CN111685234A

CN111685234A - Pig feed and method for enhancing immunity by feeding pig feed

Info

Publication number: CN111685234A
Application number: CN202010660324.3A
Authority: CN
Inventors: 中岛三博; 长岛公司
Original assignee: Ronghe Dream Fujian Biotechnology Co ltd
Current assignee: Ronghe Dream Fujian Biotechnology Co ltd
Priority date: 2020-07-10
Filing date: 2020-07-10
Publication date: 2020-09-22

Abstract

The invention relates to a pig feed and a method for enhancing immunity by feeding, wherein the pig feed comprises sparassis crispa dry powder; the sparassis crispa dry powder contains 60 wt% or more of beta-1, 3-D-glucan, and is prepared from sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221). The pig feed can effectively enhance the immunity of fattening pigs.

Description

Pig feed and method for enhancing immunity by feeding pig feed

Technical Field

The invention relates to the technical field of veterinary drug products, and in particular relates to a pig feed capable of enhancing immunity of fattening pigs.

Background

Newborn pigs gain immunological competence from the colostrum of the sow against essential nutrients in the subsequent development and against environmental pathogens. When the first-time fattening pig is fed with the medicament at the beginning of fattening, the mortality rate of infectious diseases in the period is increased, and the pneumonia is easy to occur in the period of the beginning of secondary fattening. Countermeasures against pneumonia in pigs have been a problem that has been studied. In recent years, countermeasures against respiratory syndrome and avian influenza have also become important.

The number of white blood cells in pigs is 12,000-30,000/mu l, and the number of white blood cells in pigs is increased by 2,000-8,000/mu l when the pigs are fever. Infection is more likely to occur as the number of white blood cells decreases. The immune performance of the pig will be increased in the state of active leukocyte activity. In the prior art, the practice of obtaining the immunity enhancement by injecting beta-1, 3-D-glucan is available in human clinical practice. Beta-1, 3-D-glucan from Sparassis crispa MH-3 has a simple structural type, and it has been reported in the world that mice are orally fed to obtain various immunopotentiating effects.

Experiments on the immune-enhancing effect including pneumonia and influenza in pigs were conducted by feeding first-fattened pigs with sparassis crispa MH-3 by self-oral ingestion, on the assumption that sparassis crispa MH-3 has a promoting effect on the balance of 5 kinds of white blood cells.

Japanese patent JP4183326B2 discloses a Sparassis crispa extract, which is found to contain beta-1, 3-D-glucan (6 ramifications) as a main physiologically active substance in Sparassis crispa during the study of the physiological activity of Sparassis crispa.

Furthermore, OHNO et al (OHNO Dayeshanren, Naohito Yuntai Liang, Miura Mitsu san Bo et al, anti tumor 1, 3-. beta. -Glucan free from Cultured blood Body of spark crassa [ J ]. Biological & Pharmaceutical Bulletin,2000,23(7):861 872.) reported that dry powder of Sparassis crispa-3 (accession number FERM P-17221) has an immune function-enhancing effect by establishing an experimental model of leukopenia mice.

Therefore, at present, the feed containing the active ingredient beta-1, 3-D-glucan for improving the immunity of the fattening pigs cannot be provided.

Disclosure of Invention

Therefore, there is a need to provide a pig feed containing sparassis crispa powder with high concentration of beta-1, 3-D-glucan and a feeding method thereof, so as to enhance the immunity of fattening pigs and reduce the occurrence of diseases.

In order to achieve the above object, the present invention provides a pig feed comprising dry powder of Sparassis crispa; the dry powder of Sparassis crispa contains 60 wt% or more of beta-1, 3-D-glucan, and is prepared from Sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221).

Specifically, the preparation method of the sparassis crispa dry powder comprises the following steps:

(1) a first culture step: inoculating the sparassis crispa MH-3 into an agar culture medium containing nutrient components for culturing to obtain a first generation culture strain;

(2) a second culture step: filling 400 g-500 g of culture base material into a first mushroom cultivation bottle, inoculating the first generation culture strain into the first mushroom cultivation bottle for cultivation to obtain a second generation culture strain, wherein the culture base material comprises fine granular scraps of conifers, water and mycelium active nutrient;

(3) a third culturing step: filling 2000 g-3000 g of culture medium in a first fungus bed bag, inoculating the second generation culture strain into the first fungus bed bag for culture to obtain a third generation culture strain;

(4) a fourth culturing step: inoculating the Sparassis crispa MH-3, the hypha and the fruiting body of the second generation culture strain and the mixture of the hypha and the fruiting body of the third generation culture strain into an agar culture medium containing nutrient components for culture to obtain a first generation complex strain;

(5) a fifth culturing step: filling 400 g-500 g of culture medium into a second mushroom cultivation bottle, inoculating the first generation of compound bacteria into the second mushroom cultivation bottle for cultivation to obtain a second generation of compound bacterial strain;

(6) a sixth culturing step: filling 2000 g-3000 g of culture medium in a second fungus bed bag, inoculating the second generation compound strain into the second fungus bed bag for culture to obtain a third generation compound strain;

(7) a drying procedure: and drying the third generation compound strain to obtain the dry powder of the sparassis crispa.

Further, the wood chip is also included.

Further, the sawdust is red pine pieces.

A method for enhancing immunity by feeding pig feed comprises feeding 400-600mg of Sparassis crispa dry powder to each pig with primary fattening for 2 times per day; feeding 1000-1500mg of sparassis crispa dry powder to each fattening pig twice, and feeding for 2 times a day; the sparassis crispa dry powder contains 60 wt% or more of beta-1, 3-D-glucan, and is prepared from sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221).

