acids, accelerating protein synthesis, increasing cellular amino acid content, and fully exerting its nutritional regulatory role. Research has found that these oligopeptides present in fermented milk can exert antioxidant, anti-inflammatory, lipid-lowering, blood pressure-lowering, and mood-regulating effects, including anti-anxiety and anti-depressant effects, contributing to improved human health.

[0085] ② Carbohydrates and their derivatives.

[0086] Sugars and their derivatives are important sources of carbohydrates and energy in dairy products. Groups A and E, after 14 days of storage, showed higher levels of certain sugars and their derivatives compared to other groups, including melibiose, galacto-oligosaccharides, sedoheptulose, and N-acetylgalactosamine. Among these, galacto-oligosaccharides are functional oligosaccharides found in animal milk / breast milk, formed by 1-7 galactosylgalactose or glucose molecules linked by glycosidic bonds. They have effects such as regulating intestinal flora, improving intestinal function, and enhancing human immunity. Furthermore, galacto-oligosaccharides can promote the proliferation of bifidobacteria, improve mineral absorption, prevent osteoporosis, prevent and treat constipation, improve lipid metabolism, and lower serum cholesterol levels.

[0087] Merbiose is a reducing disaccharide composed of galactose and glucose. It possesses prebiotic properties and has potential applications in disease treatment and the development of functional foods. It can promote the intestinal absorption of calcium. 2+ It improves the gut environment by promoting the absorption of quercetin and the growth of Bifidobacteria and Lactobacillus. Furthermore, melibiose can reduce neuroinflammation, making it a potential treatment for Alzheimer's disease; it can also modulate helper T cell responses and improve oral tolerance, thereby preventing and alleviating allergy symptoms.

[0088] Sedum heptaketose, an important intermediate in the non-oxidative reaction of the pentose phosphate pathway, is a rare heptaketose in nature. Studies have shown that its derivatives have multiple effects, including anti-type 2 diabetes, lowering triglycerides, and protecting the liver and kidneys.

[0089] N-acetylgalactosamine, also known as GalNAc, has significant applications in biomedicine, drug delivery, and microbial metabolism. Some studies suggest it can promote the growth of specific beneficial bacteria. In liver cancer research, it can precisely target the desialylate glycoprotein receptor (ASGPR) on the surface of hepatocytes. Conjugating nucleic acid drugs to GalNAc allows for efficient and specific drug delivery to the liver, improving efficacy and reducing side effects. This technology has been successfully applied to marketed drugs. Furthermore, GalNAc is a key biomarker, particularly as part of the Tn antigen; its abnormal glycosylation pattern is closely related to tumor development and metastasis. Studies have shown that GalNAc, as a component of extracellular polysaccharides (EPS) produced by certain lactic acid bacteria, also helps improve the viscosity and mouthfeel of fermented milk.

[0090] ③ Fatty acids and their derivatives.

[0091] Fatty acids are carboxylic acid compounds and are the basic building blocks of lipids such as fats and phospholipids. Groups A and E, after 14 days of storage, showed higher levels of certain fatty acids and their derivatives compared to other groups, including palmitamide, mevalonic acid, and phosphatidic acid. Palmitamide, an amide derivative of palmitic acid, has been shown in studies to potentially regulate cellular metabolism by enhancing host cell glycolysis and also has the ability to induce apoptosis in breast cancer cells. Furthermore, palmitamide is not only a predictive biomarker for 5-FU resistance but also has the potential to reverse 5-FU resistance in colon cancer cells.

[0092] Mevalonic acid, as a precursor of the mevalonic acid metabolic pathway (MVA pathway), is a key substance in the biosynthesis of cholesterol, terpenes, and other substances. In vitro, it can effectively inhibit the decrease in C2C12 cell viability induced by simvastatin and can be used for research on cardiomyopathy and heart failure.

[0093] Phosphatidic acid is a key precursor in the biosynthesis of glycerophospholipids, participating in the synthesis of membrane lipids and storage lipids. As a second messenger, it can also regulate various cellular functions and has wide applications in the food and pharmaceutical fields. Furthermore, studies have shown that phosphatidic acid supplementation can enhance muscle strength.

