阅读说明：本技术 多糖的制备方法、保健口服液及制备方法 (Preparation method of polysaccharide, health-care oral liquid and preparation method ) 是由 肖丹 吕兆丰 王楠 陈世珩 白翠 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种多糖的制备方法、保健口服液及其制备方法,该方法包括：利用含人参的培养基对蝙蝠蛾拟青霉菌种进行液体深层发酵,获得发酵液；对所述发酵液进行处理,获得多糖。通过这种方式,本发明能够得到营养价值丰富的多糖,进而得到营养价值丰富的保健口服液。(The invention discloses a preparation method of polysaccharide, a health-care oral liquid and a preparation method thereof, wherein the method comprises the following steps: carrying out liquid submerged fermentation on paecilomyces hepiali strain by using a culture medium containing ginseng to obtain a fermentation liquid; and treating the fermentation liquor to obtain polysaccharide. By the method, the polysaccharide with rich nutritive value can be obtained, and the health-care oral liquid with rich nutritive value can be further obtained.)

1. A method of producing a polysaccharide, comprising:

carrying out liquid submerged fermentation on paecilomyces hepiali strain by using a culture medium containing ginseng to obtain a fermentation liquid;

and treating the fermentation liquor to obtain polysaccharide.

2. The method according to claim 1, wherein said paecilomyces hepiali species is a paecilomyces hepiali mutant strain, wherein said paecilomyces hepiali mutant strain is capable of producing higher polysaccharides than an unmutagenized paecilomyces hepiali species;

wherein, before the liquid submerged fermentation of the paecilomyces hepiali strain by using the culture medium containing the ginseng, the method comprises the following steps:

providing the paecilomyces hepiali mutant strain.

3. The method of claim 2, wherein said providing said paecilomyces hepiali mutant strain comprises:

mutagenizing a paecilomyces hepiali strain which is not subjected to mutagenesis treatment by adopting a chemical mutagen to obtain the paecilomyces hepiali mutant strain;

and culturing the paecilomyces hepiali mutant strain by using a first culture medium under a first culture condition to obtain a paecilomyces hepiali mutant strain.

4. The method of claim 3, wherein the chemical mutagen is nitrosoguanidine;

wherein the components of the first medium comprise: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O and vitamin B1;

wherein the components of the first medium comprise: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O、ZnSO₄·7H₂O, vitamin B1;

wherein the first culture conditions are: the pH value range of the first culture medium is 5.5-7.5, the culture temperature range is 22-28 ℃, and the rotating speed range of the shaking table is 120-.

5. The method as claimed in claim 1, wherein the submerged fermentation of Paecilomyces hepiali strain with a ginseng-containing medium comprises:

seed culture is carried out on paecilomyces hepiali strain by utilizing a seed culture medium to obtain a seed culture solution;

performing first fermentation culture on the inoculated seed culture solution by using a ginseng fermentation culture medium to obtain a first fermentation culture solution;

performing fed-batch amplification second fermentation culture on the inoculated first fermentation culture solution by using a ginseng fermentation culture medium to obtain a second fermentation culture solution;

wherein the components of the seed culture medium comprise: glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and calcium chloride; the total nitrogen of the seed culture medium is greater than or equal to 9%;

the p H value range of the seed culture medium is 5.0-7.0;

wherein the ginseng fermentation medium is obtained by adding ginseng powder to the fermentation medium in a predetermined volume ratio;

the components of the fermentation medium comprise: corn starch, glucose, corn steep liquor, magnesium sulfate, potassium dihydrogen phosphate, zinc sulfate and vitamin B1;

the pH value range of the fermentation medium is 5.0-7.0;

the inoculation amount of the first fermentation culture solution is 6-8% of the volume of the ginseng fermentation culture medium.

6. The method of claim 1, wherein the treating the fermentation broth to obtain a polysaccharide comprises:

concentrating the fermentation liquor;

and (3) treating the concentrated fermentation liquor by adopting an ethanol precipitation method to obtain a precipitate, wherein the precipitate is polysaccharide.

7. A health oral liquid, comprising a polysaccharide prepared by the method of any one of claims 1 to 6.

8. The oral liquid of claim 7, wherein the oral liquid further comprises taurine, honey, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate, and citric acid.

9. A preparation method of a health-care oral liquid is characterized by comprising the following steps:

providing a polysaccharide extraction mother liquor after purification of the polysaccharide according to any one of claims 1 to 6;

the health oral liquid is prepared by the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor.

10. The method of claim 9, wherein the other health oral liquid components comprise: taurine, honey, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate and citric acid;

wherein the health oral liquid prepared by the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor comprises:

pouring the polysaccharide extraction mother liquor and the first other health oral liquid components into a mixing tank, adding purified water to a first preset volume, uniformly mixing, and heating to boil for sterilization;

cooling the sterilized solution, adding a second other health oral liquid component, and adding purified water to a second predetermined volume to obtain the health oral liquid.

