CN108998398A - A method of using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type - Google Patents

Abstract

The invention discloses a kind of methods using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, using Pasteur acetobacter Acetobacter pasteurium as bacterial strain, 6~8g glucose to be added in every 100mL distilled water, 1~3mL dehydrated alcohol, 3~5g yeast extract, 1~3g beef extract, the mixture that 0.2~0.3g sodium citrate and citric acid mass ratio are 1:1, 5~7.5g sodium chloride, 2.5~5g sodium acetate, 0.75~1.25g Tween-20, the solution that pH value is 6~7 is fast training liquid culture medium, using the trapezoidal closed culture apparatus High Density Cultivation acetic acid bacteria of micro- waterfall type, it can cultivate to obtain a large amount of thallus.Device therefor of the present invention is at low cost, mechanization degree is high, human input is few, culture effect is good, it can be achieved that totally-enclosed serialization automation culture, disposably exporting bacteria suspension after culture both can be obtained finished product.The liquid circulation loop that the present apparatus is formed, improves the utilization rate and bacteria concentration of raw material, improves utilization rate of equipment and installations to greatest extent.

Description

A method for rapidly cultivating acetic acid bacteria using a micro-waterfall trapezoidal closed culture device

technical field

The invention belongs to the technical field of acetic acid bacteria cultivation, and in particular relates to a method for quickly cultivating acetic acid bacteria using a micro-waterfall trapezoidal closed acetic acid bacteria cultivation device.

Background technique

The main reason for the high-density cultivation of microorganisms is that many microbial products are stored in the cells of the microorganisms. To obtain a large amount of microbial products, it is necessary to carry out centralized and large-scale industrial production of microorganisms. With the deepening of research, the physiological characteristics of many important microorganisms in industrial production have been thoroughly studied, coupled with the continuous development of bioengineering technology, high-density cultivation of microorganisms has been applied in many productions. The so-called high-density culture refers to obtaining a higher cell density, and generally refers to a culture method that obtains a cell density higher than that obtained by conventional culture can be called high-density culture. It belongs to a relative concept, and all treatments that can significantly increase the concentration of bacteria can be called high-density culture. From a microscopic point of view, the consumption of nutrients in the culture medium and the accumulation of microbial growth and metabolic length are the key to the cessation of cell growth during the culture process, and the high-density culture technology of microorganisms advocates a sustainable continuous culture method through cycles. Continuous culture improves the utilization rate of equipment, and maintains a high growth rate of microorganisms by controlling the concentration of nutrients and metabolites, so as to achieve high-speed, high-concentration culture.

The general high-density culture methods of microorganisms mainly include: dialysis culture, that is, the use of membrane separation technology to effectively separate metabolites in the medium and at the same time, nutrients enter the medium through the membrane, so as to achieve nutritional supplementation on the basis of not causing cell loss The culture method for the purpose of metabolites and elimination of metabolites. Fed-batch culture, that is, in the initial culture process, nutrients with a low concentration are added, and new nutrients are gradually added during the culture process to further grow and metabolize the cells until the end of the culture. Among them, the research on fed-batch culture is the most mature and widely used. However, there are relatively few related researches on equipment design and transformation for high-density cultivation of acetic acid bacteria.

Contents of the invention

In order to overcome the above problems, the present invention provides a method for cultivating yeast with low equipment cost, high degree of mechanization, less manpower input, good cultivating effect and fast.

The technical solution taken to solve the above technical problems consists of the following steps:

1. Cultivation of acetic acid bacteria strains

Inoculate the activated Acetobacter pasteurium strains in the acclimatization medium at 30±1°C for 48 hours, wherein the acclimatization medium is to add 2-6g of glucose, 0.5-2g of yeast extract, 0.5 ～2g peptone, 0.3～0.4g Na ₂ HPO ₄ 2H ₂ O, 0.1～0.2g sodium citrate, 1～2g agar, add 1～3mL absolute ethanol and 0～3mL glacial acetic acid after sterilization; Repeat the acclimation culture for 0-4 times according to the above method, and increase the amount of glacial acetic acid added to every 100mL of distilled water in the acclimatization medium by 0-1mL successively; after the acclimatization culture, isolate the dominant acetic acid bacteria and carry out the first-level expansion culture and the second-level expansion culture , to obtain the seed culture solution.

2. Device disinfection

Under airtight conditions, fumigate the micro-waterfall trapezoidal airtight culture device with glacial acetic acid aqueous solution with a mass fraction of 95% for 24 hours, and at the same time use a 55W ultraviolet sterilizing lamp to irradiate for 2 hours, and the mass fraction in each cubic meter of airtight space is 95%. The amount of glacial acetic acid aqueous solution used is 100-200mL.

3. Circular culture of acetic acid bacteria

Under sterile conditions, inoculate the seed culture solution in step 1 with the quick-cultivation liquid medium, and the resulting culture solution flows into the culture tank from the injection port, and flows from the uppermost stepped deflector into the lowermost stepped deflector. The deflector, then flows into the heating tank through the liquid outlet at the bottom of the culture tank, and the heated culture solution flows into the culture tank through the peristaltic pump for cultivation. After culturing for 5 to 9 days, turn off the constant flow pump and heating tank, recover the bacterial suspension in the culture tank, collect the bacteria after centrifugation, and obtain acetic acid bacteria.

