CN109825533B - Fermentation medium for preparing high-activity phellinus igniarius fermentation product and fermentation culture method thereof - Google Patents
Fermentation medium for preparing high-activity phellinus igniarius fermentation product and fermentation culture method thereof Download PDFInfo
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Abstract
The invention discloses a fermentation medium for preparing a high-activity phellinus igniarius fermentation product and a fermentation culture method thereof, wherein the fermentation medium comprises the following components in parts by weight: 20-30 parts of soybean sprout pulp, 5-10 parts of wheat cellulose, 5-10 parts of radix tetrastigme zymolyte, 2-3 parts of mulberry twig and cyclocarya paliurus extract, 0.05-0.1 part of calcium chloride, 0.3-0.5 part of lactose, 1-3 parts of glucose, 0.02-0.05 part of salicylic acid and 80-100 parts of water. The fermentation medium of the invention is beneficial to improving the content and activity of active ingredients in the final product phellinus igniarius fermentation product. The prepared high-activity phellinus igniarius fermentation product has an obvious protective effect on D-galactose-induced subacute oxidative damage of mice.
Description
Technical Field
The invention relates to a fermentation medium for preparing a high-activity phellinus igniarius fermentation product and a fermentation culture method thereof.
Background
Phellinus linteus (Inonotus sanghuanghuang) is parasitic on mulberry tree, and its fruiting body mostly overlaps together, with horseshoe shape or irregular shape, and makes lemon yellow to golden yellow when young, and earthy yellow or yellow brown when old. Phellinus linteus is a precious large perennial medicinal wood rot fungus, which usually grows on Morus plants. Phellinus linteus is called as "forest gold" by modern people. Phellinus igniarius is a medicinal fungus with the highest effective rate in the current internationally recognized biological cancer treatment medicaments. China is a large export country of wild medicinal fungi, but the research on phellinus igniarius is still in the initial stage at present, and the technology is not mature. Because the limited phellinus igniarius wild resources in China have long growth period and poor stability of effective components, are excessively collected and are less and less, the phellinus igniarius wild resources are difficult to become stable industrial product sources, and the market requirements can not be met far away. The biological product prepared by the biological fermentation technology is the basis for overcoming the best way of utilizing wild resources with limited biomass and realizing the development and utilization of useful metabolites. Compared with the traditional sporocarp cultivation, the method for preparing the secondary metabolite of phellinus igniarius through liquid fermentation has the advantages of short production period, more metabolites, easy quality control, large-scale production and the like. The fermentation culture medium for preparing the secondary metabolite of the phellinus igniarius by liquid fermentation has important significance, and can directly influence the activity of the secondary metabolite of the phellinus igniarius.
Disclosure of Invention
In view of the above, the present invention aims to provide a fermentation medium for preparing a high-activity phellinus igniarius fermentation product and a fermentation culture method thereof, which can effectively improve the activity of the phellinus igniarius fermentation product.
The adopted technical scheme is as follows:
a fermentation medium for preparing a high-activity phellinus igniarius fermentation product comprises the following components in parts by weight: 20-30 parts of soybean sprout pulp, 5-10 parts of wheat cellulose, 5-10 parts of radix tetrastigme zymolyte, 2-3 parts of mulberry twig and cyclocarya paliurus extract, 0.05-0.1 part of calcium chloride, 0.3-0.5 part of lactose, 1-3 parts of glucose, 0.02-0.05 part of salicylic acid and 80-100 parts of water.
Further, the soybean sprout pulp is prepared by the following method: selecting soybean with complete grain shape, placing into a full-automatic bean sprouting machine, spraying for sprouting, and stopping sprouting when the bean sprout head is exposed to 4-5 mm; mixing soybean sprout and water at a weight ratio of 1:1, and pulping at high speed to obtain soybean sprout pulp.
Further, the ramulus mori and cyclocarya paliurus extracts are prepared by the following method: respectively crushing ramulus mori and cyclocarya paliurus leaves, mixing the ramulus mori and the cyclocarya paliurus leaves according to a weight ratio of 1:1, adding 20-30 times of 70 vol% ethanol solution, performing shake extraction for 10 hours in a shaking table at 40-50 ℃, filtering to obtain a supernatant, performing reduced pressure concentration on the supernatant, and performing vacuum freeze drying to obtain the ramulus mori and cyclocarya paliurus extracts.
Further, the radix tetrastigme zymolyte is prepared by the following method: drying and powdering radix tetrastigme rhizome, adding 10 times of deionized water, adding amylase with volume mass ratio of 0.1-0.2ml/100g, and performing enzymolysis at 70 ℃ for 3-5 hours to obtain radix tetrastigme zymolyte.
