CN114214217A - Kluyveromyces marxianus high-density fermentation medium and use method thereof - Google Patents
Kluyveromyces marxianus high-density fermentation medium and use method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly provides a safe and efficient kluyveromyces marxianus high-density fermentation medium and a using method thereof, optimal fermentation conditions are obtained by optimizing a fermentation process, the method provided by the invention can be used for remarkably increasing the number of live bacteria in a fermentation tank of kluyveromyces marxianus by adjusting the material supplementing speed, and the thallus concentration of the final fermentation liquor in the tank can reach 1.2 multiplied by 1010About cfu/mL, solves the problem that the number of live bacteria of yeast in the fermentation liquid of the tank-placed fermentation liquid is generally low in the fermentation process of the Kluyveromyces marxianus at present, and realizes high-quality industrial production conversion.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a kluyveromyces marxianus high-density fermentation medium and a using method thereof.
Background
Kluyveromyces marxianus belongs to Kluyveromyces of Saccharomyces, and is widely present in natural environment such as fermented mare milk, fruit, etc. Kluyveromyces marxianus is food safety grade yeast, the safety in food and medical industry is confirmed by the Ministry of health, food safety supervision of European Union and the United states food and drug administration, and the Kluyveromyces marxianus can be eaten by human beings. As a novel yeast, kluyveromyces marxianus also has application potential in feed. In Europe, the food safety agency has listed Kluyveromyces marxianus in the range of safe biological preparations for food and feed addition, indicating the safety and important value of Kluyveromyces marxianus, and in China, researchers have recognized the commercial value of Kluyveromyces marxianus
As a living bacteria preparation product, the number of the living bacteria of the Kluyveromyces marxianus fermentation liquor directly has great influence on the number of the living bacteria of a final product. The Chinese invention patent (application No. CN201910827684.5, patent name: a propagation culture medium of Kluyveromyces marxianus and preparation method thereof) provides a fermentation culture medium for rapid proliferation of Kluyveromyces marxianus, which can make the strain concentration of Kluyveromyces marxianus in a tank reach 6.8 × 108About cfu/mL. However, for commercial production, the number of the fermentation viable bacteria of Kluyveromyces marxianus is still to be increased, so that the number of the viable bacteria of the final viable bacteria preparation product is ensured to be higher, and the market competitiveness of the product is improved.
Disclosure of Invention
The invention aims to solve the problem that the concentration of Kluyveromyces marxianus fermentation thallus in the prior art is generally low.
Therefore, the invention provides a Kluyveromyces marxianus high-density fermentation medium, which comprises: 0.5-2% of anhydrous glucose, 0.5-2% of sucrose, 0.5-2% of glycerol, 0.01-0.1% of fructo-oligosaccharide, 0.01-0.1% of galacto-oligosaccharide, 0.01-0.1% of resistant dextrin, 0.01-0.1% of inulin, 0.5-1.5% of (NH)4)2SO40.1-1% of MgSO (MgSO)4·7H2O, 0.5-2% KH2PO40.05-0.2% of CaSO4·2H2O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution and 0.01-0.1% of natural killer.
Specifically, the 500x trace element liquid storage bagComprises 1-10g/L KCl, 5-10g/L EDTA, and 1-5g/L ZnSO4·7H2O, 0.1-1g/L MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid.
The invention also provides a Kluyveromyces marxianus high-density fermentation method, which comprises the following steps:
(1) activating strains: inoculating kluyveromyces marxianus into a solid culture medium for activation to obtain a purified strain, and inoculating the purified strain into the solid culture medium for constant-temperature culture for later use;
(2) preparing a seed solution: inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture to obtain a first-stage seed liquid; inoculating the first-stage seed liquid into a seed fermentation tank for fermentation culture to obtain a second-stage seed liquid;
(3) culturing in a fermentation tank: preparing the Kluyveromyces marxianus high-density fermentation medium, placing the Kluyveromyces marxianus high-density fermentation medium in a fermentation tank, and inoculating the secondary seed solution into the fermentation tank for fermentation culture; feeding materials according to the rebound of dissolved oxygen without reduction, and controlling the feeding speed according to the fermentation time and the dissolved oxygen until the fermentation is finished.
Specifically, in the step (1), the activation temperature is 28-32 ℃, the activation time is 48-60h, the constant-temperature culture temperature is 28-32 ℃, and the culture time is 35-48 h; the fermentation culture temperature of the first-stage seed liquid in the step (2) is 28-32 ℃, the rotating speed of a shaking table is 150-.