[0094] ④ Others.

[0095] Many bioactive small molecules, including nicotinic acid glycine, lipoamide, and dephosphorylated coenzyme A, have been identified in the metabolites of fermented milk. Nicotinic acid glycine, formed by the combination of nicotinic acid and glycine, is a metabolite of vitamin B3 in vivo and is considered a quantitative indicator of liver function in processing nicotinic acid. Studies have shown that after oral supplementation with nicotinamide, nicotinic acid glycine can serve as a biomarker for monitoring nicotinic acid metabolism in the body and can be used in research on mitochondrial dysfunction-related diseases such as glaucoma.

[0096] Lipoic acid amide is the active amide form of lipoic acid. Its main function is as a core coenzyme in a complex of several key enzymes, including pyruvate dehydrogenase, linking carbohydrate metabolism and the tricarboxylic acid cycle. These substances exhibit significant antioxidant and anti-inflammatory effects by activating signaling pathways such as Nrf2. Studies have shown that they can protect nerve cells and kidney cells, resist oxidative stress damage, and inhibit the formation of stress granules, demonstrating potential therapeutic value in models of neurodegenerative diseases such as ALS.

[0097] Dephosphorylated coenzyme A (CoA), as a precursor to CoA, plays a crucial role in CoA synthesis. Maintaining normal CoA levels in the body helps support the normal metabolism of fatty acids, glucose, and amino acids, and is of great significance for research on metabolic diseases such as mitochondrial dysfunction. Studies have shown that dephosphorylated CoA may enhance CoA synthesis and improve metabolic function.

[0098] 3) KEGG enrichment analysis of differentially metabolites.

[0099] By performing KEGG pathway enrichment analysis on the screened differential metabolites, metabolic pathways that are significantly altered under experimental conditions can be identified, thus providing important clues for understanding their underlying biological mechanisms. For example... Figure 11 As shown, the vertical axis represents the metabolic pathways where these metabolites are enriched, and the horizontal axis represents the enrichment rate of that metabolic pathway. The intensity of the color represents the P-value; the redder the color, the smaller the P-value, indicating a significant enrichment of that metabolic pathway. A total of 19 metabolic pathways were enriched for the differentially expressed metabolites, among which 5 pathways were particularly significant: glycolysis, fructose and mannose metabolism, pentose phosphate pathway, galactose metabolism, and taurine and hypotaurine metabolism.

[0100] In fermented milk systems, probiotics metabolize lactose into glucose via the glycolysis pathway (EMP), releasing energy (ATP) for their growth and reproduction. Simultaneously, the resulting lactic acid acidifies the environment, inhibiting unwanted bacteria and promoting milk coagulation, thus creating the unique texture and flavor of fermented milk. The activity level of the glycolysis pathway is also directly used as a metabolomics indicator to measure the quality of fermented milk; a recent study used key metabolites from the glycolysis / gluconeogenesis pathway as references for the quality of fermented milk products.

[0101] The metabolism of fructose and mannose is a core pathway for probiotics to utilize different carbon sources. It is activated when fructose or mannose is present in the environment. For example, when glucose is restricted, Lactobacillus casei Zhang will significantly upregulate proteins related to this pathway to maintain survival. This pathway can also generate mannitol, which plays an important role in improving the quality of fermented milk.

[0102] The pentose phosphate pathway is a crucial metabolic bypass for cells when utilizing glucose and a major source of ribose-5-phosphate, a key glycosyl backbone required for cellular genetic material synthesis. Furthermore, this pathway generates large amounts of NADPH, providing reducing power for various intracellular synthetic reactions and antioxidant defense systems. The pentose phosphate pathway is fundamental to heterolactic fermentation in some lactic acid bacteria, allowing strains to grow in more complex carbohydrate environments and produce flavor compounds. Studies have confirmed that this pathway is significantly enriched in probiotic fermented milk beverages, making it an important component for probiotics to function.