In the technical scheme, the soybean sprout pulp has the following functions: of proteins of germinated soybeansVB2、VB6、VCThe content is obviously improved; the anti-nutritional factors such as trypsin inhibitor, phytic acid and the like are decomposed due to germination, and the contents of soybean isoflavone, lecithin, saponin and the like are increased to different degrees, so that the nutritional value and the medicinal value of the germinated soybeans are improved;
the radix tetrastigme zymolyte has the following effects: the amylase is utilized to carry out enzymolysis saccharification on a large amount of starch in the radix tetrastigme, and the starch is hydrolyzed into maltose and glucose which are easily absorbed by strains, so that the effective components are better released.
The mulberry twig and cyclocarya paliurus extract has the following effects: mycelium biomass can be increased.
The role of lactose is: can promote the synthesis of polysaccharide.
The effects of calcium chloride and salicylic acid are: can promote the synthesis of triterpenes.
A fermentation culture method for preparing high-activity phellinus igniarius fermentation products comprises the following steps:
(1) inoculating the activated phellinus igniarius strain into a seed liquid culture medium to carry out phellinus igniarius fermentation culture for 3-7 days to obtain phellinus igniarius liquid seed liquid;
(2) inoculating the phellinus igniarius liquid seed solution accounting for 1-5% of the volume of the fermentation medium into the fermentation medium in any one of the schemes, culturing for 5-8 days at the temperature of 24-27 ℃, and after the fermentation is finished, carrying out vacuum freeze drying on the fermentation liquid to obtain a high-activity phellinus igniarius fermentation product.
Further, the seed liquid culture medium in the step (1) comprises the following components in parts by weight: 20 parts of germinated soybean, 5 parts of glucose, 0.05 part of magnesium sulfate and 0.02 part of potassium dihydrogen phosphate.
Further, the phellinus linteus strain in the step (1) is selected from i.sanghuanghuang of Qiandao lake, Hangzhou, Zhejiang.
The invention has the beneficial effects that:
(1) the nutrient components and the effective components in the fermentation culture solution are better biodegraded and converted through fermentation, which is beneficial to improving the content and the activity of the active components in the final product phellinus igniarius fermentation product.
(2) The prepared high-activity phellinus igniarius fermentation product has an obvious protective effect on D-galactose-induced subacute oxidative damage of mice.
Drawings
FIG. 1 is a graph showing the effect of fermentation products of Phellinus linteus on mouse serum oxidation index MDA.
FIG. 2 is a graph showing the effect of fermentation products of Phellinus linteus on mouse serum oxidation index T-AOC.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the use and purpose of these exemplary embodiments are merely to exemplify the present invention, and do not set forth any limitation on the actual scope of the present invention in any form, and the scope of the present invention is not limited thereto.
Example 1
The method for preparing the high-activity phellinus igniarius fermentation product comprises the following steps of:
(1) preparing a fermentation medium, wherein the fermentation medium comprises the following components: 20g/L of soybean sprout pulp, 5g/L of wheat cellulose, 5g/L of radix tetrastigme zymolyte, 2g/L of mulberry twig and cyclocarya paliurus extract, 0.05g/L of calcium chloride, 0.3g/L of lactose, 1g/L of glucose, 0.02g/L of salicylic acid and 80g/L of water.
Wherein, the preparation of soybean sprout pulp: selecting soybean with complete grain shape, placing into a full-automatic bean sprouting machine, spraying for sprouting, and stopping sprouting when the bean sprout head is exposed to 4-5 mm; preparing the soybean sprouts: and (5) pulping at a high speed for later use according to the water mass ratio of 1: 1.
Preparing the extracts of mulberry twigs and cyclocarya paliurus: respectively crushing ramulus mori and cyclocarya paliurus leaves, mixing the crushed ramulus mori and the cyclocarya paliurus leaves according to the weight ratio of 1:1, adding 20-30 times (by weight) of 70 vol% ethanol solution into a shaking table at 40-50 ℃, performing shaking extraction for 10 hours, filtering to obtain a supernatant, performing reduced pressure concentration on the supernatant, and performing vacuum freeze drying to obtain the extract.
Radix tetrastigme zymolyte: drying and powdering radix tetrastigme rhizome, adding 10 times of deionized water, adding amylase with volume-mass ratio of (0.1-0.2ml/100g), and performing enzymolysis at 70 ℃ for 3-5 hours for later use.
