CN112481327A - Fermentation regulation and control method of water-soluble monascus yellow pigment - Google Patents
Fermentation regulation and control method of water-soluble monascus yellow pigment Download PDFInfo
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Abstract
The invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation regulation and control method of water-soluble monascus yellow pigment, which comprises the following steps: performing test tube slant activation amplification culture on monascus strains, inoculating spore and mycelium eluent into a primary seed culture medium, performing seed amplification culture, and culturing to logarithmic phase to obtain a seed solution; secondly, proportionally inoculating the seed solution obtained in the first step into a secondary seed culture medium, carrying out seed expansion culture, and culturing to logarithmic phase to obtain a secondary seed solution; inoculating the second-stage seed culture solution obtained in the step II into a fermentation culture medium according to the proportion of 5-20% for fermentation regulation and control. Under the effective combined regulation and control of temperature and pH, aerobic fermentation is carried out, and fed-batch feed which is favorable for forming products is added into fermentation liquor to obtain the high-yield water-soluble yellow pigment.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation regulation and control method of water-soluble monascus yellow pigment.
Background
The monascus pigment is one of natural pigments and is obtained by fermenting monascus. Most of pigments obtained by liquid fermentation of monascus are intracellular pigments, and the production process and application of the pigments have safety problems due to extraction of organic solvents. The water-insoluble coloring matter has limitations in use as an edible coloring matter. The water-soluble monascus yellow pigment is an extracellular water-soluble pigment, is obtained by submerged liquid fermentation and extraction of monascus, has the characteristic of being natural and non-toxic, and has wide application prospects in the aspects of deep processing of beverages, foods, grains and the like. At present, microbial fermentation and plant extraction are the main methods for producing natural yellow pigment. The monascus yellow pigment produced by the fermentation method in China is still in the development and research stage so far, the water-soluble monascus yellow pigment sold on the market is prepared by a semi-synthesis method, and the monascus red pigment produced by the fermentation method is prepared by sulfonation reaction and is prepared by non-natural fermentation. The Wu professor team of southern China university has deeper research on the fermented monascus yellow pigment, and the level of extracellular water-soluble yellow pigment can reach about 250 u/ml. The intracellular yellow pigment level of fermentation of a 50L self-control fermentation tank researched by a professor team of Jiangnan university reaches 397 u/ml. Other literature data show that the fermentation level of the extracellular water-soluble yellow pigment is low.
Disclosure of Invention
Aiming at the technical defects, the invention provides a fermentation regulation and control method of water-soluble monascus yellow pigment.
In order to solve the technical problems, the technical scheme of the invention is as follows: a fermentation regulation method of water-soluble monascus yellow pigment comprises the following steps: performing test tube slant activation amplification culture on monascus strains, inoculating spore and mycelium eluent into a primary seed culture medium, performing seed amplification culture, and culturing to logarithmic phase to obtain a seed solution; secondly, proportionally inoculating the seed solution obtained in the first step into a secondary seed culture medium, carrying out seed expansion culture, and culturing to logarithmic phase to obtain a secondary seed solution; inoculating the secondary seed culture solution obtained in the step II into a fermentation culture medium according to the proportion of 5-20% (V/V) for fermentation, wherein the dissolved oxygen is more than or equal to 20%, the fermentation is carried out for 2-4 days, when the wet weight of thalli reaches 25-30g/L, the fermentation temperature is controlled at 31 ℃, and the pH is regulated and controlled at 4.0-4.5 by 20% ammonia water; fermenting for 5-7 days, controlling the fermentation temperature at 33 deg.C, and regulating pH to 3.0-3.5 with 20% (WT) ammonia water; fermenting for 8-10 days, controlling the fermentation temperature at 35 deg.C, regulating pH to 2.5-3.0 with 20% (WT) ammonia water, and controlling reducing sugar at 40-50g/L by feeding sugar-supplementing liquid during fermentation. The fermentation time is 8-10 days, which is the process control point, and the final tank placing period can reach 13 days, namely the aerobic fermentation time is 13 days. After fermenting for 2-4 days, controlling pH and temperature in a proper growth range of the thallus in order to stabilize the thallus growth condition. Then the thalli is shifted from the growth stage to the production stage by a gradient regulation method, and products are secreted. The dissolved oxygen is a relative value, under a certain fermentation control condition, the calibration value of fermentation for 0h is 100%, and the oxygen consumption of microorganisms in the fermentation process is determined relative to the calibration value.
