A culture medium for streptococcus thermophilus and its use

Abstract

The application discloses a culture medium of Streptococcus thermophilus and application thereof. The culture medium is whey as the only or main nutrition, and the specific composition is cheese whey powder 3.5-10.5 g / L, sodium acetate is used to adjust pH to 6.0-6.7, and water is used as a solvent; or supernatant obtained by using lactic acid bacteria to ferment skim milk powder culture medium, and pH is 6.0-6.7. The culture medium is used to culture Streptococcus thermophilus, and the stable period is significantly prolonged, and after vacuum freeze-drying, the viable count of the bacterial powder is as high as 11.11 lg (CFU / g); non-targeted metabolomics analysis finds that the supernatant is rich in dipeptide Pro-His with antioxidant activity and characteristic peptide segments with ACE inhibition and DPP-IV inhibition potential, and taste nucleotides are significantly up-regulated, so that the fermented liquid is endowed with natural freshening characteristics. The application can obtain bacterial powder with high viable count by using whey, and can also harvest functional whey fermentation beverage rich in bioactive peptides.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a culture medium for Streptococcus thermophilus and its application. Background Technology

[0002] Streptococcus thermophilus is an important industrial lactic acid bacteria. It is oval-shaped, occurs in pairs or long chains, is non-motile, Gram-positive, and facultatively anaerobic. Its optimal growth temperature is approximately 42–45°C, and it exhibits strong tolerance to high temperatures. This strain primarily performs homolactic fermentation, rapidly breaking down lactose to produce lactic acid, lowering the pH of the fermentation system, and promoting milk coagulation to form a gel structure. Its metabolism produces flavor compounds such as acetaldehyde and diacetyl, giving yogurt its unique aroma. Due to its efficient acidification capacity and stable fermentation characteristics, Streptococcus thermophilus is often used as a starter strain for yogurt fermentation, effectively shortening fermentation time and stabilizing product texture. Based on these excellent fermentation properties, Streptococcus thermophilus has been widely used in dairy fermentation, probiotic preparations, and the development of functional foods.

[0003] In yogurt fermentation systems, *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. bulgaricus constitute a classic core strain combination, achieving a synergistic fermentation effect through a precise interaction mechanism. *Streptococcus thermophilus* efficiently metabolizes lactose, rapidly generating lactic acid, CO2, and formate metabolites, significantly lowering the pH of the fermentation system and creating a suitable acidic environment for the growth of *Lactobacillus delbrueckii* subsp. bulgaricus, in turn, efficiently hydrolyzes casein, generating various free amino acids and bioactive peptides. These substances can be efficiently utilized by *Streptococcus thermophilus*, thereby promoting its proliferation and metabolic efficiency. Therefore, *Lactobacillus delbrueckii* subsp. bulgaricus is often used as an auxiliary strain in *Streptococcus thermophilus* culture to provide essential amino acids and small peptide nutrients for its growth.

[0004] Currently, M17 broth medium is commonly used for the culture of Streptococcus thermophilus. While M17 broth medium can effectively support the growth of Streptococcus thermophilus, its complex composition and high cost make it difficult to meet the economic and sustainability requirements of large-scale industrial production. Therefore, developing a low-cost, high-efficiency industrial culture medium for Streptococcus thermophilus has significant economic value and application prospects.

[0005] Whey is the liquid obtained after separating the curd from raw milk and / or dairy products during the production of cheese, casein, or other similar products through methods such as rennet curdling or acidification curdling. Whey is a major byproduct of the dairy industry (especially cheese production), produced in enormous quantities. It is estimated that approximately 9 kg of whey is generated for every 1 kg of cheese produced. Whey is rich in lactose, whey protein, minerals, and various growth factors, possessing nutritional potential as a microbial fermentation substrate. However, large quantities of whey are directly discharged without effective utilization, resulting in resource waste and serious environmental pollution. Therefore, using whey as a fermentation substrate for Streptococcus thermophilus can achieve resource utilization of whey waste, reduce fermentation production costs, and meet the industrial demands of green manufacturing and sustainable development.

