阅读说明：本技术 一种去壁灵芝孢子粉的制备方法 (Preparation method of ganoderma lucidum spore powder with wall removed ) 是由 程俊文 贺亮 李肖娟 金李洁 方良珍 李其 于 2020-03-30 设计创作，主要内容包括：本发明公开了一种去壁灵芝孢子粉的制备方法。本发明根据灵芝孢子壁壳成分的理化特性,通过在发酵阶段先接入乳酸克鲁维酵母发酵,再配合交变磁场短时发酵,然后再接入罗伊氏乳杆菌发酵,利用特定的发酵过程在降解灵芝孢子壁壳的同时将壁壳中的几丁质等成分进行发酵转化,又能很好地使孢子粉内的三萜和多糖类有效成分释放出来,制得的去壁灵芝孢子粉无壁壳残留,壁壳成分全部生物转化,生物活性得到显著提高。(The invention discloses a preparation method of ganoderma lucidum spore powder with removed wall. According to the physicochemical characteristics of ganoderma spore exine components, kluyveromyces lactis is firstly inoculated into a fermentation stage for fermentation, then the kluyveromyces lactis is matched with an alternating magnetic field for short-time fermentation, and then lactobacillus reuteri is inoculated for fermentation, the specific fermentation process is utilized to degrade the ganoderma spore exine and simultaneously ferment and convert chitin and other components in the exine, the triterpene and polysaccharide effective components in the spore powder can be well released, the prepared exine-removed ganoderma spore powder has no exine residue, the exine components are completely biologically converted, and the biological activity is obviously improved.)

1. A preparation method of ganoderma lucidum spore powder without wall is characterized by comprising the following steps:

(1) pretreatment: removing impurities from the raw material of the ganoderma lucidum spore powder and drying to obtain dry ganoderma lucidum spore powder;

(2) preparing a kluyveromyces lactis seed solution: inoculating the activated kluyveromyces lactis strain into a liquid seed culture medium A, and culturing for 5-24 h to obtain kluyveromyces lactis seed liquid;

(3) preparation of lactobacillus reuteri seed solution: inoculating the activated lactobacillus reuteri strain into a liquid seed culture medium B to culture for 5-24 h to obtain lactobacillus reuteri seed liquid;

(4) and (2) biodegradation of ganoderma spore powder exine, namely mixing the dried ganoderma spore powder in the step (1) with water to prepare a liquid fermentation culture medium with the concentration of the ganoderma spore powder of 5 g/L-20 g/L, sterilizing and cooling, inoculating kluyveromyces lactis seed liquid into the liquid fermentation culture medium, culturing for 10h-60h, then placing in an alternating magnetic field environment for culturing for 1h-10h, sterilizing and cooling, then inoculating lactobacillus reuteri seed liquid, culturing for 10h-60h, and drying the obtained fermentation liquid to obtain the sporoderm-removed ganoderma spore powder.

2. The method according to claim 1, wherein the amount of the Kluyveromyces lactis seed liquid used in step (4) is 1-15% by volume of the liquid fermentation medium.

3. The method according to claim 1, wherein the lactobacillus reuteri seed solution is used in an amount of 1-10% by volume of the liquid fermentation medium in the step (4).

4. The production method according to claim 1, wherein in the step (4), the conditions of the alternating magnetic field are: the magnetic field intensity is 3mT-10mT, and the magnetic field frequency is 20Hz-50 Hz.

5. The method according to claim 1, wherein the Kluyveromyces lactis seed liquid is cultured in the liquid fermentation medium at a temperature of 20-35 ℃ in step (4).

6. The method according to claim 1, wherein the Lactobacillus reuteri seed solution is cultured in the liquid fermentation medium at a temperature of 25-37 ℃ in step (4).

7. The method according to claim 1, wherein the liquid seed culture medium A is composed of, in terms of 1 liter, 2g to 8g of glucose, 2g to 10g of Ganoderma lucidum spore powder, and the balance of water.

8. The method according to claim 1, wherein the liquid seed culture medium B is composed of 2g to 8g of glucose, 2g to 10g of Ganoderma lucidum spore powder and the balance of water in 1 liter.

9. The preparation method according to claim 1, wherein in the step (1), the raw material of the ganoderma lucidum spore powder is one or more of ganoderma lucidum spore powder without wall breaking, ganoderma lucidum spore powder with wall breaking and residue of extracted ganoderma lucidum spore powder.

10. A sporoderm-removed ganoderma lucidum spore powder, characterized in that the sporoderm-removed ganoderma lucidum spore powder is prepared according to the preparation method of any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of ganoderma lucidum spore powder with a removed wall.

