A strain of aroma-producing Lactobacillus bulgaricus isolated from Dangxiong milk of Tibet and its application

Abstract

The present invention provides a flavor-producing Lactobacillus bulgaricus isolated from Tibet Dangxiong milk and its application. The Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 2022316. The Lactobacillus bulgaricus provided by the present invention metabolizes to produce abundant flavor substances such as aldehydes, ketones, alcohols, esters, etc. during yogurt fermentation.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and specifically relates to a fragrance-producing Lactobacillus bulgaricus isolated from Tibetan Dangxiong Naila and an application thereof. Background Art

[0002] The strain of yogurt starter is the core technology of yogurt product production. Among them, Lactobacillus bulgaricus, as the main component of yogurt starter, is crucial to the formation of yogurt flavor. The flavor substances acetaldehyde and diacetyl produced mainly give yogurt a fresh and fragrant taste.

[0003] Lactobacillus delbrueckii subspecies bulgaricus, commonly known as Lactobacillus bulgaricus, is one of the main lactic acid bacteria used in yogurt production. Lactobacillus bulgaricus has a very important health care effect on the human body. It has the functions of promoting the growth and colonization of beneficial bacteria, clearing the intestines, resisting diarrhea, maintaining gastrointestinal health, promoting digestion and absorption, increasing immunity, and anti-cancer, anti-tumor and other important physiological functions. It is widely used in food fermentation, industrial lactic acid fermentation, feed industry and medical care. Summary of the invention

[0004] The strains of fermented yogurt in the prior art have poor performance in producing flavor substances and cannot meet people's requirements for food nutrition and taste. In view of the problems existing in the prior art, the present invention provides a Lactobacillus bulgaricus.

[0005] In a first aspect, the present invention provides a Lactobacillus bulgaricus Dangxiong LBⅢ strain, which is deposited in China Center for Type Culture Collection (CCTCC), the depository address of which is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO:M 2022316.

[0006] Preferably, the 16Sr DNA sequence of the strain is as shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention also provides a fermentation preparation method of the Lactobacillus bulgaricus agent, the method comprising the following steps: culturing the Lactobacillus bulgaricus strain.

[0008] Preferably, the preparation method comprises the following steps:

[0009] (1) amplifying and culturing the Lactobacillus bulgaricus strain;

[0010] (2) adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing the mixture at 32-34°C.

[0011] In a third aspect, the present invention further provides a bacterial agent, wherein the bacterial agent contains the Lactobacillus bulgaricus Dangxiong LBⅢ strain.

[0012] Preferably, the bacterial agent is obtained by the fermentation preparation method.

[0013] A fourth aspect is the use of the Lactobacillus bulgaricus or the bacterial agent in food or food additives.

[0014] Preferably, the food is a fermented dairy product, preferably one or a combination of two or more of yogurt, cheese, and fermented dairy beverage.

[0015] In a fifth aspect, the present invention provides a fermented dairy product, wherein the starter of the fermented dairy product contains the Lactobacillus bulgaricus Dangxiong LBⅢ strain.

[0016] Preferably, the fermented dairy product contains furfural.

[0017] Preferably, the fermented dairy product further contains acetaldehyde and / or diacetyl.

[0018] Preferably, the fermented dairy product contains furfural, acetaldehyde and diacetyl.

[0019] Preferably, the fermented dairy product contains 2.9-3.0 mg / L diacetyl, or preferably, the fermented dairy product contains 29-30 mg / L acetaldehyde.

