Use of lactobacillus paracasei zfm54 to improve yogurt quality and nutritional value

Abstract

The application belongs to the field of dairy products, and particularly relates to the use of commercial bacteria and lactobacillus paracasei for co-fermentation of dairy products. The application discloses application of lactobacillus paracasei ZFM54 in improving quality and nutritional value of yogurt, and the lactobacillus paracasei ZFM54 and starter VEGE 033 are used together for fermentation of the yogurt. The lactobacillus paracasei ZFM54 is added to the commercial bacteria for co-fermentation, and the yogurt prepared by adding the lactobacillus paracasei ZFM54 for co-fermentation can significantly improve the quality (including acidity, water retention, lactic acid bacteria content and flavor substances) and nutritional value (including increased lactic acid bacteria content and increased non-essential amino acid substances such as N-ethyl glycine, 4-(1S)-1-hydroxyethyl phenol, L-lysine, 4-amino benzoic acid and Leu-Asn) of the yogurt.

Description

Technical Field

[0001] This invention belongs to the field of dairy products, specifically involving the co-fermentation of dairy products using commercial bacteria and Lactobacillus paracasei. Background Technology

[0002] The gut microbiota is crucial for human health. Imbalances in the gut microbiota during the symbiotic relationship with the host can lead to various systemic diseases, such as inflammatory bowel disease and hepatic encephalopathy. Lactic acid bacteria are recognized as food-grade microorganisms and are widely used in the fermented food industry. They not only form probiotic films to protect the gastrointestinal mucosa but also metabolize and produce various natural nutrients and antibacterial substances, such as short-chain fatty acids, folic acid, and bacteriocins, thus optimizing the gut microbiota structure and maintaining intestinal microecological balance. *Lactiplantibacillus paracasei* ZFM54 is a bacteriocinogenic and folic acid-producing lactic acid bacterium previously screened in our laboratory. It exhibits good environmental tolerance and tolerance to gastrointestinal simulated fluids, and can inhibit various foodborne pathogens. This invention explores how to use this strain to ferment yogurt and conduct quality analysis. Furthermore, it investigates the volatile and non-volatile metabolomic characteristics of yogurt fermented with *Lactiplantibacillus paracasei* ZFM54, laying the foundation for the commercial application of *Lactiplantibacillus paracasei* ZFM54.

[0003] The invention "Isolation and Purification Method of Bacteriocin from Lactobacillus paracasei ZFM54" published on 201911342111.X screened Lactobacillus paracasei ZMF54, which has antibacterial effects against both Gram-positive and Gram-negative bacteria and has strong antibacterial activity.

[0004] The invention "Lactobacillus paracasei and its application" dated 201911341915.8 discloses that the bacteriocin ZFM54 can be used for antibacterial activity in acidic environments and can be used as a food biological preservative. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new use for Lactobacillus paracasei ZFM54, namely, to provide the application of Lactobacillus paracasei ZFM54 in improving the quality and nutritional value of yogurt.

[0006] To address the aforementioned technical problems, this invention provides the application of Lactobacillus paracasei ZFM54 in improving the quality and nutritional value of yogurt: Lactobacillus paracasei ZFM54 and starter culture VEGE 033 are used together in yogurt fermentation.

[0007] This invention also provides a method for preparing yogurt that improves its quality and nutritional value, comprising the following steps:

[0008] 1) Preparation of raw materials:

[0009] The raw materials consist of 12-13% (preferably 12.4%) of skim milk powder, 6-7% (preferably 6.5%) of sucrose, and the balance being distilled water;

[0010] 2) After homogenizing the raw materials, sterilize them (pasteurize) and cool them to 4±0.5℃ to obtain aseptic skim milk;

[0011] 3) Add (1±0.1)mL of L. paracasei ZFM54 bacterial suspension and 1.0~1.1mg of starter culture VEGE 033 to 99mL of aseptic skim milk, ferment at (42±1)℃ for (8±0.5)h, and cool the resulting yogurt to 4±0.5℃ for refrigeration.

[0012] As an improvement to the yogurt preparation method of the present invention that enhances quality and nutritional value: the cell concentration of the L. paracasei ZFM54 bacterial suspension is (1±0.2)×10⁻⁶. 7 CFU / mL.

[0013] As an improvement to the yogurt preparation method of the present invention that enhances quality and nutritional value: Lactobacillus paracasei ZFM54 cells are resuspended in aseptic skim milk, and the cell concentration is adjusted to (1±0.2)×10⁻⁶. 7 CFU / mL, to obtain L. paracasei ZFM54 bacterial suspension.

