CN115197976A - Method for preparing polygonatum cyrtonema prebiotics by microbial fermentation - Google Patents
Method for preparing polygonatum cyrtonema prebiotics by microbial fermentation Download PDFInfo
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- CN115197976A CN115197976A CN202210639014.2A CN202210639014A CN115197976A CN 115197976 A CN115197976 A CN 115197976A CN 202210639014 A CN202210639014 A CN 202210639014A CN 115197976 A CN115197976 A CN 115197976A
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- fermentation
- polygonatum cyrtonema
- prebiotics
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- polygonatum
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- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
Abstract
The invention discloses a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, belonging to the technical field of fermentation engineering. The polygonatum cyrtonema fermentation liquid is prepared by fermenting polygonatum cyrtonema with aspergillus oryzae and then carrying out water extraction on the polygonatum cyrtonema after fermentation, and fermenting the water extract again with saccharomycetes. Compared with the sealwort aqueous extract obtained by the traditional hot water extraction method, the sealwort fermentation liquid prepared by the invention has obviously improved contents of active ingredients such as polysaccharide, polyphenol, beta-glucan and the like. Meanwhile, compared with the traditional nine-steaming and nine-sun method, the method of the invention has simpler operation.
Description
Technical Field
The invention relates to a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, belonging to the technical field of fermentation engineering.
Background
Polygonatum cyrtonema is a perennial herb plant of Polygonatum of Liliaceae, is one of 3 medicinal polygonatum-derived plants of Polygonatum cyrtonema, polygonatum cyrtonema and Polygonatum kingianum, wherein Polygonatum cyrtonema has the best quality and the best drug effect, and is also called as rhizoma polygonati with ginger shape because the rhizome is nodular and is slightly flat like ginger.
Polygonatum cyrtonema belongs to perennial herbaceous plants, has strong adaptability, mostly grows in the mountain shrubs and forest edge grasses or under forests in the shade, and is an under-forest interplanting crop; the rhizome of Polygonatum cyrtonema contains polysaccharide, steroid saponin and a small amount of flavonoid, and has sweet taste. The Polygonatum polysaccharide can effectively improve SOD activity of human cells and organism immunity, and has the functions of reducing blood sugar and blood fat, improving immunity, improving memory, resisting inflammation, resisting tumor, etc.
The processing technology of the ancient rhizoma polygonati is mainly 'nine times steaming and nine times drying', the ancient Chinese rhizoma polygonati is originated from the ancient times and developed in the modern times; the processing technology is evolved from unprocessed → single steaming → heavy steaming → nine times steaming and nine times drying in the sun, and can achieve the purposes of reducing toxicity, enhancing curative effect, changing meridian tropism, facilitating storage, killing germs and the like. The sealwort is steamed and dried for nine times, and the chemical components and the medicinal effect of the sealwort are obviously changed, so the sealwort is widely applied to the fields of medicaments, health-care products, foods, cosmetics and the like. However, the nine-steaming nine-processing process is long in time consumption and high in energy consumption.
Microbial fermentation can produce numerous enzymes that can cut large molecular substances into small molecular substances very efficiently. Meanwhile, many other active ingredients can be produced, such as cordycepin, adenosine, SOD and other active ingredients can be produced in the fermentation process of cordyceps militaris. Therefore, the effect of the traditional nine-steaming nine-processing can be completely realized by means of the force of microorganisms, and the effect is even better. For example, chinese patent CN 107753741A provides a method for deep processing of polygonatum and a product thereof, the invention intends to utilize microorganisms to ferment polygonatum with the help of the power of microorganisms, firstly utilizes the strong starch sugar and protein decomposition ability of aspergillus niger and aspergillus oryzae, then enzymolyzes macromolecular substances into small molecular substances, and then utilizes cordyceps militaris to carry out secondary fermentation, and compounds active ingredients such as cordycepin, polysaccharide and the like generated by cordyceps militaris, thereby endowing the fermented polygonatum with better treatment and health care effects.
However, no report on the preparation of prebiotics by fermenting polygonatum exists at present.
