CN105802858B - Culture medium for monascus fermented yeast and culture method thereof and preparation method of monascus vinegar - Google Patents
Culture medium for monascus fermented yeast and culture method thereof and preparation method of monascus vinegar Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a culture medium for monascus fermented koji, a culture method thereof and a preparation method of monascus vinegar, wherein the culture medium comprises a basic culture medium, a carbon source, a nitrogen source, a growth factor and inorganic salt; the culture method comprises the steps of taking a basic culture medium, adding a carbon source, a nitrogen source, a growth factor and inorganic salt into the basic culture medium, inoculating monascus spore suspension, adjusting the initial pH value to be 4-8, and performing shaking culture to obtain monascus fermented yeast seeds; the preparation method of the red yeast vinegar comprises the following steps: preparing monascus fermented yeast, saccharifying, fermenting, filtering and sterilizing. The invention provides a culture medium for fermenting yeast seeds by monascus, a culture method thereof and a preparation method of monascus vinegar, which are different from the existing culture method of monascus and the preparation method of monascus vinegar, select the optimal culture medium, and produce functional monascus vinegar by adopting a solid-liquid fermentation method, thereby being an innovation and an improvement of the vinegar industry.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a culture medium for monascus fermented yeast and a culture method thereof, and a preparation method of monascus vinegar.
Background
Monascus is a filamentous fungus with the traditional characteristics of China, has a plurality of kinds of metabolites, and mainly focuses on polyketide secondary metabolites. Recent research shows that monascus can also produce bioactive substances beneficial to human health, so that monascus and products thereof can be food ingredients and additives with health-care functions and new medicines for resisting fat and reducing blood pressure. Because of having certain active function, more and more medical researches in recent years show that monascus plays a very important role in protecting human health in terms of obesity, cardiovascular diseases such as coronary heart disease, hypertension and arteriosclerosis, cancer prevention and anticancer and the like. Because the types of medicines for treating cardiovascular and cerebrovascular diseases in the market at present are few, mainly statins and bepotastine acids, the acting targets of the medicines are trihydroxy trimethyl coenzyme A (HMG-CoA) reductase which has larger side effect and higher probability of causing myositis when being used as an enzyme inhibitor, and doctors and patients begin to pay more attention to the safety of the medicines after the event of the cerivastatin, the development of safe and effective new medicines is urgent. The monascus metabolic products in the monascus metabolic products can be used as sources of multiple functions such as food, medicine, health care and the like in industries such as food, medicine, chemical industry, health care, feed, cosmetics and the like, and are favored because the active substances from the monascus have the advantages of dual purposes of medicine and food and the like. The national food and drug administration has approved monascus pigment as an additive to be used in food production in 2011. At present, a few reports that monascus pigment also has other physiological activities such as antibiosis, lipid reduction and weight reduction are available. Therefore, the method has wide application prospect by carrying out efficient expanded culture fermentation on the monascus for further processing and production, but the existing culture medium for preparing the fermentation starter by fermenting the monascus has the advantages of simple components, low cost, less environmental pollution, low energy consumption and the like, but has low capability of resisting microbial pollution and high labor intensity, and meanwhile, the culture method has the defects of complex operation, low growth speed, long production period, low product quality stability and unsuitability for large-scale mechanical production.
The vinegar can stimulate appetite, inhibit pathogenic bacteria and help digestion, and is a traditional brewed condiment in China. The vinegar is prepared from starchiness such as grains (grains, cereals, potatoes, etc.) as raw materials by performing saccharification, alcoholic fermentation, acetic acid fermentation, etc. with different kinds of microorganisms. The vinegar contains acetic acid as main ingredient, and contains various amino acids, organic acids, saccharides, vitamins, alcohol, ester, and other nutrients and flavor components, and has unique color, fragrance, and taste. The vinegar production process can be divided into solid fermentation and liquid fermentation. The traditional folk vinegar mostly adopts a solid fermentation process, medium and small-sized enterprises adopt a batch liquid fermentation process, and some enterprises adopt a liquid-solid leaching and pouring circulation process. The said process is one fermentation process with great amount of raw material, and in the solid fermentation, small amount of vinegar mash is inoculated and fermented with small amount of microbe.
At present, the red yeast vinegar is mostly produced by adopting the traditional split liquid shallow fermentation process, a series of problems of nonstandard operation, difficult industrialization, unstable quality, laggard production technology and the like generally exist, the traditional production of the old vinegar utilizes natural microorganisms including harmful microorganisms, the safety of the product is also widely questioned, the quality is unstable for a long time, and the large-scale production and preparation can not be effectively realized.
Therefore, whether a culture medium for fermenting the monascus and a culture method thereof can be provided aiming at the defects in the prior art, and an improved preparation method of the monascus vinegar is provided depending on the culture medium and the culture method, so that the culture medium has the advantages of high controllability, high quality stability of the prepared monascus vinegar, and suitability for large-scale mechanical production, and becomes a technical problem to be solved by technical personnel in the field.
Disclosure of Invention
The invention aims to solve the technical problems and provides a culture medium for fermenting monascus and a culture method thereof as well as a preparation method of monascus vinegar.
In order to achieve the technical effects, the invention comprises the following technical scheme:
a culture medium for monascus fermentation yeast strains comprises 3-20 parts by weight of a carbon source, 1-10 parts by weight of a nitrogen source and 0.1-5 parts by weight of inorganic salt;
the carbon source comprises one or more of maltose, glucose, sucrose, corn flour, rice flour, sweet potato starch, wheat bran, rice bran, potato starch and glycerol;
the nitrogen source comprises peptone, yeast extract, peanut powder, fish meal, pig blood powder, silkworm pupa powder, pork liver powder, soybean powder and NaNO3And (NH)4)2SO4One or more than one of the above;
the inorganic salt comprises CaCl2、FeSO4、MnSO4、CuSO4、ZnSO4And phosphate.
Further, the carbon source comprises one or more of glucose, maltose, potato starch and sucrose; what is needed isThe nitrogen source comprises one or more of peptone, yeast extract and peanut powder; the inorganic salt comprises KH2PO4And FeSO4And/or MgSO4(ii) a The culture medium for the monascus fermented koji also comprises growth factors, and the growth factors comprise one or more of vitamin B1, vitamin B6, vitamin B12 and vitamin C.
Further, the culture medium for fermenting the koji mold by the monascus comprises the following components in percentage by weight:
the balance is the basic culture medium.
The invention takes the fermentation product of the monascus in the pigment production culture medium and the fermentation product in the basic culture medium as the contrast, takes the monascus biomass, the extracellular color value and the intracellular color false as the indexes to design an orthogonal test, selects the optimal nitrogen source, the optimal growth factor and the optimal inorganic salt, and optimizes the fermentation condition on the premise of the single-factor test optimization result of the liquid loading amount, the inoculation amount, the culture time and the initial pH. And the optimum culture medium formula is found out by utilizing SPSS software for analysis.