The above-mentioned rapid culture liquid medium is to add 6-8g glucose, 1-3mL absolute ethanol, 3-5g yeast extract, 1-3g beef extract, 0.2-0.3g sodium citrate and citric acid mass to every 100mL distilled water A mixture with a ratio of 1:1, 5-7.5g sodium chloride, 2.5-5g sodium acetate, 0.75-1.25g Tween-20, and a pH value of 6-7.

The above-mentioned micro-waterfall trapezoidal airtight culture device is: the top of the culture tank is provided with a liquid injection port, and in the culture tank, at least 4 rectangular grooves with upper surface openings are arranged sequentially from top to bottom, and each rectangular groove is provided with a stepped shape. Deflectors, the stepped deflectors in adjacent rectangular tanks are arranged alternately, the ends of the stepped deflectors are provided with deflector holes, the bottom of the culture tank is provided with a liquid outlet, and the liquid outlet passes through the pipe and the heating tank. The inlet is communicated, and the outlet of the heating tank is communicated with the liquid injection port on the top of the culture tank through a pipeline through a constant flow pump.

In the above-mentioned micro-waterfall trapezoidal airtight culture device, preferably the length of the rectangular tank is 80-120 cm, the width is 10-20 cm, and the height is 6-12 cm; the number of steps of the ladder-shaped deflector is preferably 10-30 , and the height difference between adjacent steps is 1 to 12mm, further preferably the number of steps of the ladder-shaped deflector is 15 to 25, and the height difference between adjacent steps is 3 to 6mm; preferably the guide The diameter of the orifice is 1-3 cm.

In the above step 3, preferably, the inoculum amount of the seed culture solution is 10%-14%.

In the above-mentioned step 3, it is preferable to add 7g glucose, 2mL dehydrated alcohol, 4g yeast extract, 2g beef extract, 0.25g sodium citrate and citric acid mass ratio to every 100mL distilled water of described fast culture liquid medium and be 1: 1, 6.25g sodium chloride, 3.75g sodium acetate, 1g Tween-20, pH 6.8.

In the above step 3, preferably, the circulation flow rate of the culture medium is 530-550 mL/min, and the culture temperature is 30°C.

Compared with prior art, advantage of the present invention is as follows:

1, the present invention adopts Acetobacter pasteurium Acetobacter pasteurium to be applicable to the acetic acid bacterium B-1 bacterial strain of high-density culture, has determined the culture medium and culture condition of B-1 bacterial strain high-density culture; Fermentation 7d under optimal fermentation medium, The DCW of acetic acid bacteria strain B-1 can reach 7.09g/L, and the concentration of bacteria obtained by this scheme is increased by 11.5 times compared with that before optimization.

2. The equipment used in the present invention has low cost, high degree of mechanization, less manpower input and good cultivation effect. The device uses the three-dimensional ladder structure in the ladder-shaped deflector to promote the growth of acetic acid bacteria by making full use of the advantages of the medium and equipment during the cultivation process. During the cultivation and production process, the device can realize fully enclosed continuous automatic cultivation, and after the cultivation is completed, the bacterial suspension can be exported at one time to obtain the finished product. The liquid circulation loop formed by the device improves the utilization rate of raw materials and the concentration of bacteria, and can achieve greater production efficiency in a smaller space, maximizing the utilization rate of equipment.

Description of drawings

Fig. 1 is a schematic structural view of a micro-cascade trapezoidal airtight culture device of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

Example 1

1. Cultivation of acetic acid bacteria strains

Inoculate the activated Acetobacter pasteurium strains in the acclimatization medium at 30±1°C for 48 hours, wherein the activation medium is to add 10g glucose, 1g yeast extract, 2g CaCO ₃ , 1.5 g agar, the activation culture condition is 30°C for 5 days, the acclimatization medium is to add 4g glucose, 1g yeast extract, 1g peptone, 0.34g Na ₂ HPO ₄ 2H ₂ O, 0.15g sodium citrate, 1.5g of agar, made by adding 2mL of absolute ethanol and 0.5mL of glacial acetic acid after sterilization, the acclimatization culture condition is 30°C for 3 days; then repeat the acclimatization culture for 4 times according to the above method, and add ice Acetic acid was increased by 0.5mL in equal amounts step by step; after the acclimatization and cultivation, the dominant acetic acid bacteria were isolated for primary expansion and secondary expansion to obtain seed culture solution.

2. Device disinfection

Under airtight conditions, fumigate the micro-waterfall trapezoidal airtight culture device with glacial acetic acid aqueous solution with a mass fraction of 95% for 24 hours, and at the same time use a 55W ultraviolet sterilizing lamp to irradiate for 2 hours, and the mass fraction in each cubic meter of airtight space is 95%. The amount of glacial acetic acid aqueous solution used is 100-200mL.

As shown in FIG. 1 , the micro-cascade trapezoidal airtight culture device of this embodiment is composed of a constant flow pump 1 , a heating tank 2 , a culture tank 3 , a rectangular tank 4 , and a ladder-shaped deflector 5 . The top of the culture tank 3 is processed with a liquid injection port b, and four rectangular tanks 4 with upper surface openings are stacked from top to bottom in the culture tank 3. The length of the rectangular tank 4 is 100 cm, the width is 15 cm, and the height is 10 cm. Each rectangular groove 4 is processed with a stepped deflector 5, the number of steps of the stepped deflector 5 is 20, and the height difference between adjacent steps is 3mm, the stepped deflector in the adjacent rectangular groove 4 The boards 5 are arranged in a staggered manner. A diversion hole a is processed at the end of the stepped deflector plate 5, and the diameter of the diversion hole a is 1.5 cm. The bottom of the culture tank 3 is processed with a liquid outlet, and the liquid outlet communicates with the inlet of the heating tank 2 through a pipeline, and the outlet of the heating tank 2 communicates with the liquid inlet at the top of the culture tank 4 through a pipeline through a constant flow pump 1.

3. Circular culture of acetic acid bacteria

Under sterile conditions, inoculate the seed culture solution in step 1 with the quick-cultivating liquid medium, and the inoculum size is 12%, and the described quick-cultivating liquid medium is to add 7g glucose, 2mL absolute ethanol, 4g of yeast extract, 2g of beef extract, 0.25g of a mixture of sodium citrate and citric acid with a mass ratio of 1:1, 6.25g of sodium chloride, 3.75g of sodium acetate, 1.0g of Tween-20, and a pH value of 6.8; then The resulting culture solution flows into the culture tank 3 from the liquid injection port b, flows from the uppermost stepped deflector 5 layer by layer into the lowermost stepped deflector 5, and then flows through the liquid outlet at the bottom of the culture tank 3 for heating. In tank 2, the heated culture solution is circulated into the culture tank 3 through the constant flow pump 1 for cultivation, the circulation flow rate of the culture solution is 533mL/min, and the cultivation temperature is 30°C; after 7 days of cultivation, the constant flow pump 1 and the heating tank 2 are turned off. Recover the bacterial suspension in the culture tank 3, and collect the bacterial cells after centrifugation to obtain the acetic acid bacteria suspension with a DCW concentration of 7.72 g/L.

In order to determine the processing conditions of the present invention, the inventor has carried out a large amount of research experiments, and the details are as follows:

a. Test strain

Acetobacter pasteurianus (purchased from China Common Microorganism Culture Collection and Management Center, strain number 1.41); LB acetic acid bacteria, purchased from Shaanxi Baoji Dingli Biotechnology Co., Ltd.; laboratory fermented tomato vinegar, persimmon vinegar, apricot vinegar 13 strains of acetic acid bacteria isolated in jujube vinegar, a total of 15 strains of acetic acid bacteria were used for the test, as shown in Table 1.

Table 1 Source and name of acetic acid bacteria

b. Medium

Acetic acid bacteria activation medium: 10% glucose, 1% yeast extract, 2% CaCO ₃ , 1.5% agar.

Acetic acid bacteria acclimation medium: 4% glucose, 1% yeast extract, 1% peptone, 1.5% agar, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, 2% (V/V) absolute ethanol (Add when the medium is sterilized and cooled to below 60°C). The medium used below has been sterilized, and the diameter of the petri dish used is 12cm.

c. Bacterial activation

After dissolving the freeze-dried powder of Acetobacter pasteurianus and the dry powder of LB acetic acid bacteria with physiological saline, they were inoculated on the activation medium and cultured at 30°C for 5 days.

d. Preparation of seed culture solution

The activated strains were inoculated with 13 strains of acetic acid bacteria isolated from tomato vinegar, persimmon vinegar, apricot vinegar, and jujube vinegar fermented in the laboratory into acclimatization medium for expansion, and cultured at 30°C for 3 days.

e. Surface static culture

Take 1mL of the above-mentioned seed culture solution and put them into 12cm-diameter Petri dishes filled with 70mL quick-cultivation liquid medium, and culture them at 30°C for 7 days.

f. Analytical method

Biomass determination: determined by colorimetry. The OD value of the bacterial suspension was measured at a wavelength of 600 nm using a UV-visible spectrophotometer. A standard curve was drawn with the absorbance value OD as the abscissa and the dry cell weight DCW as the ordinate.

Determination of acetic acid and ethanol content: using gas chromatography. Using n-butanol (chromatographically pure) as a solvent, the acetic acid bacteria fermentation broth was diluted 100 times and then sampled for determination. The gas chromatograph parameters were: GC-2010PLUS AF230V Shimadzu gas chromatograph equipped with FID detector and Labsolutions workstation (Shimadzu, Japan ), chromatographic column: HP-INNOWAX capillary column, chromatographic column ID: 327817H, length 30.0m, inner diameter 0.32mm, film thickness 0.25μm. Reagents: glacial acetic acid (chromatographically pure), absolute ethanol (chromatographically pure), n-butanol (chromatographically pure). Chromatographic conditions: column temperature: equilibrium temperature 60°C, equilibrium time 3min. The initial temperature is 60°C (retain for 1min), and rise to 120°C (retain for 1min) at a rate of 10°C/min. Detector temperature: 210°C. Vaporization temperature: 210°C. Injection mode: split flow. Injection time: 1min. Carrier gas: high-purity nitrogen, pressure 77.4kPa, flow rate 76.7mL/min, split ratio 28:1. Flow program: hydrogen flow: 40mL/min. Air flow: 400mL/min. Makeup: 30mL/min. Injection: 1 μL. Determination method: external standard method.

