A method for controlling the growth of bacterial strain concentration

Abstract

The invention discloses a method for controlling the growth of bacterial strain concentration, and belongs to the field of lactic acid fermentation preparation. The method comprises the following steps: preparing a culture medium, adding culture medium raw materials and sterile water; the culture medium comprises: 60-65 g / L of glucose; 15 g / L of lignocellulose hydrolysate; 5-6 g / L of yeast extract; 10-12 g / L of peptone; 1-1.5 g / L of buffer; adjusting the pH value of the culture medium to 5.5-6.0; transferring the Pichia seed liquid to the fermentation tank, and then adding the culture medium to the fermentation tank to maintain the fermentation environment at 30-35 degrees Celsius; when the bacterial strain concentration exceeds the threshold value and the lactic acid production decreases, the fermentation temperature is increased by 3 degrees Celsius to inhibit the bacterial strain concentration.

Description

Technical Field

[0001] The invention relates to the technical field of lactic acid fermentation preparation, and in particular to a method for controlling the growth of bacterial strain concentration. Background Art

[0002] In the process of lactic acid production by Pichia pastoris E1, when its reproduction enters the logarithmic phase, the concentration of the strain increases relatively quickly, the oxygen supply and osmotic pressure parameters are difficult to control, and the strain is prone to rapidly consume carbon sources without producing acid, resulting in a low sugar-acid conversion rate, low yield, and high cost. Although there are genetic engineering technologies in the prior art to overcome the above problems, the technology is costly and time-consuming. When the culture process, fermentation environment, or equipment changes, the conversion efficiency may be reduced again. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention proposes a method for controlling the growth of bacterial strain concentration.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] The first aspect of the present invention relates to a method for controlling the growth of bacterial strain concentration, characterized in that it comprises the following steps:

[0006] Prepare a culture medium by adding culture medium raw materials and sterile water; the culture medium includes:

[0007] Glucose 60-65 g / L;

[0008] Lignocellulose hydrolysate 15 g / L;

[0009] Yeast extract 5-6 g / L;

[0010] Peptone 10-12 g / L;

[0011] Buffer 1~1.5g / L;

[0012] Adjust the pH of the culture medium to 5.5 to 6.0;

[0013] Transferring the Pichia yeast seed solution to the fermentation tank, adding culture medium to the fermentation tank, and maintaining the fermentation environment at 30 to 35 degrees Celsius;

[0014] When the strain concentration exceeded the threshold and lactic acid production decreased, the fermentation temperature was increased by 3 degrees Celsius to suppress the strain concentration.

[0015] Furthermore, the culture medium also includes:

[0016] Curcuminoids 50 μg / L;

[0017] Thioredoxin 50 μg / L;

[0018] Ergothioneine 50μg / L.

[0019] Furthermore, any one or more of fructose, maltose and glycerol are included as carbon sources of the culture medium.

[0020] Furthermore, the fructose, maltose and glycerol are added in batches during the fermentation process.

[0021] Further, when the bacterial concentration exceeds a threshold value and the lactic acid production decreases, ammonium sulfate is added to inhibit the bacterial concentration.

[0022] Furthermore, the method for preparing the lignocellulose hydrolysate comprises the following steps:

[0023] Take corn cobs and heat them to 80 degrees Celsius in a 5% solution for acid pretreatment;

[0024] The pretreated lignocellulose raw material is mixed with water to form a suspension with a solid content of 10-15%;

[0025] Add cellulase and hemicellulase and adjust the pH to 4.8-5.2;

[0026] The enzymatic hydrolysis reaction was carried out at 50°C for 24-72 hours with regular stirring.

[0027] After the enzymatic hydrolysis is completed, the solid and liquid are separated by filtration, and the liquid phase is the lignocellulose hydrolyzate containing fermentable sugars such as glucose and xylose.

[0028] The second aspect of the present invention relates to a Pichia yeast fermentation medium, characterized in that it comprises the following components:

[0029] Glucose 60-65 g / L;

[0030] Lignocellulose hydrolysate 15 g / L;

[0031] Yeast extract 5-6 g / L;

[0032] Peptone 10-12 g / L;

[0033] Buffer solution 1~1.5g / L.

[0034] Beneficial effects of the present invention:

[0035] The invention increases the yield of lactic acid and reduces the production cost by adding additional ingredients and controlling the concentration of bacteria.

[0036] The present invention improves the tolerance and stress capacity of the strains through additional additives, including resistance to temperature rise, osmotic pressure changes and antioxidant capacity. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The raw materials used in the examples and comparative examples of the present invention are all commercially available raw materials.

[0039] Pichia pastoris was purchased from Beyotime Biotechnology Co., Ltd.

