Scientific Research and Essays Vol. 5(24), pp.

3945-3949, 18 December, 2010

Available online at http://www.academicjournals.org/SRE
ISSN 1992-2248 2010 Academic Journals

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. Damin Huato2, A. Garca
Espinosa2, J. F. C. Parraguirre-Lezama1, I. Hernndez Trevio1, A. Macias Lpez3 and
J. Jurez Huerta1
1

Departament of Agro-forestry Engineering, Benemerita Universidad Autonoma de Puebla, Mexico.

Departament of Agroecology and Enviroment, Benemerita Universidad Autonoma de Puebla, Mexico.
3
Postgraduate 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 agroproducts 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
about 15 species, of which the most representative are
the mushroom (Agaricus spp.), shithake (Lentinula
edodes), oyster fungus (Pleurotus spp.) and enokitake
(Flammulina velutipes) (Chang and Miles, 1989). In Latin
America, the commercial production of edible fungi
(mushrooms especially) increased by 32% in 2001, this
caused an economic spill by over 167 million dollars a
year and 34000 creating jobs in this activity. The demand
for domestic and export consumption of edible fungi in
the period 1995 to 2001 increased by 32% from 49,975 to
65,951 tons per year (Martinez-Carrera, 2002).
The countries that increased their production in the

*Corresponding author. E-mail: biol.ora@hotmail.com. Tel/Fax:

+52 7979730667.

period 1995 to 2001 were Mexico (58.6%), Chile (17.6%)

and Brazil (10.6%), constituting 86.8% of the total
production of edible mushrooms in Latin America
(Martinez -Carrera, 2002). In this manner, in Mexico the
production of edible fungi have evolved to such a degree
that is the largest producer in Latin America, occupying
the twenty-eighth place in the world, where per capita
consumption had grown slowly in recent years. Unlike the
agricultural, ranching, and forestry activities have for
centuries practiced in Mexico, biotechnology production
of edible mushrooms had only 66 years of steady
development and increasing (Martinez-Carrera et al.,
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
regions of the country.
The oyster fungus (Pleurotus spp.) shows a facility for

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cultivation around the world for its many features

(Medicinal, environmental biological remediation, enzyme
production, etc.). In addition to easy handling for
cultivation in agro-industrial waste, and quickly
decomposition of organic matter by encouraging agroecosystem, pushing small farmers because of low
production costs, employment generation and provide
additional value to their products through preserves or
jam oyster fungus (Melo et al., 2010). The mushroom
crop residues could be used as mushroom compost,
incorporated into the soil as a conditioner and or as
improver of physical properties, or be part of mixtures
made in the production of substrates for nurseries
(Varnero et al., 2010).
The cultivation of edible fungi is a process that frees up
the land resource, and you get large volumes of
production in relatively small spaces. In the early phases
of mycology, the comments played an important role in
the method of cultivation of edible fungi (Martinez-Carrera
et al., 1995). The generation of technologies is a major
aspect that contributes to solving problems of different
nature, these technologies are generated by research
centers and to demonstrate their viability must be
transferred to industry receiving them (Almanza, 1994;
Gaiska, 1997). The use of technology to the cultivation of
oyster edible fungus allows for high production in a
relatively short spaces, is widely accepted in urban and
rural areas for its nutritional properties, the fungus
mushroom is a food with 350 calories compared to red
meat only contains 150 calories or fish that contains 101
(Romero et al., 2010).
Technology transfer being proposed in this research is
the production of mushroom fungus inoculum of rural
production, as it is driving and innovating production
technology of inoculum (seed) at low costs in the rural
sector through the manufacture of a laminar flow hood
rustic, which has an area hygienically clean, sterile air
locks which can manipulate fungus mushroom mycelium
in their normal environment in which it must compete with
a host of other organisms to survive. This will allow the
optimization of its resources and making the process
more beneficial for small producers of edible fungi and is
being developed in the research center Phok Nah seed in
the LAOS (PDR) (Ogden and Prowse, 2005). Based on
the above, provides that the role of biotechnology for the
production of edible fungi inoculum, especially the
mushroom fungus (P. ostreatus) will be a pioneer in its
area to boost rural development and essential for
strengthening the role of sustainability agricultural by
using products and recycling of agro-food for a socially
acceptable, high medicinal value, protein, and business
and in addition generate significant employment
opportunities in rural communities.
The aim of this study was to demonstrate the
production of inoculum kindnesses, using a laminar flow
hood rustic to be transferred to rural communities in the
municipality of Tetela de Ocampo, Puebla-Mxico.

