Lactobacillus bulgaricus and application thereof

Abstract

The present invention belongs to the technical field of microorganisms, and specifically relates to a Lactobacillus bulgaricus and an application thereof. The Lactobacillus bulgaricus has a preservation number of CGMCC No. 25743. Vitamin K2 can be produced by fermenting cow's milk using the Lactobacillus bulgaricus as a starter, and the yield can be as high as 181.02±10.58 mg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Lactobacillus bulgaricus and application thereof. Background Art

[0002] Vitamin K2, also known as menaquinone, is a fat-soluble vitamin. Named MK-4 to MK-13, depending on the length of the isoprene side chains on its naphthoquinone ring, MK-7 (containing seven isoprene units) exhibits the strongest activity, longest-lasting effects, highest safety, lowest dosage, and longest half-life in the blood. Vitamin K2 activates osteocalcin to form "calcium claws," promoting bone mineral deposition and becoming a key component of bone health supplements. Furthermore, vitamin K2 has been found to promote blood coagulation to maintain cardiovascular health, prevent Parkinson's disease by rescuing mitochondrial dysfunction through electron transfer, promote liver function recovery, and prevent cancer and reduce the risk of type 2 diabetes by inhibiting cell cycle arrest and proliferation. Given the increasing market demand for vitamin K2 as a next-generation health supplement, the industrialization of vitamin K2 and the reduction of its production costs have become key areas of focus.

[0003] Currently, there are three main ways to obtain vitamin K2 (mk-7): (1) direct intake from fermented foods. For example, foods such as natto, fermented black beans and cheese contain trace amounts of vitamin K2 (mk-7), but the content is very low and cannot meet the normal intake of the human body; (2) chemical synthesis of vitamin K2 (mk-7). Currently, vitamin K2 (mk-7) can be artificially synthesized chemically, but the chemical synthesis reaction steps are complex and the yield is low. It also produces low-activity cis-isomers and a large amount of by-products, which seriously pollute the environment; (3) microbial fermentation to produce vitamin K2 (mk-7). Natto Bacillus has been isolated from natto. This bacterium does not produce endotoxins and is a food safety grade strain. However, the fermentation unit of this strain is very low. It is necessary to screen out high-yield strains through strain breeding and increase the fermentation unit with appropriate fermentation medium formulation and control process to meet the production standards of large-scale industrialization.

[0004] Existing research shows that many lactic acid bacteria can also produce vitamin K2. For example, Lactococcus lactis and Leuconostoc lactis are natural producers of vitamin K2. Although the production level is generally low, compared with artificially synthesized and purified compounds, the production conditions of lactic acid bacteria naturally produced by lactic acid bacteria are mild, safe and non-toxic, with few by-products. It can greatly simplify the production process and improve working conditions, reduce environmental pollution, and is conducive to resource development and comprehensive utilization.

[0005] Therefore, discovering a lactic acid bacterium that can produce vitamin K2 is of great significance for the industrial production of vitamin K2. Summary of the Invention

[0006] In response to the above problems, one of the objectives of the present invention is to provide a lactic acid bacterium that can produce vitamin K2, a Lactobacillus bulgaricus, which can use cow's milk as a fermentation substrate to produce vitamin K2, with a yield of up to 181.02±10.58 mg / L.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a Lactobacillus delbrueckii subsp. bulgaricus, which has a deposit number of CGMCC No. 25743.

[0009] Another aspect of the present invention provides a composition comprising one or more combinations of the following substances: (a) the aforementioned Lactobacillus bulgaricus; (b) a lysate of the aforementioned Lactobacillus bulgaricus; (c) a culture of the aforementioned Lactobacillus bulgaricus; and (d) a fermentation broth of the aforementioned Lactobacillus bulgaricus.

[0010] In another aspect, the present invention provides a preparation comprising the aforementioned Lactobacillus bulgaricus or the aforementioned composition, and a carrier; wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.

[0011] In another aspect, the present invention provides a method for preparing vitamin K2, comprising: using cow's milk as a fermentation substrate and fermenting with the above-mentioned Lactobacillus bulgaricus to obtain vitamin K2.

[0012] Another aspect of the present invention provides a use of the above-mentioned Lactobacillus bulgaricus in the preparation of food additives or fermented foods.

[0013] The preservation information of Lactobacillus bulgaricus in the present invention is as follows: Preservation institution: General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC); Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: September 16, 2022; Preservation number: CGMCC No. 25743; Classification name: Lactobacillus delbrueckii subsp. bulgaricus.