[0006] There are no existing reports on the use of whey as a major component in the culture of Streptococcus thermophilus. Summary of the Invention

[0007] The primary objective of this invention is to provide a culture medium for Streptococcus thermophilus, addressing the technical problems of high cost and waste of whey waste resources in existing Streptococcus thermophilus culture media. The culture medium provided by this invention is simple to prepare, uses readily available raw materials, and is low in cost. Streptococcus thermophilus cultured using this invention's culture medium can be freeze-dried to obtain a high viable count bacterial powder, and the whey culture supernatant can be used as a fermented whey beverage that combines flavor and nutrition.

[0008] Another object of the present invention is to provide a method for preparing thermophilic streptococci.

[0009] Another object of the present invention is to provide a functional beverage.

[0010] To achieve the above objectives, the present invention provides the following solution:

[0011] A culture medium for thermophilic streptococci is prepared with whey as the sole or main nutrient, and the specific composition is shown in composition (1) or composition (2).

[0012] (1) The composition is 3.5-10.5 g / L of cheese whey powder, the pH is adjusted to 6.0-6.7 with sodium acetate, and the solvent is water;

[0013] (2) The supernatant obtained by fermenting skim milk powder with lactic acid bacteria has a pH of 6.0 to 6.7.

[0014] In component (1):

[0015] The concentration of the cheese whey powder in the culture medium is preferably 6-8 g / L; more preferably 7 g / L.

[0016] The sodium acetate is preferably anhydrous sodium acetate.

[0017] The concentration of anhydrous sodium acetate in the culture medium is preferably 5-15 g / L; more preferably 9-11 g / L; and most preferably 10 g / L.

[0018] The water mentioned includes deionized water and purified water.

[0019] In component (2):

[0020] The supernatant is preferably obtained by the following steps: inoculating lactic acid bacteria into a skim milk powder culture medium, fermenting at 35-40℃ for 6-12 hours, centrifuging, and obtaining the supernatant; more preferably, it is obtained by the following steps: inoculating lactic acid bacteria into a skim milk powder culture medium, fermenting at 36-38℃ for 8-10 hours, centrifuging, and obtaining the supernatant.

[0021] The lactic acid bacteria are preferably Lactobacillus delbrueckii; more preferably Lactobacillus delbrueckii subsp. bulgaricus; and most preferably Lactobacillus delbrueckii subsp. bulgaricus LLFMX5, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 18, 2024, with accession number GDMCC No:65491.

[0022] The composition of the skim milk powder culture medium is: 20-50 g / L skim milk powder, with water as the solvent; preferably: 40 g / L skim milk powder, with water as the solvent.

[0023] The skimmed milk powder is preferably skimmed cow's milk powder.

[0024] The amount of inoculation is preferably 1-5% by volume; more preferably 2-3%.

[0025] The preferred centrifugation conditions are 2000-4000 rpm for 10-20 min; more preferably, 3000 rpm for 15 min.

[0026] The pH is preferably adjusted using sodium hydroxide; more preferably, it is adjusted using a sodium hydroxide solution with a concentration of 1 mol / L.

[0027] The preferred thermophilic streptococcus is Streptococcus thermophilus LLF17X1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 18, 2024, with accession number GDMCC No:65490.

[0028] The application of the above-mentioned thermophilic streptococcus culture medium in the preparation of thermophilic streptococcus.

[0029] A method for preparing Streptococcus thermophilus includes the following steps: inoculating Streptococcus thermophilus into a sterilized culture medium containing Streptococcus thermophilus and culturing at 40-45°C; separating the solid from the liquid, wherein the solid is Streptococcus thermophilus cells and the liquid is a functional beverage.

[0030] The above-mentioned method for preparing Streptococcus thermophilus further includes the following steps: after washing the Streptococcus thermophilus cells, freeze-drying them to obtain Streptococcus thermophilus freeze-dried powder.

[0031] The amount of inoculation is preferably 1-5% by volume; more preferably 2-3%.

[0032] The preferred method of sterilization is sterilization.

[0033] The preferred sterilization conditions are: 65℃ water bath for 30 minutes followed by pasteurization.

[0034] The culture time is preferably 5 to 10 hours; more preferably 6 to 9 hours.

[0035] The preferred method for solid-liquid separation is centrifugation.