Preferably, the culture medium for fermenting the koji mold by monascus comprises the following components in percentage by weight:
the balance is the basic culture medium.
When the culture medium for fermenting the monascus under the conditions is used for fermenting the monascus, the monascus obtained by fermentation has the highest biomass and the highest color value.
The culture medium for monascus fermentation koji, wherein the basic culture medium comprises the following components in parts by weight: 0.1-10 parts of sodium nitrate, 0.02-1 part of potassium chloride, 0.001-0.5 part of ferrous sulfate and 50-200 parts of water.
However, the minimal medium is not limited to the above components, and those commonly used in the art are within the scope of the present invention.
A method for culturing monascus fermented yeast strains by adopting the culture medium specifically comprises the following steps: adding a carbon source, a nitrogen source and inorganic salt into a basic culture medium, inoculating monascus spore suspension, adjusting the initial pH value to 4-8, and performing shaking culture to obtain monascus fermented yeast seeds.
Furthermore, growth factors are also added into the minimal medium; the liquid loading amount of the basic culture medium is 85ml, the inoculation amount of monascus is 10%, the initial pH value is adjusted to be 5, and the shake culture condition is 150r/min and shake culture is carried out at 25-30 ℃ for 11 days;
the preparation method of the monascus spore suspension comprises the following steps: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spore with sterile Tween physiological saline, shaking and scattering spore, filtering to remove mycelium to obtain final concentration of 106And (4) per mL of monascus spore suspension.
A preparation method of red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: preparing monascus fermented koji by the method of any one of claims 4 to 5;
step two: saccharification: pretreating raw materials, crushing, adding water, mixing to prepare slurry, adjusting the pH value, adding alpha-amylase, and saccharifying to obtain a saccharification fermentation substrate;
step three: fermentation: inoculating monascus fermentation yeast seeds into a saccharification fermentation substrate, sequentially performing monascus fermentation and alcohol fermentation to obtain alcohol fermentation muir, and continuously performing acetic fermentation on the alcohol fermentation muir to obtain acetic fermentation mash;
step four: filtering and sterilizing: and C, filtering, blending and sterilizing the acetic acid fermented mash obtained in the step three to obtain the red yeast vinegar.
The filtering step in the fourth step can be filtering by a filter pump, but is not limited to filtering in the mode, all modes which can realize filtering of the acetic acid fermentation mash in the technology in the field are included, the sterilization mode is to keep the temperature at 80 ℃ for 1h, the total acid of the finally obtained red yeast vinegar is more than or equal to 3.5 percent, the color value is 150-.
Further, the raw materials comprise one or more of rice, corn and medicine and food dual-purpose Chinese medicines;
the particle size of the crushed raw materials is 50-100 meshes, and the ratio of the raw materials to the water is 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be.
The acetic fermentation comprises the following steps: inoculating acetic acid bacteria slant strains to a shake flask for enlarged culture, inoculating to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with the inoculation amount of 10-11%, ventilating, stirring and fermenting for 48-60h to obtain acetic acid fermentation mash. The total acid content (in terms of acid content) was 4g/100mL, and the nonvolatile acid content (in terms of lactic acid) was 0.8g/100 mL.
The monascus fermentation and the alcohol fermentation in the step three comprise two methods:
further, in the first method, the monascus fermentation comprises the following steps: inoculating 5-10% of monascus fermentation yeast seeds into a saccharification fermentation substrate, and stirring and fermenting at 30-35 ℃ for 30-60 h to obtain monascus fermented mash;
the alcohol fermentation comprises the following steps: inoculating active yeast into fermented mash fermented by monascus, wherein the inoculation amount is 5% -10%, stirring and fermenting for 5-6 h at 25-30 ℃, then continuing to perform static fermentation for 48-60h, and adding water to adjust the alcohol concentration to 7-8% to obtain alcohol fermentation muir.
Further, in the second method, the method for fermenting monascus and fermenting alcohol comprises the following steps: uniformly transferring Monascus fermentation koji with the inoculum size of 2-20% and high-activity yeast with the inoculum size of 2-20% into a saccharification fermentation substrate, stirring and fermenting at the initial temperature of 30-35 ℃, then reducing the temperature, and continuously fermenting by adopting intermittent stirring and fermentation to obtain the alcohol fermentation muir. In the second method, further, monascus fermentation yeast seeds are uniformly and simultaneously transferred into a saccharification fermentation substrate according to the inoculation amount of 6-7% and the inoculation amount of 6-7% of high-activity yeast, and the temperature is 30-32 ℃ when the fermentation substrate is started for 8-10 hours, and the fermentation substrate is stirred and fermented; and then adjusting the temperature to be 27-28 ℃, intermittently stirring and fermenting for 5h, continuously fermenting for 64-82 h, and adjusting the alcohol concentration to 7-8% to obtain the fermented mash alcohol.
From the above, it can be seen that the monascus fermentation step and the alcohol fermentation step can be performed separately or in combination, and the combined monascus fermentation and alcohol fermentation preparation method shortens the production time by 48 to 60 hours compared with the preparation method in which the monascus fermentation step and the alcohol fermentation step are performed separately, and the quality of the red yeast vinegar is not changed. Therefore, in both methods, it is preferable to perform the monascus fermentation and the alcoholic fermentation by the second method.
By adopting the technical scheme, the method has the following beneficial effects: the culture medium for the monascus fermentation koji and the culture method thereof provided by the invention are characterized in that an orthogonal test is designed by taking monascus biomass, extracellular color value and intracellular color false as indexes, a large number of creative experiments are carried out, an optimal nitrogen source, an optimal growth factor and an optimal inorganic salt are selected, fermentation conditions are optimized on the premise of a single-factor test optimization result of liquid loading amount, inoculation amount, culture time and initial pH, and an optimal culture medium formula and a corresponding culture method are found, so that the culture medium has the advantages of gradual operation change, high monascus fermentation koji biomass, good color value, high growth speed, short growth period and high product quality stability. Compared with the traditional red yeast vinegar fermentation method, the method for producing the functional red yeast vinegar by adopting the solid-liquid fermentation method is an innovation and promotion of the vinegar industry, and the preparation method has strong controllability and high process stability and is suitable for large-scale mechanized production.
Drawings
FIG. 1 is a graph showing the effect of different liquid contents on the biomass of Monascus fermentation according to the present invention;
FIG. 2 is a graph showing the effect of different liquid contents on extracellular color number of Monascus fermentation according to the present invention;
FIG. 3 is a graph showing the effect of different liquid contents on the intracellular color number of Monascus fermentation according to the present invention;
FIG. 4 is a graph showing the effect of different cultivation times according to the present invention on the biomass of Monascus fermentation;
FIG. 5 is a graph showing the effect of different incubation times on extracellular color number of Monascus fermentation according to the present invention;
FIG. 6 is a graph showing the effect of different incubation times on the intracellular color number of Monascus fermentation according to the present invention;
FIG. 7 is a graph showing the effect of different initial pH values on the biomass of Monascus fermentation according to the present invention;
FIG. 8 is a graph showing the effect of different initial pH values on extracellular color number of Monascus fermentation according to the present invention;
FIG. 9 is a graph showing the effect of different initial pH values on the intracellular color number of Monascus fermentation according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific examples.
