NL2035198B1

NL2035198B1 - Lactobacillus delbrueckii subsp. bulgaricus and application thereof

Info

Publication number: NL2035198B1
Application number: NL2035198A
Authority: NL
Inventors: Gu Mingyu; Li Jiamin; Zhang Feng; Wang Jing; Jiang Yuanzhi; Zhu Xu; Han Ying; Chen Wei
Original assignee: Chongqing Tianyou Dairy Co Ltd
Priority date: 2023-01-05
Filing date: 2023-06-28
Publication date: 2024-01-12
Also published as: NL2035198A

Abstract

The present application belongs to the technical ﬁeld of microorganisms, and speciﬁcally, relates to a lactobacillus delbrueckii subsp. bulgaricus and an application thereof. The lactobacillus delbrueckii subsp. bulgaricus is assigned with the accession number of CGMCCNo. 25743. Using the lactobacillus delbrueckii subsp. bulgaricus as a starter to ferment cow milk may produce Vitamin K2, and a yield may reach up to 181.02i10.58 mg/L.

Description

LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS AND

APPLICATION THEREOF

TECHNICAL FIELD

3 The present application belongs to the technical field of microorganisms, and specifically, relates to a lactobacillus delbrueckii subsp. bulgaricus and an application thereof.

BACKGROUND

Vitamin K2 is also called methyl naphthoquinone, and is a lipid-soluble vitamin. mk-4 to mk-13 are named according to different lengths of isoprenoid side chains carried on a naphthoquinone main ring of the Vitamin K2; and among mk-4 to mk-13, mk-7 (that is, 7 isoprene units are contained on the side chain) is the strongest in activity, the most persistent in effect, the highest in safety, and the lowest in dosage, and has the longest half-life in the blood. The Vitamin K2 can activate osteocalcin to form a “calcium claw”, and facilitate deposition of bone mineral salts, such that the Vitamin K2 has become an important component of bone nutritional and healthy products. In addition, the Vitamin K2 has also been found to have effects of maintaining cardiovascular health by means of a procoagulant effect, treating mitochondrial dysfunction to prevent Parkinson's disease by transferring electrons, promoting the recovery of liver functions, inhibiting proliferation by blocking induction of cell cycles so as to prevent cancer, and reducing the risk of type II diabetes. By integrating biological activities and functions of the Vitamin K2, as a new generation health product, the need for the Vitamin K2 on the market is increasing day by day, such that industrialization of the Vitamin K2 and reduction of production costs have become a major concern.

Current methods for acquiring the Vitamin K2 (mk-7) are mainly classified into the following three methods. (1) The Vitamin K2 is directly ingested from fermented food, for example, food such as natto, fermented soya beans and cheese contains trace amounts of the

Vitamin K2 (mk-7); but the content of the Vitamin K2 in the food is very low and cannot meet the normal intake of a human body; (2) the Vitamin K2 (mk-7) is chemically synthesized; at present, the Vitamin K2 (mk-7) may be synthesized chemically; however, chemical synthesis reactions are complicated in process and low in yield, and low-activity cis isomers are also produced, along with a large number of by-products, and thus causing serious environmental pollution; and (3) the Vitamin K2 (mk-7) is produced by means of microbial fermentation; bacillus natto has been separated from the natto currently, and does not produce endotoxins, such that the bacillus natto is a food safety level strain; however, the strain is very low in fermentation unit, high-yielding strains need to be screened by means of strain selection, the fermentation units are improved by means of appropriate fermentation culture medium formulations and control processes, such that production standards of large-scale industrialization can be achieved.

Existing research shows that, many lactobacilli can also produce the Vitamin K2. For example, lactococcus lactis and leuconostoc lactis are natural producer for the Vitamin K2.

Although yield levels are generally low, with respect to compounds that are artificially synthesized, purified and produced, the compounds naturally produced by the lactobacilli are mild in production condition, safe and non-toxic, and less in by-product, such that production processes can be greatly simplified, working conditions are improved, and environmental pollution is reduced, thus facilitating resource development and comprehensive utilization.

Therefore, discovering a lactobacillus that can produce Vitamin K2 is of great significance for industrial production of the Vitamin K2.

SUMMARY

In view of the above problems, one of the objectives of the present application is to provide a lactobacillus, that is, a lactobacillus delbrueckii subsp. bulgaricus that may produce

Vitamin K2. Cow milk may be used as a fermentation substrate to produce the Vitamin K2, and a yield may reach 181.02+10.58 mg/L.

In order to achieve the above purpose, the present application may use the following technical solutions.

One aspect of the present application provides a lactobacillus delbrueckii subsp. bulgaricus, which is assigned with the accession number of CGMCC No. 25743.

Another aspect of the present application provides a composition. The composition includes one or a combination of a plurality of the following substances: (a) the lactobacillus delbrueckii subsp. bulgaricus; (b) lysate of the lactobacillus delbrueckii subsp. bulgaricus; (€) a culture of the lactobacillus delbrueckii subsp. bulgaricus; and (d) a fermentation broth of the lactobacillus delbrueckii subsp. bulgaricus.

Still another aspect of the present application provides a preparation. The preparation includes the lactobacillus delbrueckii subsp. bulgaricus or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.

Still another aspect of the present application provides a method for preparing Vitamin

K2. The method includes: using cow milk as a fermentation substrate, and using the lactobacillus delbrueckii subsp. bulgaricus for fermentation, so as to obtain the Vitamin K2.