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a preparation method of polysaccharide, a health-care oral liquid and a preparation method.

Background

The liquid fermentation technology was applied to health food in the 40 th of the 20 th century and to traditional Chinese medicine research in the 80 th of the 20 th century. With the deep research of microorganisms and the continuous development of separation technology and structure identification, by carrying out liquid culture on traditional Chinese medicines, not only hypha or spores can be greatly proliferated, but also various substances with physiological activity such as polysaccharide, polypeptide, alkaloid, terpenoid, enzyme, nucleic acid, amino acid and the like can be obtained in fermentation liquor, and the traditional Chinese medicines have the effects of resisting cancer, inflammation and bacteria, resisting aging, improving immunity and the like; the fermentation technology is applied to common medical and edible resources, and novel food integrating nutrition and health care functions can be prepared.

Dried hypha obtained by deep fermentation of paecilomyces hepiali (Paeilyes hepiali) has effective components and pharmacological action similar to those of cordyceps sinensis, the medicinal value of the dried hypha is verified clinically, the dried hypha is one of two earlier cordyceps fungi published by Ministry of health of China and can be used for producing health-care food, the hypha powder of the dried hypha is already marketed as a large amount of products, and the strain has the characteristics of short fermentation period and easier production process. The polysaccharide is used as a substance with important biological activity, and has good curative effect on the treatment of some difficult and complicated diseases of human beings; the polysaccharide and its derivatives have various biological functions and pharmacological effects.

However, the nutrient components of the polysaccharide finally obtained from the dried mycelium obtained by deep fermentation of paecilomyces hepiali are single.

Disclosure of Invention

The invention mainly solves the technical problem of providing a preparation method of polysaccharide, a health-care oral liquid and a preparation method thereof, and the polysaccharide with rich nutritive value can be obtained, so that the health-care oral liquid with rich nutritive value can be obtained.

In order to solve the technical problems, the invention adopts a technical scheme that: provided is a method for producing a polysaccharide, comprising: carrying out liquid submerged fermentation on paecilomyces hepiali strain by using a culture medium containing ginseng to obtain a fermentation liquid; and treating the fermentation liquor to obtain polysaccharide.

Wherein the paecilomyces hepiali strain is a paecilomyces hepiali mutant strain, wherein the paecilomyces hepiali mutant strain can produce higher polysaccharide than the non-mutated paecilomyces hepiali strain; wherein, before the liquid submerged fermentation of the paecilomyces hepiali strain by using the culture medium containing the ginseng, the method comprises the following steps: providing the paecilomyces hepiali mutant strain.

Wherein, the step of providing the paecilomyces hepiali mutant strain comprises the following steps: mutagenizing a paecilomyces hepiali strain which is not subjected to mutagenesis treatment by adopting a chemical mutagen to obtain the paecilomyces hepiali mutant strain; and culturing the paecilomyces hepiali mutant strain by using a first culture medium under a first culture condition to obtain a paecilomyces hepiali mutant strain.

Wherein the chemical mutagen is nitrosoguanidine; wherein the components of the first medium comprise: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O and vitamin B1; wherein the components of the first medium comprise: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O、ZnSO₄·7H₂O, vitamin B1; wherein the first culture conditions are: the pH value range of the first culture medium is 5.5-7.5, the culture temperature range is 22-28 ℃, and the rotating speed range of the shaking table is 120-.

Wherein, the liquid submerged fermentation of the paecilomyces hepiali strain by using the culture medium containing the ginseng comprises the following steps: seed culture is carried out on paecilomyces hepiali strain by utilizing a seed culture medium to obtain a seed culture solution; performing first fermentation culture on the inoculated seed culture solution by using a ginseng fermentation culture medium to obtain a first fermentation culture solution; performing fed-batch amplification second fermentation culture on the inoculated first fermentation culture solution by using a ginseng fermentation culture medium to obtain a second fermentation culture solution; wherein the components of the seed culture medium comprise: glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and calcium chloride; the total nitrogen of the seed culture medium is greater than or equal to 9%; the p H value of the seed medium is 5.0-7.0; wherein the ginseng fermentation medium is obtained by adding ginseng powder to the fermentation medium in a predetermined volume ratio; the components of the fermentation medium comprise: corn starch, glucose, corn steep liquor, magnesium sulfate, potassium dihydrogen phosphate, zinc sulfate, and vitamin B1; the pH value of the fermentation medium is 5.0-7.0; the inoculation amount of the first fermentation culture solution is 6-8% of the volume of the ginseng fermentation culture medium.