The minimal medium in the following examples comprises the following components in parts by weight: 0.1-10 parts of sodium nitrate, 0.02-1 part of potassium chloride, 0.001-0.5 part of ferrous sulfate and 50-200 parts of water.
However, the minimal medium is not limited to the above components, and any minimal medium conventionally used in the art and directly obtained based on the above components is within the scope of the present invention.
The first embodiment is as follows: a culture medium for monascus fermentation koji comprises a basic culture medium, 3 parts by weight of a carbon source, 10 parts by weight of a nitrogen source and 0.1 part by weight of inorganic salt;
the carbon source comprises maltose, glucose, sucrose, corn flour, rice flour, sweet potato starch, wheat bran, rice bran, potato starch and glycerol;
the nitrogen source comprises peptone, yeast extract, peanut powder, fish meal, pig blood powder, silkworm pupa powder, pork liver powder, soybean powder and NaNO3And (NH)4)2SO4；
The inorganic salt comprises CaCl2、FeSO4、MnSO4、CuSO4、ZnSO4And a phosphate salt.
Example two: a culture medium for monascus fermentation koji comprises a basic culture medium, 10 parts by weight of a carbon source, 5 parts by weight of a nitrogen source and 1 part by weight of inorganic salt;
the carbon source comprises maltose, glucose, sucrose, corn flour and glycerol;
the nitrogen source comprises peptone, yeast extract, peanut powder and (NH)4)2SO4；
The inorganic salt comprises CaCl2、FeSO4、MnSO4And a phosphate salt.
The medium for the monascus fermentation of the koji species further comprises growth factors, and the growth therefore comprises vitamin B1.
Example three: a culture medium for monascus fermentation koji comprises a basic culture medium, 18 parts by weight of a carbon source, 3 parts by weight of a nitrogen source and 4 parts by weight of inorganic salt;
the carbon source comprises glucose, maltose, potato starch and sucrose; the nitrogen source comprises peptone, yeast extract and peanut powder; the growth factor is vitamin B1; the inorganic salt comprises KH2PO4And FeSO4。
Example four:
a culture medium for monascus fermentation koji comprises a basic culture medium, and also comprises 20 parts by weight of a carbon source, 1 part by weight of a nitrogen source and 5 parts by weight of inorganic salt;
the carbon source comprises glucose, maltose, potato starch and sucrose;
the nitrogen source comprises peptone, yeast extract and peanut powder;
the inorganic salt comprises KH2PO4、FeSO4And MgSO4；
The culture medium for the monascus fermented koji mold further comprises growth factors, and the growth factors accordingly comprise vitamin B1, vitamin B6, vitamin B12 and vitamin C.
Example five: a method for culturing monascus fermented yeast strains by adopting the culture medium specifically comprises the following steps: and (2) taking a basic culture medium, adding 20 parts by weight of carbon source, 1 part by weight of nitrogen source and 5 parts by weight of inorganic salt into the basic culture medium, continuously adding growth factors into the basic culture medium, inoculating monascus spore suspension, adjusting the initial pH value to be 5, and carrying out shake culture to obtain monascus fermentation yeast seeds.
Example six: a culture medium for monascus fermentation yeast strains comprises the following components in percentage by weight:
the balance is the basic culture medium.
A method for culturing monascus fermented yeast seeds specifically comprises the following steps:
the method comprises the following steps: preparing monascus spore suspension: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spore with sterile Tween physiological saline, transferring into sterile triangular flask with glass beads, shaking, scattering spore, filtering with 4 layers of sterile lens paper to remove mycelium to obtain final concentration of 106Per mL monascus spore suspension;
step two: preparing monascus fermented yeast: taking 85ml of the minimal medium with liquid filling, adding 3% of glucose, 5% of peanut powder and 0.1% of KH2PO41% MgSO 24The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total sum of the two components is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to be 4, the monascus inoculation amount is 10%, and the monascus is shake-cultured at the temperature of 25-30 ℃ at a speed of 150r/min for 11 days.
Example seven: a culture medium for monascus fermentation yeast strains comprises the following components in percentage by weight:
the balance is the basic culture medium.
A method for culturing monascus fermented yeast seeds specifically comprises the following steps:
the method comprises the following steps: preparing monascus spore suspension: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL;
step two: preparing monascus fermented yeast: taking 85ml of the minimal medium with liquid loading, adding 10% of glucose, 1% of peanut powder and 1% of KH2PO40.1% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total sum of the two components is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to be 8, the monascus inoculation amount is 10%, and the monascus is shake-cultured at the temperature of 25-30 ℃ at a speed of 150r/min for 11 days.
Example eight: a culture medium for monascus fermentation yeast strains comprises the following components in percentage by weight:
the balance is the basic culture medium.
A method for culturing monascus fermented yeast seeds specifically comprises the following steps:
the method comprises the following steps: preparing monascus spore suspension: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL;
step two: preparation of Monascus fermentation starter: taking 85ml of the minimal medium with liquid filling, adding 4 weight percent of glucose, 1.5 weight percent of peanut powder and 0.8 weight percent of KH2PO40.12% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total sum of the two components is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to be 5, the monascus inoculation amount is 10%, and the monascus is shake-cultured at the temperature of 25-30 ℃ at a speed of 150r/min for 11 days. Example nine: a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL; taking 85ml of a basic culture medium with liquid filling amount, adding a carbon source, a nitrogen source, a growth factor and inorganic salt into the basic culture medium, inoculating monascus spore suspension, adjusting the initial pH value to 4-8, and performing shaking culture to obtain monascus fermentation yeast seeds;
step two: saccharification: pretreating raw materials, crushing, adding water, mixing to prepare slurry, adjusting the pH value, adding alpha-amylase, and saccharifying to obtain a saccharification fermentation substrate;
step three: fermentation: inoculating monascus fermentation yeast seeds into a saccharification fermentation substrate, sequentially performing monascus fermentation and alcohol fermentation to obtain alcohol fermentation muir, and continuously performing acetic fermentation on the alcohol fermentation muir to obtain acetic fermentation mash;
step four: filtering and sterilizing: and C, filtering, blending and sterilizing the acetic acid fermented mash obtained in the step three to obtain the red yeast vinegar.
