Lactobacillus delbrueckii subsp. lactis having high ability to form biofilm and strong stress resistance and use thereof

Abstract

The present invention relates to the technical fields of microbiology and fermentation. Specifically provided are Lactobacillus delbrueckii subsp. lactis having a high ability to form a biofilm and strong stress resistance and the use thereof. The strain is Lactobacillus delbrueckii subsp. lactis Dangxiong LBVH, and is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC No.: M2023396. The strain has the following characterisctis that the ability to form a biofilm is high, the yogurt fermented with the strain has the fruity flavor, and the ability to resist adverse environment is extremely strong. When used in the fermentation of food under a variety of high stress conditions, such as high salt, high sugar and high acid, a large number of viable bacteria and fermentation viability can be maintained, thereby endowing the fermented food with excellent product characteristics.

Description

Lactobacillus delbrueckii subspecies lactis with high biofilm formation and strong stress resistance and its application

[0001] Related applications

[0002] This application claims priority to the prior application document with application number 202310802241.7 filed in China on June 25, 2023, and incorporates its entire contents into this document. Technical Field

[0003] The present invention relates to the technical field of microbial screening and fermentation, and in particular to a Lactobacillus delbrueckii subspecies lactis with high biofilm formation and strong stress resistance and an application thereof. Background Art

[0004] Lactic acid bacteria are Gram-positive bacteria widely used in the fermentation of fruits, vegetables, fish, meat, and dairy products. They have become a focus of international probiotic research because they facilitate digestion, prevent diarrhea, lower cholesterol levels, create a healthy intestinal environment, and extend the shelf life of foods. Lactobacillus delbrueckii subsp. lactis, in particular, is widely used as a starter culture in the dairy industry and as a probiotic in health products. However, during the fermentation process, lactic acid bacteria often face numerous adverse environmental stresses, which limits their further application. This is why research on highly stress-resistant lactic acid bacteria is crucial. Currently, domestic research on lactic acid bacteria faces a number of challenges, including production technology that lags behind advanced technologies abroad, resulting in low product quality; an inability to guarantee the specific efficacy of the strains used, a lack of superior strains; and low survival rates of probiotics. Research on the mechanisms underlying the stress-resistant properties of lactic acid bacteria is also relatively rare. Therefore, research on highly stress-resistant lactic acid bacteria is of great significance.

[0005] A biofilm is an organized bacterial colony that adheres to the surface of living or inanimate objects and is coated with extracellular macromolecules. It is also called a biofilm. Biofilm is a common way of life for most bacteria and may be found in places such as medical devices, pathological tissues, food industry raw materials, food machinery, and pipeline surfaces. As a widespread lifestyle for microorganisms in nature, biofilms are an effective way for bacteria to resist various environmental stresses. When bacteria encounter threats from extreme environments (temperature, pH, osmotic pressure, metal ions, antibiotics, and other microorganisms), they aggregate to form a biofilm to resist the adverse environment. Therefore, the ability to form a biofilm is often closely related to the stress resistance of microorganisms.

[0006] Patent application CN 109576182 A discloses a highly stress-resistant Lactobacillus rhamnosus A-4 and its use: the Lactobacillus rhamnosus A-4 has a preservation number of CGMCC No. 16550. The present invention also relates to the use of Lactobacillus rhamnosus A-4 in the preparation of beverages. The Lactobacillus rhamnosus A-4 involved in the present invention can withstand a high temperature of 75°C, and its survival rate after 2h of pH 2.0 acid treatment is as high as 96.88% ± 1.4%; it can still grow and reproduce under high osmotic pressure stress of 4.5% NaCl, low nutritional stress of 8-fold culture medium dilution, pH 3.5 acid stress, and 5-45°C temperature stress, and has broad application prospects in the preparation of room-temperature live bacteria lactic acid bacteria beverages.

[0007] Patent application CN 110656061 A discloses a strain of Lactobacillus paracasei subsp. paracasei RP38, which was deposited with the China Center for Type Culture Collection on May 5, 2019, under the accession number CCTCC NO: M2019322. Lactobacillus paracasei subsp. Paracasei RP38 has the following properties: ① It can metabolize sugars as a carbon source to produce volatile flavor compounds in a nitrogen-deficient environment, adapting to the physical properties of raw materials such as grapes and Tremella and Longan juice. The fermented aroma produced by metabolism is rich and harmonious with the raw material aroma, making it an ideal starter for lactic acid fermented juice products. ② It can be used for the biological deodorization of kelp products, biotransforming and significantly reducing the presence of fishy substances such as 1,3-octadiene, E,Z-3-ethylidenecyclohexene, n-nonanol, n-heptanal, and iodooctane, producing secondary metabolites with fruity and flavorful aromas, such as β-phenylethanol, peach aldehyde, ethyl tetradecanoate, dodecanal, and E-2-nonen-1-ol. ③ It exhibits strong stress resistance, such as withstanding environmental stresses such as high acidity at pH 2.0 and high osmotic pressure at 30% glucose concentration.

[0008] Patent application CN 111280355 A discloses Lactobacillus plantarum P_17 and a method for producing fermented apple juice using the same. Apple juice is fermented using Lactobacillus plantarum P_17, which has a CGMCC accession number of 19251. Lactobacillus plantarum P_17 exhibits outstanding acid, sugar, and low-temperature resistance, strong pepsin and acid resistance, and the ability to withstand artificial intestinal fluid stress, bile salts, and high osmotic pressure. The fermented apple juice has a uniform color, a smooth texture, and a unique fermentation aroma and apple flavor. The fermented apple juice produced by this method has a higher viable count than other strains, and significantly higher gamma-aminobutyric acid, total acid, and antioxidant values ​​than conventional apple juice. This method simplifies the production process and utilizes readily available and abundant raw materials, making it a significant advance in the industrial production of lactic acid fermented fruit and vegetable juice beverages.