Wherein the treatment of the fermentation broth to obtain a polysaccharide comprises: concentrating the fermentation liquor; and (3) treating the concentrated fermentation liquor by adopting an ethanol precipitation method to obtain a precipitate, wherein the precipitate is polysaccharide.

In order to solve the technical problem, the invention adopts another technical scheme that: provides a health-care oral liquid, which comprises the polysaccharide prepared by any one of the preparation methods.

Wherein the health oral liquid further comprises taurine, honey, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate and citric acid.

In order to solve the technical problem, the invention adopts another technical scheme that: provides a preparation method of a health-care oral liquid, which comprises the following steps: providing a polysaccharide extraction mother liquor after purification of a polysaccharide as described in any one of the above; the health oral liquid is prepared by the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor.

Wherein the other health oral liquid comprises the following components: taurine, honey, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate and citric acid; wherein the health oral liquid prepared by the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor comprises: pouring the polysaccharide extraction mother liquor and the first other health oral liquid components into a mixing tank, adding purified water to a first preset volume, uniformly mixing, and heating to boil for sterilization; cooling the sterilized solution, adding a second other health oral liquid component, and adding purified water to a second predetermined volume to obtain the health oral liquid.

The invention has the beneficial effects that: different from the situation of the prior art, the invention utilizes the culture medium containing the ginseng to carry out liquid submerged fermentation on the paecilomyces hepiali strain, and the obtained mycelium in the fermentation liquor also comprises the nutrient components of the ginseng, so that the polysaccharide obtained after the fermentation liquor is treated also comprises the nutrient components in the ginseng, such as rare ginsenoside and the like, besides the single polysaccharide nutrient component of the original paecilomyces hepiali mycelium. By the method, the polysaccharide with rich nutritive value can be obtained, and technical support is provided for preparing the nutritive oral liquid with rich nutritive value.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:

FIG. 1 is a schematic flow diagram of one embodiment of a process for producing a polysaccharide of the present invention;

FIG. 2 is a schematic flow diagram of another embodiment of a process for producing a polysaccharide of the present invention;

FIG. 3 is a schematic flow diagram of a process for producing a polysaccharide according to another embodiment of the present invention;

FIG. 4 is a schematic flow diagram of a process for producing a polysaccharide according to another embodiment of the present invention;

FIG. 5 is a schematic flow diagram of another embodiment of the process for producing a polysaccharide of the present invention.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Prior to describing the present invention in detail, a prior art related to the present invention will be understood.

In the present society, the phenomena of physical decline and frequent occurrence of chronic diseases generally exist in high-pressure white-collar workers, frequently-going-on-business workers, sportsmen with large amount of exercise and easy fatigue and student groups ready for interview all the time, and the phenomena are in a transition state between health and diseases, namely a 'third state' -sub-health state. However, the real reason of sub-health is not completely clarified at present, and a targeted treatment measure is lacked. Therefore, the health food for enhancing immunity is developed, has wide market space and meets the requirements of life and work of people at present. Therefore, the research has higher practical application value.

The liquid fermentation technology is a process of dissolving nutrient substances in liquid as a culture medium, then inoculating strains, and obtaining a target product through culture. Liquid fermentation, large scale, high yield and capability of industrial continuous production; the production efficiency is high, and the method is suitable for mechanized and automatic production and is the main mode of the current microbial fermentation industry.

The liquid fermentation technology was applied to health food in the 40 th of the 20 th century and to traditional Chinese medicine research in the 80 th of the 20 th century. The traditional Chinese medicinal materials and the auxiliary materials are stirred and placed at a certain temperature and humidity, and the aims of improving the efficacy of the traditional Chinese medicine, changing the property of the medicine, reducing toxic and side effects, expanding the variety of the medicine and the like are fulfilled through the fermentation of microorganisms. With the intensive research on microorganisms and the continuous development of separation technology and structural identification, by carrying out liquid culture on traditional Chinese medicines, not only hypha or spores can be proliferated in large quantity, but also various substances with physiological activity, such as polysaccharide, polypeptide, alkaloid, terpenoid, enzyme, nucleic acid, amino acid and the like, can be obtained in fermentation liquor. The physiologically active substance has anticancer, antiinflammatory, antibacterial, antiaging, and immunity enhancing effects. The fermentation technology is applied to common medical and edible resources, and novel food integrating nutrition and health care functions can be prepared.

In summary, the liquid fermentation technology is still in the beginning stage of the research on health food, and some problems inevitably exist. The research on drug toxicity and pharmacology is less; the change of chemical components after fermentation and the related research methodology standard of pharmacodynamic evaluation of fermentation products are not established, and the development of the traditional Chinese medicine liquid fermentation technology is seriously influenced. The liquid fermentation technology can degrade the medicine into small molecular active substances through the action of microorganisms, is easier to combine with human body cell protein through the blood brain barrier, and can be directly utilized. Therefore, the degraded micromolecular active substance has the advantages of quicker and more complete absorption in human bodies, better effect and the like.

