CN115141774B

CN115141774B - Acetobacter aceti, acquisition method, preparation method of composite freeze-dried starter and application of composite freeze-dried starter in vinegar preparation

Info

Publication number: CN115141774B
Application number: CN202210811343.0A
Authority: CN
Inventors: 何二平; 赵祖艳; 董霞; 吴荣书
Original assignee: Chengjiang Laocang Vinegar Food Technology Co ltd
Current assignee: Chengjiang Laocang Vinegar Food Technology Co ltd
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2024-01-30
Anticipated expiration: 2042-07-11
Also published as: CN115141774A

Abstract

The invention discloses Acetobacter aceti, an acquisition method, a preparation method of a composite freeze-drying starter and application thereof in vinegar preparation, wherein a dominant strain Acetobacter aceti (Acetobacter aceti) is screened out on the basis of a single factor experiment by taking Yunnan old bin vinegar as a raw material, the optimal preparation process parameters of the acetic acid fermentation condition of the old bin vinegar are optimized and determined by adopting a response surface method, and the freeze-drying composite starter of the Acetobacter aceti is optimized and screened out; the prepared acetobacter aceti composite freeze-dried starter bacterial powder with higher activity is combined with a composite freeze-dried starter prepared by a laboratory and is preliminarily applied to the small-scale production of Yunnan old bin vinegar in the laboratory, and has important significance in shortening the production process period of the old bin vinegar and improving the stability of the old bin vinegar product.

Description

Acetobacter aceti, acquisition method, preparation method of composite freeze-dried starter and application of composite freeze-dried starter in vinegar preparation

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a Yunnan old bin vinegar dominant 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.

Background

The Yunnan old bin 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 palatable 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 reserved as seed liquor and added into fresh fermentation liquor for continuous fermentation. The method has the advantages of easy operation, low energy consumption, high raw material utilization rate, easy control of fermentation process, rapid and convenient subsequent treatment, and the like. The key point of the success and failure of fermentation is the microbial film generated on the surface of the liquid, and the complete film can accelerate the fermentation and shorten the fermentation period.

The Yunnan old bin vinegar belongs to a black tea fungus beverage, has the health care effect, contains a large amount of organic acid and beneficial microorganisms, can promote digestion and improves delicate flavor. Black tea fungus is the traditional sea treasure and stomach treasure, also called jellyfish fungus in foreign countries, and is the symbiont of membrane vinegar fungus and saccharomycete.

The production of the old bin vinegar 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, fermenting, adding water, blending, deoxidizing, secondary blending, filtering, homogenizing, pasteurizing, canning, inspecting, and obtaining the final product

The fermentation process of the Yunnan old bunker vinegar can be divided into 3 stages, the traditional fermentation period is calculated, the 1 st to 13 th days are the alcoholization stages, and saccharomycetes are used as dominant strains at the stages, and the sugar consumed by fermentation liquid is used for producing alcohol as the main material; the 13 th to 25 th days are acetification stages, the dominant strain of the acetification acetobacter in the stages is mainly that the acetification acetobacter consumes alcohol and reducing sugar, and the acetification acetobacter is converted into acetic acid, and the process of improving the acidity of fermentation liquor is adopted; after 25 days, the post-maturation stage is performed, nutrient components in the fermentation broth are insufficient, and the alcoholization and the acetification are stopped, mainly the alcohols and the organic acids generated in the fermentation broth react to generate flavor substances.

The conventional preservation method of acetic acid bacteria is inclined plane preservation and liquid preservation, and requires periodic passage. However, periodic passaging of acetic acid bacteria increases the probability of degradation and mutation, thereby wasting the precious strain resources which are preferably obtained, making later studies difficult to carry out, and even causing great loss and harm to industrial production. Therefore, it is extremely important to select an appropriate preservation method in order to maintain the characteristics and viability of acetic acid strains.

In recent years, with the expansion of the old yunnan vinegar market, the old yunnan vinegar is deeply favored by consumers, but enterprises have a plurality of problems in the process of producing the old yunnan vinegar: the old bin vinegar is unstable in fermentation, low in resource utilization rate, long in traditional fermentation time, the fermented product has no definite standard, the quality of batches of the product is non-uniform, the acid yield difference is large, the fermentation process is greatly influenced by temperature, and particularly, the acid yield is low in winter, so that serious economic loss and resource waste are caused for fermented vinegar production enterprises.

Disclosure of Invention

In order to simplify the production process flow of the Yunnan old bin vinegar, solve the phenomena of mixed bacteria pollution, strain degradation, low acid yield and the like, the dominant acetification vinegar bacillus strain separated from the Yunnan old bin vinegar finished product is used as the existing condition, the strain with high activity and high acid yield is bred, and the low-temperature freeze-drying technology is combined, so that the dominant bacteria composite freeze-drying starter aiming at the old bin vinegar is explored through researching the production process, the basic technical parameters can be provided for the industrial production of the old bin vinegar, the fermentation process of the Yunnan old bin vinegar is improved, the management of the fermentation strain is facilitated, and the strength of staff is reduced, thereby reducing the investment cost, improving the quality of the Yunnan old bin vinegar product and being beneficial to the development of the Yunnan old bin vinegar industry.

In order to achieve the above purpose, the invention provides acetobacter aceti, an acquisition method, a preparation method of a composite freeze-drying starter and application thereof in vinegar preparation.

According to a first aspect, the present invention provides:

acetobacter aceti, which is the dominant fungus Acetobacter aceti (Acetobacter aceti) of Yunnan old cang vinegar, was deposited in China general microbiological culture Collection center, with a deposit address: beijing, china, the preservation number is CGMCC No.24700.

