WO2021109291A1 - 一种银耳菌种的培育方法 - Google Patents

一种银耳菌种的培育方法 Download PDF

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WO2021109291A1
WO2021109291A1 PCT/CN2019/128327 CN2019128327W WO2021109291A1 WO 2021109291 A1 WO2021109291 A1 WO 2021109291A1 CN 2019128327 W CN2019128327 W CN 2019128327W WO 2021109291 A1 WO2021109291 A1 WO 2021109291A1
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hyphae
white fungus
culture
white
medium
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PCT/CN2019/128327
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English (en)
French (fr)
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齐宁
牛贵清
张彬
宋琳琳
王娜
王学璐
林萌
高瑞
韩秀云
韩鹏
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山东安华生物医药股份有限公司
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Publication of WO2021109291A1 publication Critical patent/WO2021109291A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/40—Cultivation of spawn
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost

Definitions

  • the present disclosure relates to the technical field of strain cultivation, and specifically, is a method for cultivating white fungus strains.
  • Tremella is a fungus of Basidiomycota, Tremella, Tremella, Tremella family, Tremella (Tremella) fungus, also known as white fungus, snow fungus, Sichuan ear, etc., has the reputation of "the crown of seedlings", is a kind of edible And medicinal fungi. Wild Tremella is scarce, and artificial cultivation is generally adopted. The life history of Tremella fuciformis is complicated, and the preparation and preservation of pure mycelium of Tremella fuciformis is still quite difficult.
  • Cross-cultivation can be used to cultivate Tremella fungus strains, which are cultivated through the cross-cultivation of two hyphae, thereby cultivating Tremella fungus strains with improved characteristics, such as strong resistance, resistance to degradation, stable growth, and high yield.
  • the repeatability or reproducibility of the method used in the prior art is not high.
  • there are other shortcomings or problems in the cultivation method of Tremella fungus in the prior art such as low cellulase activity, low yield, and weak adaptability.
  • the purpose of one or more embodiments of the present disclosure includes, for example, providing a method for breeding Tremella fungus strains to solve the problems of low Tremella cellulase activity, low yield, weak adaptability, and poor repeatability in the prior art.
  • the present disclosure provides a method for cultivating white fungus strains, which includes the following steps:
  • the medium components used in step (1) are bran 100-110g/L, maltose 10-20g/L, sucrose 10-20g/L, peptone 2-3g/L, Superphosphate 2-3g/L, magnesium sulfate 0.5-1g/L and agar 20-30g/L.
  • the medium used in step (2) is composed of 20-30 g/L glucose, 3-5 g/L yeast powder, 3-5 g/L peptone, and 1.5-3 g /L KH 2 PO 4 L, Mg 2 SO 4 ⁇ 7H 2 O1.5-3g/L and vitamin B 1 0.1-0.3g/L.
  • the composition of the medium used in step (3) is 70-75g/L of phoebe wood chips, 10-15g/L of bran, 2-5g/L of rice straw powder, and sucrose 4- 5g/L, bone meal 4-5g/L, urea 2-3g/L and gypsum 2-3g/L, the ratio of material to water is 1:1.2-1.3.
  • step (3) the ratio of the white fungus pure white hyphae to the incense ash hyphae is 50:1-75:1.
  • the carbon dioxide content is maintained to be less than 0.2% during the cross-culture process in step (3).
  • the white fungus pure white hyphae selected in step (1) has a diameter of 1.5-3 microns, and the growth is good.
  • the diameter of the incense ash hyphae selected in step (2) is 3-5 microns, and the growth is good.
  • step (3) the cross ratio of the white fungus pure white hyphae and the incense ash hyphae is 40:1-70:1.
  • step (3) the crossing ratio of the white fungus hyphae and the incense ash hyphae is 45:1 to 68:1.
  • step (3) the cross ratio of white fungus pure white hyphae and incense ash hyphae is 50:1 to 65:1.
  • step (1) and step (2) can be exchanged arbitrarily.
  • the present disclosure also provides the primordium of the fruit body of Tremella obtained by the method for cultivating Tremella species described herein.
  • the present disclosure also provides a culture medium for cross-culture, including:
  • the cross culture is a cross culture of white fungus hyphae and fragrant ash hyphae.
  • the beneficial effects of the present disclosure include at least: In the cross-breeding method, the ratio of white fungus pure white hyphae to incense ash hyphae is appropriate, the incense ash hyphae grow well, can promote the growth and development of the tremella hyphae, and ensure the high yield and stability of the strains
  • Tremella is a fungus of the genus Tremella, which mainly refers to Tremella fuciformis.
