CN115851481A - Lactobacillus bulgaricus separated from swertia pseudochinensis and application thereof - Google Patents

Abstract

The invention provides a lactobacillus bulgaricus strain separated from swertia tibetana and application thereof. The Lactobacillus bulgaricus Dangxion LB III strain provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2022316. The lactobacillus bulgaricus provided by the invention is metabolized in yoghourt fermentation to generate abundant flavor substances such as aldehyde, ketone, alcohol and ester.

Description

Lactobacillus bulgaricus separated from swertia pseudochinensis and application thereof

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a lactobacillus bulgaricus strain separated from swertia pseudochinensis var tibetana and application thereof.

Background

The bacterial strain of the yoghurt starter is the most core technology for producing yoghurt products. The lactobacillus bulgaricus is used as the main component of the yoghurt starter and is important for the formation of the flavour of the yoghurt, and the produced flavour substances acetaldehyde and diacetyl mainly endow the yoghurt with faint scent taste.

Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) is commonly referred to as Lactobacillus bulgaricus (Lactobacillus bulgaricus) and is one of the major lactic acid bacteria species used for yogurt production. The lactobacillus bulgaricus has very important health care function for human body, and has the important physiological functions of promoting the growth and colonization of beneficial bacteria, clearing intestines, resisting diarrhea, maintaining the health of gastrointestinal tract, promoting the digestion and absorption, enhancing immunity, resisting cancer, resisting tumor and the like. Has wide application in the fields of food fermentation, industrial lactic acid fermentation, feed industry and medical care.

Disclosure of Invention

The strain of the fermented yoghourt in the prior art has poor flavor substance production performance and cannot meet the requirements of people on food nutrition and taste. Aiming at the problems in the prior art, the invention provides lactobacillus bulgaricus.

In a first aspect, the invention provides a Lactobacillus bulgaricus (Lactobacillus bulgaricus) Dangxiong LB III strain, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022316, wherein the preservation organization is the university of Wuhan, china.

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

In a second aspect, the invention also provides a fermentation preparation method of the lactobacillus bulgaricus microbial inoculum, which comprises the following steps: culturing the Lactobacillus bulgaricus strain.

Preferably, the preparation method comprises the following steps:

(1) Carrying out amplification culture on the lactobacillus bulgaricus strain;

(2) Adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at the temperature of 32-34 ℃.

In a third aspect, the invention also provides a microbial inoculum, wherein the microbial inoculum contains the lactobacillus bulgaricus Dangxiong LB III strain.

Preferably, the microbial inoculum is obtained by the fermentation preparation method.

In a fourth aspect, the lactobacillus bulgaricus or the microbial inoculum is applied to food or food additives.

Preferably, the food is a fermented dairy product, preferably one or a combination of more than two of yoghurt, cheese and fermented milk beverage.

In a fifth aspect, the invention provides a fermented dairy product, wherein a starter of the fermented dairy product contains the lactobacillus bulgaricus Dangxiong LB III strain.

Preferably, the fermented milk product contains furfural.

Preferably, the fermented dairy product also contains acetaldehyde and/or butanedione,

preferably, the fermented milk product contains furfural, acetaldehyde and butanedione.

Preferably, the fermented dairy product contains 2.9-3.0mg/L butanedione, or preferably, the fermented dairy product contains 29-30mg/L acetaldehyde.

Preferably, the fermented milk product contains one or more of glycidol, 4-methyl-2-hexanone, 3-methyl-2-butanone, 4-ethyloctane, dodecane, 3-methyl-3-buten-1-ol, methoxy acetone, 4-methyloctane, vinyl ethyl ether, N-nonanal, cis-2-methylcyclohexanol, furfural, acetaldehyde, butanedione, 3-p-tolyltetrahydrofuran, benzaldehyde, phenylacetaldehyde, terpineol, 3-methyl-2- (5H) -furanone, gamma-crotonolactone, N-pentanol, 2, 5-dimethylbenzaldehyde, N-N-dibenzyloxycarbonyl-L-arginine, dimethyl sulfone, 2-methylbutylhexanoate and (+) -dibenzyloxy-L-tartaric anhydride,