[0040] The preparation method of the lignocellulose hydrolysate of each embodiment and comparative example comprises the following steps:

[0041] The corn cobs were placed in a 5% solution, heated to 80 degrees Celsius, and soaked for acid pretreatment.

[0042] The pretreated lignocellulose raw material is prepared into a suspension with a solid content of 10-15% by water.

[0043] Cellulase and hemicellulase were added and the pH was adjusted to 4.8-5.2.

[0044] The enzymatic hydrolysis reaction was carried out at 50°C for 24-72 hours with regular stirring.

[0045] After the enzymatic hydrolysis is completed, the solid and liquid are separated by filtration, and the liquid phase is the lignocellulose hydrolyzate containing fermentable sugars such as glucose and xylose.

[0046] Example 1

[0047] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0048] Glucose 60g / L;

[0049] Lignocellulose hydrolysate 15 g / L;

[0050] Yeast extract 5g / L;

[0051] Peptone 10g / L;

[0052] Buffer 1g / L;

[0053] Fructose 5g / L

[0054] Maltose 5g / L

[0055] Glycerol 5g / L

[0056] Curcuminoids, ergothioneine and thioredoxin were each 50 μg / L.

[0057] Adjust the pH of the culture medium to 5.5;

[0058] Sterile water was added to bring the total volume to 1 L.

[0059] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0060] At 24h and 48h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0061] Example 2

[0062] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0063] Glucose 65g / L;

[0064] Lignocellulose hydrolysate 15 g / L;

[0065] Yeast extract 6g / L;

[0066] Peptone 12 g / L;

[0067] Buffer 1.5 g / L;

[0068] Fructose 4g / L

[0069] Maltose 3g / L

[0070] Glycerol 3g / L

[0071] Curcuminoids, ergothioneine and thioredoxin were each 60 μg / L.

[0072] Adjust the pH of the culture medium to 5.5;

[0073] Sterile water was added to bring the total volume to 1 L.

[0074] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0075] At 24h and 28h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0076] Example 3

[0077] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0078] Glucose 60g / L;

[0079] Lignocellulose hydrolysate 15 g / L;

[0080] Yeast extract 5g / L;

[0081] Peptone 10 g / L;

[0082] Buffer 1g / L;

[0083] Fructose 5g / L

[0084] Maltose 5g / L

[0085] Glycerol 5g / L

[0086] Curcuminoids, ergothioneine and thioredoxin were each 50 μg / L.

[0087] Adjust the pH of the culture medium to 5.5;

[0088] Sterile water was added to bring the total volume to 1 L.

[0089] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0090] After 24 hours of fermentation, increase the fermentation temperature to 38 degrees Celsius.

[0091] At 24h and 48h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0092] Example 4

[0093] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0094] Glucose 60g / L;

[0095] Lignocellulose hydrolysate 15 g / L;

[0096] Yeast extract 5g / L;

[0097] Peptone 10g / L;

[0098] Buffer 1g / L;

[0099] Fructose 5g / L

[0100] Maltose 5g / L

[0101] Glycerol 5g / L

[0102] Curcuminoids, ergothioneine and thioredoxin were each 50 μg / L.

[0103] Adjust the pH of the culture medium to 5.5;

[0104] Sterile water was added to bring the total volume to 1 L.

[0105] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0106] After 24 hours of fermentation, 30 g of sodium chloride was added to change the osmotic pressure in the fermentation liquid.

[0107] At 24h and 48h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0108] Example 5

[0109] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0110] Glucose 60g / L;

[0111] Lignocellulose hydrolysate 15 g / L;

[0112] Yeast extract 5g / L;

[0113] Peptone 10g / L;

[0114] Buffer 1g / L;

[0115] Fructose 5g / L

[0116] Maltose 5g / L

[0117] Glycerol 5g / L

[0118] Curcuminoids, ergothioneine and thioredoxin were each 50 μg / L.

[0119] Adjust the pH of the culture medium to 5.5;

[0120] Sterile water was added to bring the total volume to 1 L.

[0121] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0122] At 24h of fermentation, 10ml of hydrogen peroxide solution.

[0123] At 24h and 48h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0124] Example 6

[0125] The same as Example 1, except that 5 g / L of ammonium sulfate was added to the culture medium.

[0126] Comparative Example 1

[0127] Prepare the culture medium by adding culture medium raw materials and 800 ml of sterile water; the culture medium includes:

[0128] Glucose 60g / L;

[0129] Yeast extract 5g / L;

[0130] Peptone 10g / L;

[0131] Buffer 1g / L;

[0132] Fructose 5g / L

[0133] Maltose 5g / L

[0134] Glycerol 5g / L

[0135] Adjust the pH of the culture medium to 5.5;

[0136] Sterile water was added to bring the total volume to 1 L.

