Method for improving amplification culture success rate of zero-generation strain of acetic acid bacteria

Abstract

The invention discloses a method for improving the amplification culture success rate of zero-generation strains of acetic acid bacteria, and relates to the technical field of acetic acid fermentation. In view of the problem that 0-generation seeds are sensitive to the environment and the process, the rotating speed and the ventilation quantity of a seed tank in a workshop seed preparation stage are selectively controlled according to the foam forming condition in an expanding culture stage with the acidity being 0.5 g / 100mL to 1.3 g / 100mL, a polydimethylsiloxane emulsion defoaming agent is added, and the foam forming condition of the seed tank is controlled to be 0.5 g / 100mL to 1.3 g / 100mL; the negative influence on seeds caused by the fact that foam covers the surface of liquid to influence stirring and rolling of the culture solution, insufficient dissolved oxygen of fermentation liquor caused by mixing of air and the culture solution, or influence on mixing of strains and the culture solution caused by the fact that the foam takes away from the strains can be reduced, the seed delay is shortened, and the success rate and stability of workshop seed amplification culture are improved. And the propagation time can be shortened.

Description

Technical Field

[0001] The invention relates to the technical field of acetic acid fermentation, and in particular to a method for improving the success rate of expansion culture of zero-generation strains of acetic acid bacteria. Background Art

[0002] In the acetic acid fermentation industry, seed preparation is the first step in the fermentation production process, which is divided into laboratory seed preparation and production workshop seed preparation stages. Seed quality is the cornerstone of fermentation production, which is directly related to fermentation efficiency, finished product output and quality stability. Unlike the 1st generation and above bacterial strain culture and acetic acid fermentation, the 0th generation strain prepared in the laboratory is very sensitive to growth conditions and environment. When it is scaled up to provide the workshop seed tank required for large-scale fermentation process, under similar culture medium conditions, due to the differences in ventilation and stirring between laboratory shake flask culture and workshop equipment fermentation tank, the quality of the 0th generation seed in the workshop is often unstable or even fails to expand the culture.

[0003] Therefore, there is an urgent need to improve the expansion method to significantly increase the success rate of expansion of 0-generation strains prepared in the laboratory, while shortening the expansion time.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a method for improving the success rate of culturing the zero-generation strain of acetic acid bacteria, aiming to significantly improve the success rate of culturing the zero-generation strain prepared in the laboratory and shorten the culturing time.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for improving the success rate of acetic acid bacteria zero-generation strain expansion, comprising:

[0008] Adding a polydimethylsiloxane emulsion defoamer to the substrate, and then inoculating acetic acid bacteria to expand the culture;

[0009] In the expansion stage with an acidity of 0.5g / 100mL-1.3g / 100mL, if any of the situations A-D occurs, add polydimethylsiloxane emulsion defoamer:

[0010] Case A: The foam covering the liquid surface is dense and cannot break within 10 seconds;

[0011] Case B: The foam is light yellow in color;

[0012] Case C: Acidity increase is less than 0.1 g / 100 mL within 2 hours;

[0013] Case D: There are accumulations of acetic acid bacteria on the upper layer of the foam.

[0014] In an optional embodiment, the amount of polydimethylsiloxane emulsion defoamer added to the substrate is 10 ppm-35 ppm.

[0015] In an optional embodiment, during the expansion stage, a defoamer diluent having a mass fraction of 8% to 12% is added in a spraying form according to the foam situation;

[0016] Preferably, the defoaming agent diluent is prepared by mixing a polydimethylsiloxane emulsion defoaming agent and sterile water.

[0017] In an optional embodiment, during the spraying process of the defoaming agent diluent, sterile air is introduced to adjust the spray pressure, and the spray pressure is controlled to be 0.5 kg / cm 2 -1.5kg / cm 2 , the nozzle diameter is 0.4mm-0.8mm.

[0018] In an optional embodiment, during the expansion stage, the amount of polydimethylsiloxane emulsion defoamer added each time is 10ppm-20ppm;

[0019] And / or, the total amount of polydimethylsiloxane emulsion defoamer added is less than or equal to 100 ppm.

[0020] In an optional embodiment, when any one of situations A to C occurs, a polydimethylsiloxane emulsion defoamer is added until a small amount of foam floats evenly on the liquid surface.

[0021] In an alternative embodiment, when situation D occurs, a polydimethylsiloxane emulsion defoamer is added to eliminate the deposits.

[0022] In an optional embodiment, ventilation and stirring are performed during the expansion culture process, the ventilation coefficient is 0.10vvm-0.17vvm, the motor stirring speed is 600r / min-800r / min, the expansion culture temperature is controlled at 25°C-30°C, and when the acidity is greater than or equal to 1.7g / 100mL, the expansion culture is completed and enters the next level acetic acid bacteria culture tank.

