WO2019080454A1 - 一株桦褐孔菌及其应用 - Google Patents

一株桦褐孔菌及其应用

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WO2019080454A1
WO2019080454A1 PCT/CN2018/081658 CN2018081658W WO2019080454A1 WO 2019080454 A1 WO2019080454 A1 WO 2019080454A1 CN 2018081658 W CN2018081658 W CN 2018081658W WO 2019080454 A1 WO2019080454 A1 WO 2019080454A1
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fermentation
inonotus obliquus
group
fermentation product
liquid
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PCT/CN2018/081658
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English (en)
French (fr)
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张琳
吴家强
丁志勇
丁雅苓
杨少华
苏文政
黄艳艳
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山东省农业科学院畜牧兽医研究所
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Priority claimed from CN201710990292.1A external-priority patent/CN107916229B/zh
Priority claimed from CN201710990422.1A external-priority patent/CN107550946B/zh
Application filed by 山东省农业科学院畜牧兽医研究所 filed Critical 山东省农业科学院畜牧兽医研究所
Priority to KR1020187029131A priority Critical patent/KR102195870B1/ko
Publication of WO2019080454A1 publication Critical patent/WO2019080454A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention belongs to the fields of bioengineering and biotechnology, and particularly relates to a fermentation technique of fungus B. obliquus and the application thereof in enhancing poultry production and immune performance.
  • Inonotus obliquus is a kind of medicinal fungus. It is rich in polysaccharides, triterpenoids, sterols, total phenols and other active ingredients. It has been used in human diseases such as gastric cancer, intestinal cancer, heart disease and diabetes. Recent research It shows that its fruiting bodies and extracts have unique effects in human anti-tumor, anti-oxidation, immune regulation, hypoglycemic, anti-viral, anti-bacterial, anti-parasitic and other aspects. However, there are few species of Inonotus obliquus, and few fruiting bodies are formed, so wild resources are very limited. Breeding and artificial cultivation of fungi strains is an important means to obtain high-quality B.
  • the fermentation of fungi generally adopts deep-fermentation technology, which is based on the improvement of aeration process technology, and the microbial cells are cultured in a liquid substrate to obtain a large amount of mycelium and other metabolites.
  • Fungi suitable for submerged fermentation should have the following characteristics: (1) rich mycelium protein content and suitable for culture under liquid aeration conditions; (2) fast growth and high yield; (3) stable genetic characteristics and identifiable morphology feature.
  • different strains of the same fungus have different adaptability to deep-fermentation culture.
  • the first problem to be solved in the deep fermentation research and product development of fungi is the screening of suitable strains.
  • the effect of Inonotus obliquus is significant, but whether it is possible to screen suitable strains and their fermentation products can improve the production and immune performance of poultry.
  • the development of new feed additives and immunopotentiators is still unclear.
  • One of the objects of the present invention is to provide a suitable deep-fermentation of B. obliquus for the problem of rare strains suitable for submerged fermentation of P. obliquus and lack of safe, high-efficiency, green new feed additives for poultry breeding;
  • the second is to provide a preparation method of the above-mentioned fermentation product of Inonotus obliquus;
  • the third object of the present invention is to provide an application of the fermentation product of Inonotus obliquus in enhancing the performance of poultry production.
  • the fourth object of the present invention is to provide an application of the fermentation product of Inonotus obliquus as an immune enhancer in enhancing the immune performance of poultry.
  • the present invention adopts the following technical solutions.
  • a strain of Inonotus obliquus YU is deposited in the General Microorganisms Collection and Management Center of China under the accession number CGMCC No. 14549; its ITS sequence is shown in SEQ No. 1.
  • a method for obtaining the above-mentioned fermentation product of Inonotus obliquus adopts the following steps:
  • Seed culture inoculating the inoculum in a liquid medium in a seed tank, and agitating and fermenting to obtain a seed liquid;
  • Submerged fermentation the seed liquid is inoculated into the liquid medium of the fermenter, and agitated and fermented;
  • Precipitation transferring the fermentation broth into a precipitation tank, adding a flocculating agent, and precipitating and filtering to obtain a supernatant;
  • the liquid medium comprises the following components (g/L): starch 10, peptone 10, glucose 15, potassium dihydrogen phosphate 1, magnesium sulfate 0.5, vitamin B 1 0.1, soybean oil 0.2; alternatively, liquid medium
  • the pH is 6.8-7.4.
  • the slope is potato dextrose agar medium (PDA), the culture temperature is 25-28 ° C, and the culture time is 5-7 days.
  • PDA potato dextrose agar medium
  • step (2) the mycelium or spore suspension of the mother seed is inoculated; preferably the mycelium.
  • the culture temperature is 25-28 ° C; the incubation time is 3-10 days; and the shaker rotation speed is 150-200 rpm.
  • step (2) the mother seed is subjected to multi-stage shake flask culture to obtain an inoculum; the inoculum amount of the second and above shake flask culture is 8-15%; preferably, the mother seed is obtained by secondary culture to obtain an inoculum. .
  • the inoculum amount is 8-15%; the total volume of the liquid in the tank is 50-80% of the capacity of the fermenter; the culture temperature is 25-28 ° C; the culture time is 7 -15 days; stirring speed is 100-200 rev / min; ventilation is 0.1-0.5 vvm.
  • the inoculum is subjected to multi-stage seed culture to obtain a seed liquid; preferably, the inoculum is subjected to secondary culture to obtain a seed liquid.
  • the flocculating agent in the step (5) is chitosan, and the preferred amount is 0.05-0.2% by weight of the fermentation broth.
  • the biomass of the fermentation broth in the step (5) is 20 g/L or more.
  • the heating temperature is 70-95 ° C; and the holding time is 4-6 h.
  • step (6) is concentrated to a relative density of 1.035-1.065.
  • step (6) the step of drying the concentrate to a solid is further included.
  • the growth factor of the Inonotus obliquus fermentation product in poultry feed is 0.2-2 wt%; most preferably 0.8 wt%.
  • the inoculum fermentation product of the inoculum is added in an amount of 0.1 to 5% by weight, preferably 0.5 to 2.0% by weight.