Specifically, the first-time fattening pigs are fed with 500mg of sparassis crispa dry powder for 2 times a day for 15 days; feeding 1000mg of sparassis crispa dry powder to each fattening pig for 15 days, and feeding for 2 times a day.

Specifically, the single standard of feeding fattening pigs is to feed 1g of sparassis crispa dry powder per 60kg of body weight.

Compared with the prior art, the technical scheme provides the dried powder of the pig feed Sparassis crispa; the Sparassis crispa dry powder is prepared from Sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221), and contains beta-1, 3-D-dextran 60 wt% or more. Experiments show that the feed can effectively improve the content of the white blood cells in the blood of the pig, so that the immunity of the pig can be better improved.

The words used herein are defined as follows:

sparassis crispa, a white flower mushroom, grows on mountain pine stumps in summer. The natural product is a rare mushroom, rarely found in korea or taiwan in china, and cannot grow naturally. It has been found in parts of China, but is widely known because it is not registered as an herb.

β -glucan β (1,4) β (1,6) β (1,3) glucan which is a natural substance (polysaccharide) present in foods in nature. Beta- (1,4) represents cellulose (paper), whereas beta (1,6) has no effect, since its effect is unknown. Beta (1,3) glucan is widely used clinically as an immunopotentiating anticancer drug. In general, beta (1,3) glucans are referred to as beta-glucans. There are many types of beta-glucans in mushrooms, but because of their low content, their function is rarely tested.

Conventionally, it is used as an injection because it has a large molecular weight and is not absorbed by the digestive tract, but by Mina healthcare co. The study of the medicine and the university of Tokyo pharmacy proves that the mechanism and the action of the oral administration are confirmed and published in the journal of Nature.

White blood cell: there are five types of neutrophils which increase in the case of bacterial infection and inflammation, lymphocytes which increase in the case of viral infection and lymphoma, eosinophils which increase in the case of allergy and parasite, basophils which increase in the case of leukemia, and monocytes which increase in the recovery phase of infection. It is thought that the balance and motility of 5 kinds of leukocytes can exhibit an immunopotentiation effect, and thus the immune status can be known by measuring the proportion of leukocytes (five kinds of cells are measured).

The immunity enhancing feed comprises: feed for preventing infection of fattening pig. Korean pine fragments generated during the manufacture of Sparassis crispa can be used. Feed comprising a functional ingredient beta-glucan and enhancing leukocytes, thereby enhancing the immune function of pigs.

Loosening the slices: larix Gmelini pieces (finely divided pine) for culturing Sparassis crispa. Beta-glucan (a functional component of sparassis crispa) is 1.4 times as high in productivity as larch. However, since it has a strong bactericidal action and is difficult to corrode and ferment due to pine resin, it cannot be used well even if it is returned to the ground.

First-time fattening of pigs: pigs weighing about 30KG + -5 KG.

Secondary fattening of pigs: pigs weighing about 60KG + -10 KG.

Detailed Description

In order to explain technical contents, structural features, and objects and effects of the technical means in detail, the following detailed description is given with reference to specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

The test materials and reagents used in the following examples are available from commercial routes unless otherwise specified.

In the examples of the present invention, the microorganism, Sparassis crispa MH-3 strain, which belongs to the genus Sparassis and has been deposited at 17.2.1999 in NiBH (now named independent administrative agency for human product evaluation technology basic agency, patent microorganism Collection (NITE-IPOD)) under the accession number FERM P-17221, and transferred international deposit at 5.12.2019 under the Budapest treaty and assigned the International patent deposit number FERM BP-17221. Of course, the culture method of sparassis crispa provided by the invention can also be applied to the culture of other types of sparassis crispa strains, and is not limited to sparassis crispa MH-3 strains.

A pig feed comprises Sparassis crispa dry powder; the sparassis crispa dry powder contains more than or equal to 60 wt% of beta-1, 3-D-glucan, and is prepared from sparassis crispa MH-3 (International allowable microorganism collection number FERMBP-17221).

When fattening, we carried out the following experiment, 40 first-time fattening pigs (average weight of 30KG) were taken and put into a pig farm, 20 control groups were set, and the other 20 control groups were fed with immune-enhanced feed containing 500mg of beta-glucan and mainly containing Korean pine dust, and the fattening pigs were fed orally for 2 times a day for 15 days. And observing the immune enhancement effect of the fattening pigs. The index of the immunopotentiation effect is 5 kinds of white blood cells of blood extracted from pig before and after the administration of the immunopotentiating feed.

Secondly, the twice-fattening pigs (average weight of 60KG) are fed with an immune-enhanced feed containing 1000mg of beta-glucan and mainly containing Korean pine cuttings, and the twice-fattening pigs are independently taken orally for 15 days 2 times a day. Leukocytes were measured by the same method as described above.

The white blood cells include five types, namely neutrophils which are increased in the case of bacterial infection, inflammation and the like, lymphocytes which are increased in the case of viral infection, lymphoma and the like, eosinophils which are increased in the case of allergy, parasite and the like, basophils which are increased in the case of leukemia and the like, and monocytes which are increased in the recovery period of infection. The previous experiments did not compare the specific classification of leukocytes, and we considered that the balance and vitality of 5 types of leukocytes could manifest immunopotentiation, so that the classification and measurement of leukocytes were necessary.

If the lymphocyte and the monocyte are increased, the pig is proved to have the immune function enhanced by the immune strengthening feed. Mouse experiments also demonstrated that Sparassis crispa beta-glucan has a positive effect on the balance optimization of 5 types of leukocytes. After the immune-enhanced feed for enhancing the immune enhancement effect including pneumonia and influenza is taken by the pigs by self, experiments prove that the immune-enhanced feed enhances the immunity of the pigs, and the enhancement of the resistance to diseases is beneficial to reducing the death rate of the fattening pigs in the pig raising process.