[0014] The beneficial effects of the present invention include: Lactobacillus bulgaricus provided by the present invention can be used as a starter to ferment cow's milk to produce vitamin K2, and the output can be as high as 181.02±10.58 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of TY-V16;

[0016] Figure 2 This is the TY-V16 Gram stain result;

[0017] Figure 3 is the standard curve and regression equation of vitamin K2;

[0018] Figure 4 The content of vitamin K2 in the fermentation broth of 30 lactic acid bacteria strains;

[0019] Figure 5 This is the growth curve of TY-V16 under different culture conditions;

[0020] Figure 6 The content of vitamin K2 in TY-V16 fermentation broth after fermentation under different culture conditions;

[0021] Figure 7 This is a picture of TY-V16 fermented milk curd;

[0022] Figure 8 The pH value changes of TY-V16 fermented milk. DETAILED DESCRIPTION

[0023] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0024] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0025] In the present invention, the MRS liquid culture medium is as follows: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 1.0 g of Tween (Tween 80), 0.4 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0 g of calcium carbonate, and 15.0 g of agar are sequentially added with distilled water and the distilled water is added to 1000 mL, the pH value is adjusted to 6.3, the mixture is stirred and then heated, boiled for 2 minutes, and sterilized at 121° C. and 0.1 MPa for 30 minutes.

[0026] The embodiment of the present invention provides a Lactobacillus delbrueckii subsp. bulgaricus, whose deposit number is CGMCC No. 25743.

[0027] It should be noted that the Lactobacillus bulgaricus (also known as Lactobacillus bulgaricus TY-V16 or TY-V16) is derived from naturally fermented yak yogurt in the homes of herders on the Qinghai-Tibet Plateau. Gram staining results show that it is rod-shaped and is determined to be a Gram-positive bacterium (G + ); and amplifying the 16SrDNA sequence by PCR, the 16SrDNA sequence was measured to contain the sequence shown in SEQ ID NO.1, and homology analysis was performed to determine that the Lactobacillus bulgaricus was Lactobacillus delbrueckii subspecies bulgaricus.

[0028] Another embodiment of the present invention provides a composition comprising one or more combinations of the following substances: (a) the aforementioned Lactobacillus bulgaricus; (b) a lysate of the aforementioned Lactobacillus bulgaricus; (c) a culture of the aforementioned Lactobacillus bulgaricus; and (d) a fermentation broth of the aforementioned Lactobacillus bulgaricus.

[0029] It should be noted that, in the above-mentioned composition, as described above, Lactobacillus bulgaricus has a good survival rate in the digestive tract of the body, and can be prepared into a composition that can be used for food or medicine. In addition, when Lactobacillus bulgaricus is prepared into a composition, the bacteria can be directly introduced into the composition in the form of live bacteria to play a role, or the bacteria can be inactivated by existing technical means and then introduced into the composition in the form of inactivated bacteria to play a role; the lysate of the bacteria can also be introduced into the composition to play a role; the products such as proteins, peptides, secretions or metabolites obtained by culturing the bacteria can also be introduced into the composition to play a role; the fermentation liquid after the bacteria is fermented can also be introduced into the composition to play a role. In the specific use process, different forms of the bacteria can be selected according to specific needs to be prepared into a composition to play a role.

[0030] In some specific embodiments, the above composition further comprises one or more combinations of probiotics, prebiotics, dietary fiber and Chinese patent medicines.

[0031] It should be noted that the above-mentioned Lactobacillus bulgaricus and its different forms can also be used in combination with one or more of probiotics, dietary fiber and pharmaceutically active compounds; for example, Lactobacillus bulgaricus can be used in combination with Bacillus subtilis, Bifidobacterium or lactic acid bacteria so that the composition has the efficacy of Lactobacillus bulgaricus and other probiotics; for another example, Lactobacillus bulgaricus can be used in combination with prebiotics, which can provide an energy source for Lactobacillus bulgaricus, thereby enhancing the effect of Lactobacillus bulgaricus; for another example, Lactobacillus bulgaricus can be used in combination with dietary fiber, which can assist Lactobacillus bulgaricus in colonization, thereby improving the effect of Lactobacillus bulgaricus; for another example, Lactobacillus bulgaricus can also be used in combination with traditional Chinese medicine to form a composition, while exerting the effects of Lactobacillus bulgaricus and traditional Chinese medicine.

[0032] Yet another embodiment of the present invention provides a preparation comprising the aforementioned Lactobacillus bulgaricus or the aforementioned composition, and a carrier; wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.

[0033] It should be noted that the above-mentioned composition comprising Lactobacillus bulgaricus and its different forms can be added with a pharmaceutical carrier or an edible carrier to prepare a medicine or edible food or health product. Pharmaceutically acceptable carriers or edible carriers are all known in the art and may need to be selected according to the dosage form. For example, the preparation of tablets mainly involves the use of diluents (such as starch, dextrin, sucrose or glycosides), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (polyvidone, syrup or hypromellose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.); for example, the preparation of liquids mainly involves the use of bulking agents, suspending agents, emulsifiers or coloring agents, etc.

