Lactobacillus delbrueckii subsp. Bulgaricus containing preservation factor S-layer protein as well as preparation and application of lactobacillus delbrueckii subsp. Bulgaricus

Abstract

The invention provides lactobacillus delbrueckii subsp. Bulgaricus containing preservation factor S layer protein and preparation and application of the lactobacillus delbrueckii subsp. Bulgaricus. The strain is lactobacillus delbrueckii subsp. Bulgaricus inm3201, the preservation number is GDMCC NO: 66019, the preservation date is March 17, 2025, the preservation unit is Guangdong Microbial Culture Collection Center, and the preservation address is No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou. The lactobacillus delbrueckii subsp. Bulgaricus inm3201 has the advantages that not only is the preservative property of raw milk improved, but also the characteristics of aroma and taste of yoghurt and fermented milk products are effectively improved in cooperation with fermentation strains.

Description

Technical Field

[0001] The present invention relates to a strain of Lactobacillus delbrueckii subsp. bulgaricus containing a preservation factor S-layer protein, and its preparation and application. Background Art

[0002] Microbial contamination of milk is a type of abnormal milk. Mainly due to contamination before and after milking, untimely cooling, incomplete washing and sterilization of utensils, etc., fresh milk will be contaminated by a large number of microorganisms. Fresh milk is prone to peptization and alkalization by Bacillus, and abnormal flavors such as spoilage will occur; peptization and viscosity may also occur under low-temperature conditions; decomposition of fat will result in lipolytic flavor, bitterness and non-acid coagulation; moreover, Bacillus has extremely strong heat resistance and cannot be completely killed at high temperatures.

[0003] Currently, the main methods for preventing microbial contamination are as follows: (1) Udder disinfection; (2) Disinfection of milking equipment and milk buckets; (3) Hygiene requirements for milking personnel; (4) Requirements for milking environment; (5) Discarding the first few streams of milk; (6) Filtering fresh milk; (7) Cooling fresh milk; (8) The storage temperature of fresh milk should be low; (8) Dairy farmers should establish a perfect disinfection registration system and records for equipment, environment and staff.

[0004] However, these methods have high requirements for the comprehensive quality of personnel and cumbersome process operations in the actual application process. Moreover, as long as there is a slight oversight, it will cause huge economic losses and affect the brand power and credibility of the enterprise. Therefore, using biological methods to prevent raw milk from being contaminated by microorganisms can not only effectively inhibit spoilage and pathogenic microorganism contamination, but also is an innovative invention for raw milk and its dairy products.

[0005] S-layer protein refers to a special surface structure of certain bacteria. The molecular weight is about 40-200KD. It is a bioactive macromolecule coated on the cell walls of many bacteria and archaea, and the presence of S-layer protein has also been found in some lactic acid bacteria. At present, the physiological functions of S-layer protein are not well understood, and no general functions of S-layer protein have been found. The reported functions of S-layer protein are: maintaining the morphological structure of cells, protecting cells against the influence of adverse environments, adsorbing small molecules and ions, attachment sites for extracellular enzymes, attachment or surface recognition sites, etc. At present, the functions of Lactobacillus S-layer protein mainly focus on attachment, inhibition of pathogenic bacteria and immunomodulation, etc. Therefore, it also provides a theoretical basis for the present invention.

[0006] At present, there is little research on the inhibition of pathogenic bacteria by Lactobacillus S-layer protein. It is generally believed that S-layer protein is one of the adhesins of lactic acid bacteria. It exists in some Lactobacillus species, such as Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus curvatus, Lactobacillus rhamnosus, Lactobacillus amylophilus, Lactobacillus gallinarum.

[0007] Patent: An antibacterial agent against Staphylococcus aureus, its preparation method and application 2018105377163 The present invention also discloses a preparation method and application of an antibacterial agent against Staphylococcus aureus. The S-layer protein of Lactobacillus casei and nisin in the invention have a synergistic inhibitory effect on Staphylococcus aureus. The antibacterial effect of this antibacterial agent far exceeds the effect of using the S-layer protein of Lactobacillus casei or nisin alone. The inhibition rate of Staphylococcus aureus by the antibacterial agent of the present invention can be as high as 87.19%. However, this invention does not involve the inhibition of Bacillus and its application effect in raw milk and dairy products.