[0023] In an alternative embodiment, the substrate comprises fermented fruit wine and demineralized water;

[0024] The alcohol content of the fermented fruit wine is 7.0-8.5% vol, and the soluble solid content is 7.5-9.0°Bx; the alcohol content of the blended substrate is 4.0-5.5% vol, and the soluble solid content is 4.5-5.5°Bx.

[0025] In an optional embodiment, acetic acid bacteria are inoculated in a seed tank for expansion culture, the inoculation amount of acetic acid bacteria is 9%-12%, when the acidity is ≤0.5g / 100mL, the speed is controlled to be 600-650r / min, and the ventilation volume is 0.1vvm-0.15vvm; when the acidity is greater than 0.5g / 100mL, the ventilation volume is 0.15vvm-0.17vvm, and the speed is controlled to be 650-800r / min;

[0026] Preferably, before the acetic acid bacteria are inoculated into the seed tank for expansion culture, the method further comprises: inoculating into the fermentation fruit wine culture medium for shake flask expansion culture;

[0027] The substrate for the fermented fruit wine shake flask expansion culture includes fermented fruit wine and softened water, the alcohol content is 3.0% vol-4.5% vol, and the sugar content is 4.0°Bx-5.0°Bx; when the fermented fruit wine is shaken flask expansion culture, a shaker is used to provide dissolved oxygen, and the shaker speed is 180rpm-250rpm; the temperature of the fermented fruit wine shake flask expansion culture is 25℃-28℃, and the transfer acidity of the fermented fruit wine shake flask expansion culture is 1.7-2.3g / 100mL;

[0028] Preferably, before inoculating acetic acid bacteria into the fermentation wine culture medium, the method further comprises: inoculating the slant acetic acid bacteria into a liquid culture medium shake flask for expansion culture;

[0029] The liquid culture medium includes: 10g / L-35g / L of sugar source, 10g / L-35g / L of yeast extract powder and 20g / L-45g / L of anhydrous ethanol; when the liquid culture medium is shaken in a flask for expansion culture, a shaker is used to provide dissolved oxygen, and the shaker speed is 180rpm-250rpm; the temperature of the liquid culture medium for shaken in a flask for expansion culture is 25℃-28℃; the transfer acidity of the liquid culture medium for shaken in a flask for expansion culture is 1.7-2.3g / 100mL.

[0030] The invention has the following beneficial effects: in view of the problem that the 0th generation seeds are sensitive to the environment and the process, the invention selectively controls the rotation speed and ventilation volume of the seed tank in the seed preparation stage in the workshop according to the foam formation situation during the expansion and cultivation stage when the acidity is 0.5g / 100mL-1.3g / 100mL, and adds a polydimethylsiloxane emulsion defoamer, so as to reduce the negative impact on the seeds caused by the foam covering the liquid surface affecting the stirring and tumbling of the culture solution, the lack of dissolved oxygen in the fermentation liquid caused by the mixing of air and the culture solution, or the foam being carried away from the strain affecting the mixing of the strain and the culture solution, thereby shortening the seed hysteresis, improving the success rate and stability of the workshop seed expansion and cultivation, and shortening the expansion and cultivation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 This is a diagram of the acetic acid bacteria culture process;

[0033] Figure 2 The foam is white and can burst naturally; (a) and (b) represent different viewing angles;

[0034] Figure 3 This is a picture of dense, fine and non-breakable foam; (a) and (b) represent different viewing angles;

[0035] Figure 4 A small amount of foam floats evenly on the liquid surface;

[0036] Figure 5 The diagram shows the state of acetic acid bacteria agglomerating and floating on the upper layer of foam; (a) and (b) represent different viewing angles;

[0037] Figure 6 Complete elimination of acetic acid bacteria accumulation.

[0038] Figure 7 This is a real picture of foam overflowing when no defoamer is added before inoculation;

[0039] Figure 8 This is the acidity growth and acetic acid bacteria growth curve without adding defoamer before inoculation;

[0040] Fig. 9 This is the data chart of 10 batches of expansion culture in which the defoamer stock solution was added before inoculation but no defoamer was added during the process;

[0041] Fig.10 This is the data chart of 10 batches of expansion culture in which the defoamer stock solution was added before inoculation and during the process;

[0042] Fig.11 This is the data chart of 10 batches of expansion culture in which the defoamer stock solution was added before inoculation and the defoamer diluted solution was added during the process;

[0043] Fig.12 This is a schematic diagram of online acetic acid bacteria subculture;

[0044] Fig.13 This is the data diagram of each generation of expansion training for comparative example 3. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0046] Generally speaking, the main factors affecting seed culture in seed tanks include nutritional conditions, culture conditions, bacterial control, the number of seed tanks and inoculation amount, etc. The inventors found that controlling the foam generated during the fermentation process is also a key step to ensure the smooth expansion of seed culture.