The immunopotentiator for feeding and the feeding method are as follows:

we provide a dry brown powder of Sparassis crispa produced by Sparassis crispa MH-3 (International patent organism deposit number FERM BP-17221) and having a total beta-glucan content of 61.9g/100 g; and all beta-glucans are beta-1, 3-glucans. The experimental pigs 10 pigs (castrated males) were selected from each of the 20 pigs in each of the 4 columns, of which 10 × 2 were control groups and the other 10 × 2 were experimental groups. The experimental group feed is fed with 1.5kg of feed each time, and is fed with feed for free feeding for 2 times a day. The feed is added with 500mg of Sparassis crispa dry powder per head. The control group was fed with the same amount of conventional feed. In the experiment, 10 of 20 animals in each column were selected arbitrarily for testing because the intake time and intake amount in each column were not uniform. 1500mg of sparassis crispa dry powder is fed to each fattening pig for the second time.

Sparassis crispa MH-3 was produced by the Collection of microorganisms having the number of International patent publication (International patent publication) FERM BP-17221. The production method comprises inoculating MH-3 strain on large wood chips of natural Korean pine with various components, harvesting after six months, drying in warm and ventilated environment, and powdering. The main component is beta-1, 3-glucan, and has the function of improving immunity. And a Japanese patent (JP patent No. 4183326) was obtained.

In addition, beta-1, 3-glucan is a safe substance, hardly absorbed from the digestive tract or mucosa, and reacts with receptor (dectin-1) to produce immune substance (cytokine) upon contact with mucosa. Used in this test was produced Sparassis crispa MH-3 dry powder (. beta. -glucan, 61.9g/100 g). The administration was carried out at a dose of 1g x 15 days each, at a concentration ten times the data of oral administration to mice (body weight 30g, administration 60. mu.g) (β -glucan 60mg → 600 mg).

Disease of pig and efficacy of drugs for treatment and prevention

Conventionally, various antibiotics have been administered for the prevention and treatment of an infectious disease (pneumonia) that is effective. However, there is no material having a preventive effect on viruses so far, and a preventive method has been a problem.

The data on the studies of immunopotentiators and adjuvants are insufficient, and there are no results of studies on oral effects and safety as well as the administration dose and the administration days.

Potential of sparassis crispa MH-3 as pig immunity enhancing feed

In the experiment, the conditions of the pig farm, such as the self-oral ingestion of the fattening pigs, can cause residual food, quarrel and sequence (considering that the grade system of the pigs with parentage in the same fence can influence the feeding sequence). Considering that there were only 8 feeding boxes, it was possible that no MH-3 was administered to all pigs, and considering the problem of weight reduction due to the dispersion of the filler in the feed, the administration was carried out for 1g x 15 days per pig, i.e. at a concentration 10 times that of the oral administration data of mice (administered in the form of 60. mu.g as beta-glucan to 30g weight mice) (60 mg → 600mg as beta-glucan concentration in 30kg weight pigs). The concentration of mycelium-containing pine wood chips (pine tree powder) is planned to be added to future feeds at about 0.01.

The test period of the scheme is 14 days after the fattening pigs live. In 14 days from 4 months and 3 days to 17 days in 2009, a secondary fattening pig test was performed on a primary fattening pig with an average body weight of 60 kg. For 40 primary fattening pigs, 20 of the 40 primary fattening pigs are used as a control group to be placed in a pig farm when arriving, and the rest 20 primary fattening pigs are fed with 500mg of the dry Sparassis crispa powder twice a day for 15 consecutive days. After free oral ingestion, the immunopotentiating effect on the finishing pigs was observed. The index of immunopotentiation was determined by measuring leukocytes in blood collected from pigs before and after administration of "Dry Sparassis crispa powder". The result shows that the oral administration of the 'embroidered coccus dry powder' to the first-time fattening pig can enhance the whole body immune function of the pig and can be safely used for preventing infectious diseases. Thereafter, the twice-fattened pigs were allowed to freely take 1000mg of "sparassis crispa dry powder" twice daily continuously, and observed.

The data for porcine white blood cells are shown in the table below,

unit: x 100/. mu.L

Test numbers 1-20 in the table are control group pigs, 21-40 are experimental group pigs, and Δ each represents the difference from D1. D1: 2, 19 days in month; d2: 3, month and 4 days; d3: 4, month and 3 days; d4: 4 months and 17 days. As can be seen, D1-D2 are primary fattening pigs, and D3-D4 are secondary fattening pigs. It can be seen from the table that the immunity of the fat pigs can be effectively enhanced by the fattening method.

Sparassis crispa extract is used as a nutritional ingredient added to Japanese red pine. In addition to the conventional methods, the temperature and humidity are also regulated. As a cultivation method, we have devised a production method which does not require the use of steam for "shiitake mushroom" cultivation and does not directly contact air, and successfully produced (5 times as large as the conventional method) Sparassis crispa on a large scale.

Compared with the red pine pot culture method developed by Mina Health Co., Ltd. and containing 43.6g/100g of the active ingredient, the content of the beta-glucan as the functional ingredient is 61.9g/100g, which is 1.42 times of the content of 43.6g/100 g. However, a significant bed of Korean pine fungi is produced during the harvest of Sparassis crispa. Up to now, the fungal bed has been restored back to the farmland and used as fertilizer, while the method using pinus densiflora cannot return to the farmland as fertilizer, but it is incinerated and destroyed, and cannot be decomposed in the soil. We see how our solution optimizes reuse from the CO2 emission point of view.

