CN116814509B - Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii - Google Patents
Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an acetobacter pasteurism strain which is resistant to ethanol, acid and high in acid yield. Compared with the current common industrial strains Shanghai brewing 1.01 and AS1.41, the acetic acid bacteria SP021 has good tolerance to high-concentration ethanol and can tolerate ethanol with the concentration of 14%; can grow under the condition of pH 2.0 and is suitable for fermentation of high-acidity products. Under the same culture condition, the SP021 has higher total acid, butyric acid and lactic acid yield than Shanghai brewing 1.01 and AS1.41, and has higher industrial application value.
Description
Technical Field
The invention belongs to the field of microorganisms and fermentation industry, and particularly relates to ethanol-resistant acid-resistant high-acid-yield acetobacter pastoris.
Background
Acetic acid bacteria are an important industrial microorganism, and are indispensable in the process of producing vinegar and fruit vinegar. In addition, acetic acid bacteria are also commonly used in the production of fermented products from fruits and vegetables to convert ethanol to acetic acid. Acetic acid bacteria strain is one of key factors affecting the quality of fruit and vegetable fermented products. Acetic acid bacteria such AS AS1.41 and Shanghai brewing 1.01 commonly used in the industry at present have the problems of weak ethanol tolerance, weak acid production capacity and the like, and the optimal growth pH of the strains is 3.5-6.5, so that the strain is not suitable for the production of high-acidity products.
Therefore, the screening of acetic acid bacteria strains which are tolerant to high concentration ethanol and high acidity and produce acid with high acid yield has important significance for industrial application such as fruit and vegetable fermentation.
Disclosure of Invention
The invention provides an ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii strainAcetobacter pasteurianusSP021. Compared with the current common industrial strains Shanghai brewing 1.01 and AS1.41, the acetic acid bacteria have good tolerance to high-concentration ethanol and can tolerate ethanol with the concentration of 14 percent; can grow under the condition of pH 2.0 and is suitable for fermentation of high-acidity products. Under the same culture condition, the SP021 has higher total acid, butyric acid and lactic acid yield than Shanghai brewing 1.01 and AS1.41, and has higher industrial application value.
Acetobacter pasteurism of the inventionAcetobacter pasteurianus) SP021 was deposited at 28, 9, 2022 with the chinese microbiological bacterial culture collection center (address: the microbiological institute of China Beijing China academy of sciences, beijing, kogyo area, north Chen West Lu No. 1, 3) with the preservation number of CGMCC No. 25794.
In one aspect, the invention provides acetobacter pasteurism which is preserved in China general microbiological culture Collection center with a preservation number of CGMCC No. 25794.
In one aspect, the invention provides a composition comprising acetobacter pasteurisation as described herein.
In one embodiment, the composition is a microbial inoculant composition for fermentation.
In one aspect, the invention provides the use of acetobacter pasteurisation as described herein in the production of a ferment from fruit and vegetable. In one embodiment, the fruit and vegetable is selected from the group consisting of a medicinal and edible source, citrus or berry fruit. In one embodiment, the fruit and vegetable is selected from litchi, apple, wolfberry, mulberry, citrus, grapefruit, lemon, blueberry, raspberry, cranberry, elderberry, or pomegranate.
In one aspect, the invention provides the use of the acetobacter pasteurisation described herein in the production of an organic acid, wherein the organic acid comprises acetic acid, propionic acid and/or butyric acid.
In one aspect, the invention provides the use of the acetobacter pasteurism described herein in brewing vinegar and/or fruit vinegar.
In one aspect, the invention provides a method of preparing a fruit and vegetable ferment comprising the steps of pressing fruit and vegetable into fruit and vegetable juice and fruit and vegetable marc, and mixing acetobacter pasteurii as described herein with the fruit and vegetable juice and/or fruit and vegetable marc, and performing fermentation.
In one embodiment, the fruit and vegetable is selected from the group consisting of a medicinal and edible source, citrus or berry fruit. In one embodiment, the fruit and vegetable is selected from litchi, apple, wolfberry, mulberry, citrus, grapefruit, lemon, blueberry, raspberry, cranberry, elderberry, or pomegranate.
In one aspect, the invention provides fruit and vegetable ferments produced by the methods described herein.