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-buten-1-ol, methoxy acetone, 4-methyloctane, vinyl ethyl ether, dimethyl trisulfide, 2-nonanone, N-nonanal, cis-2-methylcyclohexanol, acetic acid, furfural, acetaldehyde, butanedione, o-dichlorobenzene, isopropanol, 3-p-tolyltetrahydrofuran, benzaldehyde, N-octanol, nitrous acid amyl ester, 2-undecanone, isovaleric acid, phenylacetaldehyde, one or a combination of more than two of dimethyl silanediol, N-nonanol, isovaleric acid, terpineol, 3-methyl-2- (5H) -furanone, naphthalene, isovaleric acid, gamma-crotonolactone, N-pentanol, 2, 5-dimethylbenzaldehyde, 2-tridecanone, phenethylacetate, 3-methylvaleric acid, N-N-dibenzyloxycarbonyl-L-arginine, dimethyl sulfone, phenethyl alcohol, 2-methylbutyl hexanoate, 2-pentadecanone, octanoic acid, 3-methylvaleric acid, delta-decalactone, decanoic acid, delta-dodecalactone and (+) -dibenzyloxy-L-tartaric anhydride,

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-buten-1-ol, methoxy acetone, 4-methyloctane, vinyl ethyl ether, dimethyl trithione, 2-nonanone, N-nonanal, cis-2-methylcyclohexanol, acetic acid, furfural, acetaldehyde, butanedione, o-dichlorobenzene, isopropanol, 3-p-tolyltetrahydrofuran, benzaldehyde, N-octanol, amyl ester, 2-undecanone, isovaleric acid, phenylacetaldehyde, dimethylsilanediol, N-nonanol, isovaleric acid, terpineol, 3-methyl-2- (5H) -furanone, naphthalene, isovaleric acid, γ -crotonolactone, N-pentanol, 2, 5-dimethylbenzaldehyde, 2-tridecanone, phenethylacetate, 3-methylpentanoic acid, N-dibenzyloxycarbonyl-L-arginine, dimethylsulfone, phenethyl alcohol, 2-methylbutyl hexanoate, 2-pentadecanone, 3-methyl-decanoic acid, L-decanoic acid anhydride, L-decanoic acid and (+) -methylbutanoic acid.

In a sixth aspect, the invention provides a method for preparing the fermented dairy product, which comprises the following steps: preparing a leavening agent by using the Lactobacillus bulgaricus Dangxiong LB III strain or the microbial inoculum, and then adding the leavening agent into the emulsion for fermentation.

Preferably, the emulsion is an animal or vegetable emulsion;

preferably, the animal milk is one or the combination of more than two of cow milk, goat milk or horse milk;

preferably, the vegetable emulsion is soy milk.

The Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention is separated from dairy products made by herdsmen in Dangxiangxong county (altitude 4350 m) in Tibet. The strain is preserved in China Center for Type Culture Collection (CCTCC) at 3 months and 25 days in 2022, and the preservation number is M2022316. The Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention is metabolized in yoghourt fermentation to generate abundant flavor substances such as aldehyde, ketone, alcohol, ester and the like, and compared with a commercially available yoghourt leavening agent, the Lactobacillus bulgaricus Dangxiong LB III strain exclusively produces furfural which is one of tomato flavor characteristic substances, so that the yoghourt is endowed with unique and obvious tomato flavor; the strain can simultaneously produce acetaldehyde which is a characteristic flavor substance of the yoghourt in a high yield manner, and endows the yoghourt with fresh and delicious characteristic flavor. The yogurt starter prepared by the strain provided by the invention can endow yogurt with pleasant flavor and has a nutritional function.

Information on the preservation of strains

The Lactobacillus bulgaricus (Lactobacillus bulgaricus) Langxiong LB III strain provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 3 and 25 months in 2022, and the preservation number is CCTCC NO: m2022316, deposit address: china, wuhan university, zip code: 430072; telephone: 027-68754052.