Example ten: a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into sterile triangular flask with glass beads, shaking, scattering spores sufficiently, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mLLiquid; taking 85ml of the minimal medium with liquid filling, adding 3% of glucose, 5% of peanut powder and 0.1% of KH2PO41% MgSO 24The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total sum of the two components is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to be 7, the monascus inoculation amount is 10%, and the monascus spore suspension is shake-cultured at the temperature of 25-30 ℃ at the speed of 150r/min for 11 days;
step two: saccharification: pretreating one or more of rice, corn and medicine-food dual-purpose traditional Chinese medicines, pulverizing into powder with a particle size of 50 meshes, and mixing the powder with water according to a material-water ratio of 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be;
step three: fermentation:
fermenting monascus: inoculating 5% of monascus fermentation yeast seed into a saccharification fermentation substrate, and stirring and fermenting at 30 ℃ for 60 hours to obtain fermented mash after monascus fermentation;
alcohol fermentation: inoculating active yeast into fermented mash fermented by monascus, wherein the inoculation amount is 5 percent, stirring and fermenting for 6 hours at 25 ℃, then continuing to perform static fermentation for 48 hours, and adding water to adjust the alcohol concentration to 7 percent to obtain alcohol fermentation muir;
acetic acid fermentation: transferring acetic acid bacteria slant strains to a shake flask for enlarged culture, transferring to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with the inoculation amount of 10%, ventilating, stirring and fermenting for 48h to obtain acetic acid fermentation mash; the total acid content (meaning acid content) is 4g/100mL, and the nonvolatile acid content (calculated by lactic acid) is 0.8g/100 mL;
step four: filtering and sterilizing: filtering the acetic acid fermentation mash obtained in the step three by using a filter pump, preserving the temperature of the filtrate at 80 ℃ for 1h, blending to obtain a finished product, wherein the total acid is more than or equal to 3.5%, the color value is 150-.
Example eleven: a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL; taking 85ml of the minimal medium with liquid loading, adding 10% of glucose, 1% of peanut powder and 1% of KH2PO40.1% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total sum of the two components is 100 percent, monascus spore suspension is inoculated, the monascus inoculation amount is 10 percent, the initial pH value is adjusted to be 5, and shaking culture is carried out at the temperature of 25-30 ℃ at 150r/min for 11 days;
step two: saccharification: pretreating one or more of rice, corn and medicine-food dual-purpose traditional Chinese medicines, pulverizing into 100-mesh powder, and mixing the powder with water according to a material-water ratio of 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be;
step three: fermentation:
monascus fermentation and alcoholic fermentation: transferring Monascus fermentation starter with inoculation amount of 2% and high activity yeast with inoculation amount of 20% into saccharification fermentation medium, fermenting at 30 deg.C under stirring, cooling, and fermenting under intermittent stirring to obtain alcohol fermentation muir.
Acetic acid fermentation: transferring acetic acid bacteria slant strains to a shake flask for enlarged culture, transferring to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with the inoculation amount of 10%, ventilating, stirring and fermenting for 60h to obtain acetic acid fermentation mash; the total acid content (meaning acid content) is 4g/100mL, and the nonvolatile acid content (calculated by lactic acid) is 0.8g/100 mL;
step four: filtering and sterilizing: filtering the acetic acid fermentation mash obtained in the step three by using a filter pump, preserving the temperature of the filtrate at 80 ℃ for 1h, blending to obtain a finished product, wherein the total acid is more than or equal to 3.5%, the color value is 150-.
Example twelve: a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL; taking 85ml of the minimal medium with liquid filling amount, and adding 4 weight percent of glucose, 1.5 weight percent of peanut powder and 0.1-0.2 weight percent of KH2PO40.12% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total amount of the monascus fermentation yeast is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to 4-8, and monascus fermentation yeast seeds are obtained through shaking culture. The inoculation amount of the monascus is 10%, the initial pH value is adjusted to be 5, and the monascus is shake-cultured at the temperature of 25-30 ℃ for 11 days at the speed of 150 r/min;
step two: saccharification: pretreating one or more of rice, corn and medicine-food dual-purpose traditional Chinese medicines, pulverizing into powder with a particle size of 80 meshes, and mixing the powder with water according to a material-water ratio of 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be;
step three: fermentation: monascus fermentation and alcoholic fermentation: transferring Monascus fermentation starter with 6% inoculum size and high activity yeast with 7% inoculum size into saccharification fermentation substrate, starting at 32 deg.C for 8 hr, stirring, and fermenting; then adjusting the temperature to 27 ℃, intermittently stirring and fermenting for 5 hours, continuously fermenting for 64-82 hours, and adjusting the alcohol concentration to 7-8% to obtain fermented mash alcohol;
acetic acid fermentation: transferring acetic acid bacteria slant strains to a shake flask for enlarged culture, transferring to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with the inoculation amount of 10%, ventilating, stirring and fermenting for 48h to obtain acetic acid fermentation mash; the total acid content (meaning acid content) is 4g/100mL, and the nonvolatile acid content (calculated by lactic acid) is 0.8g/100 mL;
step four: filtering and sterilizing: filtering the acetic acid fermentation mash obtained in the step three by using a filter pump, preserving the temperature of the filtrate at 80 ℃ for 1h, blending to obtain a finished product, wherein the total acid is more than or equal to 3.5%, the color value is 150-.
Example thirteen: a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL; taking 85ml of the minimal medium with liquid filling amount, and adding 4 weight percent of glucose, 1.5 weight percent of peanut powder and 0.1-0.2 weight percent of KH2PO40.12% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total amount of the monascus fermentation yeast is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to 4-8, and monascus fermentation yeast seeds are obtained through shaking culture. The inoculation amount of the monascus is 10%, the initial pH value is adjusted to be 5, and the monascus is shake-cultured at the temperature of 25-30 ℃ for 11 days at the speed of 150 r/min;
step two: saccharification: pretreating one or more of rice, corn and medicine-food dual-purpose traditional Chinese medicines, pulverizing into powder with a particle size of 90 meshes, and mixing the powder with water according to a material-water ratio of 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be;
step three: fermentation: monascus fermentation and alcoholic fermentation: transferring Monascus fermentation starter with inoculum size of 7% and high activity yeast with inoculum size of 6% into saccharification fermentation substrate, starting at 30 deg.C for 10 hr, stirring, and fermenting; then adjusting the temperature to 28 ℃, intermittently stirring and fermenting for 5 hours, continuously fermenting for 64-82 hours, and adjusting the alcohol concentration to 7-8% to obtain fermented mash alcohol;
acetic acid fermentation: transferring acetic acid bacteria slant strains to a shake flask for enlarged culture, transferring to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with the inoculation amount of 10-11%, ventilating, stirring and fermenting for 48-60h to obtain acetic acid fermentation mash; the total acid content (meaning acid content) is 4g/100mL, and the nonvolatile acid content (calculated by lactic acid) is 0.8g/100 mL;
step four: filtering and sterilizing: filtering the acetic acid fermentation mash obtained in the step three by using a filter pump, preserving the temperature of the filtrate at 80 ℃ for 1h, blending to obtain a finished product, wherein the total acid is more than or equal to 3.5%, the color value is 150-.
