Lactobacillus delbrueckii subsp. Indica and application thereof

Abstract

The invention discloses a lactobacillus delbrueckii subsp. Indicus and application of the lactobacillus delbrueckii subsp. Indicus. The lactobacillus delbrueckii subsp. Indicus is preserved in Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC No: 64946, and the lactobacillus delbrueckii subsp. Indicus is preserved in Guangdong Province Microbial Culture Collection Center. The lactobacillus delbrueckii subsp. Indicus has good safety, gastrointestinal environment adaptability and antibacterial effect, the fermented milk prepared by compounding and fermenting the lactobacillus delbrueckii subsp. Indicus and streptococcus thermophilus according to 1: 1-1: 6 has the characteristics of low post-acidification, good texture and flavor and the like, the titration acidity after 4-10 hours of fermentation is 60-90 DEG T, the titration acidity change in the middle and later periods of storage is less than 5 DEG T, the lactobacillus delbrueckii subsp. Indicus has good rheological properties, and the lactobacillus delbrueckii subsp. The beverage contains characteristic flavor substances such as octanoic acid and dimethyl disulfide.

Description

Technical Field

[0001] This invention relates to a type of Lactobacillus delbrueckii, specifically to a subsp. indicus of Lactobacillus delbrueckii and its application in the preparation of yogurt starter and fermented milk; it belongs to the field of microbial application technology. Background Technology

[0002] Yogurt, as an important component of dairy products, is widely loved by consumers for its unique flavor, excellent nutritional value, and significant probiotic effects. With the increasing demand for food safety and functional foods, research on yogurt starter cultures has become a core area in the dairy industry. Yogurt starter cultures are key factors in ensuring the stable quality of yogurt during production and in achieving its excellent sensory qualities and texture. However, my country started relatively late in starter culture preparation technology, and existing yogurt starter cultures still have certain limitations, such as a limited selection of strains, unstable fermentation performance, and inability to fully meet consumers' needs for specific nutritional and functional benefits. Currently, the import ratio of starter cultures in China is as high as 90%-95%, and core strains (such as exopolysaccharide-producing strains and acid-resistant probiotics) are basically monopolized by international brands, resulting in high starter culture costs and dependence on the international supply chain.

[0003] Traditional yogurt starter cultures mainly use Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for fermentation, which have basic fermentation capabilities, but are still lacking in functionality. Lactobacillus delbrueckii, an important member of the Lactobacillus genus, can currently be divided into six subspecies: the Bulgarian subspecies (Lactobacillus delbrueckii subsp. bulgaricus) has become the most representative and dominant subspecies in the genus due to its efficient application in the fermented food industry and in-depth exploration in the field of basic research; the lactic acid subspecies (Lactobacillus delbrueckii subsp. lactis) is often used in the production of cheese; the Indian subspecies (Lactobacillus delbrueckii subsp. indicus) was isolated from traditional fermented dairy products in India; and the subspecies Lactobacillus delbrueckii, Jakobsenii, and allosunkii are all isolated from plant sources and used in plant-based fermentation.

[0004] Chinese invention patent CN110484477B discloses a strain of *Lactobacillus delbrueckii* subsp. bulgaricus and its applications. The accession number for *Lactobacillus delbrueckii* subsp. bulgaricus Ouya-D-L5 is CGMCC No. 17959. This subsp. bulgaricus Ouya-D-L5 has a high capacity for producing extracellular polysaccharides. Fermenting skim milk at 38℃ for 18 hours can produce up to 268.9 mg / L of extracellular polysaccharides. The extracellular polysaccharides produced by this strain are not easily destroyed by shear force, resulting in high viscosity, weak acid production, mild sourness, and a mild astringency in stirred yogurt. This bacterium also has certain antioxidant and free radical scavenging abilities. As a yogurt starter, this strain produces stirred yogurt with excellent flavor and texture, providing a new fermentation source for yogurt production and preventing post-fermentation acidification. This technology is the same as the subsp. bulgaricus strain of this invention and has certain functional similarities. Please explain the problems existing in this technology in light of its characteristics, and be sure to analyze the reasons for the problems in conjunction with its characteristics. However, this technology has a weak acid-producing capacity of Lactobacillus delbrueckii subsp. bulgaricus, which leads to a slow increase in the acidity of yogurt, reduces production efficiency, and causes large changes in acidity during storage, which is not conducive to maintaining the quality stability of yogurt during storage.

[0005] Chinese invention patent CN118562684B discloses an acid- and bile-resistant *Lactobacillus delbrueckii* subsp. bulgaricus LB34 and its applications, with accession number CGMCC NO.24665 and accession date April 11, 2022. This strain exhibits good resistance to gastric acid and bile salts, inhibits numerous intestinal pathogens, and improves the intestinal microecological environment. When used in yogurt fermentation, it results in a short acid production cycle, high fermentation efficiency, and a yogurt with a high-viscosity, silky texture, reducing the need for stabilizers. Simultaneously, the yogurt contains a diverse range of volatile flavor compounds and has a rich aroma. However, the gastrointestinal adhesion ability of this *Lactobacillus delbrueckii* subsp. bulgaricus needs improvement, and adhesion is a key characteristic for its colonization and probiotic effects in the intestines. Summary of the Invention

[0006] To address the problems existing in the prior art, the primary objective of this invention is to provide a *Lactobacillus delbrueckii* subsp. India that possesses antibiotic sensitivity, gastrointestinal adaptability, antibacterial efficacy, and excellent fermentation and functional properties.

[0007] Another objective of this invention is to provide the application of Lactobacillus delbrueckii subsp. indicus LLFMY24 in the preparation of yogurt starter and fermented milk.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A subsp. indicus of Lactobacillus delbrueckii has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:64946.

[0010] The application of *Lactobacillus delbrueckii* subsp. India in the preparation of yogurt starter.

[0011] The application of *Lactobacillus delbrueckii* subsp. indicus in the preparation of yogurt starter: *Lactobacillus delbrueckii* subsp. indicus and *Streptococcus thermophilus* were inoculated separately into broth medium and cultured at 37–42°C for 12–24 hours. After centrifugation to collect the cells, they were washed and resuspended. The bacterial counts of the two resuspensions were adjusted to 1 × 10⁻⁶. 8 ~1×10 10 CFU / mL, the two resuspensions are mixed at a ratio of 1:10 to 1:1 to obtain the yogurt starter.

[0012] Preferably, the broth culture medium is MRS / M17 broth culture medium; the washing is done with sterile water.

[0013] A fermented milk, produced by fermentation with the aforementioned yogurt starter.

[0014] The yogurt starter culture is prepared by inoculating sterilized milk with the starter culture; fermenting at 42±2℃ for 5 to 8 hours until the titratable acidity reaches 60 to 90°T; and then maturing after cooling.

[0015] Preferably, the sterilized milk is prepared by sterilizing one or more of the following raw materials: raw cow's milk, raw sheep's milk, concentrated milk for food industry, and milk powder, at a temperature of 90-98°C for 5-10 minutes.

[0016] Preferably, the inoculation temperature is 40-45°C, and the starter culture is inoculated into the sterilized milk at a rate of 0.01-1.0% per gram per milliliter.

[0017] Preferably, the fermented milk has a titration acidity of 60–90°T, a water-holding capacity of 55–58%, and a viscosity of 1000–2000 cP.

[0018] Preferably, after the fermented milk is stored at 4-8℃ for 3 to 21 days, the titratable acidity change is ≤5°T and the pH change is ≤0.3.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] 1) The *Lactobacillus delbrueckii* subsp. *indicus* disclosed in this invention has simple culture conditions, is sensitive to antibiotics such as ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin, and chloramphenicol, and does not produce biogenic amines such as histamine, tyramine, cadaverine, or putrescine, nor does it produce nitroreductase, indole, or hemolysin. Whole-genome information shows that this strain lacks complete gene clusters related to biogenic amine formation and genes encoding typical virulence factors, exhibiting good safety and great application potential in industries such as fermented foods.

[0021] 2) The Lactobacillus delbrueckii subsp. indicus disclosed in this invention has strong gastrointestinal tolerance and adhesion. After entering the gastrointestinal fluid for 15 hours, its viable bacterial count only decreased by 0.74 log CFU / mL, and its adhesion rate to small intestinal epithelial cells was 32.01%. It also has a good inhibitory effect on foodborne pathogens and has excellent probiotic potential.