Background

Ganoderma lucidum (Gaoderma L ucidum Karst), also known as Rue, Shen Zhi, Xiancao, Yao Cao, Huanyang Cao, Linzhong Ling, Jun Ling, Wannian mushroom, Ling Cao, Chi Zhi, Dan Zhi, Qin Zhen, etc., is a fruiting body of Ganoderma lucidum, a fungus of Polyporaceae, the shape of which is umbrella-shaped, pileus kidney-shaped, semicircular or nearly circular.

Ganoderma spore is the seeds of Ganoderma lucidum which is the tiny egg-shaped germ cells ejected from Ganoderma lucidum pileus during the growth and maturation stage. A large number of modern pharmacological studies and clinical cases prove that the ganoderma lucidum spore powder has good regulating effect on an immune system, a nervous system, an endocrine and metabolic system, a cardiovascular system and the like. The Ganoderma spore contains abundant active components such as ganoderan, triterpenes, and Ganoderma spore oil, and has all genetic materials and health promotion effects of Ganoderma.

From the view of cell structure, the ganoderma lucidum spore has a double-wall structure, wherein the double-wall structure is formed by chitin and glucan, and the texture is tough and accounts for 50% -70%. Chitin is a nitrogenous polysaccharide biological polymer widely existing in nature. Experiments show that the ganoderma lucidum spore wall can block the dissolution of effective components and influence the absorption in vivo, so that most of the products at present adopt different wall breaking processes to crush the spore wall so as to improve the bioavailability of the active components in vivo and enhance the treatment effect. The effective components of the ganoderma lucidum spores which are not subjected to wall breaking can be absorbed by human bodies only by about 12 percent, and the absorption rate of the ganoderma lucidum spores after wall breaking can reach more than 95 percent, so that the ganoderma lucidum spores have more remarkable anti-tumor, anti-oxidation and immunoregulation effects.

The existing wall breaking methods of ganoderma lucidum spore powder comprise physical, chemical and biological methods. The method for physically breaking the walls, such as mechanical crushing, is simple and easy to operate, has high wall breaking rate, but has long wall breaking time and high energy consumption, can cause partial heat-sensitive substances to be inactivated and effective substances to be lost and damaged due to a large amount of heat generated in the wall breaking process, and can easily cause heavy metal pollution and the like due to the fact that mechanical impurities are easily mixed in the wall-broken product. Chemical methods also tend to result in loss and destruction of the active substance. The biological method adopts mild means such as enzymolysis and microbial fermentation, can effectively preserve the functional components of the ganoderma lucidum spores, and has the advantages of low energy consumption, good wall breaking effect and the like. However, the existing biological enzymolysis method adopts microbial fermentation to break the wall of ganoderma lucidum spore powder, and the problems of long fermentation period, low wall shell removal rate, low content of effective components and the like generally exist. In the existing wall-breaking method of ganoderma lucidum spore powder, chitin and the like in a double-layer wall still exist after wall breaking, are mixed with effective components in the wall and are difficult to remove, and the problem of low content of active components in spores is still not solved fundamentally.

Chinese patent CN 104013652B discloses a refining process and a comprehensive utilization method of ganoderma lucidum spore powder and application thereof, wherein the refining process comprises the following steps: wall-breaking ganoderma lucidum spore powder is subjected to wall-removing refining after wall breaking, the wall-breaking ganoderma lucidum spore powder is soaked in 20-95% ethanol solution and water, the wall shell is separated, wall-removed ganoderma lucidum spore powder filtrate and ganoderma lucidum spore powder wall shell are obtained, the wall ganoderma lucidum spore powder filtrate is concentrated to obtain concentrated solution, and the concentrated solution is dried to obtain refined ganoderma lucidum spore powder, or the concentrated solution is directly prepared into a refined ganoderma lucidum spore powder preparation or a preparation intermediate by adopting a one-step granulation method; the ganoderma lucidum spore powder exine is subjected to a comprehensive recycling process of chemical extraction to prepare dichloromethane extraction solid powder, ethyl acetate extraction solid powder, n-butanol extraction solid powder, exine water extract solid powder and chitosan. The process has the advantages of complex operation, more organic solvents and high industrial production cost.

Disclosure of Invention

The invention aims to provide a high-efficiency, quick, safe and controllable preparation method of wall-broken ganoderma lucidum spore powder aiming at the defects of the existing preparation process of the wall-broken ganoderma lucidum spore powder, so as to improve the quality and the added value of ganoderma lucidum spore powder products and provide a feasible thought and method for industrial development.

