Key Technology for Liquid Spawn Production and Green and High-yield Cultivation of Pleurotus eryngii

Yali ZHANG, Shuguang ZHAO, Yu LI, Zhengpeng LI, Cuie ZHANG, Xiaotao SUN

1. Agricultural Technology Extension Center of Guannan County, Guannan 222500, China; 2. Vegetable Office of Guannan County, Guannan 222500, China; 3. Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China; 4. Jiangsu Lisha Fungus Industry Co., Ltd., Guannan 222500, China

Abstract [Objectives] Pleurotus eryngii is currently the second largest edible fungus variety cultivated in China, and it mainly adopts the bag cultivation mode. This study aims to effectively solve the problems of high cost of solid spawn, long spawn production cycle, low application level of green and high-yield cultivation technology and equipment, and unstable output and quality in the factory production process and to further promote industrial upgrading. [Methods] Since June, 2018, a series of research and technical optimizations had been carried out by a technical research team consisting of personnels from Vegetable Office of Guannan County, Institute of Edible Fungi of Shanghai Academy of Agricultural Sciences and Jiangsu Xiangru Biotechnology Co., Ltd., around the production of liquid spawn, introduction of excellent varieties of P. eryngii, medium pretreatment, autoclaving, clean room inoculation, digital cultivation, harvesting, packaging, etc. [Results] The key technology of industrial production, liquid spawn production and green and high-yield cultivation of P. eryngii are summarized. [Conclusions] The economic benefit of liquid spawn of P. eryngii is extremely considerable compared with liquid spawns such as branch spawn.

Key words Pleurotus eryngii, Liquid spawn, Factory cultivation, Green, High yield

1 Introduction

Pleurotuseryngii, also known as Ganbeigu and Ciqin Ce’er, is a new breed of rare edible fungus that integrates edible, medicinal and food therapeutic value in recent years. The pileus ofP.eryngiiis brown or light gray and hemispherical; the stipe is milky white, stick-shaped or bowl-shaped; and the context is fleshy and crisp, rich in protein, carbohydrates, vitamins, calcium, magnesium, zinc and other minerals, with light almond fragrance, abalone taste, and functions of anti-cancer, lowering blood fat, lowering cholesterol, promoting gastrointestinal digestion and beauty.P.eryngiiis suitable for a variety of cooking methods such as frying, roasting and deep frying, and is popular among consumers. It has developed into the second largest industrial edible fungus variety in China afterFlammulinavelutipes. Due to the relatively short cultivation history ofP.eryngiiin China and the absence of a stable technical system, there are mainly technical bottlenecks in production, such as high cost of solid spawn, long spawn production cycle, low integration and application level of green and high-yield technical equipment, and unstable output and quality. Since 2018, starting from the implementation of Jiangsu Modern Agricultural (Vegetables) Industrial Technology System Construction Project, the Vegetable Office of Guannan County has built a technical research team with the Institute of Edible Fungi of Shanghai Academy of Agricultural Sciences and Jiangsu Xiangru Biotechnology Co., Ltd. and carried out a series of experimental studies on the production of liquid spawn, introduction of excellent varieties, pretreatment and autoclaving of culture materials and digital control of cultivation environment ofP.eryngii. As a result, the key technology of industrialized liquid strain production and green and high-yield cultivation ofP.eryngiiare summarized. The technology has achieved significant promotion and application results in northern Jiangsu. Related technical points are now introduced as follows.

2 Introduction and screening of excellent varieties

The technical research team introduced five excellent varieties of Suxing No.1, Riyin No.1, Xingda No.1, Guosen No.1 and Xingbaogu No.1 from scientific research institutes and production enterprises in Fujian, Jiangsu and other main producing areas of edible fungi. Through the cultivation experiment and production practice, the agronomic traits such as mycelial growth and fruit body commerciality of different varieties were analyzed and compared. Finally, Riyin No.1 is determined as the major variety to be promoted in the local and surrounding factory production ofP.eryngii.

3 Production of liquid spawn

In the experiment, Riyin No.1 was used as the test variety. The stock spawn uses the conventional PDA medium) The first-level liquid spawn (stock in conical flask) is formulated with 200 g of potato, 20 g of glucose and 1 000 mL of purified water, with natural pH). The formula of the second-level liquid spawn (cultivation in fermentor) is based on the reference to the research results of liquid spawns in domestic edible fungus research institutes and some industrial plants ofF.velutipesin recent years, consisting of 2% soybean meal, 1% peptone, 2% corn flour, 0.5% yeast extract, 0.5% KH2PO4, 0.3% MgSO4and 2% soft white sugar) The culture material for bag cultivation ofP.eryngiiis composed of 30% wood chips, 20% corn cob, 30% bran, 3% corn flour, 5% soybean meal, 1% light calcium carbonate and 1% lime. Before sterilization, the moisture content and pH of the culture material are 64%-66% and 8, respectively. A total of 3 000 bags were prepared for each variety. At different stages, the characteristics of mycelial pellets and the growth and agronomic traits of mycelia were observed and recorded[1-2].