[0036] The preferred centrifugation conditions are 2000-4000 rpm for 10-20 min; more preferably, 3000 rpm for 15 min.

[0037] The preferred cleaning solvent is a sodium chloride solution of 0.85–0.90% (w / v).

[0038] The preferred freeze-drying conditions are pre-freezing at -80°C followed by vacuum freeze-drying.

[0039] The preferred conditions for vacuum freeze drying are a temperature of -60 to -55°C and a vacuum degree of 6 to 8 Pa.

[0040] The vacuum freeze-drying time is preferably at least 16 hours; more preferably 20 to 30 hours; and most preferably 24 hours.

[0041] A functional beverage is obtained by the above preparation method.

[0042] The present invention discloses the following technical effects:

[0043] (1) The whey culture medium provided by the present invention is simple to prepare, the raw materials are easy to obtain and the cost is low. By using whey by-products to replace the expensive M17 culture medium, the culture cost of Streptococcus thermophilus is significantly reduced.

[0044] (2) Using whey medium to culture Streptococcus thermophilus can significantly prolong the stable period of the strain (the stable period is up to 6 hours in cheese whey medium and up to 12 hours in fresh whey medium), which is beneficial to the control of fermentation process in industrial production.

[0045] (3) Using whey culture medium to culture Streptococcus thermophilus, high viable count bacterial powder can be obtained after vacuum freeze drying. The whey culture medium for cheese reaches 11.11 lg (CFU / g), and the fresh whey culture medium reaches 10.09 lg (CFU / g).

[0046] (4) Through non-targeted metabolomics analysis, it was found that the culture supernatant is rich in characteristic functional peptides such as dipeptide Pro-His with antioxidant activity, Val-Pro-Gln with ACE inhibition potential, and Phe-Pro-Ile with DPP-IV inhibition potential, as well as natural flavor nucleotides such as 5'-cytidine monophosphate (CMP). It has both functional activity and excellent flavor potential, and has development value as a functional whey fermented beverage.

[0047] The innovative aspects of this invention:

[0048] (1) This invention achieves efficient culture of Streptococcus thermophilus using a formula of cheese whey powder and anhydrous sodium acetate. The culture medium has a very simple composition and the cost is significantly reduced compared to M17 broth medium.

[0049] (2) In this invention, the supernatant of skim milk fermented by Lactobacillus delbrueckii subsp. bulgaricus (neutralized with NaOH) is used as a natural culture medium for Streptococcus thermophilus, and the metabolic interaction between strains is utilized to achieve a recycling model.

[0050] (3) The fermented whey supernatant of the present invention does not require preparation, has both functional activity and excellent flavor, and can be used directly as a functional whey beverage or as a food ingredient for the development of functional foods. Attached Figure Description

[0051] Figure 1 This is a growth curve of Streptococcus thermophilus LLF17X1.

[0052] Figure 2 This is a bar chart showing the top 30 metabolites of Streptococcus thermophilus LLF17X1 before and after whey fermentation in cheese, ranked by fold change.

[0053] Figure 3 This is a bar chart showing the top 30 metabolites of Streptococcus thermophilus LLF17X1 before and after fermentation of fresh whey, ranked by fold change. Detailed Implementation

[0054] To better understand this invention, the following detailed description is provided in conjunction with embodiments. The inventors have conducted in-depth creative research and experimentation on this invention, resulting in many successful examples, which are listed below as specific embodiments and comparative examples.

[0055] In the following examples and comparative examples:

[0056] (1) Activation of the strain

[0057] Experimental strains: *Streptococcus thermophilus* LLF17X1, isolated and purified from Xinjiang yogurt, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 18, 2024, with accession number GDMCC No:65490; *Lactobacillus delbrueckii subsp. bulgaricus* LLFMX5, isolated and purified from Xinjiang yogurt, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 18, 2024, with accession number GDMCC No:65491.

[0058] Streptococcus thermophilus LLF17X1 from cryopreserved tubes was streaked onto M17 solid medium, and Lactobacillus delbrueckii subsp. bulgaricus LLFMX5 from cryopreserved tubes was streaked onto MRS solid medium. After culturing for 24 h, single colonies were picked and inoculated onto M17 / MRS broth medium and cultured at 42 °C and 37 °C for 9 h respectively. After activation by subculturing twice at an inoculum rate of 2% (v / v), the culture was ready for use.

