Lactobacillus delbrueckii lactic acid subspecies YN-01 and products prepared therefrom and applications thereof

Abstract

The present invention provides a Lactobacillus delbrueckii lactic acid subspecies YN-01 and a product and application prepared therefrom, belonging to the field of biomedical technology. The Lactobacillus delbrueckii lactic acid subspecies YN-01 was deposited in the China Center for Type Culture Collection on October 22, 2024, with a deposit address of Wuhan University, Wuhan, China; the deposit number is CCTCC NO: M 20242299; the Latin name is Lactobacillus delbrueckii subsp.lactis YN‑01; the gene sequence of 16srRNA of the Lactobacillus delbrueckii lactic acid subspecies YN‑01 is shown in SEQ ID NO. 1. The present invention adopts the above-mentioned Lactobacillus delbrueckii lactic acid subspecies YN‑01 and the products and applications prepared therefrom. Lactobacillus delbrueckii lactic acid subspecies YN‑01 has good gastrointestinal fluid and bile salt tolerance, and has high protease activity, and has great application value in fermented foods.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a Lactobacillus delbrueckii lactic acid subspecies YN-01 and products prepared therefrom and applications thereof. Background Art

[0002] Probiotics are live microorganisms that are beneficial to the health of the host and are widely used in many fields. They regulate the intestinal microecology, enhance the immune system, promote digestion and absorption, and improve gastrointestinal problems such as constipation and diarrhea. In addition, probiotics have anti-inflammatory, anti-allergic and anti-infective effects, which help maintain the intestinal barrier function and prevent chronic diseases. Its application range is not limited to food and beverages, such as yogurt, fermented milk and probiotic supplements, but also includes medical fields, such as treating intestinal diseases, affecting mental health and improving the health of newborns. With the deepening of research, the potential of probiotics in improving overall health and improving the quality of life is constantly being explored.

[0003] The role of proteases in probiotics is mainly reflected in enhancing their digestion and metabolism. Probiotics can secrete a variety of proteases, which break down proteins in food, help release amino acids and other nutrients, and thus promote nutrient absorption by the host. In addition, proteases can also regulate intestinal microecology, promote the growth of beneficial bacteria, inhibit the reproduction of harmful bacteria, and maintain intestinal health. At the same time, proteases participate in the regulation of immune function, affect immune responses by degrading specific peptide chains, and enhance the host's immunity. These effects together promote the importance of probiotics in maintaining intestinal balance and promoting overall health.

[0004] Proteases play a key role in the preparation of fermented foods, mainly by breaking down proteins in ingredients to improve the nutritional value and flavor of foods. During the fermentation process, proteases can release amino acids and small peptides, which not only enhance the umami flavor of the food but also improve its taste. In addition, the activity of proteases helps promote the reproduction and fermentation of microorganisms, thereby accelerating the maturation and fermentation process of food. In the production of certain fermented foods, such as fermented tofu, soy sauce and cheese, the role of proteases is particularly significant, which can form a unique flavor and texture and improve the overall quality of the product. Therefore, the rational use of the role of proteases in fermented foods can effectively improve the flavor, nutrition and preservation of the products.

[0005] Lactobacillus delbrueckii includes 6 subspecies, including Lactobacillus delbrueckii subspecies bulgaricus ( L. delbrueckii subsp. bulgaricus ), Lactobacillus delbrueckii subsp. lactis ( L. delbrueckii subsp. lactis )、Lactobacillus delbrueckii subsp. delbrueckii( L. delbrueckii subsp. delbrueckii )、Lactobacillus delbrueckii subsp. jasminogen activator( L. delbrueckii subsp. jakobsenii )、Lactobacillus delbrueckii subsp. Sunkii( L. delbrueckii subsp. sunkii) and Lactobacillus delbrueckii subsp. indica ( L. delbrueckii subsp. indicus ), the 16srRNA sequence similarity of the six strains of Lactobacillus delbrueckii subspecies was 99.21-99.54%. Lactobacillus delbrueckii has acquired genetic diversity and increased its genome size through horizontal gene transfer between subspecies. This adaptability makes it an economic strain with wide industrial applications, such as Lactobacillus delbrueckii lactic acid subspecies is widely used in the production of mozzarella and Parmesan cheese. The proteolytic system of Lactobacillus delbrueckii plays a key role in the growth of strains. When the number of live bacteria increases during the fermentation process, the proteolytic system is needed to provide the strain with the amino acids and peptides required for growth. The differences in the proteolytic system genes between Lactobacillus delbrueckii strains will cause differences between the bovine milk fermentation peptide groups. At the same time, the characterization of the protease corresponding to the proteolytic system gene is related to the formation of bioactive peptides.