Dried hypha obtained by deep fermentation of paecilomyces hepiali (Paeilyes hepiali) has effective components and pharmacological action similar to those of cordyceps sinensis, the medicinal value of the dried hypha is verified clinically, the dried hypha is one of two earlier cordyceps fungi published by Ministry of health of China and can be used for producing health-care food, and a large amount of hypha powder of the dried hypha is sold on the market. The strain has the characteristics of short fermentation period and easy production process. As the paecilomyces hepiali hypha extract, the polysaccharide is a substance with important biological activity in the extract, and has good curative effect on the treatment of some difficult and complicated diseases of human beings. The polysaccharide and the derivatives thereof have various biological functions, such as cell specificity identification, receptors of cell surfaces to various antigens and medicines, immune cells activation and the like, so that the polysaccharide has various pharmacological effects, such as blood pressure reduction, blood fat reduction, stomach and liver strengthening, oxidation resistance, aging delay, immunity improvement, fatigue relief and the like, and the polysaccharide becomes a hot spot of the current medicinal fungus research.

The polysaccharide extracted from the paecilomyces hepiali mycelia has single nutrient component and low nutrient value. The invention adds ginseng into the culture solution to carry out liquid submerged fermentation on the paecilomyces hepialid strain, so that the variety of nutrient components in the fermentation solution is increased; the content of the polysaccharide is improved by optimizing and cultivating the varietal paecilomyces hepiali strain with high polysaccharide yield and optimizing a culture medium and culture conditions; the fermentation liquor is treated to obtain crude polysaccharide, the extraction rate of the crude polysaccharide is increased, and the oral liquid prepared from the extracted crude polysaccharide has sour and sweet taste, slightly bitter and astringent taste, high nutritional value, and can relieve fatigue and improve human immunity.

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application.

Referring to FIG. 1, FIG. 1 is a schematic flow diagram of one embodiment of a process for producing a polysaccharide of the present invention, the process comprising:

step S101: carrying out liquid submerged fermentation on paecilomyces hepiali strain by using a culture medium containing ginseng to obtain fermentation liquor.

Step S102: and treating the fermentation liquor to obtain the polysaccharide.

Ginseng is the best product of nourishing yin and tonifying life, strengthening body resistance and consolidating constitution, and contains various saponins and polysaccharides. The succulent root of ginseng is a well-known tonic, is suitable for regulating blood pressure, recovering heart function, neurasthenia and asthenia, and also has expectorant, stomach invigorating, diuretic and exciting effects.

Submerged liquid fermentation, also known as submerged culture, is a stirred culture in which a liquid medium is used in a fermenter. The liquid submerged fermentation has the advantages that: first, it is the optimal growth environment for many microorganisms; secondly, the thalli, the nutrients and the products are easy to diffuse, are carried out under the condition of homogenization or quasi-homogenization, are convenient to control and are easy to enlarge the production scale; thirdly, the liquid is convenient to convey and easy to mechanically operate; fourthly, the factory building area is small, the production efficiency is high, the automatic control is easy, and the product quality is stable; fifthly, the product is easy to extract, refine and the like.

The submerged fermentation mode of the liquid includes batch fermentation, continuous fermentation and fed-batch fermentation. The batch fermentation is that nutrients and strains are added at one time for culture until the culture is finished, and the medium has no material exchange with the outside except air inlet and tail gas exhaust. Continuous fermentation means that fresh culture medium is added into a fermentation tank at a certain speed, and culture solution flows out at the same speed, so that the liquid amount in the fermentation tank is kept constant, and microorganisms grow in a stable state; this approach can effectively extend the log phase in batch cultures. Fed-batch fermentation, also known as semi-continuous fermentation, is a fermentation technology between batch fermentation and continuous fermentation, and refers to a culture technology for supplementing a certain amount of materials to a culture system in a certain way in microbial batch fermentation; the method can keep the concentration of the nutrient in the culture solution within a certain range for a long time, thereby not only ensuring the growth requirement of the microorganism, but also not causing adverse effect, and achieving the purpose of improving the yield.

Because the paecilomyces hepiali strain is subjected to liquid submerged fermentation by using the culture medium containing the ginseng, the obtained mycelium in the fermentation liquor also comprises the nutrient components of the ginseng, so that the polysaccharide obtained after the fermentation liquor is treated also comprises the nutrient components in the ginseng, such as rare ginsenoside and the like, besides the single polysaccharide nutrient component of the original paecilomyces hepiali mycelium. By the method, the polysaccharide with rich nutritive value can be obtained, and technical support is provided for preparing the nutritive oral liquid with rich nutritive value.