[0020] Preferably, the fermented dairy product contains one or a combination of two or more of glycidol, 4-methyl-2-hexanone, 3-methyl-2-butanone, 4-ethyloctane, dodecane, 3-methyl-3-butene-1-ol, methoxyacetone, 4-methyloctane, vinyl ethyl ether, n-nonanal, cis-2-methylcyclohexanol, furfural, acetaldehyde, diacetyl, 3-p-tolyltetrahydrofuran, benzaldehyde, phenylacetaldehyde, terpineol, 3-methyl-2-(5H)-furanone, γ-crotonyl lactone, n-pentanol, 2,5-dimethylbenzaldehyde, NN-dibenzyloxycarbonyl-L-arginine, dimethyl sulfone, 2-methylbutylhexanoate and (+)-dibenzyloxy-L-tartaric anhydride,

[0021] Preferably, the fermented dairy product contains glycidol, 4-methyl-2-hexanone, 3-methyl-2-butanone, 4-ethyloctane, butyl acetate, 2-heptanone, dodecane, 3-methyl-3-butene-1-ol, methoxyacetone, 4-methyloctane, ethyl vinyl ether, dimethyl trisulfide, 2-nonanone, nonanal, cis-2-methylcyclohexanol, acetic acid, furfural, acetaldehyde, diacetyl, o-dichlorobenzene, isopropanol, 3-p-tolyltetrahydrofuran, benzaldehyde, n-octanol, amyl nitrite, 2-undecanone, isovaleric acid, phenylacetaldehyde, dimethylsilanediol, n-nonanol, isovaleric acid, terpineol, 3-methyl-2-(5H)-furanone, naphthalene, isovaleric acid, γ-crotonyl lactone, n-pentanol, 2,5-dimethylbenzaldehyde, 2-tridecanone, phenylethyl acetate, 3-methylvaleric acid, NN-dibenzyloxycarbonyl-L-arginine, dimethyl sulfone, phenylethyl alcohol, 2-methylbutyl hexanoate, 2-pentadecanone, octanoic acid, 3-methylvaleric acid, butyl-decanoic acid, decanoic acid, butyl-dodecalactone and (+)-dibenzyloxy-L-tartaric anhydride, one or a combination of two or more thereof,

[0022] Preferably, the fermented dairy product contains glycidol, 4-methyl-2-hexanone, 3-methyl-2-butanone, 4-ethyloctane, butyl acetate, 2-heptanone, dodecane, 3-methyl-3-butene-1-ol, methoxyacetone, 4-methyloctane, ethyl vinyl ether, dimethyl trisulfide, 2-nonanone, nonanal, cis-2-methylcyclohexanol, acetic acid, furfural, acetaldehyde, diacetyl, o-dichlorobenzene, isopropanol, 3-p-tolyltetrahydrofuran, benzaldehyde, n-octanol, amyl nitrite, 2-undecanone, isopentyl nitrite, 2-undecone, isopentyl nitrite, 2-undecone, 2-undecane ... Acid, phenylacetaldehyde, dimethylsilanediol, n-nonanol, isovaleric acid., terpineol, 3-methyl-2-(5H)-furanone, naphthalene, isovaleric acid, γ-crotonyl lactone, n-pentanol, 2,5-dimethylbenzaldehyde, 2-tridecanone, phenylethyl acetate, 3-methylvaleric acid, NN-dibenzyloxycarbonyl-L-arginine, dimethyl sulfone, phenylethyl alcohol, 2-methylbutylhexanoate, 2-pentadecanone, octanoic acid, 3-methylvaleric acid, butyl-decanoic acid, decanoic acid, butyl-dodecanolactone and (+)-dibenzyloxy-L-tartaric anhydride.

[0023] In a sixth aspect, the present invention provides a method for preparing the fermented dairy product, the method comprising the following steps: preparing a starter culture using the Lactobacillus bulgaricus Dangxiong LBⅢ strain or the bacterial agent, and then adding the starter culture into the emulsion for fermentation.

[0024] Preferably, the emulsion is an animal emulsion or a plant emulsion;

[0025] Preferably, the animal emulsion is one or a combination of two or more of cow's milk, goat's milk or horse's milk;

[0026] Preferably, the plant emulsion is soy milk.

