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PROSPECTS OF THE NEW SUBSTRATES INTRODUCING IN CULTIVATION TECHNOLOGY OF OYSTER MUSHROOM [PLEUROTUS OSTREATUS] IN ARMENIA

INTRODUCTION Lack of nutrient resources has become a vital issue worldwide. Simultaneously with the world population increase, the significant increase in food supply per capita in developing countries is being observed (1). It’s known that some species of mushrooms have significant nutritional value and are widely used by the population. The diet of 30% of the world population is protein deficient, and the recent analysis has proved that 200g of mushrooms can efficiently replace 100g of meat as a protein source (2). Nowadays, more than 6 billion kg of edible mushrooms are cultivated each year (Fig. 2). Nutritional value of fruit bodies can be considered high due to the presence of essential amino acids (valine, leucine, isoleucine, methionine, tryptophan, etc.) and vitamins - A, B1, B2, C, D, PP, and a number of elements - potassium, phosphorus, calcium, iron, zinc, etc. (3).

In Armenia, two species - oyster mushrooms (Pleurotus ostreatus) (Fig. 3a, b) and champignon (Agaricus bisporus) – are generally used as the food sources, which makes up a significant portion of the cultivated mushrooms. In addition to the high nutritional value, many of the edible mushrooms have medicinal properties (Fig. 4) and are widely used due to the presence (in mycelium and fruit bodies) of biologically active compounds. The cultivation on different substrates makes it possible to get a non-waste technology, resulting in protein-rich food (fruit bodies), fodder, and organic fertilizer (mycelial biomass) (4,5). Oyster mushrooms have the certain advantages over the other cultivated mushrooms: rapid growth rate of the mycelium, high degree of competitiveness in relation to the competing micromycetes, resistance to infections and, mostly important, oyster mushrooms grow on different substrates containing cellulose and lignin (more than 200 substrates). The latter feature is explained by the presence of strong exoenzyme system capable of degrading lignin and cellulose that could be promising in the treatment of certain agricultural wastes (6). As it was marked, oyster mushrooms also have therapeutic properties, mostly notable antifungal and anticancer activity (7). A water extract of P. ostreatus exhibited significant cytotoxicity by inducing apoptosis of human carcinoma cells (8). It is commonly known that the oyster mushroom is a producer of lavastatin (trade name - mevinolin), which inhibits biosynthesis of cholesterol. Among the cardiovascular patients, who included oyster mushroom in their daily ration, a significant decline of the cholesterol rate (by 20 – 40%) was observed. It was noted that serum triglyceride and total cholesterol levels were lowered in hyperlipidaemic rats supplemented with the P. ostreatus powdered fruit bodies (9). Methanol extracts of P. ostreatus fruiting bodies possessed antioxidant activity (10). It’s also reported about antimicrobial activities of P. ostreatus crude extracts from fermentation broth on gram-positive and gram-negative bacteria (11).

Introducing the prospects of application of the used substrates, we consider it important to

note that the used substrates are ecological safe and have high nutritional value and also, due to their bioactive compounds, have nematocidal properties (Fig.5,6). The used substrates could also be applied in cultivating of the other fungal species, in particular the fungus Stropharia rugoso-annulata (12).

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Considering the noted advantages, we state that the expanding of cultivation of this type of fungus in Armenia makes it possible to solve a number of challenges facing the agriculture and the population.

Scientific Classification of Pleurotus ostreatus (Jacq.: Fr.) Kumm          

               

Fig. 1. Pleurotus ostreatus (Jacq.: Fr.) Kumm.

*Description:

Common names: The oyster mushroom, Oyster shelf, Tree Oyster Straw Mushroom, Hiratake or Tamogitake (Japanese name).

Ecology: Saprobic; growing in shelf-like clusters on dead logs and living trees; causing a white rot; winter, and early spring; common.

Distribution: Distributed throughout the suitable temperature zones and the tropical forests of the world.

Kingdom: Fungi

Phylum: Basidiomycota

Class: Agaricomycetes

Order: Agaricales

Family: Tricholomataceae

Genus: Pleurotus

Species: P. ostreatus (Fig. 1)

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Cap: 4-15 cm; convex, becoming flat or somewhat depressed; somewhat greasy when young and fresh; smooth; pale brown to dark brown.

Gills: Running down the stem; close; whitish or with a gray tinge, sometimes yellowish in age.

Stem: Usually rudimentary and lateral (or absent) when the mushroom is growing from the side of a log or tree.

Flesh: Thick; white.

Odor and Taste: Odor distinctive but hard to describe; taste mild.

Spore Print: Whitish to grayish or lilac.

Microscopic Features: Spores 8-10.5 x 3-3.5 µ; smooth; cylindrical to narrowly kidney-shaped.

*Description on basis of www.mushroomexpert.com

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Nutritional Content On the average, fresh mushrooms contain about 80-95% moisture, 3% protein, 0.3-0.4% fat and 1% minerals and vitamins. The data on nutritional content of oyster mushrooms are represented in the Tables 1 and 2. Table 1. Ratios of essential amino acids in Oyster mushroom (Pleurotus ostreatus) to those in cows’ milk (13, 20).

Amino acid Pleurotus ostreatus (14) Pleurotus ostreatus (15) isoleucine 0.634 0.506

leucine 0.502 0.424 lysine 0.413 0.478 phenylalanine 0.485 0.600 tyrosine 0.444 1.344 threonine 0.730 0.693 tryptophan 0.630 0.563 valine 0.917 1.000 cystine 1.333 0.407 methionine 0.530 0.599 Table 2. Proximate composition of Oyster mushroom (Pleurotus ostreatus) g per 100 g fresh weight. Minerals mg per 100 g fresh weight (20).

Species and references Moisture Ash Nitrogen N x 5.0

Protein Fat Crude fiber

Na K Ca Fe P

P. ostreatus (16) 92.0 0.64 0.40 2.00 0.35 2.40 1.00 296 0.1 0.43 111.0

P. ostreatus (17) 88.6 0.87 0.50 2.50 0.25 0.61

P. ostreatus “Florida” (18)

91.5 0.79 0.25 1.25 0.14 1.01

P. ostreatus (19) 88.9 0.44 4.71 0.20 1.23 8.21 30 1.9 0.4 0.89 117.8

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Cultivation process of Oyster mushroom (Pleurotus ostreatus) Growth parameters (21) Spawn Run: Temperature: 24ºC Relative Humidity: 85-95% Duration: 12-21 days CO2: 5000-20.000 ppm Fresh air exchanges: 1 per hour Light requirements: n/a Primordia Formation Temperature: 10-15,6ºC Relative Humidity: 95-100% Duration: 3-5 days CO2: < 1000 ppm Fresh air exchanges: 4-8 per hour Light requirements: 100-1500 (2000) lux Fruit body development Incubation Temperature: 10-21ºC Relative Humidity: 85-95% Duration: 4-7 days CO2: < 1000 ppm Fresh air exchanges: 4-8 per hour Light requirements: 100-1500 (2000) lux Cropping Cycle 3-4 crops, every 7-14 days , over 45-55 days

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BODY

Nowadays, oyster mushrooms are widely cultivated (using intensive technology) in the Republic of Armenia. Straw is used as the main substrate. However, throughout the world the wide range of substrates, which are agricultural and timber industry waste, is successfully applied. The basis of our proposal is the idea of using the waste substrate (characteristic for the Armenian agriculture and industry) in the technology of oyster mushrooms industrial production. Proposed substrates Straw and hulls

Wheat is grown almost throughout the territory of Armenia, bringing the straw that is widely used in the cultivation of oyster mushrooms (white chalk, gypsum, etc. are used as the additives). We propose to use the wheat straw and hulls as the main part (30-50%) of the substrate, and to replace the rest with one of the following substrates. Corn stаlks and corncob

Production of maize is centralized in the north-east Tavoush (from 30% to 35% of the grown corn in corn Armenia), as well as in the province of Ararat and Armavir (grown from 43% to 48% of local maize) (1). The presence of the agricultural waste brings up the opportunity to develop the cultivation of oyster mushrooms in the aforementioned areas, reducing the substrate transportation costs. Corn stаlk and corncob can be used to grow mushrooms as the part of the substrate. Studies conducted in the Laboratory of Mycology, Department of Botany, YSU, showed rapid growth of mycelium of oyster mushrooms, which was grown on corncob (Fig. 8) and highest yield (10 kg of substrate - 5 kg of fruit bodies) (23). We also want to note that the use of corn stalks as supplements to the main substrate leads to increase in fruiting, up to 8% (24).

Sugar cane and sugar beet waste

This substrate has good prospects, given the fact that in the 2009-2010 it is scheduled to put into operation a sugar producing factory, currently under construction, in the Shirak area. It has been stipulated to conclude contracts with more than 3000 agricultural farms for the processing of beet. The combination of sugar cane (Fig. 9) and sawdust substrates ( 75% and 25% respectively) showed a high rate of the fruit body yield , Pleurotus ostreatus (biological efficiency - 107.61%) and is economically profitable (25).

Sawdust

Sawdust is a woodworking industry waste and could be considered as the most natural substrate for oyster mushrooms. According to some experimental data, BE was 64.69% in the case

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of sawdust substrate applying. High fruiting when combined with other substrates (BE: Sawdust + leaves - 62.9%; sawdust + sugar cane - an average of 106-107%) has also been noted. (25, 26).

Grapes pulp

In Armenia, due to the large volume of viticulture and wine industry, this substrate in the cultivation of oyster mushrooms technology has great potential. In particular, there are 25 wine and 7 brandy producing companies in Armenia, which, according to the 2004, bought 79.100 tons of grapes (1). Substrate consisting of grapes pulp showed biological efficiency 16-38,8%, however, it is important to note that the substrate composed of vine and grapes pulp was marked by rapid growth of mycelia, and intensive fruiting (27).

Paper waste

Paper waste is also applicable in the context of Armenia. Note that the waste paper in combination with other substrates showed rapid growth of mycelium and remarkable fruiting (28, 29).

Other substrates and waste based spawn

In the technology of oyster mushroom growing the other residues, in particular, the pod sunflower, rice straw (Fig. 10) soybean and cotton stalks, which showed 60-70% of biological efficiency or, in other words, the 600-700 g fruit body per 1 kg of substrate (30), are also widely applied. In developing Asian countries such as India, Indonesia, as well as in some African states, the waste inherent in their agriculture, such as banana leaves and other plant residues are used in cultivating of oyster mushrooms.

Oyster mushrooms are grown from mycelium propagated on the basis of steam-sterilized

cereal grain. This cereal grain/mycelium mixture is called spawn and is used to seed mushroom substrate. It is important to note that in the everyday technology the Lemke method (22), based on using of wheat and barley (grain spawn), is being applied, which makes the mycelia (Fig. 7) production unprofitable. Thus, we offer to introduce the waste based spawn in the oyster mushroom cultivating, aiming to decrease the cost of the product.

Research work

It is necessary to carry out some research work to implement this proposal and to ensure high productivity. Note that to ensure high fertility of the mushroom, using of industrial strains is suggested. At present, mainly the strain Pleurotus ostreatus HK-35 (Fig. 11) is used in Armenia, but applying of the new substrates leads to the obtaining of new strains and to identifying of their production values. In particular, it is proposed to establish a research laboratory where the research work will be carried out to obtain new strains and their selection. Note also that the industrial strains

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could be sold to the foreign countries and bring additional profit. Oyster mushroom in nature grows almost throughout the entire territory of Armenia and has a great potential for the identification of new industrial strains. Establishing of the laboratory will provide an opportunity to research new substrates and to compose prescriptions for the agricultural waste substrates. Laboratory experiments are exceptionally important in the development of technology of this species of fungi.

International Mycological Resources on Internet

Russia

Mushroom cultivation school - http://www.gribovod.ru/index.html Industrial Strains of Oyster mushroom - http://www.tomi63.ru/s2.php

Ukraine

Industrial Strains of Oyster mushroom - http://www.ukrmiceliy.donbass.com/sh_veshenka.htm

Griboed - http://www.griboed.net.ua/55

Gribnoy dom - http://www.gribovod.com.ua/

Poland

Konwektor - equipment for cultivating - http://www.konwektor.pl/rus/

Belgium

Industrial Strains of Oyster mushroom - http://www.combiness.com/mycalg.htm

USA

Fungi Perfecti® - www.fungi.com

Mycosource Inc. - http://www.mycosource.com/

Mycoweb - http://www.mykoweb.com/

International Organizations

International Mycological Association - http://www.ima-mycology.org/ European Mycological Association - http://www.euromould.org/ Sylvan - http://www.sylvaninc.com/ Mushroom Business – International Independent Information for growers http://www.mushroombusiness.com/

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Crediting

For the development of the oyster mushroom industrial cultivation and the application of the proposed substrates it is necessarily to undertake a program of lending, which will focus on funding of the joint projects of different agricultural industries, factories and organizations involved in the cultivation of oyster mushrooms. Programs should encourage the involvement of agricultural waste in the production and cultivation as a basis for mutually beneficial cooperation. Since the used substrates are considered as the fodder enriched in amino acids, it is important also to encourage cooperation with the livestock farms and other agricultural organizations, which could be interested in purchasing of the used oyster mushroom substrate.

CONCLUSION

Summarizing, we note that Armenia's agricultural waste could be used in cultivating of oyster mushrooms. Particularly actual could be using of grapes pulp, corncob, waste sugar cane and sugar beet as a co-substrate with already extensively used straw, bran, sawdust etc. We’d also wish to note that the development of oyster mushrooms industrial production and the introduction of new technologies will provide the impetus for our study of the agriculture waste and introducing of the new substrates. Thus, we propose the following plan:

• Assessment of the total amount of waste (agricultural production, agriculture and production enterprises etc.). • Implementation of research work to obtain the optimal substrate for oyster mushrooms growing on the proposed waste. • Lending business programs to encourage the cultivation of oyster mushrooms using agriculture waste as the substrate.

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APPENDICES: FIGURES, ACKNOWLEDGMENTS, LIST OF ABBREVIATIONS, REFERENCES, TABLE OF CONTENTS Figures

Fig. 2. Cultivated species of edible fungi

Fig. 3.a,b. The growth of fruit bodies of oyster mushrooms (Pleurotus ostreatus)

on the straw substrate

a/

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b/

Fig. 4. Biological active supplements on the fungal basis

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**Fig. 5.A nematode paralysed by the nematotoxin ostreatin produced by secretory appendages on the hyphae of Pleurotus ostreatus (Oyster Mushroom).

 

**Fig. 6. Microscopic field showing several microcolonies of bacteria under attack by the hyphae of Pleurotus ostreatus

 

 

**George Barron's micro-pictures;http://www.uoguelph.ca/~gbarron/ZBiodiversity/pleutoxd.html 

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Fig. 7. Growing oyster mushroom mycelium (Pleurotus ostreatus) on the grain substrate (laboratory conditions)

Fig. 8. The growth of mycelium of oyster mushrooms (Pleurotus ostreatus) on the corncob substrate

 

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Fig. 9. Dry sugarcane bagasse (Photo of Dewraj Taurachand)

Fig. 10. Oyster mushroom cultivated on rice straw

(Photo of Chang-Hyun You)

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Fig. 11. Oyster mushroom strain (Pleurotus ostreatus HK-35) mycelium on agar media

List of Abbreviations YSU - Yerevan State University BE - Biological Efficiency

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References

1. Snapshots: Framing the "next level up" of Sustainable Agriculture in Armenia. 2008. III Arm Agroforum / IV Fruitfull Armenia Joint International Conference, Yerevan, Armenia, 292.

2. Souci S.W., Fachman W. and Krant H. 1975-1989. Food Composition and Nutrition Tables. Wissenschaftliche Verlagsgesellschaft mbh, Stuttgart.

3. Zaikina N.A., Kovalenko A.E., Galinkin V.A., Dyakov Yu.K., Tishenkov A.D. 2007. Basis of Biotechnology of Higher Fungi. S. Petersburg, 336.

4. Delcaire J. R. 1978. Economics of cultivated mushrooms. The biology and cultivation of edible mushrooms. Acad. press, 728 – 792.

5. Declaire J. R. 1981. Place et role des champignons cultives comme source de proteines humaines en 1an 2000. Mushroom, 11:1 – 8.

6. Bano Z., Ghosh P. K., Rajarathman S., Shashirekha M. N. 1996. Biotransformation efficiencies of lignocellulose wastes by mushrooms. Journal of Scientific and Industrial Research, 55(5 – 6): 400 – 407.

7. Wasser S.P., Weis A.L. 1999. Medicinal properties of substances occuring in higher basidiomycetes mushroom. Current perspectives (review). Inter. J. Medicinal Mushrooms, 1: 31 – 62.

8. Gu Y.H., Sivam G. 2006. Cytotoxic effect of oyster mushroom Pleurotus ostreatus on human androgen-independent prostate cancer PC-3 cells, J. Med. Food., 9:196–204.

9. Hossain S., Hashimoto M., Choudhury E.K., Alam N., Hussain S., Hasan M., Choudhury S.K., Mahmud I. 2003. Dietary mushroom (Pleurotus ostreatus) ameliorates atherogenic lipid in hypercholesterolaemic rats, Clin. Exp. Pharmacol. Physiol., 30:470–475.

10. Yang J.H., Lin H.C., Mau J.L. 2002. Antioxidant properties of several commercial mushrooms, Food Chem., 77:229–235.

11. Gerasimenya V.P., Efremenkova O.V., Kamzolkina O.V., Bogush T.A., Tolstych I.V., Zenkova V.A. 2002. Antimicrobial and antitoxical action of edible and medicinal mushroom Pleurotus ostreatus (Jacq.: Fr.) Kumm. extracts, Int. J. Med. Mush. 4:48–54.

12. Poppe J., Höfte M. 1995. Twenty wastes for twenty cultivated mushrooms. Mushroom Science, 14(1): 171-179.

13. Food and Agriculture Organization, U.N. 1970. Amino acid Content of Foods and Biological Data on Proteins. FAO, Rome. 278.

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14. Kalberer P. and Kuunsch U. 1974. Amino acid composition of the oyster mushroom (Pleurotus ostreatus), Food Sci. Technol. 7: 242-244.

15. Mattila P., Salo-Väänänen P., Könkö K., Aro H. and Jalava T. 2002. Basic composition and amino acid contents of mushrooms cultivated in Finland, J. Agric. Food Chem. 50:6419-6422.

16. Mattila P., Könkö K., Eurola M., Pihlava J-M., Astola J, Vahteristo L., Hietaniemi V.,

Kumpulainen J., Valtonen M. and Piironen V. 2001. Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms, J. Agric. Food Chem. 49:2343- 2348.

17. Yang J-H., Lin H-C., Mau J-L. 2001. Non-volatile taste components of several commercial

mushrooms, Food Chem. 72: 465-471. 18. Bano Z., Bhagya S., Srinivasan K. S. 1981. Essential amino acid composition and proximate

analysis of the mushroom Pleurotus eous and P. florida, Mushroom Newsletter of the Tropics 1 (3):6-10.

19. Kawai H., Matsuzawa M., Tsutagawa Y., Sasaki H., Kasuga A., Aoyagi Y. 1994. Relationship

between fruiting body composition and substrate in hiratake and maitake mushrooms cultivated on sawdust substrate beds, Nippon Shokuhin Kogyo Gakkaishi. 41:419-424.

20. Kurtzman R. H., Jr. 2005. Mushrooms: sources for modern western medicine, Micologia Aplicada International. 17(2):21-33.

21. Stamets P. 1993. Growing gourmet & medicinal mushrooms, Ten Speed Press, Berkley, 554.

22. Lemke G. 1972. Mezelwachstumsteste mit vier Champignonstammen . Champignon, 12(128): 1-5.

23. Nanagulian S.G., Taslakchian M.G., Kirakossian N.J. 1995. Waste – free technology of growing Pleurotus ostreatus in Armenia. Proceedings of 3rd International Congress “Science and Practice in Mushroom Cultivation”, Kashira, Russian Federation, 31-32.

24. Bugarski D., Djuro G., Takac A., Dragan J. 1997. Influence of substrates on fructification of oyster mushroom (Pleurotus ostreatus) strain NS-16. Acta Hort. (ISHS), 462:891-894.

25. Vetayasuporn S. Chutichudet P. Cho-Ruk K. 2006. Bagasse as a possible substrate for Pleurotus ostreatus (Fr.) Kummer cultivation for the local mushroom farms in the Northeast of Thailand. Pakistan Journal of Biological Sciences, 13(9): 2512-2515.

26. Shah Z.A., Ashraf M., Ishtiaq M.Ch. 2004. Comparative study on cultivation and yield performance of oyster mushroom (Pleurotus ostreatus) on different substrates (wheat straw, leaves, saw dust). Pakistan Journal of Nutrition, 3 (3): 158-160.

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27. Sánchez A., Ysunza F., Beltrán-García M. J., Esqueda M. 2002. Biodegradation of viticulture wastes by Pleurotus: A source of microbial and human food and its potential use in animal feeding. J. Agric. Food Chem., 50 (9): 2537–2542.

28. Hashimoto K., Takahashi Z. 1976. Studies on the growth of Pleurotus. Mushroom Science 9(2):585-593.

29. Baysal E., Peker H. 2006. An alternate to waste paper recycling; mushroom cultivation. Teknoloji, 3-4: 9-13.

30. Platt M., Chet I., Henis Y. 1982. Growth of Pleurotus ostreatus on cotton straw. Mushroom Journal 20: 425-426.

Acknowledgments I would like to express my gratitude to my supervisor, Professor, Dr. of Sci, Nanagulyan Siranush for her help and support in carrying out this work.  

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Table of Contents

Introduction ………………………………………………………………………1

Scientific Classification of Pleurotus ostreatus (Jacq.: Fr.) Kumm …………………….. 2 Description ………………………………………………………………………………. 2 Nutritional Content ………………………………………………………………………. 4 Cultivation process of Oyster mushroom (Pleurotus ostreatus) ………………………… 5 Body ……………………………………………………………………………… 6 Proposed substrates ………………………………………………………………... 6 Other substrates and waste based spawn ……………………………………………. 7 Research work ……………………………………………………………………... 7 International Mycological Resources on Internet ………………………………………... 8 Crediting …………………………………………………………………………………. 9 Conclusion ………………………………………………………………………. 9 Appendices ............................................................................................................10 Figures .............................................................................................................................. 10 List of Abbreviations ……………………………………………………………... 15 References ……………………………………………………………………………… 16