CN115141774A - Acetobacter aceti, obtaining method thereof, preparation method of composite freeze-dried leaven and application of composite freeze-dried leaven in vinegar preparation - Google Patents
Acetobacter aceti, obtaining method thereof, preparation method of composite freeze-dried leaven and application of composite freeze-dried leaven in vinegar preparation Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
- C12J1/04—Vinegar; Preparation or purification thereof from alcohol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/02—Acetobacter
Abstract
The invention discloses Acetobacter aceti and an acquisition method thereof, a preparation method of a composite freeze-drying starter and application of the composite freeze-drying starter in vinegar preparation, wherein the method comprises the steps of taking Yunnan old vinegar as a raw material, screening out a dominant strain Acetobacter aceti (Acetobacter aceti) on the basis of a single-factor experiment, optimizing and determining the optimal preparation process parameters of the acetic acid fermentation conditions of the old vinegar by adopting a response surface method, and optimizing and screening out the freeze-drying composite starter of the Acetobacter aceti; the prepared high-activity acetobacter aceti composite freeze-drying starter bacteria powder is combined with a composite freeze-drying yeast composite freeze-drying starter prepared in a laboratory, is preliminarily applied to small-scale production of Yunnan mature vinegar in the laboratory, and has important significance for shortening the production process period of the mature vinegar and improving the stability of the mature vinegar product.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to dominant acetobacter aceti of Yunnan old-warehouse vinegar, an acquisition method, a preparation method of a composite freeze-drying starter and application of the composite freeze-drying starter in vinegar preparation.
Background
The Yunnan old-style vinegar is prepared by taking brown sugar and ancient tree tea dust as raw materials and utilizing natural fermentation, and no additive is added in the preparation process. The produced vinegar is clear and transparent, has proper sour and sweet taste, and has fresh sour taste and special fragrance. The fermentation process adopts a continuous surface static fermentation method, and after the fermentation is finished, a part of fermentation liquor is remained as seed liquor and added into fresh fermentation liquor for continuous fermentation. The method has the advantages of easy operation, low energy consumption, high utilization rate of raw materials, easy control of fermentation process, rapid and convenient subsequent treatment, and the like. The key of the success or failure of the fermentation is a microbial film generated on the surface of the liquid, and the complete microbial film can accelerate the fermentation and shorten the fermentation period.
The Yunnan old vinegar belongs to a black tea fungus beverage, has a health care effect, contains a large amount of organic acid and beneficial microorganisms, can promote digestion and improve delicate flavor. The black tea fungus is the 'Haibao' and 'Weibao' in the past, is also called jellyfish fungus abroad, and is a symbiont of membrane vinegar fungus and saccharomycetes.
The production of the vinegar in the old storehouse adopts a surface static fermentation method, the vinegar yield is high, the labor intensity is low, the energy consumption is low, the operation is suitable for large-scale production, the fermentation time is short, and the subsequent treatment is convenient. The production process comprises the following steps:
boiling water → adding raw materials (brown sugar and tea) → cooling → fermentation → adding water mixing → deoxidation → secondary mixing → filtration → homogenization → pasteurization → canning → inspection → finished product
The fermentation process of Yunnan mature vinegar can be divided into 3 stages, the traditional fermentation period is calculated, the 1 st to 13 th days are the alcoholization stage, the yeast is taken as the dominant strain in the stage, and the sugar of the fermentation liquor is consumed to produce alcohol as the main material; the 13 th to 25 th days are acetification stages, the acetification bacillus is the dominant strain of the stage, and the process is mainly that the acetification bacillus consumes alcohol and reducing sugar and converts the alcohol and the reducing sugar into acetic acid, and the process is used for improving the acidity of fermentation liquor; after 25 days, the post-maturation stage, the fermentation liquor has insufficient nutrients, and the alcoholization and acetification are stopped, mainly the reaction of alcohols and organic acids generated in the fermentation liquor is carried out to generate flavor substances.
The conventional preservation method of the acetic acid bacteria is slant preservation and liquid preservation, and regular passage is required. However, regular passage of acetic acid bacteria increases the probability of degeneration and mutation, and further wastes precious strain resources which are preferably obtained, so that the subsequent research is difficult to perform, and even great loss and harm are caused to industrial production. Therefore, it is very important to select an appropriate preservation method in order to maintain the characteristics and viability of the acetic acid strain.
In recent years, with the expansion of the market of Yunnan aged vinegar, the Yunnan aged vinegar is popular with consumers, but enterprises have a plurality of problems in the process of producing the Yunnan aged vinegar: the vinegar in the old storehouse has unstable fermentation, low resource utilization rate and longer traditional fermentation time, the fermented product has no clear standard, the batch quality of the product is not uniform, the difference of the acid production rate is larger, the fermentation process is greatly influenced by the temperature, the acid production amount is lower particularly in winter, and serious economic loss and resource waste are caused to the fermented vinegar production enterprises.
Disclosure of Invention
In order to simplify the production process flow of Yunnan old-storehouse vinegar and solve the problems of mixed bacteria pollution, strain degeneration, low acid yield and the like, dominant acetobacter aceti strains separated from Yunnan old-storehouse vinegar finished products are taken as the existing conditions, strains with high activity and high acid yield are bred, a low-temperature freeze-drying technology is combined, and a dominant bacteria composite freeze-drying leaven for the old-storehouse vinegar is researched by researching the production process, so that basic technical parameters can be provided for industrial production of the old-storehouse vinegar, the Yunnan old-storehouse vinegar fermentation process is improved, fermentation strain management is facilitated, and meanwhile, the intensity of workers can be reduced, so that the investment cost is reduced, the quality of the Yunnan old-storehouse vinegar products is improved, and the development of the Yunnan old-storehouse vinegar industry is facilitated.
In order to achieve the purpose, the invention provides acetobacter aceti, an acquisition method, a preparation method of a composite freeze-drying starter and application of the composite freeze-drying starter in vinegar preparation.
According to a first aspect, the technical solution of the present invention:
the Acetobacter aceti strain is an Acetobacter aceti (Acetobacter aceti) which is a dominant bacterium of Yunnan old-warehouse vinegar, is preserved in the China general microbiological culture Collection center at 18.04.2022, and has the preservation address as follows: china Beijing, the preservation number is CGMCC No.24700.
According to a second aspect, the technical solution of the present invention: an Acetobacter aceti obtaining method comprises the following steps:
s1: sampling, and preserving vinegar liquid at low temperature in the acetification stage of 13-25 days in a Yunnan old-warehouse vinegar fermentation tank;
s2: preparing a bacterial suspension: weighing 1g of vinegar sample, pouring the vinegar sample into 15mL of sterilized centrifuge tube, adding 9mL of sterile water, and oscillating for 5-10 min to fully disperse the sample, namely preparing 10 -1 A concentrated suspension.
S3: diluting: will be 1mL 10 -1 The suspension is sucked into a centrifuge tube containing 9mL of sterile water by a sterile pipette, namely 10 -2 The sample dilution of (2), repeating the process to make 10 in sequence -3 、10 -4 、10 -5 、10 -6 The diluent (2).