Drawings

FIG. 1 shows a microscopic image of a Lactobacillus bulgaricus Dangxiong LB III strain;

FIG. 2 shows a phylogenetic tree constructed from Lactobacillus bulgaricus Dangxion LB III strain;

FIG. 3 shows acetaldehyde and diacetyl content of fermented yogurt with different strains;

FIG. 4 shows the flavor substance detection peak of the yogurt fermented by different strains, wherein FIG. 4 (a) shows the flavor substance detection peak of the yogurt fermented by the Lactobacillus bulgaricus Dangxion LB III strain of the invention, FIG. 4 (b) shows the flavor substance detection peak of the yogurt fermented by the HS-2 strain, and FIG. 4 (c) shows the flavor substance detection peak of the yogurt fermented by the YR-4 strain.

Detailed Description

The strain Dangxiong LB III provided by the invention is lactobacillus bulgaricus which has been preserved in China center for type culture Collection in 3 month and 25 month in 2022, and the preservation number is CCTCC NO: M2022316. The strain provided by the invention can meet the requirements of vast consumers on continuously improving the nutritive value, flavor and taste of fermented yogurt.

The lactobacillus provided by the invention is separated from the delavay self-made by herdsmen in the county (altitude 4350 m) of Tibet, is identified as Lactobacillus delbrueckii subsp bulgaricus by 16Sr RNA, is a natural wild type microbial strain, belongs to the food microorganism of the 'strain list for food' issued by Ministry of health in 2010, can be widely applied to various fermented milk products, and has the food safety attribute.

In one embodiment of the invention, the preparation of the fermented dairy product by fermenting the lactobacillus bulgaricus Dangxiong LB III strain provided by the invention comprises the following steps:

(1) Activating the Lactobacillus bulgaricus Dangxion LB III strain;

(2) Preparing a fermented dairy product starter;

(3) And (4) preparing a fermented dairy product.

The invention provides a specific embodiment for detecting the flavor substances in the fermented dairy product by a GC-MS method. Through GC-MS flavor substance detection of the yoghourt, the metabolism of the Lactobacillus bulgaricus Dangxiong LB III strain is found to generate 50 flavor substances, the flavor substances are rich in aldehydes, ketones, alcohols, acids, esters and the like, and the flavor substances of 2 control strains (commercial yoghourt leavening agents) are 36 and 39 respectively; and furfural, a key substance containing the characteristic flavor of the tomatoes, is detected only in the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LB III strain; the strain can simultaneously produce acetaldehyde with high yield, which is a characteristic flavor substance of the yogurt, and the acetaldehyde content is 1.2 times that of the acetaldehyde content of the yogurt prepared by using a commercial starter, so that the yogurt is endowed with characteristic flavor of being more refreshing and delicious.

The reagents and instrument source information used in the examples of the present invention are shown in table 1 below.

TABLE 1

Example 1 isolation and characterization of the strains

The separation and identification of the Lactobacillus bulgaricus Dangxiong LB III strain comprises the following steps:

(1) Strain enrichment: taking 5g of a delavay sample self-made by a herdsman in Dangsheng Dangxong county (altitude 4350 m), crushing the sample, adding 45ml of sterile normal saline to disperse uniformly, taking 2ml of the dispersion, adding the dispersion into 20ml of sterilized litmus milk culture medium, placing the culture medium in an incubator at 37 ℃ for static culture, and taking out the culture medium when the culture medium is acidified, solidified and pink.

(2) Culturing a single colony: diluting the litmus milk culture medium bacterial liquid to 10 ^-5 And sucking 100 mul of the suspension, coating the suspension on a BL solid medium plate, and culturing in an incubator at 37 ℃ until bacterial colonies are formed. On the BL plate, the bacterial colony is smooth and slightly convex, moist, the edge is not smooth, colorless to slightly white, the diameter is 1-3mm, and the bacterial colony back is yellowish.