Still another aspect of the present application provides an application of the lactobacillus delbrueckii subsp. bulgaricus in preparation of food additives or fermented food.

The collection information of the lactobacillus delbrueckii subsp. bulgaricus in the present application includes the following. The lactobacillus delbrueckii subsp. bulgaricus is deposited with the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Yard 1, BeiChen West Road, Chaoyang District, Beijing on September 16, 2022 and is assigned with the accession number of CGMCC No. 25743, with the classification name being actobacillus delbrueckii subsp. bulgaricus.

The present application has the following beneficial effects. The lactobacillus delbrueckii subsp. bulgaricus provided in the present application may produce the Vitamin K2 by being used as a starter to ferment the cow milk, and the yield may reach up to 181.02+10.58 mg/L.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows colonial morphology of TY-V16.

Fig. 2 shows a gram staining result of TY-V 16.

Fig. 3 shows a Vitamin K2 standard curve and a regression equation.

Fig. 4 shows the content of Vitamin K2 in 30 lactobacillus fermentation broths.

Fig. 5 shows growth curves of TY-V 16 under different culture conditions.

Fig. 6 shows the content of Vitamin K2 in fermentation broths of TY-V16 fermented under different culture conditions.

Fig. 7 shows a curd picture of TY-V16-fermented cow milk.

Fig. 8 shows changes in a pH value of TY-V16-fermented cow milk.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The embodiments are given to better describe the present application, but the content of the present application is not limited only to the embodiments given. Therefore, non-essential improvements and adjustments to the embodiments made by a person skilled in the art in accordance with the content of the above present application still fall within the scope of protection of the present application.

The terms used herein are only intended to describe specific embodiments and are not intended to limit the present disclosure. Expressions in the singular form include those in the plural form unless the expressions have a distinctly different meaning in the context. As used herein, it is to be understood that terms such as "include", "have", "contain", and the like are intended to indicate the presence of features, figures, operations, components, parts, elements, materials, or combinations. The terms of the present application are disclosed in the specification and are not intended to exclude the possibility that one or more other features, figures, operations, components, parts, elements, materials, or combinations thereof may exist or may be added. As used here, "/" may be interpreted as "and" or "or", as appropriate.

In the present application, an MRS liquid culture medium is made by successively adding 10.0 g of peptone, 10.0 g of a beef extract, 5.0 g of a yeast extract, 20.0g of glucose,

5.0 g of sodium acetate, 2.0g of citric acid diamine, 1.0g of Tween (Tween 80), 0.4 g of potassium phosphate dibasic, 0.58 g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0 g of calcium carbonate, and 15.0 g of agar to distilled water, making up the distilled water to 1000 mL, adjusting a pH value to 6.3, performing stirring, then performing heating, performing boiling for 2 min, and performing sterilization for 30 min at 0. 1Mpa.

An embodiment of the present application provides a lactobacillus delbrueckii subsp. bulgaricus, which is assigned with the accession number of CGMCC No. 25743.

It is to be noted that, the lactobacillus delbrueckii subsp. bulgaricus (which is also called lactobacillus delbrueckii subsp. bulgaricus TY-V16 or TY-V16) is derived from naturally-fermented yak yogurt from a herdsman on the Qinghai-Tibet Plateau. After being detected by means of gram staining results, the lactobacillus delbrueckii subsp. bulgaricus is found to be in a rod-like shape, and determined as gram-positive bacteria (G+). In addition, by means of PCR amplification of a 16SrDNA sequence, the 16SrDNA sequence is detected to include a sequence shown as SEQIDNO.1, and by means of homology analysis, it indicates that the strain is lactobacillus delbrueckii subsp. bulgaricus.

Another embodiment of the present application provides a composition. The composition includes one or a combination of a plurality of the following substances: (a) the lactobacillus delbrueckii subsp. bulgaricus; (b) lysate of the lactobacillus delbrueckii subsp. bulgaricus; (Cc) a culture of the lactobacillus delbrueckii subsp. bulgaricus; and (d) a fermentation broth of the lactobacillus delbrueckii subsp. bulgaricus.

It is to be noted that, in the composition, as described above, the lactobacillus delbrueckii subsp. bulgaricus has desirable survival rate in the digestive tract of the body, such that the lactobacillus delbrueckii subsp. bulgaricus may be prepared into an edible or medicinal composition. In addition, when the lactobacillus delbrueckii subsp. bulgaricus is prepared into the composition, the lactobacillus delbrueckii subsp. bulgaricus may achieve an effect by being directly introduced into the composition in the form of viable bacteria, may achieve an effect by being introduced into the composition in the form of inactivated bacteria after inactivation by means of the existing technology, may achieve an effect by introducing the lysate of the lactobacillus delbrueckii subsp. bulgaricus into the composition, may achieve an effect by introducing products such as proteins, peptides, secretions or metabolites obtained from the culture of the lactobacillus delbrueckii subsp. bulgaricus into the composition, or may achieve an effect by introducing the fermentation broth from the fermentation of the lactobacillus delbrueckii subsp. bulgaricus into the composition. In a specific use process, different forms of the lactobacillus delbrueckii subsp. bulgaricus may be selected for the preparation of the composition according to specific requirements.

In some specific embodiments, the composition further includes one or a combination of probiotics, prebiotics, dietary fiber and traditional Chinese drugs.