[0047] There are many methods for eliminating foam. The composition of the culture medium can be adjusted to reduce the chance of foam formation. Mechanical or chemical defoaming can also be used to eliminate the foam that has been formed, but mechanical defoaming cannot fundamentally eliminate the factors that cause foam stability. The fermentation industry generally adopts mechanical defoaming and chemical defoaming or two kinds of simultaneous use to carry out. When chemical defoaming is used in the fermentation process, its defoaming effect is closely related to the defoamer type, property, molecular weight, etc., and is also closely related to the defoamer concentration, adding method, etc. The above-mentioned research is all for the foam control in the vinegar fermentation process, mainly reducing the material loss in the fermentation. The present invention creatively selects the appropriate time to add the polydimethylsiloxane emulsion defoamer according to the foam situation in the 0th generation seed expansion culture process, which can shorten the seed delay and improve the success rate and stability of the strain expansion culture.

[0048] The embodiment of the present invention provides a method for improving the success rate of acetic acid bacteria zero-generation strain expansion, comprising:

[0049] S1. Before vaccination

[0050] Before inoculating acetic acid bacteria, adding polydimethylsiloxane emulsion defoamer to the substrate can effectively prevent foam from overflowing from the seed tank in the early stage of expansion, maintain the continuous increase in acidity, and improve the success rate of expansion. If polydimethylsiloxane emulsion defoamer is not added before inoculating acetic acid bacteria, foam will easily overflow from the seed tank in the early stage of expansion, and acetic acid bacteria in most of the expansion mash will overflow with the foam, and the acidity will not increase, resulting in expansion failure.

[0051] Specifically, the polydimethylsiloxane emulsion defoamer is a commercially available raw material, and its main components include water, polydimethylsiloxane, polyoxyethylene sorbitan tristearate or sorbitan monostearate, and the like.

[0052] In some embodiments, the amount of polydimethylsiloxane emulsion defoamer added to the substrate is 10ppm-35ppm, such as 10ppm, 15ppm, 20ppm, 25ppm, 30ppm, 35ppm, etc. The amount of polydimethylsiloxane emulsion defoamer added is preferably within the above range, which can effectively prevent the foam from overflowing the seed tank in the early stage of expansion cultivation, and at the same time will not make the amount of defoamer added too large.

[0053] In some embodiments, the substrate includes fermented fruit wine and softened water; the alcohol content of the fermented fruit wine is 7.0-8.5% vol (such as 7.0% vol, 7.5% vol, 8.0% vol, 8.5% vol), and the soluble solid content is 7.5-9.0°Bx (such as 7.5°Bx, 8.0°Bx, 8.5°Bx, 9.0°Bx, etc.); by adjusting the amount of softened water, the alcohol content of the prepared substrate is 4.0-5.5% vol (such as 4.0% vol, 4.5% vol, 5.0% vol, 5.5% vol, etc.), and the soluble solid content is 4.5-5.5°Bx (such as 4.5°Bx, 5.0°Bx, 5.5°Bx, etc.).

[0054] In an optional embodiment, if Figure 1 As shown. Acetic acid bacteria are inoculated in the seed tank for expansion culture. The inoculation amount of acetic acid bacteria is 9%-12%. When the acidity is ≤0.5g / 100mL, the speed is controlled to be 600-650r / min, and the ventilation volume is 0.1vvm-0.15vvm; when the acidity is greater than 0.5g / 100mL, the ventilation volume is 0.15vvm-0.17vvm, the speed is controlled to be 650-800r / min, and low-speed stirring is initially used to prevent the culture solution from generating a large amount of foam. Specifically, the parameter specifications of the seed tank are as follows: 100L, power 2.0-2.2KW, current 6A, speed 600r / min-800r / min, frequency 40-55HZ, voltage 380V.

[0055] In some embodiments, before the acetic acid bacteria are inoculated into the seed tank for expansion culture, the method also includes: inoculating into the fermented fruit wine culture medium for shake flask expansion culture; the substrate for the fermented fruit wine shake flask expansion culture includes fermented fruit wine and softened water, with an alcohol content of 3.0% vol-4.5% vol and a sugar content of 4.0°Bx-5.0°Bx; when the fermented fruit wine is shaken for expansion culture, a shaker is used to provide dissolved oxygen, and the shaker speed is 180rpm-250rpm; the temperature for the fermented fruit wine shake flask expansion culture is 25℃-28℃, and the transfer acidity of the fermented fruit wine shake flask expansion culture is 1.7-2.3g / 100mL.

