CN113559022A - Fermentation method for fermenting traditional Chinese medicine 'Xinqibai' by using probiotics - Google Patents
Fermentation method for fermenting traditional Chinese medicine 'Xinqibai' by using probiotics Download PDFInfo
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Abstract
The invention relates to a fermentation method for fermenting a traditional Chinese medicine 'Xinqibai' by probiotics, wherein the raw materials of the method comprise white poria, cortex mori radicis, radix paeoniae alba, radix ampelopsis, cortex dictamni, bletilla striata and white ginseng, and the method specifically comprises the steps of pulverizing the raw materials, sieving, mixing with glutinous rice flour to form a mixed raw material; soaking the mixed raw materials in water to form a fermentation material; putting the fermentation material into a fermentation tank for fermentation; and performing solid-liquid separation on the obtained fermentation liquor, decoloring and deodorizing by using active carbon, and filtering the active carbon to obtain a new heptaalba lactic acid bacteria fermentation stock solution. The method overcomes the defects of troublesome operation, incomplete extraction of effective components and low absorption degree of the traditional method. Through the fermentation treatment of microorganisms, the microorganisms produce various extracellular enzymes such as cellulase and pectinase in the metabolic process, and the enzymes can decompose the cell wall of plant cells. In addition, various enzymes generated by microbial metabolism can reduce the molecular weight of effective components, and simultaneously remove various macromolecular impurities, so that the enzymes can be better absorbed and utilized by human bodies.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a fermentation method for fermenting a traditional Chinese medicine 'Xinqibai' by using probiotics.
Background
The functional raw materials obtained by fermenting traditional Chinese medicines have wide application in the field of cosmetics. The release of active ingredients in cytoplasm of the cells is blocked because the plant cells have cell walls with compact structures; secondly, the molecular weight of the effective components of the traditional Chinese medicine is generally larger, so that the effective components are not easy to break through the skin barrier and be absorbed by the human body. The utilization rate of effective components of the traditional Chinese medicinal materials can be greatly improved by microbial fermentation treatment, because microorganisms can generate a plurality of extracellular enzymes such as cellulase, pectinase and the like in the metabolic process, and the extracellular enzymes can decompose cell walls of plant cells, so that the cells of the traditional Chinese medicinal materials are broken, and the effective components are exposed. In addition, various enzymes generated by microbial metabolism can reduce the molecular weight of effective components, and simultaneously remove various macromolecular impurities, so that the enzymes can be better absorbed and utilized by human bodies.
Probiotic bacteria refer to microorganisms beneficial to body health, and as of 2019, probiotic bacteria species available in the food field including 35 species or subspecies are published in China on "list of probiotic bacteria species available for health food" (No. 2001/84 by law), "list of fungal bacteria species available for health food" (No. 2001/84 by law), "list of bacteria species available for food" (No. 2010) and "list of bacteria species available for infant food" (No. 2011/25 by Ministry of health).
Lactic acid is one of three major organic acids recognized in the world, and the application of the lactic acid is extremely wide. Lactic acid, lactate and derivatives thereof are widely applied to the fields of food, medicine, feed, chemical industry and the like. The human body contains only L-lactate dehydrogenase and metabolizes only L-lactate. The use of L-lactic acid in the food industry is absolutely safe, so the world health organization advocates the use of L-lactic acid in the food and pharmaceutical industries instead of DL-lactic acid, which is widely used at present.
Among many lactic acid-producing microorganisms, Rhizopus oryzae (Rhizopus oryzae/AS3.819) is an ideal strain for producing L-lactic acid with high optical purity due to its high optical purity, simple nutrient consumption, and easy separation. The rice fermentation liquor produced by the rhizopus oryzae fermentation has the efficacy of promoting skin penetration, has high safety to organisms, and also has the efficacy of moisturizing, whitening and removing wrinkles.
The invention name of publication No. CN101463370B is a method for preparing L-lactic acid by fermenting potato starch with Rhizopus oryzae, which comprises (1) preparing Rhizopus oryzae spores; (2) preparing rhizopus oryzae spores into rhizopus oryzae spore emulsion suspension; (3) fixing the rhizopus oryzae spore emulsion suspension on an immobilization carrier to obtain immobilized rhizopus oryzae seeds; and (4) inoculating the immobilized rhizopus oryzae seeds into a fermentation medium for immobilized fermentation. The method of the invention cultivates high-yield rhizopus oryzae strains and fixes the rhizopus oryzae strains on a cotton cloth carrier to obtain immobilized rhizopus oryzae seeds; the method uses an immobilization carrier to immobilize rhizopus oryzae seeds for fermentation.