[0027] The Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention is isolated from homemade dairy products of herders in Dangxiong County, Tibet (4350m above sea level). The strain has been deposited in the China Center for Type Culture Collection on March 25, 2022, with a deposit number of CCTCC NO:M 2022316. The Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention metabolizes to produce abundant flavor substances such as aldehydes, ketones, alcohols, esters, etc. in yogurt fermentation, and compared with commercially available yogurt starter, it produces furfural, one of the characteristic tomato flavor substances, giving yogurt a unique and obvious tomato flavor; the strain also produces acetaldehyde, a characteristic yogurt flavor substance, in high yield, giving yogurt a refreshing and delicious characteristic flavor. It can meet the ever-increasing requirements of consumers for the nutritional value and flavor taste of fermented yogurt. The yogurt starter prepared using the strain provided by the present invention can give yogurt a pleasant flavor and have nutritional functions.

[0028] Strain deposit information

[0029] The Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on March 25, 2022, with a deposit number of CCTCC NO: M2022316, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is the microscopic examination of Lactobacillus bulgaricus Dangxiong LBⅢ strain;

[0031] Figure 2 Shown is the phylogenetic tree constructed for the Lactobacillus bulgaricus Dangxiong LBⅢ strain;

[0032] Figure 3 Shown are the acetaldehyde and diacetyl contents of yogurt fermented with different strains;

[0033] Figure 4 The figure shows the peak diagram of flavor substances detected in yogurt fermented by different strains, among which: Figure 4 (a) shows the peak diagram of flavor substances detected in yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain of the present invention, Figure 4 (b) shows the peak diagram of flavor substances detected in yogurt fermented by HS-2 strain. Figure 4 (c) shows the peak diagram of flavor substance detection in yogurt fermented by YR-4 strain. DETAILED DESCRIPTION

[0034] The strain Dangxiong LBⅢ provided by the present invention is Lactobacillus bulgaricus, which has been deposited in the China Center for Type Culture Collection on March 25, 2022, with a deposit number of CCTCC NO: M 2022316. The strain provided by the present invention can meet the ever-increasing requirements of consumers for the nutritional value and flavor of fermented yogurt.

[0035] The lactic acid bacteria provided by the present invention are isolated from homemade milk pulled by herders in Dangxiong County, Tibet (4350m above sea level). 16Sr RNA identification shows that it is Lactobacillus delbrueckii subspecies bulgaricus, a natural wild microbial strain, and belongs to the food microorganisms listed in the "List of Bacteria That Can Be Used in Food" promulgated by the Ministry of Health in 2010. It can be widely used in various fermented dairy products and has food safety attributes.

[0036] In one embodiment of the present invention, the preparation of fermented dairy products by fermentation using the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention comprises the following steps:

[0037] (1) Activation of Lactobacillus bulgaricus Dangxiong LBⅢ strain;

[0038] (2) Preparation of fermented dairy product starter;

[0039] (3) Production of fermented dairy products.

[0040] In the specific embodiment provided by the present invention, the flavor substances in fermented dairy products are detected by GC-MS method. The flavor substances in yogurt were detected by GC-MS, and it was found that the Bulgarian Lactobacillus Dangxiong LBⅢ strain metabolized 50 flavor substances, which were rich in flavor substances such as aldehydes, ketones, alcohols, acids, and esters, while the flavor substances of the two control strains (commercially available yogurt starters) were 36 and 39 respectively; and only the Bulgarian Lactobacillus Dangxiong LBⅢ strain fermented yogurt was detected to contain the key substance of tomato characteristic flavor - furfural; the strain also produced a high amount of acetaldehyde, a characteristic flavor substance of yogurt, and its acetaldehyde content was 1.2 times that of yogurt made by commercial starters, giving the yogurt a more refreshing and delicious characteristic flavor.

[0041] The source information of reagents and instruments used in the examples of the present invention is shown in Table 1 below.

[0042] Table 1

[0043]

[0044]

[0045] Example 1 Isolation and identification of strains

[0046] The isolation and identification of Lactobacillus bulgaricus Dangxiong LBⅢ strain was carried out as follows:

[0047] (1) Strain enrichment: Take 5 g of homemade milk sample from herders in Dangxiong County, Tibet (4350 m above sea level), grind it and add 45 ml of sterile saline to disperse it evenly. Take 2 ml of the above dispersion and add it to 20 ml of sterilized litmus milk medium. Place it in a 37°C incubator and culture it. When the medium is acidified and solidified and turns pink, take it out.