[0056] In some embodiments, before the acetic acid bacteria are inoculated into the fermentation wine culture medium, the method further includes: inoculating the slant acetic acid bacteria into a liquid culture medium shake flask for expansion culture. The liquid culture medium includes: 10g / L-35g / L of sugar source, 10g / L-35g / L of yeast extract powder, and 20g / L-45g / L of anhydrous ethanol; when the liquid culture medium is shaken in a flask for expansion culture, a shaker is used to provide dissolved oxygen, and the shaker speed is 180rpm-250rpm; the temperature of the liquid culture medium shake flask for expansion culture is 25℃-28℃; the transfer acidity of the liquid culture medium shake flask for expansion culture is 1.7-2.3g / 100mL.

[0057] Specifically, the shaking table culture uses 500mL or 1L conical flasks, with a liquid volume of 10-20%, and a shaking table speed of 180-250r / min. Because it has just been transferred from the slope, due to the small amount, 500mL conical flasks are usually used in the shaking table culture in the liquid culture medium; while the fruit wine culture medium will use 500mL conical flasks and 1L conical flasks to hold the strains cultured in the liquid culture medium respectively. Using two mixed shaking bottles at the same time also helps to improve the success rate of the strains cultured in the fruit wine culture medium in the workshop seed tank.

[0058] Specifically, in the liquid culture medium, the content of the sugar source can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, etc., and the type of the sugar source is not limited, such as glucose; the content of the yeast extract powder can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, etc.; the content of anhydrous ethanol can be 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, etc.

[0059] The specific type of fermented fruit wine is not limited, such as apple wine, hawthorn wine, grape wine, etc. The sugar content of the fermented fruit wine can be 7.5°Bx, 8.0°Bx, 8.5°Bx, 9.0°Bx, etc., and the alcohol content can be 7.0%vol, 7.5%vol, 8.0%vol, 8.5%vol, etc.

[0060] S2, expanding training

[0061] After step S1, the acetic acid bacteria strain is inoculated for expansion culture, and a polydimethylsiloxane emulsion defoamer is added according to the foam situation during the expansion culture process. In the expansion culture stage with an acidity of 0.5g / 100mL-1.3g / 100mL, if any of the situations A-D occurs, a polydimethylsiloxane emulsion defoamer is added:

[0062] Case A: The foam covering the liquid surface is dense and cannot break within 10 seconds; dense means that the foam completely covers the liquid surface, the liquid surface cannot be seen rolling, and more than 80% of the visible area is densely arranged with a diameter of less than 1 cm;

[0063] Case B: The foam is light yellow in color;

[0064] Case C: The acidity increase is less than 0.1 g / 100 mL within 2 hours (acidity increases slowly);

[0065] Case D: There are accumulations of acetic acid bacteria on the upper layer of the foam.

[0066] It should be noted that any of the situations A to D requires the addition of a polydimethylsiloxane emulsion defoamer, otherwise it will affect the smooth progress of the expansion culture process and reduce the success rate of the expansion culture. During the expansion culture process, the focus is on the expansion culture stage when the acidity is 0.5g / 100mL-1.3g / 100mL (i.e., the logarithmic growth period of acetic acid bacteria). After the acidity is greater than 1.3g / 100mL (i.e., the late logarithmic growth period to the early stationary period of acetic acid bacteria), it is generally not necessary to add a defoamer.

[0067] Specifically, Figure 2 As shown, during the expansion culture process, the liquid surface is covered with foam, which is white in color. The top layer of foam gradually becomes larger and can burst naturally within 10 seconds, so there is no need to add a defoaming agent.

[0068] Specifically, during the expansion culture process, the liquid surface is covered with foam. When situations A to C occur, it is necessary to defoam until a small amount of foam floats evenly on the liquid surface. Figure 3 As shown in the figure, it is necessary to defoam until at least a small amount of foam floats evenly on the liquid surface. Figure 4 "A small amount" means that the foam partially or evenly covers the liquid surface, the liquid surface can be seen rolling, and the foam in the visible range can burst naturally within 10 seconds.

[0069] Specifically, Figure 5 As shown in the figure, during the expansion culture process, the liquid surface is covered with foam, and acetic acid bacteria are accumulated on the upper layer of the foam, with a color of light yellow to dark yellow. It is necessary to defoam until all the foam disappears and the liquid surface rolls and is completely exposed, such as Figure 6 shown.

[0070] In some embodiments, during the expansion stage, a defoamer diluent with a mass fraction of 8%-12% is added in the form of spraying according to the foam situation. The defoamer diluent is added in the form of spraying to make the defoamer disperse more evenly and improve the defoaming effect. The defoamer diluent is prepared by mixing a polydimethylsiloxane emulsion defoamer and sterile water. By adjusting the amount of sterile water, the mass fraction of the polydimethylsiloxane emulsion defoamer can be 8%, 9%, 10%, 11%, 12%, etc.

