NewBioWorld A Journal of Alumni Association of Biotechnology (2021) 3(1):1-7
Review on production
technique and nutritional status of Calocybe
Sharon Khalkho, Deepali Koreti, Anjali Kosre, S.K. Jadhav, Nagendra Kumar
S.o.S. in Biotechnology, Pt.
Ravishankar Shukla University, Raipur (C.G.) 492 010, India.
Received in revised form
Calocybe indica or milky white mushroom, it’s an indigenous edible mushroom originates
from India and apart from India, it is also gaining considerable importance
in other tropical countries like Bangladesh, Malaysia, China and Singapore.
It has an attractive fruiting body, pleasant white color, highly nutritious,
long shelf life, require less time to grow, its cultivation process is easy
and economical. C. indica is rich in metabolites including protein, lipid, carbohydrate, and
dietary fibers. C. indica is a micro-fungus, they accumulate
nutrients as well as minerals from the substrate in which they grow. Because
of these feature, C. indica can be a good candidate for making and use
it as an enriched food source. Enrichment of C. indica is the new way
for mushroom quality improvement. By using a different type of renewable and
cheaper supplementation substrates, we can minimize the overall production
cause and makes a better quality of C. indica for consumption, as well
as nutraceutical development etc. Enriched mushroom cultivation and
production can helpful for sustainable development.
are a diverse group of an organism which play a very important role in the
environment, health and plays a major role in soil weathering, organic
substrate decomposition, and elemental recycling. Mushroom is a macro fungus
with a fruiting body and they can be either hypogenous or epigeous by nature
(Chang and Miles, 2005). Mushrooms are chlorophyll less, saprophytic which
grows on dead and decaying matter. They play a vital role in the environment as
they degrade the substance on which they grow. Mushrooms have a long history of
use not less than 2000 years. Mushroom is highly nutritional food, it has been
used as a food source as well as medicine for a very long time (Wasser, 2002).
Mushrooms are being consumed since ancient times as it has good taste and
flavor (Das et al. 2010; Barman et al. 2015). Mushrooms are
widely used as food and food supplements and as its important food concerning
human health, nutrition and disease prevention. They are rich in protein,
minerals and vitamins and they contain amino acids (Sandler, 2013). Mushrooms
are consumed to fulfill nutritional needs has been one of the major factors in
the history of mankind (Chang, 2006; Krishnamoorthy and Venkatesh, 2015).
are about 1.5 million different species of mushrooms. Mushroom is one of the
cheapest sources of proteins (Barman et al. 2015). Recycling is the need of
modern times and mushroom is an ideal candidate to harness its potentialities.
To fulfil the food requirements of the world mushroom can be a good and
commercially beneficial option. Mushrooms have a varied range of applications
in bioremediation of soil, bioconversion of wastewater, medicine and
agricultural waste disposal etc. (Kumar et al. 2017;
Chakraborty et al. 2019). Lignocellulosic agricultural and forest
residues are converting into a protein-rich mushroom to increase the protein
demand of the world is the most economically viable and sustainable
biotechnology process (Hawksworth, 2002; Krishnamoorthy and Venkatesh, 2015).
C. indica is commonly known as milky white mushroom this mushroom is grown in subtropical
and temperate zones of India and South Asia. C. indica was reported in West Bengal, India by Purkayatha and
Chandra in 1974. The name Calocybe was derived from a Greek word kalos
means ‘pretty’ and cabos meaning ‘head’ (Krishnamoorthy and Muthuswamy, 1997). Calocybe has
become the third most commercially grown mushroom after oyster and button
mushroom (Krishnamoorthy, 2003). It is an indigenous tropical mushroom which
can cultivated during summer and rainy season. According
to Purkayartha and Chandra, (1974) C.
indica is a popular mushroom due to its big size, attractive colour, good
texture, sustainable yield and delicious taste (Joshphine et al. 2014).
Recycling is the need of modern times and mushroom is an ideal candidate to
harness its potentialities (Kanta et al. 2014). The lack of food and
malnutrition in a world of rising food prices, mushroom cultivation is very
good option as it has high nutritive value.