[0034] In some specific embodiments, the above-mentioned preparations may include pills, capsules, powders, gels, and granules in addition to tablets and liquids. It should be noted that the solid dosage forms such as tablets, pills, granules, or capsules may be in the form of probiotic tablets, probiotic sugar pills, probiotic powders, or probiotic capsules; the liquids may be in the form of probiotic beverages; and the gels may be in the form of probiotic jelly, probiotic milk caps, or solidified yogurt.

[0035] Another embodiment of the present invention provides a method for preparing vitamin K2, comprising: using cow's milk as a fermentation substrate and fermenting with the above-mentioned Lactobacillus bulgaricus to obtain vitamin K2.

[0036] It should be noted that cow's milk is a nutrient-rich natural culture medium. Lactic acid bacteria can produce large amounts of lactic acid from cow's milk and are recognized as safe food-grade microorganisms. Therefore, the Lactobacillus bulgaricus in the present invention can use cow's milk as a fermentation substrate to produce vitamin K2 and also produce fermented products. It should also be noted that the cow's milk can be either compound milk or raw milk.

[0037] In some specific embodiments, in the above-mentioned method for preparing vitamin K2, Lactobacillus bulgaricus is enriched using a culture medium before fermentation; the culture medium is MRS liquid culture medium supplemented with 55g / L-65g / L glycerol and 8g / L-12g / L sucrose.

[0038] It should be noted that culturing Lactobacillus bulgaricus after adding 55g / L-65g / L glycerol and 8g / L-12g / L sucrose to MRS liquid culture medium can increase the number of viable bacteria and the production of vitamin K2; compared with culturing in MRS liquid culture medium, the number of viable bacteria can be increased by about 31.7%, and the subsequent vitamin K2 production can be increased by about 58.9%.

[0039] In some specific embodiments, in the above-mentioned method for preparing vitamin K2, glycerol and sucrose are added to the MRS liquid culture medium, and the peptone in the MRS liquid culture medium can be replaced with an equal amount of soy peptone, tryptone, bacterial peptone, casein peptone or Peptone can also increase the number of viable bacteria and the production of vitamin K2 compared to the original MRS liquid medium; among them, replacing it with an equal amount of trypsin and The effect of peptone is similar to that of live bacteria without replacement, and the production of vitamin K2 is slightly increased. The effects of peptone and soy peptone were significantly improved compared with the number of live bacteria without replacement, and the production of vitamin K2 was significantly improved; preferably, soy peptone was replaced, and the number of live bacteria increased by about 5% compared with the number without replacement, and the production of vitamin K2 in the process could be increased by about 18.6%, reaching 341.29±31.05 mg / L; in addition, the culture medium replaced with soy peptone and added with glycerol and sucrose increased the production of vitamin K2 (181.02±10.58 mg / L) by about 88.54% compared with the ordinary culture medium after bacterial enhancement; and the fermentation time of the late fermentation of the culture medium can be shortened from 40h of the ordinary culture medium to 24h to achieve a vitamin K2 production of 341.29±31.05 mg / L, and the fermentation time is shortened by about 40%, shortening the production cycle, and can effectively reduce the costs of raw materials, water, electricity and steam used for fermentation, with high economic benefits.

[0040] Another embodiment of the present invention provides a use of the above-mentioned Lactobacillus bulgaricus in preparing a food additive or a fermented food. It should be noted that the Lactobacillus bulgaricus of the present invention can be made into a food additive for fermentation, or the Lactobacillus bulgaricus of the present invention can be used in the preparation of a fermented food. The fermented food can be any known in the art, such as kimchi, fermented bean products, or fermented dairy products.

[0041] In some specific embodiments, in the above application, the fermented food includes fermented milk, cheese or fermented milk beverage.

[0042] It should be noted that the lactobacillus bulgaricus in the present invention is with pasteurized milk, HUT sterilized milk and reconstituted milk as fermentation substrate, and the fermented curd state is all good and high-yield vitamin K2, has the potentiality of making fermented milk beverage, composite with thermophilic streptococcus and has preparation and produces vitamin K2 yogurt starter;And the lactobacillus bulgaricus in the present invention and commercial starter collaborative fermentation all do not have significant impact on aspects such as the tissue states such as pH value, acidity, water separation rate, clarification index, texture, etc., also can not affect the overall acceptance of consumers of yogurt, and has good application prospect.In addition, this lactobacillus bulgaricus also has calcium supplementation effect, so it is applied in the preparation of fermented milk, cheese or fermented milk beverage, can also increase the calcium supplementation effect of fermented milk, cheese or fermented milk beverage.