It is to be noted that, the lactobacillus delbrueckii subsp. bulgaricus and different forms thereof may also be used in combination with one or the combination of probiotics, dietary 5 fiber and a pharmacologically active compound. For example, the lactobacillus delbrueckii subsp. bulgaricus may be used in combination with bacillus subtilis, bifidobacterium or lactobacillus, so as to cause the composition to simultaneously have the effects of the lactobacillus delbrueckii subsp. bulgaricus and other probiotics. For another example, the lactobacillus delbrueckii subsp. bulgaricus may be used in combination with prebiotics, and the prebiotics may provide an energy source for the lactobacillus delbrueckii subsp. bulgaricus, such that the effect of the lactobacillus delbrueckii subsp. bulgaricus may be improved. For another example, the lactobacillus delbrueckii subsp. bulgaricus may be used in combination with the dietary fiber, and the dietary fiber may assist in colonization of the lactobacillus delbrueckii subsp. bulgaricus, such that the effect of the lactobacillus delbrueckii subsp. bulgaricus may be improved. For another example, the lactobacillus delbrueckii subsp. bulgaricus may be used in combination with the traditional Chinese drugs to form the composition, such that the effects of the lactobacillus delbrueckii subsp. bulgaricus and the traditional Chinese drugs may be simultaneously achieved.

Still another embodiment of the present application provides a preparation. The preparation includes the lactobacillus delbrueckii subsp. bulgaricus or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.

It is to be noted that, drugs or edible food or health care products may be prepared by adding the medicinal carrier or the edible carrier to the composition including the lactobacillus delbrueckii subsp. bulgaricus and different forms thereof. The medicinal carrier or the edible carrier is known in the art and may be selected according to the dosage form as needed. For example, the preparation of tablets mainly uses a diluent (such as starch, dextrin, sucrose or sugar), an absorbent (such as calcium sulfate, calcium hydrogen phosphate or light magnesium oxide), an adhesive (such as povidone, syrup or hydroxypropyl methylcellulose), a wetting agent (such as water), or a disintegrating agent (such as dry starch, sodium hydroxymethyl starch or cross-linked povidone). For example, the preparation of the liquid preparation mainly uses a bulking agent, a suspending agent, an emulsifying agent, a colorant, or the like.

In some specific embodiments, in addition to the tablets and the liquid preparation, the preparation may further include pills, capsules, powder, gel, and granules. It is to be noted that, solid dosage forms such as the tablets, the pills, the granules or the capsules may be product forms such as probiotic tablets, probiotic sugar pills, probiotic powder or probiotic capsules.

The liquid preparation may be a product form such as a probiotic beverage. The gel may be product forms such as probiotic jelly, probiotic milk foam or solidified yogurt.

Still another embodiment of the present application provides a method for preparing

Vitamin K2. The method includes: using cow milk as a fermentation substrate, and using the lactobacillus delbrueckii subsp. bulgaricus for fermentation, so as to obtain the Vitamin K2.

It is to be noted that, the cow milk is a nutrient-rich natural culture medium. The lactobacilli can use the cow milk to produce a large amount of lactic acid, and are recognized as safe food-level microorganisms. Therefore, the lactobacillus delbrueckii subsp. bulgaricus in the present application may produce the Vitamin K2 by using the cow milk as the fermentation substrate, and fermented products can also be obtained. It is further to be noted that, the cow milk may be mixed milk or raw milk.

In some specific embodiments, in the method for preparing Vitamin K2, before fermentation, enrichment is performed on the lactobacillus delbrueckii subsp. bulgaricus by using a culture medium; and the culture medium is made by adding 55 g/L-65 g/L of glycerol and 8 g/L-12 g/L of sucrose in the MRS liquid culture medium.

It is to be noted that, after 55 g/L-65 g/L of the glycerol and 8 g/L-12 g/L of the sucrose are added to the MRS liquid culture medium, culturing the lactobacillus delbrueckii subsp. bulgaricus may increase the number of viable bacteria and the yield of the Vitamin K2; and compared with culture using the MRS liquid culture medium, the number of viable bacteria may be increased by approximately 31.7%, and the yield of the Vitamin K2 may be increased by approximately 58.9%, subsequently.

In some specific embodiments, in the method for preparing Vitamin K2, on the basis of addition of the glycerol and the sucrose in the MRS liquid culture medium, peptone in the

MRS liquid culture medium may also be replaced with an equal amount of soybean peptone, tryptone, bacterial peptone, casein peptone, or proteose peptone, and the number of viable bacteria and the yield of the Vitamin K2 may also be increased compared with an original

MRS liquid culture medium. Compared with the peptone in the liquid culture medium being not replaced, the number of viable bacteria when the peptone in the liquid culture medium is replaced with an equal amount of the tryptone and the proteose peptone is similar, and the yield of the Vitamin K2 is slightly increased; the number of viable bacteria when the peptone in the liquid culture medium is replaced with an equal amount of the bacterial peptone, the casein peptone and the soybean peptone and the number of viable bacteria when the peptone in the liquid culture medium is not replaced are both obviously increased, and the yield of the

Vitamin K2 is obviously increased; and preferably, compared with the peptone in the liquid culture medium being not replaced, the number of viable bacteria when the peptone in the liquid culture medium is replaced with the soybean peptone is increased by approximately 5%, and the yield of the Vitamin K2 may be increased by approximately 18.6%, subsequently, reaching 341.29+31.05 mg/L. In addition, compared with a common culture medium, in the culture medium with the peptone being replaced with the soybean peptone and added with the glycerol and the sucrose, the yield (181.02+10.58 mgL) of the Vitamin K2 prepared after enrichment is increased by approximately 88.54%. The fermentation time of the culture medium may be shortened from 40h of the common culture medium to 24h at later fermentation, such that 341.29+31.05 mg/L of the yield of the Vitamin K2 may be reached, the fermentation time is shortened by approximately 40%, thereby shortening a production cycle. Therefore, costs of raw materials, water, electricity and steam for fermentation may be effectively reduced, and the preparation method has high economic benefits.