[0008] Patent: An antibacterial agent for meat preservation 2017100423654 The present invention discloses an antibacterial agent for meat preservation. The antibacterial agent for meat preservation in the invention can utilize the synergistic effect of the S-layer protein of Lactobacillus crispatus and nisin to enhance the inhibitory effect on Staphylococcus saprophyticus, a common spoilage bacterium in meat, for meat preservation and extend the shelf life. However, this invention does not involve the inhibition of Bacillus and its application effect in raw milk and dairy products.

[0009] Currently, there is no related technology involving the application of lactic acid bacteria S-layer protein in raw milk and dairy products, and it is increasingly resisted by consumers in the ingredient list presented in the form of adding preservatives in raw milk or dairy products. Thus, it can be seen that developing a probiotic to improve the preservation of raw milk and dairy products has a broad application prospect. Summary of the Invention

[0010] In view of the above problems, the present invention provides a Lactobacillus delbrueckii subsp. bulgaricus containing a preservation factor S-layer protein, its preparation and application.

[0011] The invention purpose is achieved through the following scheme: A Lactobacillus delbrueckii subsp. bulgaricus containing a preservation factor S-layer protein, This strain is Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) inm3201, with the preservation number GDMCC NO: 66019, the preservation date: March 17, 2025, and the preservation unit: Guangdong Provincial Microbial Culture Collection Center, with the preservation address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0012] Furthermore, the colony characteristics of Lactobacillus delbrueckii subsp. bulgaricus inm3201 are as follows: Cultured on solid MRS plate medium for 48 h, it is a straight bacillus with rounded ends, usually a long rod, the cell body is 2 μm - 9 μm in length and 0.5 μm - 0.8 μm in width, Gram-positive, non-spore-forming; facultative anaerobic, the surface colony diameter is 1 - 2 mm, convex, round, with a smooth, fine surface and white color.

[0013] A preparation method of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein is as follows: Screen and isolate lactic acid bacteria from dairy product samples. Process the dairy products into a pulp, add sterile water, dilute, evenly coat dairy products with different dilution concentrations on solid MRS medium, place them in an incubator for cultivation, select white colonies on the solid MRS medium, and screen lactic acid bacteria that meet the morphology; Lactic acid bacteria screening: Cultivate the obtained lactic acid bacteria until the logarithmic growth phase, then inoculate them at an inoculation amount of 1%, centrifuge to remove the supernatant, wash with sterile physiological saline, inoculate into LB medium, and at the same time inoculate Bacillus licheniformis, and observe the antibacterial results.

[0014] Furthermore, MRS medium: peptone, beef extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, ammonium citrate hydrogen, sodium acetate, magnesium sulfate, manganese sulfate, Tween 80, dissolved in distilled water; solid MRS adds agar on the basis of the liquid.

[0015] A probiotic preservation preparation prepared from Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein.

[0016] An anti-corrosion application of a strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein.

[0017] Advantages of the present invention: (1) For the first time, it is found that the S-layer protein in Lactobacillus delbrueckii subsp. bulgaricus inm3201 can effectively improve the anti-corrosion and preservation function of raw milk.

[0018] (2) The Lactobacillus delbrueckii subsp. bulgaricus inm3201 described in the present invention not only improves the anti-corrosion of raw milk, but also cooperates with fermentation strains to effectively improve the product aroma and taste characteristics of yogurt and fermented milk.

[0019] (3) The screening method of Lactobacillus delbrueckii subsp. bulgaricus inm3201 of the present invention has universality and can be widely applied to the screening of anti-corrosion bacteria for raw milk and dairy products.

[0020] (4) The Lactobacillus delbrueckii subsp. bulgaricus inm3201 provided by the present invention can improve the preservation of raw milk, fermented milk and yogurt, and there will be no peculiar smell in the raw milk and dairy products added with Lactobacillus delbrueckii subsp. bulgaricus inm3201. Later, it can be mixed with other food raw materials, which can not only improve the nutrition and flavor of fermented milk, but also have no safety risk. Description of the Drawings

[0021] Figure 1 It is the antibacterial rate effect diagram of 9 strains of bacteria.

[0022] Figure 2 It is the physical diagram of antibacterial verification of 4 strains of bacteria.