[0022] 3) The Lactobacillus delbrueckii subsp. indicus disclosed in this invention, when combined with Streptococcus thermophilus, exhibits strong acid-producing ability during yogurt fermentation. Compared to yogurt prepared with commercial starter cultures, the resulting yogurt exhibits better rheological properties and lower post-acidification. The titratable acidity change during the later stages of storage is less than 5°T, and it produces characteristic flavor components such as caprylic acid and dimethyl disulfide. There is no whey separation, the texture is delicate, the taste is moderate, and the overall sensory acceptance is high. Attached Figure Description

[0023] Figure 1 The colony morphology (a) and microscopic image (b) of Lactobacillus delbrueckii subsp. indicus of this invention are shown.

[0024] Figure 2 This is a circulated genome diagram of Lactobacillus delbrueckii subsp. indicus, the species of Lactobacillus indicus of this invention.

[0025] Figure 3 This is a graph showing the change in the number of viable bacteria of Lactobacillus delbrueckii subsp. indicus in simulated gastrointestinal fluid.

[0026] Figure 4This is a growth curve of Lactobacillus delbrueckii subsp. indicus and Streptococcus thermophilus in MRS / M17 broth.

[0027] Figure 5 This is a graph showing the acid production curves of Lactobacillus delbrueckii subsp. indicus and Streptococcus thermophilus in whole milk.

[0028] Figure 6 The images show (a) and (b) microscopic images of the colony morphology of Streptococcus thermophilus of this invention.

[0029] Figure 7 This is a genomic diagram of the thermophilic streptococcus of this invention;

[0030] Figure 8 This is a graph showing the changes in titration acidity and pH of fermented milk prepared with the starter culture Y-2425 of this invention and the control group (LS) during storage, i.e., the post-acidification curve.

[0031] Figure 9 Viscosity curves of fermented milk prepared with the starter culture Y-2425 of this invention and the control group (LS);

[0032] Figure 10 Frequency scan curves of fermented milk prepared with the starter culture Y-2425 of this invention and the control group (LS). Detailed Implementation

[0033] To better understand the present invention, it will be further described below with reference to the accompanying drawings and specific embodiments. However, the implementation of the present invention is not limited thereto. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In this invention, Lactobacillus delbrueckii subsp.indicus, Lactobacillus delbrueckii subsp.indicus, LLFMY24 and LLFMY24 all refer to Lactobacillus delbrueckii subsp.indicus, with LLFMY24 being an abbreviation.

[0035] In this invention, Streptococcus thermophilus, Streptococcus thermophilus, Streptococcus thermophilus LLF17Y25 and LLF17Y25 all refer to Streptococcus thermophilus, and LLF17Y25 is an abbreviation.

[0036] The inventors isolated a strain of Lactobacillus delbrueckii subsp. indicus from traditional fermented milk. This Lactobacillus delbrueckii subsp. indicus was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 2, 2024, with accession number GDMCCNo:64946, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0037] Tests revealed that *Lactobacillus delbrueckii* subsp. *indicus* exhibits rapid growth, high viability, and simple culture conditions. It is sensitive to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin, and chloramphenicol. It does not produce four biogenic amines: cadaverine, histamine, putrescine, and tyramine. It also does not produce nitroreductase, indole, or hemolysin, and lacks complete gene clusters related to biogenic amine formation and typical virulence factor encoding genes, demonstrating good safety. It exhibits strong tolerance to the gastrointestinal environment and strong adhesion to Caco-2 cells. Its bacterial suspension shows good inhibitory effects on common intestinal pathogens in solid culture media, indicating excellent probiotic potential. The prepared yogurt starter Y-2425 demonstrates high efficiency in yogurt fermentation and produces yogurt with significantly better texture, flavor, and lower post-acidification compared to the control group (LS) starter.

[0038] In the following examples and comparative examples:

[0039] (1) Preparation of MRS / M17 broth culture medium

[0040] 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 1L of distilled water, and sterilize at 121℃ for 15min. For MRS / M17 solid culture medium, add 1.8g / 100mL agar powder to the broth culture medium.

[0041] (2) Gram staining and morphological observation methods refer to "Classification, Identification and Test Methods of Lactic Acid Bacteria" edited by Ling Daiwen (1999 edition).

[0042] (3) 16S rDNA sequencing and identification

[0043] The most commonly used universal bacterial primers were used: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 2492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction mixture consisted of: 2 μL DNA template, 2 μL primer 27F, 2 μL primer 1492R, 20 μL 2×PCR Mix, and 24 μL ultrapure water. The PCR program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 46℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and 72℃ final extension for 10 min. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Homology comparison of the sequencing results was performed using the BLAST tool in NCBI.

[0044] (4) Whole genome sequencing

[0045] The activated bacterial strain was centrifuged at 8000xg for 5 min to collect the bacterial cells. High-quality genomic DNA was extracted and its purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. Large DNA fragments were recovered using the BluePippin automated nucleic acid recovery system. Library construction was performed using the SQK-LSK109 ligation kit, including DNA damage repair and end repair, magnetic bead purification, adapter ligation and magnetic bead purification, Qubit library quantification, and finally, the high-quality library DNA was sequenced. The raw data underwent quality control, filtering out low-quality and excessively short reads (<2000bp). The filtered reads were assembled de novo using Canu v1.5, and the assembly results were corrected using Racon v3.4.3 software. Circularization and start site adjustment were performed using Circlator v1.5.5, and error correction was performed on the assembled draft genome using Pilon v1.22. Gene prediction was performed using Prodigal v2.6.3, and repetitive sequence alignment was performed using RepeatMasker v4.0.5. Twelve strains with >98% similarity to the experimental strain were downloaded from the NCBI database as reference strains for genome-wide similarity analysis to further determine strain classification (see Table 2). Average Nucleotide Identity (ANI) analysis between the strain and closely related type species was performed using the JSpeciesWS online database (https: / / www.ribocon.com / jspeciesws.html), with BLAST+ used to calculate ANI. Digital DNA-DNA hybridization (dDDH) values ​​between the strain and closely related strains were calculated using Formula 2 of the online tool Genome-to-Genome Distance Calculator (http: / / ggdc.dsmz.de / ggdc.php).The strain's gene sequences were aligned with protein homologous sequences in databases including the Cluster of Orthologous Groups of Proteins (COG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Carbohydrate-Active Enzymes (CAZy), the Comprehensive Antibiotic Resistance Database (CARD), and the Virulence Factors of Pathogenic Bacteria (VFDB) database to annotate genes related to carbohydrate biotransformation, protein hydrolysis, amino acid synthesis and metabolism, and fatty acid synthesis, as well as antibiotic resistance, virulence factors, and biogenic amine synthesis and metabolism. BLASTP was used to obtain annotation information for all involved enzymes in the non-redundant protein (Nr) and Swissprot databases, with an e-value cutoff of 1e. -5 .

[0046] (5) Antibiotic resistance

[0047] According to the European Food Safety Authority (EFSA) regulations on drug susceptibility testing, the microbroth dilution method was used for drug resistance phenotype testing. The selected antibiotics were ampicillin, vancomycin, gentamicin, kanamycin, chloramphenicol, erythromycin, clindamycin, tetracycline, and chloramphenicol, prepared with gradient concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. The strains stored at -80℃ were activated twice consecutively, and the bacterial suspension concentration was adjusted to 8 log CFU / mL. 100 μL of each gradient concentration of antibiotics and 100 μL of bacterial suspension were added to 96-well plates. A positive control was set up with no antibiotics added, only the test bacterial suspension and culture medium containing the highest concentration of antibiotic solvent; a negative control was set up with neither antibiotics nor test strains added, only culture medium. The plates were incubated at 37℃ for 48 h, and the absorbance was measured at 625 nm. The drug resistance phenotype of different strains was determined based on the minimum inhibitory concentration (MIC) inflection point specified by EFSA.

[0048] (6) Produced amines

[0049] The amino acid culture media used for detecting tyramine, cadaverine, putrescine, and histamine were prepared by adding 0.5% (w / v) L-tyrosine, L-lysine, L-ornithine, and L-histidine to the amino acid decarboxylase culture medium, respectively. The media were then dissolved in distilled water and the pH was adjusted to 6.8. The activated bacterial suspensions were inoculated into the four amino acid culture media at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. The color changes of the culture media were observed. Uninoculated culture media and culture media inoculated with bacterial suspensions but without any added amino acids served as controls. A turbid and purple culture medium indicated a positive result; otherwise, a negative result was observed.