(3) Screening acid-producing strains: subjecting the single colony obtained by the preliminary screening to a concentration of 0.2% CaCO ₃ Performing streak culture on BL solid culture medium plate, culturing at 37 deg.C for 36h, observing whether transparent ring is generated around colony after culturing, and selectingAnd selecting a single colony with a large transparent circle for further separation and purification.

(4) Primary identification of gram stain: gram staining is carried out on the re-screened strain, if the staining of the strain is purple, the strain is gram-positive bacteria, the gram-positive bacteria are left for subsequent research, and if the staining is red, the strain is directly eliminated as gram-negative bacteria. The identification result shows that: the obtained re-screened strain is gram-positive bacterium, does not move, has no spore, flagella and capsule, has a fine rod-shaped thallus, presents a single rod or forms a chain, and is the target strain. The morphological characteristics of the strain are shown in figure 1.

(5) Molecular biological identification of 16S rRNA: 16S rDNA sequencing and strain identification are carried out on the strain to be identified by using high-fidelity enzyme. The information of the primers used and the PCR amplification reaction system and conditions are shown in tables 2 and 3.

TABLE 2 primer information

TABLE 3 PCR amplification reaction systems and conditions

After the PCR product is detected to be qualified, the PCR product is sent to a sequencing company for sequencing, and the sequence of the 16S rRNA gene is shown as SEQ ID NO.1, and specifically comprises the following steps:

GATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACACCATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAAACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAAAACCTTACCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGTGCAGTACAACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGCTCTCAGATCGGACTGCGGGCTGCAACTCGCCTGCACGAAACTGGAATCGCTAGTAATCTCGGATCACCACGCCGCGGTGAATACGTTCCCGCGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGTCGGTGAGATAACCTTTATAGGAGTCAGCCGCCTAA

the sequencing results were aligned with NCBI-BLAST and phylogenetic trees were constructed as shown in FIG. 2. The strain is identified as Lactobacillus bulgaricus (Lactobacillus bulgaricus) according to the phylogenetic tree shown in FIG. 2, and is named as Lactobacillus bulgaricus (Lactobacillus bulgaricus) Dangxiong LB III strain, and the obtained Lactobacillus bulgaricus Dangxiong LB III strain is preserved in China Center for Type Culture Collection (CCTCC) at 3 and 25 months in 2022 with the preservation number of M2022316 being CCTCC NO.

Example 2 preparation of Lactobacillus bulgaricus Dangxion LB III powder

(1) Activation of lactobacillus bulgaricus Dangxiong LB iii strain: taking a lactobacillus bulgaricus Dangxiong LB III strain preserved in a frozen glycerol tube at the temperature of-80 ℃, culturing for 12h at the temperature of 37 ℃, and continuously transferring for 2 times to complete strain activation;

(2) Expanding culture of the lactobacillus bulgaricus Dangxiong LB III strain: inoculating the activated strain to 70ml of MRS liquid culture medium according to 2 percent, and culturing for 12h at 37 ℃;

(3) Culture of Lactobacillus bulgaricus Dangxiong LB III Strain: inoculating 2% of the expanded culture strain to 3500ml of MRS liquid culture medium, and culturing at 37 ℃ for 12h;

(4) Separating the thallus of the lactobacillus bulgaricus Dangxiong LB III: centrifuging the culture solution at 5000rpm for 10min, pouring off the supernatant, and retaining the precipitate;

(5) Preparing a freeze-drying solution: 10g of skimmed milk powder, 5g of maltodextrin, 5g of trehalose, 3g of glycerol, 12g of thallus precipitate and 65g of pure water are mixed uniformly to prepare thallus freeze-dried liquid, and the thallus freeze-dried liquid is transferred into a freeze-drying bottle to be pre-frozen for 2 hours at the temperature of minus 40 ℃.