[0006] However, although multiple subspecies of Lactobacillus delbrueckii have been extensively studied, research on a specific subspecies such as Lactobacillus delbrueckii lactis subspecies YN-01 has not been reported. Summary of the invention

[0007] The purpose of the present invention is to provide a Lactobacillus delbrueckii lactic acid subspecies YN-01 and products and applications prepared therefrom. The Lactobacillus delbrueckii lactic acid subspecies YN-01 has good gastrointestinal fluid and bile salt tolerance and high protease activity, and has great application value in fermented foods.

[0008] To achieve the above object, the present invention provides a Lactobacillus delbrueckii lactic acid subspecies YN-01, which was deposited in the China Center for Type Culture Collection on October 22, 2024, with a deposit address of Wuhan University, Wuhan, China; the deposit number is CCTCC NO: M 20242299; the Latin name is Lactobacillus delbrueckii subsp. lactis YN-01;

[0009] The gene sequence of 16srRNA of the Lactobacillus delbrueckii lactic acid subspecies YN-01 is shown in SEQ ID NO.1.

[0010] The present invention also provides a use of the above-mentioned Lactobacillus delbrueckii lactic acid subspecies YN-01 in the preparation of fermented food.

[0011] The present invention also provides a food comprising the above-mentioned Lactobacillus delbrueckii lactic acid subspecies YN-01, wherein the number of viable bacteria of Lactobacillus delbrueckii lactic acid subspecies YN-01 in the food is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0012] The present invention also provides a beverage comprising the above-mentioned Lactobacillus delbrueckii lactic acid subspecies YN-01, wherein the viable cell count of Lactobacillus delbrueckii lactic acid subspecies YN-01 in the beverage is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0013] Therefore, the present invention adopts the above-mentioned Lactobacillus delbrueckii lactic acid subspecies YN-01 and the products and applications prepared therefrom, and the beneficial technical effects are as follows:

[0014] (1) The Lactobacillus delbrueckii subspecies lactis YN-01 of the present invention has good tolerance to gastrointestinal fluid and can survive stably and function. Lactobacillus delbrueckii subspecies lactis YN-01 has multiple genes related to acid, heat, cold, and bile salt tolerance, which enhances its ability to survive in different environments.

[0015] (2) Lactobacillus delbrueckii subspecies lactis YN-01 has significant protease activity, which can efficiently decompose the protein in food ingredients and improve the nutritional value and flavor of food. Protease activity helps promote the reproduction and fermentation of microorganisms and accelerate the maturation and fermentation process of food.

[0016] (3) Lactobacillus delbrueckii subspecies lactis YN-01 can be used to make foods such as yogurt, fermented milk, probiotic supplements, etc., and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the phylogenetic tree of Lactobacillus delbrueckii subsp. lactis YN-01;

[0018] Figure 2 is the activity diagram of the protease of Lactobacillus delbrueckii subsp. lactis YN-01;

[0019] Figure 3 This is the effect of fermentation time on ACE inhibitory activity and peptide concentration in fermented milk;

[0020] Figure 4 This is the metabolite composition diagram of fermented beef jerky;

[0021] Figure 5 This is the PCA diagram of metabolites before and after fermentation of beef jerky; Figure 5 (a) is in the positive ion mode; Figure 5 (b) is in negative ion mode;

[0022] Figure 6 This is the PCA diagram of metabolites of yellow persimmon paste before and after fermentation; Figure 6 (a) is in the positive ion mode; Figure 6 (b) is in negative ion mode;

[0023] Figure 7 This is the differential metabolite composition diagram of yellow persimmon paste before and after fermentation;

[0024] Figure 8 Comparison of the main active peptides in yellow persimmon paste before and after fermentation; Figure 8 (a) in it is Ser Thr lle; Figure 8 (b) is Leu Thr Val; Figure 8 The (c) in is lle lle His; Figure 8 Where (d) is lle lle Arg; Figure 8 (e) in it is Val Arg Val; Figure 8 The (f) in the figure is Val Pro Phe. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0026] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0027] Embodiment 1

[0028] Isolation of Lactobacillus delbrueckii lactis subsp. lactis YN-01.

[0029] The yogurt sample was diluted on site in multiples and 10 -5 and 10 -6 200 μL of the dilution gradient sample was evenly spread on the MRS broth medium (solid) plate, and then brought back to the laboratory and cultured at 42°C for 48 hours. Single colonies with different morphology, size, and color were picked and inoculated into MRS broth medium (liquid), and cultured in a 42°C constant temperature incubator for 24-36 hours. After the strains grew well, Gram staining and microscopic examination were performed. The isolated strains were preserved, and the genomic DNA of the strains was extracted for subsequent determination and analysis.