S4: preparing culture medium with yeast extract 1%, glucose 1% and agar 2%, adding calcium carbonate 2% and anhydrous ethanol 4% before pouring, standing in a constant temperature incubator at 35 deg.C after the plate is solidified, and recording dilution times and corresponding colony number after 72 hr
S5: separating and purifying, selecting typical colonies on an acetobacter aceti plate, streaking, performing inverted culture at 30 ℃ for 72h, selecting single colonies, and performing colony characteristic observation, gram staining, microscopic cell morphology observation and acetobacter aceti qualitative test. Selecting a strain which is gram-negative and can form a reddish brown precipitate with FeCl3 solution, carrying out plate streaking pure separation on the strain, transferring the pure separated strain onto a slant solid culture medium, and storing the pure separated strain in a refrigerator at 4 ℃.
S6: screening, inoculating activated acetobacter aceti strains into a basic liquid culture medium, performing static culture at 30 ℃ for 48 hours to prepare acetobacter aceti seed liquid, sucking 2% of the acetobacter aceti seed liquid into 100mL of liquid fermentation culture medium, performing static culture at 30 ℃, measuring the acid yield once every 24 hours, continuously measuring for 10 days, measuring three groups of strains in parallel, determining the acid production capacity of the strains, drawing an acid yield curve according to the test result, and screening the acetobacter aceti strains.
According to a third aspect, the technical solution of the present invention: a preparation method for preparing a composite freeze-dried starter culture by utilizing acetobacter aceti comprises the following preparation steps:
step one, preparing strain seed liquid: selecting a small amount of acetobacter aceti strains from the strain preservation slant, inoculating the strains into a basic culture medium, and performing static culture at the temperature of 30 ℃ for 48 hours to prepare an acetobacter aceti strain seed solution;
step two, compounding the strains, wherein the inoculation amount of the acetobacter aceti is 1.0%, the inoculation amount of the acetobacter vinelans is 0.03%, and the acetobacter vinelans is inoculated into a culture medium and fermented for 9 days to obtain the compounded strain.
Step three, centrifugal collection of thalli: collecting bacterial sludge under the conditions that the centrifugal rotation speed is 5000r/min, the centrifugal time is 20min and the centrifugal temperature is 8 ℃;
step four, preparing a protective agent: the protective agent comprises milk powder and sugar alcohol, and the ratio of the sugar alcohol to the skimmed milk powder is 1:1; the sugar alcohol comprises mannitol and lactose, the lactose to mannitol ratio is 1:1;
step five, freeze-drying: vacuum freeze-drying according to the ratio of the bacterial sludge to the protective agent 1:3 to obtain the acetobacter aceti composite freeze-dried starter.
Further, the culture medium in the step 1 comprises a liquid culture medium and a solid culture medium, and the liquid culture medium and the solid culture medium are respectively: 1% of yeast extract, 1% of glucose and 4% of absolute ethyl alcohol.
Solid medium: 1% of yeast extract, 1% of glucose, 4% of absolute ethyl alcohol, 2% of agar and 2% of calcium carbonate.
Further, the concentration of each component of the protective agent in the third step is as follows: the milk powder concentration is 14.314%, the mannitol concentration is 2.831%, and the lactose concentration is 9.347%.
Further, the maximum thickness of the bacterial suspension in the step five freeze-drying process is 0.75cm.
According to a fourth aspect, the technical solution of the present invention: an application of a composite freeze-dried starter of acetobacter aceti in preparing aged vinegar.
Further, the preparation method is used for preparing the application of the acetobacter aceti composite freeze-drying starter combined with the yeast composite freeze-drying starter in preparing the aged vinegar.
Further, the addition amount of the acetobacter aceti composite freeze-drying starter is 1%.
Further, the specific steps of preparing the mature vinegar by jointly using the leavening agent comprise:
1) The composite freeze dried yeast starter is activated in potato glucose water for 2 generations, and the concentration of the yeast is adjusted to 3 multiplied by 10 6 CFU/mL for standby;
2) And (3) an old storehouse vinification stage: adding 10 kilograms of tea and 20 kilograms of brown sugar into 200 kilograms of water, boiling, filtering, cooling, putting into a soil jar with old fermentation liquor, and mixing the bacterial liquid in the step 1) according to a ratio of 100:1, adding the mixture into sugar tea water for fermentation in a alcoholization stage, and sealing and fermenting for 6 days;
3) And (3) vinegar acetification stage of the old warehouse: step 2) directly adding a composite freeze-drying leaven into fermentation liquor which completes the alcoholization stage, and according to the wine: adding the composite freeze-dried starter according to the proportion of 100.
Compared with the prior art, the invention has the beneficial effects that:
(1) The prepared composite vinegar acetified vinegar bacillus starter is applied to vinegar in a mature storehouse for acetic fermentation, the prepared vinegar liquid is obviously shortened in acetic fermentation time compared with acetic fermentation time of a factory, the acetic fermentation time is shortened from 13 days to 9 days, and the acid yield can reach 3.13g/100mL after the starter is inoculated and fermented for 9 days at 30 ℃;
(2) The acetobacter aceti powder is inoculated into vinification fermentation liquor prepared from yeast powder for acetic fermentation, the acidity can reach 6.27g/100mL after fermentation for 9 days at 30 ℃, the sensory score is 91 minutes, the vinegar liquid is mellow and long in taste, has obvious raw material color, is finer and slightly sweet than the commercial vinegar in a warehouse, and is similar in body form.
(2) And (3) antioxidant results: the best capacity of removing free hydroxyl groups by adding the acetobacter aceti powder into the wine liquid of the yeast powder is 48.33 percent, 58.33 percent, 72.67 percent and 68 percent of the capacity of removing free hydroxyl groups by the vinegar liquid prepared from the vinegar plant liquid, the bacterial liquid, the composite bacteria powder and the composite acetobacter aceti powder. The four sample solutions have better effect on the removal capacity of DPPH free radicals, and the DPPH free radical removal capacities of vinegar solutions prepared from four groups of vinegar plant solutions, bacterial solutions, compound bacteria powder and compound acetobacter aceti powder are respectively 71.67%, 84.33%, 95% and 90.33%; the iron ion reducing power was 0.43, 0.49, 0.6, 0.56, respectively.