[0009] Patent application CN 109777748 A is a strain of Lactobacillus brevis CD0817 with high γ-aminobutyric acid production: the Lactobacillus brevis CD0817 was deposited in the China Center for Type Culture Collection on July 9, 2018, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, abbreviated as CCTCC, and the deposit number is CCTCC NO: M2018462. The Lactobacillus brevis CD0817 of the present invention has the characteristics of high GABA production, richer gene functions, and can grow in a high osmotic pressure environment.

[0010] However, there is no report in the prior art that Lactobacillus delbrueckii subsp. lactis has stress resistance.

[0011] Summary of the Invention

[0012] In view of the technical problem that lactic acid bacteria are faced with adverse environmental stress in many current fermented food production processes, resulting in the death or low vitality of lactic acid bacteria in the fermentation environment, which ultimately affects the quality of fermented foods, the present application provides a kind of Lactobacillus delbrueckii subspecies lactis with strong stress resistance. This strain has high biofilm formation ability, fermented yogurt has fruity flavor, and is extremely strong in resisting adverse environments. It is applied in fermented foods under various high stress conditions such as high salt, high sugar, and high acid, and can maintain a high number of viable bacteria and fermentation activity, giving fermented foods excellent product characteristics (good flavor, physicochemical properties, functional ingredients, etc.).

[0013] Specifically, this application proposes the following technical solutions.

[0014] The present application provides a Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII strain, wherein the strain is Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No.: M2023396.

[0015] In some embodiments of the present application, for the above-mentioned Lactobacillus delbrueckii subsp. lactis, the 16s rRNA sequence of the strain comprises or consists of the sequence shown in SEQ ID NO: 3.

[0016] In some embodiments of the present application, for the above-mentioned Lactobacillus delbrueckii subspecies lactis, the Lactobacillus delbrueckii subspecies lactis carries a gene expressing a biofilm.

[0017] In some embodiments of the present application, for the above-mentioned Lactobacillus delbrueckii subsp. lactis, the gene expressing the biofilm comprises the sequence shown in SEQ ID NOs: 4-7 or consists of the sequence shown in SEQ ID NOs: 4-7.

[0018] The present application provides a bacterial preparation, which contains the above-mentioned Lactobacillus delbrueckii subsp. lactis.

[0019] The present application provides the use of the above-mentioned Lactobacillus delbrueckii subspecies lactis or the above-mentioned bacterial preparation in the field of fermentation, preferably in the field of food fermentation.

[0020] In some embodiments of the present application, for the above-mentioned application, the food is a dairy product, a soy sauce product, a pear juice product or a vinegar product, and more preferably, the dairy product is yogurt or cheese.

[0021] The present application provides the use of the above-mentioned Lactobacillus delbrueckii subspecies lactis or the above-mentioned bacterial preparation in biological preservation or in the preparation of anti-caries products.

[0022] The present application provides the use of the above-mentioned Lactobacillus delbrueckii subspecies lactis or the above-mentioned bacterial preparation in the preparation of animal feed or in the preparation of products for regulating intestinal flora.

[0023] The present application provides a genome of a biofilm-forming Lactobacillus delbrueckii subsp. lactis, wherein the genome comprises one or more genes selected from the sequences shown in SEQ ID NOs: 4-7.

[0024] In some embodiments of the present application, for the genome described above, the genome is tolerant to NaCl, sugar and / or acid.

[0025] The present application provides a protein tolerant to NaCl, sugar or acid, which is encoded by one or more of the proteins shown in SEQ ID NOs: 4-7, or obtained by the aforementioned Lactobacillus delbrueckii subsp. lactis or the aforementioned bacterial agent.

[0026] The present application provides a food obtained by fermenting food materials with the above-mentioned Lactobacillus delbrueckii subsp. lactis.

[0027] In some embodiments of the present application, for the food described above, the food is a soy sauce product, a dairy product, a pear juice product or a vinegar product.

[0028] In some embodiments of the present application, for the above-mentioned food, the soy sauce product is obtained by mixing soybean meal, flour, and bran and then inoculating the mixed strain of Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae, wherein the mixing ratio of the Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae is 1:0.5-1; and / or

[0029] The dairy product is obtained by fermenting raw cow's milk with the above-mentioned Lactobacillus delbrueckii subsp. lactis; and / or

[0030] The pear juice product is obtained by fermenting pear juice with the above-mentioned Lactobacillus delbrueckii subsp. lactis; and / or

[0031] The vinegar product is obtained by fermenting vinegar by mixing the above-mentioned Lactobacillus delbrueckii subspecies lactis and acetic acid bacteria.

[0032] In some embodiments of the present application, the food described above is an anti-caries or caries prevention food or a food that regulates intestinal flora.

[0033] Beneficial effects achieved by this application:

[0034] The strain Lactobacillus delbrueckii subsp. lactis described in the present application contains 4 unique genes related to biofilm formation compared with the 14 strains of Lactobacillus delbrueckii subsp. lactis (Control_14) whose complete genomes have been published in the NCBI database, and has a high biofilm formation ability. This characteristic makes Lactobacillus delbrueckii subsp. lactis have strong resistance to osmotic pressure, acid, gastrointestinal digestive fluid, etc., and has strong stress resistance; the strain can maintain strong fermentation activity when used in a variety of high-stress fermentation environments such as dairy fermentation, soy sauce fermentation, vinegar fermentation, and fruit juice fermentation, and gives fermented foods excellent product characteristics (good flavor, physical and chemical properties, functional ingredients, etc.).