Further: in the method for regulating and controlling the fermentation of the water-soluble monascus yellow pigment, the step three is to supplement a carbon source aqueous solution with 60g/L of sugar solution. The carbon source is at least one of starch, glucose, maltose and glycerol. The carbon source is preferably at least one of glucose and maltose. The Monascus strain is Monascus ruber (Monascus ruber) CGMCC NO.10910 or Monascus ruber (Monascus anka) GIM 3.592. The test tube slant culture medium comprises 5g of corn flour, 10mL of bean concentrate and KH per 100mL of slant culture medium2PO4 0.5g，MgSO40.2g, agar 2g, and drinking water with constant volume of 100mL and natural pH.
The first-class seed culture medium comprises 5g of soluble starch, 2g of corn flour, 10mL of bean concentrate and KH per 100mL of culture medium2PO4 0.5g，MgSO40.2g, 100mL of drinking water with natural pH.
The activation method comprises the following steps: inoculating lyophilized bacteria powder or glycerol-preserved bacteria liquid into test tube slant culture medium, and culturing at 28 deg.C for 7-14 days; the slant test tube seed is eluted with 10ml of sterile water to obtain the eluent, 5nl of the eluent is absorbed and inoculated into the first-class seed culture medium, and the culture is carried out for 24 to 32 hours at 30 ℃ and 100 rpm.
The second-stage seed culture medium of the step II comprises 2g of glucose, 5g of corn flour, 10mL of bean concentrate and NaNO in every 100mL of seed culture medium3 0.5g，KH2PO4 0.5g，MgSO40.2g, the volume of drinking water is 100mL, and the pH is natural; the second-stage seed culture condition is that the first-stage seed liquid of the first-stage seed culture medium is inoculated into the second-stage seed culture medium according to the proportion of 0.01-0.1% (v/v), the temperature is 28-32 ℃, and the temperature is 30 DEGCulturing at 0rpm for 40-60 hr, and aerobic fermenting to obtain seed liquid in logarithmic phase. The composition of the fermentation medium is that every 100mL of the medium contains 5g of carbon source, 0.5g of corn flour, 1mL of bean concentrate, 5g of ammonium salt, 3g of nitrate and KH2PO4 0.3g，MgSO4 0.1g,MnSO4·H2O5.0 mg, 100mL of drinking water with natural pH.
In the invention, natural pH means that pH value does not need special acid-base regulation and control, and the natural meaning is achieved. Aerobic fermentation refers to fermentation that requires oxygen for growth of the bacteria.
Compared with the prior art, the method has the advantages that the fermentation conditions in the fermentation process are effectively regulated, the fermentation pH and temperature are changed in a gradient manner according to the metabolic growth characteristics in the fermentation process of monascus, and the thalli are controlled to metabolize the natural pigment mainly comprising the yellow pigment in the low-pH and high-inorganic-nitrogen environment by adopting an artificial means. Stimulating the thallus to enhance the expression level of the yellow pigment gene and enhance the secretion intensity of the yellow pigment through temperature. The two are effectively combined and regulated, aerobic fermentation and fed-batch are carried out, so that the supplementary material which is favorable for forming a product is added into the fermentation liquor, and the high-yield water-soluble yellow pigment is obtained.