The fungal bed of Sparassis crispa was characterized by a high concentration of beta-glucan (0.7g/100g), which was not observed in other fungal beds. Usually, up to now, several times of artificially produced "mushrooms" (5 times for shiitake mushrooms) can be harvested by inoculating one fungal bed once. Our Sparassis crispa can only be harvested once from a fungal bed with an inoculum. Therefore, the required number of Korean pine beds is 5 times as much as that of "shiitake mushrooms". Therefore, one characteristic of our Sparassis crispa, unlike other Lentinus edodes, is that even after harvesting, the fungal bed contains a large amount of Sparassis crispa mycelia and has a large water content, but can be stored at room temperature for several months while inhibiting the generation of mold, and can be stored for a long period if dried.

In the embodiment of the invention, the culture method of sparassis crispa comprises the following steps:

(1) a first culture step: sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221) was inoculated into an agar medium containing nutrients and cultured to obtain a first generation culture strain.

In the first culturing step, the nutrient component added to the agar medium may be one or more of peptone, epithidium (EBIOS, beer yeast tablet), banana powder, honey powder, wheat flour, calcium chloride, agar powder, needle-leaved tree hot water extract, and the like. In addition, mycelium active nutrients such as wheat, bran, barley and corn bran can be added into the agar culture medium.

Examples of the needle-leaved tree hot water extract include: an extract obtained by hot water extraction of fragments of coniferous leaves such as larch and red pine with hot water of 40 to 80 ℃. The agar medium is preferably a slant medium to increase the medium area of the mycelium. In addition, the pH value of the agar medium preferably ranges from 6.0 to 7.0. Preferably, the agar medium is placed in a test tube in an inclined manner, sterilized, formed into a slant solid medium, inoculated with Sparassis crispa MH-3, and cultured.

(2) A second culture step: filling 400 g-500 g of culture medium into a bottle for mushroom cultivation, inoculating the first generation culture strain into the bottle for mushroom cultivation, and culturing to obtain a second generation culture strain, wherein the culture medium comprises fine granular scraps of conifers, water and mycelium active nutrients.

In the second culturing process, fine-grained conifer chips include, for example, chips obtained by pulverizing raw wood of conifers (e.g., larch, red pine), and preferably have a particle size of about 0.1mm to 10 mm. The mycelium-active nutrients include, for example, wheat, bran, barley and corn bran, etc. Preferably, the content of the mycelium active nutrient is 20% by weight or less of the whole culture substrate.

The specific structure of the bottle for mushroom cultivation is not limited, and a heat-resistant plastic bottle which can contain 400g to 500g of culture medium and is commercially available can be used. In a bottle for mushroom cultivation, fine-grained sawdust (150g to 450g) of conifer, water and mycelium active nutrients are filled to form a culture substrate, and after heat sterilization, a first-generation culture strain is inoculated into the bottle for mushroom cultivation by using an inoculator for cultivation.

The culture conditions may be appropriately set within a range suitable for the growth of the strain, for example, a temperature of 18 to 25 ℃ and a humidity of 55 to 75%. Further, the oxygen concentration in the culture chamber is preferably 210,000ppm or more, and the carbon dioxide concentration in the culture chamber is preferably 1,000ppm or less. The culture time may be appropriately set, and the production of mycelia is used as an index, and the culture time may be, for example, 60 to 90 days.

(3) A third culturing step: and (3) filling 2000 g-3000 g of culture base material into the fungus bed bag, and inoculating the second generation culture strain into the fungus bed bag for culture to obtain a third generation culture strain.

In the third culturing step, the same fine-grained clasts and mycelium active nutrients of conifer as in the second culturing step can be used as the culture substrate. The material and form of the fungal bed bag are not particularly limited, and for example, a commercially available fungal bed bag capable of containing 2000g to 3000g of culture medium can be used. Filling fine granular scraps (such as 1000-2000 g) of coniferous trees, water and mycelium active nutrients into a fungus bed bag to serve as a culture medium material; after heat sterilization, the second generation culture strain was inoculated into a fungal bed bag using an inoculator for culture.

The culture conditions are not particularly limited, and may be appropriately set within a range suitable for the growth of the strain, for example, a temperature of 18 to 25 ℃ and a humidity of 55 to 75%. Further, the oxygen concentration in the culture chamber is preferably 210,000ppm or more, and the carbon dioxide concentration in the culture chamber is preferably 1,000ppm or less. The culture time may be appropriately set, and the generation of mycelia and primordia of fruit body may be used as an index, and for example, a time of about 60 to 130 days may be set.

(4) A fourth culturing step: inoculating Sparassis crispa MH-3, mycelium and/or fruiting body of the second generation culture strain, and mycelium and/or fruiting body of the third generation culture strain into agar culture medium containing nutrient components, and culturing to obtain the first generation complex strain.

In the fourth culturing step, the nutrient components of the agar medium may be the same as those in the first culturing step, and preferably, the agar medium is a slant agar medium. In the mixture consisting of Sparassis crispa MH-3, hyphae and/or fruit body of the second generation cultured strain, and hyphae and/or fruit body of the third generation cultured strain, fruiting body obtained by cutting the inner surface and outer surface of the fruit body into square of 2mm to 10mm and decomposing the tissue can be used. The culture conditions are not particularly limited, and the culture temperature may be set to 18 to 25 ℃ and the number of culture days may be set to about 45 to 90 days.

Compared with sparassis crispa MH-3, the mixture of the above strains contains stronger mutant strains, evolved strains and excellent strains. By using such a mixture, Sparassis crispa containing high-purity β -1, 3-D-glucan (6-mers) at a high concentration (60 wt% or more) can be stably obtained.