  • the fruiting bodies of Tremella are widely used as traditional Chinese medicine.
  • pure white mycelium refers to pure white fungus hyphae. It is generally believed that the cultivation of pure white fungus hyphae requires the assistance of companion bacteria because of their weak ability to decompose lignin and cellulose.
  • fragrant ash mycelium generally refers to the companion bacteria that can assist the growth of Tremella. It has a strong ability to decompose cellulose and lignin. It can symbiotically better with pure Tremella hyphae, and even synergistically decompose and utilize the lignin matrix. , It is helpful to the cultivation of Tremella, especially the fruiting bodies of Tremella.
  • Co-cultivation refers to the co-cultivation of pure white hyphae and incense ash hyphae, and generally includes extraction of pure white hyphae, extraction of incense ash hyphae and co-cultivation.
  • the primordium of the fruit body of Tremella is the product of the mycelium twisted into a mass in the process of mycelium culture, which can be gelatinized by continuing the culture, and then form a mature fruit body.
  • ear wood refers to the wild or artificially cultivated segment trees that are longer than white fungus.
  • One or more embodiments of the present disclosure provide a method for cultivating white fungus strains, which includes the following steps:
  • steps (1) and (2) can be implemented sequentially, for example, step (1) is performed and step (2) is performed, or step (1) is performed after step (2) is performed. In one or more embodiments, steps (1) and (2) can also be performed simultaneously, for example, during step (2), step (1) is performed, or during step (1), step ( 2).
  • the isolation and culture of white fungus hyphae include: selecting ear wood, air-drying and sterilizing, and shaving wood dust from the wood at the base of the ear, inoculating the culture medium, and placing Culturing at room temperature, such as 25-28° C., for the first time period, such as 8-10 days, to obtain pure white fungus hyphae.
  • (2) the separation and culture of the incense ash hyphae includes: after sterilization, cutting off, for example, sawing off the most part of the fungus segment, where there are marble-like markings on the cross section, containing the mycelium.
  • the sawdust is inoculated on the culture medium and cultured for a second period of time, for example, 5-6 days, the hyphae are selected and transferred to the tube to cultivate the incense ash hyphae.
  • (3) cross-culture includes: cultivating the white fungus pure white hypha obtained in step (1) and the incense ash hypha obtained in step (2) by using a sawdust medium mixing method.
  • (3) cross-culture includes: first pick out the white fungus pure white hyphae and culture medium from the test tube, put it on the sawdust medium, then extract a little fragrant ash hyphae, put them next to the white fungus pure white hyphae, and cultivate To generate the primordium of Tremella fruit body.
  • (3) the temperature of the cross-culture is 20-22°C.
  • (3) the humidity of the cross-culture is 85%-95%, such as 87%-93%, or 88%-91%.
  • (3) the light intensity of the cross-culture is 50-100 lux, such as 60-90 lux, or 70-80 lux, or 73-77 lux.
  • One or more embodiments of the present disclosure provide a method for cultivating white fungus strains, which includes the following steps:
  • steps (1) and (2) can be implemented sequentially, for example, step (1) is performed and step (2) is performed, or step (1) is performed after step (2) is performed. In one or more embodiments, steps (1) and (2) can also be performed simultaneously, for example, during step (2), step (1) is performed, or during step (1), step ( 2).
  • the medium used in step (1) may be a medium suitable for culturing white fungus pure white hyphae, for example, a conventional white fungus pure white hypha medium in the art.
  • the medium components used in step (1) are bran 100-110g/L, maltose 10-20g/L, sucrose 10-20g/L, peptone 2-3g/L, Superphosphate 2-3g/L, magnesium sulfate 0.5-1g/L and agar 20-30g/L.
  • the white fungus pure white mycelium grows densely, vigorously, and grows well.
  • the medium used in step (2) may be a medium suitable for cultivating incense ash hyphae, for example, an incense ash hypha medium conventional in the art.
  • the medium used in step (2) is composed of 20-30 g/L glucose, 3-5 g/L yeast powder, 3-5 g/L peptone, and 1.5-3 g /L KH 2 PO 4 L, Mg 2 SO 4 ⁇ 7H 2 O1.5-3g/L and vitamin B 1 0.1-0.3g/L.
  • Different medium conditions have certain differences in the growth rate and mycelial morphology of the mycelium. With this medium, the growth rate is fast, the mycelium is strong and dense, and the back of the mycelium is obviously blackened due to the production of melanin.