Drawings
FIG. 1 is a line graph showing the change of acidity of a fermentation solution according to different factors;
wherein A1, the influence of fermentation time on the acidity of the fermentation liquor; b1, influence of the addition amount of the vinegar brewing bacteria on the acidity of the fermentation liquor; influence of the addition amount of C1 to B10 on the acidity of the fermentation liquor;
FIG. 2 is a line graph showing the centrifugal yield of ghost bacteria according to different factors;
a2, influence of centrifugal speed on centrifugal yield; b2, influence of centrifugal time on centrifugal yield; c2, influence of centrifugal temperature on centrifugal yield;
FIG. 3 is a bar graph of the effect of different sugar alcohols on the survival of lyophilized bacteria;
FIG. 4 is a line graph showing the effect of various factors on the survival rate of the lyophilized bacteria;
a4, influence of the concentration of the skim milk powder on the survival rate of the freeze-dried bacteria; b4, the influence of the concentration of the mannitol on the survival rate of the freeze-dried bacteria; c4, influence of lactose concentration on the survival rate of the freeze-dried bacteria;
FIG. 5 is a bar graph of the effect of sugar alcohol ratio on the viability of freeze-dried bacteria;
FIG. 6 is a line diagram illustrating the influence of various factors on the survival rate of the lyophilized bacteria;
a6, influence of the ratio of sugar alcohol to milk powder on the survival rate of the freeze-dried bacteria; b6, influence of the ratio of the bacterial sludge to the composite protective agent on the survival rate; c6, influence of the thickness of the bacterial suspension on the survival rate;
FIG. 7 is a curved surface diagram and a contour diagram of the effect of milk powder concentration and mannitol concentration on survival rate;
FIG. 8 is a curved surface diagram and a contour diagram of the effect of milk powder concentration and lactose concentration on survival rate
FIG. 9 is a curved surface diagram and a contour diagram of the effect of the milk powder concentration and the ratio of the bacterial sludge protectant on the survival rate;
FIG. 10 is a graph of the surface and contour plot of the effect of mannitol concentration and lactose concentration on survival rate
FIG. 11 is a plot of the surface and contour plot of the effect of mannitol concentration and bacterial sludge protectant ratio on survival;
FIG. 12 is a plot of the effect of lactose concentration and ratio of bacterial sludge protectant on survival and a contour plot;
FIG. 13 shows the effect of bacterial powder addition on vinegar production in old warehouses;
FIG. 14 shows the variation of total sugar in the acetification stage of different fermentation modes
FIG. 15 shows the variation of reducing sugars during the acetification stage of different fermentation modes
FIG. 16 shows the change of total acid and pH during the acetification stage of different fermentation modes;
FIG. 17 shows a comparison of DPPH radical scavenging capacity;
FIG. 18 is a comparison of hydroxyl radical scavenging capacity;
FIG. 19 is a comparison of the acid production of dominant acetobacter aceti strains in vinegar solution of old storehouse;
FIG. 20 is a growth curve of the dominant strain (B10).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following detailed description and the accompanying drawings. It is to be understood that these descriptions are only illustrative and are not intended to limit the scope of the present invention. Moreover, in the following invention, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Example 1: method for obtaining dominant bacteria acetobacter aceti of vinegar in old storehouse
S1: sampling, and preserving vinegar liquid at low temperature in the acetification stage of 13-25 days in a Yunnan old-warehouse vinegar fermentation tank;
s2: preparing a bacterial suspension: weighing 1g of vinegar sample, pouring the vinegar sample into 15mL of sterilized centrifuge tube, adding 9mL of sterile water, and oscillating for 5-10 min to fully disperse the sample, namely preparing 10 -1 Concentration ofA suspension of (a).
S3: diluting: will be 1mL 10 -1 The suspension is sucked into a centrifuge tube containing 9mL of sterile water by a sterile pipette, namely 10 -2 The sample dilution of (1) can be prepared sequentially by repeating the process to obtain 10 -3 、10 -4 、10 -5 、10 -6 The diluent (2).
S4: preparing a culture medium by using 1% of yeast extract, 1% of glucose and 2% of agar, adding 2% of calcium carbonate and 4% of absolute ethyl alcohol before pouring by adopting a pouring method, inversely placing the plate in a constant-temperature incubator at 35 ℃ for culture after the plate is solidified, and recording dilution times and corresponding colony number after 72 hours
S5: separating and purifying, selecting typical bacterial colony on an acetobacter aceti plate, streaking, carrying out inverted culture at 30 ℃ for 72h, selecting single bacterial colony, and carrying out bacterial colony characteristic observation, gram staining, microscopic cell morphology observation and acetobacter aceti qualitative test; selecting a person which is gram-negative and can form a reddish brown precipitate with FeCl3 solution, carrying out plate streaking pure separation on the strain, transferring the pure separated strain onto an inclined solid culture medium, and storing in a refrigerator at 4 ℃ after culture;
s6: screening, inoculating activated acetobacter aceti strains into a basic liquid culture medium, performing static culture at 30 ℃ for 48 hours to prepare acetobacter aceti seed liquid, sucking 2% of the acetobacter aceti seed liquid into 100mL of liquid fermentation culture medium, performing static culture at 30 ℃, measuring the acid yield once every 24 hours, continuously measuring for 10 days, measuring three groups of strains in parallel, determining the acid production capacity of the strains, and screening a high-yield acetobacter aceti strain B10.
As shown in FIG. 19, the acid production amounts of three different Acetobacter aceti strains were significantly different when they were fermented in the medium. The B10 strain has higher acid production capacity than the other two strains, is a dominant strain, and the acid production of the B10 strain reaches 2.9g/100mL. Screening acetic acid bacteria from Fujian red yeast vinegar by checking Jing, etc., wherein the acid yield of the strain Y5054 reaches 5.7g/100mL on day 7. Compared with the literature, the acetobacter aceti separated in the experiment has a low acid production value, and the analysis reason may be that the acid production capacity of the B10 dominant strain in the old vinegar is weak, so that the experiment needs to compound a small amount of acetobacter aceti to improve the acid production capacity besides the dominant acetobacter aceti B10 in the old vinegar.
As can be seen from FIG. 20, the OD value of the strain B10 slowly increased from 0 to 32 hours, rapidly increased from 32 to 50 hours, and the OD value of the strain hardly changed after 50 hours. This is because the bacteria are in the lag phase at the beginning, which is the phase of the acetobacter aceti adapting to the environment, and the bacteria grow slowly; 32-50 h, the thallus is in the logarithmic phase of growth, the quantity of the thallus is rapidly increased when the thallus is propagated; after 50h, the B10 acetobacter aceti grows into a stable period, the growth of the thalli is in dynamic balance, and the thalli generally tend to be stable. The number of viable bacteria in the stationary phase reaches the highest, and in order to obtain a large number of viable bacteria by high-density culture, 50h of bacteria are selected for freeze drying test in the test.
Example 2: strain compounding
1. Single factor test
(1) Determining the addition amount of vinegar strain in the old storehouse: as can be seen from C1 in FIG. 1, the acid-producing capacity of Acetobacter aceti B10 tends to increase first and then decrease with increasing inoculation amount, and when the inoculation amount of Acetobacter aceti B10 is 1.0%, the acidity reaches a maximum of 2.93g/100mL. When the inoculation amount is low, fermentation liquor is not sufficiently fermented, the generated acidity is low, and when the inoculation amount is excessive, the fermentation liquor which contains more nutrition is used for metabolism and propagation of acetobacter aceti autologous cells [i] Since the acid-producing effect of Acetobacter aceti was suppressed, the inoculum size of Acetobacter aceti B10 was selected to be 1.0%.
