CN106010996B - Acetobacter and culture separation method, screening method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of microorganisms, in particular to acetobacter, a culture separation method, a screening method and application thereof, wherein the acetobacter is classified and named as: acetobacter passarianus is preserved in China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC M2016036, and the preservation date is 2016, 1 and 14. According to the bacillus aceticus and the culture separation method, the screening method and the application thereof, the culture separation method and the screening method of the bacillus aceticus are reasonably designed, and finally high-quality bacillus aceticus which is high in acid production capacity and alcohol resistance in mixed fruit and vegetable juice is effectively obtained.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to acetobacter aceti, and a culture separation method, a screening method and application thereof.
Background
The fruit vinegar is a new generation of acidic condiment or beverage which is made by using fruits or leftovers of fruit processing as main raw materials and fermenting the fruits and acetic acid through alcohol fermentation and acetic acid fermentation and has rich nutrition, good flavor and outstanding health care effect. The fruit vinegar in China has a long history, is almost the same as fruit wine, and is recorded as early as the summer. Compared with grain vinegar, the fruit vinegar is rich in nutrient components, and contains more than 10 kinds of organic acids, and various amino acids, vitamins, inorganic salts, minerals, carbohydrates and the like required by human bodies. With the continuous improvement of living standard of people, the health care function of the fruit vinegar is more and more emphasized by people, and researches show that: the fruit vinegar has the functions of dietotherapy health care, weight reduction, skin whitening and protection, disease resistance, aging resistance, fatigue resistance and the like, and is known as food in the 21 st century. The hot tide of drinking fruit vinegar is also being raised in China. At present, researches on fruit vinegar strain breeding, fruit vinegar process and fruit vinegar blending are more and more favored by researchers, and the strain for brewing the fruit vinegar is one of important factors influencing the taste and the quality of the fruit vinegar.
At present, two acetic acid bacteria are mainly used in China in the fruit vinegar brewing process, one is a strain used in liquid state fermentation of fruit vinegar, namely Asl.41 (A.racemse L.) foul Acetobacter turbidized variety, the other is a strain separated from Dandong fast brewed vinegar by Shanghai brewing scientific research institute and Shanghai vinegar factory, the acetic acid bacteria are bacteria capable of oxidizing alcohol into acetic acid, the characteristic understanding of the acetic acid bacteria is already known in China before 1400 years, and [23] is successfully utilized in Pasteur 1868 to illustrate that the acetic acid bacteria are microorganisms in the vinegar, according to the physiological and biochemical characteristics of the acetic acid bacteria, the acetic acid bacteria can be divided into Acetobacter (Acetobacter) and Gluconobacter (Gluconobacter), the acetic acid bacteria are mainly used for oxidizing alcohol into acetic acid and are used in the vinegar industry, the Gluconobacter is mainly used for producing the acetic acid, the gluconic acid and the gluconic acid can be further applied to the vinegar brewing industry, and the vinegar industry can be further used for producing the acetic acid, the gluconic acid and the Vc Vc..
The selection of fruit vinegar strains is one of the key factors influencing the quality of fruit vinegar. The turbid variety of the acetobacter putida and the Shanghai brewing 1.01 acetobacter aceti (A.lovaniense L) which are added into the strains currently used for producing the fruit vinegar have the defects of low acid production capability and alcohol resistance capability and insufficient fragrance of the formed fruit vinegar.
Therefore, in order to overcome the defects in the prior art, an effective screening method and a cultivation and separation method for acetobacter are urgently needed to obtain acetobacter with excellent quality, strong acid production capacity and high alcohol resistance.
Disclosure of Invention
The invention aims at the culture and separation method and the screening method of the acetobacter aceti, and has the advantages of easy operation and simple method, and the finally screened acetobacter aceti has high acid yield and fast growth rate.
In order to achieve the technical effects, the invention comprises the following technical scheme:
a acetobacter species, which is classified and designated as: acetobacter passarianus is preserved in China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC M2016036, and the preservation date is 2016, 1 and 14.
The 16s DNA sequence of the acetobacter is shown in the amino acid sequence shown in SEQ ID NO. 1.
A bacteriocin is prepared by fermenting the above acetobacter.
The method for culturing and separating the acetobacter comprises the following steps: taking a vinegar culture sample of naturally fermented vinegar in mixed fruits and vegetables or grains, putting the vinegar culture sample into a container filled with a basic culture medium, carrying out shake culture, taking a diluted proliferation solution, inoculating the vinegar culture sample onto a separation culture medium by a coating method for culture, selecting a single colony with a transparent ring after the culture is finished, transferring the single colony to an inclined plane preservation culture medium, and preserving to obtain the acetobacter.