(5) A fifth culturing step: 400 g-500 g of culture medium is filled in a bottle for mushroom cultivation, and the first generation of complex bacteria is inoculated into the bottle for mushroom cultivation for cultivation to obtain a second generation of complex bacterial strain.

In the fifth culturing step, the fine-grained clasts of conifer and the active nutrients of mycelia constituting the culture substrate can be prepared by the same method as in the second culturing step. In addition, the same type of culture bottle as that used in the second culture step can be used as the mushroom culture bottle. In addition, the culture conditions and the culture time may be set in the same manner as in the second culture step.

(6) A sixth culturing step: and filling 2000 g-3000 g of culture base material in a second fungus bed bag, and inoculating the second generation compound strain into the second fungus bed bag for culture to obtain a third generation compound strain.

In the sixth culturing step, the fine-grained clasts and mycelium active nutrients of conifers can be prepared in the same manner as in the third culturing step. In addition, the same type of fungal bed bag as that used in the third culturing step may be used. In addition, the culture conditions and the culture time may be set in the same manner as in the third culture step.

In the embodiment of the invention, the preparation method of the sparassis crispa dry powder comprises the following steps:

a drying procedure: drying the third generation compound strain obtained by the culture method to obtain dry powder of Sparassis crispa.

Preferably, in the drying step, the third generation complex strain used for drying is a fruit body of the third generation complex strain. The drying method and conditions may be appropriately set, and for example, a method of drying at a temperature of 65 to 80 ℃ or a method of freeze-drying may be used. By pulverizing the dried Sparassis crispa into a desired size by such a method, Dry Sparassis crispa powder can be obtained.

The dry sparassis crispa powder prepared in the examples of the present invention contains 60% by weight or more of β -1, 3-D-glucan (6 diverged).

In the present example, a sparassis crispa extract containing β -1, 3-D-glucan (6 ramifications) was obtained by extracting sparassis crispa dry powder with a solvent extraction method.

When the dry powder of Sparassis crispa is extracted, an aqueous solvent or a nonaqueous solvent can be used as a solvent, and preferably, an aqueous solvent is used. The aqueous solvent includes water or alkaline water obtained by adding a base or other alkaline substance to water, an aqueous solvent obtained by adding an organic solvent compatible with alcohol, acidic water containing an acid or acidic substance, and the like. In addition, examples of the non-aqueous solvent include polar organic solvents such as dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF).

When the dry powder of Sparassis crispa is extracted with water, hot water extraction is preferably used. In this case, the temperature of the hot water is preferably controlled to be in the range of 80 to 125 ℃.

The physiologically active agent of the present invention comprises a sparassis crispa extract obtained by extracting sparassis crispa dry powder containing 60 wt% or more of beta-1, 3-D-glucan (6 diverged) as a first active ingredient.

Beta-1, 3-D-glucan (6 divergent) is known to have physiological functional activities such as cancer prevention and treatment, diabetes, hypertension treatment, anticancer (tumor) action, hypoglycemic action, immunostimulating action, antihypertensive action, and the like.

Therefore, the active ingredient of the pig feed provided by the embodiment of the invention, namely the sparassis crispa extract, can be directly used, and can also be prepared by adding sparassis crispa powder.

The carrier as referred to above means a pharmaceutical carrier which is conventional in the pharmaceutical field, and includes diluents, excipients, fillers, binders, wetting agents, disintegrants, dissolving agents, dissolution aids, colorants, deodorizing agents, stabilizing agents, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, adsorption carriers and the like, and can be suitably used according to a conventional method.

The present invention will be described in detail with reference to specific examples, but the present invention is not limited to the following embodiments.

EXAMPLE 1 preparation of Dry powder of Sparassis crispa

(1) First culture Process (test tube culture)

The main instrument used a test tube and a slant agar medium (hereinafter referred to as "K-2").

Specifically, K-2 was composed of 1L purified water, 1.2g peptone, 3.6g of Epimedium (EBIOS, beer yeast tablet, available from Korea food Co., Ltd., Japan), 0.6g calcium chloride, 15g powder agar, 10mL of a needle-leaved tree hot water extract, 3.0g mycelium active nutrient (KN-1), and a slant culture medium having a pH of 6.0 to 7.0.

The K-2 was stored in a test tube. In addition, the sterilization condition of K-2 is set to be 110-115 ℃, and the pressure is 2 air pressure; after sterilizing for 30 minutes, it was left to cool. Sparassis crispa MH-3 (International patent microorganism Collection No. FERM BP-17221) was inoculated into K-2 and cultured to obtain a first generation test tube culture strain (hereinafter referred to as N-1).

(2) Second culture step (bottle culture)

A flask container of the culture medium and a culture substrate were prepared. The bottle container is a heat-resistant plastic bottle (commercially available) for mushroom cultivation capable of containing 400-500 g of culture medium.

The strain culture medium as culture medium comprises fine-grained shaped pieces of conifer such as Larix Gmelini and Pinus densiflora (loading weight of 150-450 g), purified water 65%, and mycelium active nutrient (KN-2) 20% or less (total loading weight of 400-500 g).

The strain culture medium is sterilized at 120-121 ℃ under 2 atm for 3 hours, and then is cooled in an environment below 18 ℃ for 12 hours. Thereafter, N-1 was inoculated on the strain culture medium using an inoculator in a clean room. The temperature of the culture chamber is kept between 18 ℃ and 25 ℃ and the humidity is kept between 55% and 75%. In addition, the oxygen concentration in the culture chamber was carefully maintained at 210,000ppm or less, and the carbon dioxide concentration in the chamber was not more than 1,000 ppm. Culturing the N-1 in a culture room for 60-90 days to obtain mycelium. Hereinafter, the second generation culture strain obtained in the second culture step is referred to as "MH-819".

(3) Third culture Process (fungal bed culture)

A fungal bed bag capable of containing 2000g to 3000g of a culture medium was prepared for culturing Sparassis crispa cells.

A culture medium for a bacterial bed as a culture substrate comprises fine-grained chips (having a packing weight of 1000g to 2000g) of conifers such as larch and red pine, 65% of water, and 20% or less of a mycelium active nutrient (KN-3) (having a total packing weight of 2000g to 3000 g). The culture medium in the bacterial bed is sterilized at 120-121 ℃ and 2 atm for 3 hours, and then is cooled after being placed in an environment below 18 ℃ for 12 hours.

Thereafter, MH-819 was inoculated into a fungal bed medium using an inoculator in a clean room. The temperature of the culture chamber is maintained at 18-25 ℃ and the humidity is maintained at 55-75%. Further, the oxygen concentration in the culture chamber was carefully maintained at 210,000ppm or less, and the oxygen dioxide concentration in the chamber was not more than 1,000 ppm. MH-819 is cultured in a culture room, mycelium can be generated after 60 to 90 days of culture period, and sporophore primordium is generated within 10 to 40 days after the mycelium is generated. Hereinafter, the third-generation cultured strain obtained in the third culture step is referred to as "MH-835".

(4) Fourth culture Process (test tube culture)

Slant agar medium K-2 (test tube) was prepared. The slant agar culture medium comprises 1L purified water, 1.2g peptone, 3.6g epi-ingot, 0.6g calcium chloride, 15g powder agar, 10mL needle-leaved tree hot water extract, and 3.0g mycelium active nutrient (KN-1). Meanwhile, the pH value of the slant agar culture medium of the test tube is 6.0-7.0. The sterilization environment of the slant agar culture medium is set to be 110-115 ℃, and the pressure is 2 air pressure; after sterilizing for 30 minutes, it was left to cool.

The first generation of complex strains was obtained by inoculating a mixture of Sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221), MH-819 mycelium and MH-835 fruiting body (tissue decomposition) in a clean room in an agar slant medium K-2 (test tube) using an inoculator. The culture temperature is controlled between 18 ℃ and 25 ℃. Mycelium can be produced after 45-90 days of culture. Hereinafter, the first generation of the complex strain obtained in the fourth culturing step is referred to as "MH-78".

(5) Fifth culture Process (bottle culture)

A flask container of the culture medium and a culture substrate were prepared. The culture medium bottle container is a heat-resistant plastic bottle for mushroom cultivation.

The strain culture medium as culture medium comprises fine-grained scraps (loading weight of 420-480 g) of conifer such as Larix Gmelini and Pinus densiflora, purified water 65%, and mycelium active nutrient (KN-4) 20% or less. The strain culture medium is sterilized at 120-121 ℃ under 2 atm for 3 hours, and then cooled in an environment below 18 ℃ for 12 hours. The temperature of the culture room is kept between 18 ℃ and 25 ℃ and the humidity is kept between 55% and 75%. Meanwhile, the oxygen concentration in the culture chamber was carefully maintained below 210,000ppm, and the carbon dioxide concentration did not exceed 1,000 ppm.

The MH-78 was inoculated into a strain medium using an inoculator in a clean room for culture to obtain a second generation of complex strain. Hyphae are produced after 60 to 90 days of culture. Hereinafter, the second generation strain obtained in the fifth culturing step is referred to as "MH-78A".

(6) Sixth culturing step (fungal bed culture)

A fungal bed bag for culturing Sparassis crispa cells was prepared.

A culture medium for a bacterial bed as a culture substrate comprises fine-grained chips (the weight of the chips is 2.4 kg-2.8 kg) of conifers such as larch and red pine, 65% of purified water, and 20% or less of mycelium active nutrients (KN-5). The culture medium in the bacterial bed is sterilized at 120-121 ℃ and 2 atm for 3 hours, and then is cooled after being placed in an environment below 18 ℃ for 12 hours. The temperature of the culture room is kept between 18 ℃ and 25 ℃ and the humidity is kept between 55 percent and 75 percent. Meanwhile, the oxygen concentration in the culture chamber was carefully maintained below 210,000ppm, and the carbon dioxide concentration was not more than 1,000 ppm.

Thereafter, MH-78A was inoculated into a culture medium of a bacterial bed using an inoculator in a clean room to culture, and a third generation of a complex strain was obtained. Mycelium can be produced after 60 to 90 days of culture period, and sporophore primordium is produced within 10 to 40 days after the mycelium is produced. Hereinafter, the third generation complex strain obtained in the sixth culturing step is referred to as "MH-78B".

(7) Powdering step

MH-78B is dried in an environment of 65-80 ℃ for 12-18 hours and then passes through a 100-mesh sieve or a 200-mesh sieve to obtain dry powder of the sparassis crispa, which is recorded as MH-3-A.

Example 2 analysis of the composition of Dry powder of Sparassis crispa

A component analysis (per 100g) was performed at the Nippon food analysis center of the consortium Law, using the dry powder of Sparassis crispa (MH-3-A) obtained in example 1 as a test sample A, B. The results are shown in Table 1.

TABLE 1 composition analysis table of Dry Sparassis crispa powder (MH-3-A)

Analytical test items	A (MH-3-A) result value	B (MH-3-A) result value
			Moisture content	4.9g	3.0g
Protein	3.1g	3.4g
			Lipid	1.4g	1.1g
Ash content	1.1g	1.0g
			Carbohydrate compound	89.5g	91.5g
Energy of	192kcal	195kcal
			Sodium salt	3.1mg	3.6mg
β -1, 3-D-Glucan (6 divergence)	61.9g	63.2g

As shown in Table 1, the dry powders (MH-3-A) A and B of Sparassis crispa obtained in example 1 contained 60g or more (60 wt% or more) of β -1, 3-D-glucan (6 split) per 100 g.