[0035] The strain described in this application was isolated from the milk of a herdsman's home at an altitude of about 4,350 meters in Dangxiong County, Tibet. It is a natural wild strain. After identification, it was found to belong to the lactobacillus delbrueckii subspecies lactis, which is included in the "List of Probiotics that can be used in Health Foods" and has food safety attributes.

[0036] The strain described in this application can grow in MRS liquid culture medium with a mass concentration of 10% NaCl, a mass concentration of 45% glucose, and a pH of 3.0, and can also survive in artificial gastric juice and artificial intestinal juice. It has high stress resistance, and its biofilm formation ability was measured and it was found that it has a strong biofilm formation ability.

[0037] The strain described in this application can maintain high activity in a variety of high-stress fermentation environments, and can be used in food fermentation in a variety of environments such as dairy products, soy sauce, vinegar, and fruit juice, giving fermented foods excellent product characteristics.

[0038] Strain deposit information

[0039] The strain Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII used in the present invention was deposited in the China Center for Type Culture Collection on March 23, 2023, with the deposit number CCTCC NO: M 2023396, and the deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: 02768754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic diagram of the microscopic morphological characteristics of the strain.

[0041] FIG2 is a schematic diagram of gel electrophoresis of amino acids encoded by four genes.

[0042] FIG3 is a schematic diagram showing the biofilm formation of two Lactobacillus delbrueckii subspecies lactis under different NaCl concentrations.

[0043] Figures 4A to 4C are schematic diagrams of the growth curves of Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII (DB-8) at different concentrations of NaCl, sugar, and different pH values, wherein Figure 4A is a schematic diagram of the growth curve of DB-8 at different concentrations of NaCl, Figure 4B is a schematic diagram of the growth curve of DB-8 at different sugar concentrations, and Figure 4C is a schematic diagram of the growth curve of DB-8 at different pH values.

[0044] FIG5 is a schematic diagram of the expression of biofilm-forming genes in DB-8 under stress conditions. DETAILED DESCRIPTION

[0045] As described above, the present application provides Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII (DB-8 for short), which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No.: M2023396.

[0046] The strain described in this application was isolated from the milk of a herdsman's home at an altitude of about 4,350 meters in Dangxiong County, Tibet. It is a natural wild strain with good stress resistance and the ability to withstand strong adverse environments.

[0047] In some embodiments, the 16s rRNA sequence of the strain comprises or consists of the sequence shown in SEQ ID NO: 3.

[0048] The sequence of SEQ ID NO:3 is as follows:

[0049] In some embodiments, the DB-8 carries a gene expressing a biofilm. In some embodiments, the gene expressing a biofilm comprises or consists of a sequence as shown in SEQ ID NOs: 4-7.

[0050] In the present application, the DB-8 has four unique genes related to biofilm formation compared with Control_14, thereby making the strain have stronger stress resistance.

[0051] In this application, Control_14 refers to 14 strains of Lactobacillus delbrueckii subsp. lactis whose complete genomes have been published in the NCBI database.

[0052] In this application, the sequence of SEQ ID NO: 4 (i.e., DB-8 GL000445) is:

[0053] The sequence of SEQ ID NO:5 (i.e., DB-8 GL001087) is:

[0054] The sequence of SEQ ID NO:6 (i.e., DB-8 GL001801) is:

[0055] The sequence of SEQ ID NO:7 (DB-8 GL001802) is:

[0056] In some embodiments, the amino acid sequences encoded by SEQ ID NOs: 4-7 are shown in SEQ ID NOs: 8-11.

[0057] Wherein, the sequence of SEQ ID NO:8 is:

[0058] The sequence of SEQ ID NO:9 is:

[0059] The sequence of SEQ ID NO: 10 is:

[0060] The sequence of SEQ ID NO:11 is:

[0061] Since the strain described in the present application carries the above-mentioned specific genes related to biofilms, when the bacteria encounter extreme environmental stress (temperature, pH value, osmotic pressure, metal ions, antibiotics and other microorganisms, etc.), they will aggregate to form a biofilm to resist the adverse environment. Therefore, the strain has high stress resistance. For example, it can grow in MRS liquid culture medium with a mass concentration of 10% NaCl, a mass concentration of 45% glucose, and a pH of 3.0, and can also survive in artificial gastric juice and artificial intestinal fluid.

[0062] The present application provides a bacterial preparation, which contains the above-mentioned Lactobacillus delbrueckii subsp. lactis.

[0063] The present application provides the use of the above-mentioned strain or the above-mentioned bacterial preparation in the field of fermentation, preferably in the field of food fermentation. Preferably, the food is a dairy product, a soy sauce product, a pear juice product or a vinegar product. Further preferably, the dairy product is yogurt or cheese.

[0064] For example, when the strain described above is used to ferment yogurt, the resulting yogurt has a rich aroma, a fruity flavor, a refreshing taste, and a stable after-acidity.

[0065] When the above-mentioned strain is used to ferment soy sauce, the total acid, total ester content and alcohol content are significantly increased, which promotes the flavor of the soy sauce and makes the aroma more intense.

[0066] When the strain is used to ferment pear juice, the obtained pear juice is more refreshing, the fermentation time can be shortened, and the pear juice contains a higher number of viable bacteria.