Further, the shaking culture condition is that the shaking culture is carried out for 48 hours at the rotating speed of 120r/min and the temperature of 30 ℃; diluting the proliferation solution with sterile water 100000 times, 1000000 times and 10000000 times, inoculating vinegar culture sample onto separate culture medium by coating method, culturing at 30 deg.C for 72 hr, and finishing the culture.
Further, the preparation method of the basic culture medium comprises the following steps: heating and dissolving yeast extract and glucose with the same mass in water, adjusting pH to 4.5, fixing volume to 1L, subpackaging, and sterilizing at 121 deg.C for 20 min;
the preparation method of the separation culture medium comprises the following steps: adding agar into 100ml of the basic culture medium, sterilizing at 0.1MPa and 121 deg.C, and addingSterile CaCO3And absolute ethyl alcohol, respectively filling into sterile culture dishes to prepare separation culture media; the agar accounts for 2 percent of the volume of the separation culture medium, and the sterile CaCO3The volume percentage of the absolute ethyl alcohol in the separation culture medium is 2%, and the volume percentage of the absolute ethyl alcohol in the separation culture medium is 3%;
the preparation method of the slant preservation culture medium comprises the following steps: and adding agar into the prepared basic culture medium, sterilizing at the temperature of 121 ℃ under the pressure of 0.1MPa, and adding sterile CaCO3 and absolute ethyl alcohol to obtain the slant preservation culture medium.
The screening method of the acetobacter comprises the following steps:
the method comprises the following steps: culturing and separating the acetobacter strains: taking a vinegar culture sample of naturally fermented vinegar in mixed fruits and vegetables or grains, putting the vinegar culture sample into a container filled with a basic culture medium, carrying out shake culture, taking a diluted proliferation solution, inoculating the vinegar culture sample onto a separation culture medium by a coating method for culture, selecting a single colony with a transparent ring after the culture is finished, transferring the single colony to an inclined plane preservation culture medium, and preserving to obtain acetobacter;
step two: screening of acetobacter: transferring the acetobacter obtained in the first step into a basic culture medium, performing shake culture to obtain acetobacter strain seed liquid, respectively inoculating the prepared acetobacter strain seed liquid into a basic culture medium containing 0%, 5%, 9%, 11%, 13% and 15% alcohol by volume percentage, performing shake culture, then determining the OD value and the acid yield, and screening the acetobacter according to the determination result.
Further, in the first step, the shaking culture condition is that the shaking culture is carried out for 48 hours at the rotating speed of 120r/min and the temperature of 30 ℃; diluting the proliferation solution to concentrations of 10-5, 10-6 and 10-7 with sterile water, inoculating vinegar culture samples to a separation culture medium by coating method, culturing at 30 deg.C for 72 hr, and finishing the culture.
Further, in the first step, the preparation method of the basic culture medium comprises the following steps: heating and dissolving yeast extract and glucose with the same mass in water, adjusting pH to 4.5, fixing volume to 1L, subpackaging, and sterilizing at 121 deg.C for 20 min;
the preparation method of the separation culture medium comprises the following steps: adding agar into 100ml of the basic culture medium, sterilizing at 0.1MPa and 121 ℃, and adding sterile CaCO3And absolute ethyl alcohol, which are respectively filled in a sterile culture dish to prepare a separation culture medium; the agar accounts for 2 percent of the volume of the separation culture medium, and the sterile CaCO3The volume percentage of the absolute ethyl alcohol in the separation culture medium is 2%, and the volume percentage of the absolute ethyl alcohol in the separation culture medium is 3%;
the preparation method of the slant preservation culture medium comprises the following steps: adding agar into the prepared basic culture medium, sterilizing at 121 deg.C under 0.1MPa, adding sterile CaCO3And absolute ethyl alcohol to obtain the slant preservation culture medium.
Further, in the second step, the shaking culture condition is 30 ℃, 200r/min, and the shaking culture is 24 hours; the shake culture conditions are 30 ℃, 200-220r/min and 48h of shake culture; the OD value is determined by measuring the absorbance at a wavelength of 600nm with a spectrophotometer and calculating the OD value.
The application of acetobacter is used for fermenting grain mixed alcohol or acetic acid of fruit and vegetable juice.
By adopting the technical scheme, the method has the following beneficial effects: according to the bacillus aceticus and the culture separation method, the screening method and the application thereof, the culture separation method and the screening method of the bacillus aceticus are reasonably designed, and finally, high-quality bacillus aceticus which is strong in acid production capacity, high in alcohol resistance and high in growth rate in mixed fruit and vegetable juice is effectively obtained.
Drawings
FIG. 1 is a diagram of a colony of Acetobacter of the present invention;
FIG. 2 is a 100-fold magnified micrograph of Acetobacter strains according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific examples.
The first embodiment is as follows: a acetobacter species, which is classified and designated as: acetobacter pasteianus, preserved in China Center for Type Culture Collection (CCTCC), with the preservation number of CCTCC M2016036 and the preservation date of 2016, 1 and 14. The preservation address is Wuhan, Wuhan university.