Detailed Description
The present invention is further illustrated by the following specific examples. The following procedures, which are not described in detail, can be performed according to the molecular biology laboratory manual.
Example 1
Preparation of seed liquid: inoculating lyophilized powder of Monascus ruber (Monascus ruber) CGMCC NO.10910, or GIM 3.592 of Monascus ruber (Monascus anka) or glycerol stock solution into test tube slant culture medium (each 100mL slant culture medium contains 5g of semen Maydis powder, 10mL of semen glycines powder, and KH2PO4 0.5g，MgSO40.2g, agar 2g, and drinking water with constant volume of 100mL and natural pH. ) Culturing at 28 deg.C for 7-14 days. Eluting spore and mycelium with 10mL sterile water, inoculating 5nl of first-class seed culture medium (each 100mL culture medium contains soluble starch 5g, corn flour 2g, bean concentrate 10mL, KH)2PO4 0.5g，MgSO40.2g, 100mL of drinking water with natural pH. ) Culturing at 30 deg.C and 100rpm for 24-32 hr. Inoculating 0.01-0.1% (V/V) of first-class seed liquidInoculating to secondary seed culture medium (each 100mL seed culture medium contains glucose 2g, corn flour 5g, soybean concentrate 10mL, NaNO)3 0.5g，KH2PO4 0.5g，MgSO40.2g, 100mL of drinking water with natural pH. ) Culturing at 28-32 deg.C and 300rpm for 40-60h, and aerobically fermenting to obtain seed liquid in logarithmic growth phase.
Fermentation: inoculating the secondary seed culture solution into fermentation medium (each 100mL of the culture medium contains soluble starch 5g, corn flour 0.5g, soybean concentrate 1mL, ammonium salt 5g, nitrate 3g, and KH)2PO4 0.3g，MgSO4 0.1g,MnSO4·H2O5.0 mg, 100mL of drinking water with natural pH. ) The fermentation regulation and control are as follows: 300-600rpm, the ventilation ratio is 0.1-0.3, and the dissolved oxygen is more than or equal to 20 percent. Fermenting for 2-4 days, controlling the fermentation temperature at 31 ℃ and the pH value at 4.0-4.5 by 20% (WT) ammonia water when the wet weight of the thallus reaches 25-30 g/L; fermenting for 5-7 days, controlling the fermentation temperature at 33 deg.C, and regulating pH to 3.0-3.5 with 20% (WT) ammonia water; fermenting for 8-10 days, controlling the fermentation temperature at 35 deg.C, and regulating pH to 2.5-3.0 with 20% (WT) ammonia water. The reducing sugar is controlled at 4-5% by feeding sugar-supplementing liquid (60% glucose liquid) in the fermentation process.
Detecting water-soluble monascus yellow pigment: centrifuging fermentation liquor in a tank at 10000rpm for 10min to obtain supernatant, diluting with pure water by 1000 times, measuring the light absorption value at the characteristic wavelength of 350nm in an ultraviolet spectrophotometer to be 0.500, and calculating to obtain water-soluble monascus yellow pigment of 500 u/mL.
Example 2
Preparation of seed liquid: the procedure of example 1 was repeated.
Fermentation: the operation is the same as that of the step II in the example 1, except that the carbon source is changed from soluble starch to 5% glucose. The sugar supplementing liquid is replaced by 50% maltose solution.
Detecting water-soluble monascus yellow pigment: same as example 1 step c. The absorbance at the characteristic wavelength of 350nm was measured by an ultraviolet spectrophotometer to be 0.509, and the result showed that the water-soluble monascus yellow pigment was 509 u/mL.
Example 3:
preparation of seed liquid: the procedure of example 1 was repeated.
Fermentation: the operation was performed as in EXAMPLE 1 except that the carbon source was changed from soluble starch to 5% maltose. 60% glucose solution is used instead for the blood sugar supplement solution.