According to a second aspect, the present invention provides: an acquisition method of acetobacter aceti, the acquisition step comprising:

s1: sampling, and taking vinegar liquid in a vinegar fermentation tank of the old bin of Yunnan for 13-25 days for low-temperature preservation;

s2: preparing a bacterial suspension: weighing 1g of vinegar sample, pouring the vinegar sample into a 15mL sterilized centrifuge tube, adding 9mL of sterile water, and oscillating for 5-10 min to fully disperse the sample, namely preparing 10 ^-1 Suspension at a concentration.

S3: dilution: 1mL 10 ^-1 The suspension was sucked into a centrifuge tube containing 9mL of sterile water, 10 ^-2 Is sequentially prepared into 10 by repeating the process ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Is a diluent of (a).

S4: preparing culture medium with yeast extract 1%, glucose 1%, agar 2%, adding calcium carbonate 2% and absolute ethanol 4% before pouring, solidifying, culturing in 35 deg.C constant temperature incubator, and recording dilution and corresponding colony number after 72 hr

S5: separating and purifying, namely picking a typical colony on an acetobacter aceti plate, inversely culturing for 72 hours at 30 ℃ after streaking, picking a single colony, and performing colony characteristic observation, gram staining, microscopic cell morphology observation and acetobacter aceti qualitative tests. Selecting gram-negative and capable of forming a reddish brown precipitate with FeCl3 solution, carrying out flat streak pure separation on the strain, transferring the pure separated strain to a slant solid culture medium, culturing, and then preserving in a refrigerator at 4 ℃.

S6: screening, inoculating activated Acetobacter aceti strains into a basic liquid culture medium, standing 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, standing at 30 ℃ for culture, measuring acid yield once every 24 hours, continuously measuring for 10 days, measuring three groups of parallelism of each strain, determining acid production capacity of the strain, drawing an acid yield curve according to test results, and screening the Acetobacter aceti strains.

According to a third aspect, the present invention provides: a preparation method for preparing a composite freeze-dried starter by using acetobacter aceti, which comprises the following preparation steps:

step one, preparing strain seed liquid: selecting a small amount of Acetobacter aceti strain from the strain preservation inclined plane, inoculating the strain into a basic culture medium, and performing stationary culture for 48 hours at the temperature of 30 ℃ to prepare Acetobacter aceti seed liquid;

step two, strain compounding, wherein the inoculum size of the acetobacter aceti is 1.0%, the inoculum size of the acetobacter aceti is 0.03%, and the acetobacter aceti is inoculated in a culture medium for fermentation for 9d to obtain a compound strain.

Step three, centrifugal collection of thalli: collecting bacterial sludge under the conditions that the centrifugal rotating speed is 5000r/min, the centrifugal time is 20min and the centrifugal temperature is 8 ℃;

step four, preparation of a protective agent: the protective agent comprises milk powder and sugar alcohol, wherein the ratio of the sugar alcohol to the skim milk powder is 1:1; the sugar alcohol comprises mannitol and lactose, and the ratio of lactose to mannitol is 1:1;

step five, freeze-drying: vacuum freeze-drying according to the ratio of the bacterial sludge to the protective agent of 1:3 to obtain the acetobacter aceti composite freeze-dried starter.

Further, the culture medium in the step 1 includes a liquid culture medium and a solid culture medium, which are respectively the 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.

Further, the concentration of each component of the protective agent in the third step is as follows: the concentration of milk powder is 14.314%, the concentration of mannitol is 2.831%, and the concentration of lactose is 9.347%.

Further, the thickness of the bacterial suspension at the time of the freeze-drying in the step five is 0.75cm.

According to a fourth aspect, the present invention provides: application of Acetobacter aceti composite freeze-dried ferment in preparing aged warehouse vinegar.

Further, the preparation method is an application of the acetobacter aceti composite freeze-dried starter and saccharomycete composite freeze-dried starter in preparation of aged bin vinegar.

Further, the adding amount of the acetobacter aceti composite freeze-drying fermenting agent is 1%.

Further, the specific steps for preparing the aged vinegar by using the fermenting agent in combination include:

1) Activating yeast composite freeze-dried ferment in potato dextrose water for 2 generations, and adjusting the concentration of bacteria to 3×10 ⁶ CFU/mL for standby;

2) Stage of aging bin vinegar alcoholization: adding 10 kg of tea and 20 kg of brown sugar into 200 kg of water, boiling, filtering, cooling, putting into a soil jar with old fermentation liquor, and mixing the bacterial liquid in 1) according to the bacterial liquid of 100:1, inoculating into sugar tea water for fermentation in a alcoholization stage, and sealing and fermenting for 6 days;

3) Stage of acetification of old bin vinegar: and 2) directly adding a composite freeze-drying fermenting agent into the fermentation liquor after the alcohol-forming stage, wherein the fermenting liquor is prepared from the following steps of: the composite freeze-drying starter is added in a ratio of 100:1, and the fermentation is carried out for 9 days.

Compared with the prior art, the invention has the beneficial effects that:

(1) The prepared composite acetobacter aceti starter for the aged storehouse vinegar is applied to the aged storehouse vinegar for acetic fermentation, the acetic acid fermentation time of the prepared vinegar liquid is obviously shortened compared with that of factory acetic acid, the acetic acid fermentation time is shortened to 9 days from 13 days, and the acid yield of the starter after being inoculated can reach 3.13g/100mL after fermentation for 9 days at 30 ℃;

(2) The aceti powder is added with the vinification fermentation liquor prepared from the saccharomycete 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 is mellow and long-lasting in taste, has obvious raw material color and luster, and has a fine and slightly sweet taste compared with the prior art of the old bin vinegar, and the taste is similar to the state of the old bin vinegar.