[0071] Furthermore, during the spraying process of the defoaming agent dilution, sterile air was introduced to adjust the spray pressure, and the spray pressure was controlled to be 0.5 kg / cm 2 -1.5kg / cm 2The nozzle diameter is 0.4mm-0.8mm. The defoaming effect can be improved by adjusting the spray pressure and the diameter of the spray droplets.

[0072] Furthermore, in the expansion stage, the amount of polydimethylsiloxane emulsion defoamer added each time is 10ppm-20ppm, such as 10ppm, 12ppm, 14ppm, 16ppm, 18ppm, 20ppm, etc. The total amount of polydimethylsiloxane emulsion defoamer added is less than or equal to 100ppm, and the amount of defoamer is reduced while ensuring the defoaming effect.

[0073] In some embodiments, the inoculation amount of acetic acid bacteria is 9%-12%, and the inoculation amount of acetic acid bacteria is preferably controlled within the above range to improve the success rate of acetic acid bacteria expansion. The type of acetic acid bacteria is not limited, such as A.orleanense, A.schutzenbachii, A.rancens, AS1.41 acetic acid bacteria, and thermostable acetic acid bacteria CGMCC No.8189, but is not limited thereto.

[0074] Furthermore, ventilation and stirring are performed during the expansion process, and the expansion temperature is controlled to be 25°C-30°C. When the acidity is greater than or equal to 1.7g / 100mL, the expansion is completed. Specifically, the expansion temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0075] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0076] It should be noted that the composition of the laboratory expansion stage substrate used in the following examples and comparative examples is as follows: the substrate includes two stages of culture, namely, a liquid culture medium and a fermented fruit wine culture medium, wherein the liquid culture medium includes: 25 g / L of a sugar source (glucose), 25 g / L of yeast extract powder and 35 g / L of anhydrous ethanol; the fermented fruit wine is apple wine, with a sugar content of 8.5°Bx and an alcohol content of 7.8%vol, and the fermented fruit wine culture medium has an alcohol content of 4.3%vol and a sugar content of 4.5%vol. The shaking speed is 220rpm and the culture temperature is 27°C. The transfer acidity is greater than 1.7g / 100mL.

[0077] The composition of the seed tank substrate used in the following examples and comparative examples is as follows: fermented fruit wine and softened water. After preparation, the substrate has an alcohol content of 5.0% vol and a sugar content of 5.4%.

[0078] The acetic acid bacteria used in the following examples and comparative examples are: thermostable acetic acid bacteria CGMCC No. 8189. The inoculation amount is 10%.

[0079] The polydimethylsiloxane emulsion defoamer used in the following examples and comparative examples was purchased from DOW, model: AFE-1520.

[0080] The expansion conditions in the following examples and comparative examples are as follows: the expansion temperature is controlled to be 28° C., and the expansion is completed when the acidity is greater than or equal to 1.7 g / 100 mL.

[0081] Comparative Example 1 (no defoamer added before inoculation)

[0082] After the culture substrate in the seed tank was prepared and sterilized, no polydimethylsiloxane emulsion was added before inoculation. After inoculation of acetic acid bacteria, ventilation at 0.11vvm and stirring by a motor at 650r / min were started.

[0083] The results showed that within half an hour after the ventilation and stirring inoculation were turned on, the foam overflowed the seed tank (see Figure 7 ), most of the acetic acid bacteria in the mash overflowed with foam, the acidity did not increase, and the expansion failed. The acidity increase and acetic acid bacteria growth curve of this comparative example process are shown in the figure below: Figure 8 As shown, it can be seen that the acidity increases slowly.

[0084] It should be added that if defoamers are not added before inoculation, foam will overflow within 30 minutes, and acetic acid bacteria will overflow with it, or even all acetic acid bacteria will overflow, and the expansion culture will inevitably fail. Even if defoamers are added during the process, some acetic acid bacteria will return to the fermentation liquid, but due to the low concentration of bacteria in the fermentation liquid, the expansion time will be greatly extended. Figure 8 As shown, the acidity does not increase for a long time. On the other hand, overflow will also increase the chance of infection with foreign bacteria, affecting the subsequent acetic acid fermentation production.

[0085] Comparative Example 2 Zero-generation acetic acid bacteria (adding defoamer stock solution before inoculation, and no defoamer during the process)

[0086] After the seed tank culture substrate is prepared and sterilized, polydimethylsiloxane emulsion is added to the culture solution at 20ppm before inoculation. After the laboratory zero-generation acetic acid bacteria liquid seeds are inoculated, 0.11vvm ventilation and motor 650r / min stirring are turned on, and no defoaming agent is added later. When the acidity is greater than 0.5g / 100mL, the ventilation is adjusted to 0.15vvm and the motor is adjusted to 700r / min.