[0059] Weigh 54g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312) or 42.3g of M17 broth powder (Beijing Solarbio Technology Co., Ltd., LA0610), dissolve in distilled water, and bring the volume to 1L. Sterilize at 121℃ for 15min to obtain MRS broth culture medium and M17 broth culture medium. MRS / M17 solid culture medium is made by adding 1.8g / 100mL of agar powder to the broth culture medium.

[0060] (2) Determination of viable bacteria count

[0061] Refer to GB 4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria".

[0062] (3) Vacuum freeze-drying of Streptococcus thermophilus LLF17X1

[0063] The activated Streptococcus thermophilus LLF17X1 bacterial suspension was inoculated at 2% (v / v) into sterile M17 broth medium, 7 g / L cheese whey solution (commercially available, pH adjusted to 6.5 with anhydrous sodium acetate), and fresh whey medium with pH adjusted to 6.0 using 1 mol / L sodium hydroxide solution. After being incubated at 42℃ until the stationary phase, the supernatant was removed by centrifugation at 3000 rpm for 15 min to obtain bacterial cells. The bacterial cells were washed twice with 0.85% (m / v) sterile physiological saline. After centrifugation, the bacterial cells were pre-frozen at -80℃ and then freeze-dried under vacuum (temperature -60~-55℃, vacuum degree 6~8 Pa) for 24 h.

[0064] (4) Non-targeted metabolomics analysis

[0065] ① Sample preparation and ultra-high performance liquid chromatography-mass spectrometry analysis

[0066] Take 100 mg of lyophilized sample into a centrifuge tube, add 400 μL of extraction buffer (prepared by mixing methanol and acetonitrile in a 1:1 volume ratio), vortex for 1 min, and extract by low-temperature sonication (5℃, 40 kHz) for 30 min. Freeze the sample at -20℃ for 30 min, then centrifuge at 4℃ and 14000 rpm for 15 min. Transfer the supernatant to a new centrifuge tube, dry the supernatant using a nitrogen blower, add 100 μL of reconstitution solution (prepared by mixing acetonitrile and water in a 1:1 volume ratio), and extract by low-temperature sonication (5℃, 40 kHz) for 5 min. Centrifuge at 4℃ and 14000 rpm for 15 min, and analyze the supernatant. Prepare a quality control (QC) sample by mixing equal volumes of all samples. During instrumental analysis, insert one QC sample every 5–10 samples to examine the repeatability of the analysis process.

[0067] Ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS, model: Orbitrap Exploris 240, Thermo Fisher Scientific) was used for detection. Chromatographic conditions: 3 μL samples were separated using an HSST3 column (2.1 mm × 100 mm, 1.8 μm) before mass spectrometry detection. Mobile phase A was an aqueous solution of acetonitrile containing 0.1% (v / v) formic acid, prepared by mixing acetonitrile and water at a volume ratio of 95:5; mobile phase B was an aqueous solution of acetonitrile / isopropanol containing 0.1% (v / v) formic acid, prepared by mixing acetonitrile, isopropanol, and water at a volume ratio of 47.5:47.5:5. The flow rate was 0.40 mL / min, and the column temperature was 40 °C. Mass spectrometry conditions: Electrospray ionization (ESI) was used, and data were acquired in both positive and negative ion modes. Mass scan range m / z: 70~1050. Positive ion mode spray voltage 3500V, negative ion mode spray voltage -3000V, sheath gas 50arb, ion source temperature 450℃, collision energy gradient cyclic 20V, 40V, 60V.

