TWI662132B - Solid-state culture method for raising Hericium erinaceus A (Erinacine A) containing protective nerve component - Google Patents

Abstract

Case provided a solid culture medium and high yield culturing method, characterized in that the components for culturing neuroprotection Hericium Hericium element A (Erinacine A), adding a line Hericium mycelium (Hericiumerinaceus) in a solid medium cultured in a condition of 20-30 ° C; the solid culture medium comprises a whole grain, a nutrient solution, wherein the nutrient solution comprises a sucrose, a casein peptone, Optionally adding a nutrient solution having a pH of 5.0-6.5, wherein the sucrose has a weight percentage ratio of 0.75-2.2%, the casein percentage of the cheese protein is 3-4%; and a neuroprotection The composition comprising the culture product of the aforementioned solid state culture method, comprising cephalosporin A.

Description

Solid-state culture method for raising Hericium erinaceus A (Erinacine A) containing protective nerve component

The invention relates to the field of mushroom cultivation, mainly in the high-yield solid state culture method and the optimization of the culture medium.

Hericium erinaceus ( Hermitage genus Hericium erinaceus) is a taxonomic, fungal, phylogenetic , polyporous , dentate , and genus Hericium in taxonomy; also known as Hericium, Hericium erinaceus, and locust fungus. Named after its shape, it is distributed in the eucalyptus trunks of broad-leaved forests in the temperate regions of the world. The fungi, mycelium and fruiting bodies can be used as medicines. The fruit bodies themselves are rich in polysaccharides, fatty acids, ergosterols, compounds, and monkey heads. Ketone, double linseed oil phospholipid vinylamine, amino acid, trace elements and other active ingredients, Chinese medicine records can help digestion, benefit the five internal organs, run six lungs and tonic loss, has been reported to have anti-tumor, block cancer cell proliferation, Immune regulation, anti-oxidation, anti-inflammatory, memory enhancement, blood sugar lowering and other effects.

Erinacine, which is present in the mycelium of Hericium erinaceus, stimulates the secretion of rat stellate cells or rat adrenal medulla chromaffin cells (PC12) with the dual effects of neurotrophic nutrition and neurotrophic growth. Nerve growth factor (NGF), which prevents neuronal apoptosis and increases neuroprotective effects such as synaptic growth, can prevent neurodegenerative diseases such as Alzheimer's disease.

Erinacine A in Hericium is currently reported as the main mode of "liquid culture" production. It is mainly due to the rapid growth of liquid culture and easy control of reaction conditions. High-level advantages. The literature (Eng. Life Sci. 2010, 10, No. 5, 446-457) teaches that through liquid culture, mycelium can reach 13.3g / L, and the content of cephalosporin A is 14.4mg / g; and Taiwan Patent Publication No. TW I516598 discloses a method for culturing mycelium of Hericium erinaceus to prevent rapid degradation of Hericium A, and teaches that the mycelium obtained by liquid culture is 4.48 g/L, and the content of cephalosporin A is 29.65 mg/g.

However, due to the high sterility requirements of liquid culture, various chemical agents are added depending on the culture, and the waste after cultivation is likely to cause environmental pollution. Therefore, there is still room for improvement in the manpower, equipment, management, and cost of the cultivation process; However, solid-state culture has the advantages of simple operation, no space occupation, low production cost, simple waste disposal and no pollution concerns. However, it is difficult to optimize due to the heterogeneous conditions of solid-state culture, and the parameters are not as good as the liquid culture. Therefore, there are still restrictions on the use.

Therefore, it is a problem to be solved by the present invention to provide a method for producing Hericium A with high yield, easy operation, high reproducibility, low cost, and no pollution.

In view of this, the inventors of this case have a deep understanding of the shortcomings and shortcomings of the previous case, and they have improved and innovated, and finally succeeded in the development of this medium and high-yield, high-industry utilization of the solid-state culture medium and its method.