Status of Calocybe
indica in India
C. indica are generally grown
in human rich soil in agricultural fields on tropical and sub-tropical parts of
India. It is commonly called as milky mushroom, was first reported in 1981 by
Purkayastha and Chandra. It is mainly found in West Bengal and also has been
reported in plains of Tamil Nadu and Rajasthan (Doshi et al. 1989;
Krishnamoorthy, 1995). From the last decade it has been commercially cultivated
in southern parts of India, Tamil Nadu, Andhra Pradesh, and Karnataka and
recently been cultivated in North India (Singh et al. 2017). A new variety of
milky mushroom APK 2 was reported by Krishnamoorthy (2003), in the sugarcane
field near Coimbatore. It is a popular mushroom among mushroom grower and
consumers because of its large pleasant milky white sporocarps, long shelf life,
high biological efficiency, good taste, simple cultivation techniques and can
be grown on various agricultural wastes etc.
The agricultural waste constitute mainly of cellulose, hemicellulose and lignin
etc. Lignin fraction which is recalcitrant in nature, but in mushroom it
possesses the specific type of hydrolytic enzyme system with capacity of
utilizing lignin for fruit body production (Bokaria et al. 2014; Barman et al. 2015).
However during spawn run stage, mushroom metabolizes mainly the lignin, the
hemicellulose, cellulose and other sugar derivatives were metabolized on
Summary of Cultivation
and paddy straw are the most common substrate for cultivation of C. indica. Milky
mushroom for the first time was cultivated at North Bengal. It’s a summer
mushroom and it grows on the temperature range of 25-35°C, so summer season is
the best season for the cultivation. The time required for growth of its mycelia
in potato dextrose agar or malt extract agar is generally 8-10 days and the
optimum pH range is between 5.5 and 8.5 (Krishnamoorthy and Venkatesh, 2015) in
mycelium production in liquid broth. Size of mushroom depends upon the
availability of light (Krishnamoorthy and Venkatesh, 2015). Mycelium growth favoured
low intensity of 800 lux or below, but for higher mushroom yield high intensity
1600 lux or more is needed. The crop production process following these
Spawn and spawning
Spawn is the seed or vegetative
mycelium required for the cultivation of mushroom. It is the first stage of
mushroom production. The culture of mycelium depends on several conditions like
growing media, pH, temperature, nutrient elements, humidity, light intensity,
CO2 and oxygen (Calam, 1971). Mycelium grows around distinct grains
and provides improved spawning in grain type spawn. The first industrial method
of spawn production was given by Huhnke et al. (1973), which were based on a fermented substrate in Pleurotus
spp. Zadrazil (1978) developed a “super grain spawn” which serves the
purpose of “active mycelium” as a seed material for cultivation. In this, the
mycelium spread over the mushroom substrate for a few days, which reduced the
cost of spawn considerably. Another factor, such as pH 6.5- 6.7 is the
maintained for the growth of mycelium in the spawn. Wheat grains are the best
substrate for C. indica spawn
production. In the spawn preparation process, wheat grains are boiled in the
water and when grains become soft. Excess water drain out and after cooling,
grains were mixed with CaCO3 keep
the grain loose and to maintain pH (Joshphine et al. 2015 and Krishnamoorthy
and Venkatesh 2015). After cooling of the substrate it is kept in polypropylene
bags and it’s autoclaved at 121°C and 15 lbs psi for 20 minutes and after that
the bags are aseptically inoculated with mushroom mycelium and are incubated at
30°C (Krishnamoorthy et al. 2003). The complete colonization of substrate by
mushroom mycelium can be seen in 15-20 days, which means that it is ready for
culture bed inoculation (Pandey et al. 2000; Krishnamoorthy et al. 2015). Age
of spawn is an important factor which influences the yield of mushroom and
flushing pattern. Pani, (2011) reported the best milky white mushroom was
reported using 21 day old spawn. According to Doshi et al. (1993) three-layered
spawning method gives increased sporophore yield in C. indica.As compared to spawning, which takes 20
days, layer spawning takes less colonization time in a substrate (15 days).
Substrate and supplements
quality depends on a substrate for nutrition which helps in the growth,
development, and fruiting of mushroom etc.
(Chang and Miles, 2004). C. indica was cultivated on wide ranges of
substrates. C. indica was cultivated
for the first time in 1981 on unsterilized paddy straw and wheat bran substrate
(Kumar et al. 2007). Krishnamoorthy and Muthusamy (1998) used different
substrates such as paddy straw, paddy straw compost, maize stalks, palmrosa
grass, sugarcane bagasse, vetiver grass, groundnut haulms, and soybean straw
for the cultivation of C. indica. Among this paddy straw and maize stems
gave higher yield by 94 and 99 % bio-efficiencies respectively. They reported
that the paddy straw compost was not appropriate for cultivation.
Krishnamoorthy and Venkatesh, (2015) reported high colonization rate by using
the substrates like paddy straw, and sorghum stalks for C. indica cultivation.