EXAMPLE 3 solvent extraction of Dry Sparassis crispa powder (MH-3-A)

Dry powder of Sparassis crispa (MH-3-A) was extracted, and the amount of the components was measured in terms of the glycoprotein ratio.

The sparassis crispa extract was extracted from sparassis crispa dry powder by the following method and the amount of components was measured.

1. The method of "hot water extraction 1 time" is as follows: dried powder 50g obtained by drying Sparassis crispa fruiting body with warm air, and sieving with 100 mesh sieve. To 50g of the dried powder, 700mL of EtOH (ethanol) was added, and left to stand for 2 days for degreasing treatment to obtain an extract and a residue fraction (or a precipitate fraction). Then, 500mL of hot water (121 ℃) was added to the residue fraction, and automatic autoclaving was performed for 2 hours to obtain (i) an extract and (ii) a residue fraction. Subsequently, the extract (r) and the residue fraction (c) are separated. Adding 4L of ethanol into the extract (r), stirring at 3000rpm for 5 minutes at 4 ℃, adding 400mL of 50% ethanol (200mL of water +200mL of ethanol) into the obtained precipitation component, stirring at 3000rpm for 5 minutes, adding acetone, and standing for 1 day to obtain a hot water extract (c).

2. The method of hot water extraction 4 times comprises the following steps: 4L of ethanol was added to extract (r), stirring was carried out at 3000rpm for 5 minutes at 4 ℃, 400mL of 50% ethanol (200mL of water +200mL of ethanol) was added to the obtained precipitate fraction, stirring was carried out at 3000rpm for 5 minutes, 800mL of ethanol was added to the obtained supernatant, stirring was carried out at 3000rpm for 5 minutes, and then acetone was added thereto and left to stand for 1 day, to obtain a hot water extract 4 times.

3. "Cold alkali extraction (1)" method: adding 500mL of 10% NaOH/5% urea into the residue component, standing at 4 deg.C for 2 days, and stirring at 3000rpm for 10 min to obtain cold alkali extract (1) -iv.

4. The cold alkali extraction (2) method, 500mL 10% NaOH/5% urea is added into the residue component of the fourth step, and the mixture is placed at 4 ℃ for 2 days and stirred at 3000rpm for 10 minutes, thus obtaining the cold alkali extract (2) - (-) -c).

5. The hot alkali extraction method comprises the following steps: adding 500mL of 10% NaOH/5% urea into the residue, treating at 65 deg.C for 1 hr, and stirring at 3000rpm for 10 min to obtain thermokalite extract, — o.

And (3) measuring the component amount of each group of extracts:

the sugar content was determined by the phenol-sulfuric acid method using glucose as a standard substance, and the protein content was determined by the BCA method using Bovine Serum Albumin (BSA) as a standard substance. Meanwhile, regarding the sugar composition, 2N-trichloroacetic acid was completely hydrolyzed, reduced, acetylated, and then analyzed by gas chromatography. The measurement results are shown in Table 2.

TABLE 2 table of the measurement results of the component amounts of each extract

Sparassis crispa dry powder (50g), and sieving with 100 mesh sieve

As is clear from the analysis of the ratio of glycoproteins, the dry powder of Sparassis crispa prepared in example 1 was a high-purity polysaccharide containing 90% of sugars. Wherein the extract obtained by the method of "hot water extraction 4 times" has high purity, and the content of beta-1, 3-D-glucan (6 bifurcations) in the extract obtained by the method of "cold alkali extraction (1)" is as high as 8.97g/50g of dry powder of Sparassis crispa. However, OHNO et al (OHNO Dayeshanren, Naohito Yuanqianliang, Miura Mitsugami, et al, Antitumor 1, 3-beta-Glucan from Cultured reagent Body of Sparassis crispa [ J ]. Biological & Pharmaceutical Bulletin,2000,23(7):861 872.) extracted from Sparassis crispa dry powder, obtained extract had sugar content of only 64-70%, protein content of only 0.8-3.0%, beta-1, 3-D-Glucan (6-split) content of only 0-3.68 g/25g Sparassis crispa dry powder, which was much lower than that of Sparassis crispa extract prepared in this example.

Comparative example preparation and composition analysis of Dry powder of Sparassis crispa

1. Preparation of Sparassis crispa dry powder

The microorganism used in this comparative example was a natural Sparassis crispa (hereinafter referred to as "MH-1") collected from mountain, and fruiting bodies of Sparassis crispa were obtained by two-stage culture as described below.

(1) In stage 1, inoculation and culture of MH-1 was performed. The main apparatus uses test tube and slant agar culture medium.

The agar culture medium used in the test tube is prepared by mixing 1L of purified water, 1.2g of peptone, 3.6g of EBIOS, 0.6g of calcium chloride, 0.6mL of HYPONEX, 48g of banana powder, 18g of honey powder and 20g of agar powder, and the pH value is 6.0-7.0. Meanwhile, the sterilization conditions of the agar medium are as follows: sterilizing at 110-115 deg.C under 2-atmosphere pressure for 30 min, and cooling. Hereinafter, this agar medium is described as "K-1".

MH-1 was inoculated into the slant agar medium K-1 prepared by the above method, and the culture of the strain was started. MH-1 was grown in an agar medium, and when mycelium could be produced after culturing at 20 ℃ for 45 to 90 days, it was confirmed that Sparassis crispa (MH-1) was cultured. Thereafter, MH-1 inoculation was repeated every 65 to 120 days. Hereinafter, this test tube cultured strain is described as "M-1".