[0014] During the invention process:

[0015] 1. The optimal fermentation conditions for Lactobacillus paracasei ZFM54 yogurt were determined through single-factor and response surface methodology experiments.

[0016] 2. Physicochemical properties of yogurt LS and yogurt S were determined.

[0017] 3. The differences in volatile metabolites in yogurt LS and yogurt S were analyzed using SPME-GC-MS.

[0018] 4. The differences in nonvolatile metabolites in yogurt LS and yogurt S were analyzed using UPLC-Q-TOF-MS.

[0019] Compared with the prior art, the present invention has the following technical advantages:

[0020] 1. By screening and optimizing from more single-factor perspectives and combining response surface methodology, more accurate fermentation process conditions can be obtained.

[0021] 2. Adding Lactobacillus paracasei ZFM54 to commercial cultures for co-fermentation significantly improves the quality (including acidity, water-holding capacity, lactic acid bacteria content, and increased flavor compounds) and nutritional value (including increased lactic acid bacteria content and increased levels of non-essential amino acids such as N-ethylglycine, 4-(1S)-1-hydroxyethylphenol, L-lysine, 4-aminobenzoic acid, and Leu-Asn). Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 This is a graph showing the effects of bacterial strain ratio, inoculum size, fermentation time, fermentation temperature, and sucrose addition on the sensory properties and acidity of yogurt.

[0024] Figure 1 middle:

[0025] A represents the effect of the ratio of bacterial strains on the sensory properties and acidity of yogurt.

[0026] B represents the effect of inoculum size on the sensory properties and acidity of yogurt.

[0027] C represents the effect of fermentation time on the sensory properties and acidity of yogurt.

[0028] D represents the effect of fermentation temperature on the sensory properties and acidity of yogurt.

[0029] E represents the effect of sucrose addition on the sensory properties and acidity of yogurt.

[0030] Specifically:

[0031] In Figure A, the yogurt with the highest sensory score has a delicate texture and suitable taste when the bacterial strain ratio is 2:1. As the relative proportion of *Lactobacillus paracasei* ZFM54 increases, the acidity of the yogurt first increases and then decreases. This may be because an excessively high proportion of *Lactobacillus paracasei* ZFM54 would accelerate acid production, but excessive acidity would inhibit the growth of cocci, thus reducing the overall acid production capacity. Therefore, a bacterial strain ratio of 2:1 indicates the best compatibility and symbiotic effect among the fermentation bacteria in the yogurt; hence, a bacterial strain ratio of 2:1 was chosen.

[0032] In Figure B, as the inoculum amount increases, the overall sensory score of the yogurt gradually decreases. The inoculum amount has little effect on the color and aroma of the yogurt, but it has a significant impact on the texture and taste. When the inoculum amount is 1.5%, the yogurt has a uniform texture and a balanced sweet and sour taste, representing the best quality. The acidity of the yogurt increases with the inoculum amount. At an inoculum amount of 1.5%, the acidity of the yogurt is greater than 70°T. Subsequently, as the inoculum amount increases, the change in acidity tends to level off. Therefore, the inoculum amount was determined to be 1.5%.

[0033] In Figure C, the fermentation time significantly affects the overall sensory quality of yogurt. As fermentation time increases, the acidity of the yogurt gradually rises, and the overall sensory score first increases and then decreases. At 4 hours of fermentation, the yogurt has a soft texture but a poor taste. The highest overall score is achieved at 8 hours, when the yogurt has a smooth and uniform surface, a delicate texture, and a suitable sweet-sour ratio. After 8 hours of fermentation, the bacterial strain produces more acid, resulting in a more sour taste. Furthermore, prolonged fermentation damages the yogurt's gel structure, especially after 12 hours, when surface cracks appear and water seeps out. Therefore, the fermentation time was determined to be 8 hours.

[0034] As shown in Figure D, the coagulation effect and texture of the yogurt deteriorate with increasing temperature. This is likely because temperature affects the growth of the bacterial strains, thus influencing the coagulation and acid production processes. Yogurt cultured at 42℃ exhibits the best flavor and texture, receiving the highest overall score. At fermentation temperatures of 37℃ and 40℃, the yogurt exhibits low acidity, a milky consistency, and is not viscous enough, with a sweeter taste. At a fermentation temperature of 45℃, the yogurt develops cracks, and whey separates. Therefore, a fermentation temperature of 42℃ was determined.