The invention discloses an invention patent application with a publication number of CN101153297A, in particular to a novel process method for rhizopus oryzae sphere single-pot semi-continuous high-intensity fermentation of L-lactic acid with high optical purity, which comprises the following steps: (1) preparing high-density high-activity rhizopus oryzae spore emulsion suspension, (2) transferring rhizopus oryzae spherical cells to a 500L mechanical stirring tank in an aseptic mode for propagation, (3) performing primary fermentation in a 500L tank in a single tank, (4) performing repeated fermentation in a 500L tank in a single tank, and (5) continuously performing 15-batch semi-continuous high-strength fermentation in a 500L tank in a single tank. The shape and the size of the bacterial spheres formed by the suspension culture system in the fermentation process are regular and uniform; repeated fermentation can be successfully realized, N source and other inorganic salts consumed by thalli are saved, and the fermentation cost is reduced; the method adopts semi-continuous fermentation, and has the advantages of short fermentation period and high fermentation strength.
The invention of publication No. CN101085982 is an invention patent application of a preparation method for producing microbial polysaccharide preparation by using lactobacillus and yeast, the disclosed method comprises liquid culture by using beneficial strains; centrifugally collecting cell walls of the thalli; then the flora is subjected to solid amplification culture, and cell walls rich in polysaccharide are added and collected in the culture. The invention has the advantages that the high-concentration lactic acid bacteria in the product can obviously improve the digestion and absorption functions of aquatic animals, and meanwhile, the produced peptidoglycan increases the immunity of the aquatic animals to pathogens; the yeast with high concentration in the product can improve rich single-cell protein required by fish growth, simultaneously, the yeast cells are fully broken, a large amount of beta-1, 3 glucan is released, and the immunity to gram-positive and gram-negative bacteria is obviously improved.
The long-circulating Qibai ointment is one of the most popular whitening prescriptions, the classic Qibai ointment appears in Taiping Shenghui prescription of Song dynasty at the earliest time and later in Yuan Yao prescription of the Ministry, and comprises the components of largehead atractylodes rhizome, white poria, Japanese ampelopsis root, bletilla, dahurian angelica root, giant typhonium rhizome, manchurian wildginger and egg white. Experiments show that the external seven-white ointment can obviously reduce the level of skin melanin index, inhibit the activity of melanocyte, reduce melanogenesis and relieve skin pigmentation caused by UVB. Modern researches show that angelica dahurica contains imperatorin which is a substance with photosensitivity, and can really moisten skin when being used in the absence of light, but can cause skin to blacken and grow spots once being illuminated for a long time, and can also cause convulsion, hypertension, abortion of pregnant women and the like when being serious, so that the angelica dahurica is forbidden in the current cosmetic category.
The traditional usage comprises pulverizing rhizoma paridis into fine powder, concocting with ovum gallus Domesticus album to obtain pill with size of ovum gallus Domesticus flavus or thumb shape or cake shape, and drying in the shade. Cleaning face every night, grinding the pill in warm serous fluid in a porcelain, and coating face with the medicinal liquid. The traditional method is troublesome to use, and the effective components in the traditional Chinese medicine are incompletely extracted and have low absorption degree. The traditional solvent extraction method can not be heated, so that the oxidation and polymerization of active ingredients are easily caused. Therefore, the fermentation method for gently extracting the effective components of the traditional Chinese medicine and degrading the macromolecules of the traditional Chinese medicine has important significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a fermentation method for fermenting traditional Chinese medicine 'Xinqibai' high-yield lactic acid probiotics by using probiotics; overcomes the problem of utilization efficiency of the traditional Chinese medicine Qibai ointment.
In the technical scheme of the invention, the raw materials of the method, Xinqibai, comprise: poria, cortex Mori, radix Paeoniae alba, radix Ampelopsis, cortex Dictamni Radicis, rhizoma Bletillae and radix Ginseng alba.
The fermentation method for fermenting the traditional Chinese medicine 'Xinqibai' high-yield lactic acid probiotics by using the probiotics comprises the following steps:
step one, pulverizing the raw materials, sieving the pulverized raw materials by a 50-mesh sieve, and mixing the raw materials with glutinous rice flour according to the weight ratio of 1:1 to form mixed raw materials;
step two, soaking the mixed raw materials in water, wherein the ratio of the mixed raw materials to the water is 3:7 by weight;
step three, putting the fermentation material formed by soaking the mixed raw material in the step two in water into a fermentation tank for fermentation;
and step four, performing solid-liquid separation on the fermentation liquor obtained in the step three, decoloring and deodorizing by using 200-mesh active carbon, and filtering the active carbon to obtain a new heptaalba lactic acid bacteria fermentation stock solution.