[0030] The formula of MRS broth medium (solid) is: 10g soy peptone, 5g beef extract, 4g yeast powder, 20g glucose, 1mL Tween-80, 2g disodium hydrogen phosphate, 5g anhydrous sodium acetate, 2g triammine citrate, 0.02g manganese sulfate, 0.1g magnesium sulfate, 15g agar powder, and distilled water to make up to 1L. Adjust the initial pH to 6.20 and sterilize at 121℃ for 15min.

[0031] The formula of MRS broth medium (liquid) is: soy peptone 10g, beef extract 5g, yeast powder 4g, glucose 20g, Tween-80 1mL, disodium hydrogen phosphate 2g, anhydrous sodium acetate 5g, triammine citrate 2g, manganese sulfate 0.02g, magnesium sulfate 0.1g, distilled water to make up to 1L, adjust the initial pH to 6.20, and sterilize at 121℃ for 15min.

[0032] The present invention has no particular limitation on the sources of soy peptone, beef extract, yeast powder, glucose, Tween-80, disodium hydrogen phosphate, anhydrous sodium acetate, triammonium citrate, manganese sulfate, magnesium sulfate, and agar powder, and conventional commercial products well known to those skilled in the art can be used.

[0033] Strain identification was carried out by physiological and biochemical identification, 16srRNA molecular identification and whole genome sequencing.

[0034] The frozen test strain was inoculated into the preculture medium, cultured at 42°C for 24-36 hours, and subcultured for 2-3 generations. Then, 30 mL of the bacterial culture at the end of logarithmic growth was placed in a sterile tube for centrifugation. After centrifugation at 8000×g for 5 minutes (4°C), the bacteria were collected, the supernatant was discarded, and the genomic DNA of the strain was extracted using the CTAB freeze-thaw method specifically for lactic acid bacteria.

[0035] The composition of the preculture medium: peptone 10g, yeast extract powder 4g, beef extract powder 8g, glucose 20g, anhydrous sodium acetate 5g, triammine citrate 2g, dimethyl phosphate 2g, Tween-80 1mL, magnesium sulfate heptahydrate 0.2g, manganese sulfate tetrahydrate 0.05g, distilled water 1L, cysteine ​​salt 0.5g, pH adjusted to about 6.2, sterilized at 21°C for 15min.

[0036] The isolated strain was subjected to physiological and biochemical identification and 16srRNA molecular identification according to the Bergey Manual of Bacterial Identification, using bacterial API50CH (BioMérieux). DNA samples of the strain were extracted and 16srRNA molecular identification was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The molecular sequence was compared with NCBI database blastn, confirming that YN-01 was Lactobacillus delbrueckii lactis subsp. phylogenetic tree can be found in Figure 1 .

[0037] The whole genome of Lactobacillus delbrueckii subsp. lactis YN-01 was sequenced using the Illumina HiSeq sequencing platform. After assembly and alignment, its genome length was 2007516bp, the G+C content was 49.7%, and the chromosome genome contained 3531 coding genes.

[0038] Colony morphology characteristics.

[0039] The Lactobacillus delbrueckii lactic acid subspecies YN-01 of the present invention forms milky white opaque colonies with a diameter of 0.5 to 1 mm, round and flat or micro-papillary, flat edges, on an MRS solid culture medium.

[0040] In the clean bench, the purified Lactobacillus delbrueckii lactic acid subspecies YN-01 sludge was picked up with an inoculation loop for smearing, and Gram staining was performed after it dried. The morphology and staining characteristics of the strain were observed and recorded under a microscope. The morphology of this strain under the microscope was Gram-positive bacteria, with round or oval bodies, mostly single, paired, chained or agglomerated; on the modified MRS solid medium, the colony diameter and colony edges were neat, creamy white and opaque.

[0041] The Lactobacillus delbrueckii lactic acid subspecies YN-01 of the present invention has the following biological characteristics: the bacteria are rod-shaped under a microscope, and the arrangement state is also single, paired, chain or clustered, but not in an obvious chain structure, without flagella or cilia, and will not move.

[0042] Gastrointestinal fluid tolerance of Lactobacillus delbrueckii subsp. lactis YN-01.