  • a poultry feed and an immunopotentiator comprising the fermentation product of Inonotus obliquus prepared by the above method.
  • the YU strain of Inonotus obliquus obtained by the invention has stable genetic property, fast growth rate and high yield, and is suitable for deep fermentation, and can be used as a deep fermentation strain.
  • the addition of the fermentation product obtained from this strain to the poultry diet can significantly enhance the production performance of the organism, effectively increase the average daily gain of poultry, and reduce the feed consumption rate and feed-to-meat ratio.
  • the fermentation product provided by the invention can realize efficient breeding of poultry, reduce production cost, and effectively reduce or even replace the use of additives such as hormones and antibiotics.
  • the body's immune organ growth, non-specific immune response and specific immune response can significantly enhance the body's immune organ growth, non-specific immune response and specific immune response, including the improvement of immune organ index, the promotion of non-specific immune-related indicators, the enhancement of peripheral blood lymphocyte proliferation and differentiation, antibody and neutralization. Increase in antibody titer, increase in cytokine content, and improvement in challenge protection rate.
  • Figure 1 shows the effect of feeding of fermentation products of Inonotus obliquus on daily weight gain of chickens
  • Figure 2 shows the effect of feeding of fermentation products of Inonotus obliquus on chicken meat ratio
  • Figure 3 shows the effects of the fermentation products of Astragalus polysaccharides and Inonotus obliquus on the daily gain of chickens
  • Figure 4 shows the effect of the fermentation products of Astragalus polysaccharides and Inonotus obliquus on the ratio of chicken meat to meat.
  • Figure 5 shows the effect of fermentation products of Inonotus obliquus on immune organ index (21 days);
  • Figure 6 shows the effect of fermentation products of Inonotus obliquus on immune organ index (42 days);
  • Figure 7 shows the effect of fermentation products of Inonotus obliquus on total antioxidant capacity
  • Figure 8 shows the effect of fermentation products of Inonotus obliquus on superoxide dismutase
  • Figure 9 is the effect of fermentation products of Inonotus obliquus on serum lysozyme
  • Figure 10 shows the results of HI antibody detection
  • FIG. 11 shows the results of detection of neutralizing antibodies
  • Figure 12 shows the results of IL-2 detection
  • Figure 13 shows the results of IL-4 detection
  • Figure 14 shows the results of IFN- ⁇ detection
  • FIG. 15 shows the results of IFN- ⁇ /IL-4 detection.
  • Example 1 Isolation and identification of Inonotus obliquus.
  • the H. sinensis JX strain was introduced from the Institute of Biology of the Hebei Academy of Sciences and has been identified as B. obliquus.
  • the YU strain of Inonotus obliquus is a wild strain collected from Dali City, Yunnan province. During the collection, the fruiting insect-free fruiting bodies were removed from the surface of the tree, and the depth of collection was 1-3 cm on the surface of the tree. The specimens were placed in the specimen box and returned to the laboratory for further separation and purification.
  • Separation strains adopt agaric separation method, first remove the wood chips and impurities on the surface of the fruit body with distilled water, then surface disinfection with 75% ethanol, transfer to the ultra-clean table, open the fruit body, and aseptically dissect the deep tissue, quickly put The tiger red solid medium was cultured in a constant temperature incubator (28 ° C) for 3 days, and then the purified bacteria were transferred to a PDA solid medium, and the colony growth condition was observed daily.
  • the ITS-rDNA universal primers ITS1: 5'TCCGTAGGTGAACCTGCGG3' and ITS2: 5'TCCTCCGCTTATTGATATGC3' were PCR-amplified for specific primers, and the strains were identified.
  • the ITS sequence obtained by amplification is shown in SEQ No. 1, which has 99% or more homology with the ITS-rDNA sequence of Inonotus obliquus (DQ103883) in GeneBank, and the isolated wild strain is determined to be Inonotus obliquus.
  • the strain is named YU.
  • the strain was stored in the China General Microorganisms Collection and Management Center on September 5, 2017, and the storage number is CGMCC No.: 14549.
  • Example 2 Obtainment of a fermentation product of Inonotus obliquus.
  • liquid medium According to the amount of liquid medium, weigh the following components (g / L): peptone 10, glucose 15, potassium dihydrogen phosphate 1, magnesium sulfate 0.5, vitamin B 1 0.1, soybean oil 0.2, sterile water Make up to the required volume, adjust the pH to 7.0, sterilize at 121 ° C for 30 min, and set aside.
  • Example 1 The strain preserved by Inonotus obliquus in Example 1 was inoculated into a test tube slant potato dextrose agar medium (PDA), and transferred twice, each time at 25 ° C for 7 days. For the mother, spare;
  • PDA potato dextrose agar medium
  • Seed culture The liquid inoculum was inoculated into 7 L liquid medium in a 10% amount and placed in a 10 L fermentor, incubated at 25 ° C for 10 days, and then transferred to 100 L of fermentation containing 70 L of liquid medium. In the tank, the mixture was incubated at 25 ° C for 10 days;
  • Submerged fermentation the fermented product was transferred into a 1000 kg fermentor containing 700 kg of liquid medium, and mixed and incubated at 25 ° C for 10 days;
  • Example 3 Submerged fermentation performance of Inonotus obliquus.
  • Example 1 The inoculum YU and JX strains of Example 1 were subjected to submerged fermentation according to the steps (1) to (4) in Example 2, and 100 mL of each fermentation broth was taken, centrifuged, and the precipitate was collected and washed with deionized water. After centrifugation, the precipitate was collected three times, and the mixture was dried at 105 ° C to measure the biomass.
  • Table 1 According to the data in Table 1, the strain YU strain has the best fermentation index, the mycelium shape is excellent, the growth potential is strong, and the biomass growth is obvious; while the JX strain has almost no growth and is not suitable for deep fermentation.
  • Example 4 Enhancement of poultry production performance by fermentation products of Inonotus obliquus.
  • Average Daily Weight Gain (ADG) (Ending Weight - Initial Weight) / Feeding Days
  • ADFI Average daily feed intake
  • Feed to meat ratio (F/G) average daily feed intake / average daily gain.