The new heptaalba lactic acid bacteria fermentation stock solution can be used as a skin brightening and whitening raw material for cosmetics.
In the technical scheme of the invention, the raw materials comprise, by weight, 220 parts of poria cocos wolf 180, 45-55 parts of white mulberry root-bark, 45-55 parts of white paeony root, 45-55 parts of ampelopsis japonica, 45-55 parts of cortex dictamni, 45-55 parts of bletilla striata and 45-55 parts of white ginseng; preferably, the raw materials comprise, by weight, 200 parts of white poria, 50 parts of white mulberry root-bark, 50 parts of white paeony root, 50 parts of Japanese ampelopsis root, 50 parts of dittany bark, 50 parts of bletilla striata and 50 parts of white ginseng.
Further, the microorganism used for fermentation is rhizopus oryzae and lactobacillus
The lactobacillus is selected from one of lactobacillus acidophilus, lactobacillus casei, lactobacillus jensenii and lactobacillus raman.
Further, the fermentation in the third step of the method is as follows:
inoculating granular rhizopus oryzae into the fermentation material in a volume percentage of 3-8%, controlling the pH value by sodium carbonate at 35 ℃ and 200r/min, controlling the pH value at 4.0-5.0, fermenting for 24-36h, and ensuring that the yield of lactic acid reaches 3-5 g/L. Adjusting pH to 7.5-8.0 with ammonia water, maintaining the temperature at 35 deg.C, inoculating lactobacillus suspension at 2-5% volume, co-culturing with Rhizopus oryzae, and fermenting for 24-36 hr to obtain lactic acid yield of 10-13 g/L. The lactobacillus suspension is prepared by 10mmol/L phosphate buffer solution (pH8.0), and the ratio of lactobacillus to phosphate buffer solution is (by weight parts) 1-15: 100.
Further, in the third step, fermentation is carried out in a fermentation tank, 25L of fermentation materials are filled in every 50L of fermentation tank, the pH value of the fermentation is 4.0-5.0, the fermentation temperature is 32-34 ℃, and the fermentation period is 60 hours; controlling the aeration flow rate to be 0.3L/L.min and the rotation speed to be 50-80rpm from the beginning of fermentation to the 20 th hour; controlling the aeration flow rate to be 0.6L/L.min and the rotation speed to be 450rpm from the 20 th hour to the 40 th hour of self-fermentation; controlling the aeration flow rate to be 0.6L/L.min and the rotation speed to be 50rpm from the 40 th hour to the 60 th hour of self-fermentation; adding an antifoaming agent at the initial stage of fermentation, wherein the addition amount of the antifoaming agent is 0.0 lwt%, and gradually adding to 0.1 wt% from the 30 th hour of fermentation;
wherein, the culture of the rhizopus oryzae and the lactobacillus comprises the following steps:
(1) respectively inoculating rhizopus oryzae and lactobacillus to a liquid fermentation culture medium for culture to obtain seed solutions of two strains.
(2) Inoculating the seed liquid of the two strains into a culture medium for fermentation culture.
(3) And (3) drying the fermented material obtained in the step (2).
Further, after the lactobacillus is subjected to seed culture, the lactobacillus is subjected to amplification culture; wherein, the enlarged culture is firstly fermented and cultured for 36h at 30 ℃, and then is continuously fermented and cultured for 36h at 32 ℃;
further, according to the technical scheme, rhizopus oryzae is subjected to seed culture to form spore suspension, and then is subjected to fermentation basic culture until granular thalli are formed; inoculating rhizopus oryzae subjected to basic fermentation culture in a fermentation culture medium, controlling the pH value to be 4.0-5.0 by using sodium carbonate, fermenting at 35 ℃ for 24-36h, adjusting the pH value to be 7.5-8.0 by using ammonia water, inoculating the lactobacillus obtained in the step (1), maintaining the temperature at 35 ℃, and co-culturing with the rhizopus oryzae.
In the step (1), after the lactobacillus is subjected to seed culture and amplification culture, the thallus density is more than 30g wet thallus/L.
In the step (1), the lactobacillus is cultured in the seed culture medium and the seed culture method, which are conventional lactobacillus seed culture medium and conventional lactobacillus seed culture method, for example, the seed culture medium preferably has the following formula: 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent; the seed culture method is preferably: lactobacillus seeds are inoculated into a seed culture medium and cultured for 24 hours at the temperature of 30 ℃ and under the condition of 200 r/min.