Detecting water-soluble monascus yellow pigment: the detection method is the same as the step III of the embodiment 1. The absorbance at the characteristic wavelength of 350nm was measured with an ultraviolet spectrophotometer to be 0.481, and the result showed that the water-soluble monascus yellow pigment was 481 u/mL.
It should be understood that the examples are merely for illustrative purposes and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A fermentation regulation method of water-soluble monascus yellow pigment comprises the following steps:
performing test tube slant activation amplification culture on monascus strains, inoculating spore and mycelium eluent into a primary seed culture medium, performing seed amplification culture, and culturing to logarithmic phase to obtain a seed solution;
secondly, proportionally inoculating the seed solution obtained in the first step into a secondary seed culture medium, carrying out seed expansion culture, and culturing to logarithmic phase to obtain a secondary seed solution;
inoculating the secondary seed culture solution obtained in the step II into a fermentation culture medium according to the proportion of 5-20% (V/V) for fermentation, wherein the dissolved oxygen is more than or equal to 20%, the fermentation is carried out for 2-4 days, when the wet weight of thalli reaches 25-30g/L, the fermentation temperature is controlled at 31 ℃, and the pH is regulated and controlled at 4.0-4.5 by 20% ammonia water; fermenting for 5-7 days, controlling the fermentation temperature at 33 deg.C, and regulating pH to 3.0-3.5 with 20% (WT) ammonia water; fermenting for 8-10 days, controlling the fermentation temperature at 35 deg.C, regulating pH to 2.5-3.0 with 20% (WT) ammonia water, and controlling reducing sugar at 40-50g/L by feeding sugar-supplementing liquid during fermentation.
2. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: and the sugar solution is supplemented with 60g/L carbon source aqueous solution.
3. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 2, wherein: the carbon source is at least one of starch, glucose, maltose and glycerol.
4. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 3, wherein: the carbon source is preferably at least one of glucose and maltose.
5. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: the Monascus strain is Monascus ruber (Monascus ruber) CGMCC NO.10910 or Monascus ruber (Monascus anka) GIM 3.592.
6. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: the test tube slant culture medium comprises 5g of corn flour, 10mL of bean concentrate and KH per 100mL of slant culture medium2PO4 0.5g，MgSO40.2g of agar and 100mL of drinking water with natural pH;
the first-class seed culture medium comprises 5g of soluble starch, 2g of corn flour, 10mL of bean concentrate and KH per 100mL of culture medium2PO4 0.5g，MgSO40.2g, 100mL of drinking water with natural pH.
7. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: the activation method comprises the following steps: inoculating lyophilized bacteria powder or glycerol-preserved bacteria liquid into test tube slant culture medium, and culturing at 28 deg.C for 7-14 days; eluting spore and mycelium with 10ml sterile water to obtain eluate, absorbing 5nl of eluate, inoculating into first-stage seed culture medium, and culturing at 30 deg.C and 100rpm for 24-32 hr.
8. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein:the second-stage seed culture medium of the step II comprises 2g of glucose, 5g of corn flour, 10mL of bean concentrate and NaNO in every 100mL of seed culture medium30.5g，KH2PO4 0.5g，MgSO40.2g, 100mL of drinking water with natural pH.
9. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: the second-stage seed culture condition is that the first-stage seed liquid in the first-stage step is inoculated into a second-stage seed culture medium according to the volume ratio of 0.01-0.1%, cultured for 40-60h at 28-32 ℃ and 300rpm, and aerobically fermented to obtain the seed liquid in the logarithmic phase.
10. The method for regulating fermentation of water-soluble monascus yellow pigment according to claim 1, wherein: the composition of the fermentation medium is that every 100mL of the medium contains 5g of carbon source, 0.5g of corn flour, 1mL of bean concentrate, 5g of ammonium salt, 3g of nitrate and KH2PO4 0.3g，MgSO4 0.1g,MnSO4·H2O5.0 mg, 100mL of drinking water with natural pH.
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