[0050] (7) Indigo-producing matrix

[0051] The activated bacterial suspension was inoculated into indigo-based medium at 3% (v / v) and incubated at 37°C for 48 hours. Kovacs' reagent was slowly added dropwise, and the color change was observed. Escherichia coli ATCC 25922 was used as a positive control, and uninoculated indigo-based medium served as a blank control. A deep red color on the surface of the medium indicated a positive result; otherwise, a negative result was indicated.

[0052] (8) Nitroreductase activity

[0053] The activated bacterial culture was inoculated into nitrate medium at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. Then, 1-2 drops of nitrate reducing reagent solution A and solution B were added to the culture medium, and the results were observed. *Escherichia coli* ATCC25922 was used as a positive control, and the uninoculated culture medium served as a blank control. A positive result for nitrate reductase detection was indicated by a cloudy, deep red culture medium; a negative result was indicated by a cloudy, yellow, or pale pink culture medium.

[0054] (9) Hemolytic activity

[0055] The activated bacterial suspension was inoculated onto Columbia blood agar medium using the streak plate method and incubated at 37°C for 48 hours. The presence of hemolysis zones was observed. Staphylococcus aureus ATCC 25923 was used as a positive control, and the uninoculated medium served as a blank control. A grass-green hemolysis zone indicated α-hemolysis; a clear hemolysis zone indicated β-hemolysis; and the absence of either indicated no hemolysis (γ-hemolysis).

[0056] (10) Tolerance to simulated gastrointestinal fluid

[0057] Simulated gastric fluid containing 3 mg / mL pepsin was prepared using 0.01 mol / L PBS buffer (pH 3.0), and simulated intestinal fluid containing 1 mg / mL trypsin was prepared using 0.01 mol / L PBS buffer (pH 8.0). Both simulated gastric and intestinal fluids were filtered through a 0.22 μm filter and stored at 4°C for later use. The overnight culture was centrifuged, washed, and the bacterial concentration was adjusted to 10⁻⁶ with PBS. 9 CFU / mL, 1 mL of bacterial culture was added to 9 mL of simulated gastric fluid. After incubation at 37°C for 3 hours, 1 mL of the bacterial-containing gastric fluid was added to 9 mL of simulated intestinal fluid and incubated at 37°C for another 12 hours. Samples were taken at 0h, 1h, 3h, 5h, 7h, 11h, and 15h, and the viable count was calculated using the plate dilution method.

[0058] (11) Adhesion to small intestinal epithelial cells

[0059] Caco-2 cell culture: After resuscitation, Caco-2 cells were added to DMEM complete culture medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin mixture, and incubated statically at 37°C and 5% CO2. The cell culture medium was changed approximately every 2 days. When the cells grew well and adhered to the culture medium to the wall at approximately 80%, they were passaged using 0.25% trypsin-EDTA digestion. Caco-2 cells were passaged approximately 5 times for later use.

[0060] Preparation of bacterial suspension: Centrifuge the activated bacterial solution at 8000xg for 5 min to collect the bacterial cells. Wash 2-3 times with sterile PBS and resuspend in antibiotic-free DMEM culture medium. Adjust the concentration of the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, for later use.

[0061] Adhesion count: Caco-2 cells were counted at a concentration of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured at 37°C with 5% CO2. The culture medium was changed every other day. When the cells grew to a monolayer, the culture medium was removed, and the prepared bacterial suspension was added and incubated at 37°C for 2 hours. After incubation, unattached bacteria were washed away with sterile PBS, and the cells were then lysed with 0.05% Triton X-100 solution to obtain a bacterial suspension adhering to the cells. The adhering bacteria were counted using the agar dilution method.

[0062] (12) Inhibitory effect on pathogenic bacteria

[0063] The inhibitory effects of the bacterial strain on six common enteropathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium ATCC 14028, Listeria monocytogenes CMCC 54002, Enterobacter sakazakii ATCC 29544, Streptococcus hemolyticus ATCC21059, and Staphylococcus aureus ATCC 12598) were determined using the Oxford cup method. The indicator pathogens were centrifuged at 8000 x g for 5 min, the supernatant was discarded, and the bacterial concentration was adjusted to 10⁻⁶ with sterile physiological saline. 6 -10 7 CFU / mL, take 100 μL and spread it on an LB agar plate. Place a sterile Oxford cup parallel to the plate, and add 200 μL of the test bacteria suspension in the logarithmic growth phase to the Oxford cup. Incubate at 37°C until a clear inhibition zone appears, and measure the diameter of the inhibition zone with calipers. Use sterile MRS / M17 broth medium as a blank control.

[0064] (13) Growth capacity

[0065] Centrifuge the activated bacterial suspension at 8000xg for 5 min, collect the bacterial cells, wash 2-3 times with sterile phosphate-buffered saline (PBS), and resuspend in 5 mL of sterile PBS for later use. Inoculate the bacterial suspensions of *Lactobacillus delbrueckii* and *Streptococcus thermophilus* at 5% (v / v) into MRS / M17 broth medium and incubate at 37°C. Take samples at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 20 h, and 24 h of culture to determine their OD values. 600 With culture time as the x-axis, OD 600 Plot the growth curve of the strain on the ordinate. Use the culture medium containing the inoculated bacterial solution as a blank control. The experiment was repeated three times.

[0066] (14) Determination of acidity in fermented lactic acid

[0067] The titratable acidity and pH of fermented milk were determined in accordance with GB 5009.239-2016, "National Food Safety Standard - Determination of Acidity in Food".

[0068] (15) Determination of water-holding capacity of fermented milk

[0069] Take 12g of fermented milk sample into a 15mL centrifuge tube, centrifuge, discard the supernatant, and record the mass. Repeat the measurement three times for each sample and take the average value. Calculate the water-holding capacity of the sample according to formula (1).

[0070] WHC=(M2-M0) / M1×100% Formula (1)

[0071] In the formula: WHC (%) is the water-holding capacity of the fermented milk, M0 (g) is the mass of the centrifuge tube, M1 (g) is the mass of the fermented milk before centrifugation, and M2 is the mass of the centrifuge tube containing the fermented milk after centrifugation.

[0072] (16) Determination of the viscosity of fermented milk

[0073] Take an appropriate amount of fermented milk sample and place it in a beaker. Use a viscometer to measure its viscosity. The measurement conditions are: #4 rotor, torque 20-100%, temperature 25℃, and measurement time 30s. Each sample is measured three times.

[0074] (17) Sensory evaluation

[0075] A sensory evaluation team composed of 10 food science professionals with relevant sensory evaluation experience was formed. Evaluation standards were developed based on the sensory requirements of GB19302-2010 "National Food Safety Standard for Fermented Milk" and RHB 103-2004 "Detailed Rules for Sensory Quality Evaluation of Yogurt" (see Table 1). The color, aroma, and texture of the fermented milk samples were evaluated after 24 hours of post-ripening.

[0076] Table 1 Sensory Evaluation Criteria for Fermented Milk

[0077]

[0078] (18) Rheological properties

[0079] The rheological properties of fermented milk were determined using a rotational rheometer. A 50 mm diameter stainless steel plate was used, with a spacing of 1.0 mm, an equilibration time of 2 min, and a test temperature of 25 °C.

[0080] Viscosity curve: The shear rate was measured in the range of 0.1 to 100 s⁻¹, and each sample was measured three times.

[0081] Frequency scanning: First, stress scanning tests were performed to determine the linear viscoelastic range. The frequency scanning test range was 0.1-100 rad / s, with a controlled stress of 0.1%, to obtain the changes in the storage modulus (G') and loss modulus (G”) of the fermented milk as a function of frequency. Each sample was measured three times.

[0082] (19) Volatile flavor compounds

[0083] Volatile flavor compounds in the control group and compound fermented milk were detected using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fermented milk samples aged 24 hours were collected for further analysis of volatile flavor compounds.

[0084] Extraction conditions: Accurately weigh 1g of fermented milk sample, 0.25g of sodium chloride, and 10μL of internal standard (2-methyl-3-heptanone, 10μg / mL) into a 20mL headspace vial. Equilibrate at 60℃ for 15min with a vibration speed of 500rpm. Place the extraction head (50 / 30μm DVB / CAR / PDMS) at the inlet of the gas chromatograph and age it at 250℃ for 5min. Insert it above the gas chromatograph vial to extract flavor compounds from the sample. The extraction conditions are: temperature 60℃, magnetic stirrer speed 500rpm, extraction time 30min. Then desorb at 250℃ for 5min at the inlet of the gas chromatograph.