(6) Freeze-drying the lactobacillus bulgaricus Dangxiong LB III powder: the freeze-drying is carried out by adopting a Labconco vacuum freeze-drying machine, and the freeze-drying procedure is as follows:

step (ii) of	Temperature (. Degree.C.)	Time (h)	Degree of vacuum (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

Example 3 preparation of yogurt by fermentation Using Lactobacillus bulgaricus Dangxiong LB III Strain

Preparation of yogurt

Taking fresh cow milk, adding 6.5 wt% of edible sucrose, mixing, preheating, homogenizing and sterilizing; cooling to 40-50 deg.C, adding into a fermentation tank, inoculating 50mg/L of the lyophilized powder of Lactobacillus bulgaricus Dangxiong LB III strain prepared in example 2 and commercial yogurt starter YR-4 and HS-2 (domestic yogurt starter purchased from Tianmao on the Internet), and fermenting at 37 deg.C for 12 hr; and after the fermentation is finished, refrigerating the mixture at 4 ℃ for 24 hours to obtain the yoghourt.

Sensory evaluation of yogurt

The sensory evaluation of the yogurt refers to the sensory quality evaluation rule of Chinese Dairy products industry Specification RHB 103-2004 yogurt, 7 persons are invited to taste yogurt samples fermented by different strains and then score, and the final result is averaged. The evaluation index and the scoring criteria of yogurt are shown in table 4.

TABLE 4 sensory Scoring standards for yoghurts

The different strains for fermenting the yoghourt are shown in the table 5, and the steps and the process for preparing the yoghourt by the strains are the same as those for preparing the yoghourt by fermenting the lactobacillus bulgaricus Dangxiong LB III strain.

TABLE 5 sensory scores for fermented yogurts with different strains

	Dangxiong LBⅢ	HS-2	YR-4
				Color	9.71	10.00	9.43
Sweet and sour ratio	10.00	9.57	8.86
				Flavor (I) and flavor (II)	39.29	34.71	34.00
Taste and texture state	39.00	38.71	37.86
				Composite score	98	92.99	90.15

As shown in table 5, the results of the experiment: the yoghourt fermented by the Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention has the highest comprehensive score, and particularly, the score of the Lactobacillus bulgaricus Dangxiong LB III strain is far higher than that of a control strain in the aspect of flavor.

The yogurt fermented by the Lactobacillus bulgaricus Dangxiong LB III strain is subjected to 7-person group sensory evaluation, the yogurt has the characteristics of tomato flavor and refreshing and delicious yogurt by feedback, the yogurt fermented by the Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention has the highest comprehensive score, and particularly the score of the Lactobacillus bulgaricus Dangxiong LB III strain is far higher than that of a control strain (a commercial yogurt starter) in the aspect of flavor.

(III) detection of main flavor substance acetaldehyde and diacetyl content in yoghourt

1. Determination of acetaldehyde content

(1) Preparing a reagent: weighing 12.70g iodine into a 500mL beaker, adding 40g potassium iodide, adding a proper amount of water for dissolving, transferring to a 1 000mL brown volumetric flask, and shaking up with constant volume to prepare a 0.1mol/L iodine standard solution. Then, 10.00mL of 0.1mol/L iodine standard solution is accurately measured and put into a 100mL brown volumetric flask, the volume is determined by distilled water, and the solution is shaken up to the point where the two solutions are used.

(2) And (3) sample determination: taking 1% of NaHSO ₃ Placing 5mL of the solution in a 250mL conical flask, adding 25mL of supernatant of 6000rpm sample (yogurt prepared in step one) after centrifugation for 20min, mixing, standing at room temperature for 1h, adding 1mL of 1% starch solution, and adding 0.1mol/L iodineTitrating the solution to be close to light blue purple, titrating the solution to light blue purple by using 0.01mol/L iodine solution, and not fading the light blue purple within 30s, wherein the titrations are not counted. Then 20mL of 1mol/L NaHCO is added ₃ And (3) fully shaking and uniformly mixing the solution to ensure that the blue-violet color of the solution disappears, finally titrating the solution to a blue-violet end point by using 0.01mol/L iodine solution, recording the volume of the consumed standard iodine solution, wherein each sample has 2 parallels, and simultaneously carrying out a blank experiment.