Example fourteen:
a method for preparing red yeast vinegar comprises the following steps:
the method comprises the following steps: preparing monascus fermented yeast: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, oscillating, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into monascus spore suspension with final concentration of 106/mL; taking 85ml of the minimal medium with liquid filling amount, adding 8% of glucose, 3% of peanut powder and 0.5% of KH2PO40.6% MgSO4The basic culture medium, glucose, peanut powder and KH2PO4And MgSO4The total amount of the monascus fermentation yeast is 100%, monascus spore suspension is inoculated, the initial pH value is adjusted to 4-8, and monascus fermentation yeast seeds are obtained through shaking culture. The inoculation amount of the monascus is 10%, the initial pH value is adjusted to be 5, and the monascus is shake-cultured at the temperature of 25-30 ℃ for 11 days at the speed of 150 r/min;
step two: saccharification: pretreating one or more of rice, corn and medicine-food dual-purpose traditional Chinese medicines, pulverizing into powder with a particle size of 80 meshes, and mixing the powder with water according to a material-water ratio of 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate; the saccharification fermentation substrate iodine is light orange yellow; DE value is 25% -30%; the acidity is 0.2%, and the sugar degree reaches 18-20 DEG Be;
step three: fermentation: monascus fermentation and alcoholic fermentation: transferring Monascus fermentation starter with inoculation amount of 20% and high activity yeast with inoculation amount of 2% into saccharification fermentation medium, fermenting at 35 deg.C under stirring, cooling, and fermenting under intermittent stirring to obtain alcohol fermentation muir.
Acetic acid fermentation: inoculating acetic acid bacteria slant strain to shake flask for amplification culture, inoculating to seed culture medium according to 8% inoculum size, performing shake culture to obtain acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermentation muir with inoculum size of 11%, ventilating, stirring, and fermenting for 48h to obtain acetic acid fermented mash; the total acid content (meaning acid content) is 4g/100mL, and the nonvolatile acid content (calculated by lactic acid) is 0.8g/100 mL;
step four: filtering and sterilizing: filtering the acetic acid fermentation mash obtained in the step three by using a filter pump, preserving the temperature of the filtrate at 80 ℃ for 1h, blending to obtain a finished product, wherein the total acid is more than or equal to 3.5%, the color value is 150-.
Screening experiments for the culture media for Monascus fermentation koji:
different nutritional factors such as carbon and nitrogen sources are respectively added into the basic culture medium or fermentation conditions such as pH of fermentation liquor are changed to investigate the monascus biomass in the fermentation system by the single factors, and suitable types of the single factors are screened out.
Preparing a spore suspension: culturing an aspergillus oryzae strain at 28 ℃ for 7-11 days on a slope; washing spores with sterile Tween physiological saline, transferring into a sterile triangular flask with glass beads, shaking, sufficiently scattering spores, filtering with 4 layers of sterile lens paper to remove mycelia, and making into uniform spore suspension with final concentration of 106/mL.
The following are the methods for color value detection:
(1) measurement of color number of mycelia
Extracting with ethanol at 60 deg.C in water bath at a ratio of 1g dried mycelium and 50mL 70% ethanol for 2 times. The extracts were combined and filtered, diluted appropriately, the volume was determined and the optical density was measured at 505nm wavelength. The optical density value is multiplied by the dilution factor and multiplied by 100 and divided by the gram number of the sample to obtain the color value (U/g) of the mycelium (GB 4926-85).
(2) Determination of color value of fermentation liquor
And (3) absorbing the fermentation liquor, diluting the fermentation liquor by using 70% ethanol solution (pH is 6-7), shaking up, standing, using unfermented Chinese medicine juice diluted by the same times as a blank control, and measuring the absorbance at the wavelength of 505 nm. And multiplying the light absorption value by the dilution factor of the fermentation liquor to obtain the color value (U/mL) of the fermentation liquor.
1. Screening of carbon sources:
the experimental method comprises the following steps: respectively adding maltose, glucose, sucrose, corn flour, rice flour, sweet potato starch, wheat bran, rice bran, potato starch and glycerol into a basic culture medium according to the addition of 3%, inoculating monascus, performing shaking culture at 30 ℃ and 125rmp for 11d, and measuring biomass and color value.
The experimental results are as follows:
the effect of the addition of different carbon sources on the fermentation of monascus is shown in table 1. According to the biomass, the monascus biomass (3.91 g/L, 3.78g/L and 3.85g/L respectively) is increased most obviously after adding glucose, maltose and sucrose, and is increased by 25.3%, 23.4% and 21.2% respectively. The carbon source in the forms of glucose, sucrose and maltose has a simple structure and is easily decomposed and utilized by monascus rapidly; the carbon source in the form of wheat bran has a complicated structure, and the monascus cells lack enzymes for decomposing the carbon source into simple carbon sources, so that the carbon source cannot be effectively utilized.
The effect of the addition of different carbon sources on the fermentation of monascus is shown in table 1. Aiming at biomass color value, adding a carbon source can obviously improve the intracellular color value of monascus, and the most improved matters are glucose, maltose and potato starch (3060U/g, 2290U/g and 1340U/g respectively), which are respectively improved by 518.2%, 362.6% and 170.7%. After the sweet potato starch, the corn flour and the cane sugar are added, the extracellular color value (790.0U/100 mL, 654.0U/100mL and 603.0U/100mL respectively) of the monascus is obviously improved, and is respectively improved by 240.5%, 181.9% and 159.9% compared with the value before the carbon source is added.
Analysis of variance the influence of different carbon sources on the content of total flavone F is 106.967, P is 0.000; the effect of different carbon sources on biomass F45030 and P0.000. For biomass differences, there were significant differences between the carbon sources. Analysis of variance the influence of different carbon sources on intracellular color number is 1452000, P is 0.000; the influence of different carbon sources on extracellular color number F1.333E 7, P0.000; that is, the difference is significant, and it is considered that the addition of different carbon sources causes the extracellular color number and the intracellular color number to be different. Further LSD multiple comparisons show that significant differences exist between carbon sources for both intracellular and extracellular color number differences. The above analysis shows that the variety of carbon sources is different and the influence on each index of the fermentation system is generally significant, so that the selection of the variety of carbon sources to be added to the fermentation system for producing vinegar by using monascus is very important. Glucose, sucrose and maltose are selected as carbon source species participating in the monascus fermentation orthogonal test optimization.