(2) Antioxidation results: the wine added with the aceti powder into the yeast powder has the best capability of scavenging hydroxyl radicals, and the capability of scavenging hydroxyl radicals of the vinegar prepared by four groups of vinegar factory liquid, bacterial liquid, composite bacterial powder and composite aceti powder is 48.33%, 58.33%, 72.67% and 68% respectively. The four sample solutions have better effect on the DPPH free radical scavenging capability, and the vinegar liquid DPPH free radical scavenging capability prepared by four groups of vinegar factory solution, bacterial solution, composite bacterial powder and composite acetobacter aceti powder is 71.67%, 84.33%, 95% and 90.33% respectively; the iron ion reducing capacities were 0.43, 0.49, 0.6 and 0.56, respectively.

Drawings

FIG. 1 is a diagram showing the acidity variation lines of fermentation broths with different factor shadows;

wherein A1 is the influence of fermentation time on acidity of fermentation liquor; b1, influence of the addition amount of vinegar brewing bacteria on acidity of fermentation liquor; C1:B10 addition amount affects acidity of fermentation liquor;

FIG. 2 is a plot of the centrifuge yields for different factor shadow cells;

a2, influence of centrifugal rotation speed on centrifugal yield; b2, influence of centrifugation time on centrifugation 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 rate of the lyophilizate;

FIG. 4 is a line graph of the influence of different factors on the survival rate of the freeze-dried bacteria;

a4, influence of the concentration of the skim milk powder on the survival rate of the freeze-dried bacteria; b4, influence of mannitol concentration on the survival rate of the freeze-dried bacteria; c4, influence of lactose concentration on survival rate of freeze-dried bacteria;

FIG. 5 is a bar graph showing the effect of sugar alcohol ratio on the survival rate of lyophilized bacteria;

FIG. 6 is a graph of the effect of different factors on the survival rate of the freeze-dried bacteria;

a6, influence of the proportion of sugar alcohol and milk powder on the survival rate of the freeze-dried bacteria; b6, the influence of the proportion of the bacterial sludge and 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 graph of the effect of milk powder concentration and mannitol concentration on survival and a contour plot;

FIG. 8 is a graph showing the effect of milk powder concentration and lactose concentration on survival and a contour plot

FIG. 9 is a graph of the curve and contour plot of the effect of milk powder concentration to bacterial mud protectant ratio on survival;

FIG. 10 is a graph showing the effect of mannitol concentration and lactose concentration on survival and a contour plot

FIG. 11 is a graph of the effect of mannitol concentration and slime protectant ratio on survival and contour plot;

FIG. 12 is a graph of the effect of lactose concentration to slime protectant ratio on survival and contour plot;

FIG. 13 shows the effect of the amount of added bacteria powder on the acid production of old bunker vinegar;

FIG. 14 shows the variation of total sugar in the acetification stage of different fermentation modes

FIG. 15 shows the change of reducing sugar in the acetification stage of different fermentation modes

FIG. 16 shows the change in total acid and pH during the acetification stage of the different fermentation modes;

FIG. 17 is a comparison of DPPH radical scavenging ability;

FIG. 18 is a comparison of hydroxyl radical scavenging ability;

FIG. 19 is a graph showing acid production comparison of dominant acetic acid-producing strain of Acetobacter aceti in aged bunker vinegar;

FIG. 20 is a growth curve of the dominant strain (B10).

Detailed Description

The objects, technical solutions and advantages of the present invention will become more apparent by the following detailed description of the present invention when taken in conjunction with the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following invention, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present invention.

Example 1: method for obtaining acetobacter aceti of dominant bacteria of aged cang vinegar

S5: separating and purifying, namely picking a typical colony on an acetobacter aceti flat plate, inversely culturing for 72 hours at 30 ℃ after streaking, picking a single colony, and performing colony characteristic observation, gram staining, microscopic cell morphology observation and acetobacter aceti qualitative tests; selecting gram-negative and capable of forming a reddish brown precipitate with FeCl3 solution, carrying out flat streak pure separation on the strain, transferring the pure separated strain to a slant solid culture medium, culturing, and then preserving in a refrigerator at 4 ℃;

s6: screening, inoculating activated Acetobacter aceti strains into a basic liquid culture medium, performing stationary 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 stationary culture at 30 ℃ for measuring acid yield once every 24 hours, continuously measuring for 10 days, measuring three groups of parallelism of each strain, determining the acid production capacity of the strain, and screening the high-yield Acetobacter aceti strain B10.

As shown in FIG. 19, three different Acetobacter aceti strains were fermented in the medium with significant differences in acid production. B10 strain has higher acid-producing ability than the other two strains, is a dominant strain, and the acid-producing amount of the strain B10 reaches 2.9g/100mL. Screening acetic acid bacteria from Fujian monascus vinegar by Jing et al, wherein the acid yield of the strain Y5054 reaches 5.7g/100mL at the 7 th day. Compared with the literature, the acid yield values of the acetobacter aceti separated in the experiment are low, and the analysis reasons are probably that the acid yield of the B10 dominant strain in the old bin vinegar is weak, so that the experiment utilizes the dominant acetobacter aceti B10 in the old bin vinegar, and a small amount of acetobacter aceti is additionally compounded to improve the acid yield.

As can be seen from FIG. 20, the OD of strain B10 increased slowly during 0-32 h, increased rapidly during 32-50 h, and the OD of strain changed little after 50 h. The bacteria are in a delay period just at the beginning and are in a stage of adaptation to the environment by the acetobacter aceti, so that the growth of the bacteria is slow; the bacterial cells are in the logarithmic phase of growth for 32-50 h, and the bacterial cells are propagated faster and the number is increased rapidly; after 50 hours, the B10 acetobacter aceti grows into a stable period, the growth of thalli is in dynamic balance, and the thalli overall tends to be stable. The number of viable bacteria in the stationary phase is the highest, and in order to obtain a large number of viable bacteria, the test was performed by selecting 50 hours of cells for freeze-drying test.