According to the invention, according to the physicochemical characteristics of the ganoderma spore exine components, kluyveromyces lactis is firstly inoculated in a fermentation stage for fermentation, then the kluyveromyces lactis is matched with an alternating magnetic field for short-time fermentation, then lactobacillus reuteri is inoculated for fermentation, and the specific fermentation process is utilized to degrade the ganoderma spore exine and simultaneously ferment and convert the chitin and other components in the exine, so that the triterpene and polysaccharide effective components in the spore powder can be well released, the prepared wall-removed ganoderma spore powder has no exine residue, all the exine components are biologically converted, and the biological activity is obviously improved.

In order to achieve the above purpose, the invention adopts the technical scheme that:

a method for preparing Ganoderma spore powder with removed wall comprises the steps of:

(1) pretreatment: removing impurities from the raw material of the ganoderma lucidum spore powder and drying to obtain dry ganoderma lucidum spore powder;

(2) preparing a kluyveromyces lactis seed solution: inoculating the activated kluyveromyces lactis strain into a liquid seed culture medium A, and culturing for 5-24 h to obtain kluyveromyces lactis seed liquid;

(3) preparation of lactobacillus reuteri seed solution: inoculating the activated lactobacillus reuteri strain into a liquid seed culture medium B to culture for 5-24 h to obtain lactobacillus reuteri seed liquid;

(4) and (2) biodegradation of ganoderma spore powder exine, namely mixing the dried ganoderma spore powder in the step (1) with water to prepare a liquid fermentation culture medium with the concentration of the ganoderma spore powder of 5 g/L-20 g/L, sterilizing and cooling, inoculating kluyveromyces lactis seed liquid into the liquid fermentation culture medium, culturing for 10h-60h, then placing in an alternating magnetic field environment for culturing for 1h-10h, sterilizing and cooling, then inoculating lactobacillus reuteri seed liquid, culturing for 10h-60h, and drying the obtained fermentation liquid to obtain the sporoderm-removed ganoderma spore powder.

The invention mainly utilizes the fermentation of the Kluyveromyces lactis, the short-time fermentation of the alternating magnetic field and the fermentation of the lactobacillus reuteri to realize the complete biotransformation of the components of the ganoderma spore exine, and the complete degradation of the ganoderma spore exine, so that the effective components such as ganoderma triterpene, polysaccharide and the like in the exine are released, and the ganoderma spore powder without exine residue is obtained. Therefore, the parameters in the preparation method of the invention are changed within the range, and the preparation of the ganoderma lucidum spore powder without affecting the preparation of the ganoderma lucidum spore powder and the invention effect thereof are not affected, so that the preparation of the ganoderma lucidum spore powder without wall can be realized by the combination of any parameter in the preparation method of the invention, and the effect of the invention can be achieved. The raw materials used in the invention are all available, and any replacement in the range has no obvious influence on the invention effect. In order to achieve a better effect of the invention, the following preferable steps are performed:

in the step (1), the ganoderma lucidum spore powder raw material is screened to remove impurities, then washed and centrifuged to remove impurities to obtain wet ganoderma lucidum spore powder, and dried to obtain the dry ganoderma lucidum spore powder.

The ganoderma lucidum spore powder raw material is selected from the existing ganoderma lucidum spore powder (containing ganoderma lucidum spore wall shell), the ganoderma lucidum spore powder raw material containing the ganoderma lucidum spore wall shell can be subjected to wall removal by adopting the method of the invention, can be directly prepared from the commercial products or can be prepared according to the existing method, for example, one or more than two of the ganoderma lucidum spore powder without wall breaking, the existing wall-broken ganoderma lucidum spore powder (such as the commercial wall-broken ganoderma lucidum spore powder), the residue of the extracted ganoderma lucidum spore powder and the like can be selected. The dosage ratio of more than two selected ganoderma lucidum spore powder raw materials is not particularly limited and can be any ratio.

In the step (2), the strains can adopt any one Kluyveromyces lactis (Kluyveromyces lactis) to achieve the effect of the invention; commercially available products can be used, for example: kluyveromyces lactis (Kluyveromyces lactis) ACCC20054, purchased from China agricultural microorganism culture Collection management center.

In the step (3), the Lactobacillus reuteri strain can adopt any Lactobacillus reuteri (L Lactobacillus reuteri) strain, can achieve the effect of the invention, and can adopt a commercially available product, such as Lactobacillus reuteri (L Lactobacillus reuteri) ACCC00652, which is purchased from China agricultural microorganism strain preservation management center.

The culture temperature of the activated Kluyveromyces lactis strain and the activated Lactobacillus reuteri strain in the liquid seed culture medium is natural environment temperature, preferably 20-37 ℃, more preferably 20-35 ℃ and 25-37 ℃, and the culture temperature of the activated Kluyveromyces lactis strain is 1cm to 1-1L ℃ in general, and the culture temperature of the activated Lactobacillus reuteri strain in the liquid seed culture medium is 1cm to 37 ℃²-4cm²The size of the activated Kluyveromyces lactis strain block or 1cm²-4cm²Activated lactobacillus reuteri in large and small sizesAnd (5) strain block.

