Preparation of Inoculum of Pleurotus Ostreatus in Laminar Flow Hood Rustic

Full Length Research Paper

Preparation of inoculum of Pleurotus ostreatus in laminar flow hood rustic

Omar Romero-Arenas1*, I. Tello Salgado1, M. Huerta Lara 2, M. A. Damián Huato2, A. García Espinosa2, J. F. C. Parraguirre-Lezama1, I. Hernández Treviño1, A. Macias López3 and J. Juárez Huerta1

1Departament of Agro-forestry Engineering, Benemerita Universidad Autonoma de Puebla, Mexico. 2Departament of Agroecology and Enviroment, Benemerita Universidad Autonoma de Puebla, Mexico. 3Postgraduate College, Campus Puebla, Mexico.

Accepted 15 November, 2010

The oyster fungus (Pleurotus ostreatus) is more cultivated by producers due to their low investment requirements compared to other cash crops in order to ease cultivation, adaptation to the local agro- products and the necessary facilities. However, the technology used so far by the small and medium producers, is based on the continued acquisition of inoculum (seed) primarily to educational institutions and agro-industrial laboratories, making dependent activity on the availability of the inoculum, transportation, in addition to increasing production costs due to the high cost of the inoculum, which is why the rural production of oyster fungus inoculum through a laminar flow hood homemade (rustic) is a tool that is being developed at the School of Engineering Agro forestry, BUAP and most importantly, be a model that is capable of being transferred to the region northern of Puebla State, representing an alternative appropriate, technology to promote the cultivation of edible fungi, as well as employment generation in the region. The laminar flow hood rustic is efficient to produce the innoculum and reduce the production cost in about 40%.

Key words: Oyster fungus, seed, agro-industrial laboratories, laminar flow hood rustic.

INTRODUCTION

World production of edible mushroom is represented by period 1995 to 2001 were Mexico (58.6%), Chile (17.6%) about 15 species, of which the most representative are and Brazil (10.6%), constituting 86.8% of the total the mushroom (Agaricus spp.), shithake (Lentinula production of edible mushrooms in Latin America edodes), oyster fungus (Pleurotus spp.) and enokitake (Martinez -Carrera, 2002). In this manner, in Mexico the (Flammulina velutipes) (Chang and Miles, 1989). In Latin production of edible fungi have evolved to such a degree America, the commercial production of edible fungi that is the largest producer in Latin America, occupying (mushrooms especially) increased by 32% in 2001, this the twenty-eighth place in the world, where per capita caused an economic spill by over 167 million dollars a consumption had grown slowly in recent years. Unlike the year and 34000 creating jobs in this activity. The demand agricultural, ranching, and forestry activities have for for domestic and export consumption of edible fungi in centuries practiced in Mexico, biotechnology production the period 1995 to 2001 increased by 32% from 49,975 to of edible mushrooms had only 66 years of steady 65,951 tons per year (Martinez-Carrera, 2002). development and increasing (Martinez-Carrera et al., The countries that increased their production in the 1991b). Considering the current national and international context, it is essential to promote a more basic and applied research, linked directly to the rural sector and commercial production of edible mushrooms in strategic *Corresponding author. E-mail: [email protected]. Tel/Fax: regions of the country. +52 7979730667. The oyster fungus (Pleurotus spp.) shows a facility for 3946 Sci. Res. Essays

cultivation around the world for its many features MATERIALS AND METHODS (Medicinal, environmental biological remediation, enzyme The experimental work was conducted at the Department of production, etc.). In addition to easy handling for Agroforestry Engineering, Academic Unity Regional of Tetela, cultivation in agro-industrial waste, and quickly Benemerita Universidad Autonoma de Puebla (BUAP), specifically decomposition of organic matter by encouraging agro- in research pilot plant production of edible mushroom in University ecosystem, pushing small farmers because of low Avenue, Barrio Benito Juarez, Tetela de Ocampo, Puebla, Mexico production costs, employment generation and provide (Figure 1). additional value to their products through preserves or jam oyster fungus (Melo et al., 2010). The mushroom Substrates and Cepas crop residues could be used as mushroom compost, incorporated into the soil as a conditioner and or as The strain (CP-50) of P. ostreatus (Jacq.ex Fr.) Kumm used in the study comes from the genetic resources center edible fungi improver of physical properties, or be part of mixtures (CREGENHC) postgraduate College, and is deposited in the strain made in the production of substrates for nurseries bank of Edible Fungi Campus Puebla, Mexico. The strain is (Varnero et al., 2010). maintained in a medium composed of potato agar and dextrose The cultivation of edible fungi is a process that frees up (PDA) Bioxon mark, at room temperature (Sobal et al., 2007). the land resource, and you get large volumes of The substrates used in the preparation of inoculum (seed) were production in relatively small spaces. In the early phases wheat (Triticum aestivum L.) and maize (Zea mays L.) acquired in Tetela de Ocampo, Puebla-México. The inocolum preparation was of mycology, the comments played an important role in performed in the pilot plant research in production of edible the method of cultivation of edible fungi (Martinez-Carrera mushrooms in the community of Benito Juarez, in the municipality et al., 1995). The generation of technologies is a major of Tetela de Ocampo, located in the Sierra Norte of Puebla State, aspect that contributes to solving problems of different its geographical boundaries are: 19 °43 '00 "and 19º 57' 06" north nature, these technologies are generated by research latitude and 97º 38 '42"and 97º54'06" west longitude. Their centers and to demonstrate their viability must be adjacencies are the north by the municipalities of Tepetzintla and Cuautempan, South with Ixtacamaxtitlán, to the west with Zautla transferred to industry receiving them (Almanza, 1994; and Xochiapulco, and the East with Aquixtla, Zacatlán and Gaiska, 1997). The use of technology to the cultivation of Ixtacamaxtitlán (Encyclopedia of the municipalities of Puebla, oyster edible fungus allows for high production in a 2006). relatively short spaces, is widely accepted in urban and rural areas for its nutritional properties, the fungus DEVELOPMENT mushroom is a food with 350 calories compared to red meat only contains 150 calories or fish that contains 101 The inoculum or seed term usually refers to fungal mycelium used (Romero et al., 2010). to inoculate the final substrate where the fungus will bear fruit, that Technology transfer being proposed in this research is is, the material used to "seed" when growing edible mushrooms. However, there are two types of seeds: The mother and the the production of mushroom fungus inoculum of rural secondary seed or seed for planting. The seed mother comes production, as it is driving and innovating production directly from fungal mycelium grown on a nutrient medium; this technology of inoculum (seed) at low costs in the rural means that for a substrate, preparation is inoculated with a piece of sector through the manufacture of a laminar flow hood agar. To produce the secondary seed, however, the substrate is rustic, which has an area hygienically clean, sterile air inoculated with seed mother in a state of active growth. The locks which can manipulate fungus mushroom mycelium substrate used for either the mother seed or grain or seed are secondary forest residues such as sawdust. Often, the first in jars or in their normal environment in which it must compete with bottles and plastic bags second thermo-resistant (Chemo, 2002). a host of other organisms to survive. This will allow the To carry out the process of inoculum preparation, vertical laminar optimization of its resources and making the process flow hood is required, as it protects the sample from contamination. more beneficial for small producers of edible fungi and is During operation, the air is drawn through a pre-filter located at the being developed in the research center Phok Nah seed in top of the cabinet to trap large particles, then air is blown through a the LAOS (PDR) (Ogden and Prowse, 2005). Based on HEPA filter (Hight Efficiency Particulate Arrestance) with efficiency of 99.99% and is projected vertically to the work area, the air the above, provides that the role of biotechnology for the filtering through the filter HEPA prevents contaminants from production of edible fungi inoculum, especially the entering particles, providing a work environment free of particles mushroom fungus (P. ostreatus) will be a pioneer in its that reduces cross-contamination, which is why the manufacture area to boost rural development and essential for was proposed in a vertical rustic laminar flow hood, to strengthen strengthening the role of sustainability agricultural by the rural production of mushroom fungus in the region of the Sierra Norte of Puebla State, with the aim of transferring this technology at using products and recycling of agro-food for a socially affordable costs to producers and agro build successful businesses acceptable, high medicinal value, protein, and business in the production of edible fungi. and in addition generate significant employment The rustic laminar flow hood was manufactured under test with opportunities in rural communities. materials of the region, in this case, use of wood and iron 5×3×1, 6 The aim of this study was to demonstrate the mm, anti rust paint finish white (Figure 2), only acquired the HEPA production of inoculum kindnesses, using a laminar flow filter with an authorized dealer, a turbine built in the upper middle part driven by a ¼ hp electric motor at 3000 rpm, that extracts air hood rustic to be transferred to rural communities in the from the room by pre filter at the top of the cabinet and then through municipality of Tetela de Ocampo, Puebla-México. the HEPA filter whose function is to remove 99.99% of all particles Romero-Arenas et al. 3947

Figure 1. Location of the experimental work in Puebla, Mexico.

racks in front and in back of the cabinet and taken outside where there is a clean area within the bank by the inflow of air into the front. The average speed obtained from the downward flow is 50 fpm (17.8 m/s), the average speed flow admission is 75 fpm (23.6 m/s), and the discharge volume is 398 cfm (12.9 m3/min). Subsequent to the tests mentioned, above we proceeded to the preparation of the inoculum with the substrates in the region mentioned above.