TABLE 1 influence of carbon sources on the Monascus biomass
| Carbon source | Biomass (g/L) | Intracellular color number (U/g) | Extracellular color number (U/100mL) |
| Glucose | 3.91 | 3060 | 518.0 |
| Sucrose | 3.85 | 1300 | 603.0 |
| Maltose | 3.78 | 2290 | 219.2 |
| Rice flour | 2.59 | 1120 | 571.0 |
| Corn flour | 1.05 | 1220 | 654.0 |
| Rice bran | 0.98 | 1090 | 426.0 |
| Wheat bran | 0.78 | 1010 | 377.0 |
| Potato starch | 3.47 | 1340 | 168.2 |
| Sweet potato starch | 3.16 | 1310 | 790.0 |
| Glycerol | 3.55 | 1100 | 202.0 |
2. And (3) screening of nitrogen sources:
the experimental method comprises the following steps: adding peptone 1%, yeast extract 1%, peanut powder 1%, fish powder 1%, pig blood powder 1%, silkworm chrysalis powder 1%, pork liver powder 1%, soybean powder 1%, NaNO into basic culture medium30.2％，(NH4)2SO40.2%, inoculating Monascus purpureus went, shaking at 30 deg.C and 125rmp for 11d, and measuring color number and biomass.
The experimental results are as follows: the effect of the addition of different nitrogen sources on the fermentation of monascus is shown in table 2.
The addition of the nitrogen source can increase the biomass of the monascus and improve the extracellular color number of the monascus, wherein the biomass of the monascus (5.26 g/L, 5.10g/L and 4.99g/L respectively) is increased most obviously after the silkworm chrysalis powder, the NaNO3 and the peanut powder are added, and the biomass of the monascus is increased by 68.6%, 63.5% and 59.9% respectively. Analysis of variance the effect of different nitrogen sources on biomass F1.435E 4 and P0.000, the addition of different nitrogen sources differing the biomass values. Further multiple comparisons with LSD showed significant differences between the various nitrogen sources for biomass differences. Aiming at the color value, the most obvious improvement is that peptone, yeast extract and peanut powder are respectively improved by 339.7%, 284.5% and 653.4%. Except that the intracellular color number of the monascus is increased by 70.7% after the peanut powder is added, the intracellular color number is reduced by adding other nitrogen sources in the table.
The three nitrogen sources of peptone, yeast extract and peanut powder can better meet the fermentation requirement by integrating the analysis of the influence of various forms of nitrogen sources on the monascus biomass, the raw material source, the cost, whether the monascus is suitable for eating and the like.
TABLE 2 influence of nitrogen sources on fermentation
3. Screening of growth factors:
the experimental method comprises the following steps: respectively adding trace vitamin B1, vitamin B6, vitamin B12 and vitamin C into Chachi's culture by aseptic technique, and inoculating Monascus spore suspension for culture. Monascus was inoculated, cultured at 30 ℃ under shaking at 125rmp for 11d, and biomass and color number were measured.
The experimental results are as follows: the addition of different growth factors to monascus is shown in table 3.
The addition of the growth factors improves the monascus biomass and reduces the extracellular color value of monascus.
Wherein the increase of the biomass is the largest after the vitamin B1 is added, the intracellular color number is obviously increased after the vitamin B1 is added, and F is 7654 and P is 0.000 which are obviously different when the influence of different growth factors on the biomass is analyzed by variance. Further multiple comparison of LSD showed significant differences between growth factors for differences in biomass. Analysis of variance the effect of different growth factors on intracellular color number F ═ 1.958E6, P ═ 0.000; the effect of different growth factors on extracellular color number F542 and P0.000. That is, the difference is significant, so it is believed that the addition of different growth factors causes the extracellular color number and intracellular color number to differ. Further multiple comparison of LSDs showed that differences between the various growth factors were significant for differences in extracellular color number, except that there was no significant difference between vitamin B1 and vitamin B12. For the intracellular color value difference, the growth factors have significant difference. The addition of vitamin B1 was most effective.
TABLE 3 Effect of growth factors on fermentation
| Growth factor | Biomass (g/L) | Intracellular color number (U/g) | Extracellular color number (U/100mL) |
| Vitamin B1 | 4.85 | 586.5 | 134.0 |
| Vitamin B6 | 4.64 | 492.5 | 159.0 |
| Vitamin B12 | 3.75 | 393.4 | 133.0 |
| Vitamin C | 4.08 | 455.5 | 130.0 |
4. Screening inorganic salts:
the experimental method comprises the following steps: respectively adding CaCl into the minimal medium2、FeSO4、MnSO4、CuSO4、ZnSO4The cells were inoculated with Monascus purpureus, cultured at 30 ℃ under shaking at 125rmp for 11d, and the biomass and color number were measured.
The experimental results are as follows: the effect of the addition of the inorganic salt on the fermentation of vinegar prepared from monascus is shown in table 4.
Addition of FeSO4、MnSO4、CuSO4Then the biomass of the Monascus is increased compared to when no inorganic salt is added, CaCl2、ZnSO4The addition of (b) reduces the biomass of monascus. Analysis of variance the effect of different inorganic salts on biomass was significant, F2.318E 4 and P0.000. Further multiple comparisons of LSDs showed differences in biomass. As can be seen from Table 4, FeSO4The inorganic salt is added into the ginkgo leaf juice culture medium to obtain the best effect. After inorganic salts in the table are respectively added into the basic culture medium for fermentation, the extracellular color value of monascus in the fermentation liquid is improved, and FeSO4The addition of (2) obviously improves the extracellular and intracellular color number, and the addition of other inorganic salts reduces the intracellular color number.
TABLE 4 Effect of inorganic salts on fermentation
| Inorganic salt | Biomass (g/L) | Extracellular color number (U/100mL) | Intracellular color number (U/g) |
| CaCl2 | 3.05 | 489.0 | 420 |
| FeSO4 | 4.31 | 631.0 | 685 |
| MnSO4 | 3.59 | 444.0 | 431 |
| CuSO4 | 4.69 | 358.0 | 376 |
| ZnSO4 | 2.55 | 380.0 | 412.5 |
5. Screening of liquid loading amount:
the experimental method comprises the following steps: the liquid contents of 25mL, 40mL, 55mL, 70mL, 85mL and 100mL were graded and the liquid contents were dispensed into a 250mL Erlenmeyer flask for cultivation, and the flask was inoculated with Monascus purpureus went, cultured at 30 ℃ under 125rmp shaking for 11d, and the biomass and color number were measured.
The experimental results are as follows: the effects of different liquid contents on the fermentation of monascus are shown in fig. 1-3.
The measured value of biomass gradually increased as the liquid loading amount increased, and the value of biomass reached the maximum when the liquid loading amount increased to 85mL, after which the biomass began to decrease. The reason why the biomass is decreased may be that the amount of liquid held increases nutrients in the shake flask and also decreases dissolved oxygen in the fermentation liquid, and when the amount of liquid held increases to a certain extent, the decrease in oxygen becomes a main limiting factor in the fermentation, and thus the index detection value begins to decrease. The color value gradually increased with the increase in the liquid holding amount, and the color value reached the maximum when the liquid holding amount was increased to 85mL, after which the color value began to decrease. Considering that the restriction of the solution aeration amount is large at 100mL, the liquid loading amount for the Monascus fermentation was determined to be 85 mL.