In one embodiment, the paecilomyces hepiali strain is a paecilomyces hepiali mutant strain, wherein the paecilomyces hepiali mutant strain can produce higher polysaccharide than an unmutagenized paecilomyces hepiali strain.

Referring to fig. 2, before the step S101, performing submerged fermentation on paecilomyces hepiali strain by using the culture medium containing ginseng, the method specifically includes:

step S103: providing a paecilomyces hepiali mutant strain.

If there is the existing paecilomyces hepiali mutant strain, it can be activated and used directly. If there is no existing paecilomyces hepiali mutant strain, the paecilomyces hepiali strain without mutagenesis treatment needs to be subjected to mutagenesis treatment. Mutagenesis technology is an important tool for breeding. The mutagenesis is generally carried out by physical mutagenesis and chemical mutagenesis. The physical mutagenesis comprises ultraviolet rays, laser, microwaves, X rays, gama rays, alpha rays, beta rays, fast neutrons and the like, wherein the ultraviolet rays, the microwaves, the X rays and the gama rays have good mutagenesis effect on microorganisms and are widely applied.

Referring to fig. 3, in an embodiment, step S103 may specifically include: sub-step S1031 and sub-step S1032.

Substep S1031: and (3) mutagenizing the paecilomyces hepiali strain which is not subjected to mutagenesis treatment by adopting a chemical mutagen to obtain the paecilomyces hepiali mutagenized strain.

Chemical mutagens (chemical substances with a mutagenesis effect) used in the chemical mutagenesis are many, mainly comprise alkylating agent, nitrosoguanidine, diethyl sulfate, nitrogen mustard hydrochloride, 5 bromouracil and the like, have strong toxicity to cells, generally cannot be utilized by microorganisms, and cause the microorganisms to generate mutation after the action so as to play a role in mutagenesis breeding. Chemical mutagens tend to be specific, with one mutagen acting on a certain site of a gene and little effect on the rest.

Wherein the chemical mutagen is Nitrosoguanidine (NTG). NTG is used for mutation breeding and is a chemical super-mutagenic agent with a recognized remarkable effect. The main principle of NTG for mutation breeding is as follows: nitrosoguanidine belongs to the alkylating agent, and the alkylating agent is a quite effective chemical mutagen in mutation, and the mutagen has 1 or more active alkyl groups, and the active alkyl groups are easy to replace active hydrogen atoms in DNA molecules, so that base and phosphate parts on the DNA molecules are alkylated, and base pairing errors are caused when DNA is replicated, thereby causing mutation.

Step S1032: culturing the paecilomyces hepiali mutant strain by using a first culture medium under a first culture condition to obtain the paecilomyces hepiali mutant strain. In this way, sufficient paecilomyces hepialid strain can be obtained and subcultured continuously.

Wherein the first medium comprises the following components: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O and vitamin B1.

Wherein, the firstA medium comprising: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O、ZnSO₄·7H₂O, vitamin B1.

In one particular application, the components of the first medium include: 10g/L glucose, 5g/L yeast extract powder, 1g/L KH₂PO₄0.5g/L MgSO₄·7H₂O, 50mg/L ZnSO₄·7H₂O, 10mg/L vitamin B1.

Wherein the first culture conditions are: the pH of the first medium is in the range of 5.5-7.5, for example: 5.5, 6.5, 7.5, etc., and the culture temperature is in the range of 22-28 ℃, for example: 22 ℃, 25 ℃, 28 ℃, etc., and the range of the shaking table rotation speed is 120-: 120 rpm, 150rpm, 180 rpm, etc.

Referring to fig. 4, in an embodiment, in step S101, performing submerged fermentation on paecilomyces hepiali strain by using a culture medium containing ginseng may specifically include:

substep S1011: seed culture is carried out on the paecilomyces hepiali strain by utilizing the seed culture medium to obtain a seed culture solution.

The seed culture medium is used for spore germination, growth and mass propagation of mycelium, and makes the mycelium grow stout to become a 'seed' with strong activity. Therefore, the seed culture medium has rich and complete nutrient components, high nitrogen source and vitamin content and low total concentration, and may have high dissolved oxygen for the growth and propagation of great amount of thallus. The composition of the seed medium is determined by the physiological characteristics of the different species, considering that a stable pH is maintained during the metabolism of the microorganism. Seed culture media generally use natural organic nitrogen sources that are rich and complete in nutrients, since some amino acids stimulate spore germination. However, since inorganic nitrogen sources are easily available and are advantageous for the rapid growth of cells, organic and inorganic nitrogen sources are often included in the seed medium. The last stage of seed culture medium preferably has components close to the fermentation medium, so that the seeds can adapt to the fermentation medium quickly and grow quickly after entering the fermentation medium.