[0023] Figure 3 The result diagram of the S-layer protein extracted from strain AB0101 on SDS-PAGE.

[0024] Figure 4 The microscopic observation diagram of strain AB0101 after Gram staining. Specific implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments: Unless otherwise specified, the experimental methods used in the following implementation examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0026] Example 1, referring to the appendix Figures 1-4 A strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein, This strain is Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) inm3201, with the preservation number of GDMCC NO: 66019, the preservation date is: March 17, 2025, and the preservation unit is: Guangdong Microbial Culture Collection Center, and the preservation address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0027] A strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein, the colony characteristics of Lactobacillus delbrueckii subsp. bulgaricus inm3201 are as follows: cultured on solid MRS plate medium for 48 h, round-ended straight bacilli, usually long rods, with the cell body length of 2 μm - 9 μm and the width of 0.5 μm - 0.8 μm, Gram-positive, non-spore-forming; facultative anaerobic, the surface colony diameter is 1 - 2 mm, convex, round, smooth, fine, and white in color.

[0028] The preparation method of a strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein is as follows: Isolate and screen lactic acid bacteria from dairy product samples, process the dairy products into a paste, add sterile water, and dilute the concentration to 10 5 -10 6 , evenly coat dairy products with different dilution concentrations on solid MRS medium, place them in an incubator at 37 °C for 48 h, and then select white colonies on the solid MRS medium, and screen the lactic acid bacteria that meet the morphology; Lactic acid bacteria screening: Cultivate the obtained lactic acid bacteria to the logarithmic growth phase, and then inoculate them at an inoculation amount of 1%, with the final concentration of the strain being 1×10 6 cfu / mL, centrifuge to remove the supernatant, wash twice with sterile physiological saline, inoculate into LB medium, and at the same time inoculate 1×10 4Bacillus licheniformis at cfu / mL, placed in static culture at 37°C for 2 days, and the antibacterial results were observed.

[0029] A method for preparing Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein. Solid MRS medium: Weigh 10 g of peptone, 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of ammonium citrate hydrogen, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 1 g of Tween 80, dissolve in 1000 mL of distilled water, sterilize at 0.1 MPa and 121°C for 15 min; Solid MRS adds 2% agar on the basis of the liquid; The LB medium formula is: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, sterilize at 121°C for 15 min; Solid LB adds 2% agar on the basis of the liquid.

[0030] A method for preparing Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein 1. Treatment of raw materials Take dairy raw materials, add sterile normal saline and dilute to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Concentrations to make a bacterial cell dilution solution. Take 0.1 mL from each concentration dilution solution and evenly coat it on a solid MRS medium petri dish, culture at 37°C for 48 h, select single colonies with white colonies, convex colonies, smooth surfaces, and neat colony edges, streak the colonies on the solid MRS medium for secondary purification, culture at 37°C for 48 h, pick single colonies for glycerol preservation, and name them successively AB0101~AB0109.

[0031] 2. Primary screening of strains Inoculate these 9 strains at an inoculation amount of 1% into MRS liquid medium and culture at 37°C for 24 h. Then inoculate at an inoculation amount of 1% into MRS liquid medium and statically culture at 37°C for 12 h until the final cell density reaches 1×10 6 cfu / mL. Then centrifuge at 8000 g for 10 min, remove the supernatant, add an equal volume of sterile normal saline, mix evenly, and repeat 2 times to obtain a bacterial suspension which is the sample for physical and chemical index detection. The results are as Figure 1 shown.

[0032] Antibacterial detection method: The well plate method is used for detection. The total system in each well is 2 mL, and the final concentration of Bacillus licheniformis is 1×10 4cfu / mL. The amount of the added sample is 80 μL, with 3 parallels for each sample. It is placed at 37 °C and incubated for 24 h. Then it is put into an ELISA reader to detect the absorbance, and the detection wavelength is OD 600 , and an antibacterial rate greater than 50% is considered effective antibacterial. The results are as Figure 1 .

[0033] Antibacterial rate (%) = (OD of the control group 600 - OD of the sample group 600 ) / OD of the control group 600 3. Re-screening of strains The 4 strains of lactic acid bacteria AB0101, AB0104, AB0107, and AB0108 initially screened out are reactivated and cultured, and the S-layer protein is extracted.