The present invention proposes a solid culture substrate characterized in that the neuroprotective component Erinacine A for cultivating the Hericium erinaceus comprises a whole grain, a nutrient solution, wherein the nutrient solution comprises a sucrose. a casein peptone, optionally added a nutritional supplement, the nutrient solution has a pH of 5.0-6.5, wherein the sucrose has a weight percentage ratio of 0.75-2.2%, and the weight percentage of the cheese peptone Between 3-4%.

Wherein, the weight ratio of the whole grains to the nutrient solution is between 1:1 and 2.

Wherein the preferred percentage by weight of the sucrose is 1.42%, and the preferred percentage by weight of the casein is 3.35%.

Wherein, the whole grains may be present alone or in a mixture type selected from the group consisting of buckwheat, wheat, brown rice, millet, coix seed, red bean and black bean.

Wherein, the nutritional supplement may be selected from the group consisting of: ascorbic acid, oleic acid, citric acid, zinc sulfate heptahydrate (ZnSO ₄ .7H ₂ O), sodium chloride (NaCl) Among the groups formed, oleic acid or citric acid is preferred.

In this case, a solid-state culture method of high-yield neuroprotective ingredient, Erinacine A, is proposed, which comprises the steps of: adding a Hericium erinaceus mycelium ( Herbicium erinaceus ) to the above solid medium, and culturing at 20-30 ° C In the conditions.

Among them, preferred conditions are a temperature of 25 to 27 ° C and a pH of 5.5 to 6.0.

Wherein, the culture days are between 40 and 70 days, and preferably the culture days are between 50 and 60 days.

Among them, it can be used for cultivating the active ingredient other than the cephalosporin A in the mycelium of the Hericium erinaceus.

The embodiment of the present invention adopts the screening of the whole grains and the screening of the nutrient solution, and includes the single factor test, the partial factor test in the response surface method, and the central mixing test to obtain the optimized solid medium culture method and method of the monkey head A. From a variety of carbon sources and nitrogen sources, sucrose and casein were identified as key factors, and the optimal ratio and regression model were obtained. Nursing value verification.

The present invention further provides a neuroprotective composition comprising a solid state culture product obtained by a solid culture method, comprising a cephalosporin A, wherein the solid culture product can be directly used, or subjected to coarse screening, grinding, and extraction. Or the purification treatment; the solid culture method is as follows: adding a Hericium erinaceus ( Hernicium erinaceus ) in a solid medium, cultured at 20-30 ° C; wherein the solid medium contains a grain a multi-grain, a nutrient solution, wherein the nutrient solution comprises a sucrose, a casein peptone, and optionally a nutritional supplement, the nutrient solution having a pH of 5.0-6.5, wherein the weight of the sucrose The percentage ratio is between 0.75 and 2.2%, and the weight percentage of the cheese peptone is between 3-4%.

Compared with the previous culture method of Hericium A, this case has the advantages of solid and liquid culture. It not only overcomes the difficulties of optimizing the parameters of solid culture, but also has the disadvantage that the yield is not as high as that of liquid culture, and it has the advantage of solid culture. The potential of industrial development is detailed below:

(1) High yield

It has been optimized to limit the high yield of solid-state cultures in the past, and the content of the cephalosporin A is comparable to or better than that of the liquid culture.

(2) Simple operation

Compared with liquid culture, it is necessary to configure and regulate the proportion of each additive, and monitor the change of the parameters of the fermentation tank in the process. The operation is complicated. The case can be statically cultured only after the initial addition.

(3) does not take up space

Compared with liquid fermentation tanks, pipelines, and aseptic devices, the mycelium cultured in solid state can grow on the culture medium and save space.

(4) Low production cost

Compared with the production cost of aseptic operation device, fermentation tank system, and addition liquid for liquid fermentation, solid-state fermentation can achieve a relatively high yield of raw materials at a lower cost.