[0043] Acid resistance test: The fermentation liquid of the activated two-generation Lactobacillus delbrueckii subspecies YN-01 strain was centrifuged at 4°C and 4000 r / min for 10 min, the bacteria were collected, and the bacteria were washed once with sterilized PBS buffer. Then the bacterial concentration was adjusted to OD 600 =1, PBS was used as blank control. The bacterial suspension was inoculated into MRS liquid medium with a pH of 2.0 at 3%, mixed, and cultured at 37°C. The bacterial suspension at 0h and 3h was taken to determine the number of viable bacteria and to determine the bile salt tolerance of the strain. Three replicates were set for each group.

[0044] Bile salt tolerance test: Lactobacillus delbrueckii subspecies YN-01 was activated for two generations, and the fermentation liquid of the second generation strain was centrifuged at 4°C and 4000r / min for 10 minutes to collect the cells, and the cells were washed once with sterilized PBS buffer. The cell concentration was adjusted to OD 600 =1, PBS was used as blank control. The prepared bacterial suspension was inoculated into MRS liquid medium with a bile salt concentration of 0.3% at 3%, mixed evenly, and the viable bacterial counts were determined at 0, 3, 6, and 9 hours at 37°C to determine the bile salt tolerance of the strains. Each group was replicated 3 times.

[0045] Gastric juice resistance test: Take 2mL of 1mol / L hydrochloric acid and adjust the pH to 2.0 with distilled water. Add pepsin at a ratio of 1g / 100mL, fully dissolve, and sterilize with a 0.20μm microporous filter membrane. Inoculate the activated bacterial solution into the treated artificial gastric juice at a volume fraction of 10%, mix well, and culture in a constant temperature shaker at 37℃ and 100r / min. Use the spreading dilution method to determine the survival rate of high-yield GABA lactic acid bacteria after 3h of culture in artificial gastric juice, and repeat the determination three times.

[0046] Intestinal fluid resistance test: Take 0.85g KH 2 PO 4 Add 62.5L of distilled water, adjust the pH to 7.6 with 1mol / L NaOH solution, sterilize the prepared solution with high pressure, add trypsin at a ratio of 1g / 100mL on a clean bench, fully dissolve, and sterilize with a 0.20μm microporous filter membrane. Inoculate the activated bacterial solution into the treated artificial intestinal fluid at an inoculum volume of 10%, mix well, and culture in a constant temperature shaker at 37℃ and 100r / min. Determine the number of viable bacteria at 0 and 3h by the pouring method, and repeat the determination three times.

[0047] Tolerance effect: After the pH of Lactobacillus delbrueckii subspecies lactic acid YN-01 was 2.0, the bile salt concentration was 0.3%, and it was treated with simulated gastric fluid and simulated intestinal fluid, the results are shown in Table 1 below:

[0048] Table 1 Survival of Lactobacillus delbrueckii subsp. lactis YN-01 in simulated environment

[0049] ;

[0050] As shown in Table 1, Lactobacillus delbrueckii lactic acid subspecies YN-01 has good tolerance characteristics, with a survival rate of 79.5% in 0.3% bile salt, 86% at pH 2.0, 78% after 3 h of digestion in artificial gastric juice, and 75% after 3 h of treatment in simulated intestinal fluid.

[0051] According to the acid resistance, bile salt resistance and gastrointestinal fluid tolerance effects of this example, it can be seen that Lactobacillus delbrueckii lactis subspecies YN-01 has good tolerance to gastrointestinal fluid in the intestine and has probiotic properties.

[0052] Functional gene characteristics of Lactobacillus delbrueckii subsp. lactis YN-01.

[0053] The KEGG database was used to perform functional annotation on the whole genome of Lactobacillus delbrueckii subsp. lactis YN-01. KEGG is a professional database for comparing and analyzing bacterial genes and metabolic pathways at the molecular level. It can explore the molecular level of bacteria and study the relationship between functional genes and biological molecules in various biological pathways. The KEGG database divides the biological pathways of microorganisms into six categories: Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Human Diseases.

[0054] The tolerance-related functional genes of Lactobacillus delbrueckii subspecies lactis YN-01 were annotated, and 7 acid tolerance-related genes were annotated ( argH , murE , lysC , argR , lysS , argS , dapA ); 8 genes related to heat tolerance ( dnaA , dnaN , dnaB , dnaE , hrC , dnaJ , dnaG , dnaK ); 2 cold tolerance related genes ( cspA , C ); 1 bile salt tolerance-related gene ( d ).

[0055] Genes related to probiotic functions were detected in the genome of Lactobacillus delbrueckii subsp. lactis YN-01 and annotated to obtain functional genes related to the production of riboflavin and lactic acid, the synthesis of bioactive peptides, and acid and bile resistance. The specific genes are shown in Table 2.