In one embodiment, the components of the seed medium include: glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and calcium chloride; the total nitrogen of the seed culture medium is greater than or equal to 9%; the p H values for the seed medium range from 5.0 to 7.0, for example: 5.0, 6.0, 7.0, etc.

In one embodiment, the seed medium comprises the following components: 3% of glucose, 2% of yeast extract, 0.2% of magnesium sulfate, 0.1% of dipotassium hydrogen phosphate, 0.2% of monopotassium phosphate and 0.03% of calcium chloride; the total nitrogen of the seed culture medium is greater than or equal to 9%; the p H value for the seed medium was 6.0.

Sub-step S1012: and performing first fermentation culture on the inoculated seed culture solution by using a ginseng fermentation culture medium to obtain a first fermentation culture solution.

In this example, the ginseng fermentation medium is obtained by adding ginseng powder to the fermentation medium at a predetermined volume ratio. Wherein the predetermined volume ratio of the ginseng powder to the fermentation medium is 1: 50.

The fermentation medium is used for the growth, propagation and synthesis of the strain. It not only ensures that the seeds can grow rapidly after being inoculated to reach a certain hypha concentration, but also ensures that the grown bacteria can rapidly synthesize the required products. Thus, the composition of the fermentation medium contains, in addition to elements and compounds necessary for the growth of the bacteria, specific elements, precursors, promoters, etc. required for the product. However, if the total concentration of carbon source, nitrogen source, phosphorus source, etc. required for the growth and biosynthesis of the product is too high or the optimum conditions required for each of the two stages of growth and synthesis are different, it is considered that the medium is satisfied by fed-batch.

In one embodiment, the components of the fermentation medium include: corn starch, glucose, corn steep liquor, magnesium sulfate, potassium dihydrogen phosphate, zinc sulfate and vitamin B1; the pH of the fermentation medium is in the range of 5.0-7.0, for example: 5.0, 6.0, 7.0, etc.

In one particular application, the components of the fermentation medium include: 40g/L of corn starch, 5g/L of glucose, 25g/L of corn steep liquor, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1.5g/L of zinc sulfate and 25mg/L of vitamin B1; the pH of the fermentation medium was 7.0.

Substep S1013: and (3) performing fed-batch amplification second fermentation culture on the inoculated first fermentation culture solution by using the ginseng fermentation culture medium to obtain a second fermentation culture solution.

Wherein the inoculation amount of the first fermentation culture solution is 6-8% of the volume of the ginseng fermentation culture medium, such as: 6%, 7%, 8%, etc.

Referring to fig. 5, in an embodiment, in step S102, processing the fermentation broth to obtain a polysaccharide may specifically include:

step S1021: and concentrating the fermentation liquor. The concentration treatment mode includes but is not limited to: centrifugation, boiling with heat, and the like.

Step S1022: and (3) treating the concentrated fermentation liquor by adopting an ethanol precipitation method to obtain a precipitate, wherein the precipitate is polysaccharide. The steps roughly comprise enzymolysis extraction, heating extraction, supernatant taking, ethanol precipitation of the supernatant, precipitate taking and drying constant weight, and the polysaccharide is obtained.

The fermentation liquor is a large amount of cultured paecilomyces hepiali mycelia. The polysaccharide is extracted from mycelium.

The method for producing the above polysaccharide will be described in detail below as a specific practical application.

Step 1, preparing a first culture medium of a constant-temperature shaking table:

the first medium contains the following components: glucose, yeast extract powder, KH₂PO₄、MgSO₄·7H₂O and vitamin B1; further, 10g/L glucose, 5g/L yeast extract powder, 1g/L KH₂PO₄0.5g/L MgSO₄·7H₂O, 50mg/L ZnSO₄·7H₂O, 10mg/L vitamin B1.

Range values of the first culture conditions: the pH value of the first culture medium is 5.5-7.5, the culture temperature is 22-28 ℃, and the rotation speed of a shaking table is 120-; specifically, the pH value of the first medium is 6.0, the culture temperature is 25 ℃, and the rotation speed of a shaking table is 150 rpm.