Disclosure of Invention
The invention aims to provide a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation.
In order to achieve the purpose, the invention provides a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, which comprises the following steps:
mixing Polygonatum cyrtonema powder with water to obtain a mixture;
inoculating Aspergillus oryzae into the mixture, and performing a first fermentation to obtain a first fermentation product;
extracting the first fermentation product with water to obtain an extract;
and (3) sterilizing the extract, inoculating saccharomycetes, and performing secondary fermentation to obtain polygonatum cyrtonema prebiotics.
Preferably, the mass ratio of polygonatum cyrtonema powder to water in the mixture is 1:1 to 3.
Preferably, the inoculation amount of the aspergillus oryzae is 0.05-1% of the mass of polygonatum cyrtonema powder.
Preferably, the temperature of the first fermentation is 30-35 ℃; the time of the first fermentation is 24-72 h.
Preferably, the temperature of the water extraction is 80-100 ℃; the number of water extractions is 1-3; the time of each water extraction is 1-3 h.
Preferably, the inoculation amount of the yeast is 1-5% of the mass of polygonatum cyrtonema powder.
Preferably, the temperature of the second fermentation is 25-35 ℃; the time of the second fermentation is 48-96 hours;
the second fermentation is shaking table fermentation, and the rotation speed of the shaking table fermentation is 100-180 r/min.
The invention also provides polygonatum cyrtonema prebiotics prepared by the method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, wherein the polygonatum cyrtonema prebiotics comprises beta-glucan with the concentration of 0.276-0.284 mg/mL and polyphenol with the concentration of 0.0814-0.0846 mg/mL.
The invention also provides application of the polygonatum cyrtonema prebiotics in food, cosmetics or promotion of growth of probiotics.
Preferably, the application in preparing food for resisting oxidation and/or regulating intestinal flora or preparing cosmetics for resisting oxidation is included.
Preferably, when the polygonatum cyrtonema prebiotic is applied to food or cosmetics, the polygonatum cyrtonema prebiotic can be used for completely or partially replacing the amount of water in the food or cosmetic formula.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, wherein aspergillus oryzae is adopted to ferment polygonatum cyrtonema and then carry out water extraction, saccharomycetes is used to carry out secondary fermentation on a water extract to prepare polygonatum cyrtonema fermentation liquor, and the obtained polygonatum cyrtonema fermentation liquor contains polygonatum cyrtonema prebiotics, has good effect of promoting the growth of probiotics and can be used as prebiotics to promote the growth of the probiotics;
(2) Compared with the sealwort aqueous extract obtained by the traditional hot water extraction method, the sealwort polysaccharose fermentation liquid prepared by the invention has the advantages that the content of active ingredients such as polysaccharide, polyphenol, beta-glucan and the like is obviously improved, and the sealwort fermentation liquid has good oxidation resistance; in addition, compared with the traditional nine-steaming and nine-sun method, the method of the invention has simpler operation.
Drawings
FIG. 1 is a flow chart of an experiment of example 1;
FIG. 2 shows the effect of different fermentation broths on DPPH removal;
FIG. 3 shows ABTS clearance effect of different fermentation broths;
FIG. 4 shows the effect of different fermentation broths on superoxide anion removal;
FIG. 5 shows the scavenging effect of different fermentation liquids on hydroxyl radicals;
FIG. 6 shows the effect of different fermentation broths on hydrogen peroxide removal;
FIG. 7 is a graph of the effect of different treatments on the OD600 of Bifidobacterium lactis BL-99;
FIG. 8 is the log CFU/mL of Bifidobacterium lactis BL-99 as a function of different treatments;
FIG. 9 is a graph of the effect of different treatments on the pH of Bifidobacterium lactis BL-99;
FIG. 10 is a graph of the effect of different treatments on the OD600 of Lactobacillus paracasei K56;
FIG. 11 is a graph of the log CFU/mL of Lactobacillus paracasei K56 effect of different treatments;
FIG. 12 is a graph of the effect of different treatments on the pH of Lactobacillus paracasei K56;
FIG. 13 is a graph of the effect of different treatments on the OD600 of Lactobacillus paracasei ET-22;
FIG. 14 is a graph of the log CFU/mL of Lactobacillus paracasei ET-22 as a function of different treatments;
FIG. 15 is a graph of the effect of different treatments on the pH of Lactobacillus paracasei ET-22.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
The invention provides a method for preparing polygonatum cyrtonema prebiotics by microbial fermentation, which comprises the following steps:
mixing Polygonatum cyrtonema powder with water to obtain a mixture;
inoculating aspergillus oryzae into the mixture, and performing first fermentation to obtain a first fermentation product;
performing water extraction on the first fermentation product to obtain an extract;
and (3) sterilizing the extract, inoculating saccharomycetes, and performing secondary fermentation to obtain polygonatum cyrtonema prebiotics.