(5) Simple waste disposal and no pollution concerns

Because liquid fermentation requires the addition of chemical agents to regulate the reaction, and after the end of the labor process, the waste needs additional treatment, which not only takes time and increases the cost. The solid culture medium in this case is simply processed.

Figure 1 shows the implementation architecture and test flow.

Figure 2 is a three-dimensional spatial perspective view of the relative concentration of sucrose and casein peptone corresponding to the content of Erinacine A.

Figure 3 is a contour plot of the relative concentration of sucrose and casein corresponding to the amount of cephalosporin A.

Fig. 4 is a graph showing the analysis of a high performance liquid chromatograph of the culture product for the treatment of cephalosporin A.

All of the technical and scientific terms used in this specification, unless otherwise defined, are the meanings that are common to the field of the skilled person, the singular terms "a", "an", "the", "said" Unless otherwise stated, it may refer to more than one object. In addition, the terms "including" and "including" are open-ended terms; in addition, unless otherwise stated, the materials used in this case are commercially available.

As used herein, the term "solid state culture" means that there is no or substantially no tour. Microbial culture on a solid substrate that is separated from water; the term PDA for PDA plate medium refers to Potato dextrose agar, the potato glucose medium; the term "single factor test" refers to a test that only arranges one cause; the term "response surface method" (Response surface method; RSM), also known as the response surface method, combines the optimization of the function and the prediction in the regression analysis in combination with the mathematical and statistical extension methods for optimal experimental design or operating conditions. The concept, by constructing a functional relationship between the control factor and the response value of the problem, predicts the corresponding results produced by different control factor settings, and is often applied to determine the optimal operational settings in multiple factors; the term "partial factor" Two-level Factorial Design refers to the experimental design of two or more variables; the term "central mixing test" is also known as the central synthetic design, which is an experimental design method in the second-order reaction zone; the term "high-performance liquid layer High performance liquid chromatography (HPLC) is a chromatographic technique used to separate The mixture, in order to confirm and quantify the ratios of the individual components.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Strains of the following Examples, is Hericium Hericiumerinaceus BCRC 36470, purchased from Food Industry Research Institute, Hsinchu Health funding center; before performing a subsequent solid culture, activated species, through the activated species of The strain was cultured in PDA plate medium.

The solid medium comprises a whole grain and a nutrient solution, wherein the nutrient solution comprises a carbon source, a nitrogen source, and optionally a nutrient additive, and the pH is between 5.5 and 6.0, wherein the grain is a grain. The miscellaneous grains are subjected to preliminary screening tests, and preferably selected from the group consisting of buckwheat, wheat, brown rice, millet, coix seed, red beans and black beans, either alone or in combination.

In the following examples, the weight ratio of the whole grains to the nutrient solution is between 1:1 and 2. After steam sterilization, the solid state culture condition of Hericium erinaceus is set at 25-27 ° C, in culture 50. At -60 days, the content of cephalosporin A was determined. The architecture and test procedure are shown in Figure 1 .

Example 1 Single factor test

In order to explore a solid-state culture method for optimizing the yield of Erinacine A (hereinafter referred to as Hericium A), first, a single-factor test of various carbon sources, multiple nitrogen sources, and various nutrient additives is performed. To screen for greater influence on the output of the cephalosporin A content in each group, the classification groups and results are as follows:

(1) Carbon source test

Adding 3% glucose, fructose, sucrose, maltose, or lactose as test groups, solid-state culture, and detecting solid-state cultured monkeys under various variables The content of cephalosporin A, which was 3.85 mg/g in the sucrose group and 3.2 mg/g in the glucose group, was the most excellent yield.

(2) Nitrogen source test

Adding 1% peptone, meat peptone, yeast extract, polypeptone, or casein peptone as experimental groups for solid-state culture and detection The content of the solid-state culture product, cephalosporin A, was measured, and the yield was the most excellent in 15.45 mg/g of the casein peptone group and 14.25 mg/g of the polypeptone group.