Example 2: strain compounding

1. Single factor test

(1) Determining the addition amount of the old bin vinegar strain: as can be seen from C1 of FIG. 1, the inoculum size of Acetobacter aceti B10 was increased, and the acid production capacity showed a tendency of a rising and falling, and the acidity reached a maximum value of 2.93g/100mL when the inoculum size of Acetobacter aceti B10 was 1.0%. When the inoculum size is low, the fermentation liquid is insufficiently fermented, the acidity generated is low, and when the inoculum size is excessive, the nutrient in the fermentation liquid is more used for the metabolism and propagation of the acetobacter aceti autologous cells ^[i] The acidogenic effect of Acetobacter aceti was suppressed, and therefore, the inoculum size of Acetobacter aceti B10 was selected to be 1.0%.

(2) The addition amount of the commercial strain is determined: as can be seen from the analysis of B1 in FIG. 1, the acid production capacity of the fermentation broth increases and decreases during acetic acid fermentation with increasing addition of vinegar brewing bacteria in the same fermentation time, and the acidity reaches 4.1g/100mL at the maximum when the inoculation amount is 0.03%. When the inoculum size of Acetobacter aceti is higher 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 increase of the inoculation amount can improve the alcohol conversion rate and the acid production capacity; excessive inoculation amount, excessive nutrient consumption, reduced acid production capacity, and reduced total acid content caused by aging of thallus in the late fermentation stage ^[ii] Therefore, the inoculation amount of vinegar brewing bacteria should be selected to be 0.03%.

(3) Determination of fermentation time: as can be seen from A1 of FIG. 1, the acidity gradually increases with the increase of the fermentation time, and the acidity obviously increases when the fermentation time is 6-9 days, and then the acid production capacity gradually becomes gentle with the increase of the fermentation time. The fermentation time affects the growth of microorganisms and the synthesis of products, the fermentation is incomplete or affects the acidity and flavor of the vinegar liquid, the fermentation time is 9 days, the acidity is basically stable, and the acidity is 2.91g/100mL at the moment, so the fermentation time in the acetification stage is preferably 9 days.

2. Orthogonal test of fermentation conditions of compound strain

According to the single factor experimental result, comprehensively considering the influence of each factor on the acidity of the fermentation broth, selecting the acidity as an investigation index, and carrying out an orthogonal test L9 (3 ³ ) The results of the orthogonal test are shown in Table 1, and the results of analysis of variance and significance test are shown in Table 2.

Table 1 L9 (3) ³ ) Orthogonal experimental results of strain compounding

TABLE 2 analysis of variance of strain complex orthogonal experiments

Dependent variables: acidity of

R ² =0.741 (post-adjustment R ² ＝-0.035)

As can be obtained by the analysis of the table 1 and the table 2, the strain compound orthogonal model is extremely remarkable (P is less than 0.01), and the fermentation time, the vinegar brewing bacteria addition amount and the B10 addition amount have remarkable influence on the acid production result (P)<0.05 The sizes of the factors influencing the acid yield of acetic acid fermentation are A in sequence>C>B, instant time>B10 addition amount>The addition amount of vinegar brewing bacteria. The orthogonal optimal combination and the K value maximum combination are the same combination, which is A ₁ B ₂ C ₂ The fermentation time is 9 days, the addition amount of the vinegar brewing bacteria is 0.03%, and the addition amount of the B10 is 1.0%. Three parallel validation experiments were performed under this condition with a total acid of up to 3.81g/100mL. In conclusion, the orthogonal experimental model can be used for strain compounding experiments.

Example 3: centrifugal test of cell

1. Single factor test:

(1) And (3) centrifugal rotation speed determination: as is clear from A2 of FIG. 2, the centrifugal yield tends to be increased and decreased with the increase of the centrifugal speed, and the centrifugal yield of the cells at a centrifugal speed of 6000r/min is the highest and is 70.39%. As the centrifugal speed increases, the centrifugal yield gradually decreases, and the centrifugal yield is largely due to the action of centrifugal force, which may cause partial death of the thalli or partial loss of the thalli in the supernatant. Therefore, the bacterial liquid is centrifuged at 6000 r/min.

(2) And (3) determining the centrifugation time: as can be seen from B2 of FIG. 2, the centrifugal yield tends to rise and then fall with the increase of the centrifugal time, and the centrifugal yield reaches 80.54% when the centrifugal time at the highest centrifugal yield is 15min. The centrifugation time is continuously increased, and the centrifugation yield is gradually reduced, mainly because the strain is easy to damage cells under high-rotation-speed centrifugation for a long time, so that the survival rate is influenced, and the centrifugation time is selected to be 15 minutes.

(3) Determination of the centrifugation temperature: as seen from FIG. 2C 2, the yield of cells was 78.26% at a centrifugation temperature of 6 ℃. The centrifugation temperature has a certain influence on the centrifugation yield, and the separation of the bacterial cells and the culture solution is influenced by the too high or the too low temperature, so that the centrifugation temperature is 6 ℃ to centrifuge the bacterial solution.

2. Cell centrifugation orthogonal test: based on the single factor test result, selecting viable count and centrifugal yield as indexes, and performing 3-factor 3-level orthogonal test L by taking centrifugal rotation speed, centrifugal time and centrifugal temperature as investigation factors ₉ (3 ³ ) Design, orthogonal test design scheme and results are shown in table 3, analysis of variance and significance test results are shown in table 4:

table 3L ₉ (3 ³ ) Results of centrifugation orthogonal experiments

TABLE 4 analysis of variance of centrifugation orthogonal experiments

Table 3-4AnOVA of centrifugal orthogonal experiment

As can be obtained by analysis in tables 3 and 4, the cell centrifugation orthogonal model is extremely remarkable (P is less than 0.01), and the influence of the centrifugation rotating speed, the centrifugation time and the centrifugation temperature on the number of living bacteria after centrifugation is more remarkable (P is less than 0.05); the influence of the centrifugal temperature on the centrifugal yield is obvious; the centrifugal speed and the centrifugal time have no obvious influence on the centrifugal yield. The number of living bacteria and the centrifugal yield are used as indexes, and the main and secondary orders of influence are the same and are B>A>C, i.e. centrifugation time>Centrifugal rotational speed>Centrifuging temperature. The optimal combination of the thallus centrifugal 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 were performed under this condition, and the number of viable bacteria after centrifugation was at most 1.587X10 ⁸ CFU/mL, yield was 98.57%.