[0056] Table 2 Probiotic-related genes

[0057] Embodiment 2

[0058] Protease activity of Lactobacillus delbrueckii subsp. lactis YN-01.

[0059] After activation for three generations, Lactobacillus delbrueckii lactis subspecies YN-01 was inoculated at a 3% inoculum into 11% sterilized reduced skim milk and fermented at 37°C for 48 h. The fermented milk was taken out and mixed by oscillation. The pH value of the fermented milk was measured and the pH was adjusted to 4.6 (isoelectric point of casein) with 1 mol / L NaOH. The mixture was centrifuged at 3000 g for 10 min, the supernatant was collected, and the pH was then adjusted to 8.3 (optimal reaction pH of ACE inhibitory peptide). The mixture was centrifuged at 3000 g for 5 min, the supernatant was collected, and stored at 4°C.

[0060] The supernatant of bacterial lysis was collected and roughly separated by ultrafiltration tube, and the components with MW>10kDa were collected. The components with MW>10kDa were purified by Sephadex G-75 dextran gel, and three peaks were eluted from large to small according to molecular weight, namely P1, P2, and P3, and their protease activity and specific activity were measured. Figure 2 As shown in the results, it was found that the enzyme activity and specific activity of crude enzyme solution with MW>10kDa were significantly higher than those of bacterial lysis supernatant (P<0.05), which was due to the removal of small molecular weight substances by ultrafiltration and the concentration of crude enzyme solution with MW>10kDa. Among the P1, P2, and P3 components, the enzyme activity and specific activity of the P3 component were significantly higher than those of P1 and P2 (P<0.05), and it was the component with the most prominent protease activity.

[0061] Genes related to the proteolytic system of Lactobacillus delbrueckii lactis subsp. lactis YN-01.

[0062] Lactic acid bacteria largely lack the ability to biosynthesize amino acids. The proteolytic system in the strain provides peptides and free amino acids for the growth of the strain during the fermentation process. The proteolytic system establishes a complete model for casein decomposition, transport of casein-derived peptide degradation and its regulation. The proteolytic system can be divided into three categories according to its function: (I) cell wall proteases, (II) peptidases, and (III) peptide transport systems, among which the peptide transport system consists of Opp, DtpT and Dpp. Opp and DtpT exist in the tripeptide transport system. DtpT is a dipeptide / tripeptide transporter of the PTR family driven by proton motive force (PMF), which has a higher affinity for hydrophilic and charged dipeptides and tripeptides. The Dpp peptide transport system depends on ATP or related energy-rich phosphorylated intermediates. The system was originally named DtpP. As shown in Table 3, two transport systems, Opp and Dpp, were found in the peptide transport system of Lactobacillus delbrueckii subsp. lactis YN-01, and Dpp contains two DppA1 genes. DppA binds with high affinity to dipeptides / tripeptides composed of basic and hydrophobic amino acid residues, and its expression also depends on the presence of other peptide transport systems (Opp or DtpT). The peptide needs to be absorbed to exert its regulatory effect. The transport capacity of the peptide is greatest when the availability of the peptide is limited.

[0063] The Opp system belongs to the oligopeptide ABC transporter superfamily and consists of five proteins: an oligopeptide binding protein (OppA), two integral membrane proteins (OppB and OppC) and two nucleotide binding proteins (OppD and OppF). The system preferentially selects hydrophobic (branched amino acid) dipeptides and tripeptides. The peptide transport system of Lactobacillus delbrueckii subsp. lactis YN-01 contains a complete Opp operon OppABCDF and 18 separate oligopeptide binding protein genes OppA (Table 3). The homologous sequence of the gene encoding the OppA protein is involved in the internalization of oligopeptides. The deletion of the DtpT gene leads to the upregulation of Opp activity. The Opp system can transport peptides of at least 19 residues at most, and the properties of these peptides will significantly affect the related transport kinetics.

[0064] Endopeptidases, aminopeptidases (PepN and PepC) and X-prolyl dipeptidyl aminopeptidase (PepX) are enzymes that act on oligopeptides. The endopeptidase encoded by PepO has a unique cleavage specificity for α-S1-casein (f1-f23), hydrolyzing Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9 and Gln-9-Gly-10. The peptides hydrolyzed by the protease encoded by PepF contain 7 to 17 amino acid residues. PepF is inhibited by nitrogen and carbon, specifically induced and regulated by medium pH. PepDA belongs to the cysteine ​​dipeptidase family C69 and has strict substrate specificity, affecting the levels of most free amino acids and cell wall structure in lactic acid bacteria. PepP is a metallopeptidase of the M24 family that prefers to cleave tripeptides carrying proline in the middle position. PepQ belongs to the M24 family of metallopeptidases. The biosynthesis of PepQ depends on the composition of the medium but not on the peptide concentration. It cleaves dipeptides carrying proline at the second position. The PepT sequence encodes an M20 peptidase, a cytoplasmic enzyme with broad substrate specificity that preferentially hydrolyzes oligopeptides carrying alanine at the P-1 position. PepT participates in the hydrolysis of oligopeptides at the late stage of the protein degradation pathway to maintain turnover.