A bacteriocin is prepared by fermenting the above acetobacter.
The acetobacter is used for acetic acid fermentation after mixed alcohol fermentation of fruit and vegetable juice and grain.
EXAMPLE I identification of Acetobacter strains
Morphological identification:
colony morphology characterization: as shown in FIG. 1, the colony is round and 1-1.5 mm, the protrusion is nearly spherical, the surface is smooth, moist, opaque and light yellowish brown.
Individual morphological characteristics: as shown in FIG. 2, the cells are short rods, 0.985 to 0.990X 1.523 to 4.031 μm, gram-negative bacteria, single-grown, spore-free, and non-motile aerobic bacteria.
16s rDNA molecular biology identification:
the acetic acid bacillus strain 16s rDNA molecule full-length sequence is obtained by PCR amplification, and is 1352 bp. A nucleic acid database is searched by a blast program, and the similarity between the full-length sequence of the acetobacter strains 16s rDNA molecules and all acetobacter strains 16s rDNA sequences in the database reaches 99-100 percent. The Acetobacter strains were preliminarily identified as Acetobacter passaurianus based on 16srDNA molecular sequence analysis.
Example two: a method for culturing and separating acetobacter comprises the following steps: taking a vinegar culture sample of naturally fermented vinegar in mixed fruits and vegetables or grains, putting the vinegar culture sample into a container filled with a basic culture medium, carrying out shake culture, taking a diluted proliferation solution, inoculating the vinegar culture sample onto a separation culture medium by a coating method for culture, selecting a single colony with a transparent ring after the culture is finished, transferring the single colony to an inclined plane preservation culture medium, and preserving to obtain the acetobacter.
A method for screening acetobacter comprises the following steps:
the method comprises the following steps: culturing and separating the acetobacter strains: obtaining acetobacter according to the preparation method;
step two: screening of acetobacter: transferring the acetobacter obtained in the first step into a basic culture medium, performing shake culture to obtain acetobacter strain seed liquid, respectively inoculating the prepared acetobacter strain seed liquid into a basic culture medium containing 0%, 5%, 9%, 11%, 13% and 15% alcohol by volume percentage, performing shake culture, then determining the OD value and the acid yield, and screening the acetobacter according to the determination result.
Example three: a method for culturing and separating acetobacter comprises the following steps: taking a vinegar culture sample of naturally fermented vinegar in mixed fruits and vegetables or grains, putting the vinegar culture sample into a container filled with a basic culture medium, carrying out shaking culture for 48 hours at the rotation speed of 120r/min and the temperature of 30 ℃, taking the multiplication liquid, diluting the multiplication liquid with sterile water by 100000 times, 1000000 times and 10000000 times respectively, taking the multiplication liquid with each dilution concentration, inoculating the vinegar culture sample onto a separation culture medium by adopting a coating method for culture, carrying out culture for 72 hours at the temperature of 30 ℃, then carrying out culture, selecting a single bacterial colony with a transparent ring after the culture is finished, transferring the single bacterial colony to a slant preservation culture medium, and preserving to obtain the acetobacter.
A method for screening acetobacter comprises the following steps:
the method comprises the following steps: culturing and separating the acetobacter strains: obtaining acetobacter according to the preparation method;
step two: screening of acetobacter: transferring the acetobacter obtained in the first step into a basic culture medium, performing shake culture to obtain acetobacter strain seed liquid, respectively inoculating the prepared acetobacter strain seed liquid into a basic culture medium containing 0%, 5%, 9%, 11%, 13% and 15% alcohol by volume percentage, performing shake culture, then determining the OD value and the acid yield, and screening the acetobacter according to the determination result.
Example four: a method for culturing and separating acetobacter comprises the following steps:
the method comprises the following steps: preparing a basic culture medium: heating and dissolving yeast extract and glucose with the same mass in water, adjusting pH to 4.5, fixing volume to 1L, subpackaging, and sterilizing at 121 deg.C for 20 min;
step two: preparing a separation culture medium: adding agar into 100ml of the basic culture medium, sterilizing at 0.1MPa and 121 ℃, and adding sterile CaCO3And absolute ethyl alcohol, which are respectively filled in a sterile culture dish to prepare a separation culture medium; the agar accounts for 2 percent of the volume of the separation culture medium, and the sterile CaCO3The volume percentage of the absolute ethyl alcohol in the separation culture medium is 2%, and the volume percentage of the absolute ethyl alcohol in the separation culture medium is 3%;
step three: preparing a slant preservation culture medium: adding agar into the prepared basic culture medium, sterilizing at 121 deg.C under 0.1MPa, adding sterile CaCO3And absolute ethyl alcohol to obtain the slant preservation culture medium.