[0043] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0044] Example 1 Isolation, purification and identification of TY-V16

[0045] (1) Experimental materials

[0046] Naturally fermented yak yogurt from herders’ homes on the Qinghai-Tibet Plateau: Use a sterile spoon to take the homemade yak yogurt from herders’ homes and put it into a 15mL sterile screw-cap centrifuge tube containing an appropriate amount of sterile calcium carbonate and soluble starch (calcium carbonate: soluble starch is 1:1). Stir evenly, tighten the screw cap, put it in a refrigerator, and transport it back to the laboratory for immediate purification and separation of lactic acid bacteria.

[0047] (2) Separation and purification

[0048] Under sterile conditions, 1 mL of the initial sample was added to 9 mL of sterile saline and vortexed to mix. -1 The sample diluent was then diluted 10-fold to 10 -7 , select 10 -5 , 10 -6 , 10 -7Evenly spread 100 μL of the dilution solution at the desired dilution on an MRS plate and incubate at 37°C for 48 hours. After incubation, observe the colony morphology on the MRS plate and select typical lactic acid bacteria colonies for plate streak purification. Repeat this streak operation until a purified strain is obtained.

[0049] (3) Morphological and structural observation

[0050] Inoculate the purified strain into 5 mL of sterile MRS broth and incubate at 37°C for 18 hours. Centrifuge 1 mL of the bacterial solution at 12,000 rpm for 1 minute, wash twice with sterile saline, and resuspend the cells in an equal volume of sterile saline. Use an inoculating loop to evenly spread a small amount onto a glass slide. After fixation, perform Gram staining, examine under a microscope, and photograph. Gram-positive bacteria (G+) stain with a blue-purple color, while Gram-negative bacteria (G-) stain with a red color. Observe and record cell morphology and Gram stain results.

[0051] The cell morphology after purification of the strain is as follows Figure 1 As shown, it forms single colonies in solid culture medium. The colonies are large, round, translucent, with smooth and convex surfaces, irregular edges, and white color.

[0052] The results after Gram staining are as follows Figure 2 As shown ( Figure 2 Grayscale processing was done, the original image was purple). Under the microscope, the cells all appeared purple, were Gram-positive bacteria (G+), and had a rod-shaped morphology.

[0053] The cell morphology of the above strains is consistent with the characteristics of lactobacillus, and the morphological structure is uniform, indicating that the strains are pure.

[0054] (4) PCR amplification of 16S rDNA sequence

[0055] PCR amplification was performed using a 25 μL reaction system: 1 μL template, 1 μL each of primers (27F, 1492R), and 12.5 μL of 2× TaqPCR Master Mix, with the volume made up to 25 μL with sterile ultrapure water. PCR amplification conditions included 94°C pre-denaturation for 5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by terminal extension at 72°C for 10 min. After amplification, the PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd., resulting in the sequence shown as SEQ ID No. 1. The sequence was then searched and aligned against GeneBank using BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). The results indicated that TY-V16 was Lactobacillus delbrueckii subsp. bulgaricus, hereinafter referred to as L. bulgaricus.

[0056] Example 2 Screening of TY-V16 for High Vitamin K2 Production

[0057] (1) Experimental materials

[0058] Lactobacillus bulgaricus TY-V16: isolated from naturally fermented yak yogurt from herders in the Qinghai-Tibet Plateau, and preserved in the General Microbiology Center of the China Culture Collection Administration, with the number CGMCC No. 25743.

[0059] The strain was inoculated into MRS liquid culture medium at a 5% inoculum, cultured at 37°C for 18 hours, and activated for three generations. 2 mL of the culture was centrifuged at 10,000 rpm for 15 minutes to collect the bacterial precipitate, which was washed twice with a sterile PBS solution and resuspended in an equal volume of sterile saline to prepare a bacterial suspension. The prepared bacterial suspension was added to HUT sterilized milk at a 5% inoculum, and fermented at 42°C for 48 hours to obtain a single-bacteria fermented yogurt sample. The sample was then ultrasonically disrupted for 5 minutes. After the sample was cooled, the ultrasonic disruption was repeated at least twice to completely release the menaquinones.

[0060] (2) Vitamin K2 extraction

[0061] Enzyme hydrolysis: Accurately weigh 20 mL (accurate to 0.1 mL) of the above yogurt sample, add 5 mL of phosphate buffer, mix thoroughly, add 0.2 g of lipase (accurate to 0.01 g), vortex mix thoroughly, and shake in a constant temperature water bath at 300 rpm for 2-4 hours to allow for complete enzymatic hydrolysis.