Example 4: screening optimization test of protective agent

Sugar alcohol protectant screening: the hydroxyl contained in the sugar alcohol substance can form a protective film, so that the protective film can protect cells of the bacteria, reduce the damage degree of the cells, and simultaneously the sugar alcohol substance is favorable for the cells to quickly rehydrate or repair the damaged cells. Therefore, 6 different sugar alcohol protecting agents of glucose, mannitol, fructose, lactose, sucrose and maltose which can be utilized by Acetobacter aceti are selected in the experiment, the various sugar alcohol protecting agents are dissolved by distilled water to prepare a solution with the mass concentration of 5 percent, and the solution is sterilized, wherein three protecting agents are arranged in parallel. Viable count was performed on the powder after vacuum freeze-drying, and the survival rate was calculated, and the results are shown in fig. 3 to 9: the average freeze-drying survival rates 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 sequence of 6 kinds of sugar alcohols on the thalli is lactose, mannitol, glucose, sucrose, fructose, maltose in sequence, wherein the protective effect of lactose and mannitol on acetobacter aceti is the best, and the maltose is the worst. Lactose and mannitol were therefore chosen as sugar alcohol protectants for single-factor experimental studies.

1. Single factor test

(1) Basic protectant concentration determination

The method comprises the steps of taking skimmed milk powder as a basic protective agent, respectively preparing milk powder solutions with mass concentrations of 6%, 8%, 10%, 12%, 14% and 16%, mixing the milk powder solutions with two screened sugar alcohols with mass concentrations of 5% according to a ratio of 1:1, and mixing the milk powder solutions with bacterial sludge: the protective agent is bacterial suspension washed out in a ratio of 1:4, three concentrations are parallel, and freeze-drying is carried out, wherein the freeze-drying survival rate is used as an index; as can be seen from A4 of fig. 4, the freeze-drying protection effect of the skim milk powder is most remarkable at the concentration of 14%, because the concentration of the skim milk powder is increased, the emulsion becomes viscous, the evaporation of water is unfavorable in the freeze-drying process, and the formed ice crystals are larger, so that the cell death rate is increased, and therefore, the skim milk powder with the concentration of 14% is selected as the basic protective agent.

(2) Determination of sugar alcohol concentration

Respectively preparing sugar alcohol solution solutions with mass concentrations of 1%, 3%, 5%, 7%, 9% and 11% from the two selected sugar alcohols, and mixing with 10% skimmed milk powder solution according to bacterial mud: the protective agent is bacterial suspension washed out in a ratio of 1:4, three concentrations are performed in parallel, freeze-drying is performed, and the optimal concentration of the two sugar alcohols is determined by taking the freeze-drying survival rate as an index.

As can be seen from B4 of fig. 4, the freeze-drying survival rate of the cells does not increase all the time with the increase of the mannitol concentration, and the survival rate of the freeze-dried cells reaches 67% when the mannitol concentration reaches 3%; when the concentration further increases, the number of viable colonies decreases instead, and the survival rate decreases. This is because the hydroxyl groups contained in mannitol interact with the free radicals on the cell surface of the cells, thereby reducing the exposure of the cells, and at the same time, hydrogen bonds are formed with the protein of the milk powder, so that the stability of the protein is ensured. However, when the mannitol concentration exceeds a certain level, the ability to stabilize the protein reaches a limit, and too high mannitol concentration during lyophilization may even cause deterioration of the protein. Thus, mannitol at a concentration of 3% was selected to be optimal.

As can be seen from C4 of fig. 4, when the lactose concentration is 1% -9%, the survival rate of acetobacter aceti is lower, and the tendency is improved; when the concentration reaches 9%, the survival number of the bacteria reaches the maximum value, and the survival rate reaches 69%; when the concentration further increases, the number of viable colonies decreases instead, and the survival rate decreases. Lactose is easy to dissolve in water to generate hydration, so that the growth speed of crystal nucleus is reduced, and the ice crystal formed in the pre-freezing process is reduced, so that the cells are protected from damage. However, too high lactose concentration can lead to cell death by dehydration. Therefore, lactose concentration was chosen to be optimal at 9%.

(3) Determination of sugar alcohol ratio

Mixing 5% of two sugar alcohol solutions with 10% of defatted milk powder solution according to different proportions of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4 respectively, and preparing bacterial mud: the protectant is bacterial suspension washed out in a ratio of 1:4, three bacteria are parallel to each ratio, freeze-drying is carried out, and the optimal ratio 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 at 1:1, the number of survival of the lyophilized bacteria was the largest, and the survival rate of the bacteria was 77%. Therefore, from the viewpoint of protection effect and production cost, mannitol and lactose are added in a ratio of 1:1, namely 3% mannitol and 9% lactose according to a ratio of 1:1 to be used as a sugar alcohol protective agent.

(4) Determination of sugar alcohol to milk powder ratio

Mixing 5% of two sugar alcohol solutions according to a ratio of 1:1, and mixing with 10% of defatted milk powder solution according to different ratios of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4, wherein the ratio is as follows: the protective agent is bacterial suspension washed out in a ratio of 1:4, three bacteria are arranged in parallel in each ratio, freeze-drying is carried out, and the optimal ratio of sugar alcohol to milk powder is determined by taking the freeze-drying survival rate as an index.