In the step (1), the lactobacillus is subjected to scale-up culture, wherein a culture medium of the lactobacillus is a conventional fermentation scale-up culture medium, and for example, the scale-up culture medium preferably has the following formula: 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent. The expansion culture method adopts a variable temperature culture mode, and preferably comprises the following steps: inoculating the seed liquid into a fermentation amplification culture medium according to the volume percentage of 10%, performing fermentation culture for 48h at the temperature of 33 ℃ and the speed of 200r/min, then increasing the temperature to 35 ℃, and continuing to culture for 48h, wherein the thallus density is more than or equal to 30g of wet thallus/L. After the expanded culture, the cells were recovered by filtration and stored at a low temperature of 4 ℃ for further use. When used, a lactic acid bacterium suspension was prepared with 10mmol/L phosphate buffer solution (pH 8.0).
The culture medium and the culture method of the rhizopus oryzae seed culture are the conventional seed culture medium and the conventional seed culture method of the rhizopus oryzae seed culture, and for example, the following formula is preferably adopted: 30g/L of yeast extract, 30g/L of malt extract, 30g/L of peptone, 20g/L of glycerol, 20g/L of agar and water as a solvent; the seed culture method is preferably: culturing at 35 deg.C for 5-7 days, collecting a fresh slant strain, adding a certain amount of sterile water, and making into spore suspension.
The basic culture medium and the conventional liquid culture method for the fermentation of the rhizopus oryzae are preferably adopted by the following formula: 50g/L glucose, 2g/L urea, KH2PO4 0.6g/L，MgSO4·7H2O 0.5g/L，ZnSO4·7H2O 0.0176g/L，FeSO4·7H2O0.000498 g/L, and the solvent is water; the fermentation basic culture method is preferably as follows: inoculating the spore suspension into a fermentation basic culture medium according to the volume percentage of 10%, and culturing at constant temperature for 24h at the temperature of 35 ℃ and at the speed of 200r/min, wherein the rhizopus oryzae is granular. Forming granular thallus with diameter of 0.5-2 mm.
The fermentation medium is a medium for conventional liquid culture of rhizopus oryzae, and the carbon source is cellulose hydrolysis glucose. The preferred formulation is as follows: 60-140g/L of cellulose hydrolysis glucose; (NH)4)2SO4 0.5g/L，KH2PO4 0.6g/L，MgSO47H2O 0.5g/L，ZnSO4·7H2O 0.0176g/L，FeSO4·7H2O0.00498 g/L, and the solvent is water.
Further, in the step (2), the lactobacillus is subjected to amplification culture, and stimulation culture is carried out by using an ultrasonic biological stimulation growth instrument, wherein the ultrasonic biological stimulation growth instrument has the treatment parameters of ultrasonic frequency of 30kHz, power of 10W, ultrasonic time of 10s and interval time of 20s, and ultrasonic stimulation is carried out for 30 minutes in total.
Has the advantages that: the method overcomes the defects of troublesome use, incomplete extraction of effective components in the traditional Chinese medicine and low absorption degree of the traditional Chinese medicine. The invention can improve the utilization rate of effective components by microbial fermentation treatment, the microbes can generate a plurality of extracellular enzymes such as cellulase, pectinase and the like in the metabolic process, and the extracellular enzymes can decompose the cell walls of plant cells to break the cells of the traditional Chinese medicinal materials and expose the effective components. In addition, various enzymes generated by microbial metabolism can reduce the molecular weight of effective components, and simultaneously remove various macromolecular impurities, so that the enzymes can be better absorbed and utilized by human bodies.
In order to make the purpose and technical solution of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention will be clearly and completely described below with reference to the implementation examples of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by a person skilled in the art without inventive effort based on the described embodiments of the invention, fall within the scope of protection of the invention.
Detailed description of the preferred embodiments
The technical solution of the present invention is further illustrated by the following specific examples. In the following embodiments, the raw materials are all commercial products purchased from the relevant manufacturing enterprises or commercial departments, unless otherwise specified. The processing parameters of the ultrasonic biological stimulation growth instrument are ultrasonic frequency of 30kHz and power of 10W.
Example 1:
weighing 2000g of raw materials according to the relationship of 200 parts of white poria cocos, 50 parts of white mulberry root-bark, 50 parts of white peony root, 50 parts of Japanese ampelopsis root, 50 parts of cortex dictamni, 50 parts of bletilla striata and 50 parts of white ginseng by weight, crushing the raw materials, sieving the raw materials by a 50-mesh sieve, and mixing glutinous rice powder according to the weight ratio of 1:1 to form mixed raw materials; mixing and soaking the mixed raw materials and purified water for 48 hours in a weight ratio of 3:7 to obtain a fermented material;
in this embodiment, lactobacillus is lactobacillus acidophilus.
Putting the fermentation material into a 50L fermentation tank, inoculating granular rhizopus oryzae into the fermentation material according to the volume percentage of 3%, controlling the pH value by sodium carbonate under the conditions of 35 ℃ and 200r/min, controlling the pH value to be 4.5, fermenting for 24 hours, adjusting the pH value to be 7.5 by ammonia water, maintaining the temperature at 35 ℃, inoculating lactobacillus acidophilus suspension according to the volume percentage of 2.5%, co-culturing with the rhizopus oryzae, and fermenting and culturing for 36 hours. The lactobacillus acidophilus suspension is prepared from 10mmol/L phosphate buffer solution (pH8.0), and the ratio of the lactobacillus acidophilus to the phosphate buffer solution is 2: 100. the obtained fermentation liquor is subjected to solid-liquid separation, decolorized and deodorized by active carbon of 200 meshes, and the active carbon is filtered to obtain 14.8L of new hepta-white lactic acid bacteria fermentation stock solution.
Wherein, the culture of the rhizopus oryzae and the lactobacillus acidophilus comprises the following steps:
step (1), seed culture of lactobacillus acidophilus, namely inoculating lactobacillus acidophilus seeds into a seed culture medium according to the volume percentage of 5 percent, and culturing for 24 hours at the temperature of 30 ℃ and at the speed of 200 r/min; the seed culture medium comprises the following components: 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent.
Step (2) performing amplification culture on lactobacillus acidophilus, and performing amplification culture on lactobacillus acidophilus after seed culture; wherein, the enlarged culture is firstly fermented and cultured for 36h at 30 ℃, and then is continuously fermented and cultured for 36h at 32 ℃; and (3) performing amplification culture of the lactobacillus acidophilus, and performing stimulation culture by using an ultrasonic biological stimulation growth instrument, wherein the ultrasonic biological stimulation growth instrument is used for performing ultrasonic stimulation for 30 minutes at the treatment parameters of ultrasonic frequency of 30kHz, power of 10W, ultrasonic time of 10s and interval time of 20 s. In the step (2), the lactobacillus acidophilus is subjected to amplification culture, and a culture medium comprises the following formula: 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent. The amplification culture method adopts a variable temperature culture mode, the seed liquid with the volume percentage of 10 percent is inoculated into a fermentation amplification culture medium, the fermentation culture is carried out for 48 hours under the conditions of 33 ℃ and 200r/min, then the temperature is increased to 35 ℃, the culture is continued for 48 hours, and the thallus density is more than or equal to 30g wet thallus/L. After the expanded culture, the cells were recovered by filtration and stored at a low temperature of 4 ℃ for further use. When in use, a suspension of Lactobacillus acidophilus was prepared with 10mmol/L phosphate buffer (pH 8.0).
And (3) culturing the rhizopus oryzae seeds, wherein a culture medium adopts the following formula: 30g/L of yeast extract, 30g/L of malt extract, 30g/L of peptone, 20g/L of glycerol, 20g/L of agar and water as a solvent; the seed culture method comprises the following steps: culturing at 35 deg.C for 5-7 days, collecting a fresh slant strain, adding a certain amount of sterile water, and making into spore suspension.
And (4) carrying out fermentation amplification culture on rhizopus oryzae, wherein the following formula is adopted: 50g/L glucose, 2g/L urea, KH2PO40.6g/L，MgSO4·7H2O 0.5g/L，ZnSO4·7H2O 0.0176g/L，FeSO4·7H2O0.000498 g/L, and the solvent is water; the fermentation and expanded culture method comprises the following steps: inoculating the spore suspension into fermentation amplification culture medium at volume percentage of 10%, and culturing at constant temperature at 35 deg.C and 200r/min for 24 hr to obtain granular Rhizopus oryzae. Forming granular thallus with diameter of 0.5-2 mm.
Example 2
Weighing 2000g of raw materials according to the relationship of 180 parts of white poria cocos, 55 parts of white mulberry root-bark, 50 parts of white paeony root, 45 parts of Japanese ampelopsis root, 50 parts of cortex dictamni, 55 parts of bletilla striata and 45 parts of white ginseng by weight, crushing the raw materials, sieving the raw materials by a 50-mesh sieve, and mixing glutinous rice powder according to the weight ratio of 1:1 to form mixed raw materials; mixing and soaking the mixed raw materials and purified water for 60 hours in a weight ratio of 3:7 to obtain a fermented material;
in this example, lactobacillus casei was used as lactobacillus.
Putting the fermentation materials into a 50L fermentation tank, inoculating granular rhizopus oryzae into the fermentation materials according to the volume percentage of 5%, controlling the pH value by sodium carbonate under the conditions of 35 ℃ and 200r/min, controlling the pH value to be 4.5, fermenting for 24 hours, adjusting the pH value to be 7.5 by ammonia water, maintaining the temperature to be 35 ℃, inoculating lactobacillus casei suspension according to the volume percentage of 3%, co-culturing with the rhizopus oryzae, and fermenting and culturing for 36 hours. The lactobacillus casei suspension is prepared from 10mmol/L phosphate buffer solution (pH8.0), and the ratio of lactobacillus casei to the phosphate buffer solution is (by weight parts) 5: 100. the obtained fermentation liquor is subjected to solid-liquid separation, decolorized and deodorized by active carbon of 200 meshes, and the active carbon is filtered to obtain 13.6L of new hepta-white lactic acid bacteria fermentation stock solution.
Wherein, the culture of the rhizopus oryzae and the lactobacillus casei comprises the following steps:
step (1), seed culture of lactobacillus casei, namely inoculating lactobacillus casei seeds into a seed culture medium according to the volume percentage of 6 percent, and culturing for 24 hours at the temperature of 30 ℃ and at the speed of 200 r/min; the seed culture medium comprises the following components: 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent.
Step (2), carrying out expanded culture on lactobacillus casei, wherein the lactobacillus casei is subjected to seed culture and then subjected to expanded culture; wherein, the enlarged culture is firstly fermented and cultured for 36h at 30 ℃, and then is continuously fermented and cultured for 36h at 32 ℃; and (3) performing amplification culture of lactobacillus casei, and performing stimulation culture by using an ultrasonic biological stimulation growth instrument, wherein the ultrasonic biological stimulation growth instrument is used for performing ultrasonic stimulation for 30 minutes at the treatment parameters of ultrasonic frequency of 30kHz, power of 10W, ultrasonic time of 10s and interval time of 20 s. In the step (2), the lactobacillus casei is subjected to amplification culture, and the formula of an amplification culture medium is as follows: 52g/L glucose, 1.2g/L urea, KH2PO40.6g/L, 22g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 10g/L of liquid paraffin and water as a solvent. The amplification culture method adopts a variable temperature culture mode, the seed liquid with the volume percentage of 10 percent is inoculated into a fermentation amplification culture medium, the fermentation culture is carried out for 48 hours under the conditions of 33 ℃ and 200r/min, then the temperature is increased to 35 ℃, the culture is continued for 48 hours, and the thallus density is more than or equal to 30g wet thallus/L. After the expanded culture, the cells were recovered by filtration and stored at a low temperature of 4 ℃ for further use. When used, a suspension of Lactobacillus casei was prepared with 10mmol/L phosphate buffer (pH 8.0).
The steps (3) and (4) are the same as in example 1.
Example 3
Weighing 2000g of raw materials according to the relationship of 220 parts of white poria, 50 parts of white mulberry root-bark, 55 parts of white peony root, 45 parts of Japanese ampelopsis, 55 parts of cortex dictamni, 55 parts of bletilla striata and 50 parts of white ginseng, crushing, sieving by a 50-mesh sieve, and mixing with glutinous rice flour according to the weight ratio of 1:1 to form a mixed raw material; mixing and soaking the mixed raw materials and purified water for 60 hours in a weight ratio of 3:7 to obtain a fermented material;
in this example, lactobacillus was lactobacillus raman.
Putting the fermentation material into a 50L fermentation tank, inoculating granular rhizopus oryzae into the fermentation material according to the volume percentage of 3%, controlling the pH value by sodium carbonate under the conditions of 35 ℃ and 200r/min, controlling the pH value to be 4.5, fermenting for 24 hours, adjusting the pH value to be 7.5 by ammonia water, maintaining the temperature to be 35 ℃, inoculating the lactobacillus raman suspension according to the volume percentage of 5%, co-culturing with the rhizopus oryzae, and fermenting and culturing for 36 hours. The Raman lactobacillus suspension is prepared from 10mmol/L phosphate buffer solution (pH8.0), and the ratio of the Raman lactobacillus to the phosphate buffer solution is (by weight parts) 5: 100. the obtained fermentation liquor is subjected to solid-liquid separation, decolorized and deodorized by active carbon of 200 meshes, and the active carbon is filtered to obtain 14.2L of new hepta-white lactic acid bacteria fermentation stock solution.
Wherein, the rhizopus oryzae and lactobacillus mansonii are cultured as follows:
the seed culture of the lactobacillus raman in the step (1), inoculating the lactobacillus raman seeds in a seed culture medium with the volume percentage of 8 percent, and culturing for 24 hours at the temperature of 30 ℃ and under the condition of 200 r/min; the seed culture medium comprises the following components: 56g/L glucose, 1.2g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent.