6. Screening of inoculation amount:
the experimental method comprises the following steps: the monascus spore suspension was inoculated into a 250mL flask containing 70mL of the medium at an inoculum size of 5%, 10%, 15% for culture. Monascus was inoculated, cultured at 30 ℃ under shaking at 125rmp for 11d, and biomass and color number were measured.
The experimental results are as follows: the effect of different inoculum sizes on the fermentation of monascus is shown in table 5.
The biomass of the monascus purpureus increases and then decreases with increasing inoculum size, and the biomass is the largest when the inoculum size is 10%. With the increase of the inoculation amount, the color value of the monascus in the fermented juice is increased firstly and then reduced, and the color value is the largest when the inoculation amount is 10%. Therefore, 10% was selected as the inoculum size in the fermentation of monascus.
TABLE 5 Effect of inoculum size on fermentation
7. Screening of culture time:
the experimental method comprises the following steps: culturing Monascus in a minimal medium for 8d, 9d, 10d, 11d, 12d, and 13d, inoculating Monascus, culturing at 30 deg.C and 125rmp for 11d, and measuring biomass and color value.
The experimental results are as follows: the effect of different culture times on the culture of monascus fermentation koji is shown in fig. 4-6.
The value of monascus biomass gradually increases with the increase of the culture time, but the increase rate is different, the 11d is an inflection point, the increase is faster before 11d, and the increase is slightly after 11d and tends to be gentle. The color value of the monascus gradually increases with the extension of the culture time, but the increase rate is different, the 11d is an inflection point, the increase is faster before 11d, and the increase is slightly after 11d and tends to be gentle. After analysis, the fermentation time was set to 11 days.
8. Screening of initial pH for cultivation of monascus fermentation koji:
the experimental method comprises the following steps: adjusting the initial pH of the basic culture medium to 3, 4, 5, 6, 7 and 8 respectively, inoculating monascus, culturing at 30 ℃ and 125rmp for 11d by shaking, and measuring the content, color value and biomass of total flavonoids and Monacolin K.
The experimental results are as follows: the effect of different initial pH on Monascus fermentation is shown in FIGS. 7-9.
An initial pH of 5, 6 is advantageous for increasing the biomass. An initial pH of 4, 5 is beneficial for increasing intracellular color number, and an initial pH of 5, 6 is beneficial for increasing extracellular color number. The initial pH of 5 was selected as the optimum pH for vinegar fermentation with monascus by analysis.
9. Orthogonal test screening medium formula:
the test method comprises the following steps: the mixture is subjected to concentration gradients of 1%, 2%, 3%, 4%, 5%, 6% and 7% of a carbon source and 0.1%, 0.5%, 1.0%, 1.5% and 2.0% of a nitrogen source, and KH2PO4According to a concentration gradient of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, MgSO4The maximum and minimum concentrations of these four factors involved in the orthogonal experiment were determined experimentally with concentration gradients of 0.03%, 0.06%, 0.09%, 0.12%, 0.15%, 0.18%, as shown in table 6.
TABLE 6 Upper and lower concentration limits
| Name of factor | Minimum value (%) | Maximum value (%) |
| |
4 | 6 |
| Nitrogen source | 0.5 | 1.5 |
| KH2PO4 | 0.1 | 0.2 |
| MgSO4 | 0.09 | 0.15 |
A fermentation product of monascus in a pigment production culture medium, a fermentation product in a liquid medicine culture medium and unfermented liquid medicine are used as comparison, an orthogonal test is designed by using the color value of the monascus as an index, an optimal nitrogen source, an optimal growth factor and an optimal inorganic salt are selected, and the fermentation condition is optimized on the premise of a single-factor test optimization result of liquid loading amount, inoculation amount, culture time and initial pH. And the optimum culture medium formula is found out by utilizing SPSS software for analysis.
And (3) test results: it is known that inorganic phosphorus in a certain concentration has a very important regulatory effect on fermentation of monascus, and Mg2+The KH is also indispensable to the formation of products, so that the KH is directly screened without a single-factor test2PO4And MgSO4The concentration of (2) was included in the orthogonal experiment. According to the result of the single-factor test, the vinegar making culture medium has the liquid loading of 85ml, the inoculation amount of 10 percent, the initial pH of 5 and the inorganic salt FeSO4. Selecting carbon source concentration, nitrogen source concentration and KH under the conditions of 150r/min and 28 ℃ (+ -1 ℃) for 11 days2PO4Concentration, MgSO4Six factors, i.e., the concentration, the type of carbon source and the type of nitrogen source, are shown in table 7, and an orthogonal table L18(36) was designed using SPSS software, and the optimum medium components for vinegar fermentation by monascus were screened by a multi-index orthogonal test using the intracellular and extracellular color numbers of monascus after vinegar fermentation by monascus as indexes. The results of the experimental tests are shown in Table 8, and the analysis of variance in the orthogonal test is shown in tables 9, 10 and 11.
TABLE 7 factor level table
TABLE 8 results of orthogonal experiments
TABLE 9 Biomass ANOVA results
| Factors of the fact | df | F | Sig. |
| A | 2 | 1.018 | 0.426 |
| |
2 | 4.292 | 0.082 |
| |
2 | 0.857 | 0.479 |
| |
2 | 3.161 | 0.130 |
| |
2 | 3.056 | 0.136 |
| |
2 | 0.276 | 0.772 |
TABLE 10 extracellular color number variance analysis results
TABLE 11 intracellular color number ANOVA results
| Factors of the fact | df | F | Sig. |
| A | 2 | 8.045 | 0.027 |
| |
2 | 8.347 | 0.026 |
| |
2 | 3.907 | 0.095 |
| |
2 | 10.047 | 0.018 |
| |
2 | 4.310 | 0.082 |
| |
2 | 5.110 | 0.062 |
As can be seen from the visual analysis, the importance of each factor to the biomass is B>D>E>A>C>F，A2B3C1D2E1F3The concentration of glucose is 4%, the concentration of peanut powder is 1.5%, and KH is obtained2PO4Concentration 0.1%, MgSO4Maximum biomass at a concentration of 0.12%; glucose concentration 4%, peanut powder concentration 1.5%, KH2PO4The extracellular color value is highest when the concentration is 0.2 percent and the concentration is 0.12 percent (MgSO 4); the importance of each factor to intracellular color number is D>A>B>F>E>C, when A2B3C1D2E1F3Namely, the concentration of glucose is 4 percent, the concentration of peanut powder is 1.5 percent, and KH is2PO4Concentration 0.1%, MgSO4The extracellular color number is highest at a concentration of 0.12%. As can be known from analysis of variance, the six factors have obvious influence on biomass, and the six factors have no obvious influence on the index extracellular color number; factor A, factor B, and factor D, i.e. carbon source concentration, nitrogen source concentration, MgSO4The concentration has a significant effect on intracellular color number.
