CN114214217B - Kluyveromyces marxianus high-density fermentation medium and application method thereof - Google Patents
Kluyveromyces marxianus high-density fermentation medium and application method thereof Download PDFInfo
- Publication number
- CN114214217B CN114214217B CN202210006768.4A CN202210006768A CN114214217B CN 114214217 B CN114214217 B CN 114214217B CN 202210006768 A CN202210006768 A CN 202210006768A CN 114214217 B CN114214217 B CN 114214217B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- kluyveromyces marxianus
- seed liquid
- culture
- stock solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 use method thereof, optimal fermentation conditions are obtained by optimizing a fermentation process, the method provided by the invention can obviously improve the viable count of Kluyveromyces marxianus fermentation tank release by adjusting the feeding speed, and the concentration of the fermentation liquid thallus of the tank release can reach 1.2 multiplied by 10 10 About cfu/mL, the problem that the number of viable yeasts of the fermentation liquid placed in the tank is generally low in the existing fermentation process of Kluyveromyces marxianus is solved, and the high-quality industrial production conversion is realized.
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 use method thereof.
Background
Kluyveromyces marxianus belongs to Kluyveromyces of Saccharomyces and is widely used in natural environments such as fermented mare milk, milk and fruits. Kluyveromyces marxianus is food safety-grade yeast, and the safety in the food and medical industry has been confirmed by the Ministry of health, the European Union food safety supervision and the United states food and drug administration, namely, the yeast 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 placed Kluyveromyces marxianus into the safe biological formulation range of food and feed additives, demonstrating the safety and important value of Kluyveromyces marxianus, and in China, researchers have recognized the commercial value of Kluyveromyces marxianus
As a viable bacteria preparation product, the viable bacteria number of the Kluyveromyces marxianus fermentation broth directly has great influence on the viable bacteria number of the final product. Chinese invention patent (application number: CN201910827684.5, patent name: kluyveromyces marxianus)Multiplication medium of bacteria and preparation method thereof) provides a fermentation medium for quick increment of Kluyveromyces marxianus, which can make the concentration of the Kluyveromyces marxianus put in the tank reach 6.8x10 8 cfu/mL. However, for commercial production, the number of viable bacteria of the fermentation of kluyveromyces marxianus needs to be increased, so that the number of viable bacteria of the final viable bacteria preparation product can be 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 the Kluyveromyces marxianus zymophyte is generally low in the prior art.
To this end, the invention provides a Kluyveromyces marxianus high-density fermentation medium comprising: 0.5-2% of anhydrous dextrose, 0.5-2% of sucrose, 0.5-2% of glycerin, 0.01-0.1% of fructo-oligosaccharides, 0.01-0.1% of galacto-oligosaccharides, 0.01-0.1% of resistant dextrin, 0.01-0.1% of inulin, 0.5-1.5% of (NH) 4 ) 2 SO 4 0.1-1% MgSO 4 ·7H 2 O, KH 0.5-2% 2 PO 4 0.05-0.2% CaSO 4 ·2H 2 O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 0.01-0.1% of bufomide.
Specifically, the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA, and 1-5g/L ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 CaC1 of 1-5g/L 2 ·2H 2 O, feSO of 1-5g/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000 Xvitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate, and 0.05-0.2g/L nicotinic acid.
The invention also provides a high-density fermentation method of Kluyveromyces marxianus, 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 seed liquid: inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture to obtain 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 fermentation medium in a fermentation tank, and inoculating secondary seed liquid into the fermentation tank for fermentation culture; feeding is started according to the condition that the rebound of dissolved oxygen does not decrease, and the feeding speed is controlled 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 35-48h; the fermentation culture temperature of the primary seed liquid in the step (2) is 28-32 ℃, the rotation speed of a shaking table is 150-300rpm/min, and the culture time is 18-25h.
Specifically, in the step (2), the primary seed liquid is inoculated into a seed fermentation tank for fermentation at an inoculum size 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 value is regulated to be between 5.0 and 5.5 by ammonia water in the whole fermentation process, dissolved oxygen is controlled to be more than 20% in the fermentation process, and the fermentation is ended when the wet weight of the fermentation liquid is 50-130 g/L.