(3) Nutritional additive test

Add 0.5% vitamin C (ascorbic acid), oleic acid, citric acid, zinc sulfate heptahydrate (ZnSO4.7H2O), or sodium chloride (NaCl) as the experimental group. Solid-state culture was carried out, and the content of the solid-state culture product, cephalosporin A, was determined, and the yield was most obtained in the ascorbic acid group of 7.46 mg/g and the citric acid group of 4.69 mg/g. Excellent.

Embodiment 2: Response surface method (RSM) experiment

Two-level factorial design

According to the results of single factor test, sucrose, glucose, casein 腖, polyvalent peptone, citric acid and oleic acid were selected as test factors (independent variables), and 16 groups were designed by partial factor test. The test medium (Table 1 ) was subjected to solid state culture, wherein "1" and "-1" shown in the coded table in Table 1 respectively indicate that the amount of addition was high or low; and the amount of each group was added to the monkey head. The results of A yield were statistically analyzed by Design-Expert software. The results showed the effect of each factor on the production of Hericium A (Table 2 ). Based on this, the key factor of the carbon source, sucrose and the key factor of nitrogen source, casein 腖, were selected as Follow-up main test factors.

Table 1 , partial factor test design and analysis of the content of cephalosporin A

Second, 2-response surface method center mixing test

Using sucrose and casein as the main test factors, 13 groups of different proportions of sucrose and casein were designed by the response surface method, and the formation of cephalosporin A was calculated by solid state culture. The content (Table 3 ), the data obtained using the Design-Expert software for regression statistical analysis results (Table 4 ), showing that the regression analysis model produced by the two test factors corresponding to the analyzed group will produce the production of Hericium A. There is a significant impact, where the Lack of fit is not significant, indicating that the model is appropriate.

Through the aforementioned regression model, the relationship between the relative concentration of sucrose and casein and the content of cephalosporin A is expressed in a three-dimensional spatial perspective (Fig. 2 ) and a contour map (Fig. 3 ), so that a specific amount of monkey head can be inferred. A, the relative concentration of sucrose and casein, wherein when the weight percentage of sucrose is between 0.75-2.2% and the weight percentage of casein is between 3-4%, a higher content of cephalosporin can be obtained. A.

In order to verify the applicability of this model, the content of Hericium A is 25mg/g. According to the model calculation, when the expected condition is 1.42% sucrose and 3.35% casein, the content can be obtained; solid culture is carried out in this condition, and the content of the cephalosporin A is 24.56 mg/g, which is estimated. 25 mg/g) is similar, indicating that this model is trustworthy.

During the process, the products of each group were analyzed by high performance liquid chromatography (Fig. 3 ). The analysis method was as follows: 3 mg of the monkey head A standard was dissolved in 100 ml of methanol; the powder of the solid culture product was taken. 10 g, three times with 100 ml of 50% ethanol by hot reflux continuous extraction, and finally the filtrate was combined and concentrated to 100 ml.

The solid culture product produced in the present case, comprising the neuroprotective component Hericium A, can be used as a neuroprotective composition by direct use, or by coarse screening, grinding, extraction, or purification treatment.

In this case, the screening of the optimal composition of the grains and the screening of the nutrient solution were carried out in sequence, including the single factor test, the partial factor test in the response surface method, the central blending test, and the solid state culture method for obtaining high content of cephalosporin A. From a variety of carbon sources and nitrogen sources, it is confirmed that sucrose and casein peptone are key factors, and the optimal ratio is obtained, and the constructed regression model can be trusted by numerical verification of field culture; By responding to the surface method, it is possible to obtain the correlation between the components and the reaction results in various culture materials in the solid culture, and optimize the conditions, which not only saves cost, condition test time and manpower, but also the results are consistent with The actual cultivation results are rich in industrial utilization value.