[0085] Gas chromatography conditions: A 60m × 0.25mm × 0.25μm DB-5 column (Agilent Technologies, Santa Clara, CA, USA) was used to separate volatile compounds. A temperature program was employed, starting at 40℃ and holding for 10 min, then increasing to 240℃ at a rate of 5℃ / min, and holding at 240℃ for 5 min. High-purity helium was used as the carrier gas at a flow rate of 1.0 mL / min, and splitless injection was selected.

[0086] Mass spectrometry conditions: EI ion source, ion source temperature 230℃, electron energy 70 eV, transfer line temperature 280℃. Mass scan range set to 33–500 m / z.

[0087] Qualitative and quantitative analysis: The material peaks collected by GC-MS were analyzed on the MassHunter workstation. The mass spectra of the material peaks were compared with the standard mass spectra in the NIST 20 (National Institute of Standards and Technology) standard spectral library for qualitative analysis. The relative content of each component in the sample was calculated by formula (2) using the internal standard method for quantitative analysis.

[0088]

[0089] In the formula: c i (μg / kg) represents the content of each volatile compound in the sample, c s (μg / kg) represents the content of the internal standard in the sample, A i A represents the peak area corresponding to the analyte in the sample. s This represents the peak area of ​​the internal standard.

[0090] The odor activity value (OAV) of volatile flavor compounds is calculated according to formula (3).

[0091] OAV i =ci / T i Formula (3)

[0092] In the formula: c i (μg / kg) represents the content of volatile flavor compounds in the fermented milk sample, T i (μg / kg) represents the odor threshold of the volatile flavor compounds measured in water.

[0093] Example 1: Isolation, purification and identification of Lactobacillus delbrueckii subsp. Indus LLFMY24

[0094] (1) Isolation and purification of strains and observation of colony and cell morphology

[0095] Take 25 mL of traditional fermented milk sample, add 225 mL of sterile physiological saline, mix thoroughly, and perform serial dilutions with sterile physiological saline. Take three suitable serial dilutions and spread them on MRS plates, then incubate anaerobically at 37°C for 48 h. Select single colonies with different morphologies and further purify them using the streak plate method. Repeat this process twice until single colonies with uniform morphology are obtained on the same plate. Incubate the purified single colonies anaerobically in MRS liquid medium for 48 h, then store them in a -80°C freezer with glycerol. Strain LLFMY24 appears as milky white, round colonies with a raised center on MRS solid medium, is Gram-positive, and appears as rod-shaped colonies under a microscope, arranged singly or in pairs (see...). Figure 1 ).

[0096] (2) 16S rDNA sequencing and identification

[0097] The most commonly used universal bacterial primers were used: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 2492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system consisted of: 2 μL DNA template, 2 μL primer 27F, 2 μL primer 1492R, 20 μL 2×PCR Mix, and 24 μL ultrapure water. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 46℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and 72℃ final extension for 10 min. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Homology comparison of the sequencing results was performed using the BLAST tool in NCBI. According to the BLAST tool comparison analysis in NCBI, the 16S rDNA sequence listing is shown as SEQ ID NO: 1; LLFMY24 was preliminarily identified as Lactobacillus delbrueckii.

[0098] (3) Whole genome sequencing

[0099] Lactobacillus delbrueckii LLFMY24 was activated twice in MRS broth medium, centrifuged at 8000xg for 5 min, and the supernatant was discarded. High-quality genomic DNA was extracted and its purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. Large DNA fragments were recovered using the BluePippin automated nucleic acid recovery system. Library construction was performed using the SQK-LSK109 ligation kit, including DNA damage repair and end repair, magnetic bead purification, adapter ligation and magnetic bead purification, Qubit library quantification, and finally, the high-quality library DNA was sequenced. The raw data underwent quality control, filtering out low-quality and excessively short reads (<2000bp). The filtered reads were assembled de novo using Canu v1.5, and the assembly results were corrected using Racon v3.4.3 software. Circularization and start site adjustment were performed using Circlator v1.5.5, and error correction was performed on the assembled draft genome using Pilon v1.22. Gene prediction was performed using Prodigal v2.6.3, and repetitive sequence alignment was performed using RepeatMasker v4.0.5. Twelve strains with >98% similarity to the experimental strain were downloaded from the NCBI database as reference strains for genome-wide similarity analysis to further determine strain classification (see Table 2). Average nucleotide identity (ANI) analysis between the strain and closely related type species was performed using the JSpeciesWS online database (https: / / www.ribocon.com / jspeciesws.html), with BLAST+ used to calculate ANI. Digital DNA-DNA hybridization (dDDH) values ​​between the strain and closely related strains were calculated using Formula 2 of the online tool Genome-to-Genome DistanceCalculator (http: / / ggdc.dsmz.de / ggdc.php).

[0100] Table 2. Information on the strains used in the LLFMY24 genome-wide similarity analysis.

[0101]

[0102] The genome size of *Lactobacillus delbrueckii* LLFMY24 is 1974763 bp (circular chromosome), without plasmids, and contains 1922 coding genes. The predicted total gene sequence length is 1679169 bp, with 27 predicted rRNA genes and 95 predicted tRNA genes. A circular map of the LLFMY24 genome was constructed using the assembled and predicted genome information, as shown below. Figure 2 As shown, from the outside in, the first circle indicates the genome size, with each scale representing 5kb; the second and third circles indicate the positive and negative strands of the genome, with different colors representing different COG classifications; the fourth circle indicates repetitive sequences; the fifth circle indicates tRNA (blue) and rRNA (purple); the sixth circle indicates GC content, with light yellow and blue representing portions higher and lower than the average GC content of the genome, respectively, and peak values ​​indicating the magnitude of the difference; the seventh circle indicates GC-skew, with dark gray indicating G content greater than C, and red indicating the opposite.

[0103] The ANI value of Lactobacillus delbrueckii subsp. indicus for LLFMY24 was 98.79%, while the ANI value for other subspecies was <98%. The dDDH value for Lactobacillus delbrueckii subsp. indicus was 91.0%, while the dDDH value for other subspecies was <80%. Based on the 16S rDNA sequence listing SEQ ID NO: 1, LLFMY24 can be identified as Lactobacillus delbrueckii subsp. indicus.

[0104] Lactobacillus delbrueckii subsp. indicus LLFMY24 was deposited on August 2, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:64946. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0105] Example 2: Safety of Lactobacillus delbrueckii subsp. Indus LLFMY24

[0106] (1) Antibiotic resistance

[0107] According to the European Food Safety Authority (EFSA) regulations for drug susceptibility testing, the microbroth dilution method was used for drug resistance phenotyping. The selected antibiotics were ampicillin, vancomycin, gentamicin, kanamycin, chloramphenicol, erythromycin, clindamycin, tetracycline, and chloramphenicol, prepared with gradient concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. LLFMY24, stored at -80℃, was activated twice consecutively with MRS broth medium, and the bacterial suspension concentration was adjusted to 8 log CFU / mL. 100 μL of each gradient concentration of antibiotics and 100 μL of bacterial suspension were added to 96-well plates. A positive control was set up with no antibiotics added, only the test bacterial suspension and medium containing the highest concentration of antibiotic solvent; a negative control was set up with neither antibiotics nor the test strain added, only the medium. After incubation at 37℃ for 48 h, the absorbance was measured at a wavelength of 625 nm. The drug resistance phenotypes of different strains were determined based on the minimum inhibitory concentration (MIC) inflection point specified by EFSA. The MIC inflection points for *Lactobacillus delbrueckii* were: ampicillin 1 μg / mL, vancomycin 2 μg / mL, gentamicin 16 μg / mL, kanamycin 16 μg / mL, streptomycin 16 μg / mL, erythromycin 1 μg / mL, clindamycin 1 μg / mL, tetracycline 4 μg / mL, and chloramphenicol 4 μg / mL.

[0108] The MIC values ​​for *Lactobacillus delbrueckii* subsp. *indus* LLFMY24 were: ampicillin 0.25 μg / mL, vancomycin 1 μg / mL, gentamicin 4 μg / mL, kanamycin 16 μg / mL, streptomycin 8 μg / mL, erythromycin 0.25 μg / mL, clindamycin 0.25 μg / mL, tetracycline 2 μg / mL, and chloramphenicol 1 μg / mL. All of these resistance phenotype values ​​were less than or equal to the inflection point values ​​specified by EFSA, therefore *Lactobacillus delbrueckii* subsp. *indus* LLFMY24 was sensitive to all nine tested antibiotics.