[0048] (2) Cultivate a single colony: dilute the litmus milk culture medium to 10 -5 , apply 100 μl to BL solid medium plate, place in 37℃ incubator and wait for colonies to form. On BL plate, colonies are smooth, slightly raised, moist, with rough edges, colorless to slightly white, 1-3mm in diameter, and slightly yellow on the back of colonies.

[0049] (3) Screening of acid-producing bacteria: The single colonies obtained from the initial screening were streaked on BL solid culture medium plates containing 0.2% CaCO3 and cultured in a 37°C incubator for 36 h. After the culture was completed, the colonies were observed for the presence of transparent circles around them, and the single colonies with large transparent circles were selected for further separation and purification.

[0050] (4) Preliminary identification by Gram staining: Gram staining is performed on the rescreened strains. If the bacteria are stained purple, they are Gram-positive bacteria and are retained for subsequent research. If they are stained red, they are Gram-negative bacteria and are eliminated directly. The identification results show that the rescreened strains are Gram-positive bacteria, non-motile, without spores, flagella, or capsules. The bacteria are in the shape of thin rods, either single or in chains. This strain is the target strain. The morphological characteristics of the strains under microscopic examination are as follows: Figure 1 shown.

[0051] (5) Molecular biological identification of 16S rRNA: 16S rDNA sequencing and bacterial species identification were performed on the strains to be identified using a high-fidelity enzyme. The primer information and PCR amplification reaction system and conditions used are shown in Tables 2 and 3.

[0052] Table 2 Primer information

[0053]

[0054] Table 3 PCR amplification reaction system and conditions

[0055]

[0056] After the PCR product is qualified, it is sent to a sequencing company for sequencing. The measured 16S rRNA gene sequence is shown in SEQ ID NO.1, specifically:

[0057]

[0058] The sequencing results were compared by NCBI-BLAST and the phylogenetic tree was constructed as follows: Figure 2 .according to Figure 2 The phylogenetic tree shown identified the strain as Lactobacillus bulgaricus, named Lactobacillus bulgaricus Dangxiong LBⅢ strain. The obtained Lactobacillus bulgaricus Dangxiong LBⅢ strain was deposited in the China Center for Type Culture Collection (CCTCC) on March 25, 2022, with the deposit number CCTCC NO:M 2022316.

[0059] Example 2 Preparation of Lactobacillus bulgaricus Dangxiong LBⅢ bacterial powder

[0060] (1) Activation of Lactobacillus bulgaricus Dangxiong LBⅢ strain: Take the Lactobacillus bulgaricus Dangxiong LBⅢ strain stored in a -80°C frozen glycerol tube and place it in MRS liquid culture medium, culture it at 37°C for 12 hours, and transfer it twice continuously to complete the strain activation;

[0061] (2) Expansion of Lactobacillus bulgaricus Dangxiong LBⅢ strain: inoculate 2% of the activated strain into 70 ml of MRS liquid culture medium and culture at 37°C for 12 h;

[0062] (3) Cultivation of Lactobacillus bulgaricus Dangxiong LBⅢ strain: inoculate 2% of the culture strain into 3500 ml MRS liquid medium and culture at 37°C for 12 h;

[0063] (4) Isolation of Lactobacillus bulgaricus Dangxiong LBⅢ: centrifuge the culture solution at 5000 rpm for 10 min, discard the supernatant, and retain the precipitate;

[0064] (5) Freeze-dried liquid preparation: 10 g skim milk powder, 5 g maltodextrin, 5 g trehalose, 3 g glycerol, 12 g bacterial precipitate, 65 g pure water, mix well to prepare bacterial freeze-dried liquid, transfer to freeze-drying bottle, and pre-freeze at -40°C for 2 h.