Still another embodiment of the present application provides an application of the lactobacillus delbrueckii subsp. bulgaricus in preparation of food additives or fermented food.

Itis to be noted that, the lactobacillus delbrueckii subsp. bulgaricus in the present application may be prepared into a food additive for fermentation, or the lactobacillus delbrueckii subsp. bulgaricus in the present application is applied to preparation of fermented food, and the fermented food may be known in the art, such as pickles, fermented legume products, or fermented milk products.

In some specific embodiments, in the above application, the fermented food includes fermented milk, cheese, or fermented milk beverages.

It is to be noted that, the lactobacillus delbrueckii subsp. bulgaricus in the present application uses pasteurized milk, HUT sterilized milk and reconstituted milk as fermentation substrates, with good fermentation curd status and high Vitamin K2 production, and has the potential to make the fermented milk beverages, and to prepare Vitamin K2 yogurt starters in combination with Streptococcus thermophilus. Synergistic fermentation of the lactobacillus delbrueckii subsp. bulgaricus in the present application and commercial starters has no significant effect on pH values, acidity, water-holding capacity, clarification indexes, tissue states such as texture, etc., and also has no effect on the overall consumer acceptance of yogurt, such that the lactobacillus delbrueckii subsp. bulgaricus has good application aspects.

In addition, the lactobacillus delbrueckii subsp. bulgaricus also has the effect of calcium supplementation, such that the effect of the fermented milk, the cheese, or the fermented milk beverages on supplementing calcium may also be improved by applying the lactobacillus delbrueckii subsp. bulgaricus to preparation of the fermented milk, the cheese, or the fermented milk beverages.

In order to better understand the present application, the content of the present application is further described below with reference to specific embodiments, but is not only limited to the following examples.

Embodiment 1 Separation, purification and identification of TY-V16 (1) Experimental material

Naturally fermented yak yogurt from a herdsman on the Qinghai-Tibet Plateau; the homemade yak yogurt from the herdsman was taken by using a sterile spoon, and was put into a 15 mL sterile capped centrifuge tube containing a proper amount of sterile calcium carbonate and soluble starch (a ratio of the calcium carbonate to the soluble starch being 1:1).

The tube was screwed up after the mixture was uniformly stirred, then put into a freezer, and transported back to a laboratory for immediate purification and separation of lactic acid bacteria. (2) Purification and separation

Under sterile conditions, 1 mL of an initial sample was pipetted to 9 mL of sterile saline, and a 10° sample diluent was obtained after the mixture was well mixed through vortex. Then 10-fold gradient dilution was performed to 107. 100 pL of diluent at 10%, 10%, or 107 was selected, and uniformly spread on an MRS plate for inverted culture for 48h for 37°C. After culture was completed, colonial morphology on the MRS plate was observed. Typical colonies of lactobacilli were selected to purify strains by using a streak plate method, and this streaking operation was repeated until the purified strains were obtained. (3) Morphological structure observation

The purified strains were inoculated in a 5 mL sterile MRS broth for culture for 18h at 37°C. 1 mL of bacterial fluid was taken and centrifuged for 1 min at 12000 r/min. After the bacterial fluid was washed for two times with sterile saline, isochoric sterile saline was then added to resuspend the bacteria. A small amount of the bacteria was taken and spread uniformly on a glass slide by using an inoculating loop. After fixation, Gram staining, microscopic examination, and photographing were performed. Gram-positive bacteria (G+) cells stained were blue-purple, and Gram-negative bacteria (G-) cells were red. Cell morphology and Gram staining results were observed and recorded.

Cell morphology of the purified strains was shown in Fig. 1; single colonies were formed in a solid culture medium; and the colonies were large, circular and translucent and white, and has smooth and raised surfaces and irregular edges.

Results after gram staining were shown in Fig. 2 (grey processing was performed in Fig. 2, and an original drawing was purple). The cells were all purple under a microscope, such that the strains were determined as gram-positive bacteria (G+), with rod-like shapes.

The cell morphology of the strains conformed to features of the lactobacillus, and morphological structures were uniform, such that it indicated that the strains were pure. (4) PCR amplification of I6SrDNA sequence

PCR amplification was performed by using a 25 pL of a reaction system, including 1 pL of a template, 1 uL of each of primers (27F(TACGGYTACCTTGTTACGACTT SEQ ID

NO.2), 1492R( AGAGTTTGATCMTGGCTCAG SEQ ID NO.3)), and 12.5 uL of 2xTaq

PCR Master Mix, and making up to 25 pL with sterile ultrapure water. PCR amplification conditions included: performing pre-denaturation at 94°C for 5 min, performing denaturation at 94°C for 30 s, performing annealing at 55°C for 30 s, and performing extension at 72°C for 1 min, there being a total of 35 cycles; and performing end extension at 72°C for 10 min.