M＝(V1-V2)*0.022*C*10 ⁶ /25

In the formula: m is acetaldehyde mass concentration/(mg/L); v1 is the volume/mL of iodine standard solution consumed by a titration sample; v2 is the volume/mL of the titration blank iodine consumption standard solution; c is the concentration/(mol/L) of the iodine standard solution; 25 is sample weight/mL; 0.022 is the acetaldehyde reaction chemical basic unit/g.

2. Determination of diacetyl content

(1) Drawing a butanedione standard curve: dissolving 15 mu L of butanedione in distilled water, and metering to 500mL. Respectively weighing 0.0, 2.0, 4.0, 6.0, 8.0 and 10.0mL of butanedione standard solution in 12 numbered double rows of test tubes, and adding distilled water to 10mL. 0.5mL of 1% o-phenylenediamine solution is added into 6 tubes in the front row, and no o-phenylenediamine solution is added into 6 tubes in the rear row, and the mixture is fully and uniformly mixed and then reacts in a dark place for 30min. Adding 4.0mol/L hydrochloric acid to stop the reaction after the reaction is completed (adding 2.0mL in a front row test tube and 2.5mL in a rear row test tube, shaking uniformly, using the rear row test tube as a blank control, using an ultraviolet spectrophotometer to measure absorbance at the wavelength of 335nm, drawing a standard curve by taking the concentration of butanedione as an abscissa and the absorbance as an ordinate, and performing regression according to the standard curve with the equation y =0.116 x +0.040, ² =0.9999, calculate diacetyl content.

(2) Determination of butanedione content: taking a yogurt sample to be tested (the yogurt prepared in the step one), mixing the yogurt sample with 16% trichloroacetic acid solution in equal volume, standing for 10min after mixing uniformly, and centrifuging for 10min at 5500rpm. Taking 20mL of supernatant, adding the supernatant into 2 test tubes in equal volume, adding 0.5mL of 1% o-phenylenediamine solution into the test tube No.1, adding no test tube No. 2, fully mixing the solution uniformly, reacting the solution in a dark place for 30min, adding 4.0mol/L hydrochloric acid into the test tubes 2 to terminate the reaction (adding 2.0mL into the test tube No.1 and adding 2.5mL into the test tube No. 2), mixing the solution uniformly, using the test tube No. 2 as a control solution, and measuring the absorbance at the wavelength of 335 nm. If the content of butanedione in the sample is higher, the measured absorbance exceeds the range of 0.2-1.0, and the sample can be properly diluted by 8 percent trichloroacetic acid.

The experimental results are as follows: as shown in figure 3, the acetaldehyde and diacetyl content in the fermented yoghurt by adopting the Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention (DB-3 in figure 3 is the Lactobacillus bulgaricus Dangxiong LB III strain provided by the invention) is higher than that in the yoghurt fermented by the commercial yoghurt bactericides HS-2 and YR-4. The acetaldehyde content in the yoghourt obtained by fermenting the strain provided by the invention reaches 29.11mg/L, and the diacetyl content reaches 2.993mg/L. The high content of acetaldehyde endows the yoghourt with rich flavor; researches show that when the content ratio of acetaldehyde to diacetyl is more than 3:1, the flavor of the yogurt is better.

The acetaldehyde in the yoghourt fermented by the Lactobacillus bulgaricus Dangxiong LB III strain and the commercially available yoghourt starter strain is as follows: both diacetyl are greater than 3.