Step four: culturing and separating the acetobacter strains: weighing about 20g of a vinegar mash sample of naturally fermented vinegar in mixed fruits and vegetables or grains, putting the vinegar mash sample into a 500mL three-bottle containing 100mL of a basic culture medium, carrying out shaking culture at the rotation speed of 120r/min and the temperature of 30 ℃ for 48h, diluting the proliferation solution with sterile water by 100000 times, 1000000 times and 10000000 times respectively, taking 0.2mL of the proliferation solution with each dilution concentration, and inoculating the proliferation solution on a separation culture medium by a coating method. After culturing for 72h at 30 ℃, selecting a single colony which is thick and has dominant growth, purifying, namely, after the culture is finished, selecting the single colony with a transparent ring, transferring the single colony to a slant preservation culture medium, preserving to obtain the bacillus aceticus, finally separating to obtain 9 strains of bacillus, and observing by a microscope through gram staining, spore staining and gram negative bacillus, wherein 5 strains are the bacillus, 4 strains are the gram negative bacillus, and the single colony is preliminarily identified as the bacillus aceticus by a molecular biological method. The numbers of APT1, APT10, APT18 and APT17 respectively;
a method for screening acetobacter comprises the following steps:
the method comprises the following steps: culturing and separating the acetobacter strains: obtaining acetobacter according to the preparation method;
step two: screening of acetobacter:
and (3) measuring acid resistance strength: transferring single colonies of 4 strains of acetobacter aceti APT1, APT10, APT18 and APT17 plates into a 150mL shaking flask filled with 30mL of a basic culture medium, performing shaking culture at 30 ℃ and 200r/min for 24h to respectively prepare APT1 strain seed solutions, APT10 strain seed solutions, APT18 strain seed solutions and APT17 strain seed solutions.
Inoculating the prepared strain seed liquid bacteria of APT1, APT10, APT18 and APT17 into a basal culture medium with the alcohol concentration of 0%, 5%, 9%, 11%, 13% and 15% respectively according to the inoculation amount of 3-4%, culturing for 48h at 30 ℃ at 200r/min by a shaking table, and then measuring the absorbance at the wavelength of 600nm by using a spectrophotometer according to the OD value.
Measuring the acid yield: transferring single colonies of 4 strains of acetobacter aceti APT1, APT10, APT18 and APT17 plates into a 150mL shaking flask filled with 30mL of a basic culture medium, performing shaking culture at 30 ℃ and 200r/min for 24h to respectively prepare APT1 strain seed solutions, APT10 strain seed solutions, APT18 strain seed solutions and APT17 strain seed solutions.
Inoculating the prepared APT1, APT10, APT18 and APT17 strain seed liquid bacteria into a basal culture medium with the alcohol concentration of 5%, 9%, 11%, 13% and 15% respectively according to the inoculation amount of 4-5%, carrying out shake culture at 30 ℃ for 48h at 200-.
Results of the acetobacter screening experiment in example four:
the results show that, as shown in Table 1, the strains APT10 and APT17 hardly grew normally at 5% alcohol concentration, and the strain APT1 tolerated 5% alcohol, but hardly grew at 9% alcohol. The strain APT18 shows strong alcohol tolerance and can grow in 15% alcohol.
TABLE 1 acid resistance of different strains
Note: "-" indicates that the strain did not grow at the alcohol concentration and no OD value was observed.
The results show that the strains APT1, APT10 and APT17 have the capacity of synthesizing the metabolic acid in a basic culture medium with the alcohol concentration of 5 percent, the acid production amounts are 1.189g/L, 1.2g/L and 0.058g/L respectively, and the capacity of synthesizing the metabolic acid is limited when the alcohol concentration reaches 5 percent, as shown in the table 2. The strain APT18 has strong ability of synthesizing metabolic acid, the amount of the synthesized acid is obviously higher than that of other alcohol concentrations between 11% and 13%, and the acid yield reaches 1.94 g/L.
TABLE 2 acid production of different strains
Note: "-" indicates that the strain does not produce acid at the alcohol concentration.
The acetobacter protected by the invention is the finally screened acetobacter with the strain number of APT18, and the 16sDNA sequence of the acetobacter is shown in the amino acid sequence shown in SEQ ID NO. 1.
Temperature growth test of selected APT18 strain in the four steps of the example two:
temperature growth tests show that the APT18 strain can grow at 8 ℃ and 42 ℃, and the strain has good adaptability to temperature.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. The acetobacter aceti is characterized by being classified and named as: acetobacter pasteianus, preserved in China Center for Type Culture Collection (CCTCC), with the preservation number of CCTCC M2016036 and the preservation date of 2016, 1 and 14.
2. The use of the acetobacter as claimed in claim 1, wherein said acetobacter is used for acetic fermentation after mixed alcohol fermentation of fruit and vegetable juice and grain.
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