This study also concludes that the other substrates like straw compost, coconut
coir pith compost and sawdust are not suitable for the growth of C. indica. Rice straw is the most common
lignocellulosic substrate (Mangat et al. 2008 and Joshphine et al. 2014). Paddy
straw is soaked in water for 4-6 hours, and then for 45-60 minutes heating
treatment is done. After this the substrates are kept to shade-dried to get
appropriate moisture is approximately 50-60%, before bed preparation. All
substrates were spawned by 3 to 5% weight and were kept in polypropylene bags.
These bags were then kept in the complete dark in a room at 30°C temperature
(Krishnamoorthy and Venkatesh, 2015). When we reused the substrate from the
spent bed it does not give a good yield. Due to the subsequent utilization of
mushroom mycelium there is a gradual depletion of nutrients during the
cultivation of C. indica on fresh
Beds with full mycelia growth were kept
for spawn run. Casing is an important agronomic practice for the cultivation of
milky mushroom. Casing soil protects the mushroom against pests, disease and
protects compost against desiccation; also provides gaseous exchange for the
development and growth of mushrooms (Colauto et al. 2011). The
vegetative phase changes to reproductive phase in casing which delay the
diffusion of harmful metabolic gases on the mushroom surface. pH of soil must be maintained from neutral to
alkaline in casing, as there is a high accumulation of concentrations of CO2
resulting in yield depression of soil during fructification
(Krishnamoorthy et al. 2015). As compared to chemically treated casing soil
with formalin or using heat sterilization, steam sterilized casing soil gives
better result. Amine et al. (2010) studied the effect of different
substrates and casing material on the growth. They reported that rice straw as
the most suitable substrate for the cultivation and cow dung mixed with casing
material gives better yield of C. indica. According to Krishnamoorthy and Venkatesh, (2015) partially
steamed clay loam soil yield maximum yield as compared to other media such as
peat soil, sand, biogas slurry, farmyard manure and choir pith compost as it
takes 2 days earlier for the production of pinheads.
The light should be provided frequently
for a long time, which resulted in the initiation of the fruiting body in the
form of needle shape (Joshphine et al. 2014) and harvesting of mushroom
can be done in 30-35 days after spawning (Barman et al. 2015). Mushrooms can be
done harvested by holding the cap and twisting a bit near the base (Singh et al.
2017). According to Pandey and Tewari (1993), when the first harvest is
obtained the casing material is mildly rubbed and is sprayed regularly with
water. Within 45-50 days of bed preparation second and third harvests can be
Nutritional and Volatile compounds of C.
C. indica mushroom
products are used to improve the nutritional status and to fulfil the demand
for protein (Suman et al. 2018).
Mushroom converts all the non-valuable substances into high protein food
(Crisan et al. 1978).
Despite all the nutritional qualities, medicinal values and economic benefits
the importance of mushroom is often not acknowledged (Suman et al.
2018). The protein content of this tropical mushroom is 32.3% and the crude fibre
is about 41% (Krishnamoorthy, 2003). It’s mature fruiting body contains 17%
protein on dry weight basis, 4% soluble sugars, 2.9% starch and 7.43% ash etc.
It contains 12 amino acids. It also has minerals like potassium (K), magnesium
(Mg), Sodium (Na), phosphorus (P) and Calcium (Ca) and trace elements such as Copper
(Cu), Iron (Fe), manganese (Mn) and Zinc (Zn) (Zahid et
al. 2010). Milky mushrooms contain good amount of Vitamin C, Vitamin A,
Vitamin E, Glutathione (Selvi et al. 2007; Alam et al. 2008). It is
suitable for people suffering with high hyperacidity and constipation.
Krishnamurthy (2003) reported that C. indica has higher protein than
button and oyster mushrooms. C. indica have a dry weight of 14.4% and
61.1% fibres content. The beta-glycans present in dietary fibres of mushrooms
has a stimulatory effect on the immune system with anti-mutagenic, anti-cancer
and anti-tumor activities (Crisan and Sands, 1978). Mushroom produces a variety
of different volatile compounds like alcohols, aldehydes, ketones and oxides
these are reported in milky mushroom. The major
compound in fresh milky mushrooms was 1-octen-3-ol, followed by n-octanol,
3-octanone, 2-octen-1-ol, pentanal, t-linalool oxide and 1-hexanol. For
getting volatile substance from mushroom, both dried and fresh mushrooms are
cut into smaller pieces and homogenized in distilled water for the homogenate
was subjected to simultaneous distillation and solvent extraction under reduced
pressure. The volatiles was collected at 15°C, dried over anhydrous Na2SO4 and concentrated. The drying of C. indica increases the concentration of n-hexanal, 2, 4-decadienol
and 2, 4-nonadienol. Drying of mushroom also increases the percentage of
aldehyde at all expense of alcohol and constitute substance at a higher
oxidation level (Chandravadana et al. 2005).