  • Astragalus polysaccharide is an important polysaccharide immunopotentiator with deep research and extensive use. Studies have shown that the addition of a suitable concentration of Astragalus polysaccharides to the feed can enhance the performance of poultry production. In order to more intuitively reflect the effect of Inonotus obliquus on enhancing poultry production performance, a comparative study of the two was carried out with reference to Astragalus polysaccharides.
  • group I 0.8% concentration of the fermentation product of Inonotus obliquus was added to the diet, and group II added Astragalus polysaccharide in the diet (purchased from Beijing Shengtaier Technology Co., Ltd., the recommended feeding amount was 0.2%).
  • the test method is as described in 4.2, with the measurement time as the abscissa and the weight gain as the ordinate as the graph 3; the measurement time as the abscissa and the feed-to-meat ratio as the ordinate as the graph 4.
  • Example 5 The immune enhancement effect of the fermentation product of Inonotus obliquus on poultry.
  • Astragalus polysaccharide can significantly enhance the immunity of animals.
  • Astragalus polysaccharide was selected as a positive control, and the effects of the two preparations were compared.
  • Animal experiments were divided into 7 groups (I-VII), and 20 SPF chickens of 1 day old in each group were housed in isolators.
  • the I-V group was fed with 0.2%, 0.4%, 0.8%, 1%, 2% of the feed of the fermentation product of Inonotus obliquus in Example 2, and the VI group was a blank control group (no immunopotentiator was added to the feed).
  • the VII group was fed with a feed containing 0.2% Astragalus polysaccharide (purchased from Beijing Shengtaier Technology Co., Ltd., recommended to be added in an amount of 0.2%).
  • the feeding period is 42 days, during which time it is free to eat.
  • the histogram of the thymus, spleen and bursa of the two experimental groups was made with the immune organ index as the ordinate.
  • the results of 21 days are shown in Fig. 5, and the results of 42 days are shown in Fig. 6.
  • the thymus index of the II-V group reached 52%, 51%, 51%, and 43%, respectively, which was significantly different from the other groups (P ⁇ 0.05); the spleen index of II and IV was significantly higher than the other groups; In the bursa index, the group III reached 0.7, which was significantly different from the other groups (P ⁇ 0.05).
  • the index of group I which was slightly higher than the negative control, the other concentration groups promoted the development of immune organs significantly, and were superior to the positive control.
  • the total antioxidant capacity in organ tissues is one of the important indicators reflecting the body's antioxidant activity.
  • Superoxide dismutase as a key antioxidant enzyme in the body, plays an important role in the body's oxidation and antioxidant balance.
  • the determination of these two indicators can reflect the changes in the body's non-specific immunity to some extent.
  • the lysozyme in the body mainly comes from neutrophils, monocytes and phagocytic lysosomes, so when the relevant cells are activated, the content of lysozyme will increase accordingly.
  • T-AOC, SOD and LSZ showed that when the fermentation concentration of Inonotus obliquus was lower than 0.8% (I-III group), the T-AOC value increased with the feeding time, and the higher the concentration The more significant the change.
  • the T-AOC value decreased with the continued increase in the concentration of the fermentation of Inonotus obliquus (groups IV and V), and the T-AOC value at 42 days was lower than the value of 21 days. It indicated that the total antioxidant capacity of the chicken body was positively correlated with the concentration of the low concentration (0.8%) of the growth of the inonot, and the effect was decreased when the addition amount exceeded 1%.
  • the optimal working concentration of the selected B. obliquus fermented material was used to determine the effect of its proliferation on the peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the NDV inactivated vaccine was first vaccinated, and the immunization was performed once every 3 weeks.
  • the neck was injected subcutaneously, 0.2 mL per chicken.
  • Peripheral blood was collected at 1d, 3d, 7d, 14d, 21d, 28d, and 35d after the first exemption, serum was separated, antibody titer (HI) was determined by hemagglutination inhibition test, and serum was diluted by fixed virus to determine 7d after the first exemption.
  • HI antibody titer
  • the antibody fed to the B. obliquus group was 2-3 titers higher at each measurement time point, and the Astragalus polysaccharide group (Q+V group, immunized and Compared with the Astragalus polysaccharides, it is also higher than 1-2 titers.
  • the neutralizing antibody titer the H+V group and the Q+V group were more distinct and distinct: the neutralizing antibody in the Q+V group was basically consistent with the V group, while the neutralizing antibody in the H+V group was elevated early and significantly higher. In other groups. It indicated that the effect of fermentation of Fusarium oxysporum to enhance humoral immunity was more significant than that of market-oriented products.
  • Cytokines have functions of regulating cell growth and differentiation, immune function, participating in inflammatory reaction and wound healing, and cytokines can be classified into Th1 type and Th2 type according to the type of immune response.
  • Th1 type includes IL-2, IFN- ⁇ , tumor necrosis factor (TNF), etc., which are mainly produced by cell-mediated immune responses
  • Th2 type includes IL-4, IL-5, IL-6, IL-12, etc. Cytokines produced during B cell activation.
  • IL-2 is an important indicator of cellular immunity, which stimulates Thp cells to differentiate into Th0 cells. Th0 secretes cytokines such as IL-2, IL-4 and IFN- ⁇ , so it is usually measured by IL-2.
  • IL-4 measures the level of humoral immunity
  • IFN- ⁇ measures the level of cellular immunity
  • the ratio of IFN- ⁇ /IL-4 reflects the equilibrium state of Th1/Th2 cells. From the results, it can be seen that both the fermentation of H. oxysporum and Astragalus polysaccharide can promote the expression of IL-2, IL-4 and IFN- ⁇ and the ratio of IFN- ⁇ /IL-4. The difference between the two is small, but H+V The group is slightly higher, indicating that under the premise of promoting humoral immunity, the fermentation of P. obliquus can significantly enhance the effect of cellular immunity, and effectively promote the transformation of cellular immunity from Th2 to Th1 in the later stage of the experiment. A polysaccharide enhancer effect and a more comprehensive immunopotentiator.
