CN110628662B

CN110628662B - Acetobacter strain and application thereof

Info

Publication number: CN110628662B
Application number: CN201910689925.4A
Authority: CN
Inventors: 吴笃琴; 黎亮; 黎力; 赵良忠; 李明; 李建华
Original assignee: Guizhou Lianghuanzhai Bio Tech Co ltd
Current assignee: Guizhou Lianghuanzhai Bio Tech Co ltd
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2021-04-02
Anticipated expiration: 2039-07-29
Also published as: CN110628662A

Abstract

The invention relates to the technical field of microbial engineering, in particular to acetobacter and application thereof, wherein the acetobacter is lactobacillus aceti LHZ-ST001, the preservation unit is China general microbiological culture Collection center (CGMCC for short), the preservation address is No. 3 of No.1 Xilu of Beijing Korean district, the preservation date is 09 and 10 days in 2018, and the preservation number is CGMCC No. 16448; the acetobacter has higher viable bacteria amount in a fermentation medium, has stronger capacity of producing acid and alcohol dehydrogenase, has the capacity of being compounded with other lactic acid bacteria or saccharomycetes, has better tolerance to acid and has the characteristic of high acid tolerance.

Description

Acetobacter strain and application thereof

Technical Field

The invention relates to the technical field of microbial engineering, in particular to acetobacter and application thereof.

Background

Acetic Acid Bacteria (AAB) belong to the bacterial domain (Bacteria), Proteobacteria (Proteobacteria), Alphaeomorpha (Alphaobacteria), Rhodospiriales (Rhodospirillales), Acetobacter family (Acetobacter), can oxidize ethanol or saccharides and generate organic acids such as acetic acid or gluconic acid, are important industrial microorganisms, and are mainly used for producing vinegar and fruit vinegar beverages. Acetobacter is not only applied to production of gluconic acid, vinegar, sorbic acid and the like, but also is the most important research object in biochemical reactions such as oxidation of ethanol and saccharides. At present, acetic acid bacteria play an important role in the vitamin C manufacturing industry and the vinegar brewing industry.

Acetic acid bacteria, which can grow rapidly in the presence of a small amount of organic acid in the raw material to produce acetic acid; under aerobic conditions, the acetobacter oxidizes the ethanol generated by the yeast into acetic acid through the action of a complex of ethanol dehydrogenase, acetaldehyde dehydrogenase and coenzyme oxidase combined with a membrane, the acetic acid has unique flavor, and in addition, the acetic acid can form ethyl acetate with the ethanol to increase the aromatic smell of the product. Acetic acid bacteria can produce hydroxy acids such as glycolic acid, oxalic acid, tartaric acid, adipic acid, succinic acid, mannonic acid, gluconic acid, and the like, in addition to acetic acid. The acetic acid bacteria have stronger acid resistance, take membrane protein of somatic cells as a carrier, transport acetic acid out of the cells by consuming energy, and maintain the low acetic acid level in the cells; meanwhile, the acetic acid bacteria are regulated and controlled by genes and enzymes on the basis of corresponding metabolic reactions, so that the acetic acid is decomposed into other small molecular substances, and therefore, the acetic acid bacteria have the characteristics of high acid resistance and high acid production.

At present, the fermentation rate in the traditional sour soup preparation is low, so that the prepared sour soup product has the problems of short shelf life, poor stability, easy occurrence of over-fermentation, layering, bottle-expanding peculiar smell and the like along with the prolonging of the placement time, and the commodity value of the product is seriously influenced. For example, patent No. 201810745135.9 discloses an application of a composite fermentation bacteria in preparing red sour soup, wherein the composite fermentation bacteria is composed of lactobacillus plantarum, bacillus natto and aspergillus oryzae according to a ratio of 35:23:12, the composite fermentation bacteria obtained by combining specific strains is used in preparing the red sour soup, and the red sour soup with stable quality, good flavor and long shelf life can be prepared by improving the existing red sour soup preparation process, and can be used as a sour and spicy seasoning packet to be prepared into sour and spicy instant noodles or sour and spicy rice noodles and other foods together with rice noodles or instant noodles; but has the problems of high cost, complex microbial system and unstable quality.