Firstly, polygonatum sibiricum powder is mixed with water to obtain a mixture.
In the invention, the polygonatum cyrtonema powder is preferably obtained by washing, drying, slicing and crushing rhizomes of polygonatum cyrtonema; the drying mode is preferably drying; the grain size of the polygonatum cyrtonema powder is preferably 60-100 meshes, and more preferably 80 meshes; the polygonatum cyrtonema powder is preferably stored in a drying dish in a sealing way. In the present invention, the water is preferably distilled water. In the present invention, the mass ratio of polygonatum cyrtonema powder to water in the mixture is preferably 1: (1 to 3), more preferably 1:2. in the present invention, the temperature of the mixing is preferably 121 ℃; the mixing time is preferably 15min; the mixing is preferably carried out in a sterile kettle; the mixing process can sterilize and gelatinize starch in Polygonatum cyrtonema Hua powder.
After the mixture is obtained, the aspergillus oryzae is inoculated in the mixture and is subjected to first fermentation to obtain a first fermentation product.
In the present invention, the initial temperature of the mixture is preferably 30 to 40 ℃. In the present invention, the amount of the Aspergillus oryzae to be inoculated is preferably 0.05 to 1%, more preferably 0.1 to 0.5% by mass of the Polygonatum sibiricum powder. In the present invention, the first fermentation mode is preferably solid state fermentation. In the present invention, the temperature of the first fermentation is preferably 30 to 35 ℃; the time for the first fermentation is preferably 24 to 72 hours, more preferably 36 to 48 hours.
After the first fermentation product is obtained, the invention carries out water extraction on the first fermentation product to obtain the extract.
In the present invention, before subjecting the first fermentation product to water, it is preferable to further include sterilizing the first fermentation product; the temperature of the sterilization is preferably 121 ℃; the time for the sterilization is preferably 15min. In the present invention, the mass ratio of the first fermentation product to water is preferably 1: (10 to 30), more preferably 1: (20-25); in the present invention, the temperature of the water extraction is preferably 80 to 100 ℃, and more preferably 90 ℃; the number of the water extractions is preferably 1 to 3, and more preferably 2; the time for each water extraction is preferably 1 to 3 hours, more preferably 2 hours. After the water extraction, the invention preferably further comprises the step of carrying out vacuum filtration on the water extract obtained by water extraction to obtain a filtration liquid as an extract.
After the extract is obtained, the extract is sterilized and inoculated with saccharomycetes, and secondary fermentation is carried out to obtain the polygonatum cyrtonema prebiotics.
In the present invention, the temperature of the sterilization is preferably 121 ℃; the time for the sterilization is preferably 15min. After the sterilization, it is preferable to further include cooling the sterilized extract to 30 to 40 ℃. In the invention, the inoculation amount of the yeast is 1-5% of the polygonatum cyrtonema powder by mass, and more preferably 2-3%. In the present invention, the temperature of the second fermentation is preferably 25 to 35 ℃, more preferably 30 ℃; the time of the second fermentation is preferably 48 to 96 hours, and more preferably 72 hours; the second fermentation is preferably carried out in a fermentation flask; the opening of the fermentation bottle is preferably covered with gauze; the fermentation bottle is preferably placed on a shaking table for fermentation, and the rotation speed of the second fermentation is preferably 100-180 r/min, and more preferably 150rpm. The present invention preferably comprises, after the second fermentation, sterilizing the product of the second fermentation. In the present invention, the temperature of the sterilization is preferably 121 ℃; the time for the sterilization is preferably 15min.
