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Horttech01832 82..86 mushroom [Pleurotus ostreatoroseus Immobilization of Mycelial Pellets from (Rosado et al., 2002)], paris mush- Liquid Spawn of Oyster Mushroom room (A. bisporus var. hortensis), wood blewit (Tricholoma nudum), garden Based on Carrier Adsorption morel (Morchella hortensis), yellow mo- rel (Morchella esculenta), and chante- 1 1 2 1 relle [Cantharellus cibarius (Laniece, Lanqing Wang , Yinfeng Li , Dehai Liu , Chaohui Zhang , 1966)]. Compared with the solid Yuancheng Qi1, Yuqian Gao1, Jinwen Shen1, and Liyou Qiu1,3 spawns, there are numerous advantages of adapting the liquid culture tech- nique, such as improved automation ADDITIONAL INDEX WORDS. enzyme, respiration intensity, cultivation, in the spawn plant, more uniform dis- Pleurotus ostreatus tribution of the inoculum in the sub- SUMMARY. We investigated a practical method for immobilizing liquid spawn of strate (Eyal, 1991), producing large oyster mushroom (Pleurotus ostreatus) to prolong the storage time and provide amounts of mycelia quickly at any time convenient transportation of liquid spawn of edible mushrooms. The method was of the year, a more homogeneous based on the mycelial pellets of liquid spawn adsorbed in carriers. Selected carriers fungal growth, lower cost (Rosado were similar to cultivation substrates, and the best carrier was a mixture of et al., 2002), early fruiting (Kawai cottonseed hull, corn core, and wheat bran with a ratio of 4.5:4.5:1 by weight. et al., 1995), and high fruit body yield Immobilized spawn were prepared by mixing the pellets from liquid spawn with (Kirchhoff and Lelley, 1991). carriers using a ratio of 1:8 by weight. Within the first 15 days of storage at 20–25 On the other hand, it is difficult ° C, the immobilized spawn grew strongly, respiration intensity and cellulase to store and transport liquid spawn activities rose rapidly, and the count and brightness of the isozyme bands of esterase, peroxidase, and polyphenol oxidase increased remarkably as well. From days 30 and its residual nutrients may cause to 60, the cellulase activities fell and the brightness of the peroxidase and contamination; in addition, liquid polyphenol oxidase bands gradually decreased, whereas the respiration intensity spawns of several mushrooms could and the band count of esterase and peroxidase remained constant. After 60 days, the not colonize on some cultivation sub- cultivated characteristics of the immobilized spawn were same as the fresh strates (Friel and McLoughlin , 1999; conventional solid cottonseed hull spawn. The results showed that immobilized Leatham and Griffin, 1984). These spawn on the basis of the mycelial pellets of liquid spawn adsorbed in carrier can problems limit its further application be used to extend the storage time and simplify transportation of liquid spawn in the mushroom industry. In this of edible mushroom. study, we investigate an immobiliza- tion method for liquid spawn of oyster yster mushroom is one of development vary considerably (Liu mushroom to achieve longer storage the most widely cultivated edi- et al., 2009). time and convenient transportation. Oble mushrooms in the world Since the first report on produc- (Chang,1999);itisalsousedinmed- tion of commercial meadow mush- Materials and methods icine (El-Fakharany et al., 2010; Gern room [Agaricus campestris (Humfeld, LIQUID SPAWN AND SOLID SPAWN et al., 2008), environmental remedia- 1948)] producing mycelia in sub- PREPARATION. Oyster mushroom tion (Rodrı´guez Pe´rez et al., 2008; Yan merged culture has been widely prac- TD300, a commercially cultivated strain et al., 2009), and biofuel (Okamura- ticed (Zhong and Tang, 2004). In in China, was chosen for our experi- Matsui et al., 2003). Like most other 1966, an American attained the first ments. The media used were potato edible mushrooms, its spawn for culti- patent of liquid mushroom spawn dextrose agar (PDA), potato dextrose vation is in solid state, which is com- (Laniece, 1966). Up to now, liquid broth (PDB), liquid spawn medium posed of manure-grass, sawdust, wood spawn is widely used in the cultivation (LSM), and cottonseed hull cultivated block, branch wood, and grain. Solid of many mushrooms, including but- medium (CCM). LSM was composed spawns are universally used by mush- ton mushroom [Agaricus bisporus of 10 g corn flour, 20 g soybean meal room growers throughout the world (Friel and McLoughlin, 2000)], oyster powder, 30 g sucrose, 5 g yeast extract, because they can be made with simple mushroom (Laniece, 1966; Silveira 1 g potassium dihydrogen phosphate equipments and techniques, but the et al., 2008), shiitake [Lentinus edo- (KH2PO4), 0.5 g magnesium sulfate process also needs large space and long des (Kirchhoff and Lelley, 1991; heptahydrate (MgSO4•7H2O), 2 g incubation period. Additionally, age Pellinen et al., 1987)], brazilian oyster calcium carbonate (CaCO3), and 1 L of the mycelia in different position of solid spawn is rather different; those near the inoculum age faster. Conse- Units quently, the growth rate of the mycelia To convert U.S. to SI, To convert SI to U.S., and the time course of sporophore multiply by U.S. unit SI unit multiply by 29.5735 fl oz mL 0.0338 1043.1756 fl oz/oz mLÁg–1 9.5861 · 10–4 1 3.7854 gal L 0.2642 College of Life Sciences, Henan Agricultural Univer- 2.54 inch(es) cm 0.3937 sity, 95 Wenhua Road, Zhengzhou 450002, China. 