[0103] The galactose pathway allows probiotics to hydrolyze lactose into glucose and galactose via β-galactosidase. Glucose is directly utilized, while galactose is processed by different strains through two main pathways: the Leloir pathway (gal operon), the most common pathway in lactic acid bacteria, uses a series of enzymes such as galactokinase (GalK) to convert galactose to glucose-1-phosphate, which then enters the glycolysis pathway; and the tagatose-6-phosphate pathway (lac operon), mainly found in strains such as *Lactobacillus casei* and *Lactococcus lactis*, which converts galactose to tagatose-6-phosphate for metabolism. The activity of galactokinase is the core factor leading to differences in fermented milk quality; strains with low activity will excrete galactose, causing it to accumulate in the fermented milk.

[0104] Taurine and taurine metabolism, as an important amino acid metabolic pathway, mainly metabolizes sulfur-containing amino acids into products such as taurine and taurine. Taurine, as the core substance, has health benefits such as anti-oxidation, anti-inflammation, and regulation of blood pressure and blood lipids. It also has great potential in promoting gut microbiota health and regulating gut microbiota homeostasis.

[0105] Therefore, this invention provides a starter culture composed of commercially available probiotics listed in the catalogue of bacteria that can be used in food, exhibiting good gastrointestinal tolerance, strong intestinal colonization ability, and various known probiotic functions. Fermented milk prepared using the provided starter culture can rival commercial starter cultures in terms of fermentation rate and storage quality, but the viable cell count and sensory evaluation results during storage are significantly higher than those of fermented milk prepared using commercial starter cultures. Compared to fermented milk prepared using commercial starter cultures, it contains more oligopeptides and amino acids, sugars and their derivatives, fatty acids and their derivatives, and other small molecule metabolites; it has higher nutritional value and better probiotic effects.

[0106] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A compound probiotic starter, characterized in that: The compound probiotic fermentation agent is composed of Lactobacillus rhamnosus. Lacticaseibacillus rhamnosus HN001, Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. Lactis HN019 and Lactobacillus rhamnosus Lacticaseibacillus rhamnosus LGG composition.

2. The compound probiotic fermentation agent according to claim 1, characterized in that: Lactobacillus rhamnosus in the fermentation agent Lacticaseibacillus rhamnosus HN001: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. Lactis HN019: Lactobacillus rhamnosus Lacticaseibacillus rhamnosus The live bacteria count ratio of LGG is 5:1:

1.

3. The application of the compound probiotic starter as described in claim 1 or 2 in the preparation of dairy products, characterized in that: The dairy products include fermented milk.

4. Dairy products prepared from the compound probiotic starter as described in claim 1 or 2.

5. The application of the compound probiotic starter as described in claim 1 or 2 in the preparation of oligopeptides and amino acids, characterized in that: The oligopeptides and amino acids are obtained by extraction / separation / purification of the product after fermentation with a compound probiotic fermentation agent; the oligopeptides and amino acids include isoleucine, phenylalanine, prolylproline, phenylalanine, tryptophan-tyrosine, methionine, and γ-aminobutyric acid.

6. The application of the compound probiotic starter as described in claim 1 or 2 in the preparation of sugars and their derivatives, characterized in that: The sugars and their derivatives are obtained by extraction / separation / purification of the product after fermentation with a compound probiotic fermentation agent; the sugars and their derivatives include melibiose, galactooligosaccharides, sedoheptulose, and N-acetylgalactosamine.

7. The application of the compound probiotic starter as described in claim 1 or 2 in the preparation of fatty acids and their derivatives, characterized in that: The fatty acids and their derivatives are obtained by extraction / separation / purification of the product after fermentation with a compound probiotic fermentation agent; the fatty acids and their derivatives include palmitamide, mevalonic acid and phosphatidic acid.

8. The application of the compound probiotic fermentation agent as described in claim 1 or 2 in the preparation of bioactive small molecule substances, characterized in that: The bioactive small molecules are obtained by extraction / separation / purification of the product after fermentation with a compound probiotic fermentation agent; the bioactive small molecules include nicotinyl glycine, thioctinamide and dephosphorylated coenzyme A.

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