[0062] Extraction: Remove the enzymatically hydrolyzed test solution and add 20 mL of extraction solution (n-hexane:isopropanol = 2:1) and 1 g of potassium carbonate powder. Mix thoroughly, transfer to a 150 mL separatory funnel, and extract at 300 rpm for 10 minutes. Transfer the supernatant to a 100 mL rotary evaporator. Repeat the extraction process at least once, and combine the supernatants in the rotary evaporator.

[0063] Concentration: Rotary evaporate the above n-hexane extract at 50°C until nearly dry (if there is residual liquid, blow it down with nitrogen until nearly dry), make up to volume with 1 mL of methanol, filter through a 0.22 μm filter membrane, and immediately analyze on an instrument.

[0064] (3) Vitamin K2 determination

[0065] In the following examples, vitamin K2 was determined using high performance liquid chromatography-fluorescence detection; liquid chromatography conditions: chromatographic column: C 18Column, column length 25 cm, column inner diameter 4.6 mm, filler particle size 5 μm or chromatographic column with equivalent performance; zinc powder reduction column: column length 50 mm, inner diameter 4.6 mm; detection wavelength: excitation wavelength 243 nm, emission wavelength 430 nm; injection volume: 10 μL; flow rate: 1 mL / min; mobile phase: dissolve 900 mL of methanol, 100 mL of tetrahydrofuran, 0.3 mL of glacial acetic acid, 1.5 g of zinc chloride, and 0.5 g of anhydrous sodium acetate, and filter with a 0.22 μm organic filter membrane.

[0066] Vitamin K2 standard curve drawing: Vitamin K2 (MK-7) (C 46 H 64 O2, CAS No.: 2124-57-4) standard; 98% purity, nationally certified and awarded standard substance certificate standard substance; Vitamin K2 (MK-7) standard series working solution: Accurately aspirate 20μL, 50μL, 100μL, 200μL, 500μL, and 1000μL of the vitamin K2 (MK-7) intermediate solution, dilute to 1mL with methanol, and prepare vitamin K2 (MK-7) series standard working solutions with concentrations of 20μg / L, 50μg / L, 100μg / L, 200μg / L, 500μg / L, and 1000μg / L; perform HPLC detection. The HPLC chromatogram of the vitamin K2 (MK-7) solution is shown in Figure 3 The standard curve is drawn with the peak area of ​​vitamin K2 (MK-7) as the ordinate and the concentration of the standard solution as the abscissa to obtain the standard curve regression equation. The standard curve of vitamin K2 is as follows: Figure 4 shown.

[0067] Determination of vitamin K2 content: The mass fraction of vitamin K2 (MK-7) in the sample is calculated according to the following formula:

[0068] Where:

[0069] X—Vitamin K2 content in the sample, in micrograms per milliliter (ug / mL);

[0070] ρ—the concentration of vitamin K2 in the sample solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0071] v—the final volume of the sample solution, in milliliters (mL);

[0072] V—sample volume, in milliliters (mL);

[0073] 1000—Conversion factor for converting concentration units from ng / mL to ug / mL.

[0074] The calculation result should be rounded to three significant figures.

[0075] A total of 200 lactic acid bacteria of different species (including 40 strains of Lactobacillus plantarum, 40 strains of Lactobacillus fermentum, 40 strains of Lactobacillus casei, 40 strains of Lactobacillus bulgaricus, 20 strains of Lactococcus lactis, and 20 strains of Lactobacillus helveticus) from Tianyou Dairy's own probiotic library (all strains were from yak yogurt, cheese and other samples collected from the Qinghai-Tibet Plateau, Tibet and other places, and the lactic acid bacteria in the samples were separated and purified and identified by 16sRNA sequence, and finally freeze-dried and stored in the strain library. The preserved strains are all edible and safe strains) were tested for vitamin K2 production according to the above method, and 30 strains with a certain vitamin K2 production were preliminarily screened out (vitamin K2 production ≥ 20 mg / L), among which TY-V16 (i.e. Figure 5 LDSB-7) was the best, with a yield of 181.02±10.58 mg / L. The experimental results are as follows Figure 5 As shown in the figure, the fermentation broth of strain TY-V16 contained the highest vitamin K2 content and had a stable yield, indicating that this strain has the ability to produce high amounts of vitamin K2. Therefore, strain TY-V16 was selected as the research object to explore its application in yogurt.

[0076] Example 3 Optimization of TY-V16 Fermentation Conditions for High Vitamin K2 Production

[0077] (1) Experimental materials

[0078] Lactobacillus bulgaricus TY-V16: Isolated from naturally fermented yak yogurt grown by herders on the Qinghai-Tibet Plateau, it is deposited at the General Microbiology Center of the China Culture Collection Administration under the registration number CGMCC No. 25743. It has poor growth in non-anaerobic, standard MRS medium and produces relatively low levels of vitamin K2.