  • the NDV virulent challenge was applied 35 days after the first exemption to determine the protective power of each group of vaccines.
  • the chickens in Group C were obviously loss of appetite, fluffy feathers, wilting, and all deaths (5/5); after 5 days, 2 chickens died in V group (2/5), and 1 in Q+V group Chicken died (1/5) and there was no death in the H+V group (0/5).
  • the immunopotentiator of Inonotus obliquus can effectively improve the immune efficacy of the vaccine and provide complete protection for the test animals, and the effect is remarkable, and is superior to the Astragalus polysaccharide.
  • the YU strain of Inonotus obliquus obtained by the invention has stable genetic property, fast growth rate and high yield, and is suitable for deep fermentation, and can be used as a deep fermentation strain.
  • the fermentation product obtained from the strain can realize efficient breeding of poultry and reduce production cost; effectively reduce or even replace the use of additives such as hormones and antibiotics; at the same time, can significantly enhance the immune performance of the body and can be used as a feed additive in poultry feed. Or an immunopotentiator.

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Abstract

提供了一株桦褐孔菌(Inonotus obliquus)，保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.14549。还提供了一种上述菌株深层发酵获得发酵产物的方法：菌株通过母种培育，摇瓶培养，种子培养获得种子液，将种子液进行深层发酵获得发酵液，将发酵液通过沉淀、浓缩步骤获得发酵产物。还提供了上述桦褐孔菌发酵产物作为家禽饲喂添加剂和免疫增强剂的应用。

Description

一株桦褐孔菌及其应用 技术领域
本发明属于生物工程和生物技术领域，具体涉及真菌桦褐孔菌的发酵技术及其发酵产物在增强家禽生产、免疫性能中的应用。
背景技术
随着人们生活水平的提高，对于畜产品的质量要求日益增强，近年来食品安全问题的不断显现，更是让全社会对动物源性食品安全的重视达到了前所未有的高度。如何在禽类疫病持续高发的背景下减少甚至杜绝抗生素的使用、降低禽类耐药性和其他毒副作用，实现禽类的绿色健康养殖和食品安全，寻找安全、高效、绿色新型饲料添加剂和免疫增强剂代替药物成为养殖业发展的重中之重。药用真菌以其独特的功效成为新型饲料添加剂和免疫增强剂的潜在研究目标，相关研究已表明真菌提取物或制剂能够有效促进动物的生产和免疫性能。
桦褐孔菌(Inonotus obliquus)作为药用真菌的一种，富含多糖、三萜、甾醇、总酚等活性成分，早已被人类应用于胃癌、肠癌、心脏病、糖尿病等疾病，近期研究显示其子实体和提取物在人类抗肿瘤、抗氧化、免疫调节、降血糖、抗病毒、抗细菌、抗寄生虫等方面也有独特的疗效。但桦褐孔菌品种现存较少，也很少形成子实体，故野生资源十分有限。进行真菌的菌株选育及人工培育是获得优质桦褐孔菌的重要手段，但经长期探索，始终没能取得突破性进展。发酵工程技术的进步使资源难题的破解成为可能。真菌的发酵一般采用深层发酵技术，它是在通气工艺技术的改进的基础上将微生物细胞置于液体底物里进行培养，以此获得大量菌丝体及其他代谢物质。适合深层发酵的真菌应具有以下特点：(1)菌丝体蛋白质含量丰富且适应液体通气条件下的培养；(2)生长速度快且高产；(3)具有稳定的遗传特性和可鉴别的形态特征。同时，同种真菌的不同菌株对深层发酵培养的适应能力存在差异，所以要进行真菌的深层发酵研究和产品开发首先要解决的问题就是适宜菌株的筛选。桦褐孔菌功效显著，但能否筛选到合适的菌株及其发酵产物能否提高禽类的生产和免疫性能，用于新型饲料添加剂和免疫增强剂的开发尚不清楚。
技术问题
针对桦褐孔菌适于深层发酵的菌株稀少和禽类养殖缺乏安全、高效、绿色新型饲料添加剂等问题，本发明的目的之一在于提供一株适宜深层发酵的桦褐孔菌；本发明的目的之二在于提供上述桦褐孔菌发酵产物的制备方法；本发明的目的之三在于提供一种桦褐孔菌发 酵产物在增强家禽生产性能中的应用。本发明的目的之四在于提供一种桦褐孔菌发酵产物作为免疫增强剂在增强家禽免疫性能中的应用。