[0035] Figure E shows that the amount of sucrose added has a relatively small impact on acidity but a significant impact on taste. The yogurt achieved the highest overall score when the sucrose content was 6.5%. Therefore, the sucrose addition was set at 6.5%.

[0036] Figure 2 It includes response surface methodology, analysis of variance, and optimization analysis diagrams;

[0037] Figure 2 In the response surface optimization analysis, it can be seen that the ellipticity of the contour plots AB, AC, and BC is very large, indicating that the interaction effects of AB, AC, and BC are all extremely significant (P<0.01), which is consistent with the results of the analysis of variance. Among them, the 2D contour plot of BC is elliptical, with the stable point close to the center point, and the 3D response surface graph is arched, indicating that the interaction effect between fermentation time (B) and fermentation temperature (C) is the most significant.

[0038] Figure 3 It is an analytical chromatogram of volatile metabolites;

[0039] Specifically:

[0040] Volatile metabolites in samples LS and S were detected using SPME-GC-MS. The total ion chromatograms of volatile metabolites in the two types of yogurt are shown below. Figure 3 A and Figure 3 Table 1 shows the volatile substances in the two types of yogurt (B).

[0041] Mass spectrometry analysis detected 20 substances, including 8 ketones, 5 acids, 4 alcohols, 2 alkenes, and 1 ester. The data in the table show that 2,3-butanedione, 3-hydroxybutanone, 2-heptanone, hexanoic acid, and caprylic acid were relatively abundant in both types of yogurt, and these substances are likely the main flavor components in fermented yogurt. Compared to sample S, sample LS showed a significant increase in the content of 2,3-butanedione, butyric acid, hexanoic acid, caprylic acid, and decanoic acid, and also added D-limonene flavor compounds. 2,3-Butanedione is a fatty acid formed from the oxidation of methyl ketones, giving yogurt a creamy or nutty flavor. Butyric acid, hexanoic acid, caprylic acid, and decanoic acid can regulate the acidity of yogurt, giving it a unique flavor; butyric acid can produce a cheese-like aroma, while hexanoic acid can produce a coconut oil flavor, etc.

[0042] Figure 4 This is an analysis diagram of non-volatile metabolites;

[0043] Figure 4 middle:

[0044] A shows the PCA distribution of yogurt samples with different starter cultures;

[0045] B is a heatmap of differential metabolites in yogurt produced with different starter cultures;

[0046] C is a bubble diagram showing the KEGG pathway enrichment of yogurt with different starter cultures;

[0047] Specifically:

[0048] like Figure 4 As shown in Figure A, PC1 represents the first principal component, PC2 represents the second principal component, and each point represents a sample. The five points circled on the left of the figure represent five samples of yogurt (LS), and the five points in the full figure on the right represent five samples of yogurt (S). The distance between each coordinate point represents the distance between samples; a greater distance indicates lower similarity between samples, and vice versa. Figure 4 As can be seen from A, there is a certain degree of clustering within the two groups of fermented yogurt, and the groups can be completely distinguished from each other, indicating that there are significant statistical differences in the non-volatile metabolome of yogurt samples fermented with different starter cultures.

[0049] Figure 4 B is a heatmap obtained from cluster analysis of differential metabolites in positive ion mode. The intensity of the color represents the relative content of the differential metabolites in different samples. The darker the color, the higher the content, and vice versa. As can be seen from the figure, the two groups of samples are well separated, and according to the screening criteria, a total of 49 differential metabolites were identified.

[0050] Figure 4In C, the Rich factor represents the ratio of the number of differentially expressed metabolites in the corresponding pathway to the total number of metabolites detected and annotated in that pathway; a larger value indicates a greater degree of enrichment. As shown in the figure, in the non-targeted metabolomics study of samples LS and S, the metabolic pathways enriched with significant changes include protein digestion and absorption, biosynthesis of piperidine and pyridine alkaloids, biosynthesis of secondary metabolites, ABC transport system and 2-oxycarbonyl acid metabolism, as well as tyrosine metabolism, prolactin signaling pathway, mineral absorption, glycerophospholipid metabolism, glucosinolate biosynthesis, folic acid synthesis, ether lipid metabolism, degradation of aromatic compounds, cyanoamino acid metabolism, and biosynthesis of ornithine, lysine, and nicotinic acid alkaloids. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0052] I. Setting up the experimental system:

[0053] MRS liquid culture medium: manganese sulfate 0.05g, magnesium sulfate heptahydrate 0.2g, beef meal 7g, glucose 20g, peptone 10g, yeast 5g, diammonium citrate 3g, dipotassium hydrogen phosphate 2g, anhydrous sodium acetate 5g, 1mL Tween-80, dissolved in ultrapure water and brought to a final volume of 1L, 121℃, 1×10⁻⁶ 5 Sterilize for 15 minutes.