  • the composition of the medium used in step (3) is 70-75g/L of phoebe wood chips, 10-15g/L of bran, 2-5g/L of rice straw powder, and sucrose 4- 5g/L, bone meal 4-5g/L, urea 2-3g/L and gypsum 2-3g/L, the ratio of material to water is 1:1.2-1.3.
  • Sawdust medium formula cultured strains are composed of white fungus hyphae and fragrant ash hyphae, which grow fast and grow well.
  • the ratio of the white fungus pure white hyphae to the incense ash hyphae is 50:1-75:1. Improper inoculation ratio can easily cause the white fungus pure white hyphae to become weak and inactivated, and the amount of inoculation with the white fungus pure white hyphae exceeds a certain amount in Belgium, resulting in insufficient nutrients absorbed by the fragrant ash hyphae to supply the normal growth of the white fungus pure white hyphae, and long-term starvation. If the amount of inoculation of incense ash mycelia is too small, it will also cause uneven ears, uneven size, and low yield.
  • step (3) maintaining the carbon dioxide content during the cross-culture process is less than 0.2%, which is beneficial to overcome the phenomenon of hyphae aging and inactivation.
  • the white fungus pure white hyphae selected in step (1) has a diameter of 1.5-3 microns, and the growth is good.
  • the diameter of the incense ash hyphae selected in step (2) is 3-5 microns, and the growth is good.
  • step (3) the cross ratio of the white fungus pure white hyphae and the incense ash hyphae is 40:1-70:1.
  • step (3) the crossing ratio of the white fungus hyphae and the incense ash hyphae is 45:1 to 68:1.
  • step (3) the cross ratio of white fungus pure white hyphae and incense ash hyphae is 50:1 to 65:1.
  • the present disclosure also provides the primordium of the fruit body of Tremella obtained by the method for cultivating Tremella species described herein.
  • the present disclosure also provides a culture medium for cross-culture, including:
  • the cross culture is a cross culture of white fungus hyphae and fragrant ash hyphae.
  • the inventors found that the prior art cross-breeding method lacks certainty on the hypha characteristics and cross ratio of the white fungus hyphae and fragrant ash hyphae selected for cross-fertilization, resulting in the unstable characteristics of the cultivated Tremella fungus species, and the target is obtained.
  • the reproducibility or reproducibility of white fungus strains is not high.
  • the inventors first and surprisingly discovered the above-mentioned breeding method of Tremella fungus. Through the control of various parameters and conditions in the breeding method, especially the ratio of two kinds of hyphae, it solved the problem of breeding of Tremella fungus. There are technical problems.
  • the beneficial effects of the present disclosure include at least:
  • the ratio of white fungus pure white hyphae to incense ash hyphae is appropriate, the incense ash hyphae grow well, can promote the growth and development of the tremella hyphae, and ensure the high yield and stability of the strains
  • the yield of cultivating white fungus strains is increased by 7%-8.7%; through suitable light, the cellulase activity is enhanced to 0.414 ⁇ 0.017, which ensures the strong growth of the strains.
  • a method for cultivating white fungus strains includes the following steps:
  • the wood dust is shaved from the inside of the wood where the ear base grows, and inoculated with medium (bran 100/L, maltose 20g/L, sucrose 20g/L, Peptone 3g/L, superphosphate 3g/L, magnesium sulfate 0.5g/L, and agar 20g/L), placed at 25°C for 10 days to obtain pure white fungus hyphae; select the 3 micron diameter, good growth White fungus pure white hyphae;
  • the culture medium (glucose 20g/L, yeast powder 5g/L, peptone 3g/ L, KH 2 PO 4 1.5g/L, Mg 2 SO 4 ⁇ 7H 2 O 1.5g/L and Vitamin B 1 0.1g/L) were cultured for 6 days, the hyphae were selected and transferred to the tube to cultivate the incense ash hyphae
  • the sawdust medium mixing method to cultivate the white fungus pure white hyphae and the fragrant ash hypha inoculation ratio of 50:1.
  • the white fungus pure white hyphae and the culture medium are picked out from the test tube and put into the sawdust medium (Phoebe nanmu) Sawdust 75g/L, bran 10g/L, rice straw meal 2g/L, sucrose 4g/L, bone meal 4g/L, urea 2g/L and gypsum 2g/L, material to water ratio 1:1.2 above), and then extract A small amount of fragrant ash mycelium is placed next to the white fungus hyphae, and cultured at 20°C, humidity 85%, light intensity of 50 lux, and carbon dioxide content less than 0.2%, until the primordium of Tremella fruit body is formed.