技术解决方案
为实现上述目的，本发明采用如下技术方案。
一株桦褐孔菌(Inonotus obliquus)YU，保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.14549；其ITS序列如SEQ No.1所示。
一种获得上述桦褐孔菌发酵产物的方法，采用以下步骤：
(1)母种培育：将桦褐孔菌接种于试管斜面上培养得到母种；
(2)摇瓶培养：将母种接种于液体培养基培养，得到接种液；
(3)种子培养：将接种液接种于种子罐中的液体培养基内，通气搅拌发酵获得种子液；
(4)深层发酵：将种子液接种于发酵罐的液体培养基内，通气搅拌发酵；
(5)沉淀：将发酵液转入沉淀罐，加入絮凝剂，沉淀后过滤，得上清液；
(6)浓缩：将上清液转入真空浓缩罐，浓缩至一定密度出料，即得发酵产物。
所述液体培养基包括以下组分(g/L)：淀粉10，蛋白胨10，葡萄糖15，磷酸二氢钾1，硫酸镁0.5，维生素B 1 0.1，豆油0.2；可选地，液体培养基的pH为6.8-7.4。
可选地，步骤(1)中，斜面为马铃薯葡萄糖琼脂培养基(PDA)，培养温度为25-28℃，培养时间为5-7天。
可选地，步骤(2)中，接种为母种的菌丝体或孢悬液；优选为菌丝体。
可选地，培养温度为25-28℃；培养时间为3-10天；摇床转速为150-200转/分钟。
可选地，步骤(2)中，母种经多级摇瓶培养获得接种液；二级及以上摇瓶培养的接种量为8-15％；优选地，母种经二级培养获得接种液。
可选地，步骤(3)和步骤(4)中，接种量为8-15％；罐中液体总体积为发酵罐容量的50-80％；培养温度为25-28℃；培养时间为7-15天；搅拌转速为100-200转/分钟；通气量为0.1-0.5vvm。
可选地，步骤(3)中，接种液经多级种子培养获得种子液；优选地，接种液经二级培养获得种子液。
步骤(5)中所述絮凝剂为壳聚糖，优选的添加量为发酵液重量的0.05-0.2％。
优选地，步骤(5)中发酵液的生物量为20g/L以上。
作为优化，沉淀前加热并保持一段时间；更为优化地，加热温度为70-95℃；保温时 间为4-6h。
可选地，步骤(6)浓缩至相对密度为1.035-1.065。
优选地，步骤(6)之后还包括将浓缩液干燥成固体的步骤。
一种上述方法制备的桦褐孔菌发酵产物作为家禽饲喂添加剂和免疫增强剂的应用。
可选地，桦褐孔菌发酵产物在家禽饲料中的添加量为0.2-2wt％；最优选为0.8wt％。
可选地，桦褐孔菌发酵产物在家禽饮水的添加量为0.1-5wt％，优选为0.5-2.0wt％。
一种含上述方法制备的桦褐孔菌发酵产物的家禽饲料和免疫增强剂。
有益效果
本发明筛选获得的桦褐孔菌YU菌株，遗传性能稳定、生长速度快，产量高，适于深层发酵，可作为深层发酵的菌种。由该菌种获得的发酵产物添加于家禽日粮中能够显著增强机体的生产性能，有效提升家禽的平均日增重，降低饲料消耗率和料肉比。本发明提供的发酵产物能够实现家禽的高效养殖，降低生产成本；有效减少甚至替代激素、抗生素等添加剂的使用。同时，能够显著增强机体的免疫器官生长、非特异性免疫反应和特异性免疫反应，包括免疫器官指数的提高、非特异性免疫相关指标的提升、外周血淋巴细胞增殖分化能力的增强、抗体和中和抗体滴度的增长、细胞因子含量的提升和攻毒保护率的改善。
附图说明
图1为桦褐孔菌发酵产物饲喂对鸡日增重影响；
图2为桦褐孔菌发酵产物饲喂对鸡料肉比影响；
图3为黄芪多糖与桦褐孔菌发酵产物饲喂对鸡日增重影响；
图4为黄芪多糖与桦褐孔菌发酵产物饲喂对鸡料肉比影响。
图5为桦褐孔菌发酵产物对免疫器官指数的影响(21天)；
图6为桦褐孔菌发酵产物对免疫器官指数的影响(42天)；
图7为桦褐孔菌发酵产物对总抗氧化能力的影响；
图8为桦褐孔菌发酵产物对超氧化物歧化酶的影响；
图9为桦褐孔菌发酵产物对血清溶菌酶的影响；
图10为HI抗体检测结果；
图11为中和抗体检测结果；
图12为IL-2检测结果；
图13为IL-4检测结果；
图14为IFN-γ检测结果；
图15为IFN-γ/IL-4检测结果。
菌种保存信息
桦褐孔菌(Inonotus obliquus)于2017年9月5日保存于中国普通微生物菌种保藏管理中心，保存地址为北京市朝阳区北辰西路1号院3号中国北京中国科学院微生物研究所，保存编号为CGMCC No.14549。
本发明的实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1 桦褐孔菌的分离鉴定。
桦褐孔菌JX株引自河北省科学院生物研究所，已鉴定为桦褐孔菌。桦褐孔菌YU株为野生菌株，采集自云南省大理市。采集时将无病虫害的子实体从树木表面取下，采集深度为树木表层1-3厘米，装入标本盒中返回实验室进行下一步的分离与纯化。分离菌种采用伞菌分离法，先用蒸馏水去掉子实体表面木屑及杂质，再用75％乙醇进行表面消毒，转入超净台后掰开子实体，并无菌剖取深部组织，迅速放入虎红固体培养基，置于恒温培养箱(28℃)中培养3天，后将纯化的菌转入PDA固体培养基中，每日观察菌落生长状况。随后以改良CTAB法提取的桦褐孔菌基因组为模板，ITS-rDNA通用引物ITS1：5’TCCGTAGGTGAACCTGCGG3’和ITS2：5’TCCTCCGCTTATTGATATGC3’为特异性引物进行PCR扩增，进行菌株的鉴定。扩增获得的ITS序列如SEQ No.1所示，其与GeneBank中的桦褐孔菌(DQ103883)ITS-rDNA序列具有99％以上的同源性，判定分离的野生菌为桦褐孔菌，并名为YU株，该菌株于2017年9月5日保存于中国普通微生物菌种保藏管理中心，保存编号为CGMCC No.:14549。
实施例2 桦褐孔菌发酵产物的获得。
