Increase Yield Potential with Oyster Mushroom Extract

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[2005 MushWorld Research Paper Award - Research Proposal] Increase Yield Potential with Oyster Mushroom Extract
Writer: Mungkorn Thewasingh / Date :2005-10-01 / hits: 175
I. Outline of Research Theme
_____ Product of straw mushroom is problematic in trying to enlarge scale of production. Selection of strains, substrates and environment improves yield in limited level. One problem derived from observation in practice is inconsistent fruitful. Induction of fruiting body formation leads to increase in the yield. Previous reports of fruiting inducing effect from different mushroom extracts confirm possibility to apply with straw mushrooms. Using natural extracts from edible mushrooms will be more accepted and not harmful to mushroom consumers. These extracts have also similarity to physiologic requirement of straw mushroom. Oyster mushroom (Pleurotus ostreatus) is a well known edible mushroom that has many enzymes and nutritional substances. Extracts from the oyster mushroom may have additional benefit to straw mushroom growing. This research will be emphasis on effect of extracts from some edible mushrooms on straw mushroom production.
II. Research
1. Background
_____Some researches report that extracts from bacteria (Cho et al, 2003) or from fungi (Leonard and Dick 1968 and Uno and Ishikawa, 1973) and used chemicals (Magae, 2004) can induce fruiting body formation in different species of mushrooms. The addition of organic supplements to substrates also results in earlier fructification and increases the yield of production (Domondon et al, 2003). Some of these substances don't derive from edible substances so it is hard to be accepted from mushroom consumer. Although extract from some edible mushroom induces primordial formation in some fungi (Urayama, 1969), it is not applied with edible mushrooms. The Concept that using bioactive substances from edible mushrooms can help to improve straw mushroom production should be studied in more details. Due to limitation of digestive enzymes in straw mushroom growth and differentiation may be impeded. Pleurotus ostreatus is lignocelluloic mushroom that has capability to digest various substrates. The ready prepared bioactive substances from this mushroom seem to be able to supplement important factors for growth activity and induction of primordial formation of straw mushroom. This strategy will also increase yield of product in shorter duration than conventional cultivation. Furthermore, our pilot study showed possibility of oyster mushroom extracts in increasing straw mushroom production.
Cho Y., Kim J., Crowley. D, Cho B. 2002. Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads. FEMS Microbiol Letters 218, 271-276.
Domondon D., He W., Kimpe N., Hofte M. and Poppe J. 2003. b-Adenosine,abioactive compound in grass chaff stimulating mushroom production. Phytochemistry 65,181-187.
Leonard T. J. and Dick S. 1968. Chemical induction of haploid fruiting bodies in Schizophyllum commune. Proceedings of the National Academy of Science of the USA.,59, 745-751.
Magae Y. 2004. Effect of sucrose ester of fruit body formation of Pleurotus ostreatus. WSMBMP, 117.
Uno I. and Ishikawa T. 1973. Purification and identification of the fruiting-inducing substances in Coprinus macrorhizus. Journal of Bacteriology 113, 1240-1248.
Urayama T. 1969. Stimulative effect of extracts from fruit bodies of Agaricus bisporus and some other hymenomycetes on primordiam formation in Marasmius sp. Transactions of the Japanese Mycological Society 10, 73-78.
2. Objectives
2.1. Problem
_____Inconsistent and low yield of straw mushroom production
2.2. Hypothesis
_____Bioactive substances from oyster mushroom can support growth and differentiation of straw mushroom
2.3. Verification of hypothesis
Test biologic activity of oyster mushroom extracts on enhancing growth of straw mushroom mycelia
Test effect of the oyster extracts on fructification of fully grown colonized substrates of straw mushroom.
Measure differences of product yield between supplemented and non-supplemented cultivation.
2.4. Objectives
To test biologic activity of oyster mushroom extracts on enhancing growth of straw mushroom in media with and without agar.
To test effect of the oyster mushroom extracts on fructification of fully grown colonized substrates of straw mushroom.
To measure differences of product yield between supplemented and non-supplemented cultivation in practice.