[0065] Table 3 Protease gene information of Lactobacillus delbrueckii subsp. lactis YN-01

[0066] ;

[0067] In summary, the proteolytic system of the genome of Lactobacillus delbrueckii subsp. lactis YN-01 contains abundant Pep genes encoding peptidases, which hydrolyze peptides inside and outside the bacteria, not only providing the amino acids required for bacterial growth, but also forming a large number of short peptides, and a large number of peptides have proline residues at the C-terminus and N-terminus, which indicates that Lactobacillus delbrueckii subsp. lactis YN-01 has the potential to generate bioactive peptides when used in fermented foods; at the same time, the strain has a relatively complete peptide transport system, which provides support for the function of active peptides in Lactobacillus delbrueckii subsp. lactis YN-01 fermented foods.

[0068] Embodiment 3

[0069] Lactobacillus delbrueckii subsp. lactis YN-01 enhances the ACE inhibitory activity of fermented milk.

[0070] After activation for three generations, Lactobacillus delbrueckii lactis subspecies YN-01 was inoculated at a 3% inoculum into 11% sterilized reduced skim milk and fermented at 37°C for 48 h. The fermented milk was taken out and mixed by oscillation. The pH value of the fermented milk was measured and the pH was adjusted to 4.6 (isoelectric point of casein) with 1 mol / L NaOH. The mixture was centrifuged at 3000 g for 10 min, the supernatant was collected, and the pH was then adjusted to 8.3 (optimal reaction pH of ACE inhibitory peptide). The mixture was centrifuged at 3000 g for 5 min, the supernatant was collected, and stored at 4°C.

[0071] Determination of ACE inhibitory activity by HPLC: The pH of the sample was adjusted to 8.3. The specific determination method is shown in Table 4.

[0072] Table 4 ACE inhibitory activity determination method

[0073] ;

[0074] 10 μL of the reaction solution was taken to determine the peak area of ​​hippuric acid (HA) generated by the reaction. The liquid phase conditions were set as follows: mobile phase A was 0.05% TFA aqueous solution; B was acetonitrile. The chromatographic column was balanced with 98% liquid A. The flow rate was 0.8 mL / min; the detection time was 10 min; the detection wavelength was 228 nm; the liquid phase gradient was set to 0-10 min with mobile phase A at 75% and B at 25%. The inhibition rate of ACE inhibitory peptide was calculated as follows:

[0075] ACE inhibition rate (ACEI%) = ((Aa-Ab) / Aa) × 100;

[0076] Aa: peak area of ​​HA generated without adding sample;

[0077] Ab: peak area of ​​HA generated by the reaction group;

[0078] Determination of the degree of protein hydrolysis: Prepare the tyrosine standard solution into different concentrations, take 100 μL of the standard solution, put it in a centrifuge tube, add 4 mL of OPA reagent, mix well, react at 25°C for 2 minutes, measure the absorbance at 340 nm, and draw a standard curve. Take 50 μL of the fermentation supernatant, add 2 mL of OPA reagent, mix well, react at 25°C for 2 minutes, and finally measure the absorbance at 340 nm. Determine the concentration of the polypeptide in the fermented milk according to the standard curve.

[0079] Determine the change of peptide concentration with the fermentation time, such as Figure 3As shown in the figure, the polypeptide concentration increases with the extension of fermentation time, and when the fermentation time is 48h, the polypeptide concentration is 0.966±0.028mg / mL, but when the fermentation time reaches 60h, the polypeptide concentration of fermented milk is 0.994±0.023mg / mL; at the same time, the ACE inhibitory activity of fermented milk in each time period during the fermentation period was determined, and it was found that with the extension of fermentation time, the ACE inhibitory activity of fermented milk increased. When the fermentation time was 48h, the ACE inhibitory activity of fermented milk was 80.80±0.20%, and when the fermentation time was 60h, the ACE inhibitory activity of fermented milk was 82.32±0.34%. The study showed that with the extension of fermentation time, the polypeptide concentration was positively correlated with the ACE inhibitory activity, and 48h was the optimal fermentation time, which was more conducive to the formation of ACE inhibitory peptides.