[0109] (2) Produced amines

[0110] The amino acid culture media used for detecting tyramine, cadaverine, putrescine, and histamine were prepared by adding 0.5% (w / v) L-tyrosine, L-lysine, L-ornithine, and L-histidine to the amino acid decarboxylase culture medium, respectively. The media were then dissolved in distilled water and the pH was adjusted to 6.8. The activated bacterial suspensions were inoculated into the four amino acid culture media at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. The color changes of the culture media were observed. Uninoculated culture media and culture media inoculated with bacterial suspensions but without any added amino acids served as controls. A turbid and purple culture medium indicated a positive result; otherwise, a negative result was observed.

[0111] The results for tyramine, cadaverine, putrescine, and histamine in Lactobacillus delbrueckii subsp. India LLFMY24 were all negative.

[0112] (3) Indigo-producing matrix

[0113] The activated bacterial suspension was inoculated into indigo-based medium at 3% (v / v) and incubated at 37°C for 48 hours. Kovacs' reagent was slowly added dropwise, and the color change was observed. Escherichia coli ATCC 25922 was used as a positive control, and uninoculated indigo-based medium served as a blank control. A deep red color on the surface of the medium indicated a positive result; otherwise, a negative result was indicated.

[0114] The indole test result for Lactobacillus delbrueckii subsp. India LLFMY24 was negative.

[0115] (4) Nitroreductase activity

[0116] The activated bacterial culture was inoculated into nitrate medium at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. Then, 1-2 drops of nitrate reducing reagent solution A and solution B were added to the culture medium, and the results were observed. *Escherichia coli* ATCC25922 was used as a positive control, and the uninoculated culture medium served as a blank control. A positive result for nitrate reductase detection was indicated by a cloudy, deep red culture medium; a negative result was indicated by a cloudy, yellow, or pale pink culture medium.

[0117] The nitroreductase test result for Lactobacillus delbrueckii subsp. India LLFMY24 was negative.

[0118] (5) Hemolytic activity

[0119] The activated bacterial suspension was inoculated onto Columbia blood agar medium using the streak plate method and incubated at 37°C for 48 hours. The presence of hemolysis zones was observed. Staphylococcus aureus ATCC 25923 was used as a positive control, and the uninoculated medium served as a blank control. A grass-green hemolysis zone indicated α-hemolysis; a clear hemolysis zone indicated β-hemolysis; and the absence of either indicated no hemolysis (γ-hemolysis).

[0120] The test result for Lactobacillus delbrueckii subsp. India LLFMY24 was γ-hemolysis, meaning no hemolysis.

[0121] Example 3: Potential probiotic properties of Lactobacillus delbrueckii subsp. Indus LLFMY24

[0122] (1) Tolerance to simulated gastrointestinal fluid

[0123] Simulated gastric fluid containing 3 mg / mL pepsin was prepared using 0.01 mol / L PBS buffer (pH 3.0), and simulated intestinal fluid containing 1 mg / mL trypsin was prepared using 0.01 mol / L PBS buffer (pH 8.0). Both simulated gastric and intestinal fluids were filtered through a 0.22 μm filter and stored at 4°C for later use. The overnight culture was centrifuged, washed, and the bacterial concentration was adjusted to 10⁻⁶ with PBS. 9 CFU / mL, 1 mL of bacterial culture was added to 9 mL of simulated gastric fluid. After incubation at 37°C for 3 hours, 1 mL of the bacterial-containing gastric fluid was added to 9 mL of simulated intestinal fluid and incubated at 37°C for another 12 hours. Samples were taken at 0h, 1h, 3h, 5h, 7h, 11h, and 15h, and the viable count was calculated using the plate dilution method.

[0124] Lactobacillus delbrueckii subsp. India LLFMY24 showed relatively high tolerance to simulated gastric juice at pH 3.0 and simulated intestinal juice at pH 8.0 (see [link to article]). Figure 3 After 3 hours in gastric fluid, the survival rate of LLFMY24 was 41.99%; after 12 hours in intestinal fluid, the number of viable bacteria in LLFMY24 decreased by only 0.74 log CFU / mL, with a survival rate of 18.22%. This indicates that LLMY24 has strong tolerance to the gastrointestinal environment and can successfully reach the gastrointestinal tract to exert its probiotic effects.

[0125] (2) Adhesion to small intestinal epithelial cells

[0126] Caco-2 cell culture: After resuscitation, Caco-2 cells were added to DMEM complete culture medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin mixture, and incubated statically at 37°C and 5% CO2. The cell culture medium was changed approximately every 2 days. When the cells grew well and adhered to the culture medium to the wall at approximately 80%, they were passaged using 0.25% trypsin-EDTA digestion. Caco-2 cells were passaged approximately 5 times for later use.

[0127] Preparation of bacterial suspension: Collect overnight cultured *Lactobacillus delbrueckii* subsp. *indiana* LLFMY24 by centrifugation at 8000xg for 5 min. Wash 2-3 times with sterile PBS and resuspend in antibiotic-free DMEM medium. Adjust the concentration of the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, for later use.

[0128] Adhesion count: Caco-2 cells were counted at a concentration of 2.5 × 10⁻⁶. 4Cells were seeded at a density of 1 cell / well in 24-well plates and cultured at 37°C with 5% CO2. The culture medium was changed every other day. When the cells grew to a monolayer, the culture medium was removed, and the prepared bacterial suspension was added and incubated at 37°C for 2 hours. After incubation, unattached bacteria were washed away with sterile PBS, and the cells were then lysed with 0.05% Triton X-100 solution to obtain a bacterial suspension adhering to the cells. The adhering bacteria were counted using the agar dilution method.

[0129] The initial viable count of *Lactobacillus delbrueckii* subsp. *indusinica* LLFMY24 was 9.97 × 10⁻⁶. 8 ±1.46 CFU / mL, with 3.19 × 10⁻⁶ viable bacteria adhering to Caco-2 cells. 8 ±0.34 CFU / mL, with an adhesion rate of 32.01%.

[0130] (3) Inhibitory effect on pathogenic bacteria

[0131] The inhibitory effect of *Lactobacillus delbrueckii* subsp. *indiana* LLFMY24 on six common enteropathogenic bacteria (*Escherichia coli* O157:H7, *Salmonella typhimurium* ATCC 14028, *Listeria monocytogenes* CMCC 54002, *Enterobacter sakazakii* ATCC 29544, *Streptococcus hemolyticus* ATCC 21059, and *Staphylococcus aureus* ATCC 12598) was determined using the Oxford cup method. The indicator pathogens were centrifuged at 8000 x g for 5 min, the supernatant was discarded, and the bacterial concentration was adjusted to 10⁻⁶ with sterile physiological saline. 6 -10 7 CFU / mL, take 100 μL and spread it on an LB agar plate. Place a sterile Oxford cup parallel to the plate, and add 200 μL of the test bacteria suspension in the logarithmic growth phase to the Oxford cup. Incubate at 37°C until a clear inhibition zone appears, and measure the diameter of the inhibition zone with calipers. Use sterile MRS broth medium as a blank control.

[0132] Table 3 shows the inhibition zone data of *Lactobacillus delbrueckii* subsp. *indusinica* LLFMY24 against six pathogenic bacteria. *Lactobacillus delbrueckii* subsp. *indusinica* LLFMY24 exhibited significant inhibitory effects against all six pathogenic bacteria, with inhibition zone diameters exceeding 20 mm.

[0133] Table 3. Results of antibacterial experiments on Lactobacillus delbrueckii subsp. Indosinica LLFMY24

[0134] Pathogenic bacteria Diameter of the inhibition zone (mm) Streptococcus hemolyticus ATCC21059 22.7±1.0 Enterobacter sakazakii ATCC 29544 24.7±0.8 Salmonella typhimurium ATCC 14028 23.7±0.6 Escherichia coli O157:H7 24.2±2.7 Listeria monocytogenes CMCC 54002 22.8±1.3 Staphylococcus aureus ATCC 12598 21.0±0.6 Blank control 7.8

[0135] Example 4: Growth and acid production performance of Lactobacillus delbrueckii subsp. Indus LLFMY24

[0136] (1) Growth curve

[0137] Centrifuge the activated bacterial suspension at 8000xg for 5 min, collect the bacterial cells, wash 2-3 times with sterile phosphate-buffered saline (PBS), and resuspend in 5 mL of sterile PBS for later use. Inoculate the *Lactobacillus delbrueckii* subsp. *industrialis* LLFMY24 suspension at 2% (v / v) into MRS broth medium and incubate at 37°C. Take samples at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 20 h, and 24 h of culture to determine the OD value. 600 With culture time as the x-axis, OD 600 Plot the growth curve of the strain on the ordinate. Use the culture medium containing the inoculated bacterial solution as a blank control. The experiment was repeated three times.