(2) The addition amount of commercial strains is determined as follows: from the analysis of B1 in figure 1, in the same fermentation time, along with the increase of the addition amount of the vinegar brewing bacteria, the acid production capacity of the fermentation liquor in the acetic acid fermentation process is increased firstly and then decreased, and when the inoculation amount is 0.03%, the acidity reaches the highest 4.1g/100mL. When the inoculation amount of Acetobacter aceti is more than 0.03%, the acidity of vinegar rapidly decreases. In the early stage of fermentation, the activity of the thalli is recovered, the metabolism is vigorous, and the alcohol conversion rate and the acid production capacity are improved by increasing the inoculation amount; the inoculation amount is too large, the nutrient consumption is excessive, the acid production capacity is reduced, and the total acid content is reduced due to the aging of thalli in the later fermentation stage [ii] Therefore, the inoculation amount of the vinegar brewing bacteria is 0.03 percent。
(3) Determination of fermentation time: as can be seen from A1 in fig. 1, the acidity also gradually increases with the increase of the fermentation time, and when the fermentation time is 6 to 9 days, the acidity significantly increases, and then the acid-producing capacity gradually flattens with the increase of the fermentation time. The fermentation time influences the growth of microorganisms and the synthesis of products, the fermentation is incomplete or influences the acidity and the flavor of vinegar, the fermentation time is 9 days, the acidity is basically stable, the acidity is 2.91g/100mL, and therefore the fermentation time in the acetification stage is preferably 9 days.
2. Quadrature test of fermentation condition of compound strain
According to the single-factor experiment result, the influence of each factor on the acidity of the fermentation liquor is comprehensively considered, the acidity is selected as a survey index, and a 3-factor 3-level orthogonal test L9 (3) is carried out by four factors of fermentation time, vinegar brewing bacteria addition amount and B10 addition amount 3 ) The results of the design and the orthogonal test are shown in table 1, and the results of the analysis of variance and the significance test are shown in table 2.
TABLE 1 L9 (3) 3 ) Orthogonal experimental result of strain compounding
TABLE 2 analysis of variance in strain compounding orthogonal experiments
Dependent variable: acidity of the solution
R 2 =0.741 (adjusted R) 2 ＝-0.035)
The analysis of the table 1 and the table 2 shows that the strain compound orthogonal model is very obvious (P is less than 0.01), and the fermentation time, the addition amount of the vinegar brewing bacteria and the addition amount of B10 have obvious influence on the acid production result (P)<0.05 A) the influence factors on the acid yield of acetic acid fermentation are sequentially A>C>B, i.e. time>B10 addition amount>Adding amount of vinegar-brewing bacteria. The orthogonal optimal combination and the maximum combination of K values are the same combination, and are A 1 B 2 C 2 I.e. fermentationThe time is 9 days, the addition amount of the vinegar brewing bacteria is 0.03 percent, and the addition amount of B10 is 1.0 percent. Three parallel validation experiments were performed under these conditions and the total acid was up to 3.81g/100mL. In conclusion, the orthogonal experimental model can be used for strain combination experiments.
Example 3: centrifugal test of bacteria
1. Single factor test:
(1) Determining the centrifugal rotation speed: as can be seen from A2 in FIG. 2, the centrifugal yield tends to increase first and then decrease as the centrifugal speed increases, and the centrifugal yield of the cells at a centrifugal rotation speed of 6000r/min was 70.39% at the highest. The centrifugal yield is gradually reduced along with the increase of the centrifugal rotating speed, and the death of thallus part or the loss of thallus part in the supernatant liquid can be caused to a great extent because of the centrifugal force. Therefore, the bacteria liquid is centrifuged at 6000 r/min.
(2) Determination of centrifugation time: it can be seen from B2 of fig. 2 that the centrifugal yield increases with the increase of the centrifugal time, and then increases, and the centrifugal yield reaches 80.54% when the centrifugal time is 15min when the centrifugal yield is the highest. The centrifugation time is continuously increased, and the centrifugation yield is gradually reduced, mainly because the bacterial strain is easy to damage cells under high-speed centrifugation for a long time, and the survival rate is further influenced, so the centrifugation time is selected to be 15min.
(3) Determination of centrifugation temperature: as can be seen from C2 in FIG. 2, the highest centrifugation yield of 78.26% was obtained when the cell was centrifuged at 6 ℃. The centrifugal temperature also has certain influence on the centrifugal yield, and the separation of thalli and culture solution can be influenced by overhigh or overlow temperature, so the bacteria solution is centrifuged at the centrifugal temperature of 6 ℃.
2. And (3) performing a thallus centrifugation orthogonal test: on the basis of single-factor test results, selecting viable count and centrifugal yield as indexes, taking centrifugal rotation speed, centrifugal time and centrifugal temperature as investigation factors, and performing 3-factor 3-level orthogonal test L 9 (3 3 ) Design, orthogonal test design scheme and results are shown in table 3, and results of analysis of variance and significance test are shown in table 4:
TABLE 3L 9 (3 3 ) Results of centrifugal orthogonal experiments
TABLE 4 analysis of variance in centrifugal orthogonal experiments
Table 3-4AnOVA of centrifugal orthogonal experiment
The results of the analysis in tables 3 and 4 show that the orthogonal model of the thallus centrifugation is extremely obvious (P is less than 0.01), and the influence of the centrifugation speed, the centrifugation time and the centrifugation temperature on the number of the live bacteria after centrifugation is more obvious (P is less than 0.05); the influence of the centrifugal temperature on the centrifugal yield is obvious; the centrifugal speed and centrifugal time have no significant influence on the centrifugal yield. The number of viable bacteria and the centrifugal yield are taken as indexes, the primary and secondary sequences of influence are the same, and are B>A>C, i.e. centrifuge time>Centrifugal speed of rotation>The centrifugation temperature. The optimum combination of the bacteria centrifugation conditions is A 1 B 3 C 3 Namely the centrifugal speed is 5000r/min, the centrifugal time is 20min and the centrifugal temperature is 8 ℃. Three parallel verification experiments are carried out under the condition, and the number of viable bacteria after centrifugation is the maximum and is 1.587 multiplied by 10 8 CFU/mL, yield 98.57%.
Example 4: protectant screening optimization assay
Sugar alcohol protective agent screening: because hydroxyl contained in the sugar alcohol substance can form a layer of protective film, the protective film can protect somatic cells and reduce the damage degree of the cells, and meanwhile, the sugar alcohol substance is also beneficial to the rapid rehydration of the cells or the repair of the damaged cells. Therefore, in the experiment, 6 different sugar alcohol protective agents which can be utilized by acetobacter aceti, such as glucose, mannitol, fructose, lactose, sucrose and maltose, are selected, the various sugar alcohol protective agents are dissolved by distilled water, a solution with the mass concentration of 5% is prepared and sterilized, and three protective agents are arranged in parallel. Viable bacteria count is carried out on the bacteria powder after vacuum freeze drying, and the survival rate is calculated, and the result is shown in figures 3-9: the freeze-drying survival rate averages of glucose, mannitol, fructose, lactose, sucrose and maltose are 34.99%, 42.44%, 28.99%, 48.45%, 30.02% and 26.09%, respectively. Therefore, the protective effect of 6 sugar alcohols on the bacterial cells is in the order of lactose > mannitol > glucose > sucrose > fructose > maltose, wherein the protective effect of lactose and mannitol on acetobacter aceti is the best and maltose is the worst. Therefore, lactose and mannitol were selected as sugar alcohol protective agents and subjected to one-way experimental studies.