Specifically, in the step (2), the primary seed liquid is inoculated into a seed fermentation tank for fermentation by the inoculation amount of 2-15%, the fermentation culture temperature of the secondary seed liquid is 28-32 ℃, the rotation speed is 200-400rpm/min, the tank pressure is 0.02-0.05Mpa, the pH is adjusted to be 5.0-5.5 by ammonia water in the whole fermentation process, the dissolved oxygen is controlled to be more than 20% in the fermentation process, and the fermentation is finished when the wet weight of the fermentation liquid is 50-130 g/L.
Specifically, the fermentation culture mediums of the first-stage seed liquid and the second-stage seed liquid in the step (2) are the same and both comprise: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol and 0.2-1% of (NH)4)2SO40.01-0.1% of MgSO4·7H2O, 0.05-0.2% CaSO4·2H2O, 0.1-1% KH2PO40.1-0.5% of 500x microelement stock solution and 0.1-0.5% of 1000x vitamin stock solution, wherein the pH value is 5.0-5.5;
wherein the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA and 1-5g/L ZnSO4·7H2O, 0.1-1g/L MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid.
Specifically, in the step (3), the second-stage seed solution is inoculated into a fermentation tank for fermentation in an inoculation amount of 2-15%.
Specifically, the fermentation culture temperature of the fermentation tank in the step (3) is 28-38 ℃, the rotation speed is 200-650rpm/min, the tank pressure is 0.02-0.05Mpa, the dissolved oxygen is controlled to be more than 40%, the pH is adjusted to be between 5.0-5.5 by ammonia water in the whole fermentation process, and the fermentation is finished until 22-30 h.
Specifically, the feed in the step (3) comprises 650g/L of anhydrous glucose 200-200 g/L, 500g/L of sucrose 100-200 g/L, glycerol 50-200g/L, biotin 2-8mg/L, inositol 10-100mg/L, fructo-oligosaccharide 10-50mg/L, galacto-oligosaccharide 10-50mg/L, resistant dextrin 10-50mg/L, inulin 10-50mg/L, p-aminobenzoic acid 10-100mg/L, choline 10-50mg/L, nicotinic acid 80-150mg/L and calcium pantothenate 80-150 mg/L.
Specifically, the controlling of the feed supplementing speed in the step (3) is as follows: and (3) timing after the feeding is started, controlling dissolved oxygen to be 10-50% within 0-5h, and controlling the dissolved oxygen to be 5-30% after 5h and till the fermentation is finished.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the high-density fermentation and material supplementing formula of the Kluyveromyces marxianus provided by the invention has low cost and safety, and can solve the problem that the concentration of Kluyveromyces marxianus fermentation thallus is generally low; the method provided by the invention can obviously improve the number of live bacteria in the fermentation tank of Kluyveromyces marxianus by adjusting the speed of material supplementing, and the final concentration of the thallus in the fermentation liquor in the tank can reach 1.2 multiplied by 1010About cfu/mL, solves the problem that the number of live bacteria of yeast in the fermentation liquid of the tank-placed fermentation liquid is generally low in the fermentation process of the Kluyveromyces marxianus at present, and realizes high-quality industrial production conversion.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The invention provides a kluyveromyces marxianus high-density fermentation medium, which comprises: 0.5-2% of anhydrous glucose, 0.5-2% of cane sugar and 0.5-2% of glycerol, 0.01-0.1% of fructo-oligosaccharide, 0.01-0.1% of galacto-oligosaccharide, 0.01-0.1% of resistant dextrin, 0.01-0.1% of inulin, 0.5-1.5% of (NH)4)2SO40.1-1% of MgSO (MgSO)4·7H2O, 0.5-2% KH2PO40.05-0.2% of CaSO4·2H2O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution and 0.01-0.1% of natural killer.
Wherein, the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA, and 1-5g/L ZnSO4·7H2O, 0.1-1g/L MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid.
Sterilizing for 20min at 110-115 ℃ after subpackaging and preferably at 110 ℃; the 1000x vitamin stock solution was sterilized by filtration and then added to the sterilized chilled medium.