[0138] The growth curve of Lactobacillus delbrueckii subsp. Indus LLFMY24 is as follows: Figure 4 The growth period is logarithmic from 2 to 8 hours, after which growth tends to slow down.

[0139] (2) Acid production curve

[0140] Centrifuge the activated bacterial suspension at 8000xg for 5 min, collect the bacterial cells, wash 2-3 times with sterile phosphate-buffered saline (PBS), and resuspend in 5 mL of sterile PBS for later use. Inoculate the *Lactobacillus delbrueckii* subsp. *indusinianus* LLFMY24 suspension at 2% into whole milk medium and ferment at 37℃. Collect fermented milk samples at 0, 2, 4, 6, 8, 10, 12, and 24 h. Shake thoroughly before sampling. Measure the pH and acidity of the samples and plot the acid production curve. Measure each fermented milk sample three times at each time point.

[0141] The acid production curve of Lactobacillus delbrueckii subsp. India LLFMY24 is as follows: Figure 5 It continuously produces acid from 0 to 12 hours.

[0142] Example 5: Isolation, purification and identification of Streptococcus thermophilus LLF17Y25

[0143] (1) Isolation and purification of strains and observation of colony and cell morphology

[0144] Take 25 mL of traditional fermented milk sample, add 225 mL of sterile physiological saline, mix thoroughly, and perform serial dilutions with sterile physiological saline. Take three suitable serial dilutions and spread them on M17 plates, then incubate anaerobically at 37°C for 48 h. Select single colonies with different morphologies and further purify them using the streak plate method. Repeat this process twice until single colonies with uniform morphology are obtained on the same plate. Incubate the purified single colonies anaerobically in M17 liquid medium for 48 h, then store them in a -80°C freezer with glycerol. On M17 solid medium, strain LLF17Y25 appears as milky white, translucent, smooth, round or oval colonies with regular edges. After Gram staining, the cells are spherical, arranged in pairs or chains (see...). Figure 6 ).

[0145] (2) 16S rDNA sequencing and identification

[0146] The most commonly used universal bacterial primers were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 2492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system consisted of: 2 μL DNA template, 2 μL primer 27F, 2 μL primer 1492R, 20 μL 2×PCR Mix, and 24 μL ultrapure water. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 46℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and 72℃ final extension for 10 min. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Homology comparison of the sequencing results was performed using the BLAST tool in NCBI. According to the BLAST tool comparison analysis in NCBI, the 16S rDNA sequence listing is shown as SEQ ID NO: 2; LLF17Y25 was preliminarily identified as Streptococcus thermophilus.

[0147] (3) Whole genome sequencing

[0148] Streptococcus thermophilus LLF17Y25 was activated twice with M17 broth, centrifuged at 8000xg for 5 min, and the supernatant was discarded. High-quality genomic DNA was extracted and its purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. Large DNA fragments were recovered using the BluePippin automated nucleic acid recovery system. Library construction was performed using the SQK-LSK109 ligation kit, including DNA damage repair and end repair, magnetic bead purification, adapter ligation and magnetic bead purification, Qubit library quantification, and finally, the high-quality library DNA was sequenced. The raw data underwent quality control, filtering out low-quality and excessively short (<2000bp) reads. The filtered reads were assembled de novo using Canu v1.5, and the assembly results were corrected using Racon v3.4.3 software. Circularization and start site adjustment were performed using Circlator v1.5.5, and error correction was performed on the assembled draft genome using Pilon v1.22. Gene prediction was performed using Prodigal v2.6.3, and repetitive sequence alignment was performed using RepeatMasker v4.0.5. Twelve strains with >98% similarity to the experimental strain were downloaded from the NCBI database as reference strains for genome-wide similarity analysis to further determine strain classification (see Table 4). Average nucleotide identity (ANI) analysis between the strain and closely related type species was performed using the JSpeciesWS online database (https: / / www.ribocon.com / jspeciesws.html), with BLAST+ used to calculate ANI. Digital DNA-DNA hybridization (dDDH) values ​​between the strain and closely related strains were calculated using Formula 2 of the online tool Genome-to-Genome DistanceCalculator (http: / / ggdc.dsmz.de / ggdc.php).

[0149] Table 4. Information on the strains used in the genome-wide similarity analysis of LLF17Y25.

[0150]

[0151] The genome size of *Streptococcus thermophilus* LLF17Y25 is 1933985 bp (circular chromosome), and it lacks plasmids. The complete genome contains 2027 coding genes, with a predicted total gene sequence length of 1627158 bp. There are 18 predicted rRNA genes and 67 predicted tRNA genes. A circular map of the LLF17Y25 genome was constructed using the assembled and predicted genome information, as shown below. Figure 7 As shown, Figure 7 In the diagram, the first ring from the outside in indicates the genome size, with each scale representing 5kb; the second and third rings indicate the positive and negative strands of the genome, with different colors representing different COG classifications; the fourth ring indicates repetitive sequences; the fifth ring indicates tRNA (blue) and rRNA (purple); the sixth ring indicates GC content, with light yellow and blue representing portions higher and lower than the average GC content of the genome, respectively, and peak values ​​indicating the magnitude of the difference; the seventh ring indicates GC-skew, with dark gray indicating G content greater than C, and red indicating the opposite.

[0152] The ANI values ​​between *Streptococcus thermophilus* LLF17Y25 and eight *Streptococcus thermophilus* strains were all >98%, with the highest ANI value (99.12%) compared to *Streptococcus thermophilus* MN-BM-A01. The ANI values ​​with *Streptococcus salivarius* and *Streptococcus vestibularis* were both <93%. The dDDH values ​​between LLF17Y25 and *Streptococcus thermophilus* were all >84%. Based on its 16S rDNA sequence listing (SEQ ID NO: 2), LLF17Y25 was identified as *Streptococcus thermophilus*.

[0153] Streptococcus thermophilus LLF17Y25 was deposited on August 2, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:64947. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0154] Example 6: Safety of Streptococcus thermophilus LLF17Y25

[0155] According to the European Food Safety Authority (EFSA) regulations on drug susceptibility testing, the microbroth dilution method was used for drug resistance phenotyping. The selected antibiotics were ampicillin, vancomycin, gentamicin, kanamycin, chloramphenicol, erythromycin, clindamycin, tetracycline, and chloramphenicol, prepared with gradient concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. LLF17Y25, stored at -80℃, was activated twice consecutively with M17 broth medium, adjusting the bacterial suspension concentration to 8 log CFU / mL. 100 μL of each gradient concentration of antibiotics and 100 μL of bacterial suspension were added to 96-well plates. A positive control was set up with no antibiotics added, only the test bacterial suspension and medium containing the highest concentration of antibiotic solvent; a negative control was set up with neither antibiotics nor the test strain added, only the medium. After incubation at 37℃ for 48 h, absorbance was measured at 625 nm. The drug resistance phenotypes of different strains were determined based on the minimum inhibitory concentration (MIC) inflection point specified by EFSA. The MIC inflection points for Streptococcus thermophilus were: ampicillin 2 μg / mL, vancomycin 4 μg / mL, gentamicin 32 μg / mL, kanamycin 64 μg / mL, streptomycin 64 μg / mL, erythromycin 2 μg / mL, clindamycin 2 μg / mL, tetracycline 4 μg / mL, and chloramphenicol 4 μg / mL.

[0156] The MIC values ​​for *Streptococcus thermophilus* LLF17Y25 were: ampicillin 0.5 μg / mL, vancomycin 1 μg / mL, gentamicin 8 μg / mL, kanamycin 16 μg / mL, streptomycin 16 μg / mL, erythromycin 0.5 μg / mL, clindamycin 0.5 μg / mL, tetracycline 2 μg / mL, and chloramphenicol 2 μg / mL. All of these resistance phenotype values ​​were less than or equal to the inflection point values ​​specified by EFSA, therefore *Streptococcus thermophilus* LLF17Y25 was sensitive to all nine tested antibiotics.

[0157] (2) Produced amines

[0158] The amino acid culture media used for detecting tyramine, cadaverine, putrescine, and histamine were prepared by adding 0.5% (w / v) L-tyrosine, L-lysine, L-ornithine, and L-histidine to the amino acid decarboxylase culture medium, respectively. The media were then dissolved in distilled water and the pH was adjusted to 6.8. The activated bacterial suspensions were inoculated into the four amino acid culture media at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. The color changes of the culture media were observed. Uninoculated culture media and culture media inoculated with bacterial suspensions but without any added amino acids served as controls. A turbid and purple culture medium indicated a positive result; otherwise, a negative result was observed.