Specifically, the fermentation media of the primary seed liquid and the secondary seed liquid in the step (2) are the same, and both the fermentation media comprise: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol, 0.2-1% of (NH) 4 ) 2 SO 4 0.01-0.1% MgSO 4 ·7H 2 O, 0.05-0.2% CaSO 4 ·2H 2 O, KH 0.1-1% 2 PO 4 0.1-0.5% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 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 ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 CaC1 of 1-5g/L 2 ·2H 2 O, feSO of 1-5g/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate and 0.05-0.2g/L nicotinic acid.
Specifically, in the step (3), the secondary seed liquid is inoculated into a fermentation tank for fermentation in an inoculum size of 2-15%.
Specifically, 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 value is regulated to be between 5.0 and 5.5 by ammonia water in the whole fermentation process, and the fermentation is finished for 22-30 hours.
Specifically, the feed in the step (3) comprises 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.
Specifically, the feeding speed control in the step (3) specifically includes: and (3) controlling dissolved oxygen to be 10-50% in a period of 0-5h from the beginning of feeding, and controlling dissolved oxygen to be 5-30% after 5h until fermentation is finished.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the formulation of the high-density fermentation and feed supplement of the Kluyveromyces marxianus provided by the invention has low cost and safety,the problem that the concentration of the fermentation thalli of the Kluyveromyces marxianus is generally low can be solved; the invention obtains the optimal fermentation condition by optimizing the fermentation process, the method provided by the invention can obviously improve the viable count of the Kluyveromyces marxianus fermentation tank by adjusting the feeding speed, and the concentration of the thallus in the fermentation liquid of the final tank can reach 1.2 multiplied by 10 10 About cfu/mL, the problem that the number of viable yeasts of the fermentation liquid placed in the tank is generally low in the existing fermentation process of Kluyveromyces marxianus is solved, and the high-quality industrial production conversion is realized.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in the following examples, and it is obvious that the described examples are only some examples of the present invention, but not all examples. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the invention pertains will appreciate that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the scope of the 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 the following components: 0.5-2% of anhydrous glucose, 0.5-2% of sucrose, 0.5-2% of glycerol, 0.01-0.1% of fructo-oligosaccharides, 0.01-0.1% of galacto-oligosaccharides, 0.01-0.1% of resistant dextrins, 0.01-0.1% of inulin, 0.5-1.5% of (NH) 4 ) 2 SO 4 0.1-1% MgSO 4 ·7H 2 O, KH 0.5-2% 2 PO 4 0.05-0.2% CaSO 4 ·2H 2 O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 0.01-0.1% of bufomide.
Wherein the 500 Xmicroelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA, and 1-5g/L ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 O, 1-5g/LCaC1 2 ·2H 2 O, feSO of 1-5g/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000 Xvitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate, and 0.05-0.2g/L niacin.
Packaging at 110-115deg.C, preferably 110deg.C, and sterilizing for 20min; the 1000x stock vitamin solution was sterilized by filtration and then added to the sterilized chilled medium.
The invention also provides a high-density fermentation method of Kluyveromyces marxianus, which comprises the following steps:
(1) Activating strains:
inoculating Kluyveromyces marxianus into a solid culture medium for activation, wherein the activation temperature is 28-32 ℃, preferably 30 ℃, and the activation time is 48-60 hours, so as to obtain a purified strain; inoculating the purified strain into a solid culture medium, and culturing at 28-32deg.C for 35-48 hr for use;
(2) Preparing seed liquid:
inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture, wherein the culture temperature is 28-32 ℃, preferably 30 ℃, the rotation speed of a shaking table is 150-300rpm/min, preferably 180-200rpm/min, and the culture time is 18-25h, preferably 20h, so as to obtain primary seed liquid;