Step 2, primarily breeding the paecilomyces hepiali strain:

the operation process of adopting the nitrosoguanidine chemical mutagen to induce the paecilomyces hepiali strain mutation is as follows:

fresh slant bacteria are taken, the bacteria are washed by 0.1mol/L phosphate buffer solution with the pH value of 6.0, and the bacteria are centrifugally washed and made into bacterial suspension with the buffer solution, wherein the concentration of the bacterial suspension is 106 cfu/mL. Preparing an NTG solution: because NTG is poor in water solubility, a small amount of acetone is used for dissolving in the preparation process, and then a buffer solution is added, wherein the ratio of the acetone to the buffer solution is 9: 1, preparing NTG mother solution with concentration of 1mg/mL by using 9mL of buffer solution and 1mL of nitrosoguanidine acetone solution. When the method is used, 0.2mL of mother liquor is taken and added into 1.8mL of bacterial suspension, namely a mutation treatment system with the NTG treatment concentration of 0.1mg/mL, and the mutation treatment systems with the NTG treatment concentrations of 0.2mg/mL, 0.3mg/mL, 0.4mg/mL and 0.5mg/mL are prepared in the same way. The bacterial suspension and NTG solutions with different treatment concentrations are mixed in each test tube, and the mutagenesis effect of the nitrosoguanidine with different treatment times and different concentrations is researched. When the reaction was terminated, the reaction mixture was diluted 50-fold with physiological saline, washed by centrifugation, and repeated 3 times to remove the NTG drug. Adding seed culture medium, and placing in ice water bath for 2 h. The medium was removed by centrifugation, the pellet was suspended in sterile water and diluted by a ten-fold concentration gradient method, and then spread on a potato flat medium and cultured at 37 ℃ for 48 hours in the absence of light. And taking the bacterial liquid without mutagenesis treatment to dilute and coat the plate by the same operation, and culturing for the same time in the dark as a control. And selecting the one with the larger diameter of the transparent ring and the larger diameter of the bacterial colony, transferring the one to a fermentation medium for further shaking and re-screening, and determining the polysaccharide content by adopting a phenol ammonium sulfate method to select the mutant strain with higher polysaccharide yield. Inducing the mutant paecilomyces hepiali to be activated, inoculating the mutant strains into 250mL conical flasks (filled with a first culture medium) one by using an inoculating needle, culturing for 48h in a constant-temperature shaking table at the temperature of 26 ℃ and the speed of 150rpm, and obtaining a large amount of paecilomyces hepiali mutant strains and carrying out continuous subculture.

Step 3, carrying out liquid submerged fermentation on the paecilomyces hepiali mutant strain by using the culture solution containing the ginseng:

firstly, culturing by using a seed culture medium to obtain a seed culture solution. The components of the seed culture medium comprise: 3% of glucose, 2% of yeast extract, 0.2% of magnesium sulfate, 0.1% of dipotassium hydrogen phosphate, 0.2% of monopotassium phosphate and 0.03% of calcium chloride; the total nitrogen of the seed culture medium is greater than or equal to 9%; the p H value for the seed medium was 6.0.

Preparing a fermentation medium, wherein the fermentation medium comprises the following components: 40g/L of corn starch, 5g/L of glucose, 25g/L of corn steep liquor, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1.5g/L of zinc sulfate and 25mg/L of vitamin B1; the pH of the fermentation medium was 7.0.

Adding Ginseng radix powder into the fermentation medium at a volume ratio of 1:50 to obtain Ginseng radix fermentation medium. And performing first fermentation culture on the inoculated seed culture solution by using a ginseng fermentation culture medium to obtain a first fermentation culture solution.

And 4, performing fed-batch amplification fermentation culture (namely second fermentation culture) on the paecilomyces hepiali strain on the basis of the step 3, wherein the inoculation amount of the first fermentation culture solution is 7% of the volume of the ginseng fermentation culture medium.

And 5, separating and extracting paecilomyces hepiali mycelia in the fermentation liquor to obtain the polysaccharide.

Centrifuging the fermentation broth by centrifugation (15min, 4000rpm/min), removing supernatant, heating the centrifuged fermentation broth to boil, and concentrating. The ethanol precipitation method is used, and the specific steps are as follows:

1) enzymolysis: respectively putting 26 parts of 200g of the cordyceps fungus powder into 500mL triangular flasks, and sequentially adding 300mL of distilled water and an enzyme reagent, wherein the addition amount is 0.1 percent (namely 0.3g or 0.3mL) of the amount of the distilled water. Placing the triangular flask in a reciprocating speed-regulating shaking table, setting the rotation speed at 170r/min and the temperature at 55 deg.C, and extracting for 3 h. 2) After extraction, the triangular flask is put into a water bath kettle at 100 ℃ for hot water extraction for 3h, and after the heating extraction process is finished, the triangular flask is cooled to room temperature at normal temperature and refrigerated in a refrigerator at 4 ℃ overnight. And centrifuging the extracted polysaccharide sample liquid for 20min at 4000r/min in a high-speed centrifuge the next day, and removing residues to obtain a supernatant. 3) Alcohol precipitation the supernatant was transferred to a 200mL volumetric flask and distilled water was added to a constant volume of 200 mL. And respectively transferring 200mL of the supernatant into 1000mL of numbered beakers, respectively adding 4 times of volume (namely 800mL) of absolute ethyl alcohol, uniformly mixing, and placing in a refrigerator at 4 ℃ for alcohol precipitation overnight. And on the third day, putting the overnight precipitated sample liquid into a high-speed centrifuge for centrifuging at 4000r/min for 20min, and removing the supernatant to obtain the cordyceps polysaccharide precipitated product. 4) Drying and constant weight: and (3) placing the polysaccharide alcohol precipitation product in a drying oven at 60 ℃ to dry to constant weight, and weighing to calculate the yield. The polysaccharide was measured by spectrophotometry (phenol-sulfuric acid method).