Step (2), performing amplification culture on the lactobacillus raman, namely performing seed culture on the lactobacillus raman, and performing amplification culture; wherein, the enlarged culture is firstly fermented and cultured for 36h at 30 ℃, and then is continuously fermented and cultured for 36h at 32 ℃; and (3) performing amplification culture of the lactobacillus mansonii, and performing stimulation culture by using an ultrasonic biological stimulation growth instrument, wherein the ultrasonic biological stimulation growth instrument is used for performing ultrasonic stimulation for 30 minutes at the treatment parameters of ultrasonic frequency of 30kHz, power of 10W, ultrasonic time of 10s and interval time of 20 s. In the step (2), the lactobacillus mansonii is subjected to amplification culture, and the formula of an amplification culture medium is as follows: 56g/L glucose, 1.2g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract and liquid20g/L of paraffin wax and water as a solvent. The amplification culture method adopts a variable temperature culture mode, the seed liquid with the volume percentage of 5 percent is inoculated into a fermentation amplification culture medium, the fermentation culture is carried out for 48 hours under the conditions of 33 ℃ and 200r/min, then the temperature is increased to 35 ℃, the culture is continued for 48 hours, and the thallus density is more than or equal to 30g wet thallus/L. After the expanded culture, the cells were recovered by filtration and stored at a low temperature of 4 ℃ for further use. When used, a suspension of Lactobacillus raman was prepared with 10mmol/L phosphate buffer (ph 8.0).
The steps (3) and (4) are the same as in example 1.
The above description further describes a specific embodiment of the present invention with reference to specific examples, which are intended for the detailed description of the present invention and are not intended to limit the present invention. The above-mentioned embodiments are merely descriptions of the preferred embodiments of the present invention, and do not limit the technical concept and the protection scope of the present invention, and various modifications and improvements made to the technical concept by those skilled in the art without departing from the design concept of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. A fermentation method for fermenting traditional Chinese medicine 'Xinqibai' by using probiotics comprises the following raw materials: white poria, white mulberry root-bark, white peony root, ampelopsis japonica, cortex dictamni, bletilla striata and white ginseng, and is characterized in that the method comprises the following steps:
step one, pulverizing the raw materials, sieving the pulverized raw materials by a 50-mesh sieve, and mixing the raw materials with glutinous rice flour according to the weight ratio of 1:1 to form mixed raw materials;
step two, soaking the mixed raw materials in water, wherein the ratio of the mixed raw materials to the water is 3:7 by weight;
step three, putting the fermentation material formed by soaking the mixed raw material in the step two in water into a fermentation tank for fermentation;
and step four, performing solid-liquid separation on the fermentation liquor obtained in the step three, decoloring and deodorizing by using 200-mesh active carbon, and filtering the active carbon to obtain a new heptaalba lactic acid bacteria fermentation stock solution.
2. The method for fermenting the traditional Chinese medicine 'Xinqibai' with probiotics as claimed in claim 1, wherein the raw materials comprise, by weight, 220 parts of white poria 180-.
3. The method for fermenting the traditional Chinese medicine 'Xinqibai' by using probiotics according to claim 2, wherein the raw materials comprise, by weight, 200 parts of white poria cocos, 50 parts of white mulberry root-bark, 50 parts of white peony root, 50 parts of Japanese ampelopsis root, 50 parts of cortex dictamni, 50 parts of bletilla striata and 50 parts of white ginseng.
4. The method for fermenting the traditional Chinese medicine 'Xinqibai' by using probiotics according to any one of claims 1 to 3, wherein the fermentation in the third step of the method comprises the steps of inoculating granular rhizopus oryzae into the fermentation material by the volume percentage of 3-8%, controlling the pH value by sodium carbonate at 35 ℃ and 200r/min, controlling the pH value at 4.0-5.0, and fermenting for 24-36 h; adjusting pH to 7.5-8.0 with ammonia water, maintaining the temperature at 35 deg.C, inoculating lactobacillus suspension at 2-5% volume, co-culturing with Rhizopus oryzae, and fermenting for 24-36 hr; the lactobacillus suspension is prepared by 10mmol/L phosphate buffer solution (pH8.0), and the ratio of lactobacillus to phosphate buffer solution is (by weight parts) 1-15: 100.