The advantages of the invention include:
1. the acetobacter pasteurism SP021 of the present invention can tolerate higher concentrations of ethanol.
2. The acetobacter pasteurism SP021 of the present invention can withstand the acidic conditions of lower pH.
3. The acetobacter pasteurisi SP021 of the present invention may increase the total acid and/or organic acid such as acetic acid, butyric acid or lactic acid.
4. Compared with the current common industrial strains Shanghai brewing 1.01 and AS1.41, the Acetobacter pastoris SP021 has good tolerance to high-concentration ethanol and can tolerate ethanol with the concentration of 14 percent; can grow under the condition of pH 2.0 and is suitable for fermentation of high-acidity products. Under the same culture condition, the SP021 has higher total acid, butyric acid and lactic acid yield than Shanghai brewing 1.01 and AS1.41, and has higher industrial application value.
Drawings
FIG. 1 shows the Acetobacter pasteurisis SP021 treelet. The phylogenetic tree is constructed using the NJ adjacency method.
Detailed Description
For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention and are not limiting of the invention claims. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the description of the presently disclosed subject matter.
The present invention is based in part on the inventors' discovery of new strains of acetobacter pasteurisis. The Acetobacter pastoris strain is preserved in China general microbiological culture Collection center (CGMCC) at 9 and 28 of 2022, and the preservation number is CGMCC No. 25794. The inventor finds that compared with the industrial strains Shanghai brewing 1.01 and AS1.41 which are commonly used at present, the Acetobacter pastoris strain has good tolerance to high-concentration ethanol and can tolerate ethanol with the concentration of 14 percent; can grow under the condition of pH 2.0 and is suitable for fermentation of high-acidity products. Under the same culture condition, the SP021 has higher total acid, butyric acid and lactic acid yield than Shanghai brewing 1.01 and AS1.41, and has higher industrial application value.
The acetobacter pasteurism strain of the present invention may be used as a starter for brewing vinegar and/or fruit vinegar, or may be used for the production of organic acids. The organic acid produced may include, but is not limited to, acetic acid, propionic acid, and/or butyric acid. The strain of Acetobacter pasteurisi of the present invention may also be formulated into a composition with other further strains. The composition may be a microbial inoculant composition for fermentation. Examples of other strains are not particularly limited, and may be any strain known in the art suitable for use with acetobacter pasteurisi.
The strain of Acetobacter pasteurisi of the present invention is in an isolated or pure or substantially pure form (e.g., the strain of Acetobacter pasteurisi in culture comprises more than 80%). The strain may be present or sold in lyophilized form or in culture. In these lyophilized or cultured forms, other suitable ingredients may be included in addition to the Acetobacter pasteurisus strain of the invention, such as a medium or a protectant for lyophilization, and the like. These ingredients are conventional in the art. The culture may be any suitable culture, including solid or liquid cultures.
The acetobacter pasteurism strain of the present invention may also be used in the production of fruit and vegetable ferments. The type of fruit and vegetable ferment is not particularly limited, and may be derived from fruit and vegetable ferments of fruits and vegetables such as litchi, apple and/or blueberry. Methods for producing fruit and vegetable ferments are known in the art and are not particularly limited. For example, a method of preparing a fruit and vegetable ferment may comprise the steps of pressing fruit and vegetable into fruit and vegetable juice and fruit and vegetable marc, and mixing the acetobacter pasteurium described herein with the fruit and vegetable juice and/or fruit and vegetable marc, and performing the fermentation.
The invention also encompasses fruit and vegetable ferments produced by the methods described herein. Beneficial components including short chain fatty acids, organic acids, etc. are produced during fermentation of fruits and vegetables via metabolic activity of microorganisms. Short chain fatty acids are the main products of bacterial fermentation of carbohydrates, not only involved in the energy metabolism of the organism, but also regulating immunity, protecting the intestinal mucosal barrier, playing an important role in maintaining intestinal health. Short chain fatty acids mainly include acetic acid, propionic acid and butyric acid, which are substrates for preferential utilization by intestinal epithelial cells. Organic acids are also important products of bacterial fermentation of carbohydrates. The organic acid produced by acetic acid bacteria fermentation is mainly acetic acid and then lactic acid. Lactic acid can relieve sour taste irritation caused by acetic acid, and improve taste of fermentation product. In addition, lactic acid can lower intestinal pH, thereby inhibiting colonization and growth of harmful bacteria, and thus can promote intestinal health. The acetobacter pasteurism SP021 described herein has a higher capacity for producing butyric acid and lactic acid than the usual industrial strains, exhibiting a better intestinal probiotics effect.
Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The present application will be explained in further detail with reference to examples. However, those skilled in the art will appreciate that these examples are provided for illustrative purposes only and are not intended to limit the present application.
Examples
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. All amounts listed are described in weight percent based on total weight unless otherwise indicated. The present application should not be construed as limited to the particular embodiments described.
Materials used in the examples
Culture medium:
(1) Liquid medium: 2% of yeast powder, 2% of glucose, 2% of ethanol and pH 7.0;
(2) Screening the culture medium: 2% of yeast powder, 2% of glucose, 2% of ethanol, 2% of agar, 1% of calcium carbonate and pH 7.0;
(3) Seed liquid culture medium: 2% of yeast powder, 2% of glucose, 4% of ethanol and pH 7.0;
(4) Slope preservation medium: 2% of yeast powder, 2% of glucose, 2% of ethanol, 2% of agar and pH 7.0;
(5) Viable count detection/plate medium: 2% of yeast powder, 2% of glucose, 4% of ethanol, 2% of agar and pH 5.0-5.2;
(6) Tolerance test/acid production ability test medium: yeast powder 2%, glucose 2%, ethanol 4% or according to experimental conditions, pH 5.0-5.2 or according to experimental conditions.
Strains: acetobacter pasteurisis, shanghai 1.01 and AS1.41, were purchased from the Guangdong collection of microorganisms and had strain numbers GDMCC 1.152 and GDMCC 1.67, respectively.
Example 1: separating acetic acid bacteria strain SP021 from natural fermented fruit and vegetable fermentation liquid
1. Plate primary screening: collecting natural fermented fruit and vegetable fermentation liquid, mixing the natural fermented fruit and vegetable fermentation liquid with acetic acid bacteria liquid culture medium according to the volume ratio of 1:1, and measuring the total acid mass concentration. When the total acid is obviously increased, the culture solution is diluted by a multiple ratio by using sterile water and is coated on a screening culture medium for culturing at the constant temperature of 30 ℃ for 72 hours. And (3) taking single colonies with obvious colony morphology and high growth speed for streak separation until single colonies with transparent rings and consistent morphology appear on the flat plate.
2. Qualitative experiments: sodium salt or calcium salt solution formed by acetic acid can generate reddish brown precipitate when being heated together with ferric trichloride, and blank stock solution has no precipitate. Taking bacterial liquid in a liquid culture medium, centrifuging for 20min at 6000r/min and 4 ℃, taking 5mL supernatant with bacterial cells removed, neutralizing with 1mol/L sodium hydroxide solution to pH 7.0, boiling, adding 3 drops of FeCl3 solution with mass fraction of 5%, and forming reddish brown precipitate to indicate that acetic acid is generated, wherein the corresponding strain is an acetic acid-producing strain.
3. High-yield acetic acid strain re-screening: the separated acetic acid-producing strain is inoculated into 100mL of seed liquid culture medium, the constant temperature shaking culture is carried out at 30 ℃ and 200r/min, and the total acid mass concentration is measured by sampling after fermentation for 72 hours.
Experimental results: on the screening plate, the acidogenic bacteria can produce acetic acid by using ethanol in the culture medium, and the product acetic acid reacts with calcium carbonate, so that clear transparent circles are produced around the acidogenic strain. The size of the ratio (U value) of the diameter of the transparent ring to the diameter of the colony is used as a standard. After the culture is carried out for 48 hours at the constant temperature of 30 ℃ on a primary screening culture medium, 9 single colonies are separated out, wherein the transparent circles of strains C3, C4, C7 and C9 are the largest, which indicates that the 4 strains have vigorous growth capacity and stronger acid production capacity. The four strains are taken for acid-producing qualitative tests, and only the bottoms of test tubes corresponding to the strains C3, C7 and C9 are found to generate reddish brown precipitate, so that the C4 is not acetic acid bacteria, and the strains C3, C7 and C9 are all acetic acid-producing strains. Shake flask experiments were performed on 3 acetic acid producing strains after initial screening. After 72h of constant temperature shaking culture, the acid production amounts of C3, C7 and C9 are 21.45 g/L, 48.32 g/L and 30.50g/L respectively. Therefore, the strain C7 with the largest acid yield is selected as the optimal strain separated from the naturally fermented fruit and vegetable fermentation liquid, and the next step of sequencing identification and preservation is carried out.