[0087] The results showed that during the expansion process, there were more small and dense bubbles covering the liquid surface ( Figure 3 ) or acetic acid bacteria accumulate on the foam surface ( Figure 5 ) situation. The data of 10 batches of expansion training are as follows Fig. 9 As shown, the expansion time is long and the stability is poor (the expansion time of successful batches is 21-50h, with an average of 33.51h), and the expansion success rate is only 60% (the expansion failure is determined when the acidity increase is less than 0.2g / 100mL within 6h).

[0088] Example 1 (adding defoamer before inoculation and adding defoamer stock solution during the process)

[0089] This embodiment provides a method for improving the success rate of acetic acid bacteria zero-generation strain expansion, the steps are as follows:

[0090] After the seed tank culture substrate is prepared and sterilized, polydimethylsiloxane emulsion is added to the culture solution at 20ppm before inoculation. After the laboratory seeds are connected, ventilation at 0.11vvm and stirring at 650r / min are turned on. When the acidity is greater than 0.5g / 100mL, ventilation is adjusted to 0.15vvm and motor is adjusted to 700r / min. During the expansion process (focusing on the acidity of 0.5-1.3g / 100mL), if any of the situations A to D occurs, 10ppm-20ppm polydimethylsiloxane emulsion is added for defoaming to achieve the defoaming effect corresponding to each situation (for example, when any of the situations A to C occurs, polydimethylsiloxane emulsion defoaming agent is added until at least a small amount of foam floats evenly on the liquid surface; when situation D occurs, polydimethylsiloxane emulsion defoaming agent is added to eliminate the deposits). The total amount of polydimethylsiloxane emulsion added during the whole process shall not exceed 100ppm.

[0091] The data of 10 batches of culture expansion in this example are as follows Fig.10 As shown, the expansion time was 14-27h, with an average of 20.5h, and the expansion success rate was 100%.

[0092] Example 2 (adding defoamer stock solution before inoculation and adding defoamer dilution solution during inoculation)

[0093] This embodiment provides a method for improving the success rate of acetic acid bacteria zero-generation strain expansion, the steps are as follows:

[0094] After the seed tank culture substrate is prepared and sterilized, add polydimethylsiloxane emulsion to the culture medium at 20ppm before inoculation. After the laboratory seeds are inoculated, ventilation at 0.11vvm and stirring at 650r / min are turned on. When the acidity is greater than 0.5g / 100mL, adjust to 0.15vvm ventilation and 700r / min motor. Prepare 10% polydimethylsiloxane emulsion solution with sterile pure water (the mass ratio of polydimethylsiloxane emulsion to sterile pure water is 1:9), place it in a spray device sterilized by high-temperature steam at 121℃, and keep it under pressure with sterile air for standby use. During the expansion process (focus on the acidity of 0.5-1.3g / 100mL), if any of the situations A-D occurs, turn on the spray device and evenly spray 10% polydimethylsiloxane emulsion solution to the surface of the culture medium to eliminate the foam on the surface of the liquid. Control the spray pressure to 0.5kg / cm 2 -1.5kg / cm 2The nozzle diameter is 0.6mm, and the amount of polydimethylsiloxane emulsion defoamer added each time is 10ppm-20ppm to achieve the defoaming effect corresponding to each situation. The total amount of polydimethylsiloxane emulsion added during the whole expansion process shall not exceed 100ppm.

[0095] Fig.11 The data are for 10 batches of culture expansion. The culture expansion time of the method provided in this embodiment is 10-18 hours, 13.1 hours on average, and the culture expansion success rate is 100%. The culture expansion efficiency is higher than that of adding polydimethylsiloxane emulsion stock solution in the process.

[0096] Example 3

[0097] The only difference from Example 2 is that 5 ppm of polydimethylsiloxane emulsion was added to the culture medium before inoculation.

[0098] The results showed that within 2 hours of expansion, Figure 3 The fine and dense bubbles in the solution covered the liquid surface. At 4 hours, the acidity was not much different from the initial acidity after inoculation, that is, the acidity growth stagnated.

[0099] Example 4

[0100] The only difference from Example 2 is that polydimethylsiloxane emulsion was added to the culture medium at 10 ppm before inoculation.

[0101] The results showed that within 4 hours of culture, Figure 2 The foam in the wine is white in color. The top layer of foam gradually grows larger and bursts naturally within 10 seconds. The acidity increases normally.

[0102] Example 5

[0103] The only difference from Example 2 is that 35 ppm of polydimethylsiloxane emulsion was added to the culture medium before inoculation.

[0104] The results showed that within 4 hours of culture, Figure 2 The foam in the wine is white in color. The top layer of foam gradually grows larger and bursts naturally within 10 seconds. The acidity increases normally.