1. Single factor test
(1) Basal protectant concentration determination
Preparing milk powder solutions with mass concentrations of 6%, 8%, 10%, 12%, 14% and 16% by taking skimmed milk powder as a basic protective agent, respectively mixing the milk powder solutions with two screened sugar alcohols with mass concentrations of 5% according to 1:1, and mixing the sugar alcohols with the mass concentrations of bacterial sludge: the protective agent is 1:4 proportional eluate bacterial suspension, each concentration is performed in three parallels, and freeze-drying is performed, wherein the freeze-drying survival rate is used as an index; it can be seen from A4 of fig. 4 that the freeze-drying protection effect of the skimmed milk powder is most prominent at a concentration of 14%, because the skimmed milk powder increases in concentration, the emulsion becomes viscous, and evaporation of water is not facilitated in the freeze-drying process, so that the formed ice crystals are large, and the death rate of the bacteria is increased, and therefore, the skimmed milk powder with a concentration of 14% is selected as the basic protective agent.
(2) Determination of sugar alcohol concentration
Respectively preparing sugar alcohol solution with mass concentration of 1%, 3%, 5%, 7%, 9% and 11% from the two selected sugar alcohols, mixing with skimmed milk powder solution with mass concentration of 10%, and mixing according to the following steps: the protective agent is 1:4 proportion washing bacterial suspension, each concentration is performed in parallel, freeze-drying is performed, and the optimal concentration of two sugar alcohols is determined by taking the freeze-drying survival rate as an index.
As can be seen from B4 in FIG. 4, the freeze-drying survival rate of the somatic cells is not always increased along with the increase of the mannitol concentration, and when the mannitol concentration reaches 3%, the survival rate of the freeze-dried bacteria is the greatest, and the survival rate reaches 67%; when the concentration is further increased, the number of viable bacteria decreases, and the survival rate decreases. This is because the hydroxyl group contained in mannitol interacts with free radicals on the cell surface of the cells, reducing exposure of the cells, and at the same time, hydrogen bonds are formed with proteins of milk powder, ensuring the stability of the proteins. However, when the mannitol concentration exceeds a certain level, the ability to stabilize the protein is limited, and the protein may be deteriorated even if the mannitol concentration is too high during the freeze-drying process. Therefore, mannitol was chosen optimally at a concentration of 3%.
As can be seen from C4 of FIG. 4, when the lactose concentration is 1% to 9%, the survival rate of Acetobacter aceti is low, and the increase trend is shown; when the concentration reaches 9%, the survival rate of the bacteria reaches the maximum value, and the survival rate reaches 69%; when the concentration is further increased, the number of viable bacteria decreases, and the survival rate decreases. Lactose is easily dissolved in water to hydrate, so that the growth speed of crystal nucleus is reduced, ice crystals formed in the pre-freezing process are reduced, and cells are protected from being damaged. However, lactose concentrations are too high, which leads to dehydration and death of the cells. Therefore, a lactose concentration of 9% is selected as the best.
(3) Determination of sugar alcohol ratio
Mixing 5% sugar alcohol solutions 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4 with 10% skimmed milk powder solution according to different proportions respectively, according to the ratio of bacterial sludge: the protective agent is 1:4 proportion of the eluted bacterial suspension, three of the protective agent are prepared in parallel in each proportion, freeze-drying is carried out, and the optimal proportion of the two sugar alcohols is determined by taking the freeze-drying survival rate as an index.
As can be seen from FIG. 5, when mannitol and lactose were added as 1:1, the number of viable lyophilized bacteria was the greatest, and the survival rate of the bacteria was 77%. Therefore, in view of protection effect and production cost, the ratio of the mannitol and the lactose is 1:1, namely 3% mannitol and 9% lactose are added as the sugar alcohol protective agent according to the proportion of 1:1.
(4) Determination of sugar alcohol to milk powder ratio
Mixing 5% of two types of sugar alcohol solutions according to 1:1, mixing the sugar alcohol solutions with 10% of skimmed milk powder solutions according to different proportions of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4, and mixing the sugar alcohol solutions according to the ratio of bacterial sludge: the protective agent is 1:4 proportion of the washed bacterial suspension, three of the protective agent are prepared in parallel in each proportion, freeze-drying is carried out, and the optimal proportion of sugar alcohol and milk powder is determined by taking the freeze-drying survival rate as an index.
As can be seen from A6 in FIG. 6, after the skim milk powder, mannitol and lactose are added as the composite freeze-drying protective agent, the freeze-drying survival rate of the acetobacter aceti is higher than that of the acetobacter aceti added with the single protective agent, which shows that the skim milk powder, the mannitol and the lactose have a synergistic effect with each other and the protective effect is enhanced [iii] . When sugar alcohol and milk powder are added according to 1:1, the survival rate of the freeze-dried bacteria is the highest, and the survival rate of the freeze-dried bacteria exceeds 70%. Therefore, the sugar alcohol and the milk powder are proportioned by 1:1 as the compound protective agent.
(5) Determination of the ratio of bacterial sludge to protective agent
Mixing two types of sugar alcohol solutions of 5% and a skim milk powder solution of 10%, respectively according to the ratio of the bacterial sludge: the protective agent is 1:2, 1:3, 1:4, 1:5 and 1:6 washed bacterial suspension, each proportion is made into three parallels, freeze-drying is carried out, and the optimal proportion of bacterial sludge and the protective agent is determined by taking the freeze-drying survival rate as an index.
It can be known from B6 in fig. 6 that when the bacterial sludge and the composite protective agent are added according to 1:3, the survival rate of the freeze-dried bacteria is the largest, the survival rate of the bacteria reaches 88%, the protective effect of the freeze-dried bacteria in the rest proportion is obviously enhanced, but the optimal proportion 1:3 is more suitable for the mixture ratio of the bacterial sludge and the composite protective agent in the experiment.
(6) Determination of prefreezing thickness of bacterial suspension
Mixing 5% of two types of sugar alcohol solution and 10% of skimmed milk powder solution according to 1:1, bacterial mud: the protective agent is 1:4, bacterial suspensions are washed out, the bacterial suspensions are respectively poured into prefreezing cups with different thicknesses of 0.25cm, 0.5cm, 0.75cm, 1cm and 1.25cm, three prefreezing cups are arranged in parallel in each thickness, freeze-drying is carried out, and the optimal prefreezing thickness of the bacterial suspensions is determined by taking the freeze-drying survival rate as an index.