[0080] Embodiment 4

[0081] Lactobacillus delbrueckii subsp. lactis YN-01 increases the content of active peptides in fermented beef jerky.

[0082] Take the hind leg or buttock meat of the beef, remove the fascia fat, and trim the raw meat into 10×10×10cm blocks. Put the trimmed beef blocks into the pot, add 1% scallion and 1% ginger, make the water cover the beef blocks, boil and simmer for 40 minutes. After cooling, cut into small pieces of 2×2×2cm along the direction of the muscle fiber. The ratio of meat blocks to broth is 1:1, add 1% mixed spices, 4% white sugar, 1.2% salt, 1% cooking wine, and 3% light soy sauce for stewing. Stir several times during the period to prevent the pot from burning, and simmer until the soup is dry. After cooling, inoculate Lactobacillus delbrueckii lactic acid subspecies YN-01 with an inoculation amount of 107CFU / g and ferment at 33℃ for 24h. Place the baking tray in the oven and bake at 100℃ for 30min. Turn it over and bake at 100℃ for 30min to obtain the fermented beef jerky sample.

[0083] Liquid chromatography-mass spectrometry (LC-MS) was used to detect metabolites of beef jerky before and after fermentation. Chromatographic conditions: 10 μL of sample was separated by HSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm) and then entered mass spectrometry detection. Mobile phase A was a water / acetonitrile (95 / 5, volume / volume) solution containing 0.1% formic acid, and mobile phase B was an acetonitrile / isopropanol / water (47.5 / 47.5 / 5, volume / volume / volume) solution containing 0.1% formic acid. The flow rate was 0.40 mL / min and the column temperature was 40 °C. Mass spectrometry conditions: The sample mass spectrometry signal was collected in positive and negative ion scanning mode, and the mass scanning range was m / z: 50-1000. The positive mode ion spray voltage was 5000 V, the negative mode ion spray voltage was -4000 V, the declustering voltage was 80 V, the spray gas was 50 psi, the auxiliary heating gas was 50 psi, the curtain gas was 30 psi, the ion source heating temperature was 550 ° C, the cyclic collision energy was 20-60 V, the MS1 resolution was 70000, and the MS2 resolution was 17500.

[0084] Among the metabolites of beef jerky samples before and after fermentation, a total of 1,341 metabolites were identified by MS1 and MS2. Figure 4 It can be seen that the metabolites include 460 lipids and lipid molecules, 355 organic acids and their derivatives, 211 organic heterocyclic compounds, 139 organic oxygen compounds, 138 phenylpropanoids and polyketides, 124 benzenes, 49 nucleosides, nucleotides and analogs, 28 organic nitrogen compounds, 24 alkaloids and their derivatives, etc. The quality control samples overlapped, indicating that the data in this study showed good reliability and repeatability.

[0085] The experiment used principal component analysis (PCA) combined with partial least squares discriminant analysis (PLS-DA) to determine the differences in metabolites between beef jerky and fermented beef jerky ( Figure 5 ), where B is beef jerky sample, FB is fermented beef jerky sample, and QC is quality control sample. The contribution rates of the first principal component and the second principal component in the positive ion mode were 52.50% and 18.90%, respectively, and the total contribution rate was 71.40%. The total contribution rate in the negative ion mode was 68.70%, indicating that principal component analysis can explain the overall situation of the sample. Figure 5 The samples of beef jerky and fermented beef jerky were in different quadrants, and the samples between the groups were clustered, indicating that there were obvious differences between the two groups of beef jerky and fermented beef jerky. Principal component analysis can be used to distinguish between beef jerky and fermented beef jerky samples.

[0086] The composition of active peptides increased after fermentation of beef jerky is further analyzed, as shown in Table 5. After fermentation of beef jerky with Lactobacillus delbrueckii subsp. lactis YN-01, a total of 35 active peptides were produced, giving it a stronger probiotic potential.

[0087] Table 5 Newly added active peptides after fermentation of beef jerky

[0088]

[0089] Embodiment 5

[0090] Lactobacillus delbrueckii subsp. lactis YN-01 increases the content of active peptides in fermented dried fruits and vegetables.