[0079] (2) Culture medium preparation

[0080] 60 g / L glycerol and 10 g / L sucrose were added to the MRS liquid medium as additional carbon sources, and the peptone in the MRS medium (10 g / L) was replaced with tryptone, Peptone, casein peptone, bacterial peptone and soy peptone were used as controls, with ordinary MRS liquid medium as the control. The strains were inoculated at a 5% inoculum into MRS liquid medium supplemented with carbon source and replaced with nitrogen source, cultured anaerobically at 37°C for 18 hours, and cultured for three generations.

[0081] (3) Live bacteria count

[0082] The plate count method was used to count the live lactic acid bacteria. After three generations of continuous culture, 10 μL of the bacterial solution in appropriate gradients was added to sterile MRS agar plates. The plates were anaerobically cultured at 37°C for 24-48 hours and counted. Three replicates were used for each gradient.

[0083] The difference in vitamin K2 production could be due to an increase in viable bacterial count, leading to a higher total vitamin K2 production; or it could be due to an increase in the viable bacterial count, resulting in an enhanced vitamin K2 production capacity. To investigate the cause of this enhanced vitamin K2 production by lactic acid bacteria, viable bacterial counts were performed, and the results are shown in Table 1.

[0084] Table 1 TY-V16 viable bacteria count under different fermentation conditions

[0085]

[0086]

[0087] As shown in Table 1 above, the viable bacterial count of TY-V16 increased by two orders of magnitude after adding a carbon source, avoiding light, and anaerobic culture, significantly improving its growth ability. The viable bacterial count remained on the same order of magnitude under different nitrogen source conditions, indicating that different nitrogen sources did not affect the strain's growth ability, but rather improved its ability to produce vitamin K2.

[0088] (4) Determination of strain fermentation time

[0089] 2 mL of each bacterial strain cultured under different conditions was centrifuged at 10,000 rpm for 15 minutes to collect the pellet, washed twice with sterile PBS, and resuspended in an equal volume of sterile saline to prepare a bacterial suspension. The prepared bacterial suspension was added to HUT sterilized milk at a 5% concentration and allowed to ferment at 42°C to obtain single-bacteria fermented yogurt samples. The time it took for the pH to reach 4.25 was recorded. The fermentation times of TY-V16 under different fermentation conditions are shown in Table 2.

[0090] Table 2 Fermentation time under different fermentation conditions

[0091]

[0092] Note: The peptone in the carbon source + peptone group in Table 2 above is the peptone in the original MRS liquid medium.

[0093] (5) Determination of vitamin K2 content

[0094] The cells of the single-bacteria fermented yogurt sample were disrupted by ultrasound to release menaquinones, and vitamin K2 was extracted and determined using fluorescence-high performance liquid chromatography.

[0095] The ability of the same lactic acid bacteria to produce vitamin K2 varies under different fermentation conditions. Peptone is formed by the decomposition of protein by acid, alkali or protease. It is rich in organic nitrogen compounds and can provide nitrogen source for microbial growth. Figure 6As shown in the figure, in the original MRS liquid medium, the vitamin K2 production of TY-V16 was 181.02±10.58 mg / L and the fermentation time was 40 h. After adding glycerol and sucrose as new carbon sources to the MRS liquid medium, the vitamin K2 production increased significantly and the fermentation time was significantly shortened. In addition, the peptone in the MRS medium was replaced with equal amounts of soy peptone, tryptone, bacterial peptone, casein peptone and The vitamin K2 production after adding peptone, glycerol and sucrose as new carbon sources was significantly increased compared with the original MRS liquid culture medium, and the fermentation time was also significantly shortened; among them, the vitamin K2 production after the culture medium was replaced with soy peptone and added with glycerol and sucrose reached 341.29±31.05 mg / L after enrichment, which was 88.54% higher than the original MRS liquid culture medium, and the fermentation endpoint was reached in only about 24 hours, shortening the fermentation time by about 40%.

[0096] Example 4 TY-V16 fermentation performance test

[0097] (1) Experimental materials

[0098] Lactobacillus bulgaricus TY-V16: isolated from naturally fermented yak yogurt from herders in the Qinghai-Tibet Plateau, and preserved in the General Microbiology Center of the China Culture Collection Administration, with the number CGMCC No. 25743.