After the polygonatum cyrtonema subjected to the first fermentation and the second fermentation, the antioxidant activity is obviously improved, and the removal effect on ABTS clearance and hydroxyl radical clearance is most obviously improved.
The invention also provides polygonatum cyrtonema prebiotics prepared by the preparation method of the scheme; the polygonatum cyrtonema prebiotics comprises the following components in concentration: beta-glucan 0.276-0.284 mg/mL and polyphenol 0.0814-0.0846 mg/mL.
Compared with the conventional polygonatum cyrtonema hot water extracting solution, the polygonatum cyrtonema prebiotic prepared by the method has high content of micromolecule sugar, polyphenol and beta-glucan, and the promoting effect on probiotics is also obviously improved.
The invention also provides application of the polygonatum cyrtonema prebiotics in foods, cosmetics or probiotics growth promotion; in the invention, the polygonatum cyrtonema prebiotics is preferably used for preparing food for resisting oxidation and/or regulating intestinal flora or preparing cosmetics for resisting oxidation; in the present invention, when the polygonatum cyrtonema prebiotic is applied to food or cosmetics, the polygonatum cyrtonema prebiotic in the food or cosmetics is preferably used to replace the whole or part of the water in the food or cosmetic formulation.
In the present invention, the probiotic bacteria preferably include one or both of bifidobacterium lactis and lactobacillus paracasei.
In the following examples, percentages are expressed by mass unless otherwise specified.
Example 1 preparation of Polygonatum sibiricum fermentation broth by double-strain fermentation
1. Fermentation process
The first step of Aspergillus oryzae fermentation: weighing 20g of crushed polygonatum cyrtonema powder which passes through 80 meshes, and mixing the raw materials according to the weight ratio of 1:1 adding distilled water, and sterilizing at 121 deg.C for 15min. Cooling the sterilized polygonatum cyrtonema sample at room temperature, adding 0.05% of aspergillus oryzae at 30 ℃ after the temperature is reduced to 30-40 ℃, standing and culturing for 24h, obtaining a polygonatum cyrtonema solid fermentation product a after fermentation is finished, and adding distilled water according to the mass of the initial raw materials until the ratio of material to liquid is 1:25, extracting for 2h at the extraction temperature of 80 ℃. And (4) carrying out vacuum filtration on the fermentation liquor, and fixing the volume of the filtered supernatant to 500mL to obtain polygonatum cyrtonema fermentation liquor b for the second-step fermentation.
The second step of yeast fermentation: sterilizing the fermentation liquid b after suction filtration for 15min at 121 ℃ in an autoclave. And (3) cooling the sterilized polygonatum cyrtonema fermentation liquor b at room temperature, adding 1% of Angel yeast YA300 to the fermentation liquor b after the temperature is reduced to 30-40 ℃, and culturing for 48 hours at the temperature of 30 ℃ and at the speed of 120 r/min. Obtaining polygonatum cyrtonema fermentation liquor b. See figure 1 for experimental flow chart.
2. Data relating to the experiment
2.1 active ingredients in different fermentation broths see Table 1
TABLE 1 active ingredients in different fermentation broths
In table 1, the 85% alcohol precipitation means that absolute ethanol is added to the polysaccharide solution system until the volume of ethanol is 85% of the total system volume. And (4) analyzing results: the first step of aspergillus oryzae fermentation can obtain that aspergillus oryzae can use an enzyme system generated by the aspergillus oryzae to destroy the cell wall of polygonatum cyrtonema so as to be more beneficial to dissolving out active ingredients. Meanwhile, macromolecular polysaccharide (85 percent of alcohol precipitation polysaccharide) can be converted into micromolecular polysaccharide (85 percent of alcohol precipitation supernatant) through enzymolysis, namely, the yield of the 85 percent of alcohol precipitation supernatant in the fermentation liquid b is improved by 25.02 percent compared with that in the unfermented fermentation, and meanwhile, the contents of beta-glucan and polyphenol are also improved. And through the second step of yeast fermentation, the saccharomycetes metabolize by utilizing sugar substances in the fermentation liquor b, so that the content of beta-glucan and polyphenol can be improved, and the fragrance of the fermentation liquor c is improved.