The activation method of the Kluyveromyces lactis strain and the activation method of the Lactobacillus reuteri strain are conventional strain activation methods in the field, and generally comprise the following steps: inoculating the strain preserved on the inclined plane to a PDA plate culture medium, and performing activated culture at 22-30 ℃ for 3-10 days to obtain an activated strain.

The PDA plate culture medium adopts a culture medium commonly used in seed culture in the field, and can adopt a commercial product, and further preferably, the PDA plate culture medium comprises 200g of potatoes, 20g of glucose and 15-20 g of agar, and the volume is adjusted to 1000m L by water.

The liquid seed culture medium A is composed of 2g to 8g of glucose, 2g to 10g of ganoderma lucidum spore powder and the balance of water in a 1 liter manner, the liquid seed culture medium B is composed of 2g to 8g of glucose, 2g to 10g of ganoderma lucidum spore powder and the balance of water in a 1 liter manner, 1 liter of the liquid seed culture medium A or 1 liter of the liquid seed culture medium B can be prepared according to the method that 2g to 8g of glucose and 2g to 10g of ganoderma lucidum spore powder are added with water to reach the volume of 1000m L, the liquid seed culture medium A and the liquid seed culture medium B can be the same or different, and the ganoderma lucidum spore powder dried in the step (1) is preferably adopted.

In the step (4), the dosage of the kluyveromyces lactis seed liquid is preferably 1-15% of the volume of the liquid fermentation medium. The culture temperature of the kluyveromyces lactis seed liquid in the liquid fermentation culture medium is preferably 20-35 ℃.

The dosage of the lactobacillus reuteri seed liquid is preferably 1-10% of the volume of the liquid fermentation medium. The culture temperature of the lactobacillus reuteri seed liquid in the liquid fermentation culture medium is preferably 25-37 ℃.

The conditions of the alternating magnetic field are preferably: the magnetic field intensity is 3mT-10mT, and the magnetic field frequency is 20Hz-50 Hz.

The culture medium used in the invention is used after being sterilized, and the sterilization condition adopts the conventional condition in the field, for example, the culture medium can be sterilized at 120-125 ℃ for 20-30 min.

The wall-removed ganoderma lucidum spore powder prepared by the preparation method of the wall-removed ganoderma lucidum spore powder has no residual wall shells, all wall shell components are subjected to biotransformation, the biotransformation of ineffective components (wall shells) of the spore powder and the enrichment of effective components (polysaccharide, triterpene and the like) are realized, the content of the effective components of the wall-removed ganoderma lucidum spore powder is obviously improved, and the activity is obviously enhanced. Can be used in health food or medicinal product.

Compared with the prior art, the invention has the following advantages:

1. the preparation method of the wall-removed ganoderma lucidum spore powder is environment-friendly, strong in operability and high in yield, the biotransformation of ineffective components (exine) of the spore powder and the enrichment of effective components (polysaccharide, triterpene and the like) are realized, the content of the effective components of the produced wall-removed ganoderma lucidum spore powder is obviously improved, the activity is obviously enhanced, and the quality and the added value of ganoderma lucidum spore powder products are improved.

2. The invention discovers that the Kluyveromyces lactis and the Lactobacillus reuteri which have specific degradation effect on the spore wall of the ganoderma lucidum completely degrade the spore wall shell of the ganoderma lucidum by scientifically designed staged fermentation and the action of an alternating magnetic field, so that the active ingredients such as ganoderma lucidum triterpene, polysaccharide and the like in the spore shell are released, and the obtained spore powder has no residue on the spore shell.

3. The microbial fermentation method adopted by the invention for preparing the high-quality wall-removed ganoderma lucidum spore powder has the advantages of short fermentation period, high efficiency, controllable whole fermentation process and no limitation of external environmental conditions, and is very suitable for industrial production and application and popularization.

Drawings

FIG. 1 is an electron microscope scanning picture of wall-broken Ganoderma spore powder obtained by prior art treatment;

FIG. 2 is an electron microscope scanning picture of the spore powder of Ganoderma lucidum with wall removed obtained by the method of the present invention.

Detailed Description

The present invention will be further illustrated with reference to some specific examples, but the present invention is not limited to the following examples.

Kluyveromyces lactis (Kluyveromyces lactis) ACCC20054, purchased from China agricultural microorganism culture Collection management center.

Lactobacillus reuteri (L Lactobacillus reuteri) ACCC00652, purchased from China agricultural microorganism culture Collection center.