  • a method for cultivating white fungus strains includes the following steps:
  • the wood dust is shaved from the wood at the base of the ear, and inoculated with medium (bran 100/L, maltose 10g/L, sucrose 10g/L, Peptone 2g/L, superphosphate 3g/L, magnesium sulfate 1g/L and agar 30g/L), cultured at 26°C for 9 days to obtain pure white fungus hyphae; select 3 micron diameter, good growth of white fungus Pure white hyphae;
  • a method for cultivating white fungus strains includes the following steps:
  • the wood dust is shaved from the inside of the wood where the ear base grows, and inoculated with medium (bran 100/L, maltose 20g/L, sucrose 20g/L, Peptone 3g/L, superphosphate 3g/L, magnesium sulfate 0.5g/L, and agar 30g/L), placed at 26°C for 9 days to obtain pure white fungus hyphae; select the 3 micron diameter, good growth White fungus pure white hyphae;
  • the culture medium (glucose 20g/L, yeast powder 5g/L, peptone 3g/ L, KH 2 PO 4 1.5g/L, Mg 2 SO 4 ⁇ 7H 2 O 1.5g/L and Vitamin B 1 0.2g/L) were cultured for 6 days, the hyphae were selected and transferred to the tube to cultivate the incense ash hyphae
  • the sawdust medium mixing method to cultivate the white fungus pure white hyphae and the fragrant ash hypha inoculation ratio of 50:1.
  • the white fungus pure white hyphae and the culture medium are picked out from the test tube and put into the sawdust medium (Phoebe nanmu) Sawdust 75g/L, bran 10g/L, rice straw meal 2g/L, sucrose 4g/L, bone meal 4g/L, urea 2g/L and gypsum 2g/L, material to water ratio 1:1.2 above), and then extract A small amount of fragrant ash mycelium is placed next to the white fungus hyphae, and cultured at 20°C, humidity 85%, light intensity of 100 lux, and carbon dioxide content less than 0.2%, until the primordium of Tremella fruit body is formed.
  • the ratio of white fungus pure white hyphae to fragrant ash hyphae is appropriate, and fragrant ash hyphae grow well, which can promote the growth and development of tremella hyphae and ensure the high yield and stability of the strains, which is better than using traditional methods
  • the yield of cultivating white fungus strains is increased by 7%-8.7%; through suitable light, the cellulase activity is enhanced to reach 0.414 ⁇ 0.017, which ensures the strong growth of the strains.

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Abstract

本公开涉及菌种培育技术领域，具体来说，是一种银耳菌种的培育方法。银耳纯白菌丝与香灰菌丝交合培育银耳菌种控制接种比例50:1-75:1，香灰菌丝长势好，能够促进银耳菌丝的生长发育，保证了菌种的高产以及稳定性，通过合适光照，增强了纤维素酶活力，保证了菌种的强壮发育。