After sequence amplification, Sangon Biotech (Shanghai) Co., Ltd. was entrusted to sequence

PCR amplification products, and the obtained sequence was shown as SEQIDNo. 1; and after the sequence was obtained, searching and similarity comparison were performed in GeneBank by using BLAST(http://www.ncbi.nlm.nih.gov/BLAST). Results showed that the TY-V16 was lactobacillus delbrueckii subsp. bulgaricus, which was hereinafter referred to the lactobacillus delbrueckii subsp. bulgaricus.

Embodiment 2 Screening of TY-V16 for high production of Vitamin K2 (1) Experimental material

A lactobacillus delbrueckii subsp. bulgaricus TY-V16: separated from naturally fermented yak yogurt from a herdsman on the Qinghai-Tibet Plateau, deposited with the

CGMCC, and was assigned with the accession number of CGMCC No. 25743.

The strain was inoculated in the MRS liquid culture medium according to a 5% inoculation amount and cultured at 37°C for 18 h; after activation for three generations, 2 mL was taken and centrifuged for 15 min at 10000 r/min, so as to collect a bacterial precipitate; the bacterial precipitate was washed for 2 times with a sterile PBS solution, and resuspended in isochoric sterile saline, so as to obtain bacterial suspension; the well prepared bacterial suspension was added to the HUT sterilized milk by 5%, and standing and fermentation were performed for 48h at 42°C, so as to obtain a single bacterial fermented yogurt sample; and then ultrasonication was performed on the sample for 5 min, and after the sample was cooled, ultrasonication was repeated twice, so as to complete release menaquinone substances. (2) Vitamin K2 extraction

Enzymolysis: 20 mL (accurate to 0.1 mL) of the yogurt sample was accurately weighed, 5 mL of a phosphate buffer was added, then well mixing was performed, and 0.2 g of lipase (accurate to 0.01g) was added. After vortex and well mixing, shaking was performed for 2-4h at 300 r/min in a (37£2)C thermostatic water bath oscillator, so as to achieve full enzymolysis.

Extraction: an enzymatically-hydrolyzed test solution was taken, and 20 mL of an extracting solution (n-hexane:isopropanol=2:1) and 1g of potassium carbonate powder were respectively added. After well mixing, the mixture was transferred to a 150 mL separatory funnel; vibrating extraction was performed for 10 min at 300r/min; supernatant was transferred to a 100 mL rotary evaporation bottle, and repeated extraction was performed for at least once; and the supernatant was merged into the rotary evaporation bottle.

Concentration: rotary evaporation was performed on the n-hexane extracting solution at 50°C until almost dry (if there was residual liquid, the residual liquid was blown to almost dry with nitrogen); making up was performed with 1 mL of methanol; filtration was performed by using a 0.22 um filter membrane; and measurement was immediately performed on machine. (3) Vitamin K2 measurement

In the following embodiments, the Vitamin K2 measurement was performed by means of high performance liquid chromatography with fluorescence detection; liquid chromatography conditions included: chromatographic column: C18 column with a length being 25 cm and internal diameter being 4.6 mm, a particle size of a filler being 5 um or a chromatographic column with equivalent performance; zinc powder reduction column: a column length being 50 mm, and an internal diameter being 4.6 mm; detection wavelength: an excitation wavelength being 243 nm, and an emission wavelength being 430 nm; sample size: 10 pL; flow rate: 1 mL/min, mobile phase: dissolving 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 performing filtration using a 0.22 um organic filter membrane.

Vitamin K2 standard curve plotting: a Vitamin K2 (MK-7) (CicHssO2, CAS No.: 2124-57-4) standard; standard substances certified by the state and awarded a standard substance certificate, with purity being 98%; and Vitamin K2 (MK-7) standard series working solutions: respectively accurately pipetting 20 pL, 50 pL, 100 pL, 200 pL, 500 uL and 1000 pL of a Vitamin K2 (MK-7) intermediate solution, and making up to 1 mL by using methanol, so as to prepare the Vitamin K2 (MK-7) standard series working solutions with concentrations being 20pg/L, 50 pg/L, 100 pg/L, 200 pg/L, 500 pg/L, and 1000 pg/L. High performance liquid chromatography detection was performed, and high performance liquid chromatogram of the Vitamin K2 (MK-7) solution was shown in Fig. 3. A standard curve was plotted by using the peak area of the Vitamin K2 (MK-7) as a longitudinal coordinate and the concentration of a standard solution as a horizontal coordinate, so as to obtain a standard curve regression equation, and the Vitamin K2 standard curve was shown in Fig. 4.

Measurement of content of Vitamin K2: the mass fraction of the Vitamin K2 (MK-7) in the sample was calculated according to the following formulas.

In the formula:

X-content of the Vitamin K2 in the sample, in micrograms per milliliter (ug/mL). p-concentration of the Vitamin K2 in a test solution obtained by the standard curve, in nanograms per milliliter (ng/mL). v-final fixed volume of a sample solution, in milliliter (mL).

V-sample volume, in mL. 1000-Conversion factor for converting a concentration unit from ng/mL to ug/mL.

Calculation results are kept in three valid digits.