(2) Liquid fermentation culture of phellinus igniarius; inoculating the activated Phellinus Linteus strain in seed liquid culture medium, and fermenting and culturing Phellinus Linteus for 3-7 days to obtain Phellinus Linteus liquid seed liquid; wherein the seed liquid culture medium comprises the following components: 1L of the fermentation broth contains 20g of germinated soybean, 5g of glucose, 0.05g of magnesium sulfate and 0.02g of potassium dihydrogen phosphate. And the selected phellinus linteus strain is selected from the following:
| collection site/source of strain | Host/cultivation material | ITS comparison result | GenBank accession number |
| Qiandao lake, Hangzhou, Zhejiang province | Mulberry tree | I.sanghuang | HM584808 |
Remarking: ITS (internal Transcribed spacer) identification refers to a method for obtaining information of unknown fungal species by DNA sequencing of ITS sequences and comparing the ITS sequences obtained by sequencing with known fungal ITS sequences.
(3) Liquid fermentation culture: inoculating a phellinus igniarius liquid seed solution accounting for 1-5% of the volume of the fermentation medium into the fermentation medium in the step (1), culturing for 5-8 days at the temperature of 24-27 ℃, and after the fermentation is finished, carrying out vacuum freeze drying on the fermentation liquid to obtain a high-activity phellinus igniarius fermentation product.
Example 2
With reference to example 1, the composition of the fermentation medium differs from that of example 1: 25g/L of soybean sprout pulp, 7g/L of wheat cellulose, 8g/L of radix tetrastigme zymolyte, 3g/L of mulberry twig and cyclocarya paliurus extract, 0.08g/L of calcium chloride, 0.4g/L of lactose, 2g/L of glucose, 0.04g/L of salicylic acid and 90g/L of water.
Example 3
With reference to example 1, the composition of the fermentation medium differs from that of example 1: 30g/L of soybean sprout pulp, 10g/L of wheat cellulose, 10g/L of radix tetrastigme zymolyte, 3g/L of mulberry twig and cyclocarya paliurus extract, 0.1g/L of calcium chloride, 0.5g/L of lactose, 3g/L of glucose, 0.05g/L of salicylic acid and 100g/L of water.
Comparative example 1
Referring to example 2, in contrast to example 1, in the composition of the fermentation medium, the extracts of ramulus mori and cyclocarya paliurus were changed to the extract of ramulus mori.
Preparing a mulberry twig extract: pulverizing ramulus Mori, adding 20-30 times (by weight) of 70 vol% ethanol solution, shaking and extracting at 40-50 deg.C for 10 hr, filtering to obtain supernatant, concentrating the supernatant under reduced pressure, and vacuum freeze drying to obtain extract.
Comparative example 2
Referring to example 2, in contrast to example 1, the fermentation medium was composed of a fermentation medium in which extracts of ramulus mori and cyclocarya paliurus were changed to an extract of cyclocarya paliurus.
Preparing a mulberry twig extract: pulverizing cyclocarya paliurus leaf, adding 20-30 times (by weight) of 70 vol% ethanol solution into a shaking table at 40-50 deg.C, extracting under shaking for 10 hr, filtering to obtain supernatant, concentrating the supernatant under reduced pressure, and vacuum freeze drying to obtain extract.
The yield of mycelia was calculated from the fermentation products of Phellinus linteus obtained in example 2 and comparative examples 1-2, as shown in Table 1.
TABLE 1
| Yield of mycelium | |
| Example 2 | 4.8％ |
| Comparative example 1 | 4.0％ |
| Comparative example 2 | 4.1％ |
Example 5
The high-activity fermentation product of Phellinus linteus obtained in example 2 was subjected to the following tests:
the fermentation product of phellinus linteus (hereinafter referred to as fermentation product) of example 2 is dried and crushed for standby, 60 ICR mice are randomly divided into a normal control group, a model group, a positive drug group, a fermentation product high, medium and low dose groups (18.0g/kg,12.0g/kg,6.0g/kg), 10 mice in each group are prepared by a D-galactose subcutaneous injection method, except the normal control group, the other 5 groups are used for preparing a mouse subacute peroxidation injury model by a D-galactose subcutaneous injection method, namely, 120mg/kg of D-galactose is injected into the neck and back subcutaneously at multiple points at regular time every day, and the normal control group is replaced by normal saline for injection for 42 days continuously. And (3) at the same time of molding (the intragastric administration is started the next day after the first D-galactose injection, and the intragastric administration is carried out according to the weight of 20 ml/kg), the intragastric administration corresponding dose is carried out on each group of the fermentation product, the vitamin E200 mg/kg is given to a positive control group, and the drinking water is given to a normal control group and a model group. Fasting is carried out for 24h after the last administration, and blood is taken from eyeball and liver and brain are taken. And determining the concentration of the liver protein by using a total protein quantitative determination kit. The contents of superoxide dismutase (SOD), glutathione peroxidase (GSH), total antioxidant capacity (T-AOC) and Malondialdehyde (MDA) in blood and liver tissue were determined according to the method described in the kit. The test results are shown in table 2, and fig. 1 and 2.