[0099] (2) Production of single-bacteria fermented yogurt

[0100] The above Lactobacillus bulgaricus strains were inoculated into the optimized liquid culture medium at a 5% inoculum volume, cultured at 37°C for 24 h, activated for three generations, and 2 mL of the bacterial precipitate was collected by centrifugation at 8000 r / min for 10 min. The precipitate was washed twice with sterile PBS solution and resuspended in an equal volume of sterile saline to obtain a bacterial suspension. The prepared bacterial suspension was diluted to a final concentration of 10 5 CFU / L were added into pasteurized milk, HUT sterilized milk and milk powder reconstituted milk, respectively, and fermented at 42 °C for 18 h to obtain single-bacteria fermented yogurt.

[0101] (3) TY-V16 acid production capacity test

[0102] TY-V16 was used to ferment pasteurized milk, HUT sterilized milk and milk powder reconstituted milk (milk powder: water ratio is 1:9, i.e. milk powder concentration is 10%). The results are as follows: Figure 7 The following figure shows the fermentation status of reconstituted milk, HUT sterilized milk, and pasteurized milk from left to right. TY-V16 showed good fermentation status, uniform color, and no odor in these three types of milk.

[0103] The pH changes of reconstituted milk, HUT sterilized milk, pasteurized milk and fermented broth are as follows: Figure 8 As shown, the pH of the emulsion after fermentation of powdered milk reconstituted milk, HUT sterilized milk, and pasteurized milk by Lactobacillus bulgaricus of the present invention is finally maintained at about 4.25 (a final pH value suitable for yogurt), and the acidity decreases relatively quickly, so the emulsion can be used as a starter for making yogurt.

[0104] The above data show that the Lactobacillus bulgaricus in the present invention can be used as an excellent strain for fermenting yogurt and has the potential to prepare vitamin K2 fermented milk beverages.

[0105] Example 5 Effect of TY-V16 on Yogurt Quality

[0106] (1) Experimental materials

[0107] Lactobacillus bulgaricus TY-V16: isolated from naturally fermented yak yogurt from herders in the Qinghai-Tibet Plateau, and preserved in the General Microbiology Center of the China Culture Collection Administration, with the number CGMCC No. 25743.

[0108] Traditional commercial starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus): purchased from Danisco (China)

[0109] (2) Yogurt production

[0110] TY-V16 was inoculated into the optimized liquid culture medium at a 5% inoculum volume and cultured at 37°C for 18 h. After activation for three generations, 2 mL of the bacterial precipitate was collected by centrifugation at 10,000 r / min for 15 min, washed twice with sterile PBS solution, and resuspended in an equal volume of sterile saline to prepare the bacterial suspension. One group of the bacterial suspensions was prepared by 10 5 CFU / L and 6g / L of commercial starter were added to HUT sterilized milk at the same time. Another group added 6g / L of commercial starter to HUT sterilized milk as a control. The two groups were fermented at 42℃ for 7h at the same time. After refrigeration and ripening, the state quality of the two groups of yogurt were compared.

[0111] (3) Determination of the effect of TY-V16 on the physical and chemical quality of yogurt

[0112] ① Acidity determination

[0113] The pH of yogurt was measured using a precision pH meter. Titratable acidity during yogurt storage was determined according to GB5009.239-2016, National Food Safety Standard - Determination of Acidity of Foods.

[0114] ② Determination of water separation rate of yogurt (WHC)

[0115] Accurately weigh 20g of sample, centrifuge at 4000r / min for 20min, weigh the precipitate mass, and calculate the water separation rate according to formula (1)

[0116]

[0117] Most probiotic lactic acid bacteria have strong acid production capacity and metabolize to produce different products, which will affect the properties of yogurt. Therefore, when using probiotic lactic acid bacteria to produce yogurt products, attention should be paid to their impact on product quality. In the embodiment of the present invention, the lactic acid bacteria TY-V16 with high vitamin K2 production is used as an auxiliary fermentation agent for yogurt production. By comparing the commercial fermentation agents (Lactobacillus bulgaricus and Streptococcus thermophilus), it is found that TY-V16 has substantially no effect on the clotting time, pH value, acidity, water separation rate, clarification index and tissue state of the yogurt samples, as shown in Table 3 below.

[0118] Table 3 Physicochemical properties of yogurt

[0119] pH acidity Water separation rate Clarification Index comparison 4.36±0.03a 86.27±1.01a 55.41±0.99a 0.574±0.033a TY-V16 4.40±0.02a 87.66±1.03a 54.11±0.44a 0.582±0.016a

[0120] ③ Yogurt texture determination

[0121] The texture of the yogurt samples was determined using a texture analyzer (TAXTplus, MicroStable System Co., UK). The penetration test was performed using a 10 mm diameter cylindrical probe. The samples were measured immediately after being taken out of the refrigerator (4°C). The penetration was performed through a depth of 15 mm at a speed of 1 mm / s and a trigger force of 1 g.