(IV) detection of other flavor substances in yogurt

Sample pretreatment: a Headspace solid phase microextraction (HS-SPME) is adopted to extract and enrich volatile odor compounds of a sample, and GC-MS is used for carrying out full identification on the volatile odor compounds (principle: when the odor compounds are injected into a gas chromatograph, substances separated by a chromatographic column enter an ionization chamber from a molecular separator, are bombarded by electrons to form ions, and the separated ions enter an ion detector, and mass spectrograms of the components are derived after the mass spectrometric fast scanning, so that the mass spectrograms are used as the basis of qualitative and quantitative analysis), wherein the detection parameters are shown in the following table 6. 3g of the prepared yoghurt was weighed out and 2g of water, 1.5g of sodium chloride and 1. Mu.l of the internal ortho-dichlorobenzene (concentration 185. Mu.g/ml) were added. Immediately sealing the weighed sample with a bottle cap with a silicone rubber pad, placing the sample into a rotary oscillator, balancing the sample at 50 ℃ for 30min, extracting the sample with a DVB/CAR/PDMS composite extraction head at 50 ℃ for 30min, then injecting the sample, and resolving the sample at 250 ℃ for 5min. FIG. 4 shows the flavor substance detection peak of yogurt fermented by different strains, wherein FIG. 4 (a) shows the flavor substance detection peak of yogurt fermented by Lactobacillus bulgaricus Dangxiong LB III strain of the present invention, FIG. 4 (b) shows the flavor substance detection peak of HS-2 strain fermented yogurt, and FIG. 4 (c) shows the flavor substance detection peak of YR-4 strain fermented yogurt. The results of the flavor measurement are shown in Table 7.

TABLE 6 GC-MS parameters for flavor testing

TABLE 7

The results of the tests are shown in table 7 above: the lactobacillus bulgaricus Dangxiong LB III strain provided by the invention ferments the yoghourt to generate 50 flavor substances, and the contrast strains HS-2 and YR-4 ferment the yoghourt to respectively generate 30 and 34 metabolic flavor substances; and only the lactobacillus bulgaricus Dangxiong LB III strain fermented yogurt provided by the invention contains furfural which is a key substance with the characteristic flavor of tomatoes.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims (13)

1. A Lactobacillus bulgaricus (Lactobacillus bulgaricus) Dangxion LB III strain is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2022316.

2. The lactobacillus bulgaricus Dangxiong LB iii strain of claim 1, wherein the 16Sr DNA sequence of the strain is represented by SEQ ID No. 1.

3. A fermentation preparation method of a lactobacillus bulgaricus microbial inoculum is characterized by comprising the following steps: culturing the lactobacillus bulgaricus strain of claim 1 or 2.

4. The method of claim 3, comprising the steps of:

(1) The lactobacillus bulgaricus strain of claim 1 or 2 is cultured in an enlarged manner;

(2) Adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at the temperature of 32-34 ℃.

5. A bacterial agent comprising the Lactobacillus bulgaricus Dangxiong LB III strain according to claim 1 or 2.

6. The microbial inoculum according to claim 5, which is obtained by the fermentation preparation method according to claim 3 or 4.

7. Use of the lactobacillus bulgaricus strain of claim 1 or 2 or the microbial agent of claim 5 or 6 in food or food additives.

8. The use according to claim 7, wherein the food product is a fermented dairy product, preferably one or a combination of two or more of yogurt, cheese, fermented milk drink.

9. A fermented milk product characterized in that a starter culture for said fermented milk product contains the strain Lactobacillus bulgaricus Dangxiong LB III as claimed in claim 1 or 2.

10. Fermented dairy product according to claim 9, characterized in that the fermented dairy product contains furfural;

preferably, the fermented dairy product also contains acetaldehyde and/or butanedione;

preferably, the fermented dairy product contains furfural, acetaldehyde and butanedione.

11. The fermented dairy product of claim 9 or 10, wherein the fermented dairy product comprises 2.9-3.0mg/L diacetyl,

or preferably, 29-30mg/L acetaldehyde is contained in the fermented dairy product.

12. A method of producing a fermented milk product according to any of claims 9-11, characterized in that the method of production comprises the steps of: preparing a starter by using the strain comprising Lactobacillus bulgaricus Dangxion LB III as described in claim 1 or 2 or the microbial agent as described in claim 5 or 6, and adding the starter to the emulsion for fermentation.

13. The method of claim 12, wherein the emulsion is an animal emulsion or a vegetable emulsion;

preferably, the animal milk is one or the combination of more than two of cow milk, goat milk or horse milk;

preferably, the vegetable emulsion is soy milk.

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