Disclosure of Invention

In order to solve the technical problems, the invention provides acetobacter and application thereof.

The method is realized by the following technical scheme:

the lactobacillus aceticus is lactobacillus aceticus LHZ-ST001, the preservation unit is China general microbiological culture Collection center (CGMCC for short), the preservation address is No. 3 of Xilu No.1 of Beijing market Chaoyang district, the preservation date is 09 months and 10 days in 2018, and the preservation number is CGMCC No. 16448.

The acetobacter has higher viable bacteria amount in a fermentation culture medium.

The acetobacter has stronger capacity of producing acid and alcohol dehydrogenase.

The acetobacter has the capability of being compounded with other lactic acid bacteria or yeast.

The acetobacter has better tolerance to acid and has the characteristic of high acid tolerance.

The invention also aims to provide an application of the acetobacter in the production of the Miao Dong minority sour soup.

The application of the acetobacter in the production of the Dong minority sour soup comprises the steps of preparing a bacterial liquid, preparing materials, mixing materials and fermenting, wherein the step of preparing the bacterial liquid is to dissolve the acetobacter in bean clear liquid for fermentation.

The fermentation temperature of the acetobacter is 32-36 ℃.

The method comprises the following specific steps: (1) preparing strains, namely inoculating acetobacter into a fresh and sterilized bean clear liquid matrix, and fermenting at the temperature of 32-36 ℃ for 20-24h to obtain a bacterial liquid; (2) preparing chili sauce or tomato sauce by removing stems of fresh chili and tomato, cleaning, and chopping into pieces; pulverizing into paste with colloid mill; (3) mixing, mixing the chili or tomato sauce, salt, ginger, garlic and rice wine uniformly, and inoculating 5-10% of strains; (4) and (3) fermenting, namely fermenting the product obtained in the mixing step at the temperature of 32-36 ℃ for 15-20 days to obtain the Miao Dong minority sour soup.

The sterilization temperature of the bean clear liquid in the step (1) is 121 ℃, and the sterilization time is 15 min;

in the step (2), the tomato and the pepper are ground by a colloid mill and have the particle size of 65-100 mu m.

The material component ratio in the step (3) is as follows: 6-8 parts of tomato, 2-4 parts of pepper, 0.1-0.2 part of salt, 0.4-0.6 part of ginger, 0.4-0.6 part of garlic and 0.1-0.2 part of rice wine, wherein the adding ratio of the bacterial liquid is 0.5-1 part.

Further preferably, the mixture obtained in the step (3) comprises the following materials in parts by weight: 7 parts of tomatoes, 3 parts of hot peppers, 0.1 part of salt, 0.5 part of ginger, 0.5 part of garlic and 0.1 part of rice wine, wherein the adding amount of the bacterial liquid accounts for 0.6 part.

The invention has the beneficial effects that:

the Acetobacter (Acetobacter sp. strain) LHZ-ST001 derived from the plant fermented food provided by the invention enables the Acetobacter LHZ-ST001 to grow rapidly under the condition of a fermentation culture medium to generate acetic acid; under aerobic conditions, the acetobacter LHZ-ST001 can grow rapidly to generate acetic acid under the condition that a small amount of organic acid exists in the raw material; the acetobacter LHZ001 oxidizes ethanol generated by yeast into acetic acid through the complex action of ethanol dehydrogenase, acetaldehyde dehydrogenase and coenzyme oxidase combined with a membrane, the acetic acid has unique flavor, and in addition, the acetic acid can form ethyl acetate with ethanol to increase the aromatic smell of the product. The acetobacter xylinum LHZ-ST001 can generate hydroxy acids such as glycolic acid, oxalic acid, tartaric acid, adipic acid, succinic acid, mannonic acid, gluconic acid and the like besides acetic acid. The acetobacter LHZ-ST001 has strong acid resistance, and the acetobacter LHZ001 takes membrane protein of thallus cells as a carrier, transfers acetic acid out of the cells by consuming energy, and maintains the intracellular low acetic acid level, so the acetobacter LHZ001 has the characteristics of high acid resistance and high acid production. Has high acid-producing capability, is used for fermenting food, and is practical. The acetobacter has good compounding property to other lactic acid bacteria or saccharomycetes, and can promote the stability of a microbial system, promote the proliferation of beneficial bacteria and inhibit the breeding of harmful bacteria.

The acetobacter can generate hydroxy acids such as glycolic acid, oxalic acid, tartaric acid, adipic acid, succinic acid, mannonic acid, gluconic acid and the like besides acetic acid, the content of alcohol dehydrogenase reaches 7284U/mg, and the alcohol dehydrogenase is higher than that of alcohol dehydrogenase generated by common acetobacter1-1.5 times more; the acid yield of the acetobacter aceti after 24 hours of fermentation is up to 9.1g/L, which is about 1 time (4.7g/L) higher than that of the common acetobacter aceti. The number of viable bacteria of the common acetobacter is rapidly reduced under the inhibition of high acid after fermenting for 10 hours, and the number of viable bacteria is reduced to 10 after fermenting for 24 hours²cfu/ml. The number of viable bacteria of the acetobacter LHZ-ST-001 at the initial fermentation stage is 10⁶The cfu/ml is continuously increased, and the viable count reaches the maximum of 10 after 6 hours of fermentation^8.5cfu/ml, viable count of 10 after 24 hours of fermentation⁸cfu/ml is 6 orders of magnitude higher than that of common acetobacter.

The acetobacter aceti is used for producing the Dong minority sour soup, only the clear bean liquid needs to be sterilized, the temperature is controlled to be 32-36 ℃, and the inoculation amount is less, so that the cost is low, the acetobacter aceti can have good compatibility with endogenous microorganisms in materials such as tomatoes, and the like, can inhibit harmful bacteria, promote the stability of a microorganism system, further contribute to the quality stability and the improvement of the use safety, has high sour production capability, and can shorten the fermentation time.

Drawings

FIG. 1: growth curve of acetobacter in bean serum

FIG. 2: research on acid production and acid resistance of acetobacter LHZ-ST-001

Detailed Description

The following provides a more detailed description of the present invention. The features and advantages of the present invention will be apparent to those skilled in the art from the detailed description of the invention.

Example 1 isolation and identification of the species

And (3) separation of strains:

weighing 5mL of a naturally fermented soya bean serum bacterial liquid sample, inoculating the soya bean serum bacterial liquid sample into 20mL of an enrichment liquid culture medium, standing and culturing for 24h at 36 ℃, ten-fold diluting the enriched culture liquid sample with sterile physiological saline, adding 1mL of bacterial suspension with proper dilution times into a sterilization flat dish, then carrying out MRS (MRS) plate pouring, after solidification, placing the bacterial suspension in a constant temperature incubator at 36 ℃ for 48h, observing whether an obvious calcium-dissolving ring exists, selecting a single bacterial colony for streak purification, purifying for 3-4 generations, determining the colony morphology and the thallus morphology through apparent judgment and microscopic examination, and carrying out slant preservation on the strain at 4 ℃;

and (3) identification of strains:

1. identification index

Morphological identification: gram stain, cell morphology, presence or absence of spores

Physiological and biochemical identification: catalase, oxidase, carbohydrate acid production

Molecular biological identification 16SrRNA sequence analysis

2. Identification results

(1) Physiological and biochemical characteristics and morphological characteristics:

the physiological and biochemical properties of LHZ-ST-001 are shown in Table 1

TABLE 1

(2) 16S rRNA gene sequence of the strain:

TGGGGGATAACTCTGGGAAACTGGAGCTAATACCGCATGATACCTGAGGGTCAAA GGCGCAAGTCGCCTGTGGAGGAGCCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGC CTACCAAGGCGATGATCGATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGA GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCA ACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTCG ACGGGGACGATGATGACGGTACCCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG CCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGT AGGCGGTTTGTACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGAT ACGTACAGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTC GTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATTACTGACGCT GAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAA ACGATGTGTGCTAGATGTTGGGTGACTTTGTCATTCAGTGTCGCAGTTAACGCGTTAAG CACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGG CTTGAATGTAGAGGCTGTATTCAGAGATGGATATTTCCCGCAAGGGACCTCTAACACAG GTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCCTATCTTTAGTTGCCAGCATGTTTGGGTGGGCACTCTAGAGAGACTGCCG GTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGG GCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTAGATGGTGACATCGTGCTG ATCTCTAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGA ATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACA CCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCCGCAA

(3)16S rDNA gene sequencing and identification:

the sequence alignment results of LHZ001 strain of Acetobacter sp.strain on CNBI are shown in Table 2

TABLE 2

According to the determination result (sequence table), the strain LHZ001 is identified as acetobacter.

Test example 1 Properties of Acetobacter LHZ-ST-001

1. Research on metabolic acid production pathway of acetobacter LHZ-ST-001:

the lactobacillus aceticus (Acetobacter sp. strain) LHZ-ST001 derived from the plant fermented food provided by the invention can rapidly grow under the condition of a fermentation culture medium to generate acetic acid; the lactobacillus acetate LHZ001 is a bacillus aceticus which can produce alcohol dehydrogenase with high yield, the metabolic pathways of the lactobacillus aceticus include sugar metabolism and alcohol respiratory chain metabolism, the acetic acid bacteria consume glucose to maintain cell growth, and the ethanol is oxidized to generate energy. The specific metabolic pathway is as follows: the lactobacillus acetoacidophilus LHZ-ST001 can generate energy through a TCA coupling aerobic respiration way at the initial stage of acetic fermentation; entering a rapid acetic acid accumulation stage, wherein an ethanol respiratory chain is a main energy supply metabolic pathway; and in the later fermentation period, TCA is coupled with an aerobic respiration way to supply energy by matching with an ethanol respiration chain. In the initial stage of ethanol oxidation, under the action of ethanol dehydrogenase, acetaldehyde dehydrogenase and cytochrome oxidase in lactobacillus acetate, ethanol is catalyzed and oxidized into acetic acid, and the two membrane-bound alcohol dehydrogenases, namely the ethanol dehydrogenase and the acetaldehyde dehydrogenase, are positioned outside cytoplasm of a cell membrane and take pyrroloquinoline quinone Protein (PQQ) as coenzyme; cytochrome oxidase functions to transfer electrons to oxygen and ultimately to produce water. The ethanol generated by the yeast is oxidized into acetic acid through the complex action of the ethanol dehydrogenase, the acetaldehyde dehydrogenase and the coenzyme cytochrome oxidase which are combined with the membrane, the acetic acid has unique flavor, and in addition, the ethyl acetate can be formed with the ethanol, so that the aromatic smell of the product is increased. The acetobacter xylinum LHZ-ST001 can generate hydroxy acids such as glycolic acid, oxalic acid, tartaric acid, adipic acid, succinic acid, mannonic acid, gluconic acid and the like besides acetic acid.

2. Study on ethanol dehydrogenase production capacity of acetobacter LHZ-ST-001

The method for measuring the alcohol dehydrogenase comprises the following steps: the activity of the alcohol dehydrogenase is detected by using an Alcohol Dehydrogenase (ADH) detection kit (acetaldehyde colorimetry), the principle is that acetaldehyde is used as a substrate under the weak base condition, the acetaldehyde is reduced into the alcohol by NADH under the catalysis of ADH, 1 molecule of NADH is consumed by 1 molecule of acetaldehyde catalyzed by ADH, the change of absorbance is measured by a spectrophotometric colorimetry (spectrophotometer), and the consumption rate of the NADH is calculated to further calculate the activity level of the alcohol dehydrogenase.

The content determination result of the alcohol dehydrogenase is as follows: inoculating acetic acid bacillus LHZ-ST-001 into fresh sterilized bean clear liquid matrix, and fermenting at 32-36 deg.C for 20-24h to obtain bacterial liquid. The final result shows that the content of the alcohol dehydrogenase in the bacterial liquid obtained by fermenting the acetobacter aceti LHZ-ST-001 in the sterilized soybean clear liquid at the temperature of 32-36 ℃ for 20-24h is up to 7284U/mg, which is 1-1.5 times more than that of the alcohol dehydrogenase produced by the common acetobacter aceti.

3. Research on compounding property of acetobacter LHZ-ST-001 and other lactic acid bacteria or saccharomycetes

The compounding method comprises the following steps: mixing and inoculating lactobacillus casei, saccharomyces cerevisiae, lactobacillus plantarum and leuconostoc mesenteroides which are separated from the soy whey liquid into a fresh and sterilized soy whey matrix according to different proportions, and fermenting at the temperature of 32-36 ℃ for 20-24h to prepare the compound zymophyte agent.

Compounding results are as follows: the prepared compound fermentation microbial inoculum is used for preparing the Dong minority sour soup, and the microbial abundance of the fermentation microbial inoculum in the fermented Dong minority sour soup is more than 90% of the total amount of bacteria, and the maximum microbial abundance is 94%; and the microbial abundance of lactic acid bacteria and molds in the raw material was from 2% and 11% at the initial stage of fermentation to 5% and 0.5% at the completion of fermentation, respectively. Therefore, the lactobacillus acetoacidophilus LHZ-ST001 has good compound property with other lactic acid bacteria or yeast, can promote the stability of a microbial system, and can effectively promote the growth of beneficial bacteria lactic acid bacteria and inhibit the breeding of harmful bacteria and mold.

4. Research on acid production and acid resistance of acetobacter LHZ-ST-001

The research on the acid-producing and acid-resisting properties of the acetobacter LHZ-ST-001 is shown in figure 2, and the acid-producing amount of the acetobacter LHZ-ST-001 after being fermented for 24 hours is up to 9.1g/L, which is about 1 time higher (4.7g/L) than that of the common acetobacter. The number of viable bacteria of the common acetobacter is rapidly reduced under the inhibition of high acid after fermenting for 10 hours, and the number of viable bacteria is reduced to 10 after fermenting for 24 hours²cfu/ml. The number of viable bacteria of the acetobacter LHZ-ST-001 at the initial fermentation stage is 10⁶The cfu/ml is continuously increased, and the viable count reaches the maximum of 10 after 6 hours of fermentation⁸cfu/ml, viable count of 10 after 24 hours of fermentation⁸cfu/ml is 6 orders of magnitude higher than that of common acetobacter. Experiments show that the acid production capacity and the acid resistance capacity of the acetobacter LHZ-ST-001 are both stronger than those of the common acetobacter.

Examples 2 to 3

Example 1 provides a method for applying lactobacillus acetate LHZ-ST001 in the production of the minority sour soup, which comprises the following steps: preparing a bacterial solution, inoculating acetobacter into fresh sterilized bean clear liquid, and fermenting at the temperature of 32-36 ℃ for 20-24 hours to obtain the bacterial solution; (2) preparing materials, namely cleaning fresh peppers, chopping the cleaned fresh peppers, cleaning fresh tomatoes and cutting the cleaned fresh tomatoes into tomato blocks; (3) pulverizing into paste, and grinding the above Capsici fructus and fructus Lycopersici Esculenti into paste with colloid mill; (4) mixing, namely adding a proper amount of the bacterial liquid, the salt, the ginger, the garlic and the rice wine into the crushed tomato and the mashed pepper, and uniformly mixing for later use; (5) fermenting, namely fermenting the mixed materials at the temperature of 32-36 ℃ for 10-15 days to obtain the Dong minority sour soup;

the sterilization temperature of the bean clear liquid in the step (1) is 115 ℃, and the sterilization time is 10 min;

the tomato and the pepper in the step (2) are ground by a colloid mill and have the particle size of 65-100 mu m;

the adding amounts of the tomatoes, the hot peppers, the salt, the ginger, the garlic and the rice wine in the step (3) are 7 parts, 3 parts, 0.1 part, 0.5 part and 0.1 part respectively;

the bacterial liquid adding amount in the step (3) is 3%, the fermentation temperature is 32-36 ℃, and the fermentation time is 15 days;

example 3

The difference from example 2 is that: the bacterial liquid is prepared by inoculating 3 × 10 strains of composite strains⁸CFU/mL is obtained by fermenting in bean clear liquid for 72 hours; wherein the composite strain is composed of lactobacillus acetate LHZ-ST001 and lactobacillus casei according to the inoculation amount of 1: 2;

meanwhile, in order to verify the effect of the lactobacillus acetate and the effect of the application method adopted by the invention, the inventor makes the following comparative examples and tests:

1. comparative example 1: the difference from the example 2 is that the adopted zymophyte is lactic acid bacteria;

2. comparative example 2: the difference from the embodiment 2 is that the application method comprises the following steps: (1) preparing materials, namely cleaning fresh peppers, chopping the cleaned fresh peppers, cleaning fresh tomatoes and cutting the cleaned fresh tomatoes into tomato blocks; (2) pulverizing into paste, and grinding the above Capsici fructus and fructus Lycopersici Esculenti into paste with colloid mill; (3) mixing, namely uniformly mixing the crushed tomatoes, the mashed chili, the salt, the ginger, the garlic and the rice wine, and inoculating acetobacter for later use; (4) fermenting, namely fermenting the mixed materials at the temperature of 32-36 ℃ for 10-15 days to obtain the Dong minority sour soup; wherein the inoculation amount of the acetobacter in the step (3) is 3 multiplied by 10⁸CFU/mL；

3. Analysis of sour soup ingredients

3.1 analytical methods

3.1.1 minerals: the calcium detection is carried out according to GB5009.92-2016 (determination of calcium in national food safety standard) ("determination of calcium in food); the magnesium detection is carried out according to GB5009.241-2017 determination of magnesium in food safety national standard food; the detection of sodium and potassium is carried out according to GB5009.91-2017 determination of potassium and sodium in national standard food for food safety; nitrite detection is carried out according to GB5009.33-2016 (determination of nitrite and nitrate in national food safety standard); the detection of the solid content is carried out according to GB/T10345-2007 method for analyzing national standard white spirit for food safety. Organic acid: an acid-base titration method is adopted, wherein in the lactic acid determination, a sodium hydroxide solution is 40g/L, a sulfuric acid standard titration solution is 0.5mol/L, and a phenolphthalein indicator solution is 10 g/L; in the detection of acetic acid and citric acid, the sodium hydroxide solution is 0.5mol/L, and the phenolphthalein indicator solution is 10 g/L. Functional components: the detection of the lycopene is carried out according to GB/T22249-2008 'determination of lycopene in health food'; the capsaicin detection is carried out according to GB/T30388-2013 high performance liquid chromatography for measuring the total capsaicin content in capsicum and oleoresin thereof. Volatile components: the volatile oil is extracted according to pharmacopoeia 2015 edition of the people's republic of China. Sample pretreatment: 0.2mL of volatile oil is put into a colorimetric tube, and 1.8mL of ethyl acetate is added for later use.

3.1.2 detection of Ethyl linolenate and Ethyl linoleate by gas capillary column chromatography. Chromatographic conditions are as follows: KB-Wax capillary chromatography column (30 m.times.0.32 mm.times.0.50 μm), injector temperature 230 ℃, detector temperature 250 ℃, sample volume 1 μ L, split ratio 30: 1. Preparation of a reference substance: taking 0.0526mg of ethyl linolenate, and diluting to a 2mL brown volumetric flask with ethyl acetate to prepare a ethyl linolenate reference solution with the mass concentration of 2.63 mg/mL; 0.047mg of ethyl linoleate is taken, ethyl acetate is used for metering volume to a 2mL brown volumetric flask, and a ethyl linoleate reference substance solution with the mass concentration of 2.35mg/mL is prepared.

3.1.3 detection of limonene and citral gas capillary column chromatography was used. Chromatographic conditions are as follows: the temperature of a limonene injection port is 180 ℃; the temperature of the detector is 220 ℃, the sample injection amount is 2 mu L, and the split ratio is 30: 1. The citral sample injector temperature was 230 deg.C, the detector temperature was 250 deg.C, and the sample amount was 1 μ L, both using SE-54 capillary chromatography column (30m × 0.25mm × 0.25 μm) with a split ratio of 30: 1. Preparation of a reference substance: taking 0.0494mg of limonene, using ethyl acetate to fix the volume to a 10mL brown volumetric flask, and preparing a limonene reference substance solution with the mass concentration of 4.94 mg/mL; taking 0.0552mg of citral, and diluting with ethyl acetate to a 10mL brown volumetric flask to obtain a citral control solution with a mass concentration of 5.52 mg/mL.

3.1.4 alpha-pinene detection gas capillary column chromatography was used. Chromatographic conditions are as follows: SE-54 capillary chromatography column (30 m.times.0.25 mm.times.0.25 μm), sample injector temperature 180 deg.C, detector temperature 200 deg.C, sample injection 2 μ L, and split ratio 30: 1. Preparation of a reference substance: the mass concentration of the alpha-pinene reference substance solution is 15.65 mg/mL: 0.0313mg of alpha-pinene is taken and made into a 2mL brown volumetric flask with ethyl acetate.

3.1.5 beta-pinene detection adopts high performance liquid chromatography-diode array detector method (HPLC-PDA). Chromatographic conditions are as follows: COSMOSIL5C 18-MS-ii (4.6mm i.d. × 250mm), mobile phase: acetonitrile-water-isopropanol (70: 28: 2, volume ratio), flow rate: 1mL/min, detection wavelength: 203nm, column temperature: 40 ℃, sample introduction: 10 μ L. Preparation of a reference substance: taking 0.0308mg of beta-pinene, and using ethyl acetate to fix the volume to a 5mL brown volumetric flask to prepare a beta-pinene reference substance solution with the mass concentration of 6.16 mg/mL.

3.1.6 detection of cis 3-hexenol (leaf alcohol acetate) gas capillary column chromatography was used. Chromatographic conditions are as follows: SE-54 capillary chromatography column (30 m.times.0.25 mm.times.0.25 μm), injector temperature 250 deg.C, detector temperature 250 deg.C, sample amount 1 μ L, and split ratio 30: 1. Preparation of a reference substance: taking 0.01mg of cis-3-hexenol (leaf alcohol acetate), metering the volume to a 10mL brown volumetric flask by using ethyl acetate, and preparing a cis-3-hexenol (leaf alcohol acetate) control solution with the mass concentration of 1 mg/mL.

4. Analysis results

4.1 comparison of mineral, nitrite and Total solids content in Red sour soup Table 4

TABLE 4

4.2 comparison of the content of flavor-developing substances in the Red sour soup is shown in Table 5

TABLE 5

Example sequence number	Lactic acid (mg/kg)	Acetic acid (mg/kg)	Citric acid (mg/kg)
				Example 1	18.38	21.42	24.62
Example 2	20.31	20.95	25.15
				Comparative example 1	12.17	6.89	9.57
Comparative example 2	14.61	8.54	11.08

4.3 comparison of volatile ingredients in Red sour soup the results are shown in Table 6

TABLE 6

4.4 comparative results of functional ingredients in Red sour soup are shown in Table 7

TABLE 7

	Lycopene (mug/g)	Capsaicin (mug/g)
			Example 2	118.92	45.21
Example 3	121.34	46.18
			Comparative example 1	48.87	19.42
Comparative example 2	63.74	26.33

Example 7 application of Acetobacter aceti LHZ-ST-001 in Rice sour soup

The obtained acetic acid bacillus LHZ-ST-002 is inoculated into sterilized fresh bean clear liquid and fermented for 20-24h at the temperature of 32-36 ℃ to obtain acetic acid bacillus LHZ-ST-001 bacterial liquid. Pulverizing fresh rice at high speed to obtain rice flour; gelatinizing and liquefying rice flour to obtain rice soup, and spreading for cooling; inoculating, namely adding 3 percent of prepared acetic acid bacillus LHZ-ST-001 bacterial liquid into prepared rice soup; fermenting, namely fermenting the mixed materials at the temperature of 32-36 ℃ for 10-15 days to obtain rice sour soup with acidity of 9.24g/L which is 4.2 times higher than that of the rice sour soup (2.20g/L) fermented by lactobacillus casei commonly used in the market.

It should be noted that the above examples and test examples are only for further illustration and understanding of the technical solutions of the present invention, and are not to be construed as further limitations of the technical solutions of the present invention, and the invention which does not highlight essential features and significant advances made by those skilled in the art still belongs to the protection scope of the present invention.

Sequence Listing

<110> Guizhou Lianghua Zizhai Biotechnology Co., Ltd

<120> acetobacter strain and application thereof

<160> 1

<210> 1

<211> 1276

<212> DNA

<213> Lactobacillus acetate (Acetobacter sp. strain) LHZ-ST-001

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<400> 1

TGGGGGATAACTCTGGGAAACTGGAGCTAATACCGCATGATACCTGAGGGTCAAAGGCGCAAGTCGCCTGTGGAGGAGCCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTCGACGGGGACGATGATGACGGTACCCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGTAGGCGGTTTGTACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGATACGTACAGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTAGATGTTGGGTGACTTTGTCATTCAGTGTCGCAGTTAACGCGTTAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGGCTTGAATGTAGAGGCTGTATTCAGAGATGGATATTTCCCGCAAGGGACCTCTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTAGTTGCCAGCATGTTTGGGTGGGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTAGATGGTGACATCGTGCTGATCTCTAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCCGCAA

Claims

1. An application of Acetobacter (Acetobacter sp.) LHZ-ST001 in production of HgDong minority sour soup is provided, wherein the Acetobacter is LHZ-ST001, the preservation unit is China general microbiological culture Collection center (CGMCC for short), the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo of Chaoyang district, the preservation date is 09 and 10 days in 2018, and the preservation number is CGMCC No. 16448;

2. The use of the Acetobacter sp (LHZ-ST 001) in the production of Miao Dong minority sour soup according to claim 1, wherein the Acetobacter sp has the ability to produce acetic acid and alcohol dehydrogenase.

3. The use of the Acetobacter aceti (Acetobacter sp.) LHZ-ST001 in the production of Miao Dong minority sour soup according to claim 1, wherein the Acetobacter aceti has a high acid resistance.

4. The application of the Acetobacter (Acetobacter sp.) LHZ-ST001 in the production of the Miao Dong minority sour soup according to claim 1 is characterized by comprising the steps of preparing a bacterial liquid, preparing materials, mixing materials and fermenting, wherein the prepared bacterial liquid is prepared by inoculating the Acetobacter into a bean clear liquid for fermentation.

5. The use of the Acetobacter sp LHZ-ST001 in the production of Miao Dong minority sour soup according to claim 4, wherein the fermentation temperature of the Acetobacter sp is 32-36 ℃.

6. Use of the Acetobacter sp LHZ-ST001 in the production of the Miao Dong minority sour soup according to claim 1 or 4, which comprises the following steps: (1) preparing strains, namely inoculating acetobacter into a fresh and sterilized bean clear liquid matrix, and fermenting at the temperature of 32-36 ℃ for 20-24h to obtain a bacterial liquid; (2) preparing chili sauce and tomato sauce by removing stems of fresh chili and tomato, cleaning, and chopping into pieces; pulverizing into paste with colloid mill; (3) mixing, mixing the above Capsici fructus, tomato sauce, salt, rhizoma Zingiberis recens, Bulbus Allii, and rice wine, and inoculating 5-10% strain; (4) fermenting, namely fermenting the product obtained in the mixing step at the temperature of 32-36 ℃ for 15-20 days to obtain the Dong minority sour soup;

7. The application of Acetobacter sp (LHZ-ST 001) in the production of the Miao Dong minority sour soup according to claim 6, wherein the material component ratio in the mixed material in the step (3) is as follows: 6-8 parts of tomato, 2-4 parts of pepper, 0.1-0.2 part of salt, 0.4-0.6 part of ginger, 0.4-0.6 part of garlic and 0.1-0.2 part of rice wine, wherein the adding ratio of the bacterial liquid is 0.5-1 part.