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Author(s): Sharon Khalkho1, Deepali Koreti2, Anjali Kosre3, S.K. Jadhav4, Nagendra Kumar Chandrawanshi5



    1S.o.S. in Biotechnology, Pt. Ravishankar Shukla University, Raipur (C.G.) 492 010, India.
    *Corresponding author

Published In:   Volume - 3,      Issue - 1,     Year - 2021

Cite this article:
Sharon Khalkho, Deepali Koreti, Anjali Kosre, S.K. Jadhav, Nagendra Kumar Chandrawanshi (2021) Review on production technique and nutritional status of Calocybe indica (P&C). NewBioWorld A Journal of Alumni Association of Biotechnology, 3(1):1-7.

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NewBioWorld A Journal of Alumni Association of Biotechnology (2021) 3(1):1-7


Review on production technique and nutritional status of Calocybe indica (P&C)

Sharon Khalkho, Deepali Koreti, Anjali Kosre, S.K. Jadhav, Nagendra Kumar Chandrawanshi*

S.o.S. in Biotechnology, Pt. Ravishankar Shukla University, Raipur (C.G.) 492 010, India.




Article history:



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.










Fungi 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).

There 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 decomposition.


Summary of Cultivation

Wheat straw 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 sequential steps:-

1. 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).

2. Substrate and supplements

Mushroom 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 substrate.

3. Casing

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.

4. Harvesting

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 obtained.

Nutritional and Volatile compounds of C. indica

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 nutraceutical.

Polysaccharides are 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



Body part

Extracted Compounds

Extraction method





Fresh and dry form of mushroom

Crude extract

Methanolic extraction

Non-enzymatic antioxidant

Selvi et al. (2007)


Fruiting body extract


Aqueous extraction

Immuno-enhancing and cytotoxic activity 

Mandal et al. (2011)


Fruiting body of P. florida and

C. indica


Aqueous extraction

Iimmuno-enhancing and antioxidant

Maity et al. (2011)


Cap and stipe

Crude extract

Methanolic extraction


Dandamudi et al. (2011)


Fruiting body

Water soluble glucan

Acid hydrolysis, methylation analysis, and NMR studies


Mandal et al. (2012)


Fruiting body extract

Crude polysaccharide

Solvent extraction

Antioxidant and lipid peroxidation

Govindan et al. (2014)


Methods for quality improvement- Biofortification and supplementation

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.

Biofortification 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 selenite (Na2SeO3).  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.

Fig.1. Methods used in C. indica cultivation and quality improvement through Biofortification and supplementation (Krishnamoorthy and Venkatesh, 2015)

Future prospect and Conclusion

Introducing 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

Authors 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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