25.4 inch(es) mm 0.0394 2Institute of Biology, Henan Academy of Sciences, 28 28.3495 oz g 0.0353 Huayuan Road, Zhengzhou 450008, China. 0.0069 psi MPa 145.0377 3Corresponding author. E-mail: [email protected]. (°F – 32) O 1.8 °F °C(1.8·C) + 32 82 • February 2011 21(1) tap water. CCM was a mixture of 100 g The duration from inoculation to the (Miller, 1959; Miller et al., 1960). cottonseed hulls, 1 g lime, 1 g gypsum, beginning of mycelial growth (i.e., the The enzyme activity was expressed in and 110 mL tap water (Qiu et al., lag phase) was recorded. Starting with specific activity units defined as in- 2010). TD300 was stored on PDA the steady growth of mycelia, the po- ternational units (IU) per gram dry slants at 4 °C and was transferred every sition of the advancing mycelial front weight of spawn (1 IU = quantity of 2 months. Cultures were grown on was marked on the bag at 4-d interval enzyme required to produce 1 mmol PDAplatesat25°C for 7 d. A 1-cm- to measure mycelial growth rate. of glucose per minute under the assay diameter mycelial plug from the pe- Experiments of basidiomata pro- conditions). The isozyme patterns of ripheral edge of a 7-d-old colony was duction were carried out using plastic esterase, peroxidase, and polyphenol used for the production of liquid cul- bags containing 1000 g CCM. Five oxidase of immobilized spawn and tures. The fungus was cultivated in 150 grams of immobilized spawn or solid solid spawn during storage were ana- mL PDB in a 500-mL flask at 25 °C spawn (as control) were inoculated lyzed using polyacrylamide gel electro- under 150 rpm shaking (HZQ-Q into one end of a plastic bag and cul- phoresis as described by Yan (1997). shaking incubator with temperature; tivatedasdescribedbyQiuetal. The mean of five replicates was HDL, Harbin, China). After 7–8 d, (2010). Biological efficiency (BE) was used in the result, except the basidio- the mycelia were cultivated up to the defined as the percentage of the fresh mata production experiment, which later log phase to produce an inocu- weight of harvested mushrooms over was performed with 20 replicates. lum for liquid cultures in fermentor, the dry weight of inoculated substrates. Means were separated using Duncan’s which has a total volume of 40 L Respiration intensity of immobi- multiple range tests. (BIOTECH-40JS; Baoxing, Shanghai, lized spawn and solid spawn was de- China). Twenty-eight liters of LSM was termined using the small-skep method Results and discussion added in the fermentor and the amount based on carbon dioxide (CO2)ab- Among the three tested carrier of inoculum produced was 7.5% (v/v). sorption (Chinese Society of Plant mixtures, the one composed of cotton- The culture conditions were cultiva- Physiology, 1985). Cellulase activity, seed hull, corn core, and wheat bran tion temperature at 25 °C, agitation including filter paper activity (FPA) exhibited significantly lower lag phase speed at 160 rpm, pressure in fermenter and carboxymethyl cellulase activity (P < 0.01) and higher mycelial growth at 0.04 MPa, and aeration rate at (CMCA), was determined by the rate (P < 0.05) (Table 1) and was 0.7 m3Áh–1. The mycelia were cultivated method of Miller and his colleagues selected for the experiments thereafter. for 96 h, reaching the maximum bio- mass, and then harvested to prepare immobilized spawn. Conventional solid Table 1. The lag phase and growth rate of immobilized spawn of oyster spawn was prepared as described (Qiu mushroom TD300 in different carriers. et al., 2010). IMMOBILIZATION OF LIQUID Immobilization Lag phase Mycelial growth rate z –1 y SPAWN. Three immobilization carrier carriers [mean ± SE (h)] [mean ± SD (cmÁd )] mixtures listed in Table 1 were tested. cottonseed hull + bran (w:w = 9:1) 52.8 ± 3.4 Bx 0.46 ± 0.09 bx Each was blended with pellets of cottonseed hull + corn core + wheat bran TD300 on a weight ratio of 1:10 to (w/w/w = 4.5:4.5:1) 38.4 ± 2.5 A 0.51 ± 0.03 a make immobilized spawns; as shown cottonseed hull + corn flour (w:w = 9:1) 53.4 ± 4.4 B 0.47 ± 0.04 b in Results and discussion, the mixture of zw:w is the ratio of carriers by weight. cottonseed hull, corn core, and wheat y1 cm = 0.3937 inch.
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