In this example, after the ginseng powder is added, the precipitate contains not only mycelium polysaccharide but also rare ginsenoside such as Rg3 and the like; and because the paecilomyces hepiali mutant strain is adopted, the yield of polysaccharide and mycelium are increased, the biomass of the mycelium is 20.9g/L, and the yield of the polysaccharide and the content of the polysaccharide are respectively 15.28 percent and 50.34 percent. The mycelia biomass is 15.3g/L, and the polysaccharide yield and polysaccharide content are 12.75% and 40.23% respectively by adopting the non-mutagenized paecilomyces hepialid strain.

The invention also provides a health-care oral liquid which comprises the polysaccharide prepared by any one of the preparation methods.

Wherein the health oral liquid further comprises taurine, Mel, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate and citric acid.

In one particular application, the health oral liquid comprises the following components: 9% of polysaccharide, 0.35% of taurine, 4.0% of honey, 0.1% of vitamin B1, 0.1% of vitamin B2, 0.05% of vitamin B6, 0.1% of vitamin C, 0.04% of sodium benzoate and 0.15% of citric acid.

The health oral liquid has slightly sweet and bitter taste, and is effective in relieving fatigue and enhancing immunity.

The invention also provides a preparation method of the health-care oral liquid, which comprises the following steps:

providing a polysaccharide extraction mother liquor from any one of the above purified polysaccharides; the health oral liquid is prepared from the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor.

Wherein, the other health oral liquid comprises the following components: taurine, honey, vitamin B1, vitamin B2, vitamin B6, vitamin C, sodium benzoate and citric acid.

The health oral liquid is prepared from the polysaccharide extraction mother liquor and other health oral liquid components except the polysaccharide extraction mother liquor, and specifically comprises the following components: pouring the polysaccharide extraction mother liquor and the first other health oral liquid components into a mixing tank, adding purified water to a first predetermined volume, mixing uniformly, heating to boil and sterilize; cooling the sterilized solution, adding a second other health oral liquid component, and adding purified water to a second predetermined volume to obtain the health oral liquid.

In particular, the first additional health oral liquid component comprises non-heat sensitive materials such as: sugar solutions (honey), vitamin solutions, citric acid solutions, and the like. The first other health oral liquid component may also include adjuvants. A second additional health oral liquid component comprises heat sensitive materials such as: royal jelly, and the like. The second additional health oral liquid component may also include supplemental vitamins, citric acid, and the like.

The preparation method of the above health oral liquid is described in detail below with a specific practical application.

Step 1, adopting hot-melting precipitation fine filtration to purify the extracted polysaccharide so as to obtain polysaccharide extraction mother liquor, wherein the pH value of the polysaccharide is adjusted to 5.0-6.0 by the water amount added during hot-melting precipitation, the temperature of the polysaccharide is 30 ℃, the pressure is 0.2MPa, and the aperture of a filter screen is 0.6 mu m.

Step 2, pouring the polysaccharide extraction mother liquor and other components into a mixing tank; wherein the final concentrations are respectively: 9% of polysaccharide, 0.35% of taurine, 4.0% of honey, 10.1% of vitamin B, 20.1% of vitamin B, 60.05% of vitamin B, 0.1% of vitamin C, 0.04% of sodium benzoate and 0.15% of citric acid.

And 3, adding the rest auxiliary materials according to the proportion, slowly adding purified water to the volume, and fully stirring and mixing.

And 4, heating, boiling and sterilizing for 30min, adding the honey liquid after the solution is cooled, and fully stirring and mixing.

And 5, adding the vitamin liquid and the edible flavor citric acid according to the formula proportion, and fully stirring.

Step 6, adding purified water to the liquid preparation amount, and filtering; the filtration pore size is 0.45 um.

Step 7, using 0.1mol/L HCL solution and 0.1mol/L NaOH solution to adjust the pH value of the prepared solution to be 5.0-6.0 and the relative density (relative to water) to be 1.15-1.25kg/cm³。

And 8, conveying the prepared liquid to a filling and rolling process, filling and sealing to obtain the health-care oral liquid.

In the embodiment, the raw materials are purified, mixed and the like, so that the nutritional value of the raw materials is greatly reserved, and the prepared finished product is rich in taste and high in nutritional value.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.