2.2 antioxidant Activity of different fermentation broths
2.2.1 Effect on DPPH removal see FIG. 2.
2.2.2 Effect on ABTS clearance see FIG. 3.
2.2.3 superoxide anion scavenging effect see FIG. 4.
2.2.4 hydroxyl radical scavenging effect see FIG. 5.
2.2.5 hydrogen peroxide scavenging effect see figure 6.
2.2.6 from the data of FIGS. 2-6, the IC50 of the samples for different ion scavenging effects were calculated using the span software as shown in Table 2:
TABLE 2 IC50 of samples for different ion scavenging effects
And (4) analyzing results: the IC50 of the three fermentation liquors on DPPH clearance is respectively diluted by 20.18 times, 23.31 times and 27.52 times, the IC50 on ABTS clearance is respectively diluted by 2.37 times, 6.68 times and 12.30 times, the IC50 on hydrogen peroxide clearance is respectively diluted by 39.98 times, 54.33 times and 44.31 times, the IC50 on superoxide anion clearance is respectively diluted by 17.98 times, 22.34 times and 19.12 times, and the IC50 on hydroxyl radical clearance is respectively diluted by 2.87 times, 2.62 times and 5.40 times. The three fermentation liquors are compared to find that after two-step fermentation, the antioxidant effect of the fermentation liquor c is improved compared with the effect of the fermentation liquor which is not fermented, wherein the removal effects on ABTS clearance and hydroxyl radical clearance are improved most obviously and are respectively improved by 5.19 times and 1.92 times.
2.3 growth promoting Effect on different probiotics after two-step fermentation
The method comprises the following steps: selecting Bifidobacterium lactis BL-99 (inhibiting inflammation, relieving bone loss, and promoting digestion); lactobacillus paracasei K56 (fat-reducing); lactobacillus paracasei ET-22 (inhibiting oral pathogens) is used as probiotic. Dissolving bacterial powder by using normal saline, coating bacterial liquid on a flat plate, culturing K56, ET-22 and BL-99 for 48h at 37 ℃ in an anaerobic environment, transferring the bacterial liquid to an MRS broth culture medium, culturing K56 and ET-22 for 24h and culturing BL-99 for 48h under the same condition, diluting the bacterial liquid by 1000 times by using 0.89% normal saline, absorbing 160uL to 8mL of culture medium with different carbon sources (no carbon source, 1% glucose, 1% fructo-oligosaccharide, 50% non-fermented polygonatum multiflorum extract and 50% fermentation liquid b, and recording OD values, PH and colony count for 6, 12, 24, 36 and 48h culture at 37 ℃.
2.3.1 example one: the effect of promoting bifidobacterium lactis BL-99 is shown in FIGS. 7 to 9.
2.3.2 example two: the effect of promoting lactobacillus paracasei K56 is shown in fig. 10 to 12.
2.3.2 example three: the effect of promoting Lactobacillus paracasei ET-22 is shown in FIGS. 13 to 15.