[0067] When vinegar is brewed using the strain described above, the pH and reducing sugar levels are lower than those of the control strain, indicating that the use of the strain described in this application can accelerate the fermentation process, and the resulting vinegar has higher total flavonoids, total polyphenols and DPPH clearance rates, and has better antioxidant properties, giving the vinegar higher quality and health benefits.

[0068] The present application provides the use of the above-mentioned Lactobacillus delbrueckii subspecies lactis or the above-mentioned bacterial preparation in biological preservation or in the preparation of anti-caries products, preferably in biological preservation or anti-caries to inhibit the proliferation of harmful bacteria, for example, it can inhibit the proliferation of harmful bacteria such as Salmonella, Listeria, and Streptococcus mutans, thereby achieving the functions of biological preservation and anti-caries.

[0069] The present application provides the use of the above-mentioned Lactobacillus delbrueckii subspecies lactis or the above-mentioned bacterial preparation in the preparation of animal feed or in the preparation of products for regulating intestinal flora, such as improving the nutritional value and absorption rate of fermented feed, inhibiting the proliferation of harmful bacteria in feed and the intestine, regulating the intestinal flora to compete with harmful bacteria for colonization in the intestine, thereby inhibiting harmful bacteria, lowering intestinal pH, producing metabolites such as short-chain fatty acids, inhibiting harmful bacteria, and thus regulating the intestinal flora.

[0070] The present application provides a genome of a biofilm-forming Lactobacillus delbrueckii subsp. lactis, wherein the genome comprises one or more genes selected from the sequences shown in SEQ ID NOs: 4-7.

[0071] In the present application, the nucleotide sequences of SEQ ID NOs: 4-7 are as shown above.

[0072] In some embodiments, the genome is tolerant to NaCl, sugars, and / or acids.

[0073] The present application provides a protein tolerant to NaCl, sugar or acid, which is encoded by one or more of the proteins shown in SEQ ID NOs: 4-7, or obtained by the aforementioned Lactobacillus delbrueckii subsp. lactis or the aforementioned bacterial agent.

[0074] In some embodiments, the amino acid sequence of the protein tolerant to NaCl, sugar or acid is one or more of the sequences described in SEQ ID NOs: 8-11.

[0075] The present application provides a food obtained by fermenting food materials with the aforementioned Lactobacillus delbrueckii subsp. lactis. In some embodiments, the food is a dairy product, a soy sauce product, a pear juice product, or a vinegar product.

[0076] In some embodiments, the fermented dairy product is obtained by fermenting raw cow's milk or other milk with the above-mentioned Lactobacillus delbrueckii subspecies lactis;

[0077] The soy sauce product is obtained by mixing soybean meal, flour and bran and then inoculating the mixture into the mixed strain of Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae, wherein the mixing ratio of the mixed strain of Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae is 1:0.5-1;

[0078] Wherein, the pear juice product is obtained by fermenting pear juice with the above-mentioned Lactobacillus delbrueckii subsp. lactis;

[0079] Wherein, the vinegar product is obtained by fermenting vinegar by mixing the above-mentioned Lactobacillus delbrueckii subspecies lactis and acetic acid bacteria;

[0080] In the present application, the mixing ratio of the mixed strain of Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae refers to the mass ratio of the two, which is a proportional relationship based on the percentage of the total mass of soybean meal, flour and bran. For example, when the total mass of soybean meal, flour and bran is 100g, the mass of Lactobacillus delbrueckii subspecies lactis can be 1g, and the mass of Aspergillus oryzae can be (0.5-1)g.

[0081] For example, the mixing ratio of the Lactobacillus delbrueckii subspecies lactis and the mixed strain of Aspergillus oryzae can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.

[0082] In some embodiments, the food is an anti-dental caries or dental caries prevention food or a food that regulates intestinal flora.

[0083] Example

[0084] The following describes the manufacturers of the raw materials and equipment used in this example, as well as the equipment and analysis methods used for product analysis. The chemical substances not otherwise specified are of the chemical purity level of conventional reagents.

[0085] Example 1 Screening of strains

[0086] (1) Strain enrichment: 5 g of homemade milk sample from herders in Dangxiong County, Tibet (4350 m above sea level) was crushed and evenly dispersed in 45 ml of sterile saline. 2 ml of the above dispersion was added to 20 ml of sterilized litmus milk culture medium and placed in a 37 °C incubator for static culture. The culture medium was removed when it acidified, solidified, and turned pink.

[0087] (2) Cultivate single colonies: Dilute the litmus milk culture medium to 10 -5times, aspirate 100 μl and spread on a BL solid medium plate, place in a 37°C incubator and culture until colonies form. On the BL plate, the colonies are smooth, slightly raised, moist, with rough edges, colorless to slightly white, 1-3 mm in diameter, and light yellow on the back.

[0088] (3) Screening of acid-producing bacteria: The single colonies obtained from the initial screening were streaked onto BL solid culture medium plates containing 0.2% CaCO3 by mass and cultured in a 37°C incubator for 36 h. After the culture was completed, the colonies were observed for the formation of transparent circles around them. Single colonies with a transparent circle diameter of 3-3.5 cm were selected for further separation and purification.

[0089] (4) Gram staining preliminary identification: Gram staining was performed on the strain obtained in step (3). If the bacteria stained purple, it was a Gram-positive bacterium and was retained for further study. If the bacteria stained red, it was a Gram-negative bacterium and was eliminated directly. The identification results showed that the obtained strain was a Gram-positive bacterium, non-motile, without spores, flagella, or capsule. The bacteria were in the shape of thin rods, either single rods or chains. This strain was the target strain. The microscopic morphological characteristics of the strain are shown in Figure 1.