[0054] MRS solid medium: Based on MRS liquid medium, 2% agar powder was added (i.e., 20g agar powder / 100mL), and the rest was the same as MRS liquid medium.

[0055] Activation culture of bacterial strains and preparation of bacterial suspension:

[0056] Take out the Lactobacillus paracasei strain ZFM54 stored at -80℃, thaw it at room temperature, dip the inoculation loop into the bacterial solution, streak it on the MRS solid medium plate using the three-line method, seal it with sealing film, and incubate it upside down in a 37℃ constant temperature incubator for 24 to 48 hours to activate the strain.

[0057] Liquid culture: Using an inoculation loop, pick a smooth, milky-white single colony from the streak plate and transfer it to a test tube containing 10 ml of MRS liquid medium. Incubate at 37°C for 12 to 24 hours. This is the first generation bacterial culture. Subculturing: Inoculate 1% of the first generation bacterial culture into MRS liquid medium and incubate for 24 hours to obtain the second generation bacterial culture.

[0058] Preparation of bacterial suspension: The second-generation bacterial suspension was centrifuged at 4°C and 10,000 r / min for 2 min. The bacterial pellet was collected, washed twice with PBS buffer (0.01 M, pH 6.8), and then resuspended in 12.4% sterile skim milk (preparation method detailed in Example 1 below). The bacterial concentration was adjusted to 1 × 10⁻⁶. 7 CFU / mL, this is a suspension of L. paracasei ZFM54 bacteria, used for subsequent yogurt fermentation.

[0059] Preparation of fermentation supernatant: The second-generation bacterial culture was centrifuged at 4℃ and 10000r / min for 5min, and the supernatant was collected and transferred to a new centrifuge tube.

[0060] II. Determination of the optimal fermentation process:

[0061] Using sensory evaluation as the scoring index, this study investigated the effects of five factors on the sensory quality of fermented yogurt through single-factor experiments: the ratio of fermentation strains (1:2, 1:1, 2:1, 3:1), the inoculum size (0.5%, 1%, 1.5%, 2%, 3%), fermentation time (4h, 6h, 8h, 10h, 12h), fermentation temperature (37℃, 40℃, 42℃, 43℃, 45℃), and sucrose addition (2.5%, 4.5%, 6.5%, 8.5%). Based on the results of the single-factor experiments, a three-factor, three-level Box-Behnken design was used, followed by response surface methodology to obtain the optimal fermentation process parameters, predictive values, and validation.

[0062] Sensory Evaluation: Twenty volunteers were recruited to form a sensory evaluation team to assess the yogurt. The procedure was as follows: 50mL of yogurt was placed in a beaker. Volunteers first observed the texture and color of the sample, then smelled it, rinsed their mouths with water, and finally tasted it. A sensory scoring standard was developed based on the Chinese dairy industry standard RHB 601-2005, with evaluation indicators including sweetness / acidity, color, taste, aroma, and texture. Volunteers were invited to give the yogurt a comprehensive score based on this standard, and the average score was used as the score for each yogurt.

[0063] pH measurement: Insert the pH meter probe into the sample, and read the value when the display value stabilizes. Repeat the reading three times for each sample and record the average value.

[0064] Acidity determination: The titratable acidity of the sample was determined by acid-base titration. 10 g (accurate to 0.001 g) of the well-mixed sample was weighed and placed in a 150 mL Erlenmeyer flask. 20 mL of freshly boiled and cooled water was added and mixed. 2.0 mL of phenolphthalein indicator solution was added and mixed again. The solution was then titrated with a standard sodium hydroxide solution, rotating the flask as it was added, until the color matched the reference solution and did not fade within 5 seconds. The entire titration process should be completed within 45 seconds. During the titration, nitrogen gas was blown into the flask to prevent interference from carbon dioxide. The volume of sodium hydroxide standard solution consumed (V2) was recorded and substituted into equation (1) for calculation.