MATERIALS AND METHODS

The experimental work was conducted at the Department of
Agroforestry Engineering, Academic Unity Regional of Tetela,
Benemerita Universidad Autonoma de Puebla (BUAP), specifically
in research pilot plant production of edible mushroom in University
Avenue, Barrio Benito Juarez, Tetela de Ocampo, Puebla, Mexico
(Figure 1).
Substrates and Cepas
The strain (CP-50) of P. ostreatus (Jacq.ex Fr.) Kumm used in the
study comes from the genetic resources center edible fungi
(CREGENHC) postgraduate College, and is deposited in the strain
bank of Edible Fungi Campus Puebla, Mexico. The strain is
maintained in a medium composed of potato agar and dextrose
(PDA) Bioxon mark, at room temperature (Sobal et al., 2007).
The substrates used in the preparation of inoculum (seed) were
wheat (Triticum aestivum L.) and maize (Zea mays L.) acquired in
Tetela de Ocampo, Puebla-Mxico. The inocolum preparation was
performed in the pilot plant research in production of edible
mushrooms in the community of Benito Juarez, in the municipality
of Tetela de Ocampo, located in the Sierra Norte of Puebla State,
its geographical boundaries are: 19 43 '00 "and 19 57' 06" north
latitude and 97 38 '42"and 9754'06" west longitude. Their
adjacencies are the north by the municipalities of Tepetzintla and
Cuautempan, South with Ixtacamaxtitln, to the west with Zautla
and Xochiapulco, and the East with Aquixtla, Zacatln and
Ixtacamaxtitln (Encyclopedia of the municipalities of Puebla,
2006).
DEVELOPMENT
The inoculum or seed term usually refers to fungal mycelium used
to inoculate the final substrate where the fungus will bear fruit, that
is, the material used to "seed" when growing edible mushrooms.
However, there are two types of seeds: The mother and the
secondary seed or seed for planting. The seed mother comes
directly from fungal mycelium grown on a nutrient medium; this
means that for a substrate, preparation is inoculated with a piece of
agar. To produce the secondary seed, however, the substrate is
inoculated with seed mother in a state of active growth. The
substrate used for either the mother seed or grain or seed are
secondary forest residues such as sawdust. Often, the first in jars or
bottles and plastic bags second thermo-resistant (Chemo, 2002).
To carry out the process of inoculum preparation, vertical laminar
flow hood is required, as it protects the sample from contamination.
During operation, the air is drawn through a pre-filter located at the
top of the cabinet to trap large particles, then air is blown through a
HEPA filter (Hight Efficiency Particulate Arrestance) with efficiency
of 99.99% and is projected vertically to the work area, the air
filtering through the filter HEPA prevents contaminants from
entering particles, providing a work environment free of particles
that reduces cross-contamination, which is why the manufacture
was proposed in a vertical rustic laminar flow hood, to strengthen
the rural production of mushroom fungus in the region of the Sierra
Norte of Puebla State, with the aim of transferring this technology at
affordable costs to producers and agro build successful businesses
in the production of edible fungi.
The rustic laminar flow hood was manufactured under test with
materials of the region, in this case, use of wood and iron 531, 6
mm, anti rust paint finish white (Figure 2), only acquired the HEPA
filter with an authorized dealer, a turbine built in the upper middle
part driven by a hp electric motor at 3000 rpm, that extracts air
from the room by pre filter at the top of the cabinet and then through
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
121C 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 25C 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)

Figure 2. Laminar flow hood rustic.