[0090] (5) Molecular biological identification of 16S rDNA: 16S rDNA sequencing and bacterial species identification were performed on the strain to be identified using a high-fidelity enzyme. The primer information and PCR amplification reaction system and conditions used are shown in Tables 1 and 2.

[0091] Table 1. Primer information

[0092] Table 2. PCR amplification reaction system and conditions

[0093] After the PCR product was tested and qualified, it was sent to a sequencing company for sequencing, and the 16s rRNA sequence was obtained as shown in SEQ ID NO: 3. The sequencing results were compared by NCBI-BLAST, and the strain was identified as Lactobacillus delbrueckii and named Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ (abbreviated as DB-8). It was deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M 2023396.

[0094] Example 2 DB-8 Whole Genome Sequencing Analysis

[0095] The strain DB-8 obtained in Example 1 was entrusted to Shenzhen Huada Gene Co., Ltd. for whole genome sequencing and comparative genomics analysis. The results showed that the strain contained 4 unique genes related to biofilm formation, and the gene IDs were: DB-8 GL000445 (SEQ ID NO: 4), DB-8 GL001087 (SEQ ID NO: 5), DB-8 GL001801 (SEQ ID NO: 6), DB-8 GL001802 (SEQ ID NO: 7). The four related genes have ID numbers K20374, K00694, K12990, and K12990 in KEGG. These four unique genes enable the strain to have a high biofilm formation ability and improve the stress resistance of the strain. The amino acid sequences encoded by these four genes are shown in SEQ ID NO: 8-11, respectively. The amino acids encoded by these four genes are subjected to gel electrophoresis, and the results are shown in Figure 2.

[0096] Example 3 Determination of DB-8 biofilm formation ability

[0097] The DB-8 described in Example 1 and the control strain Lactobacillus delbrueckii lactis CICC 6047 (purchased from China Industrial Microorganism Culture Collection Management Center) after activation of MRS liquid medium were diluted to OD 0.05 with MRS medium at a concentration of 0%, 2%, 4%, 6%, 8%, and 10% respectively. 600 =0.15, then added to a 96-well plate at a volume of 200 μl per well and incubated at 37°C for 24 hours to form a biofilm, with three replicates per group. The biofilm assay was as follows: the liquid in each well was removed, the wells were washed three times with 200 μl of 0.85% saline, 200 μl of 99% methanol was added, and the cells were fixed by incubation for 15 minutes, air-dried at room temperature for 5 minutes, stained with 0.1% crystal violet for 20 minutes, washed three times with 200 μl of saline to remove excess crystal violet, 200 μl of 33% acetic acid was added to each well for 20 minutes to release the bound crystal violet, and the cells were measured using a microplate reader at OD 590 The absorbance was measured at 40 nm, and the results are shown in Figure 3.

[0098] As can be seen from Figure 3, the biofilm formation of DB-8 is much higher than that of the control bacteria, and at high NaCl concentrations, DB-8 has a higher biofilm formation, indicating that the formation of biofilm improves the stress resistance of DB-8.

[0099] Example 4 Determination of DB-8's tolerance to NaCl, sugar, and pH

[0100] (1) Determination of DB-8's tolerance to NaCl

[0101] The activated DB-8 was inoculated into MRS liquid culture medium with NaCl concentrations of 2%, 4%, 6%, 8%, 10%, and 12% respectively. The inoculation amount was 2% by volume. The cells were cultured at 42°C using a Bioscreen C automatic growth curve analyzer. The growth curve was determined by sampling every 30 minutes at a wavelength of 600 nm to measure the absorbance (OD 600 nm value), with MRS liquid culture medium as a control, and the measured data were used to draw the growth curve. The growth curves of DB-8 in each culture medium are shown in Figure 4A.

[0102] As shown in Figure 4A, the growth of DB-8 was gradually inhibited with increasing NaCl concentration, but it could still grow at 10% NaCl and slightly at 12%, indicating that DB-8 has strong salt tolerance.

[0103] (2) Determination of DB-8 glucose tolerance

[0104] The activated DB-8 cells were inoculated into MRS liquid culture medium with glucose concentrations of 25%, 30%, 35%, 40%, 45%, and 50%, respectively, and cultured at 42°C using a Bioscreen C automatic growth curve analyzer. The absorbance (OD) was measured every 30 min at a wavelength of 600 nm. 600 nm value), with MRS liquid culture medium as a control, and the measured data were used to draw the growth curve. The growth curves of DB-8 in each culture medium are shown in Figure 4B.

[0105] As can be seen from Figure 4B, with the increase of glucose concentration, the growth of the strain was gradually inhibited. DB-8 could grow at a glucose concentration of 45%, and the growth of the strain was significantly inhibited when the glucose concentration was 50%, indicating that DB-8 has a strong ability to tolerate high sugar.

[0106] (3) Determination of DB-8 pH tolerance

[0107] MRS liquid medium was adjusted to different pH values ​​(2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) using hydrochloric acid. Activated DB-8 cells were inoculated into the above medium and cultured at 42°C using a Bioscreen C automated growth curve analyzer (Oy Growth Curves Ab Ltd, Finland). Samples were taken every 30 minutes at a wavelength of 600 nm to measure absorbance (OD600nm). The measured data were used to plot the growth curve, as shown in Figure 4C.

[0108] As shown in FIG4C , DB-8 can grow in a medium with a pH of 3.0, indicating that DB-8 has a strong acid resistance.