Example 2: identification of Acetobacter SP021 Strain
The C7 strain selected in example 1 was sent to Hua Dacron technologies Co., ltd for sequencing. The company uses the following primers for sequencing.
Primer information: 27F is AGAGTTTGATCCTGGCTCAG;1492R is TACGGCTACCTTGTTACGACTT.
The primer is used for cloning to obtain the 16sRNA sequence of the strain C7, namely the conserved sequence.
TGGCTCAGAGCGAACGCTGGCGGCATGCTTAACACATGCAAGTCGCACGAAGGTTTCGGCCTTAGTGGCGGACGGGTGAGTAACGCGTAGGTATCTATCCATGGGTGGGGGATAACACTGGGAAACTGGTGCTAATACCGCATGACACCTGAGGGTCAAAGGCGCAAGTCGCCTGTGGAGGAGCCTGCGTTTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCAATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTCGACGGGGACGATGATGACGGTACCCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGTAGGCGGTTTGTACAGTCAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGATACGTGCAGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTAGATGTTGGGTGACTTAGTCATTCAGTGTCGCAGTTAACGCGTTAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGGCTTGAATGTAGAGGCTGCAAGCAGAGATGTTTGTTTCCCGCAAGGGACCTCTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTAGTTGCCATCAGGTTGGGCTGGGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTAGGTGGTGACACCATGCTGATCTCTAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAAGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCGCAAGGACGC。
Phylogenetic tree was constructed using the NJ adjacency method. This method is one of the common methods for constructing phylogenetic trees. In phylogenetic classification studies, a visual tree-like branch pattern is often used to represent the evolutionary and affinitive relationships of organisms, and this pattern is called a phylogenetic tree (phylogenetic trees). The idea of the adjacency method is to calculate the minimum pairwise comparison distance and minimize the length of the whole tree, so that the topological structure of the tree is limited. The algorithm starts with a star tree, all species start from a central node, and then the two sequences of neighbors are successively found by calculating the sum of the minimum branch lengths, and the sequence pairs that minimize the overall branch length of the tree are grouped in each round by considering all possible sequence pairs, thereby generating a new distance matrix until all neighbor sequences are found.
Sequencing results show that the strain C7 is amplified to a conserved sequence 1386bp by a 27F+1499R primer, and compared with data in Genbank after sequencing, statistics show that the evolution conserved sequence of the strain and Acetobacter pasteurelloidesAcetobacter pasteurianusHomology reaches 99.93%; the evolution tree was constructed using NJ neighbor method and standard Acetobacter in NCBI database, and the results of FIG. 1 show that the strain was identical to Acetobacter pastorisAcetobacter pasteurianusLMD22.1 (NR 026107.1) closest affinity, score 66, next closest affinity is acetobacter miqAcetobacter oryzoeniB6 (MK 367404.1). Therefore, the bacteria obtained by screening by the inventor is a brand new strain of Acetobacter pasteuris, which is named as Acetobacter pasteuris @Acetobacter pasteurianus) SP021. The strain is preserved in China general microbiological culture Collection center (address: china national academy of sciences of Beijing, china) with a preservation number of CGMCC No. 25794 at 28 days of 9 months 2022.
Example 3: acetobacter pasteurii (L.) BakerAcetobacter pasteurianus) SP021 tolerant to high concentration ethanol
Experimental method
Ethanol tolerance test of acetobacter pasteurisi SP 021: acetobacter pasteurii SP021 broth (number of viable bacteria 7.55X10) 7 CFU/mL) was added to a 250mL Erlenmeyer flask containing 50mL of liquid medium (pH 5.0, ethanol concentrations of 4%, 6%, 10%, 12%, 14%, respectively)Culturing at 30deg.C in a shaking table of 150 r/min. The bacterial liquid is taken after 24 hours and 72 hours of culture to respectively measure the growth OD of the bacterial 600nm The value and the viable count of the strain.