[0105] Example 6

[0106] The only difference from Example 2 is that 45 ppm of polydimethylsiloxane emulsion was added to the culture medium before inoculation.

[0107] The results showed that within 4 hours of culture, Figure 2 The foam in the wine is white in color. The top layer of foam gradually grows larger and bursts naturally within 10 seconds. The acidity increases normally.

[0108] Comparative Example 3: Online strains of generation 3 or above (adding defoamer stock solution before inoculation, and no defoamer is added during the process):

[0109] After the expansion in the seed tank of Example 2 is completed, it is inoculated into the primary expansion tank for further expansion and cultivation. Part of the fermentation mash that has completed the expansion in the primary expansion tank (acidity ≥ 1.7 g / 100 mL) is re-inoculated into the seed tank (inoculation amount 9-12%) as the acetic acid bacteria for online propagation. Each time it is inoculated, the number of generations increases by one generation.

[0110] Schematic diagram of online acetic acid bacteria passage Fig.12 shown.

[0111] After the seed tank culture substrate is prepared and sterilized, polydimethylsiloxane emulsion is added before inoculation. After inoculating acetic acid bacteria of generation one or above, ventilation at 0.11vvm and stirring at 650r / min are started, and no defoaming agent is added subsequently. When the acidity is greater than 0.5g / 100mL, ventilation is adjusted to 0.15vvm and stirring at 700r / min.

[0112] The results showed that the expansion process Figure 2 The foam in the middle is white in color, and the top layer of foam gradually grows larger within 10 seconds and can burst naturally or Figure 3 The fine and dense foam in the liquid covers the liquid surface or the foam is light yellow. The online acetic acid bacteria were re-cultured 7 times, and the data of each generation expansion are as follows Fig.13 As shown, the expansion time was short and the stability was good (expansion time was 8-12h, average 10.43h), and the expansion success rate was 100%.

[0113] Compared with the zero-generation laboratory acetic acid bacteria, the online acetic acid bacteria of the first generation and above have better adaptability in the seed tank, and the reproduction of acetic acid bacteria is not greatly affected by foam during the expansion process.

[0114] It should be noted that the inventors have summarized the above comparisons as follows:

[0115] (1) Adding more than 10ppm of defoamer before inoculation can achieve the effect of no foaming and normal growth of acidity, and the effect difference is not large. Considering the preservative components contained in the defoamer, adding a large amount may have an adverse effect on the fermentation performance of acetic acid bacteria, so try to reduce the addition. Seed tanks generally have a small amount of liquid, and the addition amount of 10-35ppm is about 1-3g of stock solution. Because the small amount added is difficult to control, this range is set.

[0116] (2) Add 10-20ppm during the defoaming process. Spray and add at a concentration of 10%. This will not only allow the defoamer to act more evenly on the foam surface and quickly eliminate the foam, but also better control the amount of defoamer used.

[0117] (3) Since the diluent has a stronger dispersibility than the stock solution, it will quickly disperse in the substrate after being added. After the acetic acid bacteria are inoculated, it will have basically no defoaming or foaming effect. However, the stock solution can reduce the dispersion speed after being added. During the period of dissolution and dispersion, the inoculation of acetic acid bacteria can effectively inhibit the generation of foam after inoculation. Therefore, the stock solution should be added before inoculation, not the diluent.

[0118] In general, the purpose before inoculation is to inhibit the generation of foam, and the purpose during inoculation is to eliminate foam.

[0119] In summary, the present invention provides a method for improving the success rate of acetic acid bacteria zero-generation strain expansion, and the main control means are as follows:

[0120] (1) Adding an appropriate amount of defoamer concentrate before inoculation can prevent the laboratory seeds from bubbling and overflowing after inoculation, which will lead to the failure of expansion.

[0121] (2) Foam will inevitably be generated during the expansion process due to ventilation and stirring. In order to prevent the accumulation of foam and covering the liquid surface, resulting in insufficient dissolved oxygen in the culture medium, defoaming agent should be added appropriately according to the amount of foam during the expansion process. However, the operation of manually adding defoaming agent stock solution multiple times will increase the risk of introducing foreign bacteria. A certain concentration of defoaming agent dilution is placed in a sterile defoaming device, and the device is opened according to the foam situation during the expansion process. Adding it with a spray device can greatly reduce the risk of contamination.

[0122] (3) Polydimethylsiloxane emulsion has high defoaming performance. A small amount can quickly reduce the foam surface tension to a sufficiently low level. On the other hand, the polydimethylsiloxane emulsion on the market contains preservative ingredients. Therefore, the polydimethylsiloxane emulsion diluted solution is added during the expansion culture process. The flow rate can be controlled to reduce the amount of defoaming agent added during the expansion culture process, thereby minimizing the inhibitory effect of preservatives brought into the polydimethylsiloxane emulsion on the growth of acetic acid bacteria.