1. Strain screening and acetic acid acclimatization test

Under the optimal culture conditions, 15 acetic acid bacteria strains were cultured statically on the surface for 3 days, and the cell growth and acetic acid production of each acetic acid bacteria strain were studied. The concentration of glacial acetic acid in the acclimation medium was gradually increased to 2%, and the cell growth of each acetic acid bacteria strain was observed. The results are shown in Table 2.

Table 2 Cell growth of each bacterial strain, comparison of acid production ability and growth under high acidity conditions

Note: "-" indicates that the strain cannot grow under this condition.

In summary, LB-1 acetic acid bacteria in the fermentation broth had the strongest acid-producing ability, but its biomass was low. Compared with LB-1 acetic acid bacteria, Acetobacter pasteurianus strain B-1 is easy to cultivate, and it still maintains high activity when the concentration of glacial acetic acid is 2%, and has strong acid production ability. Therefore, the domesticated Acetobacter pasteurianus strain with glacial acetic acid concentration of 2% was selected as the starting strain for the study.

2. Screening of carbon sources in high-density culture of acetic acid bacteria

2.1 Effect of a single carbon source on the growth and acid production capacity of Acetobacter strain B-1 cells

Take a 150mL Erlenmeyer flask and add 70mL of water, add 1% yeast extract, 1% peptone, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, set the single carbon source as: glucose, lactose, malt extract powder, sucrose , mannose, mannitol, absolute ethanol, glycerin, fructose, maltose, xylose, the concentration is 4% (solid by mass, liquid by volume), after sterilization, the culture medium is transferred to a medium with a diameter of 12cm Petri dish. After the medium was cooled to room temperature, 1 mL of the seed culture solution was taken for inoculation, and cultured at 30°C for 7 days. After the cultivation, the influence of single carbon source on the growth and acid production (acetic acid) ability of Acetobacter strain B-1 cells was determined, and the results are shown in Table 3.

Table 3 Cell growth and acid production capacity of acetic acid bacteria strain B-1 under a single carbon source

Note: "-" indicates that the strain does not produce acid under this condition.

It can be seen from Table 3 that glucose has the most prominent effect on the growth of acetic acid bacteria strain B-1, followed by maltose and fructose. The most significant effect on the acid production of acetic acid bacteria strain B-1 was absolute ethanol. Therefore, glucose and absolute ethanol are selected as the carbon source in the quick culture liquid medium.

2.2 Effect of glucose concentration on cell growth of acetic acid bacteria strain B-1

Take 150mL Erlenmeyer flask and add 70mL water, add 1% yeast extract, 1% peptone, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% glucose were used as the single carbon source, and the same operation as in 2.1 was used to study the effect of glucose concentration on the growth of Acetobacter strain B-1 cells. The results are shown in Table 4.

The influence of table 4 glucose on the growth of acetic acid bacteria strain B-1 cells

It can be seen from Table 4 that the glucose concentration has a significant effect on the growth of Acetobacter strain B-1 cells. When the glucose concentration was 6%, the DCW of Acetobacter strain B-1 reached the highest value of 1.461g/L. Therefore, a glucose concentration of 6% was selected as the center level for subsequent optimization experiments.

2.3 Effect of ethanol concentration on cell growth of acetic acid bacteria strain B-1

Take a 150mL Erlenmeyer flask and add 70mL of water, add 1% yeast extract, 1% peptone, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8% absolute ethanol as a single carbon source, the same as 2.1 test operation, to study the influence of ethanol concentration on the growth of acetic acid bacteria strain B-1 cells , the results are shown in Table 5.

The influence of table 5 ethanol concentration on the cell growth of acetic acid bacteria strain B-1

It can be seen from Table 5 that the change of ethanol concentration will lead to the change of biomass. When the ethanol concentration was 2%, the biomass of acetic acid bacteria strain B-1 reached the highest value of 1.21g/L; when the ethanol concentration continued to increase, the biomass began to decrease. Therefore, the ethanol concentration of 2% was selected as the central level of subsequent optimization experiments.

2.4 Effects of compound carbon sources on cell growth and acid production capacity of acetic acid bacteria strain B-1

Take 150mL Erlenmeyer flask, add 70mL water, add 1% yeast extract, 1% peptone, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add 5% and 1% glucose and absolute ethanol respectively , 5% and 2%, 5% and 3%, 6% and 1%, 6% and 2%, 6% and 3%, 7% and 1%, 7% and 2%, 7% and 3% as composite The carbon source was the same as that of 2.1. The effect of the mass ratio of glucose to ethanol on the growth and acid production capacity of Acetobacter strain B-1 cells was studied. The results are shown in Table 6.