[0065] X2=[c2×(v2-v0)×100] / m2×0.1 (1)

[0066] In the formula:

[0067] X2—Acidity of the sample, in degrees (°T) [measured in milliliters of 0.1 mol / L sodium hydroxide consumed per 100g sample, in milliliters per 100g (mL / 100g)];

[0068] c2 — The molar concentration of the sodium hydroxide standard solution, expressed in moles per liter (mol / L);

[0069] V2 — The volume of sodium hydroxide standard solution consumed during titration, in milliliters (mL);

[0070] V0—The volume of sodium hydroxide standard solution consumed in the blank experiment, in milliliters (mL);

[0071] 100-100g sample;

[0072] m2 — Mass of the sample, in grams (g);

[0073] 0.1 — The acidity theory defines the molar concentration of sodium hydroxide, expressed in moles per liter (mol / L).

[0074] Determination of water holding capacity: Accurately weigh 10g of sample (M) into centrifuge tube (M1), centrifuge for 5min at 8000r / min using a high-speed centrifuge, remove the supernatant after centrifugation, and record the weight of the sample and centrifuge tube at this time (M2). Calculate water holding capacity (W) according to formula (2).

[0075] Water-holding capacity calculation formula: W=(M2-M1 / M)×100%(2)

[0076] In summary, the optimal process parameters of this invention are obtained. See Example 1 for details.

[0077] Example 1: Process flow of fermented yogurt:

[0078] The raw materials consist of 12.4% skim milk powder, 6.5% sucrose, and the balance being distilled water; the percentages mentioned above are by mass.

[0079] Skim milk powder and sucrose were placed in distilled water at 50°C and stirred until fully dissolved. Then, the mixture was homogenized once each at pressures of 15 MPa and 35 MPa. The homogenized skim milk was then pasteurized (95°C, 5 min) and rapidly cooled to 4°C in cold water to obtain aseptic skim milk (12.4% aseptic skim milk) for later use.

[0080] Add 1 mL of L. paracasei ZFM54 bacterial suspension (1×10⁻⁶) to 99 mL of sterile skim milk. 7 Both yogurt samples were inoculated with 1.09 mg of commercial starter culture VEGE 033 (CFU / mL) and labeled LS; VEGE 033 was inoculated separately with the same amount (1.09 mg of commercial starter culture per 100 ml of aseptic skim milk) and fermented (labeled S). Both yogurt samples were placed in a yogurt maker, and the fermentation temperature and time were set to 42℃ and 8 hours, respectively. After fermentation, the samples were refrigerated at 4℃.

[0081] The commercial starter culture was VEGE 033 direct-inoculation starter culture purchased from Danisco.

[0082] Experiment 1: Determination of the physicochemical properties of yogurt: According to GB19302-2010 National Food Safety Standard, the physicochemical indicators, microbiological indicators, and viable lactic acid bacteria count of two groups of yogurt (LS and S) were determined, and the differences between yogurt LS and yogurt S were compared.

[0083] Both yogurt products met national standards for physicochemical indicators. Using commercial starter cultures as a control, the addition of *Lactobacillus paracasei* ZFM54 had little effect on the fat, non-dairy fat solids, and protein content of the yogurt, but it increased the acidity and water-holding capacity. This may be because when *Lactobacillus paracasei* ZFM54 coexists with the bacteria in the commercial starter cultures, it accelerates acid production through symbiosis, promotes the hydration of lactic acid and protein, alters the structure of casein, and leads to increased viscosity in the yogurt, thus improving its water-holding capacity. This results in a more uniform texture and a smoother, more delicate taste. Furthermore, LS yogurt contains 6.0 × 10⁶ bacteria. 9 The CFU / mL lactic acid bacteria count is approximately six times that of S-grade yogurt. A higher live count of lactic acid bacteria allows for greater colonization in the gastrointestinal tract, thus fully leveraging its regulatory role in balancing the intestinal flora. In conclusion, *Lactobacillus paracasei* ZFM54 contributes to the formation of yogurt's unique sensory qualities and the improvement of probiotic levels.

[0084] Table 1. Comparison of the quality of different yogurts

[0085]

[0086]

[0087] Note: LS refers to yogurt co-fermented with Lactobacillus paracasei ZFM54 and a commercial starter culture, while S refers to yogurt fermented with a commercial starter culture. *** P < 0.001, n = 3.