By combining the visual analysis and variance analysis of six factors, A2、B3、D2、E1、F3When the glucose concentration is 4%, the peanut powder concentration is 1.5%, and MgSO is4The biomass is higher at a concentration of 0.12%; c1Namely KH2PO4The concentration of 0.1 percent is beneficial to improving biomass; finally, the optimal medium component is defined as A2、B3、C1、D2、E1、F3Namely, the concentration of glucose is 4 percent, the concentration of peanut powder is 1.5 percent, and KH is2PO4Concentration 0.1%, MgSO4The concentration is 0.12%, and the biomass can be obviously improved by 13.26 g/under the combination through verification.
By combining the visual analysis and variance analysis of six factors, A2、B3、D2、E1、F3When the glucose concentration is 4%, the peanut powder concentration is 1.5%, and MgSO is4The extracellular color value and the intracellular color value are higher when the concentration is 0.12%; c1Namely KH2PO4When the concentration is 0.1%, the intracellular color value is favorably improved; c3Namely KH2PO4The concentration of 0.2% is favorable for improving extracellular color number. Due to C1Increase of intracellular color number significantly, C3No significant increase in extracellular color number and C1And C3For extracellular color numberThe effects are similar, so the optimum culture medium component is finally defined as A2、B3、C1、D2、E1、F3Namely, the concentration of glucose is 4 percent, the concentration of peanut powder is 1.5 percent, and KH is2PO4Concentration 0.1%, MgSO4The concentration is 0.12 percent, and the combination can reach the extracellular color value of 1568U/100mL and the intracellular color value of 3909U/g through verification.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A preparation method of red yeast vinegar is characterized by comprising the following steps:
the method comprises the following steps: preparing monascus fermented yeast: inoculating monascus spore suspension into a culture medium for monascus fermented yeast strains, adjusting the initial pH value to 4-8, and performing shaking culture to obtain monascus fermented yeast strains; the culture medium for fermenting the monascus comprises a basic culture medium and the following components in percentage by weight: 3-10% of glucose, 1-5% of peanut powder, 0.1-1% of KH2PO4 and 0.1-1% of MgSO 4;
the basic culture medium comprises the following components in parts by weight: 0.1-10 parts of sodium nitrate, 0.02-1 part of potassium chloride, 0.001-0.5 part of ferrous sulfate and 50-200 parts of water;
the preparation method of the monascus spore suspension comprises the following steps: culturing the monascus strains on a slant at 28 ℃ for 7-11 days; washing spore with sterile Tween physiological saline, shaking, fully scattering spore, filtering to remove mycelium, and making into final product with concentration of 106Per mL monascus spore suspension;
step two: saccharification: pretreating raw materials, crushing, adding water, mixing to prepare slurry, adjusting the pH value, adding alpha-amylase, and saccharifying to obtain a saccharification fermentation substrate;
step three: fermentation: inoculating monascus fermentation yeast seeds into a saccharification fermentation substrate, sequentially performing monascus fermentation and alcohol fermentation to obtain alcohol fermentation mash, and continuously performing acetic acid fermentation on the alcohol fermentation mash to obtain acetic acid fermentation mash;
step four: filtering and sterilizing: and C, filtering, blending and sterilizing the acetic acid fermented mash obtained in the step three to obtain the red yeast vinegar.
2. The method for preparing red yeast rice vinegar as claimed in claim 1, wherein the raw materials are selected from rice, corn and medicinal and edible traditional Chinese medicines;
the particle size of the crushed raw materials is 50-100 meshes, and the ratio of the raw materials to the water is 1: 7-8, adding water, adjusting the pH value to 6.2-6.4, adding alpha-amylase to obtain slurry, wherein the enzyme activity of the slurry is 2000u/g, and keeping the temperature at 35 ℃ for 4-6 h to obtain a saccharification fermentation substrate;
the acetic fermentation comprises the following steps: transferring acetic acid bacteria slant strains to a shake flask for enlarged culture, transferring to a seed culture medium according to the inoculation amount of 8%, performing shaking culture to obtain an acetic acid bacteria seed solution, inoculating the acetic acid bacteria seed solution to alcohol fermented mash, wherein the inoculation amount is 10-11%, ventilating, stirring and fermenting for 48-60h to obtain the acetic acid fermented mash.
3. The method for preparing red yeast vinegar according to claim 1, wherein the fermentation of monascus comprises the following steps: inoculating 5-10% of monascus fermentation yeast seeds into a saccharification fermentation substrate, and stirring and fermenting at 30-35 ℃ for 30-60 h to obtain monascus fermented mash;
the alcohol fermentation comprises the following steps: inoculating active yeast into fermented mash fermented by monascus, wherein the inoculation amount is 5% -10%, stirring and fermenting for 5-6 h at 25-30 ℃, then continuing to perform static fermentation for 48-60h, and adding water to adjust the alcohol concentration to 7-8% to obtain alcohol fermented mash.
4. The method for preparing red yeast vinegar according to claim 1, wherein the method for monascus fermentation and alcohol fermentation comprises the following steps: uniformly transferring monascus fermentation yeast seeds with the inoculum size of 2-20% and high-activity yeast with the inoculum size of 2-20% into a saccharification fermentation substrate, stirring and fermenting at the initial temperature of 30-35 ℃, then reducing the temperature, and continuously fermenting by adopting intermittent stirring and fermentation to obtain alcohol fermentation mash.
5. The method for preparing red yeast vinegar according to claim 4, wherein monascus fermentation yeast seeds are uniformly inoculated into the saccharification fermentation substrate according to 6-7% of inoculum size and high-activity yeast is uniformly inoculated into the saccharification fermentation substrate according to 6-7% of inoculum size, and the temperature is 30-32 ℃ during the initial 8-10 h, and the fermentation is carried out with stirring; and then adjusting the temperature to be 27-28 ℃, intermittently stirring and fermenting for 5h, continuously fermenting for 64-82 h, and adjusting the alcohol concentration to 7-8% to obtain the fermented mash alcohol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101376312B1 (en) * | 2012-08-29 | 2014-03-27 | 명지대학교 산학협력단 | Functional persimmon vinegar and preparing method thereof |
| CN103865749A (en) * | 2014-04-09 | 2014-06-18 | 福建师范大学 | Method for brewing Fujian monascus vinegar |
-
2016
- 2016-04-29 CN CN201610278262.3A patent/CN105802858B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN103865749A (en) * | 2014-04-09 | 2014-06-18 | 福建师范大学 | Method for brewing Fujian monascus vinegar |
Non-Patent Citations (2)
| Title |
|---|
| 周建建等.采用响应曲面法优化红曲霉发酵培养基组分.《食品工业科技》.2012,第33卷(第23期),摘要，第153页表1，第157页右栏. * |
| 采用响应曲面法优化红曲霉发酵培养基组分;周建建等;《食品工业科技》;20121231;第33卷(第23期);摘要，第153页表1，第157页右栏 * |
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