OD 600nm Detection of values: 3mL of the bacterial liquid was taken and measured by selecting a wavelength of 600nm under an ultraviolet spectrophotometer. If the fermentation broth is too turbid (the cell density is high), the cell broth is diluted 10 times with sterile water and then measured.
Detecting the number of living bacteria: adding 0.1mL of the bacterial liquid into a sterilizing centrifuge tube, adding 0.9mL of sterile water, shaking and shaking uniformly to obtain 10 -1 Diluting the bacterial liquid. Then 0.1mL of the bacterial liquid is taken and added into a 2mL centrifuge tube containing 0.9mL of sterile water to obtain 10 -2 Diluting the bacterial liquid. By such pushing, 0.1mL of bacterial liquid with proper dilution is taken, and viable bacteria are counted by a coating method. Three dilutions were taken for each sample and each dilution was tested in duplicate.
Ethanol tolerance test of different strains: the acetic acid bacteria of Huniang 1.01, AS1.41 and SP021 (number of viable bacteria is 1×10) were inoculated in an amount of 2% 8 CFU/mL) was added to a 250mL Erlenmeyer flask containing 50mL of liquid medium (pH 5.0, ethanol concentrations of 10%, 12%, 14%, respectively) and incubated at 30deg.C in a 150r/min shaker. Culturing for 48 hr, and collecting bacterial liquid to measure the growth OD of the strain 600nm Values.
Results
The results of the ethanol tolerance test for acetobacter pasteurisi SP021 are shown in table 1. While it has been reported that Acetobacter pasteurism tolerates 9-11% alcohol (cf. CN 109735464B), the Acetobacter pasteurism SP021 of the present invention is still capable of growing under conditions of 10%, 12% and 14% high concentration ethanol, indicating tolerance to high concentration ethanol.
Table 1: results of the ethanol tolerance test of Acetobacter pasteurellosis SP021
Further, the ethanol tolerance of Acetobacter pasteurisis SP021 was compared to that of the industrial strains Huniang 1.01 and AS1.41 of Acetobacter pasteurisis. Table 2 shows the results of the experiment, tableMing industrial strains Shanghai brewing 1.01 and AS1.41 OD at 10%, 12% and 14% high concentration ethanol 600nm The value is 0, the growth cannot be realized, and the acetobacter pasteurii SP021 can still grow, and the ethanol tolerance is better.
Table 2: comparison of ethanol tolerance of Acetobacter pasteurisis SP021 with Shanghai brewing 1.01 and AS1.41
Example 4: acetobacter pasteurii (L.) BakerAcetobacter pasteurianus) SP021 tolerance to high acidity
Experimental method
Acetobacter pasteuris SP021 resistance acid test: the bacterial liquid of Acetobacter pasteurii SP021 (number of viable bacteria 7.55X10) 7 CFU/mL) was added to a 250mL Erlenmeyer flask containing 50mL of liquid medium (pH 5.0, 3.0, 2.5, 2.0, ethanol concentration 4%) and cultured at 30℃in a 150r/min shaker. The bacterial liquid is taken after 24 hours and 72 hours of culture to respectively measure the growth OD of the bacterial 600nm The value and the viable count of the strain. Bacterial OD 600nm The value and the number of viable bacteria were determined as above.
Acid resistance test of different strains: the acetic acid bacteria of Huniang 1.01, AS1.41 and SP021 (number of viable bacteria is 1×10) were inoculated in an amount of 2% 8 CFU/mL) was added to a 250mL Erlenmeyer flask containing 50mL of liquid medium (pH 2.5 and 2.0, ethanol concentration 4%) and incubated at 30℃in a 150r/min shaker. Culturing for 48 hr, and collecting bacterial liquid to measure the growth OD of the strain 600nm Values.
Results
While the prior art reports that the optimal growth pH of the fermentation industrial bacteria Shanghai 1.01 and AS1.41 is 3.5-6.5, the acetic acid bacteria SP021 of the invention can withstand the high acidity condition with the pH below 3.5, the number of viable bacteria still increases at the pH of 2.5 and can still survive at the pH of 2.0, AS shown in Table 3.