Bioactive compound and its application
Compounds that are gives biological
activity called bioactive compounds. Major bioactive
compounds in fungal groups are known as myco-chemicals, can be found in the
form of their cell wall components (polysaccharides and proteins) or as
secondary metabolites such as; phenolic compounds, terpenes and steroids, etc.
(Wasser and Weis, 2010; Patel and Goyal,
2012). These bioactive compounds are naturally found in the mycelium as well as
fruiting bodies of mushroom. Furthermore, its efficacy are depend on the type
of mushroom, the substrate for growth, substrate composition, growth
conditions, developmental stage, culture or postharvest conditions, storage and
cooking procedures etc. (Enshasy and
Hatti-Kaul, 2013). Bioactive compounds provide
medicinal or health benefits like prevention and treatment of human disease (Rathee
et al. 2012). These bioactive compounds can act as
immunomodulatory, anticarcinogenic, antiviral, antioxidant and
anti-inflammatory agents (Kwon et
al. 2009).Various mushroom species are studied for the prevention
and treatment of many diseases like hypercholesterolemia, cancers, viral
diseases and hypertension (Breene, 1990). A variety of compounds have been
isolated from various species of Pleurotus, that
have been shown to possess medicinal properties such as anti-inflammatory (Bobek,
2001), antihyperglycemic (Hu, 2006), antimicrobial (Brandt et al. 2000), antiviral (Wang et al. 2007), antitumour (Gu, 2006; Huang et al. 2013; Zhang et al.
2016), immunomodulatory (Liu et al. 2010,
2019) and antioxidant (Wong et al. 2009).
According to Rathore et al. (2018) C. indica is rich in antioxidant
properties, thus its making, one of the best choices of the development of
polymeric carbohydrates that are widely distributed in animals, plants and
microorganisms etc. Various
studies reported that mushroom polysaccharides used as potential
pharmacological agents with different bioactivities (Cheng et al. 2018; Barbosa et al. 2019; Bai et al. 2019; Rathore et al.
2019). There are some compounds showed in table 1, reported in C. indica associated
with their bioactivity. Mandal et al. (2010) isolated two new polysaccharides
from the alkaline extract of fruit bodies of C. indica and identified it
as glucans; water-soluble α, β -glucan and water-insoluble β-glucan (Calocyban).
Similar another experiment was performed by Mandal et al. (2011) for the Calocybe
indica polysaccharide identification and characterization along with its
biological activity (immune enhancing, cytotoxic activity). In this experiment,
the extracted polysaccharide identified as heteroglycan, which consist
D-glucose, D-galactose and L-fucrose (3:1:1) etc. Water-soluble polysaccharide was also isolated by Maity et al. (2011) from aqueous extract of the fruiting
body of P. florida and C. indica somatic hybrid. This polysaccharide contains galactose, fucrose and
glucose (2:1:2) and methylation an analysis, and NMR experiment showed
that it has antioxidative properties. Mandal et al. (2012) isolated glucan from
C. indica by using a hot water extraction method and observed that the
100 mg/mL of the polysaccharide can showed an efficient immune stimulatory
activity (proliferator splenocyte and thymocyte). Govindan et al. (2014)
reported that the crude polysaccharide of C. indica, significantly
helpful for the improvement of antioxidant capacity and lipid peroxidation
products in animal models.
Table no. 1. Bioactive compounds in C. indica and their application
and dry form of mushroom
et al. (2007)
and cytotoxic activity
et al. (2011)
Fruiting body of P.
Iimmuno-enhancing and antioxidant
Maity et al. (2011)
et al. (2011)
Water soluble glucan
Acid hydrolysis, methylation analysis, and NMR studies
Mandal et al. (2012)
and lipid peroxidation
et al. (2014)
Methods for quality improvement- Biofortification and
C. indica is a micro-fungus, they accumulate
nutrients as well as minerals from the substrate in which they grow. Because of
these feature, C. indica can be a good candidate for making and use it
as an enriched food source. Enrichment of C. indica is the new way for
mushroom quality improvement. Recently research also focused on sustainable
development and utilization in mushroom cultivation and production. Through
using a different type of renewable and cheaper supplementation substrates, we
can minimize the overall production cause and makes a better quality of C.
indica for consumption as well as nutraceutical development etc. There are
some researchers reported for optimizing with different supplements for
enriched or biofortified C. indica. There are various strategies use for
the enrichment of food sources such as supplementation and biofortification.