The invention also provides a Kluyveromyces marxianus high-density fermentation method, which comprises the following steps:
(1) activating strains:
inoculating Kluyveromyces marxianus into a solid culture medium for activation at 28-32 deg.C, preferably 30 deg.C for 48-60 hr to obtain purified strain; inoculating the purified strain into a solid culture medium, and culturing at constant temperature of 28-32 ℃ for 35-48h for later use;
(2) preparing a seed solution:
inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture at the culture temperature of 28-32 ℃, preferably 30 ℃, at the shaking table rotation speed of 150-;
inoculating the primary seed liquid into a seed fermentation tank for fermentation culture with the inoculation amount of 2-15%, preferably 5-8%, the culture temperature is 28-32 ℃, preferably 30 ℃, the rotation speed is 200-400rpm/min, the tank pressure is 0.02-0.05MPa, preferably 0.03MPa, the pH is adjusted to be 5.0-5.5, preferably 5.0 by ammonia water in the whole fermentation process, the dissolved oxygen is controlled to be more than 20% in the fermentation process, the fermentation is carried out for about 10-15h, preferably 12h, and when the wet weight of the fermentation liquid is 50-130g/L, the fermentation is preferably finished by 80g/L, so as to obtain secondary seed liquid;
the fermentation culture medium of the first-stage seed liquid and the second-stage seed liquid is the same and comprises: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol and 0.2-1% of (NH)4)2SO40.01-0.1% of MgSO4·7H2O, 0.05-0.2% CaSO4·2H2O, 0.1-1% KH2PO40.1-0.5% of 500x microelement stock solution and 0.1-0.5% of 1000x vitamin stock solution, wherein the pH value is 5.0-5.5;
wherein the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA and 1-5g/L ZnSO4·7H2O, 0.1-1g/L MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid;
sterilizing the culture medium for 20min at the temperature of 110-115 ℃ after subpackaging, preferably 110 ℃; wherein, the 1000x vitamin stock solution is sterilized by filtration and then is added into a sterilized cooling culture medium;
(3) culturing in a fermentation tank: preparing the Kluyveromyces marxianus high-density fermentation culture medium, placing in a fermentation tank, inoculating the secondary seed liquid into the fermentation tank at an inoculation amount of 2-15%, preferably 8-10%, and performing fermentation culture; culturing at 28-38 deg.C, preferably 32-35 deg.C, at 200-650rpm/min under 0.02-0.05MPa, preferably 0.03MPa, and adjusting pH to 5.0-5.5 with ammonia water during the whole fermentation process; in the initial stage of fermentation, dissolved oxygen is controlled to be more than 40% by alternately adjusting the rotating speed and controlling the ventilation volume; feeding materials according to the condition that the dissolved oxygen rebounds and does not fall; the feeding speed is controlled according to the fermentation time and dissolved oxygen, the dissolved oxygen is controlled to be 10-50%, preferably 15-30% within 0-5h from the timing after feeding is started, and the dissolved oxygen is controlled to be 5-30%, preferably 5-15% after 5h to the end of fermentation; ending fermentation for 22-30h, preferably 26 h;
the feeding comprises the following steps: 200-650g/L of anhydrous glucose, 100-500g/L of sucrose, 50-200g/L of glycerol, 2-8mg/L of biotin, 10-100mg/L of inositol, 10-50mg/L of fructo-oligosaccharide, 10-50mg/L of galacto-oligosaccharide, 10-50mg/L of resistant dextrin, 10-50mg/L of inulin, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 80-150mg/L of nicotinic acid and 80-150mg/L of calcium pantothenate.
The effects of the Kluyveromyces marxianus high-density fermentation medium and the method of using the same of the present invention will be examined by the following specific examples.
The Kluyveromyces marxianus used in the embodiment of the invention is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 10621.