[0034] Extraction of S-layer protein: Collect 100 mL of overnight cultured bacteria, wash them three times with distilled water, and then suspend them in 5 mL of a 5M lithium chloride solution. Incubate at 37 °C for 2 h; centrifuge at 10,000 g for 5 min, discard the bacteria, and collect the supernatant; dialyze the supernatant with distilled water at 4 °C, and continuously change the distilled water during this period; centrifuge the dialyzed suspension at 10,000 g for 20 min, collect the precipitate and freeze-dry it; dissolve the freeze-dried S-layer protein solution in the denaturing buffer, and detect the extracted S-layer protein by polyacrylamide gel electrophoresis.

[0035] Detection of protein concentration: BCA protein concentration assay kit Detection of antibacterial activity: Refer to Example 1.

[0036] Table 1 Inhibition of Bacillus licheniformis by S-layer protein Strain name AB0101 AB0104 AB0107 AB0108 Protein concentration (mg / mL) 5.32±0.21 4.67±0.18 5.34±0.17 4.35±0.24 As can be seen from the results in Table 1, all 4 strains contain S-layer protein. The S-layer protein is diluted to 100 μg / mL for antibacterial verification, and the control group is without S-layer protein. The results are shown in Figure 2 .

[0037] From Figure 2 it is shown that the inhibition rate of AB0101 is the best. Figure 3 This is the result of the detection of the S-layer protein extracted from strain AB0101 by polyacrylamide gel electrophoresis. The molecular weights of the S-layer proteins of AB0101 are 53 KDa and 95 - 130 KDa, and two relatively prominent bands. While the molecular weights of the S-layer proteins of lactic acid bacteria are 40 - 200 KDa. Thus, it can be known that the S-layer protein of AB0101 is composed of two S-layer proteins.

[0038] As a target strain for secondary screening, the morphological observation of this strain on solid MRS plate medium showed that the colony diameter was 1-2 mm, the colony was milky white, convex and round in shape, with a smooth surface and neat edges. After Gram staining and observation under a microscope, the results were as Figure 4 shown.

[0039] The AB0101 strain obtained by the above screening was identified by 16S rDNA sequencing. Through comparative analysis, the similarity between the 16S region sequence of the strain of the present invention and Lactobacillus delbrueckii subsp. bulgaricus reached 99.67%. It was determined that this strain was a Lactobacillus delbrueckii subsp. bulgaricus, which could be applied to the food fermentation industry and was named Lactobacillus delbrueckii subsp. bulgaricus inm3201. This strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 17, 2025, with the deposit number: GDMCC NO: 66019. Therefore, inm3201 and AB0101 refer to the same strain in this application. The gene sequence of the AB0101 strain of the present invention is as follows: Example 2: Detection of genetic stability of Lactobacillus delbrueckii subsp. bulgaricus inm3201 Lactobacillus delbrueckii subsp. bulgaricus inm3201 was continuously passaged 10 times on MRS liquid medium. S-layer proteins were extracted from the samples of the 1st, 5th, and 10th generations respectively, and inoculated into LB medium according to the treatment method of Example 1. The method for detecting the antibacterial rate referred to Example 1 to judge its passage stability. The specific data are shown in Table 2.

[0040] Table 2 Detection results of passage stability test

[0041] As can be seen from the results in Table 2, the antibacterial ability of Lactobacillus delbrueckii subsp. bulgaricus inm3201 at different generations was normal, and the deviation content of the antibacterial rate was within 10%. The genetic stability of the strain was good, meeting the requirements for production use.

[0042] Example 3: Application of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus inm3201 in raw milk preservation Since the extraction rate of S-layer protein alone is low and it is not conducive to industrial application, in this example, the bacterial cell method was used. When comparing the inhibition of Bacillus licheniformis, the final concentrations of the bacterial cells were 1×10 4 -10 8 CFU / mL, and AB0109 was used as a control strain. It was cultured at 37°C for 24 h to detect the preservation effect in raw milk.

[0043] Bacterial cell treatment process: Lactobacillus delbrueckii subsp. bulgaricus inm3201 was inoculated at an inoculation amount of 1% and cultured in MRS liquid medium for 24 h, centrifuged at 8000 g for 10 min, washed twice with sterile physiological saline, and then suspended with an equal volume of sterile physiological saline and left standing at room temperature for 12 h; centrifuged at 8000 g for 10 min and freeze-dried into bacterial cell powder.