Description

一种银耳菌种的培育方法
相关申请的交叉引用
本申请要求于2019年12月5日提交中国专利局的申请号为201911235928.7、名称为“一种银耳菌种的培育方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及菌种培育技术领域，具体来说，是一种银耳菌种的培育方法。
背景技术
银耳是担子菌门、银耳纲、银耳目、银耳科、银耳属(Tremella)真菌，又称白木耳、雪耳、川耳等，有“苗中之冠”的美名，是一种兼具食用和药用价值的真菌。野生银耳数量稀少，一般采用人工培育。银耳生活史较复杂，银耳纯菌丝的制备与保存目前仍相当困难。可以使用交合培育来培养银耳菌种，其通过两种菌丝的交合培育，从而培育出具有改善的特性，例如抵抗力强、不易退化、生长稳定、产量高的银耳菌种。但是在现有技术中使用的该方法的重复性或再现性不高。另外，现有技术中银耳菌种的培育方法还存在其他缺点或问题，例如银耳菌纤维素酶活力低、产量不高、适应性弱。
发明内容
本公开的一个或多个实施方式的目的包括例如提供一种银耳菌种的培育方法以解决现有技术中银耳菌纤维素酶活力低、产量不高、适应性弱、重复性差等问题。
本公开提供一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养
选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基上，置于25-28℃经8-10天培养得银耳纯白菌丝；
(2)香灰菌丝分离培养
灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基上培养5-6天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为24-26℃、光照强度为200-600lx、pH＝6.9-7.2；
(3)交合培养
利用木屑培养基混接法进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基上，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于20-22℃、湿度为 85％-95％、光照强度为50-100lux培养至生成银耳子实体原基。
在一种或多种实施方式中，步骤(1)中所用培养基组分为麸皮100-110g/L、麦芽糖10-20g/L、蔗糖10-20g/L、蛋白胨2-3g/L、过磷酸钙2-3g/L、硫酸镁0.5-1g/L和琼脂20-30g/L。
在一种或多种实施方式中，步骤(2)中所用培养基组成为葡萄糖20-30g/L、酵母粉3-5g/L、蛋白胨3-5g/L、KH 2PO 41.5-3g/L、Mg 2SO 4·7H 2O1.5-3g/L和维生素B 10.1-0.3g/L。
在一种或多种实施方式中，步骤(3)中所用培养基组成为刨花楠木木屑70-75g/L、麸皮10-15g/L,稻草秸秆粉屑2-5g/L、蔗糖4-5g/L、骨粉4-5g/L、尿素2-3g/L和石膏2-3g/L，料水比1:1.2-1.3。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-75:1。
在一种或多种实施方式中，步骤(3)中交合培养过程中保持二氧化碳含量＜0.2％。
在一种或多种实施方式中，步骤(1)中选取的银耳纯白菌丝直径为1.5-3微米，长势良好。
在一种或多种实施方式中，步骤(2)选取的香灰菌丝直径为3-5微米，长势良好。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为40:1-70:1。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为45:1-68:1。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-65:1。
在一种或多种实施方式中，步骤(1)和步骤(2)顺序可任意调换。
本公开还提供本文所述的银耳菌种的培育方法得到的银耳子实体原基。
本公开还提供一种用于交合培养的培养基，包括：
刨花楠木木屑75g/L、麸皮10g/L,稻草秸秆粉屑2g/L、蔗糖4g/L、骨粉4g/L、尿素2g/L、石膏2g/L和水，料水比为1:1.2。
在一种或多种实施方式中，所述交合培养是银耳纯白菌丝和香灰菌丝的交合培养。
本公开的有益效果至少包括：交合培育方法中，银耳纯白菌丝与香灰菌丝的比例恰当，香灰菌丝长势好，能够促进银耳菌丝的生长发育，保证了菌种的高产以及稳定性
具体实施方式
为了使本技术领域的人员更好地理解本公开中的技术方案，下面将结合本公开实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本公开一部分实施例， 而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本公开保护的范围。
除非本文另有定义，否则结合本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。以下描述示例性方法和材料，但是与本文描述的那些类似或等同的方法和材料也可以用于本公开的实践或测试中。
如本文所用，银耳是银耳属真菌，主要是指Tremella fuciformis。银耳的子实体作为传统中药被广泛应用。
如本文所用纯白菌丝是指银耳纯菌丝，一般认为，由于银耳自身对木质素和纤维素分解的能力较弱，因此银耳纯菌丝的培育需要伴生菌的协助。