[0109] Example 5 Resistance of DB-8 to Artificial Digestive Fluid

[0110] (1) Preparation of artificial digestive fluid

[0111] Artificial gastric juice: NaCl 0.20 g / 100 mL, pepsin 0.35 g / mL, adjust the pH to 2.5 with 1 mol / L HCl, filter and sterilize for later use.

[0112] Artificial intestinal fluid: Mix the following solutions A and B in a ratio of 2:1 to prepare artificial intestinal fluid. A. Pancreatic fluid: Sodium bicarbonate 1.1g / 100mL, NaCl 0.2g / 100mL, trypsin 0.1g / 100mL, adjust pH to 8.0, filter sterilize and set aside. B. Bile fluid: Bile salts 1.8g / 100mL, adjust pH to 6.8, filter sterilize and set aside.

[0113] (2) Artificial digestive fluid tolerance test

[0114] After single colonies of DB-8 were cultured overnight in MRS liquid medium, 1 mL of the culture medium was inoculated into 9 mL of artificial gastric fluid or artificial intestinal fluid and incubated at 37°C for 2 hours. A CICC 6047 control strain inoculated into 9 mL of MRS liquid medium was used as a positive control. The viable cell counts were determined, and the survival rate was calculated using the following formula. The results are shown in Table 3.

[0115] Survival rate (%) = (number of viable bacteria in artificial digestive fluid / number of viable bacteria in control) * 100%

[0116] Table 3 Survival rate of DB-8 after treatment with artificial gastric juice and artificial intestinal juice

[0117] As can be seen from Table 3, the survival rate of DB-8 bacteria in artificial gastric juice and artificial intestinal juice is high, indicating that it has high tolerance.

[0118] Example 6 RT-PCR determination of the expression of DB-8 biofilm formation genes under stress environment

[0119] The activated DB-8 were inoculated into MRS liquid medium containing 8% NaCl, 40% glucose, and 3.5 pH, and the group inoculated into MRS medium was used as the control. After culturing for 24 h, the cells were inoculated into MRS liquid medium according to the manufacturer's protocol. Max TM RNA was extracted using the Bacterial RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). TMReverse transcription and cDNA synthesis were performed using the RT reagent kit with gDNA Eraser (Perfect Real Time) (TAKARA). RT-PCR was performed using the SYBR Green PCR Master Mix in the PikoReal 96 system (Thermo Fisher Scientific). The reaction mixture contained SYBR Green premix, primers, cDNA, and RNase-free water. Additionally, 20 μL of the mixture was amplified as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 45 seconds. The reaction was analyzed using melting curve analysis to confirm the reaction. Gene expression was calculated using 16S rDNA as an internal reference gene and 2-ΔΔCt. The results are shown in Figure 5 , and the primers used are shown in Table 4 .

[0120] Table 4. RT-PCR primer information

[0121] Figure 5 shows that the expression of the four unique genes in DB-8 was upregulated under high salt, high sugar, and low pH stresses (Fold change > 1). Combined with the enhanced biofilm formation of DB-8 under high stress conditions, this suggests that the upregulated expression of these four unique genes is responsible for the enhanced biofilm formation in DB-8 and contributes to its strong stress resistance.

[0122] Example 7 Preparation of DB-8 bacterial powder

[0123] (1) Activation of DB-8 strain: Take the DB-8 strain stored in a frozen glycerol tube at -80℃ and place it in MRS liquid medium, culture it at 37℃ for 12 hours, and transfer it twice continuously to complete the strain activation;

[0124] (2) Expansion of DB-8 strain: inoculate 2% of the activated strain into 70 ml of MRS liquid medium and culture at 37°C for 12 h;

[0125] (3) Cultivation of DB-8 strain: inoculate 2% of the culture strain into 3500 ml of MRS liquid medium and culture at 37°C for 12 h;

[0126] (4) Isolation of DB-8 bacteria: Centrifuge the culture solution at 5000 rpm for 10 min, discard the supernatant, and retain the precipitate;

[0127] (5) Freeze-dried liquid: 10 g skim milk powder, 5 g maltodextrin, 5 g trehalose, 3 g glycerol, 12 g bacterial precipitate, 65 g pure water, mix well to prepare bacterial freeze-dried liquid, transfer to freeze-dried bottle, and pre-freeze at -40°C for 2 h.

[0128] (6) Freeze-drying of DB-8 bacterial powder: Freeze-drying was performed using a Labconco vacuum freeze dryer. The freeze-drying procedure is shown in Table 5.

[0129] Table 5

[0130] Example 8 DB-8 fermented yogurt

[0131] Fresh cow's milk was mixed with 6.5% edible sucrose by weight, preheated, homogenized, and sterilized. After cooling to 40-50°C, the mixture was added to a fermentation tank. The DB-8 bacterial powder prepared in Example 7, along with conventional commercially available yogurt starter 1 (purchased from the Chinese Tmall website www.tianmao.com, under the trade name Chuanxiu Yogurt Fermentation Bacteria 30-Bacteria Starter) and commercially available yogurt starter 2 (purchased from the Chinese Tmall website www.tianmao.com, under the trade name Eubit 50-Bacteria Yogurt Fermentation Bacteria Powder) were inoculated at 50 mg / L and fermented at 37°C for 12 hours. After fermentation, the milk was refrigerated at 4°C for 24 hours to produce yogurt. Yogurt sensory evaluation was conducted according to the "China Dairy Industry Standard RHB 103-2004 Yogurt Sensory Quality Evaluation Guidelines." Seven people were invited to taste and score yogurt samples fermented with different strains, and the average of the results was calculated. The sensory evaluation results are shown in Table 6.