[0044] Detection of antibacterial effect: Bacillus licheniformis was inoculated into raw milk with a final concentration of 1×10 4 cfu / mL. The plate counting method was used, with 10-fold serial dilution. 100 μL of the diluted raw milk dilutions at different dilution degrees was applied to LB solid plates and cultured at 37°C for 24 h. The total number of Bacillus licheniformis colonies was calculated. The results are shown in Table 3.

[0045] Antibacterial rate (%) = (Total number of colonies in the control group - Total number of colonies in the sample group) / Total number of colonies in the control group Table 3 Antibacterial rates of different concentrations of Lactobacillus delbrueckii subsp. bulgaricus inm3201 in raw milk

[0046] Note: The full score for each assessment is 10 points, with a scoring interval of 0.1. The comprehensive score is the sum of all items. As can be seen from the results in Table 3, adding Lactobacillus delbrueckii subsp. bulgaricus inm3201 to raw milk can significantly increase the anti-corrosion effect of raw milk. Moreover, the higher the inoculation amount, the stronger the antibacterial activity in raw milk. However, too low an addition amount cannot effectively prevent the spoilage of raw milk, and the raw milk will become sour, seriously affecting the taste. Too high an addition amount does not significantly change the anti-corrosion effect, especially in terms of total acid. Considering from the perspective of application cost, the most suitable final inoculation concentration is 1.0×10 7 CFU / mL. This concentration can not only effectively control microorganisms but also stabilize the flavor and taste of raw milk.

[0047] Example 4 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on the preservation of yogurt According to the process of Example 3, Lactobacillus delbrueckii subsp. bulgaricus inm3201 was made into a direct vat inoculum (self-made inoculum by Yiming Company), and the strain concentration was 1.0×10 11 CFU / mL.

[0048] Lactobacillus delbrueckii subsp. bulgaricus inm25LB, the preservation number of the strain is CGMCC No. 15445, the preservation date is: March 12, 2018, and the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Streptococcus salivarius subsp. thermophilus inm25-ST, the preservation number is CGMCC NO. 15446, the preservation date is: March 12, 2018, and the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; The above 2 strains have been disclosed in the application number 201910692054.1.

[0049] Sample group: A direct vat inoculum of yogurt, a mixed inoculum of Streptococcus salivarius subsp. thermophilus inm25-ST (self-made inoculum by Yiming Company) and Lactobacillus delbrueckii subsp. bulgaricus inm3201 (self-made inoculum by Yiming Company); the strain concentration is 1.0×10 11 CFU / mL; Control group: A direct vat inoculum of yogurt, a mixed inoculum of Streptococcus salivarius subsp. thermophilus inm25-ST and Lactobacillus delbrueckii subsp. bulgaricus inm25LB (self-made inoculum by Yiming Company), and the strain concentration is 1.0×10 11 CFU / mL The production standard of yogurt refers to the national standard GB 19302-2025, and the results are recorded in Table 4.

[0050] Reference Example 3 for detecting bacteriostatic effect Table 4 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on preservation in yogurt

[0051] Note: The prepared yogurt is stored at 4 °C and the shelf life is observed.

[0052] From the results in Table 4, it can be seen that during the fermentation of yogurt with the addition of Lactobacillus delbrueckii subsp. bulgaricus inm3201, as time goes by, the bacteriostatic activity of the experimental group does not change. The overall flavor of the yogurt is relatively stable, while the control group has no bacteriostatic effect, and the flavor continuously decreases during the shelf life of the yogurt. This may be due to the lack of the influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on the growth and metabolism of Streptococcus thermophilus subsp. salivarius during the fermentation process. Therefore, when the addition amount of Lactobacillus delbrueckii subsp. bulgaricus inm3201 is 1×10 7 cfu / mL, it can not only improve the anti-corrosion effect of yogurt, but also improve the flavor and taste of yogurt.

[0053] Example 5 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on preservation in flavored yogurt According to the national standard GB 19302 - 2025, flavored yogurt is prepared, and the strains used and the detection methods refer to Example 4.

[0054] Table 5 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on preservation of flavored yogurt

[0055] Note: The prepared fermented milk is stored at 4 °C and the shelf life is observed.