[0068] ② Data processing and statistical analysis

[0069] Raw LC-MS data were imported into Progenesis QI software (Waters Corporation, USA) for peak identification, peak alignment, and peak area extraction. Simultaneously, MS and MS / MS mass spectrometry information were compared with the metabolic public databases HMDB and Metlin to obtain the raw data matrix (including retention time, mass-to-charge ratio, and peak intensity information). The raw data matrix was preprocessed as follows: variables with a non-zero value ratio of more than 80% in at least one sample group were retained; missing values ​​were filled with minimum values; the mass spectrometry peak response intensity was normalized using the sum-of-the-parts normalization method; metabolite characteristic peaks with a relative standard deviation (RSD) > 30% in QC samples were removed; and log10 normalization was performed to obtain the final data matrix used for analysis. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed on the preprocessed data matrix using the R language ropls package (Version 1.6.2). Seven-fold cross-validation was used for model validation. 2 X, R 2 Y and Q 2 Evaluate model quality (Q) 2A p-value >0.5 indicates that the model has good predictive ability. Metabolites with significant differences were screened based on a variable importance in projection (VIP) >1 obtained from the OPLS-DA model and a Student's t-test p <0.05. Metabolic pathway annotation and enrichment analysis were performed on the differentially expressed metabolites using the KEGG database.

[0070] Comparative Example 1:

[0071] Streptococcus thermophilus LLF17X1 was inoculated at a 2% (v / v) inoculation rate into sterile M17 broth medium, and the viable count was determined at 0, 3, 6, 9, 12, and 15 h.

[0072] like Figure 1 As shown, in M17 broth medium, the bacterial cells grew rapidly, exhibiting a logarithmic growth phase from 0 to 6 hours, reaching a peak of 8.79 lg (CFU / mL) at 6 hours. From 6 to 9 hours, the cells entered a decline phase (8.79 lg (CFU / mL) → 8.49 lg (CFU / mL)), decreasing to 7.63 lg (CFU / mL) at 12 hours, and further decreasing to 6.31 lg (CFU / mL) at 15 hours. These results indicate that M17 broth medium supports the rapid growth of Streptococcus thermophilus LLF17X1, but the stationary phase is short and decline is rapid.

[0073] As shown in Table 1, the activated Streptococcus thermophilus LLF17X1 bacterial suspension was inoculated into M17 broth medium at an inoculation rate of 2% (v / v). After static incubation at 42℃ for 6 hours, the bacterial cells were collected by centrifugation, washed with sterile physiological saline, and then freeze-dried under vacuum. The viable bacterial count of the obtained bacterial powder was 6.20 lg (CFU / g).

[0074] Example 1: Culture of Streptococcus thermophilus in cheese whey

[0075] Cheese whey powder and anhydrous sodium acetate were dissolved in deionized water to a pH of 6.5, with a cheese whey concentration of 7 g / L and anhydrous sodium acetate concentration of 10 g / L. The mixture was pasteurized by incubating in a 65°C water bath for 30 min and then cooled to room temperature. The activated Streptococcus thermophilus LLF17X1 bacterial suspension was inoculated into the above whey culture medium at a 2% (v / v) inoculation rate and incubated at 42°C. Viable cell counts were measured at 0, 3, 6, 9, 12, and 15 h.

[0076] like Figure 1As shown, in a 7 g / L cheese whey solution (pH adjusted to 6.5 using anhydrous sodium acetate), growth was relatively slow, with a logarithmic growth phase from 0 to 9 h and a stationary phase from 9 to 15 h, showing no significant cell death. The viable cell counts at 6, 9, 12, and 15 h were 7.84, 8.40, 8.34, and 8.32 lg (CFU / mL), respectively. The results indicate that a viable cell count of 8.40 lg (CFU / mL) can be obtained in a 7 g / L cheese whey powder solution (pH adjusted to 6.5 using anhydrous sodium acetate), although lower than the peak value in M17 broth medium, exhibits the best stability.

[0077] As shown in Table 1, the activated Streptococcus thermophilus LLF17X1 bacterial suspension was inoculated into 7 g / L cheese whey solution (pH adjusted to 6.5 using anhydrous sodium acetate) at an inoculation rate of 2% (v / v). After static incubation at 42℃ for 9 h, the bacterial cells were collected by centrifugation, washed with sterile physiological saline, and then freeze-dried under vacuum. The viable count of the obtained bacterial powder was 11.11 lg (CFU / g).

[0078] The resulting fermented cheese whey supernatant is smooth on the palate, with an initial flavor of mild lactic acid followed by a refreshing sweetness and a subtle milky aroma. The acidity is rounded and not sharp, resulting in a balanced overall flavor and a refreshing and pleasant taste.