[0132] Table 6 Sensory scores of yogurt fermented with different starter cultures

[0133] As shown in the experimental results in Table 6, the yogurt fermented with the DB-8 strain provided in this application had the highest comprehensive score, especially in terms of flavor, the DB-8 strain described in this application scored much higher than the control strain.

[0134] A sensory evaluation was conducted on the yogurt produced by fermentation with the DB-8 strain by a panel of seven people, and the feedback was that the yogurt had a natural fruity flavor and was refreshing and delicious. The yogurt fermented with the DB-8 strain described in this application had the highest overall score, especially in terms of flavor, the DB-8 strain described in this application scored much higher than the control strains (commercially available yogurt starter 1 and commercially available yogurt fermentation 2).

[0135] Example 9: Brewing soy sauce with mixed bacteria of DB-8 and Aspergillus oryzae

[0136] Adopt soybean dregs, flour, wheat bran to mix back inoculation aspergillus oryzae and embodiment 7 to prepare DB-8 bacterium powder koji making, take by weighing the soybean dregs of 1kg, add 1.2kg water to moisten and soak two hours, in 115 ℃ of high pressure steam sterilization 20min, take by weighing 0.36kg flour and 0.07kg wheat bran, aspergillus oryzae (purchased from China Taobao website www.taobao.com, commodity are called bright brand soy sauce koji essence) and DB-8 bacterium powder by 0.5% and 0.5‰ inoculation of three kinds of raw materials (soybean dregs, flour and wheat bran) gross mass respectively, pack into koji frame in incubator after mixing, maintaining the temperature is 35 ℃ of koji making, turn over respectively at the 12th hour and the 17th hour, control moisture, finish koji making at the 36th hour. 120 g of koji was weighed, twice the mass of 10 wt% saline was added to a glass bottle, shaken evenly, and placed in a constant temperature incubator at 30°C for fermentation for four months. After the fermentation, the amino acid nitrogen and total acid were determined by the method of national standard GB / T18186-2000, the reducing sugar was determined by the method of GB / T5009.7-2008, the ethanol content was determined by dichromic acid colorimetry, and the total ester content was determined by continuous potentiometric titration. The results are shown in Table 7.

[0137] Table 7. Comparison of physical and chemical indicators of mixed-bacteria brewed soy sauce

[0138] As shown in Table 7, the total acid, total ester content and alcohol content were significantly increased after adding DB-8 bacterial powder, indicating that the organic acids, alcohols and lipids were significantly increased after adding DB-8 bacterial powder, which promoted the flavor of soy sauce and made the aroma more intense.

[0139] Example 10: Fermentation of pear juice with DB-8 bacterial powder

[0140] The purchased fresh crystal pears were soaked in salt water, washed, and juiced under sterile conditions. Borneol was added to adjust the sugar content of the pear juice to 35% and then divided into sterile conical flasks and sealed. After pasteurization, DB-8 bacterial powder was inoculated into the pear juice at a mass percentage concentration of 0.5‰. CICC6047 bacteria was used as a control. The juice was fermented at 30°C for 144 hours. After the fermentation, the total acid was determined by the acid-base indicator titration method according to GB 12456-2021, the viable cell count was determined by the dilution spread plate method, the pH value was determined by a pH meter, and the reducing sugar was determined by the direct titration method according to GB5009.7-2016. The results are shown in Table 8.

[0141] Table 8. Physical and chemical indicators of fermented pear juice

[0142] Table 8 shows that after 144 hours of fermentation, pear juice fermented with DB-8 bacteria powder had lower pH and reducing sugar levels, and higher total acidity. These factors contribute to a more refreshing taste and shorten the fermentation time when the same indicators are used as the endpoint. Furthermore, pear juice fermented with DB-8 bacteria powder had a higher viable bacterial count, demonstrating the excellent fermentation properties of DB-8.

[0143] Example 11: Fermentation of Vinegar by Mixing DB-8 with Acetic Acid Bacteria

[0144] Vinegar is brewed according to the following process:

[0145] After the rice was ground and sieved, twice the mass of water was added and heated for gelatinization. α-amylase (enzyme activity of 4000 U / g, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was then added for liquefaction. After cooling, saccharifying enzyme (enzyme activity of 100000 U / g, purchased from Angel Yeast Co., Ltd.) was added for saccharification. After bottling, 8‰ of the weight of the saccharified liquid was added to the commonly used brewer's yeast powder in the art (Angel brewing high-activity dry yeast, purchased from Angel's official mall https: / / shop.angelyeast.com / goods / 888) and the DB-8 powder prepared in Example 7. After 7 days of alcohol fermentation, the fermentation liquid was diluted to an alcohol content of 4.5, and acetic acid bacteria liquid (acetic acid bacteria purchased from www.taobao.com, with the trade name of Guangming brand vinegar brewing acetic acid bacteria) with a volume of 2% of the fermentation liquid was inoculated. No DB-8 was added as a control and fermented for 7 days. After the fermentation was completed, the total acid, The reducing sugar method was used to determine total acid and reducing sugar, and the total flavonoids, total polyphenols and DPPH free radical scavenging rate in vinegar were determined as follows:

[0146] Total polyphenols were determined using the Folin-phenol method: 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of a gallic acid standard solution (1000 mg / mL) were accurately pipetted into 10 mL colorimetric tubes. 5 mL of Folin-phenol reagent was added and mixed thoroughly. The mixture was allowed to stand for 3 minutes. Then, 15% Na₂CO₃ solution was added, the volume was made up to 10 mL with distilled water, and the mixture was shaken and allowed to stand for 1 hour. The absorbance was measured at a wavelength of 760 nm. A standard curve was constructed with the gallic acid content as the horizontal axis and the absorbance as the vertical axis. A 0.5 mL sample of the centrifuged supernatant and fermentation broth was taken and the absorbance was measured at a wavelength of 760 nm according to the standard curve procedure. Total polyphenol content is expressed as gallic acid equivalents (GRE / g).