Measurement of the yield of the Vitamin K2 was performed, according to the above method, on a total of 200 lactobacilli (among which had 40 strains of lactobacillus plantarum, 40 strains of lactobacillus fermentum, 40 strains of lactobacillus casei, 40 strains of a lactobacillus delbrueckii subsp. bulgaricus, 20 strains of Lactococcus lactis, and 20 strains of lactobacillus helveticus) from different species and genera in a probiotic bank owned by

Tianyou Dairy (the strains were all from samples of yak yogurt and cheese collected from places such as Qinghai-Tibet Plateau and Tibet, purification and separation and 16sRNA sequence identification were performed on the lactobacilli in the samples, and the strains were frozen, dried and preserved in a strain bank; and the strains preserved were all edible safe strains). 30 strains (20 mg/L of the yield of the Vitamin K2) with a certain amount of the

Vitamin K2 yield were preliminarily screened, and among them, the TY-V16 (that is LDSB-7 in Fig. 5) was the optimal, the yield reached 181.02+10.58 mg/L, and an experimental result was shown in Fig. 5. The TY-V16 fermentation broth had the highest content of the Vitamin

K2 and was stable in yield, such that it was determined that the strain had the capability of producing the high-yield Vitamin K2. Therefore, the strain TY-V16 was used as a research object to discover application research of the strain in yogurt.

Embodiment 3 Optimization of fermentation conditions of TY-V16 for high production of Vitamin K2 (1) Experimental material

CGMCC, and was assigned with the accession number of CGMCC No. 25743. Since growth performance was poor in a non-anaerobic and common MRS culture medium, the yield of the

Vitamin K2 was relatively low.

(2) Culture medium preparation 60g/L of glycerol and 10g/L of sucrose were added in an MRS liquid culture medium as new carbon sources, peptone in the MRS culture medium was separately replaced with tryptone, proteose peptone, casein peptone, bacterial peptone and soybean peptone in equal amounts (10 g/L), and a common MRS liquid culture medium was used as a control; and the strain was inoculated, according to a 5% inoculation amount, in the MRS liquid culture medium with the carbon source added and a nitrogen source replaced, and anaerobic culture was performed for 18h at 37°C, and culture was performed continuously for three generations. (3) Counting of viable strains

A plate count method was used for counting viable lactobacilli; after three generations of continuous culture, 10uL of bacterial fluid was added to a plate of a sterile MRS agar culture medium in a suitable gradient; anaerobic culture was performed for 24h-48h at a constant temperature of 37°C, then plate count was performed, with three parallel controls per gradient.

The reason that the yield of the Vitamin K2 was different may be an increase in the number of live bacteria, causing an increase in the total yield of the Vitamin K2, or may be the constant number of viable bacteria, such that the capability of the strain to produce the

Vitamin K2 was improved. In order to discover the reason why the capability of the lactobacillus to produce the Vitamin K2 was improved, counting of viable bacteria was performed, and results were shown in Table 1.

Table 1 Viable bacteria counting of TY-V16 under different fermentation conditions

ESE Eb

Carbon Carbon source ntation|(before source + source ~~ Hsource Hsource + source ++ bacterial conditijoptimization |original casein proteose [soybean tryptone peptone on ) peptone peptone |peptone [peptone

Viable count/l 6.3+0.1 8.3+0.1 8.4+0.0 8.9+0.2 8.840.0 8.0+0.1 8.7+0.2 g(CFUI )

From Table 1, it may be learned that, after the TY-V16 was subjected to carbon source addition, light avoidance, and anaerobic culture, the number of live bacteria has increased by two orders of magnitude, and growing capability of the TY-V16 was significantly improved.

Under different nitrogen source conditions, the number of live bacteria was on the same order of magnitude, such that it indicated that the growing capability of the strain was not affected by different nitrogen sources, but the capability of producing the Vitamin K2 was improved.

(4) Measurement of fermentation time of strain 2 mL of the strains cultured under different culture conditions was respectively taken, and centrifuged for 15 min at 10000 r/min, so as to collect a bacterial precipitate; and the bacterial precipitate was washed for 2 times with a sterile PBS solution, and resuspended in 1sochoric sterile saline, so as to obtain bacterial suspension. The well prepared bacterial suspension was added to the HUT sterilized milk by 5%, standing and fermentation were performed at 42°C, so as to obtain a single bacterial fermented yogurt sample, and the time for pH reaching 4.25 was recorded. Fermentation time of the TY-V16 under different fermentation conditions was shown in Table 2.

Table 2 Fermentation time under different fermentation conditions

Ferment [Control Carbon Carbon Carbon sourcelCarbon source/Carbon Carbon source ation (before source +source ++ bacterial+ caseinjsource ++ soybean conditio |optimizatio [peptone tryptone [peptone peptone proteose peptone

Ferment ation 40 35 33 28 27 32 24 time (h)

Note: the peptone in a Carbon source + peptone group in Table 2 is the peptone in the original MRS liquid culture medium. (5) Measurement of content of Vitamin K2

Ultrasonication was performed on the single bacterial fermented yogurt sample to crush cells, so as to release menaquinone substances, and extraction and measurement of the

Vitamin K2 were performed by means of fluorescence high performance liquid chromatography.