[0159] The results for tyramine, cadaverine, putrescine, and histamine in Streptococcus thermophilus LLF17Y25 were all negative.

[0160] (3) Indigo-producing matrix

[0161] The activated bacterial suspension was inoculated into indigo-based medium at 3% (v / v) and incubated at 37°C for 48 hours. Kovacs' reagent was slowly added dropwise, and the color change was observed. Escherichia coli ATCC 25922 was used as a positive control, and uninoculated indigo-based medium served as a blank control. A deep red color on the surface of the medium indicated a positive result; otherwise, a negative result was indicated.

[0162] The indole test result for Streptococcus thermophilus LLF17Y25 was negative.

[0163] (4) Nitroreductase activity

[0164] The activated bacterial culture was inoculated into nitrate medium at 3% (v / v) and anaerobically cultured at 37°C for 48 hours. Then, 1-2 drops of nitrate reducing reagent solution A and solution B were added to the culture medium, and the results were observed. *Escherichia coli* ATCC25922 was used as a positive control, and the uninoculated culture medium served as a blank control. A positive result for nitrate reductase detection was indicated by a cloudy, deep red culture medium; a negative result was indicated by a cloudy, yellow, or pale pink culture medium.

[0165] The test result for nitroreductase in Streptococcus thermophilus LLF17Y25 was negative.

[0166] (5) Hemolytic activity

[0167] The activated bacterial suspension was inoculated onto Columbia blood agar medium using the streak plate method and incubated at 37°C for 48 hours. The presence of hemolysis zones was observed. Staphylococcus aureus ATCC 25923 was used as a positive control, and the uninoculated medium served as a blank control. A grass-green hemolysis zone indicated α-hemolysis; a clear hemolysis zone indicated β-hemolysis; and the absence of either indicated no hemolysis (γ-hemolysis).

[0168] The test result for Streptococcus thermophilus LLF17Y25 was γ-hemolysis, meaning it was not hemolytic.

[0169] Example 7: Growth and acid production performance of Streptococcus thermophilus LLF17Y25

[0170] (1) Growth curve

[0171] Centrifuge the activated bacterial suspension at 8000xg for 5 min, collect the bacterial cells, wash 2-3 times with sterile phosphate-buffered saline (PBS), and resuspend in 5 mL of sterile PBS for later use. Inoculate the Streptococcus thermophilus LLF17Y25 bacterial suspension at 2% (v / v) into M17 broth medium and incubate at 37℃. Take samples at 0h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, and 24h of incubation to determine the OD value. 600 With culture time as the x-axis, OD 600 Plot the growth curve of the strain on the ordinate. Use the culture medium containing the inoculated bacterial solution as a blank control. The experiment was repeated three times.

[0172] The growth curve of Streptococcus thermophilus LLF17Y25 is as follows Figure 4 The growth period is logarithmic, lasting from 4 to 6 hours, after which growth tends to slow down.

[0173] (2) Acid production curve

[0174] Centrifuge the activated bacterial suspension at 8000xg for 5 min, collect the bacterial cells, wash 2-3 times with sterile phosphate-buffered saline (PBS), and resuspend in 5 mL of sterile PBS for later use. Inoculate the Streptococcus thermophilus LLF17Y25 bacterial suspension at 2% into whole milk medium and ferment at 37℃. Collect fermented milk samples at 0, 2, 4, 6, 8, 10, 12, and 24 h. Shake thoroughly before sampling. Measure the pH and acidity of the samples and plot the acid production curve. Measure each fermented milk sample three times at each time point.

[0175] The acid production curve of Streptococcus thermophilus LLF17Y25 is as follows Figure 5 It produces acid rapidly within 2-6 hours, and then the production level gradually decreases.

[0176] Example 8: Method for preparing fermented milk

[0177] (1) Preparation of fermentation agent

[0178] The activated Lactobacillus delbrueckii subsp. India LLFMY24 and Streptococcus thermophilus LLF17Y25 bacterial suspensions were centrifuged at 8000 x g / min for 5 min, and the bacterial cells were collected separately. After washing twice with sterile water, the bacterial concentration was adjusted to 10. 8 The fermentation agent was prepared at a volume ratio of CFU / mL in a 1:1 ratio and named Y-2425.

[0179] (2) Preparation of fermented milk

[0180] Cool sterilized distilled water to 50-60℃, add 12% whole milk powder and 6.5% white sugar and dissolve thoroughly. Sterilize in a 90℃ water bath for 10 minutes, then cool and refrigerate at 4℃ for later use. Mix starter culture Y-2425 and commercial starter culture (LS) at a final total bacterial count of 5×10⁻⁶. 6 Inoculate the whole milk medium with an inoculum of CFU / mL, ferment at 42°C until the titratable acidity reaches 60-70°T, and then refrigerate at 4°C.

[0181] Example 9: Flavor characteristics of double-strain fermented yogurt

[0182] (1) Determination of physicochemical properties

[0183] Y-2425 starter culture was inoculated into whole milk medium at a 1% (v / v) inoculation rate (final concentration 5 × 10⁻⁶). 6 The Y-2425 fermented milk was fermented at 42℃ for 6 hours (CFU / mL) and its titratable acidity, water-holding capacity, and viscosity were determined. The titratable acidity of Y-2425 fermented milk was 82.54±0.45°T, the water-holding capacity was 56.38±0.19%, and the viscosity was 1390.0±0.0 cP.

[0184] (2) Post-acidification

[0185] Fermentation was stopped when the titratable acidity of the fermented milk reached 60-70°T. The fermented milk was then refrigerated at 4°C, and the titratable acidity and pH of the fermented milk were measured on days 0, 1, 3, 7, 14, 21, and 28 of the storage period.

[0186] The post-acidification curve of Y-2425 is shown below. Figure 8 During the period from the end of fermentation to day 3 of storage, the acidity of Y-2425 continued to rise, increasing by 15.81°T. Over the next 3 to 28 days of storage, the acidity and pH of Y-2425 gradually stabilized, remaining between 85.00 and 87.05°T, with the pH fluctuating between 3.89 and 3.87.

[0187] (3) Sensory evaluation

[0188] The color, aroma, and texture of fermented milk prepared from Lactobacillus delbrueckii subsp. India LLFMY24 and Streptococcus thermophilus LLF17Y25 and the control group were evaluated after 24 hours of post-ripening. The results are shown in Table 5 below.

[0189] Y-2425 fermented milk scored 92.5 points in total. Its color and texture scores were both high, indicating that it has a distinct fermented flavor, no whey, a delicate texture, a moderate taste, and a thick consistency.

[0190] Table 5 Sensory scores of fermented milk

[0191]

[0192] (4) Rheological properties

[0193] The rheological properties of fermented milk were determined using a rotational rheometer. A 50 mm diameter stainless steel plate was used, with a spacing of 1.0 mm, an equilibration time of 2 min, and a test temperature of 25 °C.

[0194] Viscosity profile: The shear rate was tested in the range of 0.1–100 s. -1 Each sample was measured three times.

[0195] Frequency scanning: First, stress scanning tests were performed to determine the linear viscoelastic range. The frequency scanning test range was 0.1-100 rad / s, with a controlled stress of 0.1%, to obtain the changes in the storage modulus (G') and loss modulus (G”) of the fermented milk as a function of frequency. Each sample was measured three times.

[0196] The viscosity of the fermented milk samples all exhibited shear-thinning behavior, such as... Figure 9 As shown. This phenomenon is known as pseudoplasticity in rheology and is common in non-Newtonian fluids. The rheological properties of fermented milk are closely related to its flow in the oral cavity and the sensation of swallowing. When evaluating the rheological properties of fermented milk, the apparent viscosity at a shear rate of 50 s⁻¹ is usually used, which corresponds to the shear rate generated by the tongue against the palate in the oral cavity. The viscosity trend line of Y-2425 is above that of the control group, which to some extent reflects that its internal cross-linked network structure is more stable, has stronger shear resistance, and has a higher apparent viscosity. Figure 10 As shown, with the increase of shear frequency, both G′ and G″ of sample Y-2425 showed an increasing trend, and the G′ value was higher than the G″ value, indicating that fermented milk mainly exhibits solid-like behavior, suggesting that it has good viscoelasticity.

[0197] (5) Volatile flavor compounds

[0198] Volatile flavor compounds in Y-2425 fermented milk were detected using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fermented milk samples aged 24 hours were collected for analysis of volatile flavor compounds.