It can be seen from the above examples that the technical feature of the present invention is to provide a solid medium culture material capable of obtaining high content of cephalosporin A and a culture method thereof, in particular, an optimum carbon source and a nitrogen source combination, respectively, sucrose and casein. And reveal its optimization ratio, any further additions based on this, or adjustments to physical, chemical, time and other parameters (for example: The temperature, the number of days, the pH, etc., which are not removed from the spirit of the case, should be included in the scope of the patent application of this case; in addition, the examples in this case also reveal the composition of the solid medium culture to obtain a specific content of Hericium A. And its method, so any application of the regression model disclosed in this case for the specific content of Hericium A is also within the scope of this case; in addition, because of the active ingredient of Hericium erinaceus mycelium The general knowledge in the field knows that the content of the active ingredient is related to the mycelium. According to the optimized solid medium and method disclosed in the present application, any implementation of the other active ingredients derived from the culture of mycelium of Hericium erinaceus is also The teachings implied in this case; this case also discloses that the solid culture product of the present invention can be used for the neuroprotective composition, and any addition containing the solid culture product of the present invention as a neuroprotective composition should be included in the scope of the patent application.

The embodiments described above are merely illustrative of the technical spirit and characteristics of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the invention. Equivalent variations or modifications made in accordance with the spirit of the invention should still be included in the scope of the invention.

Claims (11)

A solid culture substrate characterized by the neuroprotective component Erinacine A for cultivating the Hericium erinaceus, comprising a whole grain, a nutrient solution, wherein the nutrient solution comprises a sucrose, Casein peptone, optionally adding a nutritional supplement, the pH of the nutrient solution is 5.0-6.5, wherein the weight percentage of the sucrose is between 0.75 and 2.2%, and the weight percentage of the cheese protein is between 3-4%. The substrate according to claim 1, wherein the weight ratio of the whole grains to the nutrient solution is between 1:1 and 2. The substrate of claim 1, wherein the preferred percentage by weight of the sucrose is 1.42%, and the preferred percentage by weight of the casein is 3.35%. The substrate of claim 1, wherein the whole grain is present in a mixture or a mixture, and the whole grain is selected from the group consisting of buckwheat, wheat, brown rice, millet, coix seed, red bean and black bean. In the group. The substrate of claim 1, wherein the nutritional additive is selected from the group consisting of ascorbic acid, oleic acid, citric acid, and zinc sulfate heptahydrate (ZnSO _{4 ).} .7H ₂ O), a group consisting of sodium chloride (NaCl). The substrate of claim 5, wherein the group is preferably oleic acid or citric acid. A solid-state culture method for a high-yield neuroprotective ingredient, Erinacine A, comprising the steps of: adding a Hericium erinaceus mycelium ( Hermitage ) to a solid medium, cultured at 20-30 ° C. Wherein the solid medium comprises a gluten, a nutrient solution, wherein the nutrient solution comprises a sucrose, a casein peptone, and optionally a nutritional supplement, the nutrient solution The pH is from 5.0 to 6.5, wherein the sucrose has a weight percentage of between 0.75 and 2.2% and the casein has a weight percentage of between 3-4%. The method of claim 7, wherein the temperature is preferably 25-27 ° C and the pH is 5.5-6.0. For example, the method described in claim 7 has a culture period of 40-70 days. The method according to claim 7 of the patent application can be used for cultivating the active ingredient other than the cephalosporin A in the mycelium of the Hericium erinaceus. A neuroprotective composition comprising a solid state culture product obtained by a solid culture method, comprising a cephalosporin A, wherein the solid culture product can be directly used, or subjected to coarse screening, grinding, extraction, or purification treatment. The solid culture method is as follows: adding a Hericium erinaceus mycelium ( Herbicium erinaceus ) in a solid medium cultured in a condition of 20-30 ° C; wherein the solid medium contains a whole grain, a a nutrient solution, wherein the nutrient solution comprises a sucrose, a casein peptone, and optionally a nutritional supplement, the nutrient solution having a pH of 5.0-6.5, wherein the weight percentage of the sucrose is between 0.75-2.2%, the weight percentage of the cheese peptone is between 3-4%.