Table 6 Effects of compound carbon sources on cell growth and acid production capacity of acetic acid bacteria strain B-1 (n=3)

Note: Comprehensive index = DCW value + acetic acid content value

It can be seen from Table 6 that glucose and absolute ethanol as composite carbon sources have significant effects on the growth and acid production capacity of Acetobacter strain B-1 cells. Among them, the DCW representing cell growth basically remained between 1.20 and 1.80 g/L, which directly proved that the composite carbon source had a more prominent effect on the cell growth of acetic acid bacteria strain B-1. After experiments and explorations, it was found that when the addition ratios of glucose and absolute ethanol were 7% and 2%, respectively, the biomass of acetic acid bacteria strain B-1 reached a higher value of 1.79g/L, the production of acetic acid was 1.78g/100mL, and the comprehensive index reached The highest value is 3.57. Therefore, the addition ratios of glucose and absolute ethanol were 7% and 2%, respectively, as the composite carbon source for the cultivation of acetic acid bacteria strain B-1.

3. Screening of nitrogen sources in high-density culture of acetic acid bacteria

3.1 Effect of a single nitrogen source on the growth and acid production capacity of Acetobacter strain B-1 cells

Take 150mL Erlenmeyer flask, add 70mL water, add 7% glucose, 2% absolute ethanol, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add: peptone, yeast extract, yeast extract, beef paste, tryptone each 1%, after sterilization, transfer the culture medium to a 12cm petri dish while it is hot. After the medium was cooled to room temperature, 1 mL of the seed culture solution was taken for inoculation, and cultured at 30°C for 7 days. After the cultivation, the effect of a single nitrogen source on the growth and acid production capacity of Acetobacter strain B-1 cells was determined, and the results are shown in Table 7.

Table 7 Cell growth and acid production capacity of acetic acid bacteria strain B-1 under a single carbon source

It can be seen from Table 7 that yeast extract can significantly promote the growth of acetic acid bacteria strain B-1, followed by beef extract and yeast extract. At the same time, no suitable nitrogen source had a significant impact on the acid production of Acetobacter strain B-1. Therefore, yeast extract and beef extract were selected as the nitrogen source in the rapid culture liquid medium.

3.2 Effect of yeast extract concentration on cell growth of acetic acid bacteria strain B-1

Take 150mL Erlenmeyer flask and add 70mL water, add 7% glucose, 2% absolute ethanol, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add 0.5%, 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, and 4% of yeast extract were used as a single nitrogen source, and the same as 2.1 test operation, the influence of yeast extract concentration on the growth of Acetobacter strain B-1 cells was studied, and the results are shown in Table 8.

The influence of table 8 yeast extract concentration on the cell growth of acetic acid bacteria strain B-1

It can be seen from Table 8 that the concentration of yeast extract has a significant effect on the growth of Acetobacter strain B-1 cells. When the concentration of yeast extract was 3.5%, the DCW index of acetic acid bacteria strain B-1 reached the highest value of 3.46g/L; then it tended to be flat, indicating that the yeast extract no longer promoted the growth of bacteria after reaching a certain concentration. Therefore, the yeast extract concentration of 3.5% was selected as the central level of subsequent optimization experiments.

3.3 The effect of beef extract concentration on the growth of acetic acid bacteria strain B-1 cells

Take 150mL Erlenmeyer flask and add 70mL water, add 7% glucose, 2% absolute ethanol, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, and add 0.5%, 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, and 4% beef extract as a single nitrogen source, with the same test operation as 2.1, to study the effect of beef extract concentration on the growth of acetic acid bacteria strain B-1 cells, the results are shown in Table 9.

The influence of table 9 beef extract concentration on the cell growth of acetic acid bacteria strain B-1

It can be seen from Table 9 that the concentration of beef extract has a significant effect on the growth of acetic acid bacteria strain B-1 cells. When the concentration of beef extract was 2%, the DCW of acetic acid bacteria strain B-1 reached a peak value of 2.90g/L; then began to decline. Therefore, the concentration of beef extract was selected as 2% as the central level of subsequent optimization experiments.

3.4 Effects of compound nitrogen sources on cell growth and acid production capacity of acetic acid bacteria strain B-1

Take 150mL Erlenmeyer flask and add 70mL water, add 7% glucose, 2% absolute ethanol, 0.34% Na ₂ HPO ₄ 2H ₂ O, 0.15% sodium citrate, the addition ratio of yeast extract and beef extract is 3% and 3% respectively. 1.5%, 3% and 2%, 3% and 2.5%, 3.5% and 1.5%, 3.5% and 2%, 3.5% and 2.5%, 4% and 1.5%, 4% and 2%, 4% and 2.5% As a composite carbon source, the same operation as in 2.1 was used to study the effect of the mass ratio of yeast extract to beef extract on the growth and acid production capacity of acetic acid bacteria strain B-1 cells. The results are shown in Table 10.

Table 10 Effects of compound nitrogen sources on cell growth and acid production ability of acetic acid bacteria strain B-1 (n=3)

Note: Comprehensive index in the table = DCW value + acetic acid content value.

It can be seen from Table 10 that yeast extract and beef extract as a compound nitrogen source have a significant impact on the growth and acid production capacity of Acetobacter strain B-1 cells. Among them, the DCW representing cell growth basically remained between 3.07 and 5.10 g/L. After experiments and explorations, it was found that when the addition ratio of yeast extract and beef extract was 4% and 2%, the biomass of acetic acid bacteria strain B-1 reached a higher value of 5.10g/L, and the production of acetic acid was 1.67g/100mL, and the comprehensive index reached The highest value is 6.77. Therefore, 4% and 2% of yeast extract and beef extract were selected as the compound nitrogen source for the cultivation of acetic acid bacteria strain B-1.