[0123] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for improving the success rate of acetic acid bacteria zero-generation strain expansion, characterized in that: include: Adding a polydimethylsiloxane emulsion defoamer to the substrate, and then inoculating acetic acid bacteria to expand the culture; In the expansion stage with an acidity of 0.5g / 100mL-1.3g / 100mL, if any of the situations A-D occurs, the polydimethylsiloxane emulsion defoamer is added: Case A: The foam covering the liquid surface is dense and cannot break within 10 seconds; Case B: The foam is light yellow in color; Case C: Acidity increase is less than 0.1 g / 100 mL within 2 hours; Case D: There are accumulations of acetic acid bacteria on the upper layer of the foam.

2. The method according to claim 1, characterized in that The amount of the polydimethylsiloxane emulsion defoamer added to the substrate is 10ppm-35ppm.

3. The method according to claim 1 or 2, characterized in that: In the expansion stage, a defoamer diluent having a mass fraction of 8% to 12% is added in a spraying form according to the foam situation; Preferably, the defoaming agent diluent is prepared by mixing the polydimethylsiloxane emulsion defoaming agent and sterile water.

4. The method according to claim 3, characterized in that During the spraying process of the defoamer dilution, sterile air was introduced to adjust the spray pressure to 0.5 kg / cm 2 -1.5kg / cm 2 , the nozzle diameter is 0.4mm-0.8mm.

5. The method according to claim 1, characterized in that In the expansion stage, the amount of the polydimethylsiloxane emulsion defoamer added each time is 10ppm-20ppm; And / or, the total amount of the polydimethylsiloxane emulsion defoamer added is less than or equal to 100 ppm.

6. The method according to claim 1, characterized in that When any one of situations A to C occurs, the polydimethylsiloxane emulsion defoamer is added until a small amount of foam floats evenly on the liquid surface.

7. The method according to claim 1, characterized in that When situation D occurs, the polydimethylsiloxane emulsion defoamer is added to eliminate the deposits.

8. The method according to claim 1, characterized in that During the expansion process, ventilation and stirring are carried out, the ventilation coefficient is 0.10vvm-0.17vvm, the motor stirring speed is 600r / min-800r / min, and the expansion temperature is controlled at 25℃-30℃. When the acidity is greater than or equal to 1.7g / 100mL, the expansion is completed and enters the next level acetic acid bacteria culture tank.

9. The method according to claim 1 or 8, characterized in that: The substrate includes fermented fruit wine and softened water; The alcohol content of the fermented fruit wine is 7.0-8.5% vol, and the soluble solid content is 7.5-9.0° Bx; the alcohol content of the prepared substrate is 4.0-5.5% vol, and the soluble solid content is 4.5-5.5° Bx.

10. The method according to claim 1, characterized in that Inoculate acetic acid bacteria in the seed tank for expansion culture, the inoculation amount of acetic acid bacteria is 9%-12%, when the acidity is ≤0.5g / 100mL, control the speed to 600-650r / min, and the ventilation volume to 0.10vvm-0.15vvm; when the acidity is greater than 0.5g / 100mL, the ventilation volume is 0.15vvm-0.17vvm, and the speed is controlled to 650-800r / min; Preferably, before the acetic acid bacteria are inoculated into the seed tank for expansion culture, the method further comprises: inoculating into the fermentation fruit wine culture medium for shake flask expansion culture; The substrate for the fermented fruit wine shake flask expansion culture includes fermented fruit wine and softened water, the alcohol content is 3.0% vol-4.5% vol, and the sugar content is 4.0° Bx-5.0° Bx; when the fermented fruit wine is shaken in the bottle for expansion culture, a shaker is used to provide dissolved oxygen, and the rotation speed of the shaker is 180 rpm-250 rpm; the temperature for the fermented fruit wine shake flask expansion culture is 25° C.-28° C., and the transfer acidity of the fermented fruit wine shake flask expansion culture is 1.7-2.3 g / 100 mL; Preferably, before inoculating acetic acid bacteria into the fermentation wine culture medium, the method further comprises: inoculating the slant acetic acid bacteria into a liquid culture medium shake flask for expansion culture; The liquid culture medium comprises: 10 g / L-35 g / L of sugar source, 10 g / L-35 g / L of yeast extract powder and 20 g / L-45 g / L of anhydrous ethanol; when the liquid culture medium is cultured in a shake flask, a shaker is used to provide dissolved oxygen, and the shaker speed is 180 rpm-250 rpm; the temperature of the liquid culture medium for cultured in a shake flask is 25° C.-28° C.; and the transfer acidity of the liquid culture medium for cultured in a shake flask is 1.7-2.3 g / 100 mL.

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