3.1 Preparation and mycelial culture of first-level liquid spawnBased on the preparation steps of conventional PDA medium, agar is removed to prepare into liquid PDA medium. In every 1 000 mL conical flask, 400 mL of culture medium is added. Subsequently, the medium is sterilized under 1.05 kg/cm2for 30 min, cooled and added with two pieces of slant fungus blocks (0.5-1.0 cm2), followed by incubation for 24 h on a still constant-temperature darkened shaker. Then, the fungi were subjected to 4-7 d of shaking culture under the conditions of 25 ℃, 150 rpm and darkness. The growth of mycelia is observed. The criterion for the end of the culture is that the culture medium is clear and transparent, and a lot of mycelial pellets are suspended in the liquid. In production, before inoculating second-level liquid spawn, the first-level liquid spawn should be sampled and inspected). The "three-look and one-smell" (looking at the color and viscosity of liquid and morphology and quantity of mycelial pellets and smelling the taste) method can be used for preliminary screening, and the conical flasks infected with miscellaneous bacteria were excluded). Total 2-3 flasks are sampled randomly from the same batch for detecting the presence of competitive mycelia under a microscope.

3.2 Preparation and mycelial culture of second-level liquid spawn

3.2.1The fermentation tank and supporting system are cleaned and checked. Newly purchased or used fermentation tanks must be cleaned before each spawn production to ensure that there are no hanging objects or mycelial pellets on the inner wall of the tank and the discharged water is clear and free of pollutants.

3.2.2Boiling for sterilization. For normal production, boiling is not required, but under the condition of changing the variety, occurrence of contamination in the previous batch and starting a new tank, boiling is essential. Specific steps are as follows: closing the inoculation valve and the intake valve, adding water from the inlet to the center line of the sight glass, closing the exhaust valve, turning on the power to raise the temperature to 100 ℃ to discharge cold air, opening gently the exhaust valve to heat up to 121 ℃ for 30-40 min, and discharging the water in the tank when the temperature in the tank drops below 100 ℃. Whiling boiling the tank, the materials are cooked and the filter element and inoculation pipette are sterilized.

3.2.3After closing the exhaust valve and the inoculation valve, various raw materials (addition amount is 70%-80% of the tank volume) and defoamer (food grade, addition amount is 0.5%-1.0% of the amount of the medium) are added to the tank through the inlet according to the formula. The height of the materials should be slightly higher than the sight glass. After adding the materials, the valve of the inlet is closed tightly.

3.2.4Sterilization and cooling. After closing all the valves, the power is turned on to heat up to 100 ℃ to discharge the cold air. In order to prevent the material liquid from gelatinizing, air is directly introduced for stirring. The temperature is raised to 121 ℃ and maintained for 30 min. The materials are discharged once every 10 min, 2 min for every time. After sterilization, circulating cooling water is used to lower the temperature to 25-28 ℃ for use.

3.2.5Inoculation. Cotton fire ring, lighter, 95% alcohol, gloves and other supplies are prepared. When the pressure in the tank drops to 0, the fire ring is placed on the inlet. Under the protection of the flame, the cover is opened. After pouring one bottle of first-level liquid spawn, the inlet is closed quickly. Under normal circumstances, spawn can be prepared after 5-7 d of culture.

3.2.6Mycelial culture and sampling and observation. The device is started, and the exhaust valve is opened to make the pressure inside the tank rise to 0.02-0.04 MPa. Under the condition of 20-22 ℃ and 1.0-1.2 m3/h, culture is performed for 24 h. Samples were taken from the inoculation port every 24 h. Each sampling operation should be carried out in an ultra-clean workbench in a 100-grade air purification room. For every time of sampling, 500 mL of the liquid is drained first, and then 100 mL of the liquid is sampled to a 250-mL sterile triangular flask, which is sealed with kraft paper rapidly.

3.3 Quality inspection of liquid spawnsThere are mainly two methods for the detection of liquid spawn in the fermentor: PDA test tube streak culture method with longer cycle and microscope inspection method with shorter cycle. The PDA test tube streak culture is carried out in the ultra-clean workbench. Using the inoculation hook, the sample is inoculated into the medium by drawing curves. The culture is performed in a constant temperature incubator at 28-30 ℃ for 24-72 h. It is preferable that there is no miscellaneous colony. The operation of the microscope inspection is the same as that of the inspection of the first-level liquid spawn. They are all completed under the microscope.