4. Screening of buffer salts in high-density culture of acetic acid bacteria

4.1 Effect of buffer salt combination on cell growth and acid production ability of acetic acid bacteria strain B-1

Take 150mL Erlenmeyer flask, add 70mL water, add 7% glucose, 2% absolute ethanol, 4% yeast extract, 2% beef extract, and add: K ₂ HPO ₄ , KH ₂ PO ₄ , NaH ₂ PO ₄ , NaH ₂ HPO ₄ , citric acid, and sodium citrate (respectively add two by two according to the mass ratio of 1:1, add the total amount of 0.2%), transfer the culture medium to a 12cm diameter petri dish while it is hot after sterilization middle. After the medium was cooled to room temperature, 1 mL of the seed culture solution was taken for inoculation, and cultured at 30°C for 7 days. After the cultivation, the effect of buffer salt on the growth and acid production ability of Acetobacter strain B-1 cells was determined, and the results are shown in Table 11.

The impact of table 11 buffer salt combination on the growth of acetic acid bacteria strain B-1 cells and the ability to produce acid

It can be seen from Table 11 that the combination of buffer salts has a significant effect on the growth of acetic acid bacteria strain B-1. Among them, sodium citrate and citric acid are the most suitable buffer salt combination for the growth of acetic acid bacteria strain B-1. Therefore, sodium citrate and citric acid are selected as the buffer salt combination in the quick culture liquid medium.

4.2 Effect of buffer salt concentration on cell growth of acetic acid bacteria strain B-1

Take a 150mL conical flask and add 70mL water, add 7% glucose, 2% absolute ethanol, 4% yeast extract, 2% beef extract, and add 0.5%, 1%, 1.5%, 2%, 2.5%, 3% , 3.5%, 4% sodium citrate and citric acid mass ratio of 1:1 as buffer salt, the same as 2.1 test operation, study the impact of buffer salt on the growth of acetic acid bacteria strain B-1 cells, the results are shown in Table 12.

The impact of table 12 buffer salt concentration on the growth of acetic acid bacteria strain B-1 cells

It can be seen from Table 12 that the concentration of sodium citrate and citrate buffer salt has a significant impact on the growth of Acetobacter strain B-1 cells. When the buffer salt concentration was 0.25%, the DCW of the test strain reached the peak value of 5.509g/L; then the biomass began to decline. Therefore, 0.25% was selected as the optimum buffer salt concentration for the cultivation of acetic acid bacteria strain B-1.

5. Screening of inorganic salts and growth factors in high-density culture of acetic acid bacteria

5.1 Inorganic salt and growth factor optimization test

Inorganic salts and growth factors have an extremely critical impact on the synthesis of target products and the growth and metabolism of microorganisms. However, due to too many test factors, traditional single-factor tests are difficult to achieve the desired effect of screening. In this experiment, 17 inorganic salts and growth factors were selected as influencing factors, and the codes and coding levels of each factor are shown in Table 13.

Table 13 Plackett-Burman experimental design level and coding

Taking 17 kinds of inorganic salts and growth factors as the investigation factors, 20 groups of experiments were carried out according to the Plackett-Burman design, and the response values were shown in Table 14 for the results of cell growth and acid production of Acetobacter strain B-1.

Table 14 Plackett-Burman test results

Carry out linear regression to the DCW of the acetic acid bacteria strain B-1 in the above table, through analysis of variance and significance test, it can be seen that the model (see formula 2-1) is extremely significant (P<0.01), and the effect on the cell biomass of the bacterial strain is obtained. The key factors are: NaCl (P<0.01), MgSO ₄ ·7H ₄ O, CaCl ₄ , LiCl and CoQ ₁₀ .

DCW (g/L)＝13.29+0.81X ₁ -0.67X ₄ +0.38X ₆ -1.27X ₇ -0.58X ₁₁ +0.41X ₁₄ -0.49X ₁₇ -0.58X ₁₈

Formula 2-1

Carry out linear regression to the average value of the acetic acid content of the acetic acid bacteria strain B-1 acid production ability in the table above, through analysis of variance and significance test, it can be seen that the model (see formula 2-2) is extremely significant (P<0.01), and obtain The key factors affecting the acid production capacity of the strains were: sodium acetate (P<0.01), Tween-20 (P<0.01), ZnSO ₄ ·7H ₂ O and CaCl ₂ . Acetic acid content = 1.75-0.031X ₁ -0.11X ₃ +0.045X ₄ +0.023X ₁₁ -0.026X ₁₂ -0.063X ₁₅ +0.055X ₁₇ +0.031X ₁₉ +0.062X ₂₀

Formula 2-2

5.2 Single factor test of optimal concentration of inorganic salts and growth factors

According to the NaCl, sodium acetate, and Tween-20 determined by the Plackett-Burman test, 5 levels were set for each, and the growth and acid production capacity of the acetic acid bacteria strain B-1 were used as indicators, and the NaCl concentration was selected as 2.5, 3.75, 5, and 6.25 , 7.5g/L, the concentration of sodium acetate is 2.5, 3.75, 5, 6.25, 7.5g/L, the concentration of Tween-20 is 0.5, 0.75, 1, 1.25, 1.5g/L, and the single factor test is carried out. Same as 2.1 test operation, study the influence of inorganic salt and growth factor concentration on the growth of Acetobacter strain B-1 cells, the results are shown in Tables 15-17.