[0065] (6) Lyophilization of Lactobacillus bulgaricus Dangxiong LBⅢ powder: Lyophilization was performed using a Labconco vacuum freeze dryer. The lyophilization procedure was as follows:

[0066] step Temperature(℃) Time (h) Vacuum degree(Pa) 1 -10 10 0 2 -8 10 0 3 -6 8 0 4 -3 6 0 5 0 4 0 6 3 4 0 7 6 4 0

[0067] Example 3 Preparation of yogurt by fermentation using Lactobacillus bulgaricus Dangxiong LBⅢ strain

[0068] (I) Preparation of yogurt

[0069] Take fresh milk, add edible sucrose with a weight ratio of 6.5%, mix well, preheat, homogenize and sterilize; cool to 40-50° C. and add to a fermentation tank, inoculate 50 mg / L of the lyophilized powder of the Lactobacillus bulgaricus Dangxiong LBⅢ strain prepared in Example 2 and commercially available yogurt starter YR-4 and HS-2 (home yogurt starter purchased on Tmall) respectively, and ferment at 37° C. for 12 h; after the fermentation is completed, refrigerate at 4° C. for 24 h to obtain yogurt.

[0070] (II) Sensory evaluation of yogurt

[0071] The sensory evaluation of yogurt was based on the "China Dairy Industry Standard RHB 103-2004 Yogurt Sensory Quality Evaluation Rules", and 7 people were invited to taste and score the yogurt samples fermented by different strains, and the final result was the average. The evaluation indexes and scoring standards of yogurt are shown in Table 4.

[0072] Table 4 Sensory scoring criteria for yogurt

[0073]

[0074]

[0075] The different strains used in the above yogurt fermentation are shown in Table 5. The steps and processes for preparing yogurt with each strain are the same as those for preparing yogurt with Lactobacillus bulgaricus Dangxiong LBⅢ strain fermentation.

[0076] Table 5 Sensory scores of yogurt fermented by different strains

[0077] Dangxiong LBⅢ HS-2 YR-4 Color 9.71 10.00 9.43 Sweet and sour ratio 10.00 9.57 8.86 Flavor 39.29 34.71 34.00 Taste and texture 39.00 38.71 37.86 Comprehensive score 98 92.99 90.15

[0078] As shown in the experimental results in Table 5, the yogurt fermented with the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention has the highest comprehensive score, especially in terms of flavor, the score of the Lactobacillus bulgaricus Dangxiong LBⅢ strain of the present invention is much higher than that of the control strain.

[0079] A sensory evaluation was conducted on the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain by a panel of 7 people, and the feedback was that the yogurt had a tomato flavor and was refreshing and delicious. The yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention had the highest comprehensive score, especially in terms of flavor, the Lactobacillus bulgaricus Dangxiong LBⅢ strain of the present invention scored much higher than the control strain (commercially available yogurt starter).

[0080] (III) Detection of acetaldehyde and diacetyl content of main flavor substances in yogurt

[0081] 1. Determination of acetaldehyde content

[0082] (1) Reagent preparation: Weigh 12.70g iodine into a 500mL beaker, add 40g potassium iodide, add appropriate amount of water to dissolve, transfer to a 1000mL brown volumetric flask, dilute to volume and shake well to prepare a 0.1mol / L iodine standard solution. Then accurately measure 10.00mL of 0.1mol / L iodine standard solution into a 100mL brown volumetric flask, dilute to volume with distilled water, shake well, and prepare the two solutions immediately before use.

[0083] (2) Sample determination: Take 5 mL of 1% NaHSO3 solution and place it in a 250 mL conical flask, add 25 mL of the supernatant of the sample (the yogurt prepared in (I)) centrifuged at 6000 rpm for 20 min, mix well and place at room temperature for 1 h, then add 1 mL of 1% starch solution, titrate with 0.1 mol / L iodine solution until it is close to light blue-purple, then titrate with 0.01 mol / L iodine solution until it is light blue-purple, and if the light blue-purple does not fade within 30 s, the above titrations are not counted. Then add 20 mL of 1 mol / L NaHCO3 solution, shake and mix thoroughly until the blue-purple color of the solution disappears, finally titrate with 0.01 mol / L iodine solution to the blue-purple endpoint, record the volume of standard iodine solution consumed, and each sample has 2 parallels, and a blank experiment is performed at the same time.