The capability of the same lactobacillus to produce the Vitamin K2 under different fermentation conditions was different. The peptone was formed by means of decomposition of proteins via acids, bases, or proteases, and was rich in organic nitrogen compound, such that the peptone may provide the nitrogen source for the growth of microorganisms. The yield of the Vitamin K2 produced by the TY-V16 under different nitrogen source conditions was shown in Fig. 6. In the original MRS liquid culture medium, the yield of the Vitamin K2 produced by the TY-V16 was 181.02+10.58 mg/L, and the fermentation time was 40h. After the glycerol and the sucrose were added in the MRS liquid culture medium as the new carbon sources, the yield of the Vitamin K2 was significantly increased, and the fermentation time was significantly shortened. In addition, the peptone in the MRS culture medium was separately replaced with an equal amount of soybean peptone, tryptone, bacterial peptone,

casein peptone, and proteose peptone; and compared with the yield of the Vitamin K2 of the original MRS liquid culture medium, the yield of the Vitamin K2 after the glycerol and the sucrose were respectively added as the new carbon sources was significantly increased, and the fermentation time was also significantly shortened. Preferably, the yield of the Vitamin K2 of the culture medium, after enrichment, with the soybean peptone replaced and the glycerol and the sucrose added reached 341.29+31.05 mg/L, which was increased by 88.54% compared with the original MRS liquid culture medium, and a fermentation endpoint was reached by only taking about 24 hours, thus shortening about 40% of the fermentation time

Embodiment 4 Test on fermentation performance of TY-V16 (1) Experimental material

CGMCC, and was assigned with the accession number of CGMCC No. 25743. (2) Single bacterial fermented yogurt preparation

The lactobacillus delbrueckii subsp. bulgaricus strains were respectively inoculated in the optimized liquid culture medium according to a 5% inoculation amount and cultured at 37°C for 24h; after activation for three generations, 2 mL was taken and centrifuged for 10 min at 8000 r/min, so as to collect a bacterial precipitate; and the bacterial precipitate was washed for 2 times with a sterile PBS solution, and resuspended in isochoric sterile saline, so as to obtain bacterial suspension The well prepared bacterial suspension was respectively added to pasteurized milk, HUT sterilized milk and milk powder reconstituted milk according to a final concentration of 105 CFU/L, and fermentation was performed for 18h at 42°C, so as to obtain single bacterial fermented yogurt. (3) Test on acid production capability of TY-V16

The TY-V16 fermented the pasteurized milk, the HUT sterilized milk and the milk powder reconstituted milk (milk powder: water being 1:9, that is, the concentration of the milk powder being 10%); and results were shown in Fig. 7, which were the fermentation of the milk powder reconstituted milk, the HUT sterilized milk and the pasteurized milk from left to right, respectively. The TY-V16 was good in fermentation tissue state and uniform in color, and had no peculiar smell in the milk powder reconstituted milk, the HUT sterilized milk and the pasteurized milk.

Changes in pH of the milk powder reconstituted milk, the HUT sterilized milk, the pasteurized milk and the fermented fermentation broth were shown in Fig. 8. The pH of milk solutions after the milk powder reconstituted milk, the HUT sterilized milk and the pasteurized milk were fermented by using the lactobacillus delbrueckii subsp. bulgaricus in the present application was finally kept at about 4.25 (which was a final pH value suitable for yogurt), and acidity was reduced quickly, such that the lactobacillus delbrueckii subsp. bulgaricus may be used as a starter for preparing the yogurt.

The above data showed that, the lactobacillus delbrueckii subsp. bulgaricus in the present application may be used as an excellent strain for making fermented yogurt, and had the potential of preparing Vitamin K2 fermented milk beverages.

Embodiment 5 Impact of TY-V16 on quality of yogurt (1) Experimental material

CGMCC, and was assigned with the accession number of CGMCC No. 25743.

A traditional commercial starter (lactobacillus delbrueckii subsp. bulgaricus and streptococcus thermophilus): purchased from Danisco (China). (2) Yogurt making

The TY-V16 was inoculated in the optimized liquid culture medium according to a 5% inoculation amount and cultured at 37°C for 18h; after activation for three generations, 2 mL was taken and centrifuged for 15 min at 10000 r/min, so as to collect a bacterial precipitate; and the bacterial precipitate was washed for 2 times with a sterile PBS solution, and resuspended in isochoric sterile saline, so as to obtain bacterial suspension. In one group, the well prepared bacterial suspension and 6 g/L of a commercial starter were simultaneously added into the HUT sterilized milk according to 10°CFU/L; in the other group, the commercial starter was added into the HUT sterilized milk according to 6 g/L as a control; and fermentation was performed on two groups for 7h at 42°C simultaneously, and the state and quality of the yogurt in the two groups was compared after cold storage and after-ripening. (3) Measurement of impact of TY-V 16 on physical and chemical quality of yogurt (D Acidity measurement

Measurement of the pH of the yogurt was performed by using a precise pH meter.

During storage of the yogurt, measurement of titratable acidity refers to GB 5009.239-2016 “National Food Safety Standard for Measurement of Food Acidity”. 2) Measurement of Water Holding Capacity (WHC) of yogurt 20 g of a sample was accurately weighed, centrifugation was performed for 20 min at 4000 r/min, the mass of a precipitate was weighed, and WHC was calculated according to an equation (1).

Mass: of precipitate:

WHC% TOG (1)

Mass of samples

Most probiotic lactobacilli had a strong acid production capability and metabolized different products, affecting the nature of the yogurt, such that when applying the probiotic lactobacilli to produce yogurt products, attention should be paid to the impact of the lactobacilli on the quality of products. In the embodiments of the present application, the lactobacillus TY-V16 for high production of the Vitamin K2 was used as an assisted starter to produce the yogurt. By means of comparison of fermentation of the commercial starter (lactobacillus delbrueckii subsp. bulgaricus or streptococcus thermophilus), it was discovered that the TY-V16 had no effect on the curding time, pH values, acidity, WHC, clarification indexes, and tissue states of yogurt samples; and details were shown in Table 3 below.