Example 1:
the kluyveromyces marxianus high-density fermentation method adopted in the embodiment comprises the following steps:
(1) activating strains:
inoculating kluyveromyces marxianus into a solid culture medium for activation, wherein the activation temperature is 30 ℃, and the activation time is 60 hours, so as to obtain a purified strain; inoculating the purified strain into YPD culture medium, and culturing at constant temperature of 30 deg.C for 35 hr;
(2) preparing a seed solution:
inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture at the culture temperature of 30 ℃, the rotation speed of a shaking table of 150rpm/min and the culture time of 20h to obtain a first-stage seed liquid;
inoculating the primary seed liquid into a seed fermentation tank by 8 percent of inoculation amount for fermentation culture, wherein the culture temperature is 30 ℃, the rotation speed is 200 plus 400rpm/min, the tank pressure is 0.03-0.05Mpa, the pH is adjusted to 5.0 by ammonia water in the whole fermentation process, the dissolved oxygen is controlled to be more than 20 percent in the fermentation process, the fermentation is carried out for about 12 hours, and when the wet weight of the fermentation liquid is 80g/L, the fermentation is finished to obtain secondary seed liquid, and the seed conversion is started;
the fermentation culture medium of the first-stage seed liquid and the second-stage seed liquid is the same and comprises: 2% of anhydrous glucose, 1% of sucrose, 1% of glycerol, 0.5% of (NH)4)2SO40.05% of MgSO4·7H2O, 0.1% CaSO4·2H2O, 0.3% KH2PO40.2% of 500x microelement stock solution and 0.1% of 1000x vitamin stock solution, wherein the pH value is 5.0;
wherein the 500x microelement stock solution comprises 5g/L KCl, 7.5g/L EDTA and 2.25g/L ZnSO4·7H2O, 0.5g/L MnCl2·7H2O, 0.15g/L CoC12·6H2O, 0.15g/L CuSO4·5H2O, 0.2g/L of Na2MoO4·2H2O, 2.25g/L of CaC12·2H2O, 1.5g/L FeSO4·7H2O, 0.5g/L H3BO30.05g/L KI; the 1000x vitamin stock solution comprises 0.1g/L biotin, 1mg/L nicotinic acid, 25mg/L inositol, 1mg/L thiamine, 1mg/L pyridoxic acid, 25mg/L p-aminobenzoic acid, 15mg/L choline, 5mg/L riboflavin, 0.1g/L calcium pantothenate, 0.1g/L niacin;
subpackaging the culture medium, and sterilizing at 110 deg.C for 20 min; wherein, the 1000x vitamin stock solution is sterilized by filtration and then is added into a sterilized cooling culture medium;
(3) culturing in a fermentation tank: preparing a Kluyveromyces marxianus high-density fermentation culture medium, placing the Kluyveromyces marxianus high-density fermentation culture medium in a fermentation tank, and inoculating the secondary seed liquid into the fermentation tank for fermentation culture in an inoculation amount of 8%; the culture temperature is 33 ℃, the rotation speed is 200-; in the initial stage of fermentation, dissolved oxygen is controlled to be more than 40% by alternately adjusting the rotating speed and controlling the ventilation volume; fermenting for about 7 hours, and feeding materials according to the condition that dissolved oxygen rebounds and does not fall; the feeding speed is controlled according to the fermentation time and dissolved oxygen, the dissolved oxygen is controlled to be 15-30% within 0-5h from the timing after feeding is started, and the dissolved oxygen is controlled to be 5-15% after 5h to the end of fermentation; ending fermentation for 25 h;
the Kluyveromyces marxianus high-density fermentation medium comprises: 1% of anhydrous glucose, 1% of sucrose, 1% of glycerol, 0.05% of fructo-oligosaccharide, 0.05% of galacto-oligosaccharide, 0.05% of resistant dextrin, 0.05% of inulin, 1% of (NH)4)2SO40.5% MgSO4·7H2O, 1% KH2PO40.1% of CaSO4·2H2O, 0.7% of 500x microelement stock solution, 0.2% of 1000x vitamin stock solution and 0.05% of natural enemy; the 500x microelement stock solution comprises 5g/L KCl, 7.5g/L EDTA and 2.25g/L ZnSO4·7H2O, 0.5g/L MnCl2·7H2O, 0.15g/L CoC12·6H2O, 0.15g/L CuSO4·5H2O, 0.2g/L of Na2MoO4·2H2O, 2.25g/L of CaC12·2H2O, 1.5g/L FeSO4·7H2O, 0.5g/L H3BO30.05g/L KI; a1000 Xvitamin stock solution comprises 0.1g/L biotin, 1mg/L nicotinic acid, 25mg/L inositol, 1mg/L thiamine, 1mg/L pyridoxic acid, 25mg/L p-aminobenzoic acid, 15mg/L choline, 5mg/L riboflavin, 0.1g/L calcium pantothenate, 0.1g/L niacin; subpackaging the culture medium, and sterilizing at 110 deg.C for 20 min; wherein, the 1000x vitamin stock solution is sterilized by filtration and then is added into a sterilized cooling culture medium;
the feeding comprises the following steps: 400g/L of anhydrous glucose, 200g/L of sucrose, 50g/L of glycerol, 5mg/L of biotin, 25mg/L of inositol, 20mg/L of fructo-oligosaccharide, 20mg/L of galacto-oligosaccharide, 20mg/L of resistant dextrin, 20mg/L of inulin, 25mg/L of p-aminobenzoic acid, 15mg/L of choline, 100mg/L of nicotinic acid and 100mg/L of calcium pantothenate; supplementing materials, sterilizing at 115 deg.C for 20 min; wherein biotin, inositol, p-aminobenzoic acid, choline, nicotinic acid and calcium pantothenate are sterilized by filtration and then added into sterilized cooling supplementary materials.