Figure no. 1 depicted the process of supplementation and strain modification.
Supplementation improves the overall nutrients composition of the mushroom and
the other hand biofortification performed for particular nutrients or minerals
or vitamins. Some organic supplementation techniques reported for
methanolic extraction cultivation and nutritional quality improvement. Sharma et
al. (2013) used organic supplements like rice bran, wheat bran, gram legume powder and
observed that 83.5% biological efficiency, which is higher as compared to
control (57.5%). Another supplementation experiment was reported by Jadhav et
al. (2013), they used casing material with bio-fertilizer including Azotobacter
and B. megaterium with Pseudomonas striata and their
different combinations. Rathor et al. (2018) performed a cultivation experiment
in methanoic extraction by supplementation of nitrogenous tree leaves. This
experiment reported that the basal substrate (wheat straw) supplemented with
25% Bauhinia variegate leave significantly improves the biological
efficiency (83%), nutraceutical compounds as well as minerals contents
of methanoic extraction etc. Kumar et al. (2020) evaluated the effect of sugar mill effluent (treated) as
moistener of the basal substrate (rice straw) for C. indica cultivation.
They observed that this supplementation was positively enhancing the overall
nutrient content and also the production in which highest bio-efficiency
recorded 127.2 g/kg with 75.3 % nutrient uptake rates.
experiment reported by Rathore et al. (2018), they cultivated C. indica on
wheat straw enriched with various concentrations of Se in the form of sodium
They found that the Na2SeO3 increases the amount
of Se the fruiting bodies, although the biomass yield inhibited above 5 mg/ml.
They also estimated that the organic forms of Se in the protein, polysaccharide
and nucleic acid extracted from fruiting bodies. The fruiting bodies were harvested
from 10 mg/ml Se substrate, have maximum protein content (25.31 g/100 g) and
amino acid found in the following concentration; glutamic acid, glycine,
aspartic acid. The Se enrichment also enhanced the antioxidant capacity of
methanolic extracts of fruiting bodies. Rathore et al. (2020) studied Vitamin
D2 fortified C. indica by natural (sunlight) and artificial (UVB light)
method and they analyzed the effect on the nutritional value in treated
mushroom. This experiment suggested
that, the maximum vitamin D2 content was found that is 78.33 μg/g in sunlight
treatment and 140.58 μg/g in UVB treatment. Also absorbed that the increased
β-glucan (22.42–44.36 g/100 g), phenols (12.46–47.38 mg GAE/g), flavonoids
(0.85–2.15 mg Quercetin/g), and antioxidant activity etc.
Methods used in C. indica cultivation and quality improvement through
Biofortification and supplementation (Krishnamoorthy and
Future prospect and Conclusion
biofortification in the mushroom industry will improve its stain quality and by
introducing minerals in the mycelium culture of C. indica, nutrient efficiency can be increased. Genetic
engineering is the field that can be used to get different stain varieties of C. indica. In addition, different substrate can be used to cultivated milky
mushroom to increase the optimization of the mushroom production. C. indica is an indigenous mushroom and
it is recently being cultivated commercially in India. After button and oyster
mushroom, C. indica is the third most
commercially grown mushroom in India. C. indica can be used as a food
supplement, because it contains various nutritive compounds and has higher
anti-oxidation properties. Besides, Indian climate conditions are suitable of
milky mushroom as they can grow on extreme temperature a condition ranging from
25-30°. C. indica is emerging as one
of the popular commercially produced mushrooms as it has a longer shelf life,
good productivity, attractive color, pleasant taste, and easy availability of
substrates of which agricultural waste as are the most promising for the
cultivation and promote sustainable development.
Conflict of interest
had no conflict of interest.
The authors are thankful to the Junior
Research Fellowship (DBT/JRF/BET18/I/2018/AL/123), Regional Centre for
Biotechnology, NCR Biotech Science Cluster 3rd Milestone, Faridabad-Gurugram
Expressway, Faridabad – 120001, for providing funding support. The authors are
also are thankful to the Head, School of Studies in Biotechnology, Pt.
Ravishankar Shukla University, Raipur for providing laboratory facilities and
to conduct the study.
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