[0122] ④ Yogurt stability determination

[0123] Measurements were made using an optical analytical centrifuge (LU MiSizer L.UM GmbH, Germany). Approximately 0.4 mL of AMD was filled into a standard cuvette. The instrument parameters were as follows: temperature 25°C; rotation speed 4000 rpm; time interval 10 seconds; and experimental time 4490 seconds. Each experiment was performed in triplicate, and each result is presented as the mean ± standard deviation of the clarification index. As shown in Table 4, TY-V16 had no significant effect on the physical and chemical properties of the yogurt samples, including hardness, viscosity, elasticity, cohesion, adhesiveness, chewiness, and rebound resilience.

[0124] Table 4 Yogurt texture

[0125]

[0126] ⑤Determination of the effect of TY-V16 on the sensory quality of yogurt

[0127] According to the Chinese dairy industry standard RHB103-2004 "Detailed Rules for Sensory Quality Evaluation of Yogurt", the sensory evaluation adopted the acceptance test method. 30 sensory evaluators (15 males and 15 females) scored the appearance, color, aroma, texture, taste and overall acceptance of the yogurt samples based on their yogurt consumption habits and interests. The total score was 9 points (1 = very dislike; 3 = dislike; 5 = neither dislike nor dislike; 7 = like; 9 = very like). Each result is shown as the mean result ± standard deviation.

[0128] Food sensory evaluation, also known as food sensory analysis, is a process of qualitative and quantitative testing and analysis of food using scientific analytical methods to perceive the characteristics or properties of food through vision, smell, taste and hearing. The different products produced by the metabolism of probiotic lactic acid bacteria can give yogurt a certain special taste, which will directly have a significant impact on consumer acceptance and purchasing intention, and is an important factor affecting product quality. The examples of the present invention use a consumer acceptance test method. The results show that there are no significant differences in the five sensory indicators of yogurt in different groups: appearance, color, texture, aroma, taste, and overall acceptance score (P>0.05), indicating that TY-V16 will not have a negative impact on the product quality of yogurt, as shown in Table 5 below.

[0129] Table 5 Sensory evaluation table

[0130] Appearance color texture aroma smell Overall acceptance comparison 6.47±1.36a 6.83±1.32a 6.63±1.52a 5.93±1.84a 6.07±1.72a 6.50±1.59a TY-V16 6.57±1.43a 6.90±1.24a 6.67±1.52a 5.80±1.77a 5.97±1.56a 6.47±1.28a

[0131] In summary, utilizing Lactobacillus bulgaricus TY-V16 of the present invention to produce vitamin K2 has the characteristics of gentle production conditions, safety and non-toxicity, and high public acceptance, and good fermentation performance simultaneously, and can cooperate with yogurt commercial starter fermentation to not affect product quality, and has good application prospects. Coordinate with optimal fermentation medium and control process, its fermentation unit can reach more than 341.29mg / L, and fermentation time can be shortened by 50%, can effectively improve the output of vitamin K2 (MK-7), reduce the costs such as raw materials, water, electricity and steam used for fermentation, and have higher economic benefit.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ), its deposit number is CGMCC No.25743.

2. Lactobacillus bulgaricus according to claim 1, characterized in that Its 16S rDNA sequence is shown in SEQ ID No.

1.

3. A composition characterized in that The invention comprises one or more combinations of the following substances: (a) the Lactobacillus bulgaricus according to claim 1 or 2; (c) the culture of the Lactobacillus bulgaricus according to claim 1 or 2.

4. The composition according to claim 3, characterized in that Also included are probiotics and / or prebiotics.

5. A preparation, characterized in that The invention comprises the Lactobacillus bulgaricus according to claim 1 or 2 or the composition according to claim 3 or 4, and a carrier; wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.

6. A method for preparing vitamin K2, characterized in that: include: Vitamin K2 is obtained by fermenting cow's milk as a fermentation substrate using the Lactobacillus bulgaricus described in claim 1 or 2.

7. The method for preparing vitamin K2 according to claim 6, characterized in that: Before fermentation, Lactobacillus bulgaricus was enriched using culture medium; The culture medium is MRS liquid medium supplemented with 55 g / L-65 g / L glycerol and 8 g / L-12 g / L sucrose.

8. The method for preparing vitamin K2 according to claim 7, wherein Replace the peptone in MRS liquid medium with an equal amount of soy peptone, tryptone, bacto peptone, casein peptone, or peptone.

9. Use of the Lactobacillus bulgaricus according to claim 1 or 2 in the preparation of food additives or fermented foods.

10. The use according to claim 9, characterized in that Fermented foods include fermented milk or cheese.

11. The use according to claim 9, characterized in that Fermented foods include fermented dairy drinks.

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