As can be seen from A6 of FIG. 6, after the skim milk powder, mannitol and lactose are added simultaneously as the composite freeze-drying protective agent, the freeze-drying survival rate of Acetobacter aceti is higher than that of the single protective agent, which proves that the skim milk powder, the mannitol and the lactose have synergistic effect with each other, and the protective effect is enhanced ^[iii] . Survival number of lyophilized bacteria when sugar alcohol and milk powder were added at 1:1At most, the survival rate of the freeze-dried bacteria exceeds 70 percent. Therefore, the sugar alcohol and milk powder are mixed according to a ratio of 1:1 to be used as a composite protective agent.

(5) Determination of the ratio of bacterial sludge to protective agent

Mixing 5% of two sugar alcohol solutions with 10% of defatted milk powder solution according to bacterial mud: the protective agent is bacterial suspension washed out in a ratio of 1:2, 1:3, 1:4, 1:5 and 1:6, three bacterial suspensions are parallel to each other, freeze-drying is carried out, and the optimal ratio of bacterial mud to the protective agent is determined by taking the freeze-drying survival rate as an index.

As can be seen from B6 in FIG. 6, when the bacterial sludge and the composite protective agent are added according to a ratio of 1:3, the survival rate of the freeze-dried bacteria is up to 88%, the protective effect of the freeze-dried bacteria in the rest proportion is obviously enhanced, but the optimal ratio of 1:3 is selected as the ratio of the experimental bacterial sludge to the composite protective agent to be more suitable.

(6) Determination of the Pre-frozen thickness of the bacterial suspension

Mixing 5% of two sugar alcohol solutions and 10% of defatted milk powder solution according to a ratio of 1:1, and bacterial mud: the protective agent is bacterial suspension washed out in a ratio of 1:4, the bacterial suspension is poured into pre-frozen cups with different thicknesses of 0.25cm, 0.5cm, 0.75cm, 1cm and 1.25cm respectively, three parallel layers are arranged on each thickness, freeze drying is carried out, and the optimal bacterial suspension pre-freezing thickness is determined by taking the freeze drying survival rate as an index.

If too little bacterial suspension is present, the lyophilization vessel will be too large in space and the cell surface will not be covered to an adequate extent, which may result in more exposed areas of cells and death during lyophilization. If the amount of the protective agent is too large, the cell permeability is insufficient, and the number of viable bacteria is also affected. As is clear from FIG. 6C 6, the number of viable lyophilized bacteria was the highest and the survival rate of the bacteria was the highest when the thickness of the bacterial suspension was 0.75cm. Thus, 0.75cm was used as the thickness of the bacterial suspension when lyophilized.

2. Box-Behnken experiment of composite protectant

(1) And according to a single factor experiment result, taking the freeze-drying survival rate as an evaluation index, carrying out formula optimization on the concentration of the skimmed milk powder, the concentration of mannitol, the concentration of lactose and the proportion of the bacterial mud protective agent, and carrying out a response surface experiment by utilizing Design-Expert10.0 software Design experiment, wherein the response surface experiment result is shown in Table 5, and the analysis of variance result is shown in Table 6.

TABLE 5 response surface test results for composite protectants

Analyzing the data by Design-Expert software to obtain a quadratic polynomial regression equation between the concentration of skimmed milk powder (A), the concentration of mannitol (B), the concentration of lactose (C), the ratio of bacterial mud protectant (D) and the freeze-drying survival rate (Y), wherein the quadratic polynomial regression equation is as follows:

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 regression analysis of variance results are shown in Table 6.

TABLE 6 regression model analysis of variance table

From the analysis of variance of table 6, the data model obtained using Design-Expert10 software was significant (p=0.0209<0.05 Interaction term AB, AC, AD, BC, BD, CD is not significant, indicating that the regression equation fitting is meaningful, the decision coefficient R of the model ² ＝0.9568，R ² _Adj The model is 0.9136, which can obtain a better fitting degree of the whole data model and reliable test results, so that the model can be used for predicting the formula of the composite protective agent.

(2) Response surface map analysis: according to response surface analysis (figures 7-12), the influence of two factors on the freeze-drying survival rate of the response value is studied by observing the inclination of the curved surface of the three-dimensional graph, and the response surface graph is arc-shaped, which shows that the concentration of the skimmed milk powder, the concentration of mannitol, the concentration of lactose and the proportion of the bacterial mud protective agent have an influence on the freeze-drying survival rate of acetobacter aceti in a certain range. The interaction between the shape of the contour map of the response surface and the factors is related, the closer the shape of the graph is to an ellipse, the more obvious the interaction between the contour map and the ellipse is, and the analysis of the graph 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 mud protective agent has obvious influence on the survival rate.

The optimal process for optimizing the compounding is as follows: the concentration of milk powder is 14.314%, the concentration of mannitol is 2.831%, the concentration of lactose is 9.347%, the ratio of bacterial mud protective agent is 1:3, and the yield is 93.197%. Under the condition, a verification experiment is carried out, the yield is 94% by three parallel experiments, the value is in a prediction interval, the experimental result is proved to be reliable, and the regression model can be used for predicting the compound protective agent formula.

Example 5: test for producing old bin vinegar by using starter

(1) Determination of the addition amount of the fungus powder

Taking 100mL of culture solution per bottle as an example, adding the freeze-dried Acetobacter aceti starter into sugar tea culture solution according to 0.1g, 0.5g, 1.0g, 1.5g and 2.0g of different gradients, wherein each gradient is three parallel, and simultaneously one contrast is provided, placing the culture solution at 30 ℃ for culturing for 9 days, taking sensory indexes and acidity as indexes, and selecting the optimal bacterial powder adding amount.