0.3 microns or large one, so that the air without particles is

projected vertically through the work area. The air is captured on

The mother seed is used to inoculate the secondary ones, which in

turn will be used as inoculum of the substrate that will produce
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
consisted of boiling 300 g of wheat and 200 g of corn for 20 min in 5
L of water, was allowed to settle for 30 min. This mixture was

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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

part is critical, its proper implementation prevents seeds too dry or
too wet will affect mycelia growth. If the grains are very dry, mycelia
growth is very slow. If the grains have a lot of water, growths is also
very slow and eventually stop when water accumulates on the
bottom. The optimum moisture for grains is about 50%. Moisture
was determined using a digital hygrometer, then were added 5 g of
lime and 20 g of gypsum with wheat and maize to reach an
alkaline pH of about 9 (Romero, 2007; Stanley, 2010).
Then placed 500 g from this mixture in jars with a capacity of
1000 ml and sterilized for 60 min at 121C. 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
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.
Preparation of secondary seed
The grains of wheat and corn can also be used to produce seed for
planting (secondary), as was done in this experiment this is called
seed to seed inoculation. Mycelia growth showed that the strain in
the substrate is 25% faster than that of the mother seed, because
the mycelium was adapted to the substrate in a short period of time
(7 days). It also had a higher amount of seed for sowing in the
substrates. The preparation consisted of inoculating the substrate
which was used to produce seed for planting, seed mother were
used for vigorous growth, inoculation was done again in the rustic,
laminar flow hood (Figure 3), and in the same way described for the
planting of grain mothers grain. The jars were shaken and a small
amount of mother seed was deposited into the bags previously
prepared as described above with the objective to obtain the
secondary seed (Stanley, 2010). In general, 1 kg of mother seed
will be enough to obtain 30 kg of secondary seed. During incubation
proceeded in the manner indicated for the mother seed.

Control of seed quality

Quality control in seed production was the constant inspection to
eliminate all those units that visibly contaminated or unacceptable
differences in appearance or growth. Here it is essential that seed
producers have a proper installation rustic where they can evaluate
the characteristics of seed production, besides being in contact with
researchers from the Mycology Laboratory of the School of
Engineering Agro forestry - BUAP, so the inconveniences of seed
production can be corrected quickly.

RESULTS AND DISCUSSION

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
present the appropriate characteristics (color, odor,
consistency and safety) for the production of the
mushroom fungus.
The proposed use of rustic laminar flow hood, in
addition to complying with the adequate production of
inoculum for the production of mushroom fungus, aim to
reduce production costs, since the cost of manufacturing
the same accounts for 40% of the cost of a laminar flow
hood similar to any trademark.
This investment for the development of a laminar rustic
flow hood is $14,000.00 (Mexican pesos), which
compared to the cost of a similar mark laminar flow hood
laboratories is $35,000.00 (Mexican pesos). This
difference in the initial investment is significant and
represents a considerable savings for producers of

Romero-Arenas et al.

mushrooms, which will help to encourage this activity in

rural communities in the municipality of Tetela de
Ocampo.
In general, the laminar flow hoods have a lifespan of 2
to 3 years depending on the use to which they are
subjected to which is determined by the HEPA filter life,
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.
The proposed use of a rustic laminar flow hood is novel
because it represents the use of a locally developed
technology with available materials in any small town and
can be very useful to make the process more profitable.
Mushroom
fungus
production
encourages
the
development of productive projects in rural areas of the
country.
As explained earlier in this research, the cultivation of
mushroom fungus implies the need for seed production
programs through the use of a rustic laminar flow hood as
a strategy for producers in the region o develop strains
adapted to local environments and substrates. In such a
situation, future research will need to focus on the
development of basic knowledge about the cultivation of
edible fungi, the appropriate characterization and
taxonomic classification of wild strains, as well as on
procedures for the conservation and replication of
specimens of a commercial nature on techniques to
preserve genotypes, crosses for the highly productive
hybrid formation and especially the production of
inoculum using agro-industrial waste in the region.
Conclusions
Based on the results of the work concludes:
1. The laminar rustic flow hood is an option for seed
production of mushroom fungus P. ostreatus at low costs
and its implementation is accessible to any producer.
2. The quality of the inoculum or seed produced from this
technological innovation, is similar to that which can be
obtained using commercial technology, but with lower
production costs.
3. The diffusion of such technologies to rural areas is a
proposal to be implemented in similar areas, in order to
encourage this activity on behalf of producers.

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ACKNOWLEDGEMENTS
The authors thank the Vicerrectoria de Investigacin y
Estudios de Posgrado (VIEP) of the Benemerita
Universidad Autonoma de Puebla (BUAP) for the
financial support of this research project.
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