If the bacterial suspension is too small, the lyophilization vessel is over-packed and the cell surfaces are not covered to a sufficient degree, which may result in more exposed areas of cells upon lyophilization and death. If the amount of the protective agent is too large, the permeability of cells is insufficient, and the number of viable bacteria in the cells is also affected. As can be seen from C6 of FIG. 6, the number of viable lyophilized bacteria was the greatest and the survival rate was the highest when the thickness of the bacterial suspension was 0.75cm. Therefore, 0.75cm was used as the thickness of the bacterial suspension during lyophilization.
2. Box-Behnken test of composite protectant
(1) According to the results of the single-factor experiment, the freeze-drying survival rate is used as an evaluation index, the formula of the skimmed milk powder concentration, the mannitol concentration, the lactose concentration and the bacterial sludge protective agent ratio is optimized, the Design-expert10.0 software is used for designing the experiment, a response surface experiment is carried out, the results of the response surface experiment are shown in table 5, and the results of the variance analysis are shown in table 6.
TABLE 5 response surface test results for composite protectant
Analyzing the data by Design-Expert software to obtain a quadratic polynomial regression equation between the concentration (A) of the skimmed milk powder, the concentration (B) of mannitol, the concentration (C) of lactose, the ratio (D) of the bacterial sludge protective agent and the freeze-drying survival rate (Y), wherein the quadratic polynomial regression equation comprises the following equations:
Y＝92.20+4.92A-2.92B+5.25C+1.08D+15.75AB+14.25AC+0.25AD-8.00BC-6.50BD-2.00CD- 19.43A 2 -13.43B 2 -23.18C 2 -9.93D 2 the regression analysis of variance results are shown in Table 6.
TABLE 6 regression model ANOVA
The analysis of variance in table 6 shows that the data model obtained by applying Design-Expert10 software is significant (P = 0.0209)<0.05 Interaction terms AB, AC, AD, BC, BD and CD have no significant influence, which shows that the fitting of the regression equation is meaningful and the determining coefficient R of the model 2 ＝0.9568，R 2 Adj The model is used for predicting the formula of the compound protective agent, and the fitting degree of the whole data model is good and the test result is reliable in the case of = 0.9136.
(2) Analysis of response surface diagram: according to the response surface analysis (fig. 7-12), the influence of the two factors on the freeze-drying survival rate of the response value is researched by observing the inclination of the curved surface of the three-dimensional graph, and the response surface graphs are all arc-shaped, which shows that the concentration of the skim milk powder, the concentration of mannitol, the concentration of lactose and the proportion of the bacterial sludge protective agent have the influence on the freeze-drying survival rate of the acetobacter aceti in a certain range. The shape of the contour map of the response surface is related to the interaction among the factors, the closer the shape of the contour map is to an ellipse, the more obvious the interaction between the contour map and the ellipse, and the analysis of the contour map shows that the interaction among the four factors of the concentration of the skim milk powder, the concentration of mannitol, the concentration of lactose and the proportion of the bacterial sludge protective agent has obvious influence on the survival rate.
The optimal process for optimizing the compound is obtained by analyzing and optimizing software and comprises the following steps: the milk powder concentration 14.314%, the mannitol concentration 2.831%, the lactose concentration 9.347%, the bacterial sludge protective agent proportion 1:3, and the yield 93.197%. The verification experiment is carried out under the condition, the yield obtained by three parallel experiments is 94%, the value is in a prediction interval, the result of the experiment is proved to be reliable, and the regression model can be used for predicting the formula of the compound protective agent.
Example 5: experiment for producing vinegar in old storehouse by using leaven
(1) The addition amount of the fungus powder is determined
Taking 100mL of culture solution per bottle as an example, adding freeze-dried vinegar bacillus leaven into sugar tea water culture solution according to different gradients of 0.1g, 0.5g, 1.0g, 1.5g and 2.0g, respectively, making three gradients in parallel, simultaneously providing a control, placing the culture solution at 30 ℃ for culturing for 9 days, and selecting the optimal addition amount of bacteria powder by taking sensory indexes and acidity as indexes.
As can be seen from FIG. 13, the addition of the freeze-dried acetified acetobacter powder directly affects the acid production speed in the production process of the vinegar acetification stage of the old warehouse, and the acid production quantity gradually increases with the increase of the addition of the powder. However, the acid production also directly affects the mouthfeel, so the optimal bacterial powder addition amount is selected as the addition amount which can not only meet the mouthfeel, but also save the cost, namely the bacterial powder addition amount is 1%, the acid production reaches 3.58g/100mL, and the national standard acidity is not less than 3.5g/100mL.
(2) Dynamic change of physicochemical indexes of vinegar in different fermentation modes
Fermentation production of vinegar in old storehouse
The dominant acetobacter aceti B10 bacterial liquid, the prepared composite freeze-drying leaven (called acetobacter aceti powder for short), and the yeast powder and the acetobacter aceti powder (called composite bacterial powder for short) prepared in the same way are applied to an old-warehouse vinegar acetification stage for fermentation as samples, and the dynamic changes of acid production, pH, total sugar and reducing sugar of each sample in the fermentation process are researched by taking the vinification liquid and the old liquid taken back by a vinegar plant for laboratory production as comparison.
First group (vinegar factory liquor): taking the final liquor and the old liquor of the vinification stage of the vinegar plant, fermenting the other raw materials according to the production of the vinegar plant except the constant temperature condition of the laboratory, and simultaneously fermenting the liquor of the vinegar plant in the laboratory as a control group of the test of the group;
second group (bacterial solution): taking liquor just finished in the alcoholization stage of the vinegar factory in the old warehouse, and mixing the liquor according to the following ratio: b10, adding the bacterial liquid in a proportion of 100;
third group: (Acetobacter aceti powder) the liquor just finished in the vinification stage of the vinegar factory in the old storehouse is taken according to the following liquor: adding the composite freeze-drying starter according to the proportion of 100;
fourth group (composite bacterial powder): taking wine fermented by the composite freeze-dried yeast starter (fermentation liquor prepared by a vinegar factory process in a laboratory), and mixing the following raw materials in percentage by weight: and adding the composite freeze-dried starter culture into the mixture according to the ratio of 100.
As shown in FIG. 14, since the vinegar factory liquor, the bacterial liquor and the aceti-acetobacter aceti powder are all subjected to the fermentation in the acetification stage in the fermentation liquor of the old-warehouse vinegar factory after the vinification is completed, the initial sugar degrees of the three groups are basically the same and are about 5.2g/100mL, while the mixed fermentation of the two bacterial powders is the liquor fermented from the raw material in a small scale in a laboratory as the fermentation liquor, and the liquor produced in the laboratory is not diluted, so the initial sugar degree of the group is higher and is 6.66g/100mL. In four experiments, the total sugar values of other groups except the vinegar factory liquid all have a rapid descending trend, and the total sugar values are slowly descended in 1-4 days of the acetification stage, mainly the amount of the acetobacter aceti is small at the time, and the acetobacter aceti existing in the fermentation liquid is producing acid by using alcohol. The consumption of total sugar is extremely fast on days 4-8, mainly because the reducing sugar is rapidly consumed because the acetobacter aceti uses alcohol to produce acetic acid and simultaneously consumes part of the reducing sugar to convert the acetic acid. As the fermentation time increases, the reducing sugar in the fermentation liquor is consumed to a very small extent and can not be utilized by the acetobacter aceti any more. The primary judgment is that the vinegar factory liquid contains a small amount of vinegar bacillus, the alcoholization stage is incomplete, and the alcohol content does not reach the concentration of acid produced by the vinegar bacillus.