制备液体培养基：根据液体培养基用量，分别称取下列组分(g/L)：蛋白胨10、葡萄糖15、磷酸二氢钾1、硫酸镁0.5、维生素B 1 0.1、豆油0.2，灭菌水补至所需体积，调整pH至7.0，121℃灭菌30min，备用。
(1)母种培育：将实施例1中桦褐孔菌保存的菌种接种于试管斜面马铃薯葡萄糖琼脂培养基(PDA)中，转接2次，每次在25℃条件下活化7天，为母种，备用；
(2)摇瓶培养：将母菌转于500mL一级培养液中，培养10天，然后转入二级培养液，继续培养5天，摇床温度28℃，转速180转/分，培养结束取少量培养物进行显微镜镜检，无杂菌进入下步；
(3)种子培养：按10％的量将液体种菌接种于7L液体培养基并装入10L发酵罐 中，25℃通气培养10天，再将其转入装有70L液体培养基的100L发酵罐中，25℃通气混合培养10天；
(4)深层发酵：将发酵物转入装有700kg液体培养基的1000kg发酵罐中，25℃通气混合培养10天；
(5)沉淀：检测发酵罐内生物量达20g/L时，停止发酵，将发酵罐升温到90度，保温4小时，再将发酵液转入沉淀罐，加入0.1％发酵液重量的壳聚糖，沉淀48小时，用半框过滤器过滤上清液；
(6)浓缩：将上清液转入真空浓缩罐进行浓缩，测得相对密度为1.035时完成发酵产物的制备，分装。
实施例3 桦褐孔菌深层发酵性能。
将实施例1中桦褐孔菌YU和JX菌种按实施例2中步骤(1)-(4)进行深层发酵，分别取发酵液各100mL，离心，收集沉淀，用去离子水水洗后再离心，重复3次后收集沉淀，在105℃条件下烘干称重，测定生物量。结果如表1所示。通过表1数据可知，菌株YU株发酵指标最优，菌丝形态优，生长势强，生物量增长明显；而JX株几乎未见生长，不适宜深层发酵。
表1 不同菌株液体培养结果
Figure PCTCN2018081658-appb-000001
实施例4 桦褐孔菌发酵产物对家禽生产性能的增强。
4.1 不同添加量桦褐孔菌发酵产物对鸡日增重与料肉比的影响
将40只1日龄SPF鸡分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组，其中Ⅰ-Ⅳ组分别在饲料中添加实施例2中的桦褐孔菌发酵产物，添加量为0.2％、0.4％、0.8％、2％，剩余一组为对照组，日粮中不添加桦褐孔菌发酵产物。分别饲养于隔离器中，饲养期42天，期间自由采食。分别于第7、14、21、28、35、42天对鸡只进行称重，称重前绝食12h。计算鸡体的增重及消耗的饲料量，进而得到不同时期的平均日增重、平均日采食量和料肉比，其计算方式为：
平均日增重(ADG)＝(期末重量-期初始重量)/饲喂天数
平均日采食量(ADFI)＝期间总耗料量/饲喂天数
料肉比(F/G)＝平均日采食量/平均日增重。
以测定时间为横坐标，以增重为纵坐标作图1；以测定时间为横坐标，以料肉比为纵 坐标作图2。结果显示，在平均增重值指标上，相比于对照组，21天之前，不同添加浓度的桦褐孔菌发酵物对鸡的增重均有促进效果；21天之后，除Ⅱ组外，其他组增重效果依然高于对照组。在料肉比指标方面，各组的料肉比均低于对照组，表明桦褐孔菌添加物可有效提高饲料的报酬率。综上所述，本发明的桦褐孔菌发酵产物能够增强家禽的生产性能，0.8％的添加浓度效果最佳。
4.2 桦褐孔菌发酵产物与黄芪多糖对鸡日增重与料肉比的影响
黄芪多糖是目前研究较为深入、使用较为广泛的重要多糖类免疫增强剂。研究表明，在饲料中添加适宜浓度的黄芪多糖能够增强家禽的生产性能。为了更直观的体现桦褐孔菌在增强家禽生产性能方面的效果，以黄芪多糖为参照，进行两者的比较研究。
设试验组2组(Ⅰ和Ⅱ)，对照组1组(Ⅲ)，每组1日龄SPF鸡20只。Ⅰ组在日粮中添加0.8％浓度的桦褐孔菌发酵产物，Ⅱ组在日粮中添加黄芪多糖(购自北京生泰尔科技股份有限公司，建议饲喂添加量0.2％)。试验方法如4.2所述，以测定时间为横坐标，以增重为纵坐标作图3；以测定时间为横坐标，以料肉比为纵坐标作图4。结果显示，两种添加物均能够促进各试验期平均体重的增加和耗料量及料肉比的下降，但相互比较，桦褐孔菌的效果更为显著：在42天试验期内，桦褐孔菌发酵物组重量增加值平均高出黄芪多糖组9.67mg，料肉比平均则降低0.156。
实施例5 桦褐孔菌发酵产物对家禽的免疫增强效果。
黄芪多糖能够显著提升动物的免疫力。为了更好的比较桦褐孔菌发酵物增强家禽免疫性能的效果，选取黄芪多糖为阳性对照，并对两种制剂的效果进行比较。动物试验共分为7个组(Ⅰ-Ⅶ)，每组1日龄SPF鸡20只，分别饲养于隔离器中。Ⅰ-Ⅴ组分别饲喂含0.2％、0.4％、0.8％、1％、2％实施例2中桦褐孔菌发酵产物的饲料，Ⅵ组为空白对照组(饲料中不添加免疫增强剂)，Ⅶ组饲喂含0.2％黄芪多糖(购自北京生泰尔科技股份有限公司，建议添加量为0.2％)的饲料作。饲养期42天，期间自由采食。
分别于饲喂的第21天和42天进行免疫器官(胸腺、脾脏和法氏囊)指数的测定：鸡称重后进行宰杀，每组3只，分别采集胸腺、脾脏和法氏囊并称重，按照免疫器官指数(％)＝免疫器官质量/活体质量×100％的计算公式得到免疫器官指数。以免疫器官指数为纵坐标作两实验组胸腺、脾脏和法氏囊的柱状图，21天结果见图5，42天结果见图6。另外，在第7、14、21、28、35和42天对各试验组5只鸡进行外周血的分离，经3000r/min离心10min分离血清，用于总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、血清溶菌酶(LSZ)的测定，以每毫升血清中的T-AOC单位数为纵坐标作T-AOC的柱状图，如图7所 示；以每毫升血清中酶含量为纵坐标作SOD、LSZ的柱状图，如图8和图9所示。图5-9中，*表示p<0.05，Duncan's检验，误差线为平均值±SD(标准差)。