[0079] Example 2: Culture of thermophilic streptococci in fresh whey

[0080] Activated Lactobacillus delbrueckii subsp. bulgaricus LLFMX5 was inoculated into a sterile 40 g / L skim milk powder solution at a 2% (v / v) inoculation rate. After static incubation at 37°C for 9 h, the supernatant was obtained by centrifugation at 3000 rpm for 15 min. The pH was adjusted to 6.0 using 1 mol / L sodium hydroxide solution to prepare fresh whey medium SSM_MX5, which was then pasteurized at 65°C for 30 min and cooled to room temperature. Activated Streptococcus thermophilus LLF17X1 was inoculated into the above whey medium SSM_MX5 at a 2% (v / v) inoculation rate and incubated statically at 42°C. Viable cell counts were measured at 0, 3, 6, 9, 12, and 15 h.

[0081] like Figure 1 As shown, *Streptococcus thermophilus* LLF17X1 grew well in the supernatant after the pH was adjusted to 6.0 with 1 mol / L sodium hydroxide solution. The logarithmic growth phase lasted from 0 to 3 hours, with the viable cell count rapidly increasing from 6.52 lg (CFU / mL) to 8.02 lg (CFU / mL). After 3 hours, it entered the stationary phase, with viable cell counts of 8.15, 8.12, 8.02, and 8.07 lg (CFU / mL) at 3, 6, 9, 12, and 15 hours, respectively. The stationary phase lasted up to 12 hours, with no significant decline observed. The maximum viable cell count reached 8.15 lg (CFU / mL).

[0082] As shown in Table 1, the activated Streptococcus thermophilus LLF17X1 bacterial suspension was inoculated into fresh whey medium SSM_MX5 at a concentration of 2% (v / v) and cultured at 42℃ for 6 hours. The bacterial cells were then collected by centrifugation, washed with sterile physiological saline, and freeze-dried. The viable count of the obtained bacterial powder was 10.09 lg (CFU / g).

[0083] The resulting fermented fresh whey supernatant has a distinct and prominent lactic acid flavor upon entry, but the acidity is pure, without any unpleasant sourness or irritation. Its acidity spreads rapidly in the mouth, accompanied by fermented aromas, resulting in a clean, direct flavor with good palatability.

[0084] Table 1. Viable bacterial count of Streptococcus thermophilus LLF17X1 before and after vacuum freeze-drying

[0085]

[0086] Note: Results are expressed as mean ± standard deviation. Different superscript lowercase letters in the same column indicate significant differences (p < 0.05).

[0087] Example 3: Differential metabolite analysis of Streptococcus thermophilus LLF17X1 before and after whey fermentation in cheese

