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 PDAplatesat25C 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. xMeans followed by the same lower and upper case letters are not significantly different by Duncan’s multiple range bran at a ratio of 4.5:4.5:1 by weight test at P < 0.05 and P < 0.01, respectively. was strongly favored. The corn core was milled to 18–20 mesh beforehand. The pellets of TD300 were collected from liquid spawn by centrifugation at 650gfor5minandthenblendedwith sterilized carrier to prepare immobi- lized spawn in a sterile room; water content of immobilized carrier was adjusted to 65% before sterilization us- ing tap water. The immobilized spawns were kept in plastic bags at 300 g per bag at 20–25 C; the plastic bag is made of polypropylene, 12 cm wide, 24 cm long, and 0.04 mm thick. Two grams of immobilized spawn were inoculated into one end of a plastic bag containing 300 g CCM Fig. 1. Effect of the ratio between pellets and immobilized carrier of oyster and then incubated at 25 C until the mushroom TD300 (by weight) on lag phase and mycelial growth rate. The carrier mycelia colonized the content of the is composed of cottonseed hull, corn core, and wheat bran. The ratio of pellets bag to determine the lag phase and to immobilization carrier varied from 1:1 to 1:21 by weight. Bars indicate SE growth rate of immobilized spawn. of measurement; 1 cm = 0.3937 inch.

• February 2011 21(1) 83 RESEARCH REPORTS

The ratio between pellets and pellets were steadily adapting and grow- spawn of edible mushroom have also immobilization carrier varied from 1:1 ing on immobilization carrier (Fig. 4A). been reported. In one study, im- to 1:21 by weight, and the lag phases Peroxidase isozyme of immobi- mobilized alginate beads of button of immobilized spawn growth displayed lized spawn displayed one band on day mushroom were prepared by entrap- a ‘‘V’’-shape change. The 1:8 ratio pro- 1 and two on day 15; the two bands ping liquid spawn with vermiculite, duced the shortest lag phase and a high were similar to that of fresh conven- hygramer, and nourishment in sodium mycelial growth rate (Fig. 1) and was tional solid spawn, but were wider and alginate; its performance was compara- used to prepare immobilized spawn. brighter, and gradually weakened there- ble to that of grain spawn in the shape To our knowledge, the changes after; an additional light band appeared and yield of fruit body (Rosado et al., in physiological and cultivated char- after day 30 (Fig. 4B). Polyphenol ox- 2002). However, Friel and McLoughlin acteristics of mushroom spawn dur- idase isozyme produced only one band (1999) reported that such a spawn, if ing storage have not been reported. on day 15, same as the isozyme pattern without nourishment, could not sur- During storage for the first 45 d, the of fresh conventional solid spawn (Fig. vive in pasteurized compost and that respiration intensity of immobilized 4C). These results revealed that the the biomass levels were significantly spawn increased linearly, probably due vigor of immobilized spawn got lost lower than that of the conventional to the gradualness in pellets’ growth on gradually after 15 d. grain spawn; with the entrapment of the immobilization carrier. The activi- Immobilized spawn was inoculated both the mycelium and a nutrient ties of lignocellulolytic enzymes rose into a plastic bag containing 1000 g source in the beads, the biomass levels and mycelial biomass subsequently en- CCM for 60 d to observe cultivation were boosted by 3-folds after cultiva- larged. Between days 45 and 60, the characteristics; its BE varied from 82.4% tion in malt extract broth for 4 d; at respiration intensified more rapidly to 88.1%. It took 49.2 to 63.1 d to reach same biomass levels, coentrapped my- (Fig. 2), likely because of the greater full mycelia colonization and another celial beads had more inocula points, mycelial biomass; high respiration in- 8.8 to 11.8 d for primordium initiation a shorter adaptation (lag) period, and a tensity was an indication of the high of immobilized spawn. The cultivation higher growth rate in pasteurized com- vigor of spawn. characteristics of immobilized spawn post than the conventional grain spawn. CMCA and FPA of immobilized after 60 d did not significantly differ Although spawn immobilization spawn surged from days 1 to 15 and from those of fresh solid spawn used as through the entrapment of alginate then FPA fell back slightly, whereas control (Table 2); the results demon- beads offers promising results, it is CMCA reduced by 22.3% to 169.1% strated that immobilized spawn could also complicated, time consuming, and duringdays30to60(Fig.3).Asimilar be stored for at least 2 months. trend was observed in oyster mush- In agriculture, immobilization room Florida f6 during solid-state fer- technique has been widely used in the mentation (Kerem et al., 1992); a partial preparation of inocula to improve its explanation was that oyster mushroom ecological competence (McLoughlin, had the higher selectivity for lignin 1994); a few studies on immobilized degradation. Three isozymograms of immobi- lized spawn, including esterase, perox- idase, and polyphenol oxidase, were analyzed during the 60-d storage. The number of esterase isozyme bands on days 1, 15, 30, and 60 were one, two, three, and six, respectively; the band of esterase isozymograms on day 60 was the same as that of fresh conventional solid spawn; the results suggested that

Fig. 3. Changes in cellulase activities of immobilized spawn during storage at 20–25 C (68.0–77.0 F). Immobilized spawn was prepared by mixing pellets of TD300 and carriers (cottonseed Fig. 4. Changes in isozyme patterns of hull + corn core + wheat bran) at a ratio immobilized spawn during storage at of 1:8 by weight. The enzyme activity 20–25 C (68.0–77.0 F). Immobilized was expressed in units of specific spawn prepared by mixing pellets of activity defined as international units TD300 and carriers (cottonseed hull + (IU) per gram dry weight of spawn (1 corn core + wheat bran) on a ratio of IU = the amount of enzyme required to 1:8 by weight: (A) esterase, (B) Fig. 2. Changes in respiration intensity produce 1 mmol of glucose per minute peroxidase, and (C) polyphenol of immobilized spawn oyster under the assay conditions). Bars oxidase. Lane C is conventional solid mushroom TD300 during storage at indicate SE of measurement; 1 IU/ spawn before storage. Lanes 0, 15, 30, 20–25 C (68.0–77.0 F). Bars indicate g = 28.3495 IU/oz. CMCA, 45, and 60 are immobilized spawn 2 SE of measurement; 1 mLÁg 1 = 9.5861 · carboxymethyl cellulase activity; PFA, stored for 0, 15, 30, 45, and 60 d, 1024 fl oz/oz. filter paper activity. respectively.

84 • February 2011 21(1) Table 2. The cultivated characteristics of immobilization spawn of oyster mushroom TD300 during storage at 20–25 C (68.0–77.0 F). Length of full Length between full mycelia mycelia colonization and Length of colonization primordium initiation Biological efficiencyy Spawnz storage (d) [mean ± SE (d)] [mean ± SE (d)] [mean ± SD (%)]y Immobilized 0 63.1 ± 4.1x 10.7 ± 1.5 87.4 ± 6.6 Control 58.5 ± 3.2 12.1 ± 2.1 85.1 ± 9.3 Immobilized 15 49.2 ± 5.4 8.8 ± 3.3 88.1 ± 4.2 Control 46.4 ± 2.9 9.2 ± 3.7 85.1 ± 5.7 Immobilized 30 50.8 ± 4.6 8.9 ± 2.5 82.4 ± 6.2 Control 53.6 ± 3.5 9.3 ± 3.6 87.5 ± 3.2 Immobilized 45 58.7 ± 4.2 10.1 ± 1.9 85.5 ± 7.5 Control 60.2 ± 4.7 11.2 ± 3.6 86.3 ± 9.1 Immobilized 60 56.6 ± 5.1 11.8 ± 2.4 86.8 ± 8.4 Control 55.1 ± 4.7 8.5 ± 2.9 84.0 ± 6.3 zImmobilized is prepared by mixing pellets of TD300 and carriers (cottonseed hull + corn core + wheat bran) on a ratio of 1:8 by weight. Control is fresh solid spawn grew on cottonseed hull with no storage. yPercentage of the fresh weight of harvested mushrooms over the dry weight of inoculation substrates. xColumns without letters following means are not significantly different according to Duncan’s multiple range test (P < 0.05). prone to contamination. In this study, Chinese Society of Plant Physiology. Pleurotus ostreatus vs Phanerocheate we developed an alternative method 1985. Manual plant physiology experi- chrysosporium. Appl. Environ. Microbiol. to immobilize mycelial pellets of liquid ments. Shanghai Science and Technology 68:1121–1127. Press, Shanghai, China. spawn based on carrier adsorption. The Kirchhoff, B. and J. Lelley. 1991. Inves- carriers are similar to those used in El-Fakharany, E.M., B.M. Haroun, tigations of shiitake [Lentinus edodes cultivation; it could effectively shorten T.B. Ng, and E.R. Redwan. 2010. Oys- (Berk.) Sing.] bag-log cultivation to in- the adaptation period and improve the ter mushroom laccase inhibits hepatitis C crease the yield in Germany, p. 509–516. growth rate after inoculation on culti- virus entry into peripheral blood cells and In: M.J. Maher (ed.). Science and cultiva- vation substrates, as demonstrated by hepatoma cells. Protein Pept. Lett. 17: tion of edible fungi. Balkema, Rotterdam, the immobilized spawn with cottonseed 1031–1039. The Netherlands. hull carrier in Table 1; pellets adsorbed Eyal, J. 1991. Mushroom mycelium grown Laniece, A. 1966. Production and use of by carriers are well spread, creating in submerged culture-Potential food appli- liquid mushroom spawn. U.S. patent more inocula points. The immobilized cations, p. 31–64. In: I. Goldberg and R. 3,286,399. U.S. Patent and Trademark spawn is convenient for storage and Williams (eds.). Biotechnology and food Office, Washington, DC. transportation and could grow on the ingredients. Van Nostrand Reinhold, carriers during storage and transporta- New York. Leatham, G.F. and T.J. Griffin. 1984. tion; the storage period is more than Friel, M.T. and A.J. McLoughlin. 1999. Adapting liquid spawn Lentinus edodes to 60 d in our study on oyster mushroom, Immobilisation as a strategy to increase the oak wood. Appl. Microbiol. Biotechnol. a very attractive feature for the industry. ecological competence of liquid cultures of 20:360–363. Agaricus bisporus in pasteurised compost. Furthermore, the production can be Liu, Y., L. Wang, Y. Gao, Y. Qi, J. Shen, FEMS Microbiol. Ecol. 30:39–46. run with simple and inexpensive ma- and L. Qiu. 2009. Studies on the liquid chines. Finally, the supernatant of liquid Friel, M.T. and A.J. McLoughlin. 2000. spawn solidification technique of Lentinus spawn is nutritious and a potential Production of a liquid inoculum/spawn edodes. Edible Fungi China 28:16–17. source for food and medicine. Overall, of Agaricus bisporus. Biotechnol. 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Measurement of car- similar results. in submerged culture. Science 107:373. boxymethylcellulase activity. Anal. Biochem. Kawai, G., H. Kobayashi, and Y. Fukushima. 1:127–132. Literature cited 1995. Liquid culture induces early fruiting Okamura-Matsui, T., T. Tomoda, in shiitake (Lentinula edodes). Mushroom Chang, S.T. 1999. World production of S. Fukuda, and M. Ohsugi. 2003. Discovery Sci. 14:825–832. cultivated edible and medicinal mush- of alcohol dehydrogenase from mush- rooms in 1997 with emphasis on Lentinus Kerem, Z., D. Freisen, and Y. Hadar. rooms and application to alcoholic bever- edodes (Berk.) Sing. in China. Intl. J. 1992. Lignocellulose degradation dur- ages. J. Mol. Catalysis B Enzyme 23:133– Medicinal Mushrooms 1:291–300. ing solid waste substrate fermentation: 144.

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