4 Pretreatment of medium

Wood chips, corn cob, bean stalks, bagasse, wheat bran, soybean meal, lime, light calcium carbonate,etc. are the main raw materials for factory cultivation ofP.eryngii. In northern Jiangsu, soft poplar wood chips are commonly used. In Shandong, hard branch chips of fruit trees are commonly used. Since the production cycle ofP.eryngiiis short, it is recommended to combine the use of fine wood chips with coarse wood chips. Just like bagasse, the wood chips need to be piled up, drenched with water and fermented for more than 2 months before use. The heap needs to be turned over every 15 d or so. Before mixing, the bark and other debris are removed. The sponge tissue of corn cob crushed material is thick, and in order to avoid incomplete sterilization due to "dry center", it can be soaked or sprayed with 1% lime water. Soybean meal, wheat bran and other high-nitrogen materials should be stacked in a dry place to ensure freshness and no moldy smell.

5 Key technology for green and high-yield cultivation

5.1 Mixing and bagging of culture materialWood chips, corn cob, bagasse, bran and other main materials are added into the mixer hopper for mechanical stirring for 20 min according to the requirements of the formula, followed by adding soybean meal and corn flour and stirring for 10 min. Then, while adding lime, light calcium carbonate and other accessories to adjust the pH, water is added while stirring for 30 min. Finally, packaging is carried out. In production, high-pressure polypropylene plastic bags with a folio caliber (17-19) cm×38 cm×0.05 mm are commonly used, and in each bag, 1.2-1.4 kg of wet material are packed, with a height of 19-21 cm, and a plastic punching rod in the center[2].

5.2 Sterilization, cooling and inoculationDuring factory production, the cultivation bags can be sterilized under high pressure at 121-123 ℃ for 2 h. After sterilization, the bags are moved into the cooling room to cool down in time. When the temperature inside the material drops below 25 ℃, 20 mL of liquid spawn is inoculated to every bag in the ultra-clean unit of a clean room.

5.3 Digital management of the environment for raising mushroomsThe cultivation of mushrooms is mainly carried out in a standardized mushroom house equipped with automatic detection and control equipment such as refrigeration, heating, ventilation, and lighting. Through equipment such as environmental parameter sensors, the temperature, relative humidity and carbon dioxide concentration in the darkened and clean environment are maintained at 20-22 ℃, 65%-70% and no more than 3‰, respectively. On the 8th to 9th day and 16th to 18th day after inoculation, whether the cultivation bags are infected with competing bacteria such asAlternariaalternateis checked. Normally, about 24 d after inoculation, the culture materials are exhausted. After 5-8 d of "post-ripening" culture, the temperature can be reduced to promote bud germination[2-3]. In the early stage of mushroom production, the temperature, relative air humidity, light duration and carbon dioxide concentration are maintained at 12-14 ℃, 80%-90%, 500 lx for 4-6 h, not exceeding 2.5%, respectively to induce the formation of primordia. When more small bumps appear on the surface, light induction is ended. At the same time, the ventilation frequency is reduced and the carbon dioxide concentration is increased to 3.5‰-4.5‰ to control the quantity of primordia[3-4]. When the mushroom buds grow to 4-6 cm long, thinning needs to be conducted. In each bag, 1-3 robust buds with good shape and good commodities are retained. After thinning, the temperature, carbon dioxide concentration and relative air humidity are maintained at 14-15 ℃, 6‰-8‰, and 80%-90%, respectively to promote the rapid extension of the stipes. When the stipes grow to 12 cm long, harvesting can be carried out.

5.4 Green prevention and control of pests and diseasesThe principle of "prevention first, comprehensive prevention and control" is adhered to. When fresh and mildew-free raw materials are used, while doing well in the management of high-pressure sterilization of culture materials, inoculation in clean room, and regulation of environmental parameters of culture, it is necessary to strengthen the sanitation management of the plant area, inside and outside the mushroom house, and timely dispose waste products. Cooling room, inoculation room, cultivation room and other areas are sterilized and disinfected with bleaching powder and other edible fungus special drugs to prevent or reduce the infection and breeding of competing bacteria and pests such asAlternariaspp. and green mold to ensure the quality and safety of fresh mushroom products.

6 Conclusions

Compared with solid spawns such as branch spawn, liquid spawn ofP.eryngiihas the advantages of mycelial rapid growth, simple operation, and so on. The production cycle of original seed can be shortened by more than 20 d, and the production cost is significantly reduced year-on-year. On average, the cost of spawn for each cultivation package decreases by 0.07 yuan. The entire cultivation cycle from inoculation to the end of fresh mushroom harvest is shortened from 54-55 d to 50-51 d. About 450 g of fresh mushrooms can be harvested from each cultivation bag on average, and the economic benefit is extremely considerable.