如本文所用香灰菌丝一般是指能够协助银耳生长的伴生菌，其有较强的纤维素和木质素分解能力，其与银耳纯菌丝共生可以更好的，甚至协同地分解利用木质素基质，有助于银耳，特别是银耳子实体的培育。
如本文所用交合培养是指纯白菌丝与香灰菌丝的共培养，一般包括提取纯白菌丝，提取香灰菌丝以及共同培养。
如本文所用，银耳子实体原基是菌丝体培养过程中菌丝扭接成块后的产物，其继续培养可以胶质化，然后形成成熟的子实体。
本文所用术语“耳木”，是指长过银耳的野生或人工栽培的段木。
本公开的一种或多种实施方式提供一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养；
(2)香灰菌丝分离培养；以及
(3)交合培养。
在一种或多种实施方式中，步骤(1)和(2)可以相继实施，例如进行步骤(1)之后进行步骤(2)，或在进行步骤(2)之后进行步骤(1)。在一种或多种实施方式中，步骤(1)和(2)也可以同时进行，例如在进行步骤(2)期间，进行步骤(1)，或在进行步骤(1)期间，进行步骤(2)。
在一种或多种实施方式中，(1)银耳纯白菌丝的分离培养包括：选取耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基上，置于常温例如25-28℃培养第一时间段例如8-10天，得银耳纯白菌丝。
在一种或多种实施方式中，(2)香灰菌丝分离培养包括：灭菌后，将银耳段木出耳最多处截断例如锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基上培养第二时间段例如5-6天，将菌丝挑选出来后转管培养得香灰菌丝。
在一种或多种实施方式中，(3)交合培养包括：利用木屑培养基混接法对步骤(1) 获得的银耳纯白菌丝和步骤(2)获得的香灰菌丝进行培育。例如，(3)交合培养包括：先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基上，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，培养至生成银耳子实体原基。在一种或多种实施方式中，(3)交合培养的温度为20-22℃。在一种或多种实施方式中，(3)交合培养的湿度为85％-95％，例如87％-93％，或88％-91％。在一种或多种实施方式中，(3)交合培养的光照强度为50-100lux，例如60-90lux，或70-80lux，或73-77lux。
本公开的一种或多种实施方式提供一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养
选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基上，置于25-28℃经8-10天培养得银耳纯白菌丝；
(2)香灰菌丝分离培养
灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基上培养5-6天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为24-26℃、光照强度为200-600lx，例如250-550lux，或300-500lux，或250-450lux，或300-400lux，或330-370lux、pH＝6.9-7.2，例如pH＝7.0或pH＝7.1；
(3)交合培养
利用木屑培养基混接法进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基上，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于20-22℃、湿度为85％-95％、光照强度为50-100lux例如60-90lux，或70-80lux，或73-77lux培养至生成银耳子实体原基。
在一种或多种实施方式中，步骤(1)和(2)可以相继实施，例如进行步骤(1)之后进行步骤(2)，或在进行步骤(2)之后进行步骤(1)。在一种或多种实施方式中，步骤(1)和(2)也可以同时进行，例如在进行步骤(2)期间，进行步骤(1)，或在进行步骤(1)期间，进行步骤(2)。
步骤(1)中所用培养基可以是适用于培养银耳纯白菌丝培养基，例如本领域常规的银耳纯白菌丝培养基。在一种或多种实施方式中，步骤(1)中所用培养基组分为麸皮100-110g/L、麦芽糖10-20g/L、蔗糖10-20g/L、蛋白胨2-3g/L、过磷酸钙2-3g/L、硫酸镁0.5-1g/L和琼脂20-30g/L。银耳纯白菌丝生长致密、旺盛、长势较好。
步骤(2)中所用培养基可以是适用于培养香灰菌丝的培养基，例如本领域常规的香灰菌丝培养基。在一种或多种实施方式中，步骤(2)中所用培养基组成为葡萄糖20-30g/L、酵母粉3-5g/L、蛋白胨3-5g/L、KH 2PO 41.5-3g/L、Mg 2SO 4·7H 2O1.5-3g/L和维生素B 10.1-0.3g/L。不同培养基条件对香灰菌丝的生长速度和菌丝形态存在一定的差异，采用该 培养基，生长速度快，菌丝健壮致密，菌丝背面因为黑色素的产生明显变黑。
在一种或多种实施方式中，步骤(3)中所用培养基组成为刨花楠木木屑70-75g/L、麸皮10-15g/L,稻草秸秆粉屑2-5g/L、蔗糖4-5g/L、骨粉4-5g/L、尿素2-3g/L和石膏2-3g/L，料水比1:1.2-1.3。木屑培养基配方培养的菌种由银耳纯白菌丝和香灰菌丝构成，长速快、长势好。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-75:1。接种比例不当容易造成银耳纯白菌丝衰弱钝化，与银耳纯白菌丝接种量超过一定比利时，导致香灰菌丝吸收的养分不够供应银耳纯白菌丝的正常生长，长期处于饥饿状态，而香灰菌丝接种量过少，也将导致出耳不齐，大小不均，产量偏低。