[0091] Select yellow persimmons with high maturity, complete appearance, bright color, uniform color and freshness, and wash them clean. Cut a cross on the top of the yellow persimmon, put it in boiling water, soak it for 40 seconds, remove it, peel it, cut it into pieces, and put it into a juicer to break it. Then sterilize the yellow persimmon pulp in a water bath at 80℃ for 25 minutes and cool it. Inoculate Lactobacillus delbrueckii lactic acid subspecies YN-01 at 0.3% (v / v) and ferment it at 33℃ for 12 hours. After the fermentation is over, mix it according to the ratio of white sugar: yellow persimmon at 1:50, put the juice into the pot for heating and concentration, and stop heating when the sauce is orange-yellow, uniform in texture, has a certain viscosity, and the soluble solids reach about 23%. During the heating and concentration process, simmer it slowly over low heat and stir it constantly to prevent sticking to the bottom of the pot. Finally, can it, sterilize it in a water bath for 30 minutes, cool it and store it, and obtain the fermented yellow persimmon sauce.

[0092] Liquid chromatography-mass spectrometry (LC-MS) was used to detect the metabolites of yellow persimmon paste before and after fermentation. A total of 3551 metabolites were identified in the metabolites of yellow persimmon paste samples before and after fermentation. Principal component analysis was used to analyze the differences in metabolites of yellow persimmon paste before and after fermentation. Figure 6 As shown, YP is the yellow persimmon paste sample and FYP is the fermented yellow persimmon paste sample. Figure 6 In (a), the first principal component PC1 contributes 67.70%, PC2 contributes 5.2%, and the total accounts for 72.9%. In the negative ion mode ( Figure 6 The total contribution of the two principal components in (b) is 76.99%, indicating that the two principal components can represent the overall information of the sample. Figure 6 The samples of yellow persimmon paste before fermentation and yellow persimmon paste after fermentation were in different quadrants, the samples within the group were clustered, and the two groups were separated from each other, indicating that there were obvious differences in the metabolite composition of the two groups of samples of unfermented yellow persimmon paste and fermented yellow persimmon paste.

[0093] like Figure 7As shown, there were 461 significantly different metabolites between yellow persimmon paste before fermentation and after fermentation (P<0.05, VIP>1), including 115 organic acids and their derivatives, 89 lipids and lipid molecules, 77 organic oxygen-containing compounds, 69 organic heterocyclic compounds, 37 benzenes, 25 nucleosides, nucleotides and analogs, 23 phenylpropanoid compounds and polyketide compounds, 10 unavailable, 7 organic nitrogen compounds, 4 alkaloids and derivatives, 2 lignans, neolignans and related compounds, 1 hydrocarbon derivative, 1 hydrocarbon, and 1 organophosphorus compound.

[0094] After fermentation, 107 short peptides increased significantly, among which tripeptides were the most abundant ( Figure 8 ). According to NCBI search, Ser-Tyr-Ile and Leu-Tyr-Val exist in the hypothetical protein sequence of Gemmatimonadota bacterium. Ile-Ile-His exists in the DinB family protein sequence of Bacteroidota bacterium. Ile-Ile-Arg exists in the amidohydrolase sequence of Leucobacter japonicus. Val-Arg-Val exists in the SDR family NAD(P)-dependent oxidoreductase sequence of Gemmatimonadota bacterium. Val-Pro-Phe exists in the aminopeptidase P familyprotein sequence of Bifidobacteriaceae bacterium. Detailed information on the newly added active peptides after fermentation of yellow persimmon paste is shown in Table 6.

[0095] Table 6 Newly added active peptides after fermentation of yellow persimmon paste

[0096] ;

[0097] ;

[0098] ;

[0099] It is worth noting that the contents not elaborated in detail in the present invention are all prior art and are well known to those skilled in the art.

[0100] Therefore, the present invention adopts the above-mentioned Lactobacillus delbrueckii subspecies lactis YN-01 and the products and applications prepared therefrom. Lactobacillus delbrueckii subspecies lactis YN-01 has good gastrointestinal fluid and bile salt tolerance and high protease activity, and has great application value in fermented foods.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A Lactobacillus delbrueckii subspecies lactic acid YN-01, characterized in that The Lactobacillus delbrueckii lactic acid subspecies YN-01 was deposited in the China Center for Type Culture Collection on October 22, 2024, with the deposit address being Wuhan University, Wuhan, China; the deposit number is CCTCC NO: M 20242299; the Latin name is Lactobacillus delbrueckii subsp. lactis YN-01; The gene sequence of 16srRNA of the Lactobacillus delbrueckii lactic acid subspecies YN-01 is shown in SEQ ID NO.

1.

2. Use of the Lactobacillus delbrueckii lactic acid subspecies YN-01 as claimed in claim 1 in preparing fermented food.

3. A food comprising the Lactobacillus delbrueckii lactic acid subspecies YN-01 as claimed in claim 1, characterized in that: The number of viable bacteria of Lactobacillus delbrueckii lactic acid subspecies YN-01 in the food is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

4. The food according to claim 3, characterized in that The food is a drink.

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