5. the method for fermenting the Chinese medicine 'Xinqibai' with probiotics according to any one of claims 1 to 3, wherein the method comprises the third step of fermenting in a fermentation tank, wherein 25L of fermentation materials are filled in every 50L of fermentation tank, the pH value of the fermentation is 4.0-5.0, the fermentation temperature is 32-34 ℃, and the fermentation period is 60 hours; controlling the aeration flow rate to be 0.3L/L.min and the rotation speed to be 50-80rpm from the beginning of fermentation to the 20 th hour; controlling the aeration flow rate to be 0.6L/L.min and the rotation speed to be 450rpm from the 20 th hour to the 40 th hour of self-fermentation; controlling the aeration flow rate to be 0.6L/L.min and the rotation speed to be 50rpm from the 40 th hour to the 60 th hour of self-fermentation; the defoaming agent is added in the early stage of fermentation, the addition amount of the defoaming agent is 0.0 lwt%, and the defoaming agent is gradually added to 0.1 wt% from the 30 th hour of fermentation.
6. The method according to any one of claims 1 to 3, wherein in the step (1), the lactobacillus is enlarged and cultured by using an ultrasonic biostimulation growth instrument, and the ultrasonic biostimulation growth instrument is used for treating parameters of 30kHz, 10W of power, 10s of ultrasonic time and 20s of interval time for 30 minutes of ultrasonic stimulation.
7. The method for fermenting a traditional Chinese medicine 'Xinqibai' with probiotics according to any one of claims 1 to 3, wherein the method specifically comprises: after the lactobacillus is subjected to seed culture, carrying out amplification culture; wherein the enlarged culture is carried out by fermenting and culturing for 36h at 30 ℃ and then continuing fermenting and culturing for 36h at 32 ℃.
8. The method for fermenting the traditional Chinese medicine 'Xinqibai' by using the probiotics according to any one of claims 1 to 3, wherein the Rhizopus oryzae is cultured by seeds to form a spore suspension, and then is cultured by fermentation base until granular thalli are formed; inoculating rhizopus oryzae subjected to basic fermentation culture into a fermentation material, controlling the pH value to be 4.0-5.0 by using sodium carbonate, fermenting at 35 ℃ for 24-36h, adjusting the pH value to be 7.5-8.0 by using ammonia water, inoculating the lactobacillus obtained in the step (1), maintaining the temperature at 35 ℃, and co-culturing with the rhizopus oryzae.
9. The method according to any one of claims 1 to 3, wherein the lactobacillus is cultured in an expanded medium according to the following formula in step (1): 60g/L glucose, 2.0g/L urea, KH2PO40.6g/L, 30g/L ammonium chloride, MgSO47H20.5g/L of O, 0.5g/L of yeast extract, 20g/L of liquid paraffin and water as a solvent; the expansion culture method adopts a variable temperature culture mode, and preferably comprises the following steps: inoculating the seed liquid into fermentation amplification culture medium at a volume percentage of 10%, and performing fermentation culture at 33 deg.C and 200r/minThe temperature is raised to 35 ℃ after 48 hours, the culture is continued for 48 hours, and the thallus density is more than or equal to 30g wet thallus/L; after the expanded culture, filtering and recovering thalli, and preserving at a low temperature of 4 ℃ for later use; when used, a lactic acid bacterium suspension was prepared with 10mmol/L phosphate buffer solution (pH 8.0).
10. The method according to any one of claims 1 to 3, wherein the rhizopus oryzae is cultured in a medium having the following formulation: 30g/L of yeast extract, 30g/L of malt extract, 30g/L of peptone, 20g/L of glycerol, 20g/L of agar and water as a solvent; the seed culture method comprises the following steps: culturing at 35 deg.C for 5-7 days, collecting a fresh slant strain, adding sterile water, and making into spore suspension.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111471727A (en) * | 2020-04-16 | 2020-07-31 | 浙江树人学院（浙江树人大学） | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
| CN115770210A (en) * | 2022-12-02 | 2023-03-10 | 江南大学 | Rice-purple sweet potato composite fermentation filtrate for cosmetics and preparation method and application thereof |
| CN116492284A (en) * | 2023-06-19 | 2023-07-28 | 妙越缇卡生物医药科技(山东)有限公司 | Whitening antibacterial lactobacillus casei SF-L-12 fermentation product and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471727A (en) * | 2020-04-16 | 2020-07-31 | 浙江树人学院（浙江树人大学） | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
| CN111471727B (en) * | 2020-04-16 | 2023-01-31 | 浙江树人学院（浙江树人大学） | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
| CN115770210A (en) * | 2022-12-02 | 2023-03-10 | 江南大学 | Rice-purple sweet potato composite fermentation filtrate for cosmetics and preparation method and application thereof |
| CN116492284A (en) * | 2023-06-19 | 2023-07-28 | 妙越缇卡生物医药科技(山东)有限公司 | Whitening antibacterial lactobacillus casei SF-L-12 fermentation product and application thereof |
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