在一种或多种实施方式中，步骤(3)中交合培养过程中保持二氧化碳含量＜0.2％，有利于克服菌丝衰老钝化的现象。
在一种或多种实施方式中，步骤(1)中选取的银耳纯白菌丝直径为1.5-3微米，长势良好。
在一种或多种实施方式中，步骤(2)选取的香灰菌丝直径为3-5微米，长势良好。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为40:1-70:1。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为45:1-68:1。
在一种或多种实施方式中，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-65:1。
本公开还提供本文所述的银耳菌种的培育方法得到的银耳子实体原基。
本公开还提供一种用于交合培养的培养基，包括：
刨花楠木木屑75g/L、麸皮10g/L,稻草秸秆粉屑2g/L、蔗糖4g/L、骨粉4g/L、尿素2g/L、石膏2g/L和水，料水比为1:1.2。
在一种或多种实施方式中，所述交合培养是银耳纯白菌丝和香灰菌丝的交合培养。
本发明人发现现有技术中的交合培育方法对进行交合选取的银耳纯白菌丝和香灰菌丝的菌丝特征以及交合比例缺乏把握，导致培育出的银耳菌种的特性不稳定，获得目标银耳菌种的重复性或重现性不高。发明人首次并令人惊讶地发现了上述银耳菌种的培育方法，通过对培育方法中各个参数和条件的控制，特别是两种菌丝的比例，很好了解决了银耳菌种培育的现有技术中存在的问题。
本公开的有益效果至少包括：交合培育方法中，银耳纯白菌丝与香灰菌丝的比例恰当，香灰菌丝长势好，能够促进银耳菌丝的生长发育，保证了菌种的高产以及稳定性，比利用 传统方法培育银耳菌种的产量提高7％-8.7％；通过合适光照，增强了纤维素酶活力，可达到0.414±0.017，保证了菌种的强壮发育。
实施例1
一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养
选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基(麸皮100/L、麦芽糖20g/L、蔗糖20g/L、蛋白胨3g/L、过磷酸钙3g/L、硫酸镁0.5g/L和琼脂20g/L)上，置于25℃经10天培养得银耳纯白菌丝；选取直径为3微米，长势良好的银耳纯白菌丝；
(2)香灰菌丝分离培养
灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基(葡萄糖20g/L、酵母粉5g/L、蛋白胨3g/L、KH 2PO 41.5g/L、Mg 2SO 4·7H 2O1.5g/L和维生素B 10.1g/L)上培养6天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为24℃、光照强度为300lux、pH＝7，选取直径为3微米，长势良好的香灰菌丝；
(3)交合培养
利用木屑培养基混接法按银耳纯白菌丝与香灰菌丝接种比例50:1进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基(刨花楠木木屑75g/L、麸皮10g/L,稻草秸秆粉屑2g/L、蔗糖4g/L、骨粉4g/L、尿素2g/L和石膏2g/L，料水比1:1.2上)，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于20℃、湿度为85％、光照强度为50lux、二氧化碳含量少于0.2％，培养至生成银耳子实体原基。
实施例2
一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养
选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基(麸皮100/L、麦芽糖10g/L、蔗糖10g/L、蛋白胨2g/L、过磷酸钙3g/L、硫酸镁1g/L和琼脂30g/L)上，置于26℃经9天培养得银耳纯白菌丝；选取直径为3微米，长势良好的银耳纯白菌丝；
(2)香灰菌丝分离培养
灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基(葡萄糖30g/L、酵母粉35g/L、蛋白胨3.5g/L、KH 2PO 43g/L、 Mg 2SO 4·7H 2O3g/L和维生素B 10.3g/L)上培养5天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为26℃、光照强度为600lux、pH＝7.2，选取直径为3微米，长势良好的香灰菌丝；
(3)交合培养
利用木屑培养基混接法按银耳纯白菌丝与香灰菌丝接种比例65:1进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基(刨花楠木木屑70g/L、麸皮15g/L,稻草秸秆粉屑5g/L、蔗糖5g/L、骨粉5g/L、尿素3g/L和石膏3g/L，料水比1:1.2上)，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于22℃、湿度为95％、光照强度为75lux、二氧化碳含量少于0.2％，培养至生成银耳子实体原基。
实施例3
一种银耳菌种的培育方法，包括如下步骤：
(1)银耳纯白菌丝的分离培养
选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基(麸皮100/L、麦芽糖20g/L、蔗糖20g/L、蛋白胨3g/L、过磷酸钙3g/L、硫酸镁0.5g/L和琼脂30g/L)上，置于26℃经9天培养得银耳纯白菌丝；选取直径为3微米，长势良好的银耳纯白菌丝；