inoculating the primary seed liquid into a seed fermentation tank for fermentation culture at an inoculum size of 2-15%, preferably 5-8%, wherein 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 value is regulated to be between 5.0 and 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 10-15 hours, preferably 12 hours, and when the wet weight of the fermentation liquid is 50-130g/L, the fermentation is finished, preferably 80g/L, so that secondary seed liquid is obtained;
the fermentation culture mediums of the primary seed liquid and the secondary seed liquid are the same, and both the primary seed liquid and the secondary seed liquid comprise: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol, 02-1% (NH) 4 ) 2 SO 4 0.01-0.1% MgSO 4 ·7H 2 O, 0.05-0.2% CaSO 4 ·2H 2 O, KH 0.1-1% 2 PO 4 0.1-0.5% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 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 ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 CaC1 of 1-5g/L 2 ·2H 2 O, feSO of 1-5g/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate and 0.05-0.2g/L nicotinic acid;
sub-packaging the culture medium, sterilizing at 110-115deg.C, preferably 110deg.C for 20min; wherein 1000x vitamin stock solution is sterilized by filtration and then added into sterilized cooling medium;
(3) Culturing in a fermentation tank: preparing the Kluyveromyces marxianus high-density fermentation medium, placing the fermentation medium into a fermentation tank, and inoculating the secondary seed liquid into the fermentation tank for fermentation culture in an inoculum size of 2-15%, preferably 8-10%; the culture temperature is 28-38deg.C, preferably 32-35deg.C, the rotation speed is 200-650rpm/min, the tank pressure is 0.02-0.05Mpa, preferably 0.03Mpa, and pH is adjusted to 5.0-5.5 with ammonia water during the whole fermentation process; at the initial stage of fermentation, controlling dissolved oxygen to be more than 40% by alternately adjusting the rotating speed and controlling ventilation; feeding the raw materials according to the condition that the rebound of dissolved oxygen does not decrease; the feeding speed is controlled according to the fermentation time and the dissolved oxygen, and the dissolved oxygen is controlled to be 10-50%, preferably 15-30% in the period of 0-5h from the timing after the feeding is started, and 5-30%, preferably 5-15% after 5h to the end of fermentation; the fermentation is finished for 22-30 hours, preferably 26 hours;
the material supplementing 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 fructooligosaccharides, 10-50mg/L of galactooligosaccharides, 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 according to the present invention are examined by specific examples as follows.
Kluyveromyces marxianus used in the embodiment of the invention is preserved in China general microbiological culture collection center (CGMCC) No.10621.
Example 1:
the high-density fermentation method of Kluyveromyces marxianus 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 30deg.C for 35 hr for use;
(2) Preparing seed liquid:
inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture, wherein the culture temperature is 30 ℃, the rotation speed of a shaking table is 150rpm/min, and the culture time is 20 hours, so that primary seed liquid is obtained;
inoculating the primary seed liquid into a seed fermentation tank for fermentation culture at the inoculum size of 8%, wherein the culture temperature is 30 ℃, the rotation speed is 200-400rpm/min, the tank pressure is 0.03-0.05Mpa, the pH value is regulated to 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 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 transfer is started;
the fermentation culture mediums of the primary seed liquid and the secondary seed liquid are the same, and both the primary seed liquid and the secondary seed liquid comprise: 2% of anhydrous glucose, 1% of sucrose, 1% of glycerol, 0.5% of (NH) 4 ) 2 SO 4 0.05% MgSO 4 ·7H 2 O, 0.1% CaSO 4 ·2H 2 O, KH 0.3% 2 PO 4 0.2% of 500x microelement stock solution, 0.1% of 1000x vitamin stock solution, and the pH value is 5.0;
wherein the 500x microelement stock solution comprises 5g/L KCl, 7.5g/L EDTA and 2.25g/L ZnSO 4 ·7H 2 O, 0.5g/L MnCl 2 ·7H 2 O, 0.15g/L CoC1 2 ·6H 2 O, 0.15g/L CuSO 4 ·5H 2 O, 0.2g/L Na 2 MoO 4 ·2H 2 O, caC1 of 2.25g/L 2 ·2H 2 O, feSO 1.5g/L 4 ·7H 2 O, 0.5g/L H 3 BO 3 KI 0.05 g/L; the 1000 Xvitamin stock solution comprises 0.1g/L biotin, 1mg/L niacin, 25mg/L inositol, 1mg/L thiamine, 1mg/L pyridoxine, 25mg/L para-aminobenzoic acid, 15mg/L choline, 5mg/L riboflavin, 0.1g/L calcium pantothenate, 0.1g/L niacin;
sterilizing the culture medium at 110deg.C for 20min after subpackaging; wherein 1000x vitamin stock solution is sterilized by filtration and then added into sterilized cooling medium;