[0088] Following fermentation of cheese whey by Streptococcus thermophilus LLF17X1, the top 30 metabolites by fold change, such as... Figure 3Shown, Ile Asn Asp, Pro-His, Glu-Thr-Ile, Phe-Glu-Leu, Leu-Glu, N-Succinyl-L, L-2,6-Diaminopimelate, Phenylalanyl-Glycine, Glu Pro Val, 4-(2-Amino-3-Hydroxyphenyl)-2,4-Dioxobutanoic Acid, Asp Val Val, Glutamylphenylalanine, N-Hydroxy-L-Tryptophan, Ile Leu Glu, Asn-Tyr, Gly-Ala-Ile, Gly Val Val, Trans-3-Hydroxy-L-Proline, L-Beta-Aspartyl-L-Phenylalanine, Leu Glu Asp, Ile Gly Val, Ala-Val, Pro Glu Ile, Deoxyguanosine, Phe-Pro-Ile, Ala Val Gln, Asp Pro Lys The abundance of IleAla, Gly Ile Gln, Leu Gln Val, Pro-Tyr-Ser, and Val Pro Gln was significantly increased. Pro-His is a typical antioxidant dipeptide with a hydrophobic Pro at the N-terminus and a hydrogen-bonding His at the C-terminus. A study published in *Nutrients* in 2022 confirmed that Pro-His has significant scavenging activity against the toxic 4-hydroxynonenal (HNE), with reactivity even higher than the well-known antioxidant dipeptide L-carnosine, and its stability in serum is also superior to L-carnosine. This study further showed that the high reactivity of Pro-His is attributed to the stronger electrophilicity of the imine intermediate formed during its reaction, and that the substitution of its primary amine with a secondary amine is beneficial to its scavenging mechanism. Peptides containing the Xaa-Pro-Yaa structure (such as Val-Pro-Gln and Phe-Pro-Ile) have a core proline residue that is a key structure ensuring ACE inhibitory or DPP-IV inhibitory activity. A 2023 study identified the hexapeptide Val-Val-Val-Pro-Gln-Asn (VVVPQN) from camellia seed cake, confirming its significant ACE inhibitory activity and stability after simulated gastrointestinal digestion. This provides direct evidence for the hypotensive function of the Val-Pro-Gln sequence. Another 2023 study also found that peptides containing the Glu-Val-Pro-Gln-Ala-Tyr sequence possess both ACE inhibitory and zinc ion chelating activities.Regarding DPP-IV inhibitory activity, multiple studies have shown that oligopeptides containing proline (Pro) at the second position are effective dipeptidyl peptidase-IV (DPP-IV) inhibitors. Ile-Pro-Ile (IPI) is one of the most potent DPP-IV inhibitory peptides known to date. In terms of flavor contribution, studies have shown that dipeptides containing glutamate (Glu) and aspartic acid (Asp) have been identified as flavor peptides with umami or umami-enhancing effects (such as Glu-Glu, Asp-Glu, etc.). Therefore, the significantly upregulated Leu-Glu, Glu-Pro-Val, and other Glu-containing peptides in this embodiment are expected to positively contribute to the umami and richness of the fermentation broth. These significantly upregulated functional peptides and flavor-related oligopeptides provide direct molecular evidence for the use of the fermentation supernatant of Streptococcus thermophilus LLF17X1 in cheese whey as a natural functional beverage base.

[0089] Example 4: Differential metabolite analysis of Streptococcus thermophilus LLF17X1 before and after fresh whey fermentation

[0090] Following the action of Streptococcus thermophilus LLF17X1 in fresh whey SSM_MX5, the top 30 metabolites by fold change were as follows: Figure 2As shown, the 29 metabolites include Pro-His, Asp Pro Lys Ile Ala, Gly Ile Gln, Pro GluIle, N-Succinyl-L, L-2,6-Diaminopimelate, Gly Val Val, Ala Ile Val, Val Leu Thr, Gly-Ala-Ile, Tyramine Glucuronide, 5'-Cytidylic Acid, Ile Gly Val, Ala-Val-Leu, Leu-Gly-Gly, Gamma-Glutamylmethionine, Leu Gln Val, Leu-Met, Leu Ser Ala, Ala-Ala-Ile, Gly-Gly-Ile, Met-Glu-Leu, Val Pro Gln, Citraconic Acid, Ile-Gly-Ser, Val-Gly-Ile, 8-Hydroxyguanosine, Ser Leu The abundance of Asp, Pro-Tyr-Ser, and 11-Beta-Hydroxyandrosterone-3-Glucuronide was significantly upregulated, while the abundance of Phe-Met was significantly downregulated. Regarding functional activity, the highly efficient antioxidant function of Pro-His has been discussed in detail in Example 3, and Val-Pro-Gln possesses ACE inhibitory potential due to the presence of the VPQ core sequence. In terms of flavor contribution, 5'-Cytidylic Acid (CMP) is a known flavor nucleotide that exhibits a synergistic umami-enhancing effect with inosinic acid and guanylic acid. The significant upregulation of CMP after fermentation directly contributes to the umami and fullness of the product, providing strong evidence for its role as a natural flavor enhancer. These significantly upregulated functional peptides and flavor nucleotides provide direct molecular evidence for the use of the fermentation supernatant of Streptococcus thermophilus LLF17X1 in fresh whey as a natural functional beverage base. In particular, the high antioxidant capacity of Pro-His and the natural umami-enhancing effect of CMP give the supernatant both functional and flavor advantages without the need for added antioxidants or umami enhancers.