Table 3: acid resistance of Acetobacter pasteurellosis SP021
Further, the acid resistance of Acetobacter pasteurisis SP021 was compared to that of industrial strains Shanghai 1.01 and AS 1.41. Experimental results show that the industrial strains Shanghai brewing 1.01 and AS1.41 have OD under the high acidity condition of pH 2.5 and pH 2.0 600nm OD with value lower than that of Acetobacter pastoris SP021 600nm The values indicate that Acetobacter pastoris SP021 grows better than Huniang 1.01 and AS1.41 under high acid conditions, and has higher acid resistance, AS shown in Table 4.
Table 4: acid resistance of Acetobacter Pasteurensis SP021 to Shanghai brewing 1.01 and AS1.41
Example 5: acid energy production detection of acetobacter pasteurii (Acetobacter pasteurianus)
Experimental method
Acid energy detection of acetic acid bacteria: shanghai brewing 1.01, AS1.41 and SP021 (number of viable bacteria is 0.2X10) 7 CFU/mL) was added to a 250mL Erlenmeyer flask containing 100mL of acid-producing medium (pH 5.0, ethanol concentration 4%) and cultured in a shaker at 30℃at 150 r/min. After 24h incubation, lmL samples were accurately removed by pipette into 150mL beakers, 50mL of distilled water was added, and after one piece of rotor was placed, 0.1mol/L NaOH standard solution was slowly dropped to ph=8.2 with stirring on a constant temperature magnetic stirrer, and the reading was recorded. The total acid (in acetic acid) content is calculated as follows: total acid content (g/L) = C x (V 1 -V 0 ) x 60 / V 2
Wherein: v (V) 0 Is the consumption of NaOH titration blank and mL;
V 1 is the consumption of NaOH titration, and is mL;
V 2 is to suck the sample volume, mL;
c is the concentration of standard NaOH, mol/L;
60 is the molar mass, g/mol, of acetic acid.
Butyric acid and lactic acid production performance of acetic acid bacteriaAnd (3) testing: shanghai brewing 1.01, AS1.41 and SP021 (number of viable bacteria is 0.2X10) 7 CFU/mL) is inoculated with 50mL of acetic acid bacteria culture medium (pH 5.0, ethanol concentration 4%), the culture is carried out in a constant temperature cabinet at 30 ℃ for 24 hours, the culture is sealed by a preservative film, 2mL of fermentation liquor is taken, the volume is fixed to 20mL by ultrapure water, the centrifugation is carried out for 20min at 9500r/min, and then the supernatant is filtered by a microporous filter membrane of 0.22 mu m. The content of lactic acid and butyric acid was determined by external standard method. The chromatographic measurement conditions are as follows: liquid phase system UItimate3000; chromatographic column sugar acid column C184.6X105 mm,5 μm; mobile phase sulfuric acid aqueous solution (273. Mu.L of concentrated sulfuric acid was added to 1L of ultrapure water); the sample injection amount is 20 mu L; the flow rate is 0.6mL/min; the detection time is 30min; detector UV210nm; column temperature: room temperature.
Results
The acetic acid bacteria have the function of converting ethanol into acetic acid in the fermentation process, and the higher the acetic acid content is, the better the fermentation performance of the acetic acid bacteria is. The experimental result shows that the acid yield of the acetobacter pastoris SP021 of the invention is about 5.6-20 times that of two strains of industrial bacteria Shanghai brewing 1.01 and AS1.41 under the acidic culture condition of pH 2.5 in the culture time of 1-7 days, and the acetobacter pastoris SP021 shows excellent acid resistance and acid production capacity.
Table 5: acid production of Acetobacter pasteurism SP021 and Industrial Shanghai brewing 1.01 and AS1.41
Beneficial components including short chain fatty acids, organic acids, etc. are produced during fermentation of fruits and vegetables via metabolic activity of microorganisms. Short chain fatty acids are the main products of bacterial fermentation of carbohydrates, not only involved in the energy metabolism of the organism, but also regulating immunity, protecting the intestinal mucosal barrier, playing an important role in maintaining intestinal health. Short chain fatty acids mainly include acetic acid, propionic acid and butyric acid, which are substrates for preferential utilization by intestinal epithelial cells. Organic acids are also important products of bacterial fermentation of carbohydrates. The organic acid produced by acetic acid bacteria fermentation is mainly acetic acid and then lactic acid. Lactic acid can relieve sour taste irritation caused by acetic acid, and improve taste of fermentation product. In addition, lactic acid can lower intestinal pH, thereby inhibiting colonization and growth of harmful bacteria, and thus can promote intestinal health.