From the variation trend of reducing sugar in the acetification stage of different fermentation modes, as can be seen from the graph 15, the reducing sugar in the fermentation liquor has a trend of decreasing with the increase of the fermentation time regardless of the fermentation mode. Wherein the initial reducing sugar of two kinds of bacteria powder fermentation is slightly lower than the other three groups, and the consumption of alcohol production by using the reducing sugar is more mainly in the alcoholization stage of the yeast powder fermentation. In the acetic acid fermentation stage, acetobacter aceti produces acetic acid by using reducing sugar mainly in days 1-8, the reducing sugar is beneficial to the growth of thalli, and the reducing sugar is consumed by the thalli in the growth process, so that the content of the reducing sugar is continuously reduced. After 8 days, the total sugar content is less, the reducing sugar decomposing capability is weakened, the reducing sugar which can be utilized by the acetobacter aceti is less, and the reducing sugar changes gently. On the 10 th day, the measured reducing sugar content of the vinegar plant liquid group is higher than that of other groups and is 3.96g/100mL, the acetic fermentation is preliminarily judged to be not finished, and the reducing sugar is slowly consumed by the acetobacter aceti. The content of reducing sugar in the mixed fermentation of the yeast powder and the acetobacter aceti powder is higher than that in the bacteria liquid and the single bacteria powder, because the alcohol content in the fermentation liquid is higher, the acid production capacity of the acetobacter aceti by using alcohol is greater than that of the acetic acid production capacity of the reducing sugar.
After the traditional vinegar in the old storehouse is fermented in the first batch, half of the original vinegar in the old storehouse is left in the fermentation vat to serve as a main strain source and a new raw material for fermentation, so that the original vinegar in the old storehouse fermented by yeast powder vinification liquid belongs to the first batch of fermentation in laboratory simulation production, and the initial acidity of the old storehouse fermented by the yeast powder vinification liquid is different from that of the old storehouse fermented by other modes. The total acid content and pH change of the aged vinegar in the acetic fermentation process are shown in FIG. 16, the total acid content is on the whole in an ascending trend, and the pH change is opposite. Wherein the total acid of the vinegar liquid fermented by adding the acetobacter aceti powder into the yeast powder vinification liquid rises fastest, the pH value drops most obviously, because enough alcohol is available in the vinification fermentation liquid of the yeast powder for the acetobacter aceti, and the acid yield reaches 6.27g/100mL when the fermentation is carried out for 9 days. And secondly, the vinegar in the old storehouse fermented by the acetobacter aceti powder has the acid production of 3.13g/100mL when the vinegar is fermented to the 9 th day, and the acetobacter aceti in the acetobacter aceti powder has insufficient alcohol and reducing sugar for producing acetic acid due to lower alcohol content in the fermentation liquid taken back by a vinegar plant, so the acid production is lower than that of the fermentation of yeast vinification liquid. As can be seen from the figure, the four groups of the acid yield capacities are composite bacteria powder, acetified acetobacter powder, bacteria liquid and vinegar plant liquid, so that the acetified acetobacter powder is matched with corresponding yeast powder vinification liquid to perform fermentation with the best effect.
Example 6: sensory evaluation of finished vinegar
The sensory requirements refer to GB/T16291.1-2012 general guide for sensory analysis, selection, training and management evaluator [iv] 20 students in food profession are selected to form a sensory evaluation group, finished vinegar produced by four different fermentation modes of vinegar plant fermentation liquor, B10 bacterial liquid, acetified vinegar bacillus powder and composite bacterial powder and market-sold old-storehouse vinegar are respectively used as 5 groups of samples for sensory evaluation, and grading of color, aroma, posture and taste is carried out [v] And the total score is 100, and finally the scores are comprehensively evaluated, wherein the scoring standard is shown in a table 7.
TABLE 7 organoleptic evaluation criteria of Vinegar
TABLE 8 sensory evaluation results of finished vinegar by different fermentation methods
The sensory evaluation results of the finished vinegar products of different fermentations in the old warehouse can be seen from table 8, the vinification liquid prepared from the yeast powder is used as the vinegar produced by the fermentation liquid, the score of the vinification liquid reaches 91 points, and the score of the vinegar produced by the vinification fermentation liquid of the acetified vinegar bacillus powder in the vinegar plant is 76 points. The wine prepared from the yeast powder is not diluted, so that the color and flavor of more raw materials of tea and brown sugar are reserved, the fermentation time is shorter than that of the traditional vinegar factory, and the volatilization of the fragrance of the raw materials is relatively less, so that the score is higher. When the basic fermentation liquor is the wine liquor of vinegar factory, the vinegar liquor prepared from the acetified vinegar bacillus powder is obviously closer to the commercial old-storehouse vinegar in color, flavor, posture and taste
Example 7: antioxidant activity of different fermentation modes
The method comprises the steps of applying the liquor obtained in the vinification stage of a vinegar factory, the dominant acetobacter aceti bacterial liquid, the prepared freeze-dried yeast powder vinification finishing liquid and the prepared freeze-dried acetobacter aceti bacterial powder as samples to the vinegar acetification stage of an old warehouse for fermentation, after culturing for 9 days at 30 ℃, detecting DPPH free radical scavenging capacity and hydroxyl free radical scavenging rate in each sample, and researching the oxidation resistance.
(1) Determination of DPPH radical scavenging Capacity: as can be seen from FIG. 17, four different fermentations had a better effect on DPPH.removing ability, and the DPPH radical removing ability of the vinegar factory liquid, the bacterial liquid, the compound bacterial powder and the acetified vinegar bacillus powder was 71.67%, 84.33%, 95% and 90.33%, respectively. The cleaning capability of the composite bacteria powder for the fermented vinegar is the best, the vinegar bacillus powder for acetification is slightly lower in the fermented vinegar, and the cleaning capability of the raw liquid fermentation of a vinegar factory is the weakest. As can be seen from table 9, significant differences were observed between the samples for DPPH radical scavenging capacity measurement.
TABLE 9 multiple comparisons
Dependent variable DPPH radical scavenging ability
LSD
* The significance level of the mean difference was 0.05.
(2) Determination of hydroxyl radical scavenging capacity: as can be seen from fig. 18, the hydroxyl radical scavenging ability of the compound bacteria powder is better, and the hydroxyl radical scavenging ability of the vinegar factory liquid, the bacteria liquid, the compound bacteria powder and the acetified vinegar bacillus powder is 48.33%, 58.33%, 72.67% and 68%, respectively, which indicates that the fermented vinegar of the compound bacteria powder and the acetified vinegar bacillus powder has better free radical scavenging ability. As can be seen from Table 10, for the determination of hydroxyl radicals, only the difference between the composite bacterium powder and the acetobacter aceti powder is not significant, and the difference between the rest samples is significant.