3. Methodology & Statistical design
3.1. Materials and method
3.1.1. Organism and inoculum
_____Straw mushroom (Volvareilla volvacea) used in this study will be received from Biotechnology Research and Development Office Department of Agriculture Thailand. This mushroom will be maintained on a potato dextrose agar (PDA) slant at 25'C. Inoculum of straw mushroom will prepare on 9 cm PDA Petri dish for 7 days at 30+2'C in room temperature. Agar plugs of 4 mm diameter will be taken from the periphery of growing colony of V. volvacea and used to inoculate into test media.
3.1.2. Preparation of aqueous oyster mushroom extraction
_____Young fruiting bodies of oyster mushroom (Pleurotus ostreatus) will be homogenized with distilled water (1:2 by w/v). The aqueous extract will be filtered through clean cheesecloth and then further removed small particles with ultracentrifugation. This supernatant will be kept for use.
3.1.3. Testing biologic activity of oyster mushroom extracts on enhancing growth of straw mushroom in media with and without agar
3.1.3.1. Substrate preparation
_____The prepared extract will be diluted with distilled water in described dilutions (1:1, 1:2 and 1:4). These dilutions will be supplemented separately (20 ml) in 100 ml of PDA agar and PDA non-agar media before sterilization. Control will be served as non-supplemented media.
Treatments
Cold water extract Non
Agar PD + 1:1 extract
(PD 30 + extract 10 + water 10) ml
PD + 1:2 extract
(PD 30 + extract 6.67 + water 13.33) ml
PD + 1:4 extract
(PD 30 + extract 4 + water 16) ml
With
Agar PDA + 1:1 extract
(PDA 30 + extract 10 + water 10 ) ml
PDA + 1:2 extract
(PDA 30 + extract 6.67 + water 13.33) ml
PDA + 1:4 extract
(PDA 30 + extract 4 + water 16) ml
Hot water extract Non Agar PD + 1:1 extract
(PD 30 + extract 10 + water 10) ml
PD + 1:2 extract
(PD 30 + extract 6.67 + water 13.33) ml
PD + 1:4 extract
(PD 30 + extract 4 + water 16) ml
With Agar PDA + 1:1 extract
(PDA 30 + extract 10 + water 10 ) ml
PDA + 1:2 extract
(PDA 30 + extract 6.67 + water 13.33) ml
PDA + 1:4 extract
(PDA 30 + extract 4 + water 16) ml
Control PDA 50 ml
PD 50 ml
PA 50 ml
DA 50ml
A 50 ml
* watter is distilled water.
3.1.3.2. Measurement of mycelial growth on PDA plate with extract supplement
_____The V. volvacea agar plugs described in 3.1.1 will be inoculated in the center of each extract incorporated media as described in 3.1.3. Their growth will be assessed at the temperature range of 30+ 2'C. Colony diameter on each plate will be measured in 1, 3, 5 and 7 days after inoculation. For non-agar culture, after 21 days of inoculation straw mushroom mycelia will be filtered and dried. Their dried weight will be recorded.
Datasheet Mean diameter (mm) Mean mycelial weight (mg)
D1 D3 D5 D7
Cold water extract PD+1:1 extract
PD+1:2 extract
PD+1:4 extract
PDA+1:1 extract
PDA+1:2 extract
PDA+1:4 extract
Hot water extract PD+1:1 extract
PD+1:2 extract
PD+1:4 extract
PDA+1:1 extract
PDA+1:2 extract
PDA+1:4 extract
Control PD
PDA
3.1.4. Testing the oyster mushroom extracts on fructification of fully grown colonized substrates of straw mushroom.
3.1.4.1. Preparation of cotton waste substrate
_____Small pieces of cotton waste will be soaked overnight in water and then mixed with lime (CaO) in 2% w/w. they will be put inside wooden flame (90x90 cm), piled up to approximately 70-90 cm in height and covered with a plastic sheet. Let them stand outdoors for two days and turn them thoroughly by hands. Water will be added when needed to maintain moisture content of 70%. The substrates will be piled up again and covered for another 2 days (Chang, 1988). A half kilogram of the fermented substrate will be packed in polypropylene bags (15x30 cm). They will be steriled at 121'C for 60 min and let them cool down to 30+ 2'C.
_____Chang S.T. 1988. Volvariella cultivation. In Food and Agriculture Organization and Department of Agriculture, Kasertsart University (Eds.), Development of button mushroom cultivation against small scale growers on Northern Thailand, Bangkok, Regional office for Asia and the Pacific (RAPA). Food and Agriculture Organization of the United Nations, pp. 79-81.
3.1.4.2. Inoculation and colonization of substrates
_____A straw mushroom colonized agar disc (5 mm in diameter) will be inoculated on the top of the substrate in plastic bags. Inoculated substrate will be incubated at 30+2'C for 12 days. Fully grown colonized substrate will be totally occupied with straw mushroom mycelia.
3.1.4.3. Supplement of oyster mushroom extracts
_____The fully grown colonized substrate will be removed from plastic bags. Each 20 ml of serial dilutions of oyster mushroom extract described in 3.1.3.1 will be sprayed on the surface of colonized substrate. The supplemented substrate will be incubated in a plastic house with 80-90% relative humidity at 30+2'C, light intensity of 1,000 lux/m2 for 12 h/d by fluorescent lamps. The cultivation house was ventilated two times a day to keep well aeration condition. After 5 days of incubation number of primordial formation will be evaluated from each substrate bag. Non-supplemented substrate will be served as control.
3.1.5. Measurement of yield differences between supplemented and non-supplemented cultivation in practice.
_____The supplemented and non-supplemented substrates described in 3.4.3 will be let to grow to early mature stage of fruiting bodies. Their wet weight will be recorded. They will be dried and recorded their dried weight.
Datasheet Number of pins (>0.5cm) after 5 days Fresh weight of fruit body Dry weight of fruit body
Cold water extract Cotton waste 1:1 extract
1:2 extract
1:4 extract
Straw 1:1 extract
1:2 extract
1:4 extract
Hot water extract Cotton waste 1:1 extract
1:2 extract
1:4 extract
Straw 1:1 extract
1:2 extract
1:4 extract
Control Cotton waste water 20 ml
Straw water 20 ml
3.2. Design for statistical analysis
_____Analysis of variances (ANOVA) and Duncan tests for mean values
3.3. Indoor cultivation with straw substrate (shelf method)
3.3.1. Spawn preparation
as described in 2.2 (used colonized cotton waste)
3.3.2 Compost preparation
Dried rice straw 100 kg will put inside a wooden box of 1.5 x 1.5 x 0.5 m. Add water, 1-1.5% urea, or ammonium sulphate, 5-10% dried animal dung and 1% limestone gradually. Left the mixture ferment for 3 days.
Take the wooden box off. The compost will be turned over and added 1-2% double super-phosphate and 1-2% of gypsum. The whole will be mixed thoroughly by means of a mechanical mixer. The mixed and supplemented compost will be then piled up and covered with plastic sheet. After two days, the fermenting compost is turned over again. It will take 5 days to prepare the compost.
Before filling the compost into shelf, the compost will be fragmented by machine. Add 1% of rice bran. The compost will be packed into the shelf (W0.8 x L3.0 x H0.6 m) x 6 shelves, 18-20 cm thick.
Blow the stream from oil-drum stream generator into the house (maintained temp. of 52'C for 8-10 h).
Wait for 2 days until white powder of Actinomycetes appear.
Blow stream again (maintained temp. of 70 'C for 4 h).
Wait until the temperature down to 35'C, inoculate the spawn on the top of the substrate
Left the substrate colonize for 7-12 day after inoculation
Fructification will be performed by spraying the effective OAE from data in 2.0 ( 3 L per shelf, 3 shelves) compared to the control (3 L of clean water/ shelf, 3 shelves)
After 7 day of OAE spraying, all fruit bodies in control and treatment will be collected.
Record wet yield of first and second flushes
Datasheet Type of treatment First flush wet weight Second flush wet weight
Control Clean water (kg) (kg)
Treatment Effective OAE
(3L/shelf, 3 shelves)
3.4. Economic and cost-effective analysis
(Yield over control X sale price of straw mushroom) - ((amount of oyster mushroom x price of oyster mushroom) + price of labor in producing OAE))
4. Expected Results
4.1. What concrete results are expected?
_____The organic supplementation of oyster mushroom extract should help to enhance straw mushroom mycelial growing. This method should also shorten duration of primordia formation and increase amount of primordia. This supplementation will support growth and increase weight of fruiting bodies. Therefore yield of product will increase over non-supplemented cultivation and reflect to improved biological efficiency.
4.2. How could those results be put into practice?
The oyster mushroom extract will be applied by supplementation after fully grown mycelia and before primordial formation. The extracts should help to increase number of primordial formation and quantity of straw mushroom product.
Effect from supplemented oyster mushroom extract should be helpful in straw mushroom cultivation regarding to different types of substrate.
Mushroom growers will make more profit from their straw mushroom cultivation. More profit will motivate the growers to enlarge scale of production because it will be worthwhile with cost of labor and time-consuming.
The mushroom growers can utilize old spent straw mushroom substrate to grow oyster mushroom and harvest its fruiting bodies to be raw material for providing the oyster mushroom extract.
With the same strategy it will be able to apply with other edible mushrooms which are difficult to grow.
4.3. How could the mushroom industry in less developed countries benefit?
Straw mushroom growers can supply their improved product quality in consistent and regular time with factory requirement and mushroom consumers
Owners of mushroom industry can be convinced with starting business of processed food from straw mushroom e.g. canning, frozen food etc.
Excess oyster mushroom from mushroom consumption can be transmuted to be oyster mushroom extracts. Further studies can give more information about useful and effective substances from the oyster mushroom extracts. These extracts can be processed to industrial product.
IV. Research Plan
Dates Activities
Outputs
Sep. - Purchase young oyster mushroom from local farm
- Prepared oyster mushroom extracts
(hot and cold water extraction)
- Preparation of supplemented
and non-supplemented media - Raw material for prepared extracts
- Aqueous extracts
- Agar and non-agar media
- Prepared crude extracts from aqueous
extracts -Yield percentage of crude extracts ( for C:N ratio)
- Straw mushroom inoculation of test media
and incubation - Data of effect of extracts on mycelial growth and recording result
Nov. Build growing house - Growing house for incubation and inducing fruiting bodies
- Preparation of cotton waste substrate - Cotton waste bag
- Inoculation and incubation - Colonized cotton waste
- Test the extracts with different concentration on primordial formation
Dec. - Prepare cotton waste and different substrates - Cotton waste and other substrate blocks
- Modify incubation house to be suitable for season (winter) - Growing house suitable for cold weather
- Inoculation and incubation - Colonized substrate
- Compare appropriate concentration of
extract with control on product yield - Product yield recording
- on primordial formation
- Analyze components of mushroom from supplemented and non-supplemented cultivation
- Analyze C:N ratio in crude extract and cotton waste
- Data of increased components in fruiting bodies from supplemented cultivation
- Benefit of the extracts on increased nutritional composition in straw mushroom fruiting body
- Collect data for economical and
cost-effective analysis - Economic and cost-effective analysis
V. Budget
The budget information will be used for us to know the estimate cost for mushroom production or research in the respective country. MushWorld will provide the selected papers with the respective prize money. You should remember it is not grant. After submission of the 1st progress report, $300 USD will be given to award candidates whose research proposals were selected.
Organism and inoculum 300 baht
Preparation of aqueous oyster mushroom extraction 10,000 baht
Substrate preparation 1,000 baht
Preparation of culture environment 20,000 baht
Nutritional analysis 5,000 baht
Total 31,300 baht
or $782.5 USD
2005 MushWorld Research Paper Award
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http://www.mushworld.com/tech/view.a...2&cata_id=1290