(3) Culturing in a fermentation tank: preparing a Kluyveromyces marxianus high-density fermentation medium, placing the fermentation medium in a fermentation tank, and inoculating the secondary seed liquid into the fermentation tank for fermentation culture in an inoculum size of 8%; the culture temperature is 33 ℃, the rotating speed is 200-650rpm/min, the tank pressure is 0.03-0.05Mpa, and the pH value is adjusted to 5.0 by ammonia water in the whole fermentation process; at the initial stage of fermentation, controlling dissolved oxygen to be more than 40% by alternately adjusting the rotating speed and controlling ventilation; fermenting for about 7 hours, and starting feeding according to the condition that the rebound of dissolved oxygen does not decrease; the feeding speed is controlled according to the fermentation time and the dissolved oxygen, the time is counted from the beginning of feeding, the dissolved oxygen is controlled to be 15-30% in the period of 0-5h, and the dissolved oxygen is controlled to be 5% -15% after 5h until the fermentation is finished; fermenting until 25h is finished;
the Kluyveromyces marxianus high-density fermentation medium comprises the following components: 1% of anhydrous glucose, 1% of sucrose, 1% of glycerol, 0.05% of fructo-oligosaccharides, 0.05% of galacto-oligosaccharides, 0.05% of resistant dextrins, 0.05%Inulin, 1% (NH) 4 ) 2 SO 4 0.5% MgSO 4 ·7H 2 O, KH 1% 2 PO 4 0.1% CaSO 4 ·2H 2 O, 0.7% of 500x microelement stock solution, 0.2% of 1000x vitamin stock solution and 0.05% of dichlord; the 500 Xmicroelement stock solution comprises 5g/L KCl, 7.5g/L EDTA, 2.25g/L ZnSO 4 ·7H 2 O, 0.5g/L MnCl 2 ·7H 2 O, 0.15g/L CoC1 2 ·6H 2 O, 0.15g/L CuSO 4 ·5H 2 O, 0.2g/L Na 2 MoO 4 ·2H 2 O, caC1 of 2.25g/L 2 ·2H 2 O, feSO 1.5g/L 4 ·7H 2 O, 0.5g/L H 3 BO 3 KI 0.05 g/L; the 1000 Xvitamin stock solution comprises 0.1g/L biotin, 1mg/L niacin, 25mg/L inositol, 1mg/L thiamine, 1mg/L pyridoxine, 25mg/L para-aminobenzoic acid, 15mg/L choline, 5mg/L riboflavin, 0.1g/L calcium pantothenate, 0.1g/L niacin; sterilizing the culture medium at 110deg.C for 20min after subpackaging; wherein 1000x vitamin stock solution is sterilized by filtration and then added into sterilized cooling medium;
the material supplementing 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 fructooligosaccharides, 20mg/L of galactooligosaccharides, 20mg/L of resistant dextrins, 20mg/L of inulin, 25mg/L of para-aminobenzoic acid, 15mg/L of choline, 100mg/L of nicotinic acid, 100mg/L of calcium pantothenate; sterilizing the feed at 115 ℃ for 20min; wherein, the biotin, inositol, para aminobenzoic acid, choline, nicotinic acid and calcium pantothenate are sterilized by filtration, and then added into the sterilized cooling feed supplement.
After the fermentation, 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 used the same Kluyveromyces marxianus high-density fermentation method as in example 1, except that the Kluyveromyces marxianus high-density fermentation medium added in the fermenter of step (3) did not contain fructo-oligosaccharides, galacto-oligosaccharides, resistant dextrins, inulin.
After the fermentation, 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:
the comparative example used the same Kluyveromyces kesii high density fermentation process as example 1, except that the feed formulation of step (3) only included: 400g/L of anhydrous dextrose, 200g/L of sucrose and 50g/L of glycerol.
After the fermentation, 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:
the comparative example used the same Kluyveromyces kesii high density fermentation process as in example 1, except that the fermentation temperature was always controlled at 28℃in step (3).
After the fermentation, 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 uses the same Kluyveromyces kesii high-density fermentation method as in example 1, except that after the feed supplement is started in step (3), the dissolved oxygen is always controlled at 15-30%.
After the fermentation, 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:
the comparative example uses the same Kluyveromyces kesii high-density fermentation method as in example 1, except that after the feed supplement is started in step (3), the dissolved oxygen is always controlled at 5-15%.
After the fermentation, 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:
the results of the fermentation culture of Kluyveromyces marxianus in the fermentation broth of China patent No. 201910827684.5 are shown in Table 1.