TABLE 10 multiple comparisons
Dependent variable-hydroxy radical scavenging ability
LSD
* Mean difference significance level was 0.05.
(3) Iron ion reducing ability: as can be seen from FIG. 14, when the vinegar factory stock solution fermented vinegar was compared, it was found that the reduction power of the fermented vinegar of the composite bacterial powder was the best, and that the four groups of the vinegar was the second to the fermentation of the vinegar with the acetobacter aceti powder, all had a certain reduction power. As can be seen from table 11, significant differences were exhibited between the four samples for the reduction force measurement.
TABLE 11 multiple comparisons
Table.3-11 multiple comparison
Dependent variable-reduction of iron ion
LSD
* The significance level of the mean difference was 0.05.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of the invention or to explain the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.
Claims (10)
1. The Acetobacter aceti is characterized in that the strain is acebacter aceti (Acetobacter aceti) which is a dominant bacterium of Yunnan old-storehouse vinegar, is preserved in 18.04.2022 years in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No.24700.
2. An acquisition method of acetobacter aceti, which is characterized in that the acquisition step comprises the following steps:
s1: sampling, and preserving vinegar liquid at low temperature in 13-25 days of an acetification stage in a Yunnan old-warehouse vinegar fermentation tank;
s2: preparing a bacterial suspension: weighing 1g of vinegar sample, pouring the vinegar sample into 15mL of sterilized centrifuge tube, adding 9mL of sterile water, and oscillating for 5-10 min to fully disperse the sample, namely preparing 10 -1 A suspension of concentration;
s3: diluting: will be 1mL 10 -1 The suspension is sucked into a centrifuge tube containing 9mL of sterile water by a sterile pipette, namely 10 -2 The sample dilution of (1) can be prepared sequentially by repeating the process to obtain 10 -3 、10 -4 、10 -5 、10 -6 The diluent of (4);
s4: preparing a culture medium by using 1% of yeast extract, 1% of glucose and 2% of agar, adding 2% of calcium carbonate and 4% of absolute ethyl alcohol before pouring by adopting a pouring method, inversely placing the culture medium in a constant-temperature incubator at 35 ℃ after a plate is solidified, and recording the dilution times and the corresponding colony number after 72 hours;
s5: separating and purifying, selecting typical bacterial colony on an acetobacter aceti plate, streaking, carrying out inverted culture at 30 ℃ for 72h, selecting single bacterial colony, and carrying out bacterial colony characteristic observation, gram staining, microscopic cell morphology observation and acetobacter aceti qualitative test; selecting a person which is gram-negative and can form a reddish brown precipitate with FeCl3 solution, carrying out plate streaking pure separation on the strain, transferring the pure separated strain onto an inclined solid culture medium, and storing in a refrigerator at 4 ℃ after culture;
s6: screening, inoculating activated acetobacter aceti strains into a basic liquid culture medium, performing static culture at 30 ℃ for 48 hours to prepare acetobacter aceti seed liquid, sucking 2% of the acetobacter aceti seed liquid into 100mL of liquid fermentation culture medium, performing static culture at 30 ℃, measuring the acid yield once every 24 hours, continuously measuring for 10 days, measuring three groups of strains in parallel, determining the acid production capacity of the strains, drawing an acid yield curve according to the test result, and screening the acetobacter aceti strains.
3. A method for preparing a composite freeze-dried starter culture by using acetobacter aceti as claimed in claim 1, wherein the preparation method comprises the following steps:
step one, preparing strain seed liquid: selecting a small amount of acetobacter aceti strains from the strain preservation slant, inoculating the strains into a basic culture medium, and performing static culture at the temperature of 30 ℃ for 48 hours to prepare an acetobacter aceti strain seed solution;
step two, compounding the strains, wherein the inoculation amount of the acetobacter aceti is 1.0%, the inoculation amount of the acetobacter vinelans is 0.03%, and the acetobacter vinelans is inoculated into a culture medium and fermented for 9 days to obtain the compounded strain.
Step three, centrifugally collecting thalli: collecting bacterial sludge under the conditions that the centrifugal rotation speed is 5000r/min, the centrifugal time is 20min and the centrifugal temperature is 8 ℃;
step four, preparing a protective agent: the protective agent comprises milk powder and sugar alcohol, and the ratio of the sugar alcohol to the skimmed milk powder is 1:1; the sugar alcohol comprises mannitol and lactose, the lactose to mannitol ratio is 1:1;
step five, freeze-drying: and (3) performing vacuum freeze-drying according to the ratio of the bacterial sludge to the protective agent 1:3 to obtain the composite freeze-dried leavening agent.
4. The method according to claim 3, wherein the culture medium in step 1 comprises a liquid culture medium and a solid culture medium, and the culture medium comprises:
liquid culture medium: 1% of yeast extract, 1% of glucose and 4% of absolute ethyl alcohol;
solid medium: 1% of yeast extract, 1% of glucose, 4% of absolute ethyl alcohol, 2% of agar and 2% of calcium carbonate.
5. The method according to claim 3, wherein the protective agent in the third step comprises the following components in concentration: the milk powder concentration is 14.314%, the mannitol concentration is 2.831%, and the lactose concentration is 9.347%.
6. The method of claim 3, wherein the maximum thickness of the bacterial suspension at lyophilization in step five is 0.75cm.
7. An application of the acetobacter aceti composite freeze-dried starter prepared according to the preparation method of claim 3 in preparing Yunnan aged vinegar.
8. An application of the Acetobacter aceti composite freeze-dried starter culture and the yeast composite freeze-dried starter culture prepared according to the method of claim 3 in preparation of Yunnan old vinegar.
9. The use of claim 8 or 9, wherein the Acetobacter aceti composite lyophilized starter is added in an amount of 1%.
10. The use according to claim 8, characterized in that the specific steps comprise:
1) The composite freeze dried yeast starter is activated in potato glucose water for 2 generations, and the concentration of the yeast is adjusted to be 3 multiplied by 10 6 CFU/mL for standby;
2) And (3) a vinification stage of the Yunnan old warehouse: adding 10 kilograms of tea and 20 kilograms of brown sugar into 200 kilograms of water, boiling, filtering, cooling, putting into a soil jar with old fermentation liquor, and mixing the bacterial liquid in the step 1) according to a ratio of 100:1, adding the mixture into sugar tea water for fermentation in a alcoholization stage, and sealing and fermenting for 6 days;
3) And (3) vinegar acetification stage of the Yunnan old warehouse: step 2) directly adding a composite freeze-drying leaven into the fermentation liquor after the alcoholization stage, and mixing the composite freeze-drying leaven with the liquor: adding the composite freeze-dried starter according to the proportion of 100.
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| CN101491362A (en) * | 2009-02-17 | 2009-07-29 | 福州大学 | Red tea fungus beverage produced by mixed culture fermentation of pure fungus |
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