Preparation CP-50 strain of P. ostreatus

The culture medium of potato dextrose agar (PDA, Bioxon) was packed in bottles of 500 ml Duran, Mark, Germany and sterilized at 121°C for 20 min. Then, under aseptic conditions in rustic laminar flow chamber, the culture medium was poured into sterile Petri dishes, plastic 90 mm in diameter, each with approximately 20 ml of PDA (Stanley, 2010). The Petri dishes were inoculated with 5 mm diameter slices of culture medium previously colonized with P. ostreatus CP-50, under aseptic conditions in rustic laminar flow hood aforementioned. The Petri dishes were incubated at 25°C until the mycelium fully colonized the culture medium it took an average of 7 days. There were 3 replicates and 2 independent experiments to corroborate the efficiency of the rustic laminar flow hood.

Preparation of the mother seed (MASTERS)

The mother seed is used to inoculate the secondary ones, which in turn will be used as inoculum of the substrate that will produce Figure 2. Laminar flow hood rustic. fruiting bodies of fungi mushroom. The mother substrate on the other hand, is inoculated with a piece of agar with mycelium from a pure culture of the strain. The inoculum was prepared using seeds of wheat (T. aestivum L.) and maize (Z. mays L.), control treatment 0.3 microns or large one, so that the air without particles is consisted of boiling 300 g of wheat and 200 g of corn for 20 min in 5 projected vertically through the work area. The air is captured on L of water, was allowed to settle for 30 min. This mixture was 3948 Sci. Res. Essays

Figure 3. Secondary seed produced in a laminar flow hood rustic.

squeezed into a plastic tray with a capacity of 10 Kg for 60 min, this Control of seed quality part is critical, its proper implementation prevents seeds too dry or too wet will affect mycelia growth. If the grains are very dry, mycelia Quality control in seed production was the constant inspection to growth is very slow. If the grains have a lot of water, growths is also eliminate all those units that visibly contaminated or unacceptable very slow and eventually stop when water accumulates on the differences in appearance or growth. Here it is essential that seed bottom. The optimum moisture for grains is about 50%. Moisture producers have a proper installation rustic where they can evaluate was determined using a digital hygrometer, then were added 5 g of the characteristics of seed production, besides being in contact with lime and 20 g of gypsum with wheat and maize to reach an researchers from the Mycology Laboratory of the School of alkaline pH of about 9 (Romero, 2007; Stanley, 2010). Engineering Agro forestry - BUAP, so the inconveniences of seed Then placed 500 g from this mixture in jars with a capacity of production can be corrected quickly. 1000 ml and sterilized for 60 min at 121°C. When the jars have cooled (24 h later), proceeded to inoculation with agar colonized of CP-50 strain P. ostreatus, for this a knife is use to cut the agar into RESULTS AND DISCUSSION small squares or pieces and transfer to jars with sterile grains into the rustic laminar flow hood where they were incubated at room temperature for 25 days. The results showed that the inoculum or seed production of P. ostreatus using rustic laminar flow hood, was efficient and showed no contamination and the inoculum Preparation of secondary seed present the appropriate characteristics (color, odor,

The grains of wheat and corn can also be used to produce seed for consistency and safety) for the production of the planting (secondary), as was done in this experiment this is called mushroom fungus. “seed to seed inoculation”. Mycelia growth showed that the strain in The proposed use of rustic laminar flow hood, in the substrate is 25% faster than that of the mother seed, because addition to complying with the adequate production of the mycelium was adapted to the substrate in a short period of time inoculum for the production of mushroom fungus, aim to (7 days). It also had a higher amount of seed for sowing in the reduce production costs, since the cost of manufacturing substrates. The preparation consisted of inoculating the substrate which was used to produce seed for planting, seed mother were the same accounts for 40% of the cost of a laminar flow used for vigorous growth, inoculation was done again in the rustic, hood similar to any trademark. laminar flow hood (Figure 3), and in the same way described for the This investment for the development of a laminar rustic planting of grain mother´s grain. The jars were shaken and a small flow hood is $14,000.00 (Mexican pesos), which amount of mother seed was deposited into the bags previously compared to the cost of a similar mark laminar flow hood prepared as described above with the objective to obtain the laboratories is $35,000.00 (Mexican pesos). This secondary seed (Stanley, 2010). In general, 1 kg of mother seed will be enough to obtain 30 kg of secondary seed. During incubation difference in the initial investment is significant and proceeded in the manner indicated for the mother seed. represents a considerable savings for producers of Romero-Arenas et al. 3949