A texture analyzer (TA XTplus, Micro Stable System Co., UK) was used to measure the texture of the yogurt samples. A penetration test was performed by using a cylindrical probe with a diameter being 10 mm; the samples were immediately measured (4°C) after being taken from a refrigerator; and a penetration of 15 mm was performed on the yogurt sampels at a speed of 1 mm/s and a trigger force of 1 g. @ Yogurt stability measurement

An optical analysis centrifugal machine (LUMi Sizer L.U.M.GmbH, Germany) was used for measurement, and about 0.4mL of AMD was filled in a standard reaction cup. Measured instrument parameters were set as follows: a temperature was 25°C; a rotary speed was 4000 rpm; a time interval was 10 seconds; experimental period was 4490s; and each experiment was made in triplicate, and each result was expressed as clarification index average result + standard deviation. Results were shown in Table 4. The TY-V16 had no significant effect on physical and chemical indexes of the yogurt samples such as hardness, viscosity, elasticity, cohesion, adhesiveness, chewiness, and resilience.

Table 4 Yogurt texture

2.364 +4 78a 0.001a 0.002a 0.347a 0.111a 0.000a 3.34a +8.67a |0.001a 0.001a 0.512a 0.163a 0.001a ©) Measurement of impact of TY-V 16 on sensory quality of yogurt

According to the Chinese dairy industry specification RHB103-2004 "Rules for Sensory

Quality Evaluation of Yogurt", sensory evaluation was conducted using an acceptance test method, with 30 sensory evaluators (15 men and 15 women) scoring the appearance, color, aroma, texture, taste and overall acceptance of the yogurt samples respectively on the basis of their habits and interests for consuming the yogurt, with a total score of 9 (1=very dislike; 3=dislike; 5=neither like nor dislike; 7=like; 9=very like); and each result was expressed as average result + standard deviation.

Food sensory evaluation, also known as food sensory analysis, was an inspection and analysis process by using scientific analysis methods to sense food characteristics or properties by means of vision, smell, taste, and hearing, and performing qualitative and quantitative analysis of food. The metabolism of the probiotic lactobacilli produced different products that give the yogurt certain special flavors, directly affecting consumer acceptance and purchase intention, such that the metabolism of the probiotic lactobacilli was an important factor affecting quality of the products. In an example of the present application, the consumer acceptance test method was used. Results showed that there were no significant differences (P>0. 05) in 5 sensory indicators, which are appearance, color, texture, aroma, taste, and overall acceptance scores, of the yogurt in different groups, such that it indicated that the TY-V16 did not have adverse effects on the quality of the yogurt products. Details were shown in Table 5 below.

Table 5 Sensory evaluation table wee fo fle oom Jie oat

To sum up, the lactobacillus delbrueckii subsp. bulgaricus TY-V16 in the present application used for producing the Vitamin K2 had the characteristics of being mild in production condition, safe and non-toxic, and high in public acceptance, was desirable in fermentation performance, and did not affect the quality of the products during co-fermentation with the commercial yogurt starters, such that the TY-V16 had good application prospects. With the optimal fermentation culture medium and control processes, the fermentation unit may reach more than 341.29 mg/L, and the fermentation time may be shortened by 50%, such that the yield of the Vitamin K2 (MK-7) may be effectively increased, costs of raw materials, water, electricity and steam for fermentation may be reduced, and therefore, the TY-V 16 has high economic benefits.

It is finally to be noted that, the above embodiments are merely for describing and not intended to limit the technical solutions of the present application. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and shall all fall within the scope defined by the claims of the present application.

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Claims

CONCLUSIONS

1. Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus delbrueckii subsp. bulgaricus) with the collection number CGMCC No. 25743.

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

3. Composition, characterized in that the composition contains one or a combination of a plurality of the following substances: (a) lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2 (b) a lysate of the lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2; (c) a culture of lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2; and (d) a fermentation broth of Lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2.

Composition according to claim 3, characterized in that the composition further contains one or more combinations of probiotics, prebiotics, dietary fiber and traditional Chinese medicine.

5. Preparation, characterized in that the preparation contains lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2 or the composition according to claims 3 or 4, and a carrier, wherein the carrier is a medicinal carrier or an edible carrier.

6. Method for preparing vitamin K2, characterized in that the method comprises: using cow's milk as a fermentation substrate, and using the lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2 prior to fermentation to obtain vitamin K2.

7. Method for preparing vitamin K2 according to claim 6, characterized in that enrichment of the lactobacillus delbrueckii subsp. is carried out before fermentation. bulgaricus using a culture medium; and the culture medium was obtained by adding 55g/L-659/L glycerol and 8g/L-12g/L sucrose to liquid MRS culture medium.

Method for preparing vitamin K2 according to claim 7, characterized in that the peptones in the liquid MRS culture medium are replaced by an equal amount of soy peptones, tryptones, bacterial peptones, casein peptones or proteose peptones.

9. Application of lactobacillus delbrueckii subsp. bulgaricus according to claim 1 or 2 in the preparation of food additives or fermented foods.

10. Application of lactobacillus delbrueckii subsp. bulgaricus according to claim 9, characterized in that the fermented foodstuffs comprise fermented milk, cheese or fermented milk drinks.