[0137] The Pichia yeast seed solution was transferred to the fermentation tank, and then the culture medium was added to the fermentation tank, and the fermentation environment was maintained at 35 degrees Celsius for fermentation. The initial strain concentration was 2×10 8 CFU / mL. Fructose, maltose and glycerol were added in batches during the 24h fermentation process.

[0138] At 24h and 48h of fermentation, the sugar-acid conversion rate was calculated by measuring the change in carbon source concentration and lactic acid concentration, and the strain concentration was measured. The results are shown in Table 1.

[0139] Comparative Example 2

[0140] The steps are the same as those in Example 3, except that curcuminoids, ergothioneine and thioredoxin are not added.

[0141] Comparative Example 3

[0142] The steps are the same as those in Example 4, except that curcuminoids, ergothioneine and thioredoxin are not added.

[0143] Comparative Example 4

[0144] The steps are the same as those in Example 5, except that curcuminoids, ergothioneine and thioredoxin are not added.

[0145] Comparative Example 5

[0146] The steps are the same as those in Example 3, except that thioredoxin is not added.

[0147] Table 1

[0148]

[0149] As can be seen from the data of Table 1, when Comparative Example 1 does not make any intervention, although the strain concentration increases faster, it consumes more carbon sources and has a lower conversion rate. Add lignocellulose hydrolysate, it can be seen that the strain concentration still keeps increasing, but the sugar acid conversion rate is appropriately increased. Add curcuminoids, so that antioxidant capacity is improved, but high temperature stress ability is not improved. Add thioredoxin and ergothioneine, antioxidant capacity is improved, high temperature stress is improved, and the ability of strain to produce by-products is specifically suppressed at high temperature, and the ability to produce lactic acid is retained. Thioredoxin is by regulating the intracellular redox state, and maintains the functional structure of Pichia pastoris specific protein to a certain extent, reduces its inactivation at high temperature, and also improves the ability to resist osmotic stress.

[0150] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0151] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for controlling the growth of bacterial strain concentration, characterized in that: The following steps are involved: Prepare a culture medium by adding culture medium raw materials and sterile water; the culture medium includes: Glucose 60-65 g / L; Lignocellulose hydrolysate 15 g / L; Yeast extract 5-6 g / L; Peptone 10-12 g / L; Buffer 1~1.5g / L; Adjust the pH of the culture medium to 5.5 to 6.0; Transferring the Pichia yeast seed solution to the fermentation tank, adding culture medium to the fermentation tank, and maintaining the fermentation environment at 30 to 35 degrees Celsius; When the strain concentration exceeded the threshold and lactic acid production decreased, the fermentation temperature was increased by 3 degrees Celsius to suppress the strain concentration.

2. The method for controlling the growth of bacterial strain concentration according to claim 1, characterized in that: The culture medium also includes: Curcuminoids 50 μg / L; Thioredoxin 50 μg / L; Ergothioneine 50μg / L.

3. The method for controlling the growth of bacterial strain concentration according to claim 1, characterized in that: It also includes any one or more of fructose, maltose and glycerol as the carbon source of the culture medium.

4. The method for controlling the growth of bacterial strain concentration according to claim 3, characterized in that: The fructose, maltose and glycerol are added in batches during the fermentation process.

5. The method for controlling the growth of bacterial strain concentration according to claim 1, characterized in that: When the bacterial concentration exceeded the threshold and the lactic acid production decreased, ammonium sulfate was added to inhibit the bacterial concentration.

6. The method for controlling the growth of bacterial strain concentration according to claim 1, characterized in that: The method for preparing the lignocellulose hydrolysate comprises the following steps: Take corn cobs and heat them to 80 degrees Celsius in a 5% solution for acid pretreatment; The pretreated lignocellulose raw material is mixed with water to form a suspension with a solid content of 10-15%; Add cellulase and hemicellulase and adjust the pH to 4.8-5.2; The enzymatic hydrolysis reaction was carried out at 50°C for 24-72 hours with regular stirring. After the enzymatic hydrolysis is completed, the solid and liquid are separated by filtration, and the liquid phase is the lignocellulose hydrolyzate containing fermentable sugars such as glucose and xylose.

7. A Pichia yeast fermentation medium, characterized in that: Includes the following ingredients: Glucose 60-65 g / L; Lignocellulose hydrolysate 15 g / L; Yeast extract 5-6 g / L; Peptone 10-12 g / L; Buffer solution 1~1.5g / L.

8. The Pichia yeast fermentation medium according to claim 7, characterized in that: The following ingredients are also included: Curcuminoids 50 μg / L; Thioredoxin 50 μg / L; Ergothioneine 50μg / L.

9. The Pichia yeast fermentation medium according to claim 7, characterized in that: It also includes any one or more of fructose, maltose and glycerol as the carbon source of the culture medium.

10. The Pichia yeast fermentation medium according to claim 7, characterized in that: Also included is ammonium sulfate.

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