(2) In stage 2, a bottle container for preparing a culture medium for Sparassis crispa fruiting bodies and a culture medium are prepared.

The main apparatus is a heat-resistant plastic bottle for mushroom cultivation sold in the market. Next, sawdust was used as a culture medium to produce Sparassis crispa fruiting bodies.

The culture medium filled in the heat-resistant plastic bottle is prepared from larch sawdust 1kg, purified water 500mL, peptone 1.0g, EBIOS 45g, calcium chloride 0.6g, banana powder 6g, honey powder 2g, wheat flour 100g, and magnesium chloride 0.5 g. Hereinafter, this bottled medium is described as "sawdust medium 1". The pH value of the sawdust culture medium 1 is adjusted to 6.0-7.0, and the sterilization conditions of the sawdust culture medium 1 are as follows: sterilizing at 120-121 deg.C under 2-atmosphere pressure for 30 min, and cooling.

Subsequently, M-1 was inoculated on the cooled sawdust culture medium 1. Hereinafter, a medium in which M-1 was inoculated on sawdust medium 1 is described as "sawdust medium 2".

The culture condition of the sawdust culture medium 2 is maintained at the temperature of 18-25 ℃ and the humidity of 55% -75%, and the hydrangea mycelium and the sporocarp begin to be cultured. The M-1 is cultured for 60 to 90 days to promote the growth of hyphae, so that primordia of the sporocarp of the sparassis crispa are gradually generated. Thereafter, the culture is carried out at a temperature of 18 to 25 ℃ and a humidity of 75 to 85% for 20 to 50 days, whereby Sparassis crispa fruiting bodies are produced after the primordial culture. Hereinafter, the Sparassis crispa fruiting body is referred to as "M-2".

Then, the obtained M-2 is dried by warm air at 80-100 ℃ for 12-18 hours to obtain a dried sparassis crispa body, and the dried sparassis crispa body is sieved by a 100-mesh sieve to be powdered to obtain sparassis crispa dried powder. Hereinafter, this dry powder of Sparassis crispa is referred to as "M-3".

2. Analysis of the composition of Dry Sparassis crispa powder M-3

Analysis of the composition of Dry Sparassis crispa powder M-3

The dry powder of Sparassis crispa (M-3) prepared in the above manner was used as a sample C to be tested, and the composition analysis was performed (per 100g) at the Nippon food analysis center of the Takara Shuzo. The results are shown in Table 3.

TABLE 3 analysis table of the components of Dry Sparassis crispa powder (M-3)

Analytical test items	C (M-3) result value
		Moisture content	8.8g
Protein	6.9g
		Lipid	0.8g
Fiber	6.3g
		Ash content	3.1g
Sugar content	74.1g
		β 1, 3-D-Glucan (6 divergence)	43.6g

As shown in Table 3, the Sparassis crispa dry powder (M-3) contained 43.6g/100g of β -1, 3-D-glucan (6-mer) and had a small content of protein and sugar. The sparassis crispa dry powder (M-3) had a lower content of beta-1, 3-D-glucan (6 ramifications) than the sparassis crispa dry powder (MH-3-A) prepared in example 1.

By the method for producing the dry powder of Sparassis crispa of example 1, a mixture of MH-819 mycelium and fruiting body (tissue decomposition) and MH-835 mycelium and fruiting body (tissue decomposition) of Sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221), which was cultured from Sparassis crispa MH-3, was obtained in the fourth culturing step. The mixture contains stronger variant strains, evolved strains and excellent strain strains. Although the strain of sparassis crispa is less adaptable to the environment and is difficult to store and culture only by the strain family of fungi, the method for producing the dry powder of sparassis crispa according to the present invention can stably obtain the dry powder of sparassis crispa containing high-purity β -1, 3-D-glucan (6-branched) at a high concentration (60 wt% or more).

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein or by using equivalent structures or equivalent processes performed in the present specification, and are included in the scope of the present invention.

Claims

1. A pig feed is characterized by comprising sparassis crispa dry powder; the sparassis crispa dry powder contains 60 wt% or more of beta-1, 3-D-glucan, and is prepared from sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221).

2. The pig feed according to claim 1, characterized in that the preparation method of the sparassis crispa dry powder comprises the following steps:

(2) a second culture step: filling 400 g-500 g of culture medium into a first mushroom cultivation bottle, inoculating the first generation culture strain into the first mushroom cultivation bottle for cultivation to obtain a second generation culture strain, wherein the culture medium comprises fine granular scraps of conifers, water and mycelium active nutrients;

3. The pig feed according to claim 1 or 2, further comprising sawdust.

4. The pig feed according to claim 3, characterized in that the sawdust is red pine wood chips.

5. A method for enhancing immunity by feeding pig feed is characterized by comprising the following steps of feeding 400-600mg of sparassis crispa dry powder to each pig with initial fattening for 2 times a day; feeding 1000-1500mg of sparassis crispa dry powder to each fattening pig twice, and feeding for 2 times a day; the sparassis crispa dry powder contains 60 wt% or more of beta-1, 3-D-glucan, and is prepared from sparassis crispa MH-3 (International patent organism depositary number FERM BP-17221).

6. The method of enhancing immunity by pig feed according to claim 5, wherein the first-fattening pigs are fed 500mg of dry powder of Sparassis crispa 2 times a day for 15 days; feeding 1000mg of sparassis crispa dry powder to each fattening pig for 15 days, and feeding for 2 times a day.

7. The method of enhancing immunity by pig feed feeding according to claim 5, wherein the single standard for feeding a finishing pig is 1g of dry powder of Sparassis crispa per 60kg of body weight.