结果表明，21日龄时，不同浓度桦褐孔菌发酵物能够显著促进免疫器官的发育。在胸腺指数中，除Ⅴ组低于阴性对照而高于阳性对照外，其余各组均高于阳性和阴性对照；各试验组的脾脏指数均显著高于阳性和阴性对照；法氏囊指数与脾脏指数相似，区别仅在于阳性对照高于阴性对照。横向比较，Ⅱ组各免疫器官指数均最高，在此时期促进器官发育最为显著。42日龄时，Ⅱ-Ⅴ组的胸腺指数分别达到52％、51％、51％和43％，与其他组差异显著(P<0.05)；Ⅱ和Ⅳ的脾脏指数明显高于其他组；而法氏囊指数中，Ⅲ组达到0.7，与其他组差异显著(P<0.05)。总体来看，除Ⅰ组的指数稍高于阴性对照外，其他添加浓度组促进免疫器官发育的效果显著，且优于阳性对照。
器官组织中的总抗氧化能力是反映机体抗氧化作用的重要指标之一，而超氧化物歧化酶作为机体关键性抗氧化酶，对生物体体内氧化与抗氧化平衡起着重要的作用。这两个指标的测定在一定程度上能够反映机体非特异性免疫里的变化。体内溶菌酶主要来自嗜中性白细胞、单核细胞及吞噬细胞溶酶体，所以当相关细胞被激活时，溶菌酶的含量会相应增加。T-AOC、SOD和LSZ的测定结果显示，当桦褐孔菌发酵物浓度低于0.8％时(Ⅰ-Ⅲ组)，T-AOC值随饲喂时间呈现升高的趋势，且浓度越高变化越显著。随着桦褐孔菌发酵物添加浓度的继续增加(Ⅳ和Ⅴ组)，T-AOC值有所降低，且42天时的T-AOC值表现低于21天的数值。表明在桦褐孔菌发酵物低浓度(0.8％)添加时，鸡体的总抗氧化能力与浓度呈正相关，而添加量超过1％时效果有所下降。与阴性对照相比，添加了桦褐孔菌发酵物各组的SOD均有明显提升；相较于阳性对照，Ⅲ组、Ⅳ组和Ⅴ组的效果更强，其中Ⅲ组效果最好。纵向比较，21天的SOD要高于42天的，各组趋势相同。在不同时间点，添加了桦褐孔菌发酵物组的LSZ均高于阴性对照。当桦褐孔菌发酵物浓度不高于0.8％时，LSZ随药物浓度的增加而升高；当药物浓度高于0.8％时，随药物浓度的增加而降低。纵向比较同样是21天的含量要高于42天。
综合免疫器官指数和非特异性免疫指标，0.8％的桦褐孔菌发酵物添加浓度是其最佳工作浓度，同时也显著优于黄芪多糖的作用效果。
然后利用筛选的桦褐孔菌发酵物最佳工作浓度，测定了其对机体外周血淋巴细胞(Peripheral blood mononuclear cell,PBMC)增殖分化效果的影响。将30只1日龄SPF鸡分为3组，其中一组在饲料中添加桦褐孔菌发酵产物(H组)，添加量为0.8％，一组添加黄芪多糖(Q组)，添加量为0.2％，第三组为对照组，日粮中不添加任何药品。分别饲养于隔离器 中，饲养期42天，期间自由采食。分别于21和42天无菌采集三组鸡的外周血并分离淋巴细胞，将PBMC密度调整至10 7个/mL后加入96孔板，每孔100μL。随后每孔加入10μL工作浓度为2μg/mL的ConA，混匀后置于37℃、5％CO 2恒温箱培养，并设置阴性对照孔(加等量的PBS)和无细胞空白对照孔，每样品重复6孔。最后在酶标仪上读取OD 490值并计算均值，刺激指数(stimulation index,SI)按如下公式进行计算：
SI＝(OD 试验组-OD 空白对照)/(OD 阴性对照-OD 空白对照)
结果如表2、表3所示，在两个时间点，试验组的刺激指数明显高于对照，可达到3.48，表明发酵产物能有效促进淋巴细胞的增殖、活化。
表2 21日龄PBMC的刺激指数
H组 Q组 对照组 阴性 空白
OD 490 1.306 1.169 0.862 0.862 0.683
SI 3.480 2.7151 1.000 \ \
表3 42日龄PBMC的刺激指数
H组 Q组 对照组 阴性 空白
OD 490 3.765 2.553 1.507 1.507 0.437
SI 3.110 1.9776 1.000 \ \
接下来结合疫苗的免疫进行了桦褐孔菌发酵物与黄芪多糖增强疫苗免疫效果的对比研究。以新城疫灭活疫苗为例，将21日龄SPF鸡分为在日粮中添加桦褐孔菌发酵产物+疫苗免疫组(H+V组)、不添加桦褐孔菌发酵产物疫苗免疫组(V组)和不免疫且不饲喂桦褐孔菌发酵产物组(C组)3组，每组10只。21日龄进行NDV灭活疫苗首免，间隔3周再加强免疫一次，颈部皮下注射，每支鸡0.2mL。于首免后1d、3d、7d、14d、21d、28d、35d采集外周血，分离血清，利用血凝抑制试验测定抗体滴度(HI)，并利用固定病毒稀释血清的方法测定首免后7d、14d、21d、28d、35d中和抗体效价；利用ELISA试剂盒测定首免后14d和28d细胞因子(IL-2，IL-4，IFN-γ)的表达量，测定方法按照试剂盒说明书进行。HI检测结果见图10，中和抗体检测结果见图11。IL-2，IL-4，IFN-γ和IFN-γ/IL-4检测结果如图12-15所示。图10-15中，*代表p<0.05，**代表p<0.01，Duncan's检验，误差线为平均值±SD(标准差)。
结果显示，与C组和V组相比，饲喂桦褐孔菌组的抗体在每个测定时间点均高出2-3个滴度，而与黄芪多糖组(Q+V组，免疫且饲喂黄芪多糖)比较，也高出1-2个滴度。对于中和抗体效价，H+V组与Q+V组强弱区分更加明显：Q+V组的中和抗体与V组基本一致，而H+V组的中和抗体提升早且显著高于其他组。表明桦褐孔菌发酵物提升体液免疫的效果较市场化产品更为显著。
细胞因子具有调节细胞生长分化、免疫功能、参与炎症反应和创伤愈合的功能，根据免疫应答类型可将细胞因子分为Th1型和Th2型。Th1型包括IL-2、IFN-γ、肿瘤坏死因子(TNF)等，主要由细胞介导的免疫反应所产生，Th2型则包括IL-4、IL-5、IL-6、IL-12等B细胞激活过程中产生的细胞因子。在免疫应答过程中，IL-2是细胞免疫的重要指标，它刺激Thp细胞向Th0细胞分化，Th0又分泌IL-2、IL-4和IFN-γ等细胞因子，故通常以IL-2衡量Th反应强度，IL-4衡量体液免疫水平，IFN-γ衡量细胞免疫水平，同时以IFN-γ/IL-4的比值反应Th1/Th2细胞的平衡状态。由结果可知，桦褐孔菌发酵物和黄芪多糖均能够促进IL-2、IL-4和IFN-γ的表达及IFN-γ/IL-4的比值，两者差异不大，但H+V组稍高，表明在能够很好的促进体液免疫的前提下，桦褐孔菌发酵物还能显著增强细胞免疫的效果，在试验后期有效促进细胞免疫从Th2型向Th1型转化，是比黄芪多糖效果更佳、作用更全面的免疫增强剂。