TABLE 2 Effect of fermentates on mouse liver tissue T-AOC, GSH and SOD content
As shown in FIG. 1 and FIG. 2, compared with the normal control group, the serum MDA of the model group is obviously increased, the T-AOC is obviously reduced, the MDA content of each dose group of the leaven is obviously reduced, and the T-AOC has no obvious change. There was no statistical difference between positive MDA and T-AOC. Compared with the model group, the MDA content in the serum of each dose group and the positive group of the leaven is obviously reduced, and the T-AOC level is obviously increased.
As shown in Table 2, compared with the normal control group, the GSH level in the liver tissue of the mouse of each dose group of the model group and the fermentation product has no obvious change, and the activity of the T-AOC and SOD enzymes of the model group shows a descending trend. SOD in the fermented product 6.0 and 12.0g/kg dose groups has no obvious change, and the SOD activity in the fermented product high dose group is reduced. The positive group T-AOC and GSH show obvious rise, and the SOD enzyme activity shows the rising trend, but no statistical difference exists. Compared with the model group, the activity of the GSH and the SOD in the positive group is obviously improved, and the T-AOC is improved, but no statistical difference exists. GSH level in liver tissues of mice of each dose group of the fermentation product has no obvious change, T-AOC of the high dose group of the fermentation product is slightly increased, SOD enzyme activity is obviously increased, and statistical difference is achieved (P is less than 0.05).
Therefore, the fermentation product of phellinus linteus has obvious protective effect on D-galactose-induced subacute oxidative damage of mice at the doses of 6.0, 12.0 and 18.0 g/kg.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (5)
1. A fermentation medium for preparing a high-activity phellinus igniarius fermentation product is characterized by comprising the following components in parts by weight: 20-30 parts of soybean sprout pulp, 5-10 parts of wheat cellulose, 5-10 parts of radix tetrastigme zymolyte, 2-3 parts of mulberry twig and cyclocarya paliurus extract, 0.05-0.1 part of calcium chloride, 0.3-0.5 part of lactose, 1-3 parts of glucose, 0.02-0.05 part of salicylic acid and 80-100 parts of water;
the mulberry twig and cyclocarya paliurus extract is prepared by the following method: respectively crushing ramulus mori and cyclocarya paliurus leaves, mixing the ramulus mori and the cyclocarya paliurus leaves according to a weight ratio of 1:1, adding 20-30 times of 70 vol% ethanol solution, performing vibration extraction for 10 hours in a shaking table at 40-50 ℃, filtering to obtain a supernatant, performing reduced pressure concentration on the supernatant, and performing vacuum freeze drying to obtain the ramulus mori and cyclocarya paliurus extracts;
the radix tetrastigme zymolyte is prepared by the following method: drying and powdering radix tetrastigme rhizome, adding 10 times of deionized water, adding amylase with volume mass ratio of 0.1-0.2ml/100g, and performing enzymolysis at 70 ℃ for 3-5 hours to obtain radix tetrastigme zymolyte.
2. The fermentation medium for producing a highly active fermentation product of Phellinus linteus according to claim 1, wherein the soybean sprout pulp is produced by: selecting soybean with complete grain shape, placing into a full-automatic bean sprouting machine, spraying for sprouting, and stopping sprouting when the bean sprout head is exposed to 4-5 mm; mixing soybean sprout and water at a weight ratio of 1:1, and pulping at high speed to obtain soybean sprout pulp.
3. A fermentation culture method for preparing a high-activity phellinus igniarius fermentation product is characterized by comprising the following steps:
(1) inoculating the activated phellinus igniarius strain into a seed liquid culture medium to carry out phellinus igniarius fermentation culture for 3-7 days to obtain phellinus igniarius liquid seed liquid;
(2) inoculating the liquid Phellinus linteus seed solution with a volume of 1% -5% of the fermentation medium into the fermentation medium of any one of claims 1-2, culturing at 24-27 deg.C for 5-8 days, and vacuum freeze drying the fermentation liquid to obtain high-activity Phellinus linteus fermentation product.
4. The fermentation culture method for preparing high-activity phellinus linteus fermentation product according to claim 3, wherein the seed liquid culture medium in step (1) comprises the following components in parts by weight: 20 parts of germinated soybean, 5 parts of glucose, 0.05 part of magnesium sulfate and 0.02 part of potassium dihydrogen phosphate.
5. The fermentation culture method for preparing a fermentation product of Phellinus linteus with high activity as claimed in claim 3, wherein the Phellinus linteus strain of step (1) is selected from I.sanghuang of Qiandao lake, Hangzhou, Zhejiang province.
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