[0084] M=(V1-V2)*0.022*C*10 6 / 25

[0085] In the formula: M is the mass concentration of acetaldehyde / (mg / L); V1 is the volume of iodine standard solution consumed by titration sample / mL; V2 is the volume of iodine standard solution consumed by titration blank / mL; C is the concentration of iodine standard solution / (mol / L); 25 is the sample weight / mL; 0.022 is the basic chemical unit of acetaldehyde reaction / g.

[0086] 2. Determination of diacetyl content

[0087] (1) Drawing of diacetyl standard curve: Dissolve 15 μL of diacetyl in distilled water and dilute to 500 mL. Measure 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of diacetyl standard solution into 12 numbered double-row test tubes, and make up to 10 mL with distilled water. Add 0.5 mL of 1% o-phenylenediamine solution to the first 6 test tubes, and do not add o-phenylenediamine solution to the last 6 test tubes. Mix thoroughly and react in the dark for 30 minutes. After the reaction is complete, add 4.0 mol / L hydrochloric acid to terminate the reaction (add 2.0 mL to the front test tube and 2.5 mL to the back test tube. After shaking, use the back test tube as a blank control. Use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 335 nm. Draw a standard curve with diacetyl concentration as the horizontal axis and absorbance as the vertical axis. According to the standard curve regression equation y = 0.116 2x + 0.040 7, R 2 =0.9999, calculate the diacetyl content.

[0088] (2) Determination of diacetyl content: Take the yogurt sample to be tested (the yogurt prepared in (I)), mix it with an equal volume of 16% trichloroacetic acid solution, let it stand for 10 minutes after mixing, and centrifuge it at 5500rpm for 10 minutes. Take 20mL of the supernatant and add equal volumes to two test tubes. Add 0.5mL of 1% o-phenylenediamine solution to test tube No. 1 and no solution to test tube No. 2. After fully mixing, let it react in the dark for 30 minutes. Then add 4.0mol / L hydrochloric acid to the two test tubes to terminate the reaction (2.0mL to test tube No. 1 and 2.5mL to test tube No. 2). After mixing, use test tube No. 2 as the control solution and measure the absorbance at a wavelength of 335nm. If the diacetyl content in the sample is high and the measured absorbance exceeds the range of 0.2 to 1.0, the sample can be appropriately diluted with 8% trichloroacetic acid.

[0089] Experimental results: Figure 3 As shown, the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention ( Figure 3 The acetaldehyde and diacetyl contents in the yogurt fermented by DB-3 (the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention) are higher than those in the yogurt fermented by the commercially available yogurt bacteria agents HS-2 and YR-4. The acetaldehyde content in the yogurt fermented by the strain provided by the present invention reaches 29.11 mg / L, and the diacetyl content reaches 2.993 mg / L. The high content of acetaldehyde gives the yogurt a rich flavor; studies have shown that when the ratio of acetaldehyde to diacetyl is greater than 3:1, the flavor of the yogurt is better.

[0090] The ratio of acetaldehyde to diacetyl in the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain of the present invention and the commercially available yogurt starter strain is greater than 3:1.

[0091] (IV) Detection of other flavor substances in yogurt

[0092] Sample pretreatment: Headspace solid-phase microextraction (HS-SPME) was used to extract and enrich the volatile odor compounds of the sample, and GC-MS was used to fully identify the volatile odor compounds (principle: when the odor compound is injected into the gas chromatograph, the substance separated by the chromatographic column enters the ionization chamber from the molecular separator and is bombarded into ions by electrons, and some of the ions enter the ion detector. After rapid mass spectrometry scanning, the mass spectrum of the component is derived as the basis for qualitative and quantitative analysis). The detection parameters are shown in Table 6 below. Weigh 3g of the prepared yogurt, add 2g of water, 1.5g of sodium chloride, and 1μl of internal standard o-dichlorobenzene (concentration 185μg / ml). The weighed sample was immediately sealed with a bottle cap with a silicone rubber pad, placed in a rotary oscillator, balanced at 50℃ for 30min, extracted at 50℃ for 30min with a DVB / CAR / PDMS composite extraction head, then injected and analyzed at 250℃ for 5min. Figure 4 The following are the peak graphs of flavor substances detected in yogurt fermented by different strains, among which: Figure 4 (a) shows the peak diagram of flavor substances detected in yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain of the present invention, Figure 4 (b) shows the peak diagram of flavor substances detected in yogurt fermented by HS-2 strain. Figure 4 (c) shows the peak diagram of flavor substances detected in yogurt fermented by YR-4 strain. The detection results of flavor substances are shown in Table 7.