As can be seen from fig. 13, the addition amount of the freeze-dried aceti powder directly affects the acid production rate during the production process of the old bin aceti stage, and the acid production amount gradually increases with the increase of the addition amount of the bacteria powder. However, the acid yield also directly affects the taste, so that the optimal bacterial powder addition amount, namely the bacterial powder addition amount of 1%, is selected to meet the taste and simultaneously save the cost, and the acid yield at the moment reaches 3.58g/100mL and also meets the national standard that the acidity is not less than 3.5g/100mL.

(2) Dynamic change of physical and chemical indexes of old bin vinegar produced by different fermentation modes in acetification stage

Fermentation production of old bin vinegar

The preparation method comprises the steps of applying dominant acetobacteria B10 bacterial liquid, a prepared composite freeze-dried starter (acetobacteria powder for short), yeast powder and acetobacteria powder (composite bacterial powder for short) prepared in the same mode as samples to an old-warehouse acetification stage for fermentation, and carrying out laboratory production by using the vinosity liquid and the old liquid retrieved from an acetification factory as a comparison to study the dynamic changes of acid yield, pH, total sugar and reducing sugar of each sample in the fermentation process.

First group (vinegar mill liquor): taking the end liquid and the old liquid of the vinification stage of the vinegar factory, fermenting other raw material steps according to the production of the vinegar factory except the constant temperature condition of a laboratory, and fermenting the vinegar factory liquid in the laboratory to serve as a control group of the test;

second group (bacterial liquid): taking wine immediately after the stage of wineization of an old-house vinegar factory, and mixing the wine with the wine: b10 bacterial liquid is added in a ratio of 100:1, and the inoculated fermentation liquid is placed in a 30 ℃ incubator for 5 days;

third group: (Acetobacter aceti powder) taking wine immediately after the alcoholization stage of the old-house vinegar factory, and according to the wine: adding the composite freeze-drying ferment at a ratio of 100:1, and placing the inoculated ferment liquid in a 30 ℃ incubator for 5 days;

fourth group (composite bacterial powder): taking wine fermented by a composite freeze-dried yeast starter (fermentation liquid prepared by a vinegar factory process in a laboratory), and mixing the wine with the wine according to the following steps: the composite freeze-drying starter is added at a ratio of 100:1, and the fermentation broth is placed in a 30 ℃ incubator for continuous fermentation for 5 days.

As shown in FIG. 14, since the vinegar factory liquid, the bacterial liquid and the acetobacter aceti powder are fermented in the stage of aceti in the complete fermentation liquid of the old stock vinegar factory, the initial sugar degree of the three groups is basically the same and is about 5.2g/100mL, and the mixed fermentation of the two bacterial powders is that the wine liquid obtained by starting fermentation from the raw materials in a small scale in a laboratory is used as the fermentation liquid, and the wine liquid produced in the laboratory is not diluted, so the initial sugar degree of the groups is higher and is 6.66g/100mL. In four groups of experiments, the total sugar values of other groups except the vinegar factory liquid have a rapid descending trend, and the descending trend is slower in the 1 st to 4 th days of the acetification stage, mainly the quantity of the acetobacter aceti is smaller at the moment, and the acetobacter aceti existing in the fermentation liquid is producing acid by utilizing alcohol. The total sugar consumption is extremely rapid at days 4-8, mainly because the acetic acid is produced by Acetobacter aceti by alcohol and partial reducing sugar is also consumed to convert the acetic acid, so the reducing sugar is rapidly consumed. With increasing fermentation time, the broth reducing sugars are consumed at least and are no longer available to acetobacter aceti. The Acetobacter aceti in the vinegar factory liquid grows slowly, the total sugar decomposition capacity is weak, and the preliminary judgment is that the quantity of the Acetobacter aceti in the vinegar factory liquid is small, the alcohol stage is incomplete, and the alcohol degree does not reach the concentration of the acid produced by the Acetobacter aceti.

As can be seen from the chart 15 showing the change trend of reducing sugar in the acetification stage from different fermentation modes, the reducing sugar in the fermentation liquid tends to decrease with the increase of the fermentation time regardless of the fermentation mode. The initial reducing sugar of two kinds of fungus powder fermentation is slightly lower than that of the other three groups, and the alcohol production consumption by utilizing the reducing sugar in the alcoholization stage of the yeast powder fermentation is more. In the acetic acid fermentation stage, acetic acid is mainly produced by acetobacter aceti by utilizing reducing sugar in 1-8 days, and meanwhile, the existence of the reducing sugar is beneficial to the growth of thalli, and the thalli consume the reducing sugar in the growth process, so that the reducing sugar content is continuously reduced. Since the total sugar content is less after day 8, the capacity of decomposing reducing sugar is reduced, the reducing sugar which can be utilized by acetobacter aceti is less, and the reducing sugar change is gentle. On day 10, the measured reducing sugar content of the vinegar factory liquid group is higher than that of other groups and is 3.96g/100mL, and the acetic fermentation is initially judged to be not finished, so that the acetic acid bacillus consumes reducing sugar slowly. The content of the reducing sugar in the mixed fermentation of the saccharomycete powder and the acetobacter aceti powder is higher than that of the fermentation of the bacterial liquid and the single bacterial powder, and the alcohol content in the fermentation liquid is higher, so that the acid production capacity of the acetobacter aceti by utilizing alcohol is higher than that of the reducing sugar.