首免后35天时利用NDV强毒进行攻毒，以测定各组疫苗的保护力。攻毒3天后C组鸡只明显食欲不振，羽毛蓬松，精神萎靡，并全部死亡(5/5)；5天后，V组有2只鸡死亡(2/5)，Q+V组有1只鸡死亡(1/5)，H+V组无死亡(0/5)。可见，桦褐孔菌免疫增强剂能够有效提升疫苗的免疫效力，为试验动物提供完全的保护，其作用效果显著，优于黄芪多糖。
本发明筛选获得的桦褐孔菌YU菌株，遗传性能稳定、生长速度快，产量高，适于深层发酵，可作为深层发酵的菌种。由该菌种获得的发酵产物能够实现家禽的高效养殖，降低生产成本；有效减少甚至替代激素、抗生素等添加剂的使用；同时，能够显著增强机体的免疫性能，可应用于家禽饲料中作为饲料添加剂或免疫增强剂。
序列表自由内容
Figure PCTCN2018081658-appb-000002
Figure PCTCN2018081658-appb-000003

Claims (10)

  1. 一株桦褐孔菌(Inonotus obliquus)，保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.14549。
  2. 一种获得如权利要求1所述的桦褐孔菌的发酵产物的方法，其特征在于，采用以下步骤：
    (1)母种培育：将桦褐孔菌接种于试管斜面上培养得到母种；
    (2)摇瓶培养：将母种接种于液体培养基培养，得到接种液；
    (3)种子培养：将接种液接种于种子罐中的液体培养基内，通气搅拌发酵获得种子液；
    (4)深层发酵：将种子液接种于发酵罐的液体培养基内，通气搅拌发酵；
    (5)沉淀：将发酵液转入沉淀罐，加入絮凝剂，沉淀后过滤，得上清液；
    (6)浓缩：将上清液转入真空浓缩罐，浓缩至一定密度出料，即得发酵产物。
  3. 根据权利要求2所述的制备方法，其特征在于，步骤(3)和步骤(4)中，接种量为8-15％；通气量为0.1-0.5vvm。
  4. 根据权利要求2所述的制备方法，其特征在于，所述步骤(2)-(4)中，液体培养基包括以下组分(g/L)：淀粉10，蛋白胨10，葡萄糖15，磷酸二氢钾1，硫酸镁0.5，维生素B 10.1，豆油0.2；pH为6.8-7.4。
  5. 根据权利要求2所述的制备方法，其特征在于，步骤(5)中发酵液的生物量为20g/L以上。
  6. 根据权利要求2所述的方法，其特征在于，步骤(5)中絮凝剂为壳聚糖。
  7. 根据权利要求2所述的方法，其特征在于，步骤(5)中沉淀前还包括发酵液加热过程，加热温度为70-95℃；保温时间为4-6h；
    步骤(6)之后还包括将浓缩液干燥成固体的步骤。
  8. 一种如权利要求2-7任一方法制备的桦褐孔菌发酵产物作为家禽饲喂添加剂和免疫增强剂的应用。
  9. 根据权利要求8所述的应用，其特征在于，桦褐孔菌发酵产物在家禽饲料中的添加量为0.2-2wt％；桦褐孔菌发酵产物在家禽饮水的添加量为0.1-5wt％。
  10. 一种含权利要求2-7任一方法制备的桦褐孔菌发酵产物的家禽饲料和免疫增强剂。
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CN110357981A (zh) * 2019-07-22 2019-10-22 湖北亿彤实业有限公司 一种具有细胞内吞介导功能的多糖载体及其制备方法
CN110357981B (zh) * 2019-07-22 2023-08-04 福建亿彤生物科技有限公司 一种具有细胞内吞介导功能的多糖载体及其制备方法
CN115181680A (zh) * 2020-11-18 2022-10-14 山西运奕道生物科技有限公司 桦褐孔菌培养物中的活性物质提取及降血糖应用
CN115181680B (zh) * 2020-11-18 2023-09-05 山西运奕道生物科技有限公司 桦褐孔菌培养物中的活性物质提取及降血糖应用
CN114010548A (zh) * 2021-11-29 2022-02-08 天津市康婷生物工程集团有限公司 层孔菌深层发酵浓缩液与菌体提取物组合物及其制备方法、应用
CN114010548B (zh) * 2021-11-29 2024-05-03 天津市康婷生物工程集团有限公司 层孔菌深层发酵浓缩液与菌体提取物组合物及其制备方法、应用
CN114587007A (zh) * 2022-02-23 2022-06-07 张淑军 一种以莲叶、绞股蓝为主原料的健康养生草本烟
CN114587007B (zh) * 2022-02-23 2023-08-04 张淑军 一种以莲叶、绞股蓝为主原料的健康养生草本烟
CN115181732A (zh) * 2022-07-21 2022-10-14 宜兴市天石饲料有限公司 一种有效去除杂质的噬菌体裂解酶源饲料添加剂制备方法
CN115737490A (zh) * 2022-11-02 2023-03-07 稀物集(广州)生物科技有限公司 一种桦褐孔菌酵母发酵液的制备方法及其在化妆品中的应用
CN116200273A (zh) * 2022-12-12 2023-06-02 黑龙江省科学院微生物研究所 一株适合快速发酵的桦褐孔菌h6及其发酵罐培养方法和应用

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