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 1:
the comparative example employed the same kluyveromyces marxianus high-density fermentation method as in example 1, except that the kluyveromyces marxianus high-density fermentation medium added to the fermentation tank in step (3) did not contain fructo-oligosaccharide, galacto-oligosaccharide, resistant dextrin, and inulin.
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 2:
this comparative example used the same kluyveromyces kluyveri high-density fermentation method as in example 1, except that the feed formula in step (3) included only: 400g/L of anhydrous glucose, 200g/L of sucrose and 50g/L of glycerol.
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 3:
this comparative example employed the same kluyveromyces kluyveri high-density fermentation method as in example 1, except that the fermentation temperature was always controlled to 28 ℃ in step (3).
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 4:
the comparative example employed the same Kluyveromyces kluyveromyces high density fermentation method as in example 1, except that the dissolved oxygen was controlled to be 15-30% at all times after the start of feeding in step (3).
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 5:
this comparative example employed the same kluyveromyces high density fermentation method as in example 1 except that the dissolved oxygen was controlled to be 5-15% at all times after the start of feeding in step (3).
After the fermentation was completed, the number of viable bacteria of Kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in Table 1.
Comparative example 6:
in this comparative example, the kluyveromyces marxianus propagation medium provided in chinese patent CN201910827684.5 was used for fermentation culture, and after the fermentation was completed, the number of viable bacteria in kluyveromyces marxianus in the fermentation broth was measured, and the results are shown in table 1.
TABLE 1 number of viable Kluyveromyces marxianus in fermentation broth
As is clear from the results of example 1 and comparative examples 1 to 6 in Table 1, in the fermentation of Kluyveromyces marxianus, the concentration of Kluyveromyces marxianus cells can be greatly increased by selecting an appropriate basal medium and supplemented material, adjusting the fermentation culture temperature, and controlling the dissolved oxygen stepwise to precisely adjust the growth rate of yeast cells.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.
Claims (10)
1. A Kluyveromyces marxianus high-density fermentation medium is characterized by comprising: 0.5-2% of anhydrous glucose, 0.5-2% of cane sugar and 0.5-2% of glycerol, 0.01-0.1% of fructo-oligosaccharide, 0.01-0.1% of galacto-oligosaccharide, 0.01-0.1% of resistant dextrin, 0.01-0.1% of inulin, 0.5-1.5% of (NH)4)2SO40.1-1% of MgSO (MgSO)4·7H2O, 0.5-2% KH2PO40.05-0.2% of CaSO4·2H2O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution and 0.01-0.1% of natural killer.
2. The Kluyveromyces marxianus high-density fermentation medium of claim 1, wherein: the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA and 1-5g/L ZnSO4·7H2O, 0.1-1g/L MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid.
3. A Kluyveromyces marxianus high-density fermentation method is characterized by comprising the following steps:
(1) activating strains: inoculating kluyveromyces marxianus into a solid culture medium for activation to obtain a purified strain, and inoculating the purified strain into the solid culture medium for constant-temperature culture for later use;
(2) preparing a seed solution: inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture to obtain a first-stage seed liquid; inoculating the first-stage seed liquid into a seed fermentation tank for fermentation culture to obtain a second-stage seed liquid;
(3) culturing in a fermentation tank: preparing the Kluyveromyces marxianus high-density fermentation culture medium of any one of claims 1 or 2, placing in a fermentation tank, inoculating the secondary seed liquid into the fermentation tank, and performing fermentation culture; feeding materials according to the rebound of dissolved oxygen without reduction, and controlling the feeding speed according to the fermentation time and the dissolved oxygen until the fermentation is finished.
4. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein: in the step (1), the activation temperature is 28-32 ℃, the activation time is 48-60h, the constant-temperature culture temperature is 28-32 ℃, and the culture time is 35-48 h; the fermentation culture temperature of the first-stage seed liquid in the step (2) is 28-32 ℃, the rotating speed of a shaking table is 150-.
5. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein: in the step (2), the primary seed liquid is inoculated into a seed fermentation tank for fermentation by the inoculation amount of 2-15%, the fermentation culture temperature of the secondary seed liquid is 28-32 ℃, the rotation speed is 200-400rpm/min, the tank pressure is 0.02-0.05Mpa, the pH is adjusted to be 5.0-5.5 by ammonia water in the whole fermentation process, the dissolved oxygen is controlled to be more than 20% in the fermentation process, and the fermentation is finished when the wet weight of the fermentation liquid is 50-130 g/L.
6. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein the fermentation medium of the primary seed liquid and the fermentation medium of the secondary seed liquid in step (2) are the same, and both comprise: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol and 0.2-1% of (NH)4)2SO40.01-0.1% of MgSO4·7H2O, 0.05-0.2% CaSO4·2H2O, 0.1-1% KH2PO40.1-0.5% of 500x microelement stock solution and 0.1-0.5% of 1000x vitamin stock solution, wherein the pH value is 5.0-5.5;
wherein the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA and 1-5g/L ZnSO4·7H2O、0.1-1g/L of MnCl2·7H2O, 0.1-0.5g/L CoC12·6H2O, 0.1-0.5g/L CuSO4·5H2O, 0.1-0.5g/L Na2MoO4·2H2O, 1-5g/L of CaC12·2H2O, 1-5g/L FeSO4·7H2O, 0.1-1g/L H3BO30.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L of biotin, 0.5-2mg/L of nicotinic acid, 10-100mg/L of inositol, 0.5-2mg/L of thiamine, 0.5-2mg/L of pyridoxic acid, 10-100mg/L of p-aminobenzoic acid, 10-50mg/L of choline, 1-20mg/L of riboflavin, 0.05-0.2g/L of calcium pantothenate and 0.05-0.2g/L of nicotinic acid.
7. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein: and (3) inoculating the secondary seed liquid into a fermentation tank for fermentation in an inoculation amount of 2-15%.
8. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein: the fermentation culture temperature of the fermentation tank in the step (3) is 28-38 ℃, the rotating speed is 200-650rpm/min, the tank pressure is 0.02-0.05Mpa, the dissolved oxygen is controlled to be more than 40%, the pH is adjusted to be between 5.0-5.5 by ammonia water in the whole fermentation process, and the fermentation is finished for 22-30 h.
9. The Kluyveromyces marxianus high-density fermentation method as claimed in claim 3, wherein the feed in step (3) comprises 650g/L of anhydrous glucose 200-650g/L, 500g/L of sucrose 100-200 g/L, glycerol 50-200g/L, biotin 2-8mg/L, inositol 10-100mg/L, fructo-oligosaccharide 10-50mg/L, galacto-oligosaccharide 10-50mg/L, resistant dextrin 10-50mg/L, inulin 10-50mg/L, p-aminobenzoic acid 10-100mg/L, choline 10-50mg/L, nicotinic acid 80-150mg/L, calcium pantothenate 80-150 mg/L.
10. The Kluyveromyces marxianus high-density fermentation method of claim 3, wherein: the step (3) of controlling the feed supplementing speed specifically comprises the following steps: and (3) timing after the feeding is started, controlling dissolved oxygen to be 10-50% within 0-5h, and controlling the dissolved oxygen to be 5-30% after 5h and till the fermentation is finished.
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| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN113046253A (en) * | 2021-04-14 | 2021-06-29 | 武汉新华扬生物股份有限公司 | Culture method for improving heat resistance of kluyveromyces marxianus |
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| CN110628653A (en) * | 2019-09-03 | 2019-12-31 | 江苏大学 | Proliferation medium of kluyveromyces marxianus and preparation method thereof |
| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN113046253A (en) * | 2021-04-14 | 2021-06-29 | 武汉新华扬生物股份有限公司 | Culture method for improving heat resistance of kluyveromyces marxianus |
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| 王亮;胡曼;王江月;巩小芬;刘朋龙;詹涛;李阳: "马克斯克鲁维酵母高密度发酵条件的优化研究", 食品工业科技, no. 017, pages 111 - 118 * |
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