After the primary fermentation of the traditional old cang vinegar is finished, half of the old cang vinegar is left in a fermentation cylinder to be used as a main strain source for fermentation with new raw materials, so that the old cang vinegar belongs to primary fermentation in laboratory simulation production, and the original acidity of the old cang vinegar by fermenting yeast powder and a wine liquor is different from that of the old cang vinegar in other modes. The total acid content and pH change of the old bunker vinegar in the acetic acid fermentation process are shown in figure 16, the total acid content integrally shows 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 alcoholic fermentation liquid of the saccharomycete powder rises fastest, and the pH value drops most obviously, because the alcoholic fermentation liquid of the saccharomycete powder has enough alcohol for the acetobacter aceti to use, and the acid yield reaches 6.27g/100mL when the fermentation is carried out to 9 days. Secondly, the acid yield of the old bin vinegar fermented by the acetobacter aceti powder reaches 3.13g/100mL when the old bin vinegar is fermented to 9 days, and the acid yield of the old bin vinegar fermented by the acetobacter aceti powder is lower than that of the fermented liquid fermented by the saccharomycete vinositae because the alcohol content in the fermented liquid retrieved by the vinegar factory is lower, and the acetobacter aceti in the acetobacter aceti powder has insufficient alcohol and reducing sugar for producing acetic acid. From the graph, the four groups of acid yield capacities are that composite bacteria powder > aceti bacteria powder > bacteria liquid > vinegar factory liquid, so that the fermentation effect of the aceti bacteria powder and the corresponding yeast powder wine liquid is best.

Example 6: sensory evaluation of the finished Vinegar

Sensory requirements reference GB/T16291.1-2012 general guidelines for sensory analysis selection, training and management of evaluator ^[iv] Selecting 20 students with food profession to form sensory evaluation group, and respectively scoring color, fragrance, physical state and taste of final vinegar and commercial aged barn vinegar produced by four different fermentation modes of vinegar factory fermentation liquor, B10 bacterial liquid, acetobacter aceti powder and composite bacterial powder as 5 groups of samples for sensory evaluation ^[v] The total score was 100 points, and finally the score was comprehensively assessed, and the scoring criteria are shown in Table 7.

TABLE 7 organoleptic criteria for vinegar

TABLE 8 sensory evaluation results of different fermentation modes of finished Vinegar

As can be seen from Table 8, the sensory evaluation results of the different fermented aged cang vinegar finished products show that the aged cang vinegar produced by using the wined liquid prepared from the saccharomycete powder as the fermentation liquid has the highest score of 91 minutes, and the vinegar produced by using the acetobacter aceti powder as the wined fermentation liquid in the vinegar factory has 76 minutes. As the wine prepared from the saccharomycete powder is not diluted, more colors and flavors of raw material tea and brown sugar are reserved, and meanwhile, the fermentation time is shorter than that of the traditional vinegar factory, and the volatilization of the raw material fragrance is relatively less, so that the score is higher. When the basic fermentation liquor is the wine liquor of the vinegar factory, the vinegar liquor prepared by the acetobacter aceti powder is obviously more similar to the commercial aged barn vinegar in terms of color, flavor, body and taste

Example 7: antioxidant Activity of different fermentation modes

And (3) applying the liquid in the alcoholization stage, the dominant acetobacter vinegared bacteria liquid, the prepared freeze-dried yeast powder alcoholization finished liquid and the prepared freeze-dried acetobacter vinegared bacteria powder taken by the vinegar factory as samples to the old warehouse acetification stage for fermentation, and detecting DPPH free radical scavenging capacity and hydroxyl free radical scavenging rate in each sample after culturing for 9 days at 30 ℃ for researching oxidation resistance.

(1) Measurement of DPPH radical scavenging ability: as shown in FIG. 17, four groups of different fermentations have better effect on DPPH and the DPPH free radical removing capacities of the vinegar factory liquid, the bacterial liquid, the composite bacterial powder and the acetobacter aceti powder are respectively 71.67%, 84.33%, 95% and 90.33%. The composite bacteria powder has the best capability of removing fermented vinegar, the acetobacter aceti powder has slightly lower fermented vinegar, and the vinegar factory has the weakest capability of removing stock solution fermentation. As can be seen from table 9, there was a significant difference between each sample for the measurement of DPPH radical scavenging ability.

Table 9 multiple comparisons

Dependent variable DPPH radical scavenging ability

LSD

* The significance level of the mean difference was 0.05.

(2) Determination of the hydroxyl radical scavenging ability: as can be seen from FIG. 18, the four groups of bacteria powder have a certain capability of scavenging hydroxyl free radicals, wherein the composite bacteria powder has the best capability of scavenging hydroxyl free radicals, and the capability of scavenging hydroxyl free radicals of vinegar factory liquid, bacteria liquid, composite bacteria powder and aceti bacteria powder is 48.33%, 58.33%, 72.67% and 68% respectively, which indicates that the fermented vinegar of the composite bacteria powder and the aceti bacteria powder has a better capability of scavenging free radicals. As can be seen from Table 10, the difference between the complex bacterial powder and the Acetobacter aceti powder was not significant for the measurement of hydroxyl radicals, and the other samples were significantly different.

Table 10 multiple comparisons

Dependent variable hydroxyl radical scavenging ability

LSD

* The significance level of the mean difference was 0.05.

(3) Iron ion reducing ability: as can be seen from FIG. 14, the reduction power of the fermented vinegar of the composite bacteria powder is best as compared with that of the fermented vinegar of the stock solution of the vinegar factory, and four groups have a certain reduction power. As can be seen from table 11, for the measurement of the reducing force, significant differences were exhibited between the four samples.

Table 11 multiple comparisons

Table.3-11 multiple comparison

Dependent variable iron ion reducing ability

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 serve to explain the principles of the invention and are not to be construed as limiting the invention. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.

Claims

1. The acetobacter aceti is characterized in that the strain is the dominant fungus acetobacter aceti of Yunnan old cang vinegarAcetobacter aceti) The strain is preserved in China general microbiological culture Collection center (CGMCC) at 18/04/2022, and the preservation number is CGMCC No.24700.

2. Use of acetobacter aceti according to claim 1 for the preparation of old yunnan cang vinegar.