The influence of table 15 sodium chloride concentration on the cell growth of acetic acid bacteria strain B-1

The influence of table 16 sodium acetate concentration on the growth of acetic acid bacteria strain B-1 cells

The influence of table 17 Tween-20 concentration on the cell growth of acetic acid bacteria strain B-1

It can be seen from Tables 15-17 that the concentrations of sodium chloride, sodium acetate and Tween-20 have a significant effect on the growth of Acetobacter strain B-1 cells. When the concentration of sodium chloride was 6.25g/L, the biomass of acetic acid bacteria strain B-1 reached the highest value of 6.54g/L; when the concentration of sodium chloride continued to increase, the biomass began to decrease. Therefore, select the sodium chloride concentration to be 6.25g/L. When the concentration of sodium acetate was 3.75g/L, the biomass of acetic acid bacteria strain B-1 reached the highest value of 6.70g/L; when the concentration of sodium acetate continued to increase, the biomass began to decrease. Therefore, the concentration of sodium acetate is 3.75g/L. When the concentration of Tween-20 was 1g/L, the biomass of acetic acid bacteria strain B-1 reached the highest value of 6.78g/L; when the concentration of Tween-20 continued to increase, the biomass tended to balance, indicating that the high concentration of Tween -20 can no longer promote the growth of acetic acid bacteria strain B-1. Therefore, the concentration of Tween-20 was selected as 1g/L.

6. Optimization experiment of initial conditions for fermentation of acetic acid bacteria

6.1 Effect of initial pH value on cell growth of acetic acid bacteria strain B-1

The initial pH values of the rapid culture liquid medium were adjusted to 6.4, 6.8, 7.2, 7.6, and 8.0, respectively, and the effects of the initial pH values on the growth of Acetobacter strain B-1 cells were studied. The results are shown in Table 18.

The influence of table 18 initial pH value on the cell growth of acetic acid bacteria strain B-1

It can be seen from Table 18 that the initial pH value of the culture medium has a significant effect on the growth of the cells. When the initial pH value is weakly acidic, it is beneficial to the cell growth of the acetic acid bacteria strain B-1, so the initial pH value of the culture medium is 6.8.

6.2 Effect of initial inoculum volume on cell growth of acetic acid bacteria strain B-1

The quick culture liquid medium was adjusted to an initial pH of 6.8, and cultured at 30°C for 7 days after inoculation according to different inoculum amounts. The effect of inoculum amount on cell growth was studied. The results are shown in Table 19.

The impact of table 19 initial inoculum size on the growth of acetic acid bacteria strain B-1 cells

It can be seen from Table 19 that with the change of inoculum amount, DCW also showed a parabolic trend. When the inoculum amount was 12%, the DCW peaked. It can be seen that the optimum inoculation amount is 12%.

6.3 Effect of culture temperature on the growth of Acetobacter strain B-1 cells

In order to explore the influence of culture temperature on the DCW of acetic acid bacteria strain B-1, 22, 26, 30, 34, and 38°C were respectively selected for culture operation in this experiment, and the results are shown in Table 20.

The influence of table 20 culture temperature on the growth of acetic acid bacteria strain B-1 cell

It can be seen from Table 20 that with the increase of culture temperature, DCW showed a trend of first increasing and then decreasing, and when the culture temperature was 30°C, DCW reached the highest. Therefore, 30°C was selected as the optimum culture temperature for acetic acid bacteria strain B-1.

7. Factors affecting the growth of acetic acid bacteria in a high-density rapid cultivation device

7.1 Effect of the height difference between adjacent steps in the stepped deflector on the growth of Acetobacter strain B-1 cells

In order to study the effect of the height difference between adjacent steps in the stepped deflector on the growth of acetic acid bacteria strain B-1 cells, this experiment set the interior of the stepped deflector to contain 20 steps, and two adjacent steps The height difference among them was 2mm, 3mm, 4mm, 5mm, 6mm, and the cultivation operation was carried out, and the results are shown in Table 21.

The height difference between adjacent steps in the stepped deflector of table 21 is on the influence of acetic acid bacteria strain B-1 cell growth

It can be seen from Table 21 that the height difference between adjacent steps in the stepped deflector is too low or too high, which is not conducive to the growth of acetic acid bacteria strain B-1 cells. As the height difference between adjacent steps increased, the biomass of acetic acid bacteria strain B-1 first increased and then tended to balance. When the height difference between adjacent steps was 3mm, the biomass reached the highest. Therefore, 3 mm is selected as the most suitable height difference between adjacent steps in the stepped deflector.