[0088] Experiment 2: Determination of volatile metabolites

[0089] volatile metabolites of yogurt LS and yogurt S were analyzed using SPME-GC-MS.

[0090] Solid-phase microextraction (SPE): Yogurt sample was placed in a 20 mL headspace vial and sealed with a cap fitted with a silicone rubber septum. The headspace vial was placed in a 50 °C water bath for 30 min. The extraction head was then inserted into the headspace vial at a depth of 1 cm from the liquid surface and allowed to adsorb at 50 °C for 30 min. After removal, the vial was inserted into the GC inlet and desorbed at 250 °C for 2 min, followed by gas chromatography-mass spectrometry (GC-MS) analysis.

[0091] Chromatographic conditions: Column: DB-WAX, 60m × 0.25mm × 0.5μm (column length × inner diameter × film thickness); Injector temperature: 250℃, splitless injection, constant flow mode, column flow rate: 1mL / min; Column temperature: 35℃ for 3min, then increased to 160℃ at 2℃ / min, then increased to 200℃ at 3℃ / min, then increased to 240℃ at 4℃ / min, and held for 5min.

[0092] Mass spectrometry conditions: Ion source: EI, ion source temperature: 230℃, quadrupole temperature: 150℃, mass scan: 33-500 amu. Data processing: The spectra obtained from gas chromatography-mass spectrometry were used for substance identification by searching the NIST14 database and comparing with standards, and the content of each volatile substance was expressed by relative peak area.

[0093] A growing body of research indicates that fatty acids such as caproic acid, caprylic acid, and capric acid help alleviate malabsorption syndrome, small intestinal dysfunction, and hereditary pancreatic diseases. D-limonene imparts a fresh orange and lemon-like aroma to yogurt. In conclusion, *Lactobacillus paracasei* ZFM54 not only assists commercial starter cultures in the fermentation of yogurt but also increases the content of volatile acids in yogurt, giving it a wider range of flavors.

[0094] Table 2 Comparison of volatile metabolite levels in different yogurts

[0095]

[0096]

[0097] Note: "-" indicates not detected. LS is yogurt co-fermented with Lactobacillus paracasei ZFM54 and a commercial starter culture, while S is yogurt fermented with a commercial starter culture. * P<0.05, ** P<0.01, n=3.

[0098] Compared to yogurt S, yogurt LS has significantly increased levels of 2,3-butanedione, 2,3-pentanedione, 2-heptanone, D-limonene, hydroxybutanone, and 1-hexanol, which enhances the content of flavor compounds and improves the flavor quality of yogurt.

[0099] Experiment 3: Determination of non-volatile metabolites

[0100] The differences in nonvolatile metabolites between samples LS and S were investigated using UPLC-Q-TOF-MS.

[0101] PCA analysis of metabolites

[0102] The two groups of fermented yogurts showed some clustering within each group and were completely distinguishable between each other, indicating that the yogurt samples fermented with different starter cultures had statistically significant differences.

[0103] Heatmap clustering analysis of metabolites

[0104] A total of 49 differentially expressed metabolites were identified, including amino acids and their metabolites: isoleucine, phenylalanine, N-ethylglycine, lysine, tyrosine, Leu-Asn, Tyr-Asp-Ala-Asp, Pro-Trp-Met, Phe-Cys-Lys, Gln-Gly-Leu, N-(2-methylbenzoyl), L-tryptophan; organic acids and their derivatives: hippuric acid, methyl acetoacetate, quinoline-2-carboxylic acid, 2'-aminobiphenyl-2,3-diol; benzene and its substituted derivatives; heterocyclic compounds; aldehydes, ketones, and esters; terpenoids; phenolic acids, and others. The contents of N-ethylglycine, 4-(1S)-1-hydroxyethylphenol, L-lysine, 4-aminobenzoic acid, and Leu-Asn were significantly upregulated, possibly because the addition of *Lactobacillus paracasei* ZFM54 promoted the breakdown of proteins into small peptides and amino acids, which is beneficial for the absorption of nutrients by the human body.

[0105] Enrichment analysis of differentially metabolites in the KEGG metabolic pathway:

[0106] The main metabolic pathway that has undergone significant changes is protein digestion and absorption. This pathway mainly breaks down large molecules such as proteins and polypeptides into small molecules such as amino acids, short peptides, and nitrogen-containing compounds that are easily absorbed by the human body, thus promoting the digestion and absorption of nutrients and serving as an important metabolic pathway for maintaining normal physiological functions.