[0056] From the results in Table 5, it can be seen that the bacteriocin preparation of Lactobacillus delbrueckii subsp. bulgaricus inm3201 provided by the present invention, when applied to the fermentation of fermented milk, can significantly improve the anti-corrosion ability in related flavored yogurt without changing the existing process. At the same time, it can also improve the aroma, taste and stability of the flavored yogurt product, providing a research and development basis for the development of flavored yogurt products.

[0057] Example 6 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on preservation of flavored fermented milk According to the national standard GB 19302 - 2025, flavored fermented milk is prepared. Lactobacillus delbrueckii subsp. bulgaricus inm3201 is added to the dairy product at a final concentration of 1.0×10 7 cfu / mL. Another group AB0109 is used as a control strain, and the bacteriostatic detection method refers to Example 4. The results are shown in Table 6 Table 6 Influence of Lactobacillus delbrueckii subsp. bulgaricus inm3201 on the preservation of flavored fermented milk

[0058] As can be seen from the results in Table 6, for the flavored fermented milk prepared by adding Lactobacillus delbrueckii subsp. bulgaricus inm3201, compared with the control group, Lactobacillus delbrueckii subsp. bulgaricus inm3201 can significantly improve the antibacterial ability of the flavored fermented milk. At the same time, it also shows that using Lactobacillus AB0109 to make flavored fermented milk in the control group will not significantly improve the antibacterial effect of the flavored fermented milk. At the same time, compared with AB01019 in the control group, Lactobacillus delbrueckii subsp. bulgaricus inm3201 can significantly improve the sensory evaluation of the flavored fermented milk.

[0059] Based on the results of the above embodiments, the preparation of Lactobacillus delbrueckii subsp. bulgaricus inm3201 can be applied to raw milk, yogurt, flavored yogurt and flavored fermented milk. Without changing the existing process, it can significantly improve the preservation ability of related products, and at the same time can also improve the aroma and taste of the products, and significantly improve the product quality.

[0060] A probiotic preservation preparation prepared from Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein.

[0061] An anti-corrosion application of a strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein.

[0062] The above embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A strain of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein, characterized in that: This strain is Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) inm3201, with the preservation number GDMCC NO: 66019, the preservation date is: March 17, 2025, and the preservation unit is: Guangdong Microbial Culture Collection Center, and the preservation address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

2. A strain of *Lactobacillus delbrueckii subsp. bulgaricus* containing the preservative factor S-layer protein according to claim 1, characterized in that: The colony characteristics of Lactobacillus delbrueckii subsp. bulgaricus inm3201 are as follows: cultured on solid MRS plate medium for 48 h, rod-shaped with rounded ends, usually long rods, 2 μm - 9 μm in length and 0.5 μm - 0.8 μm in width, Gram-positive, non-spore-forming; facultative anaerobic, surface colony diameter 1 - 2 mm, convex, round, smooth, fine, white.

3. A preparation method of Lactobacillus delbrueckii subsp. bulgaricus containing the preservative factor S-layer protein as described in claim 1, characterized in that: are as follows: Screen and select lactic acid bacteria from dairy product samples. Process the dairy products into a paste, add sterile water, dilute, evenly coat dairy products with different dilution concentrations on solid MRS medium, and place them in an incubator for cultivation. Select white colonies on the solid MRS medium, and screen lactic acid bacteria that meet the morphology. Lactic acid bacteria screening: Cultivate the obtained lactic acid bacteria to the logarithmic growth phase, then inoculate them at an inoculation amount of 1%, centrifuge to remove the supernatant, wash with sterile physiological saline, inoculate into LB medium, and at the same time inoculate Bacillus licheniformis, and place it to observe the antibacterial effect.

4. The preparation method of a strain of *Lactobacillus delbrueckii subsp. bulgaricus* containing the preservation factor S-layer protein according to claim 3, characterized in that: MRS medium: Peptone, beef extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, ammonium citrate dibasic, sodium acetate, magnesium sulfate, manganese sulfate, Tween 80, dissolved in distilled water; solid MRS adds agar on the basis of the liquid.

5. A probiotic preservation preparation prepared from Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein as described in claim 1.

6. An anti-corrosion application of Lactobacillus delbrueckii subsp. bulgaricus containing the preservation factor S-layer protein as described in claim 1.

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