7.2 Effect of culture medium circulation speed on cell growth of acetic acid bacteria strain B-1

In order to study the effect of the circulation rate of the culture medium on the growth of the acetic acid bacteria strain B-1 cells, the constant flow pump 5 was selected for the culture operation at a flow rate of 400, 533, 667, 800, and 1200 mL/min. The results are shown in Table 22.

The influence of table 22 medium circulation speed on the growth of acetic acid bacteria strain B-1 cells

From Table 22, it can be known that the circulation rate of the culture solution is too low or too high to be unfavorable for the growth of Acetobacter strain B-1 cells. With the increase of culture medium circulation rate, the biomass of acetic acid bacteria strain B-1 first increased and then decreased. When the culture medium circulation rate was 533mL/min, the biomass reached the highest. Therefore, 533mL/min was selected as the optimum culture solution circulation rate for acetic acid bacteria strain B-1.

Claims (8)

1. a kind of method using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, it is characterised in that it is by following Step composition:

(1) culture of acetic acid bacteria strain

By the strain inoculated after Pasteur's acetobacter Acetobacter pasteurium activation culture 30 ± 1 in domestication culture medium DEG C domestication culture 48 hours, wherein domestication culture medium be into every 100mL distilled water addition 2~6g glucose, 0.5~2g yeast Cream, 0.5~2g peptone, 0.3~0.4g Na₂HPO₄·2H₂O, 0.1~0.2g sodium citrate, 1~2g agar are made, sterilizing 1~3mL dehydrated alcohol and 0~3mL glacial acetic acid are added afterwards；It repeats domestication culture 0~4 time according to the above method again, and tames training Supporting the glacial acetic acid that every 100mL distilled water is added in base, gradually equivalent increases by 0~1mL；Domestication isolates advantage acetic acid after having cultivated Bacterium carries out level-one and expands culture and second level expansion culture, obtains seed culture fluid；

(2) device sterilizes

In confined conditions, the trapezoidal closed culture apparatus of micro- waterfall type is fumigated with the glacial acetic acid aqueous solution that mass fraction is 95% 24 hours, while being irradiated 2 hours using 55W ultraviolet sterilization lamp, the ice vinegar that mass fraction is 95% in every cubic metre of confined space The usage amount of aqueous acid is 100~200mL；

The trapezoidal closed culture apparatus of above-mentioned micro- waterfall type is: liquid injection port (b), culture tank (3) are provided at the top of culture tank (3) It is inside disposed with the rectangular channel (4) of at least four upper surface opening from top to bottom, is provided with stairstepping in each rectangular channel (4) Deflector (5), the stairstepping deflector (5) in adjacent rectangle slot (4) are staggered, the end setting of stairstepping deflector (5) There is deflector hole (a), the bottom of culture tank (3) is provided with liquid outlet, and liquid outlet is connected by the entrance of pipeline and heating tank (2) Logical, the outlet of heating tank (2) is connected by pipeline through constant flow pump (1) and the liquid injection port at the top of culture tank (3)；

(3) acetic acid bacteria Cyclic culture

Aseptically, the seed culture fluid in liquid culture medium inoculation step (1) will be trained fastly, gained culture solution is from liquid injection port (b) it flows into culture tank (3), successively flows into undermost stairstepping deflector (5) from the stairstepping deflector (5) of top layer, The liquid outlet through culture tank (3) bottom flows into heating tank (2) again, and the culture solution after heating recycles through peristaltic pump (1) and flows into culture Culture in slot (3), culture solution rate of circulating flow are 500~800mL/min, and cultivation temperature is 25~35 DEG C；After culture 5~9 days, close Constant flow pump (1), heating tank (2) are closed, thallus is collected after centrifugation to get acetic acid bacteria in the bacteria suspension in recycling culture tank (3)；

Above-mentioned fast training liquid culture medium be into every 100mL distilled water be added 6~8g glucose, 1~3mL dehydrated alcohol, 3~ 5g yeast extract, 1~3g beef extract, 0.2~0.3g sodium citrate and citric acid mass ratio are mixture, the 5~7.5g of 1:1 Sodium chloride, 2.5~5g sodium acetate, 0.75~1.25g Tween-20, pH value are 6~7.

2. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: a length of 80~120cm, the width of the rectangular channel (4) are 10~20cm, a height of 6~12cm.

3. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: the step number of the stairstepping deflector (5) is 10~30, and the difference in height between adjacent steps is 1~12mm.

4. the method according to claim 3 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: the step number of the stairstepping deflector (5) is 15~25, and the difference in height between adjacent steps is 3~6mm.

5. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: the diameter of the deflector hole (a) is 1~3cm.

6. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: in step (3), the inoculum concentration of the seed culture fluid is 10%~14%.

7. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: in step (3), the fast training liquid culture medium is that addition 7g glucose, 2mL are anhydrous into every 100mL distilled water Ethyl alcohol, 4g yeast extract, 2g beef extract, 0.25g sodium citrate and citric acid mass ratio are the mixture of 1:1,6.25g chlorination Sodium, 3.75g sodium acetate, 1g Tween-20, pH value 6.8.

8. the method according to claim 1 using the trapezoidal closed culture apparatus fast culture acetic acid bacteria of micro- waterfall type, Be characterized in that: in step (3), culture solution rate of circulating flow is 530~550mL/min, and cultivation temperature is 30 DEG C.