[0091] In summary, this invention provides a whey culture medium for the cultivation and application of Streptococcus thermophilus LLF17X1. This medium is simple in composition and low in cost, and can significantly extend the stability period of the strain while obtaining a viable bacterial count of up to 11.11 g (CFU / g) through vacuum freeze-drying. This invention reveals the functional material basis of the fermentation supernatant through non-targeted metabolomics. The fermentation supernatant is rich in the antioxidant dipeptide Pro-His and characteristic peptides with ACE inhibition and DPP-IV inhibition potential (such as Val-Pro-Gln, Phe-Pro-Ile, etc.). Simultaneously, flavor nucleotides such as 5'-cytidine monophosphate (CMP) are significantly upregulated, giving the fermentation broth natural umami-enhancing properties. This invention can utilize whey to obtain high-viable-count bacterial powder and also harvest functional whey fermented beverages rich in bioactive peptides, possessing environmental benefits, economic benefits, and promising industrial application prospects, providing a new technical path for the green processing of whey and the development of functional foods.

Claims

1. A culture medium for Streptococcus thermophilus, characterized in that: The culture medium is based on whey as the sole or main nutrient, and its specific composition is shown in composition (1) or composition (2). (1) The composition is 3.5-10.5 g / L of cheese whey powder, the pH is adjusted to 6.0-6.7 with sodium acetate, and the solvent is water; (2) The supernatant obtained by fermenting skim milk powder with lactic acid bacteria has a pH of 6.0 to 6.

7.

2. The culture medium for Streptococcus thermophilus according to claim 1, characterized in that: The sodium acetate mentioned in composition (1) is anhydrous sodium acetate; The concentration of anhydrous sodium acetate in the culture medium is 5-15 g / L; The supernatant described in component (2) is obtained by the following steps: lactic acid bacteria are inoculated into skim milk powder culture medium and fermented at 35-40℃ for 6-12 hours, then centrifuged to obtain the supernatant; The pH mentioned in composition (2) is obtained by adjusting with sodium hydroxide.

3. The culture medium for Streptococcus thermophilus according to claim 2, characterized in that: The concentration of the cheese whey powder in the culture medium is 6-8 g / L; The concentration of anhydrous sodium acetate in the culture medium is 9–11 g / L; The lactic acid bacteria mentioned are Lactobacillus delbrueckii; The composition of the skim milk powder culture medium is: 20-50 g / L skim milk powder, with water as the solvent; The amount of inoculation is 1-5% by volume.

4. The culture medium for Streptococcus thermophilus according to claim 3, characterized in that: The skim milk powder mentioned is skim milk powder; The centrifugation conditions are 2000-4000 rpm for 10-20 min.

5. The culture medium for Streptococcus thermophilus according to any one of claims 1 to 4, characterized in that: The thermophilic streptococcus is thermophilic streptococcus LLF17X1.

6. The use of the culture medium of Streptococcus thermophilus according to any one of claims 1 to 5 in the preparation of Streptococcus thermophilus.

7. A method for preparing thermophilic streptococci, characterized in that... The method includes the following steps: inoculating Streptococcus thermophilus into the culture medium of Streptococcus thermophilus as described in any one of claims 1 to 5, and culturing it at 40 to 45°C; separating the solid from the liquid, wherein the solid is Streptococcus thermophilus cells and the liquid is a functional beverage.

8. The method for preparing thermophilic streptococci according to claim 7, characterized in that... The process also includes the following steps: after washing the Streptococcus thermophilus cells, freeze-drying them to obtain Streptococcus thermophilus freeze-dried powder.

9. The method for preparing thermophilic streptococci according to claim 8, characterized in that: The amount of inoculation is 1-5% by volume; The sterilization method mentioned is sterilization; The culture time is 5-10 hours; The solid-liquid separation method is centrifugation; The cleaning solvent is a sodium chloride solution of 0.85–0.90% w / v; The freeze-drying conditions are as follows: pre-freezing at -80℃, temperature -60 to -55℃, vacuum degree 6 to 8 Pa, and time at least 16 hours.

10. A functional beverage, characterized in that: It is obtained by the preparation method described in claim 7.

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