[0199] The results of volatile flavor compound detection in Y-2425 fermented milk are shown in Table 6. A total of 29 volatile compounds were detected in Y-2425 fermented milk, including 4 esters, 5 aldehydes, 3 acids, 7 ketones, 4 sulfur-containing compounds, 2 nitrogen-containing compounds, and 4 aromatic and olefinic compounds. The content of volatile flavor compounds does not directly determine their importance in the overall flavor of the sample; the odor threshold of each substance must also be considered. The odor activity value (OAV) is generally used to evaluate the contribution of volatile compounds to flavor. The higher the OAV, the greater the contribution to the overall odor; that is, the ratio of the content of volatile flavor compounds in the sample to their odor threshold in water. Flavor compounds with OAV ≥ 1 are generally considered key flavor compounds that make a significant contribution to the flavor of fermented milk; substances with OAV < 1 have an important modifying effect on the overall flavor. Y-2425 had 7 volatile flavor compounds with OAV ≥ 1, as shown in Appendix Table 6. Ethyl acetate, δ-decyl lactone, 2-nonanone, hexanoic acid, and nonanal are key flavor compounds shared by both groups of fermented milk samples. Ethyl acetate is an important flavor compound in fermented milk, possessing fruity, floral, and brandy aromas. It can reduce bitterness and other off-flavors from fatty acids and amines, thus significantly enhancing the overall flavor of fermented milk. Nonanal, mainly produced by the oxidation of oleic acid, has lemony, fatty, and floral aromas. This aroma significantly enhances the flavor characteristics of fermented milk, making it richer and more unique. δ-decyl lactone, as a lactone compound, significantly contributes to the flavor of fermented milk, producing milky, coconut, and nutty flavors. 2-nonanone has a sweet, fruity, and warm milk aroma in fermented milk. Hexanoic acid imparts a sour, oily, and cheese-like flavor to fermented milk. Caprylic acid and dimethyl disulfide are key flavor compounds unique to Y-2425. In fermented milk, caprylic acid, along with other acids (such as acetic acid, butyric acid, and hexanoic acid), influences the flavor profile, imparting a cheese-like, fatty aroma and a subtle fruity tartness. Dimethyl disulfide, primarily formed during the heat treatment of dairy products through the Strecker degradation of methionine, is a major source of the cooked flavor in dairy products. Therefore, Y-2425 fermented milk possesses a more complex and richer flavor profile.

[0200] Table 6 Volatile Compounds of Fermented Milk

[0201]

[0202]

[0203] Note: "—" indicates that the relevant substance was not detected.

[0204] Comparative example: Commercially fermented yogurt

[0205] (1) Physicochemical properties

[0206] The commercial control starter culture was inoculated into whole milk medium at a 1% (v / v) inoculation rate (final concentration 5 × 10⁻⁶). 6 The sample (CFU / mL) was fermented at 42℃ for 6 hours, and its titratable acidity, water-holding capacity and viscosity were determined.

[0207] The titratable acidity of the control group (LS) fermented milk was 70.24±0.25°T, the water holding capacity was 56.21±0.14%, and the viscosity was 982.0±2.2 cP.

[0208] (2) Post-acidification

[0209] Fermentation was stopped when the titratable acidity of the fermented milk reached 60-70°T. The fermented milk was then refrigerated at 4°C, and the titratable acidity and pH of the fermented milk were measured on days 0, 1, 3, 7, 14, 21, and 28 of the storage period.

[0210] The post-acidification curve of the control group (LS) fermented milk is shown in the figure. Figure 6 During the period from the end of fermentation to day 3 of storage, the acidity continued to rise, increasing by 17.29°T. During the subsequent storage period of 3 to 28 days, the acidity and pH of the control group gradually stabilized, with the acidity varying between 76.63 and 85.58°T and the pH stabilizing between 4.12 and 4.05.

[0211] (3) Sensory evaluation

[0212] The color, aroma, and texture of the control group fermented milk prepared by commercial strain Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were evaluated after 24 hours of post-ripening. The results are shown in Table 7.

[0213] The control group LS had a total score of 88.9 points, with obvious fermentation flavor, a small amount of whey separation, delicate texture, and thick consistency.

[0214] Table 7 Sensory scores of fermented milk

[0215]

[0216] (4) Rheological properties

[0217] The rheological properties of the control group (LS) fermented milk were determined using a rotational rheometer, with the same test parameters as in Example 9.

[0218] The viscosity of the control group (LS) fermented milk sample exhibited shear-thinning behavior, such as... Figure 7 As shown, with increasing shear frequency, both G′ and G″ in the control group (LS) sample showed an increasing trend, and the G′ value was higher than the G″ value, indicating that the fermented milk mainly exhibited solids-like behavior (see...). Figure 8 ).

[0219] (5) Volatile flavor compounds

[0220] Volatile flavor compounds in Y-2425 fermented milk were detected using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fermented milk samples aged 24 hours were collected for analysis of volatile flavor compounds.

[0221] The volatile flavor compounds detected in the control group (LS) fermented milk are shown in Table 8. A total of 32 volatile compounds were detected in the control group (LS), including 5 esters, 2 alcohols, 5 aldehydes, 4 acids, 8 ketones, 1 sulfur-containing compound, 2 nitrogen-containing compounds, and 5 aromatic and olefinic compounds. The control group (LS) fermented milk contained 6 volatile flavor compounds with an OAV ≥ 1, as shown in Table 8.

[0222] Table 8 Key Flavor Compounds

[0223]

[0224] Note: The odor thresholds of each volatile flavor compound are referenced from "Compilation of Compound Odor Thresholds (Second Edition)"; the results are mean ± standard deviation (SD). Different superscript lowercase letters in the same row indicate significant differences (p<0.05), and different uppercase letters indicate extremely significant differences (p<0.001).

[0225] As can be seen from the above examples, the *Lactobacillus delbrueckii* subsp. *indusinica* strain LLFMY24 isolated from traditional fermented milk exhibits good safety (Example 2). Furthermore, this strain demonstrates high activity, ease of cultivation, good gastrointestinal adaptability, and significant antibacterial effects (Examples 3 and 4). It has application value as a probiotic and yogurt starter culture, and the fermented milk prepared from it has better texture and a unique sensory flavor than the commercial control fermented milk (Example 9 and Comparative Example).

[0226] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0227]

[0228]

Claims

1. A subsp. indicus of Lactobacillus delbrueckii, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:64946.

2. The application of Lactobacillus delbrueckii subsp. India as described in claim 1 in the preparation of yogurt starter.

3. The application of *Lactobacillus delbrueckii* subsp. *indus* according to claim 2 in the preparation of yogurt starter, characterized in that, Lactobacillus delbrueckii subsp. indicus and Streptococcus thermophilus were inoculated separately into broth medium and incubated at 37–42°C for 12–24 hours. After centrifugation to collect the cells, they were washed, resuspended, and the bacterial count of both resuspensions was adjusted to 1 × 10⁻⁶. 8 ~1×10 10 CFU / mL, the two resuspensions are mixed at a ratio of 1:10 to 1:1 to obtain the yogurt starter.

4. The application of *Lactobacillus delbrueckii* subsp. *indus* according to claim 2 in the preparation of yogurt starter, characterized in that, The broth culture medium is MRS / M17 broth culture medium; the washing is done with sterile water.

5. A fermented milk, characterized in that, It is produced by fermentation using the yogurt starter described in claim 3.

6. The fermented milk according to claim 4, characterized in that, The yogurt starter culture is prepared by inoculating sterilized milk with the starter culture; fermenting at 42±2℃ for 5 to 8 hours until the titratable acidity reaches 60 to 90°T; and then maturing after cooling.

7. The fermented milk according to claim 6, characterized in that, The sterilized milk is prepared by sterilizing one or more of the following raw materials: raw cow's milk, raw sheep's milk, concentrated milk for food industry, and milk powder, at a temperature of 90-98°C for 5-10 minutes.

8. The fermented milk according to claim 6 or 7, characterized in that, The inoculation temperature is 40-45℃, and the starter culture is inoculated into sterilized milk at a rate of 0.01-1.0% per gram per milliliter.

9. The fermented milk according to claim 6 or 7, characterized in that, The fermented milk has a titration acidity of 60–90°T, a water holding capacity of 55–58%, and a viscosity of 1000–2000 cP.

10. The fermented milk according to claim 6 or 7, characterized in that, After being stored at 4-8℃ for 3-21 days, the fermented milk exhibits a titratable acidity change of ≤5°T and a pH change of ≤0.3.

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