mushrooms, which will help to encourage this activity in ACKNOWLEDGEMENTS rural communities in the municipality of Tetela de Ocampo. The authors thank the Vicerrectoria de Investigación y In general, the laminar flow hoods have a lifespan of 2 Estudios de Posgrado (VIEP) of the Benemerita to 3 years depending on the use to which they are Universidad Autonoma de Puebla (BUAP) for the subjected to which is determined by the HEPA filter life, financial support of this research project. this rustic laminar flow hood proposal justified their initial investment as free of inoculum dependence on producers, which will increase the profits they can get. REFERENCES The proposed use of a rustic laminar flow hood is novel Almanza S (1994). Biotechnology Transfer to intermediate development because it represents the use of a locally developed countries: some general considerations. CENTURY BIOCIT, 8(9). technology with available materials in any small town and Chang ST, Miles PG (1989). Edible mushrooms and Their cultivation. can be very useful to make the process more profitable. CRC Press Inc., Boca Raton, FL. Mushroom fungus production encourages the Chemo T (2002). The Biology and Cultivation of Pleurotus spp.: Preparation of the seed. Sánchez, J. comp. Colegio de la Frontera development of productive projects in rural areas of the Sur (ECOSUR), Chiapas, Mexico, D. F., MX, Editorial Limusa S. A., country. pp. 125-137. As explained earlier in this research, the cultivation of Gaiska AB (1997). Programs of transfer of agricultural technology in mushroom fungus implies the need for seed production Mexico (Cases analysis). In: Mata GB, Perez GS, Leon F (Coord.). Transfer of agricultural technology in Mexico. Critique and proposals. programs through the use of a rustic laminar flow hood as IICA, UACH, UAM, Mexico. a strategy for producers in the region o develop strains Martinez-Carrera D (2002). Current Development of mushroom adapted to local environments and substrates. In such a biotechnology in Latin America. Int. Aplicate Micol., 14: 61-74. situation, future research will need to focus on the Martinez-Carrera D, Sobal M, Morales P, Martínez W, Aguilar A, Larqué-Saavedra A (1995). Edible Mushroom Cultivation and development of basic knowledge about the cultivation of Sustainable Agriculture in Mexico. Afr. J. Micol. Biotechnol., 3 (1): 13- edible fungi, the appropriate characterization and 18. taxonomic classification of wild strains, as well as on Martinez-Carrera D, Leben R, Morales P, Sobal M, Larqué-Saavedra A procedures for the conservation and replication of (1991b). History of the commercial cultivation of edible mushrooms in Mexico. Sci. Dev., 96: 33-43. specimens of a commercial nature on techniques to Melo de Carvalho CS, Ceci Campos S, Nogueira de Andrade MC preserve genotypes, crosses for the highly productive (2010). Mushrooms of the genus Pleurotus: a Review of Cultivation hybrid formation and especially the production of Techniques. Interciencia, 35(3): 177-182. inoculum using agro-industrial waste in the region. Ogden A, Prowse K (2005). Mushroom Growers' Manual. Gourmet Woodland Mushrooms Ltd., United Kingdom. ISSN: 1739-1377, p. 292. Romero AO (2007). Technological Development to Control Green Mold Conclusions (Trichoderma spp.) during the commercial cultivation of edible fungi (Pleurotus and Lentinula) in Mexico. M. Sc thesis. Postgraduate College. Campus Puebla, p. 136. Based on the results of the work concludes: Romero AO, Huerta LM, Damian HMA, Macías LA, Tapia Rojas AM, Parraguirre LJFC, Juarez HJ (2010). Evaluation of the productive 1. The laminar rustic flow hood is an option for seed capacity of Pleurotus ostreatus using dehydrated banana leaf (Musa production of mushroom fungus P. ostreatus at low costs paradisiaca L.) in relation to other agricultural wastes. Costa Rican Agronomy, 34(1): 53-63. and its implementation is accessible to any producer. Sobal M, Morales P, Bonilla M, Huerta G, Martínez-Carrera D (2007). 2. The quality of the inoculum or seed produced from this The Genetic Resources Center of Edible Fungi (CREGENHC) the technological innovation, is similar to that which can be Graduate College. Chapter 2.1: 14. In: The Cultivation of Mushrooms obtained using commercial technology, but with lower Pleurotus spp., Mexico JE, Sanchez D, Martínez-Carrera GM, Leal H (Eds.). ECOSUR-CONACYT, Mexico, D.F. ISBN 978-970-9712-40-7. production costs. Varnero Mary T, Madelaine SH, Quiroz Cristian A (2010). Using Forest 3. The diffusion of such technologies to rural areas is a Residues to Produce lignocellulosic Oyster Mushroom (Pleurotus proposal to be implemented in similar areas, in order to ostreatus), Inf. Technol., 21(2): 13-20. encourage this activity on behalf of producers.