[0147] The method for determining total flavonoids is as follows: accurately pipette 0.0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of a rutin standard solution (200 μg / mL) into a 10 mL colorimetric tube, dilute to 5 mL with 30% ethanol solution, then add 0.3 mL of a 5% NaNO2 solution, shake well and let it stand for 5 minutes, then add 0.3 mL of a 10% Al(NO3)3 solution, shake well and let it stand for 6 minutes, then add 2 mL of 1 mol / L NaOH, then make the volume up to 10 mL with 30% ethanol solution, shake well, let it stand for 15 minutes, and measure absorbance at a wavelength of 510 nm. Use the zero tube as a blank, and plot a standard curve with rutin content (μg) as the horizontal axis and absorbance as the vertical axis. Take 1 mL of the centrifuged supernatant fermentation broth and measure absorbance at a wavelength of 510 nm according to the standard curve operation steps. The blank control is a solution and water mixture without sample added. The total flavonoid content is expressed as rutin equivalent RE / g.

[0148] The DPPH free radical scavenging ability was determined by mixing 2.5 mL of the centrifuged supernatant fermentation broth with 2.5 mL of 0.2 mmol / L DPPH ethanol solution, keeping the mixture in the dark for 30 min, and measuring the absorbance A at a wavelength of 517 nm. i 2.5mL ethanol plus 2.5mL DPPH was used as the control, and 2.5mL ethanol plus 2.5mL sample was used as the blank. The absorbance A was measured at 517nm. j and A k .

[0149] Free radical scavenging rate (%) = [1-(A i -A k ) / A j ]×100%

[0150] The results are shown in Table 9.

[0151] Table 9. Determination of various physical and chemical indicators in fermented vinegar

[0152] Table 9 shows that vinegar fermented with DB-8 exhibits lower pH and reducing sugar levels, indicating that adding DB-8 accelerates the fermentation process. Furthermore, vinegar fermented with DB-8 exhibits higher total flavonoids, total polyphenols, and DPPH scavenging rates, demonstrating improved antioxidant properties, resulting in higher quality and health benefits.

[0153] Table 10 Sequence Listing

[0154] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any form. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A Lactobacillus delbrueckii subsp. lactis, wherein The strain is Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC No.: M2023396.

2. The Lactobacillus delbrueckii subspecies lactis according to claim 1, wherein The 16s rRNA sequence of the strain comprises the sequence shown in SEQ ID NO: 3 or consists of the sequence shown in SEQ ID NO:

3.

3. The Lactobacillus delbrueckii subspecies lactis according to claim 1 or 2, wherein The Lactobacillus delbrueckii subsp. lactis carries a gene expressing a biofilm.

4. The Lactobacillus delbrueckii subspecies lactis according to claim 3, wherein The gene expressing the biofilm comprises the sequence shown in SEQ ID NOs: 4-7 or consists of the sequence shown in SEQ ID NOs: 4-7.

5. A bacterial preparation comprising the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4.

6. Use of the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4 or the bacterial preparation according to claim 5 in the field of fermentation, preferably in the field of food fermentation.

7. The use according to claim 6, wherein: The food is a dairy product, a soy sauce product, a pear juice product or a vinegar product. More preferably, the dairy product is yogurt or cheese.

8. Use of the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4 or the bacterial preparation according to claim 5 in biological preservation or in the preparation of anti-dental caries products.

9. Use of the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4 or the bacterial preparation according to claim 5 in preparing animal feed or in preparing a product for regulating intestinal flora.

10. A genome of a biofilm forming Lactobacillus delbrueckii subsp. lactis, wherein: The genome includes one or more genes selected from the sequences shown in SEQ ID NOs: 4-7.

11. The genome of claim 10, which is tolerant to NaCl, sugars and / or acids.

12. A protein tolerant to NaCl, sugar or acid, which is encoded by one or more of the proteins shown in SEQ ID NOs: 4-7, or obtained by the Lactobacillus delbrueckii subsp. lactis described in any one of claims 1 to 4 or the bacterial agent described in claim 5.

13. A food obtained by fermenting a food material with the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4.

14. The food according to claim 13, wherein The food is a dairy product, a soy sauce product, a pear juice product or a vinegar product.

15. The food according to claim 14, wherein the soy sauce product is obtained by mixing soybean meal, flour and bran and then inoculating the mixed strain of Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae according to any one of claims 1 to 4 and fermenting, wherein: The mixing ratio of the Lactobacillus delbrueckii subspecies lactis and Aspergillus oryzae is 1:0.5-1; and / or The dairy product is obtained by fermenting raw cow milk by the Lactobacillus delbrueckii subspecies lactis according to any one of claims 1 to 4; and / or The pear juice product is obtained by fermenting pear juice with the Lactobacillus delbrueckii subsp. lactis according to any one of claims 1 to 4; and / or The soy sauce product is obtained by fermenting vinegar with the Lactobacillus delbrueckii subspecies lactis described in any one of claims 1 to 4 and acetic acid bacteria.

16. The food according to any one of claims 13 to 15, which is an anti-caries or caries-preventing food or a food for regulating intestinal flora.

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