[0093] Table 6 GC-MS parameters for flavor detection

[0094]

[0095] Table 7

[0096]

[0097]

[0098]

[0099] As shown in Table 7 above, the test results show that the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention produces 50 flavor substances, while the yogurt fermented by the control strains HS-2 and YR-4 produces 30 and 34 metabolic flavor substances, respectively; and only the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LBⅢ strain provided by the present invention contains furfural, a key substance of the characteristic tomato flavor.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus bulgaricus Dangxiong LBⅢ, characterized in that: It is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO:M 2022316.

2. The Lactobacillus bulgaricus Dangxiong LBⅢ strain according to claim 1, characterized in that The 16Sr DNA sequence of the strain is shown as SEQ ID NO.

1.

3. A fermentation method for preparing a Lactobacillus bulgaricus agent, characterized in that: The method comprises the following steps: culturing the Lactobacillus bulgaricus strain according to claim 1 or 2.

4. The preparation method according to claim 3, characterized in that: The preparation method comprises the following steps: (1) amplifying and culturing the Lactobacillus bulgaricus strain according to claim 1 or 2; (2) adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing the mixture at 32-34°C.

5. A bacterial agent, characterized in that: The bacterial agent contains the Lactobacillus bulgaricus Dangxiong LBⅢ strain according to claim 1 or 2.

6. The bacterial agent according to claim 5, characterized in that The bacterial agent is obtained by the fermentation preparation method according to claim 3 or 4.

7. Use of the Lactobacillus bulgaricus according to claim 1 or 2 or the bacterial agent according to claim 5 or 6 in the preparation of fermented dairy products.

8. The use according to claim 7, characterized in that: The fermented dairy product is cheese or a fermented dairy beverage, and the fermented dairy beverage includes yogurt.

9. A fermented dairy product, characterized in that The starter of the fermented dairy product contains the Lactobacillus bulgaricus Dangxiong LBⅢ strain according to claim 1 or 2.

10. The fermented dairy product according to claim 9, characterized in that The fermented dairy product is yogurt, and the yogurt contains furfural.

11. The fermented dairy product according to claim 10, characterized in that The yogurt also contains acetaldehyde and / or diacetyl.

12. The fermented dairy product according to claim 10, characterized in that The yogurt contains furfural, acetaldehyde and diacetyl.

13. The fermented dairy product according to claim 11, characterized in that The yogurt contains 2.9-3.0 mg / L diacetyl.

14. The fermented dairy product according to claim 12, characterized in that The yogurt contains 2.9-3.0 mg / L diacetyl.

15. The fermented dairy product according to claim 11, characterized in that The yogurt contains 29-30 mg / L of acetaldehyde.

16. The fermented dairy product according to claim 12, characterized in that The yogurt contains 29-30 mg / L of acetaldehyde.

17. The fermented dairy product according to claim 13, characterized in that The yogurt contains 29-30 mg / L acetaldehyde.

18. The fermented dairy product according to claim 14, characterized in that The yogurt contains 29-30 mg / L acetaldehyde.

19. The method for preparing a fermented dairy product according to claim 9, characterized in that: The preparation method comprises the following steps: preparing a starter using the Lactobacillus bulgaricus Dangxiong LBⅢ strain described in claim 1 or 2 or the bacterial agent described in claim 5 or 6, and then adding the starter into the emulsion for fermentation.

20. The preparation method according to claim 19, characterized in that: The emulsion is animal emulsion.

21. The preparation method according to claim 20, characterized in that: The animal emulsion is one or a combination of two or more of cow's milk, goat's milk or horse's milk.

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