And (4) analyzing results: in the promotion effect on the three probiotics, OD600 and log CFU/mL represent the increasing number of the probiotics, and the result shows that the number of the three probiotics reaches the maximum at 24h, wherein the number of the three probiotics in a control group 1% glucose group is the maximum because glucose is a monosaccharide which can be directly utilized by the probiotics and is a good carbon source, fructo-oligosaccharide is a common prebiotic and is generally selected as a positive control, and the sugar content in 50% of the polygonatum cyrtonema extracting solution added with 50% of non-fermented polygonatum sibiricum is equivalent to that in 50% of fermentation liquid b with 1% of glucose, and the result shows that the colony number of 50% of fermentation liquid c is equivalent to that of the glucose in the three probiotics and is higher than the number of 1% of fructo-oligosaccharide and 50% of non-fermented polygonatum sibiricum extracting solution, which shows that the fermentation liquid c can obviously promote the growth of the probiotics. The pH value can be used for indirectly indicating the content of short-chain fatty acid, and the short-chain fatty acid metabolized by probiotics has the effects of lowering the pH value of intestinal tracts, inhibiting colonization of pathogenic bacteria, enhancing absorption of nutrient substances and mineral substances, stimulating secretion of gastrointestinal hormone (increasing secretion of gastrin and motilin and promoting secretion of glucagon and insulin) so as to regulate lipid and sugar metabolism of organisms and the like. The pH value changes of different samples in the process of promoting the growth of the three probiotics show that the pH values of 1% glucose and 50% fermentation liquor c are the lowest and have small difference, which indicates that the contents of the short-chain fatty acid in the two samples are consistent. The above results indicate that fermentation broth c has a significant promoting effect on probiotics and contributes to the production of short chain fatty acids. Can be used as functional material for regulating intestinal flora.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.
Claims (10)
1. A method for preparing polygonatum cyrtonema prebiotics by microbial fermentation is characterized by comprising the following steps:
mixing polygonatum cyrtonema powder with water to obtain a mixture;
inoculating aspergillus oryzae into the mixture, and performing first fermentation to obtain a first fermentation product;
performing water extraction on the first fermentation product to obtain an extract;
and (3) sterilizing the extract, inoculating saccharomycetes, and performing secondary fermentation to obtain polygonatum cyrtonema prebiotics.
2. The method for preparing polygonatum cyrtonema prebiotics by microbial fermentation according to claim 1, wherein the mass ratio of polygonatum cyrtonema powder to water in the mixture is 1:1 to 3.
3. The method for preparing polygonatum cyrtonema prebiotics through microbial fermentation according to claim 1, wherein the inoculation amount of aspergillus oryzae is 0.05-1% of the mass of polygonatum cyrtonema powder.
4. The method for preparing polygonatum cyrtonema prebiotics by microbial fermentation according to claim 1 or 3, wherein the temperature of the first fermentation is 30-35 ℃; the time of the first fermentation is 24-72 h.
5. The method for preparing polygonatum cyrtonema prebiotics by microbial fermentation according to claim 1, wherein the temperature of the water extraction is 80-100 ℃; the times of water extraction are 1 to 3; the time of each water extraction is 1-3 h.
6. The method for preparing polygonatum cyrtonema prebiotics by microbial fermentation according to claim 1, wherein the inoculation amount of the yeast is 1-5% of the mass of polygonatum cyrtonema powder.
7. The method for preparing polygonatum cyrtonema prebiotics by microbial fermentation according to claim 1, wherein the temperature of the second fermentation is 25-35 ℃; the time of the second fermentation is 48-96 h;
the second fermentation is shaking table fermentation, and the rotation speed of the shaking table fermentation is 100-180 r/min.
8. The polygonatum cyrtonema prebiotic prepared by the method for preparing the polygonatum cyrtonema prebiotic by microbial fermentation of any one of claims 1 to 7, wherein the polygonatum cyrtonema prebiotic comprises beta-glucan and polyphenol with the concentrations of 0.276 to 0.284mg/mL and 0.0814 to 0.0846mg/mL respectively.
9. Use of polygonatum cyrtonema prebiotic according to claim 8 in food, cosmetics or to promote the growth of probiotics.
10. Use according to claim 9, for the preparation of a food product for combating oxidation and/or modulating the intestinal flora, or for the preparation of an anti-oxidative cosmetic product.
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| CN115261427B (en) * | 2022-09-27 | 2022-12-16 | 云南英格生物技术有限公司 | Sealwort fermented oligosaccharide and preparation method and application thereof |
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