(2)香灰菌丝分离培养
灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基(葡萄糖20g/L、酵母粉5g/L、蛋白胨3g/L、KH 2PO 41.5g/L、Mg 2SO 4·7H 2O1.5g/L和维生素B 10.2g/L)上培养6天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为24℃、光照强度为200lux、pH＝6.9，选取直径为3微米，长势良好的香灰菌丝；
(3)交合培养
利用木屑培养基混接法按银耳纯白菌丝与香灰菌丝接种比例50:1进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基(刨花楠木木屑75g/L、麸皮10g/L,稻草秸秆粉屑2g/L、蔗糖4g/L、骨粉4g/L、尿素2g/L和石膏2g/L，料水比1:1.2上)，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于20℃、湿度为85％、光照强度为100lux、二氧化碳含量少于0.2％，培养至生成银耳子实体原基。
尽管通过优选实施例的方式对本公开进行了详细描述，但本公开并不限于此。在不脱离本公开的精神和实质的前提下，本领域普通技术人员可以对本公开的实施例进行各种等效的修改或替换，而这些修改或替换都应在本公开的涵盖范围内/任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范 围之内。因此，本公开的保护范围应所述以权利要求的保护范围为准。
工业实用性
本公开的交合培育方法中，银耳纯白菌丝与香灰菌丝的比例恰当，香灰菌丝长势好，能够促进银耳菌丝的生长发育，保证了菌种的高产以及稳定性，比利用传统方法培育银耳菌种的产量提高7％-8.7％；通过合适光照，增强了纤维素酶活力，可达到0.414±0.017，保证了菌种的强壮发育。

Claims (14)

  1. 一种银耳菌种的培育方法，其特征在于，包括如下步骤：
    (1)银耳纯白菌丝的分离培养
    选取子实体生长多、杂菌少的耳木，风干灭菌后于耳基着生处木材内部剃取木屑粉末，接种培养基上，置于25-28℃经8-10天培养得银耳纯白菌丝；
    (2)香灰菌丝分离培养
    灭菌后，将银耳段木出耳最多处锯断，在断面上有大理石状斑纹处取含有菌丝体的木屑，接种至培养基上培养5-6天，将菌丝挑选出来后转管培养得香灰菌丝；接种培养条件为：温度为24-26℃、光照强度为200-600lx、pH＝6.9-7.2；
    (3)交合培养
    利用木屑培养基混接法进行培育，先把银耳纯白菌丝同培养基从试管中挑取出来，放入木屑培养基上，然后提取少许香灰菌丝，放入银耳纯白菌丝旁边，于20-22℃、湿度为85％-95％、光照强度为50-100lux培养至生成银耳子实体原基。
  2. 根据权利要求1所述的银耳菌种的培育方法，其特征在于，步骤(1)中所用培养基组分为麸皮100-110g/L、麦芽糖10-20g/L、蔗糖10-20g/L、蛋白胨2-3g/L、过磷酸钙2-3g/L、硫酸镁0.5-1g/L和琼脂20-30g/L。
  3. 根据权利要求2或1所述的银耳菌种的培育方法，其特征在于，步骤(2)中所用培养基组成为葡萄糖20-30g/L、酵母粉3-5g/L、蛋白胨3-5g/L、KH 2PO 41.5-3g/L、Mg 2SO 4·7H 2O1.5-3g/L和维生素B 10.1-0.3g/L。
  4. 根据权利要求1-3中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中所用培养基组成为刨花楠木木屑70-75g/L、麸皮10-15g/L,稻草秸秆粉屑2-5g/L、蔗糖4-5g/L、骨粉4-5g/L、尿素2-3g/L和石膏2-3g/L，料水比1:1.2-1.3。
  5. 根据权利要求1-4中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-75:1。
  6. 根据权利要求1-4中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为40:1-70:1。
  7. 根据权利要求1-4中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为45:1-68:1。
  8. 根据权利要求1-4中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中银耳纯白菌丝与香灰菌丝的交合比例为50:1-65:1。
  9. 根据权利要求1-8中任一项所述的银耳菌种的培育方法，其特征在于，步骤(1) 中选取的银耳纯白菌丝直径为1.5-3微米，长势良好，步骤(2)选取的香灰菌丝直径为3-5微米，长势良好。
  10. 根据权利要求1-9中任一项所述的银耳菌种的培育方法，其特征在于，步骤(3)中交合培养过程中应保持二氧化碳含量＜0.2％。
  11. 根据权利要求1-9中任一项所述的银耳菌种的培育方法，其特征在于，步骤(1)和步骤(2)顺序可任意调换。
  12. 根据权利要求1-11中任一项所述的银耳菌种的培育方法得到的银耳子实体原基。
  13. 一种用于交合培养的培养基，包括：
    刨花楠木木屑75g/L、麸皮10g/L,稻草秸秆粉屑2g/L、蔗糖4g/L、骨粉4g/L、尿素2g/L、石膏2g/L和水，料水比为1:1.2。
  14. 根据权利要求13所述的培养基，所述交合培养是银耳纯白菌丝和香灰菌丝的交合培养。
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