[0107] In summary, compared to yogurt S, LS yogurt exhibits significantly higher acidity, water-holding capacity, and lactic acid bacteria concentration, especially the lactic acid bacteria concentration, which is almost 6 times that of the control yogurt S. Regarding volatile metabolites, compared to yogurt S, LS yogurt shows a significant increase in the content of 2,3-butanedione, 2-heptanone, D-limonene, butyric acid, hexanoic acid, and N-quinic acid, thus enhancing the content of flavor compounds and improving the flavor quality of the yogurt. Regarding non-volatile metabolites, LS yogurt shows a significant increase in the content of N-ethylglycine, 4-(1S)-1-hydroxyethylphenol, L-lysine, 4-aminobenzoic acid, and Leu-Asn, and can alter many other metabolites, improving the nutritional value of the yogurt. Therefore, it can be concluded that yogurt prepared by co-fermentation with Lactobacillus paracasei ZFM54 can significantly improve the quality (acidity, water-holding capacity, lactic acid bacteria content, and flavor compounds) and nutritional value (increased lactic acid bacteria content and increased non-essential amino acids such as N-ethylglycine, 4-(1S)-1-hydroxyethylphenol, L-lysine, 4-aminobenzoic acid, and Leu-Asn).

[0108] Comparative Example 1: The amount of L. paracasei ZFM54 bacterial suspension used in Example 1 was changed to 0.75 mL of 1×10⁻⁶. 7 CFU / mL bacterial suspension; the amount of commercial starter VEGE 033 was changed to 1.635 mg; the rest was the same as LS in Example 1. The resulting product was named Yogurt LSⅠ. The acidity, water-holding capacity, and lactic acid bacteria content of Yogurt LSⅠ were significantly lower than those of the Yogurt LS of the present invention. The content of 2-heptanone, D-limonene, etc. in Yogurt LSⅠ was also significantly lower than that in the Yogurt LS of the present invention.

[0109] Comparative Example 2: The amount of L. paracasei ZFM54 bacterial suspension used in Example 1 was changed to 1.125 mL of 1×10 7 CFU / mL bacterial suspension; the amount of commercial starter VEGE 033 was changed to 0.8175 mg; the rest was the same as LS in Example 1. The resulting product was named Yogurt LSII. The acidity, water-holding capacity, and lactic acid bacteria content of Yogurt LSII were significantly lower than those of the Yogurt LS of the present invention. The content of 2-heptanone, D-limonene, etc. in Yogurt LSII was also significantly lower than that in the Yogurt LS of the present invention.

[0110] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The application of Lactobacillus paracasei ZFM54 in improving the quality and nutritional value of yogurt, characterized by: Lactobacillus paracasei ZFM54 and starter culture VEGE 033 were used together for yogurt fermentation; Includes the following steps: 1) Preparation of raw materials: The raw materials consist of 12-13% skim milk powder, 6-7% sucrose, and the remainder distilled water. 2) After homogenizing the raw materials, sterilize them and cool them to 4±0.5°C to obtain aseptic skim milk; 3) Add (1 ± 0.1) mL to 99 mL of sterile skim milk. L. paracasei Lactobacillus paracasei ZFM54 suspension and 1.0~1.1 mg of starter culture VEGE 033 were fermented at (42±1)°C for (8±0.5) h, and the resulting yogurt was cooled to 4±0.5°C and refrigerated. L. paracasei The bacterial concentration of Lactobacillus paracasei ZFM54 suspension was (1±0.2)×10⁻⁶. 7 CFU / mL; Improving yogurt quality includes increasing its acidity, water-holding capacity, lactic acid bacteria content, and flavor compounds. Enhancing the nutritional value of yogurt includes increasing the content of lactic acid bacteria and increasing N-ethylglycine, 4-(1S)-1-hydroxyethylphenol, L-lysine, 4-aminobenzoic acid and leucyl-asparagine (Leu-Asn); The increase in flavor compounds includes increased levels of 2,3-butanedione, 2,3-pentanedione, 2-heptanone, and D-limonene.

2. The application according to claim 1, characterized in that: Lactobacillus paracasei ZFM54 cells were resuspended in sterile skim milk and the cell concentration was adjusted to (1±0.2)×10⁻⁶. 7 CFU / mL, L. paracasei Lactobacillus paracasei ZFM54 bacterial suspension.

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