TABLE 1 number of Kluyveromyces marxianus viable bacteria in tank-released fermentation broth
As is clear from the results of example 1 and comparative examples 1 to 6 in Table 1, the concentration of Kluyveromyces marxianus cells during fermentation can be greatly improved by selecting a proper basic medium and a proper feed, and by controlling dissolved oxygen stepwise and precisely adjusting the growth rate of yeast cells at a proper fermentation culture temperature.
The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.
Claims (9)
1. A kluyveromyces marxianus high-density fermentation medium, comprising: 0.5-2% of anhydrous glucose, 0.5-2% of sucrose, 0.5-2% of glycerol, 0.01-0.1% of fructo-oligosaccharides, 0.01-0.1% of galacto-oligosaccharides, 0.01-0.1% of resistant dextrins, 0.01-0.1% of inulin, 0.5-1.5% of (NH) 4 ) 2 SO 4 0.1-1% MgSO 4 ·7H 2 O, KH 0.5-2% 2 PO 4 0.05-0.2% CaSO 4 ·2H 2 O, 0.1-1% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 0.01-0.1% of dichlormid; the 500x microelement stock solution comprises 1-10g/L KCl, 5-10g/L EDTA, and 1-5g/L ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 CaC1 of 1-5g/L 2 ·2H 2 O、1-5gFeSO of/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate and 0.05-0.2g/L nicotinic acid.
2. The high-density fermentation method of Kluyveromyces marxianus is characterized by comprising the following steps of:
(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 seed liquid: inoculating the activated Kluyveromyces marxianus strain into a shake flask seed liquid culture medium for fermentation culture to obtain 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 according to claim 1, placing the fermentation medium in a fermentation tank, and inoculating the secondary seed liquid into the fermentation tank for fermentation culture; feeding is started according to the condition that the rebound of dissolved oxygen does not decrease, and the feeding speed is controlled according to the fermentation time and the dissolved oxygen until the fermentation is finished.
3. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, wherein: the activation temperature in the step (1) is 28-32 ℃, the activation time is 48-60h, the constant temperature culture temperature is 28-32 ℃, and the culture time is 35-48h; the fermentation culture temperature of the primary seed liquid in the step (2) is 28-32 ℃, the rotation speed of a shaking table is 150-300rpm/min, and the culture time is 18-25h.
4. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, wherein: in the step (2), the primary seed liquid is inoculated into a seed fermentation tank for fermentation according to 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 value is regulated 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.
5. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, wherein the fermentation media of the primary seed liquid and the secondary seed liquid in the step (2) are the same, and the method comprises the following steps: 0.5-3% of anhydrous glucose, 0.5-3% of sucrose, 0.5-3% of glycerol, 0.2-1% of (NH) 4 ) 2 SO 4 0.01-0.1% MgSO 4 ·7H 2 O, 0.05-0.2% CaSO 4 ·2H 2 O, KH 0.1-1% 2 PO 4 0.1-0.5% of 500x microelement stock solution, 0.1-0.5% of 1000x vitamin stock solution, and 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 ZnSO 4 ·7H 2 O, 0.1-1g/L MnCl 2 ·7H 2 O, 0.1-0.5g/L CoC1 2 ·6H 2 O, 0.1-0.5g/L CuSO 4 ·5H 2 O, na 0.1-0.5g/L 2 MoO 4 ·2H 2 CaC1 of 1-5g/L 2 ·2H 2 O, feSO of 1-5g/L 4 ·7H 2 O, 0.1-1g/L H 3 BO 3 0.01-0.1g/L KI; the 1000x vitamin stock solution comprises 0.05-0.2g/L biotin, 0.5-2mg/L niacin, 10-100mg/L inositol, 0.5-2mg/L thiamine, 0.5-2mg/L pyridoxine, 10-100mg/L para-aminobenzoic acid, 10-50mg/L choline, 1-20mg/L riboflavin, 0.05-0.2g/L calcium pantothenate and 0.05-0.2g/L nicotinic acid.
6. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, wherein: and (3) inoculating the secondary seed liquid into a fermentation tank for fermentation in an inoculum size of 2-15%.
7. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, 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 value is regulated to be between 5.0 and 5.5 by ammonia water in the whole fermentation process, and the fermentation is finished until 22-30 hours.