The inventors further compared the ability of acetic acid bacteria SP021 to produce specific short chain fatty acids and organic acids with common industrial strains. The results showed that under the same culture conditions, SP021 produced butyric acid 5.7 times AS high AS that of industrial strain Shanghai 1.01 and 1.3 times AS high AS that of AS 1.41; the amount of lactic acid produced was 2.1 times that of Shanghai brewing 1.01 and 2 times that of AS1.41, AS shown in Table 6. SP021 has higher butyric acid and lactic acid production capacity than common industrial strains, and shows better intestinal probiotics.
Table 6: butyric acid and lactic acid production of Acetobacter pastoris SP021 and Industrial Shanghai brewing 1.01 and AS1.41
The invention provides an ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii strainAcetobacter pasteurianusSP021. Compared with the current common industrial strains Shanghai brewing 1.01 and AS1.41, the acetic acid bacteria have good tolerance to high-concentration ethanol and can tolerate ethanol with the concentration of 14 percent; can grow under the condition of pH 2.0 and is suitable for fermentation of high-acidity products. Under the same culture condition, the SP021 has higher total acid, butyric acid and lactic acid yield than Shanghai brewing 1.01 and AS1.41, and has higher industrial application value.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (17)
1. Acetobacter pasteurii (Acetobacter pasteurianus) SP021 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 25794.
2. A composition comprising the acetobacter pasteurisi SP021 of claim 1.
3. The composition of claim 2 which is a microbial inoculant composition for fermentation.
4. The composition of claim 2, which is in lyophilized form or in culture.
5. Use of acetobacter pasteurisi SP021 according to claim 1 in food products.
6. The use according to claim 5, wherein the food product is a dietary supplement.
7. Use of acetobacter pasteurism SP021 according to claim 1 in the production of a ferment from fruit and vegetable.
8. The use according to claim 7, wherein the fruits and vegetables are of the pharmaceutical and food homology.
9. Use according to claim 7, wherein the fruit and vegetable is selected from citrus or berry fruits.
10. The use according to claim 7, wherein the fruit and vegetable is selected from litchi, apple, matrimony vine, mulberry, citrus, grapefruit, lemon, blueberry, raspberry, cranberry, elderberry or pomegranate.
11. A method of preparing a fruit and vegetable ferment comprising the steps of pressing fruit and vegetable into fruit and vegetable juice and fruit and vegetable marc, and mixing the acetobacter pasteurii according to claim 1 with the fruit and vegetable juice and/or fruit and vegetable marc, and performing fermentation.
12. The method of claim 11, wherein the fruit and vegetable are of the pharmaceutical and food homology.
13. The method of claim 11, wherein the fruit and vegetable is selected from citrus or berry fruits.
14. The method of claim 11, wherein the fruit and vegetable is selected from litchi, apple, medlar, mulberry, citrus, grapefruit, lemon, blueberry, raspberry, cranberry, elderberry, or pomegranate.
15. A ferment produced by the method of claim 12.
16. Use of acetobacter pasteurism SP021 according to claim 1 in the brewing of table vinegar and/or fruit vinegar.
17. Use of acetobacter pasteurisi SP021 according to claim 1 in the production of organic acids comprising acetic acid, propionic acid and/or butyric acid.
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| CN103614318A (en) * | 2013-11-08 | 2014-03-05 | 天地壹号饮料股份有限公司 | High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation |
| KR20230022672A (en) * | 2021-08-09 | 2023-02-16 | 경북대학교 산학협력단 | Novel traditional vinegar-derived strains and uses thereof |
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| CN102433268A (en) * | 2011-08-08 | 2012-05-02 | 丽水市鱼跃酿造食品有限公司 | Acetobacter pasteurium and its application method in producing citrus vinegar |
| CN103614318A (en) * | 2013-11-08 | 2014-03-05 | 天地壹号饮料股份有限公司 | High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation |
| KR20230022672A (en) * | 2021-08-09 | 2023-02-16 | 경북대학교 산학협력단 | Novel traditional vinegar-derived strains and uses thereof |
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