8. The method of high-density fermentation of kluyveromyces marxianus according to claim 2, wherein the feed in step (3) comprises 200-650g/L of anhydrous dextrose, 100-500g/L of sucrose, 50-200g/L of glycerol, 2-8mg/L of biotin, 10-100mg/L of inositol, 10-50mg/L of fructooligosaccharides, 10-50mg/L of galactooligosaccharides, 10-50mg/L of resistant dextrins, 10-50mg/L of inulin, 10-100mg/L of para-aminobenzoic acid, 10-50mg/L of choline, 80-150mg/L of niacin, 80-150mg/L of calcium pantothenate.
9. The high-density fermentation method of kluyveromyces marxianus as claimed in claim 2, wherein: the feeding speed control in the step (3) specifically comprises the following steps: and (3) controlling dissolved oxygen to be 10-50% in a period of 0-5h from the beginning of feeding, and controlling dissolved oxygen to be 5-30% after 5h until fermentation is finished.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210006768.4A CN114214217B (en) | 2022-01-05 | 2022-01-05 | Kluyveromyces marxianus high-density fermentation medium and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210006768.4A CN114214217B (en) | 2022-01-05 | 2022-01-05 | Kluyveromyces marxianus high-density fermentation medium and application method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114214217A CN114214217A (en) | 2022-03-22 |
| CN114214217B true CN114214217B (en) | 2023-08-08 |
Family
ID=80707817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210006768.4A Active CN114214217B (en) | 2022-01-05 | 2022-01-05 | Kluyveromyces marxianus high-density fermentation medium and application method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114214217B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118374369B (en) * | 2024-06-26 | 2024-09-06 | 天津科技大学 | Kluyveromyces marxianus and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2022
- 2022-01-05 CN CN202210006768.4A patent/CN114214217B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 马克斯克鲁维酵母高密度发酵条件的优化研究;王亮;胡曼;王江月;巩小芬;刘朋龙;詹涛;李阳;食品工业科技(017);111-118 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114214217A (en) | 2022-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11434467B2 (en) | Aspergillus niger seed continuous culture and method for producing citric acid therefrom | |
| CN109370933B (en) | Co-culture method for improving acid resistance of saccharomycetes | |
| CN114214217B (en) | Kluyveromyces marxianus high-density fermentation medium and application method thereof | |
| CN109971663A (en) | One plant of heat-resisting sub- sieve solution rouge yeast and its application | |
| CN107217020B (en) | Culture medium suitable for lactobacillus acidophilus and preparation method thereof | |
| CN112608861B (en) | Composite preparation containing clostridium butyricum and pediococcus acidilactici as well as preparation method and application of composite preparation | |
| CN112940945B (en) | Method for fermenting hirsutella sinensis | |
| CN111500508A (en) | Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans | |
| CN108713558B (en) | Growth promoting preparation for increasing content of total flavonoids in ginkgo leaves | |
| CN106754524A (en) | Lactobacillus paracasei N1115 culture mediums and its application | |
| CN116649578A (en) | Metagen, preparation method thereof and application thereof in antibacterial products | |
| CN109234215B (en) | Lactobacillus rhamnosus low-salt culture medium and culture method | |
| CN113046253B (en) | Culture method for improving heat resistance of kluyveromyces marxianus | |
| CN107058414B (en) | Method for preparing L-alanine | |
| JP2017514526A (en) | Co-culture of propionic acid bacteria and yeast | |
| CN107022583B (en) | Method for producing L-alanine by feeding fermentation | |
| CN108641961A (en) | A kind of method of High Density Cultivation Guava Leaf endophyte | |
| CN109355221B (en) | Culture medium and method for preparing vibrio cholerae ghost through high-density fermentation | |
| CN103740779B (en) | Method for producing Monacolin K by using fermentation of soybean molasses | |
| CN113005051A (en) | Lactobacillus paracasei and application thereof | |
| Pai | Biotin uptake in biotin regulatory mutant of Escherichia coli | |
| CN110951651A (en) | Selenium-enriched lactobacillus preparation and preparation method thereof | |
| CN111349578A (en) | Preparation method of lactobacillus solid microbial inoculum | |
| CN114292893B (en) | Method for shortening fermentation period and improving acid yield by adding various microelements into VC mixed strain seed tank | |
| CN103614424A (en) | Method of improving fermentation unit of gulconic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |