Waste Biomass Valor (2012) 3:175–189 DOI 10.1007/s12649-012-9107-9 SHORT COMMUNICATION Potential Applications of Mahua (Madhuca indica) Biomass Aditi Gupta • Rohit Chaudhary • Satyawati Sharma Received: 20 October 2011 / Accepted: 21 January 2012 / Published online: 2 March 2012 Ó Springer Science+Business Media B.V. 2012 Abstract The genus Madhuca, belonging to the family of Sapotaceae, is a multipurpose tree with its species, Madhuca latifolia, M. longifolia and M. butyracea being the most prevalent. The tree is economically important because of widespread uses of almost its every part. The present paper reviews information about ecology, distribution, species of mahua, different parts and their uses focusing on flowers, oil and seed cake. Flowers are used for the treatment of skin diseases, preparation of intoxicating liquor and various nutraceuticals. Production of biodiesel from mahua seed oil is recently gaining considerable importance. After the extraction of oil, approximately 60% is left as de-oiled seed cake. The paper discusses at length the composition of cake and its various applications. The cake is a rich source of sugars, nitrogen and proteins. But, the presence of toxic saponins in it limits its extensive usage. The cake has been used as a low grade fertilizer, bio-pesticide, included in animal feed (up to 20%) and in dye removal from waste waters. Various detoxification methods have also been tried for the use of cake as an improved animal feed only. Not many applications of the raw and detoxified cake have been explored. The paper also deals with the experiments performed to evaluate the use of raw as well as detoxified cake (produced as a result of a simple water treatment) for biogas production and mushroom cultivation. Significant enhancement in the biogas (93%) as well as the mushroom yield (128%) could be obtained. A. Gupta
S. Sharma (&) Centre for Rural Development and Technology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India e-mail: satyawatis@hotmail.com R. Chaudhary Department of Chemical Engineering, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India Keywords Mahua
Seed cake
Detoxified Introduction Madhuca indica is one of the most important Indian forest trees belonging to the family of Sapotaceae. It is found in abundance in the forests of Asian and Australian continents [1–4]. Adapted to arid environments, it is a prominent tree in tropical mixed deciduous forests [1, 5] of West Bengal, Bihar, Orissa, Madhya Pradesh, Punjab and Uttar Pradesh [6, 7] and sub mountainous region of the Himalayas [8] in India. It is much planted in the plains of Northern India and Deccan peninsula [9] and propagates mostly by seeds [10]. This tree species has been domesticated by tribal people in India and Pakistan [11, 12] for its wide variety of uses from its flower, leaves, bark, seed oil, seed cake, etc. since the ancient times [13–15]. Its various parts have been used as food, fuel, fertilizer, cattle feed, in preparation of alcoholic beverage, oil and its derivatives such as soaps, detergents, butter, etc. The tree has a very rich medicinal application in the treatment of rheumatism, ulcers, bronchitis, dressing wounds and heart diseases [11]. Many other uses are continuously being explored. Biological and Chemical Aspects Taxonomy and Ecology Mahua is a slow-growing species, found commonly in deciduous forest of Indian sub-continent [6], attaining a mean height of 0.9–1.2 m at the end of the fourth year but may attain a height of 20 m on full growth [16, 17]. It matures from 8 to 15 years, fruits up to 60 years and 123 176 Waste Biomass Valor (2012) 3:175–189 Table 1 Species of Madhuca in India [11] Species Distribution Distinguishing characteristics M. latifolia Uttar Pradesh, Madhya Pradesh and Hyderabad Leaves elliptic or oblong elliptic, shortly acuminate, calyx rusty tomentose, anthers 20–30, 3-seriate subsessile. M. longifolia South India Leaves lanceolate, narrowed at both ends, glabrous distinctly nerved, anthers 16, 2-seriate subsessile, lips 3-toothed, young fruit globose densely hirsute. M. butyracea (Local name: Phulwara) Sub-Himalayan tracks with maximum abundance in kumaon and Gharwal regions Leaves obovate or obovate-oblong, stamens 30–40, filaments glabrous as long as anthers M. neriifolia/ M.malabarica Bombay, South Karnara, Madras and Mysore Branchlets glabrous, leaves lanceolate or oblong obtuse or scarcely acute, glabrescent, distinctly nerved, stamens 16, in two series, subsessile, connective, excurrent lanceolate-linear, young fruit oblong-lanceolate, glabrous M. bourdillonii Mysore and Western Ghats Branchlets glabrous, leaves very narrowly oblong, coriaceous, nerves obscure anthers 16, 2-seriate, subsessile, connective, excurrent, lanceolate-linear, young fruit oblonglanceolate, glabrous possesses evergreen or semi-evergreen foliage [1, 5]. It is known by its various names as mahua and mohwa in North India, illipi in South, etc. [11]. and be scientifically classified as follows: Kingdom: Plantae—Plant Subkingdom: Tracheobionta—Vascular Plants Superdivision: Spermatophyta—Seed plants Division: Magnoliaphyta—Flowering Plants Class: Magnoliopsida—Dicotyledons Subclass: Dilleniidae Order: Ericales Family: Sapotaceae—Spodilla family It grows well on a wide variety of soils especially on alluvial soil in Indo-gangetic plains. For its better growth and productivity, a deep loamy or sandy-loam soil with good drainage is best suitable. It can also occur on shallow bouldery, clayey and calcareous soils [18]. It grows up to an average altitude of 1,200 m whereas in Himalayan regions it can be found up to 4,500 m. A mean annual temperature of 2–46°C and mean annual rainfall of 550–1,500 mm is suitable for its growth. The mean relative humidity in its natural habitat varies from 40 to 80% in January and from 60 to 90% in July [18]. Species and Distribution The genus Madhuca belongs to the family of Sapotaceae, which includes more than 800 tree species many of which are used for the production of latex (e.g. Guttapercha) [1]. Royan [19] listed around 84 species of mahua out of which Madhuca latifolia, M. longifolia, M. butyracea, M. neriifolia and M. bourdillonii are commonly found in India. Few species have also been isolated from Malaysia, [3, 4] 123 Pakistan [12], Sri-Lanka [20], Australia [2] and New Guinea [1]. Recently four new species i.e. M. chai-ananii, M. chiangmaiensis, M. klackenbergii and M. smitinandii have also been isolated from Thailand [5]. According to a survey carried out by the Indian Central Seeds Committee, there are about 8.5 million Madhuca trees in India [21]. The distribution of five common species in India has been summarized in Table 1. These five species can also be differentiated by their leaf structure which has also been listed in the same table. Different Parts and Their Uses Mahua is an economically important tree because of widespread uses of almost it’s every part. The tree has a large spreading superficial root system that holds the soil together and prevents soil erosion. Vesicular–arbuscular mycorrhizal associations and root colonization have been observed in mahua which helps in nitrogen fixation. Wood is hard and reddish brown in colour, very heavy (929 kg/ cu. m) and takes a fine finish. It is used for house construction, naves and felloes of cartwheels, door and window frames. Leaves are generally oblong-shaped, thick and firm, exuding a milky sap when broken. Young leaves are pinkish and woolly underneath. Flowers are cream colored, fleshy and clustered at the end of branches. Fruits are ovoid, fleshy greenish 3–5 cm long containing 1–4 shiny elliptical brown seeds. Most of the leaves fall from February to April and at the same time, flower appears. A couple of months after flowering period, the fruit opens. The fruit is an egg-shaped king of berry. Mature seeds can be obtained during June to July. The different parts and their various applications have been summarized in Table 2. Waste Biomass Valor (2012) 3:175–189 177 Table 2 Uses of different parts of mahua Part Use(s) References(s) Roots Root paste used to expel intestinal worms [22] Bark Treatment of rheumatism, ulcers, itches, bleeding and spongy gums, tonsilitis, leprosy and diabetes [11, 23, 24] Leaves Flower Fruit Seed Seed oil Dyeing and tanning [11] Insect repellant Building structures and furniture [25] [11] Fuel wood and timber [26] As a poultice to relieve eczema [24] Dressing for wounds and burns (Leaf ash ? Ghee (clarified butter)) [24] Fodder for cattle, Green mannure [27] As coolant, aphrodisiac, galactagogue, expectorent and carminative [6, 11] Beneficial in heart diseases, burning sensation, biliousness and ear complaints [6, 11] Making sugar syrup (after decolorizing it with 3.5–5% Activated Charcoal) [6] Preparation of bakery and confectionary goods (candy, biscuits, jam, jelly and cake) Fomentation in cases of orchitis [28] [11] Flowers fried in ghee (clarified butter) are eaten by people suffering from piles [23] Preparation of liquor [2, 3, 6, 15, 26, 29–36] Ethanol fermentation [27, 31] Antibacterial activity against Escherichia coli [37] Effective against rice pest diseases [4] Treatment of chest problem such as bronchitis [24] Used in making vinegar [24] Spent flowers (after fermentation) are also used as animal feed [3, 6] New anti-bacterial mahua Flower agar medium [38] Production of organic mannure by addition to the waste organic matter [39] Increases the production of breast milk [24, 40] Employed as a lotion in chronic ulcer, in acute and chronic tonsillitis and pharyngitis [41] Consumption and curing blood diseases [11, 42] As galactagogue [11] Seed husk contains active carbon with good bleaching [11] Seed shells for removal of Cr(VI) [43] Insect repellant [25] Nutritional tonic and stimulant [44] Curing bronchitis, snake bites, fish poison and scorpian sting [44, 45] Beneficial in skin diseases, rheumatism, bilious fevers and burning sensation Good emollient [11, 23, 24, 46] [11] Biodiesel production [16, 17, 47, 48] Seeds fat used as galactagogue [16, 17] Manufacture of laundry soaps and lubricants [16, 17, 49] Used in cooking, adulteration of ghee, and manufacturing chocolates [16, 17] Controlling pulse beetle Callosobruchus maculatus c [50] Used as bakery fat due to absence of trans fatty acid [23, 46, 51] Fat used in making margarine [52] Making cocoa butter substitute [9, 53] Poultry rations [49] Used in cosmetic and pharmaceutical industries [3, 49] 123 178 Waste Biomass Valor (2012) 3:175–189 Table 2 continued Part Use(s) References(s) Seed cake Hair wash along with Shikakai (Acacia concinna) [9, 25, 54] Fungicide [49, 55] Insecticidal and pesticidal property [9, 17, 25] Dye removal from waste waters [56] Nematicide [57] Livestock feed and manure (rich in nitrogen) [3, 48, 58] Mahua flowers, seed oil and seed cake due to their manifold uses have been discussed in detail Mahua Flowers Table 3 Composition of mahua flowers (?; trace amounts) Constituents The annual production of mahua flowers in India during 1997–1998 was about 45,000 metric tons which has remained more or less constant during 2003–2004 and 2004–2005 [31]. Flowering of an average size mahua tree occurs during March–April each year [6, 59, 60]. The flowers are fleshy, off white in color, and emit attractive sweet fragrance when the plant is in full bloom [61]. Collection of mahua flower is an important source of employment for the poor in India [6]. Storage of flowers for its various uses is a difficult task as being hygroscopic in nature, they tend to absorb atmospheric moisture and get spoiled [18]. Table 3 lists the composition of mahua flowers as reported by various authors for different species. Being a rich source of sugars, proteins, vitamins and minerals, mahua flowers are eaten raw or when cooked [6]. Massive use of flower is implemented in making distillated liquor [3, 11, 31, 62]. The freshly prepared liquor has a strong, smoky, fetid odour, which disappears on ageing. It is reported to excite gastric irritation and produce other unpleasant effects whereas the redistilled one can be as good in quality as Irish liquor [23]. Mahua flower being very rich in fermentable sugars (28.1–36.3 g per 100 g/ 66–72% of dry weight) are considered as the most important raw material, next to cane molasses, in production of alcohol [11, 31]. About 90 per cent of the theoretical amount of ethyl alcohol can be obtained by the fermentation of Madhuca flowers using endogenous natural yeast strain. The fermentation is facilitated by the addition of ammonium phosphate and dilute sulphuric acid [30]. One ton of dried mahua flowers are reported to produce 90 gallons of 95% ethyl alcohol (rectified spirit). Mahua flowers have also been tried for acetone fermentation. Flowers as well as spent flowers generated after fermentation also find application as a feed for livestock without affecting their yield and quality of milk [3, 11]. It has been observed that the flesh of animals, particularly pigs, fed on mahua flowers acquire a delicate flavor [9]. About 80% of juice extracted from flowers is concentrated to produce honey like liquid sweetener. This sweetener can be used 123 [65] [30] [18] 54.06 Total sugars (%) 72.9 60.7 (Sugars) Arabinose Pentoses Rhamnose Dextrose Fructose Levulose Glucose Maltose Sucrose Sucrose Fat (%) 0.6 Fibre (%) 1.7 Moisture (%) 18.6 19.8 Ash (%) 2.7 4.36 Starch (%) Vitamins (per 100 g) 3.6 3.6 Vitamin A 39 IU Thiamine 32 lg Nicotinic acid 5.2 mg Riboflavin 878 mg Ascorbic acid 7.0 mg Pentothenic acid ? Folic acid ? 0.5 Minerals Phosphorous 0.09 1.9 11.61 0.028 mg 0.87 g Niacin Biotin [66] 4.8 mg ? 1.140 mg/ 100 g 2 mg/l Calcium 0.140 mg 8 ppm Iron 15.0 lg Magnesium ? Copper ? Enzymes Catalase Catalase Maltase Emulsin Oxidase Invertase Oxidase further as syrup suitable for making good quality jams, candies, jelly, sauce, confectionery goods and as a substitute for brown sugar [6, 11]. The flowers can also be Waste Biomass Valor (2012) 3:175–189 used in making vinegar and other products which are conventionally made from cane sugar [11]. Flowers possess several medicinal value as well- coolant, aphrodisiac, galactagogue, expectorant, demulcent, anthelmintic, carminative and laxative properties [7, 9, 11]. They are also reported to be beneficial in heart diseases, burning sensation, biliousness and ear complaints [11]. Distilled spirit from flower is a powerful diffusible stimulant and astringent [25] and considered to be a tonic and salutary in diarrhea and colitis. The flowers promote the removal of catarrhal matter and phlegm from the bronchial tubes and therefore used in cough, cold and bronchitis. In skin diseases, the juice of flower is rubbed for oleation as they also exercise a soothing effect on the skin and mucous membranes [63]. The decoction of flower is a valuable remedy for pitta disease. Flower juice is also beneficial as a nasya (nasal drops) in diseases of the head due to pitta like sinusitis [64]. Fresh juice of flower is used in great benefits to arrest bleeding. The flowers have also been reported to play an important role in augmenting the breast milk in lactating mothers and in boosting the quantity of seminal fluids [64]. Some antibacterial properties have also been reported [9]. Flower powder in mixture with ghee and honey has been found to be beneficial for people suffering from piles [11]. Dried flower are used as a fomentation in cases of orchitis for their sedative effect [11]. The honey from flower is edible and is reported to be used for eye diseases [9]. Mahua Oil Seed Madhuca flowers produce green fleshy fruits containing three to four seeds which are ellipsoidal in shape. Unlike many other tropical fruit seeds, the seeds of Madhuca show a good commercial potential as a source of vegetable fat [20]. The potential availability of mahua seeds is 4.9–5.1 lakhs tones [52] which are being collected in organized sectors and utilized for oil [3, 49]. Harvesting period of mahua seeds is at the time of peak rainfall (July–August) [67]. Depending on the size and age of the tree, yield of mahua sees varies from 5 to 200 kg/tree [48]. The average yield of sun dried mahua seed is about 1.6 kg/tree. The seed yield of the tree can increase tenfold starting from an age of 10 years up to an age of 60 years [48]. The tree is valued for its fat/oil and is therefore also known as ‘The Indian Butter tree’. The composition of mahua seed as reported by various authors has been listed in Table 4. Mahua has an estimated oil production potential of 18 lakhs metric tones per year in India [48, 68]. It is by far considered the most important tree seed oils for the tribals of India [18]. Fresh oil is pale yellow fluid at room temperature with an unpleasant taste and odour [3, 18]. On the basis of iodine number, mahua oil can be classified as non- 179 drying oil [3]. The peroxide value of mahua seed oil indicates that mahua seed may have low levels of oxidative and lipolytic activities. It tends to go rancid during storage, mainly due to the presence of oxidable constituents in the unsaponification fraction [3]. Palmitic acid is the major saturated fatty acid present. The total unsaturated fatty acid content have a major portion of oleic and linoleic acid. This might focus interest on the use of mahua seed oil, as unsaturated vegetable oils have an ability to reduce serum cholesterol levels [3]. The high oil content indicates the suitability of mahua seeds for industrial purposes as it reduces the production cost by avoiding hydrogenation and transesterification. Unlike other tree borne seeds which have the presence of toxic principles like curcurin in jatropha, ricin in castor and pongamin and karakjiin flavonoid in karanj, mahua oil is fit for human consumption [69]. Table 5 lists the composition and various properties of mahua seed oil. The oil is edible and is used for cooking in some rural areas of India. As reported by National Seed and Vegetable Oil Development Board of the Ministry of Agriculture, Govt. of India, oil is used for edible purpose by blending 5% of it in other edible oils [67]. Mahua oil (MO) finds application in the manufacturing of soaps, particularly laundry soaps. The oil is sometimes used as an adulterant for ghee; for this purpose it is clarified with buttermilk to mask the odour. Refined oil finds use in cooking, confectionery, chocolate making and manufacturing of lubricating greases and fatty alcohols [67]. The oils have also been tested as potential cocoa butter extenders [47, 70, 71]. The oil is also used for candle making, as batching oil in jute and as a raw material for the production of stearic acid [9]. Mahua oil has been reported to be beneficial in curing skin diseases, rheumatism, bilious fevers and burning sensation [11]. It acts as a good emollient. It also possesses pesticidal value. Application of M. longifolia oil at 0.5 ml/100 g of seeds has been recommended as the optimum dose to protect seeds without affecting their organoleptic properties or germination or any other damage. Utilization of M. longifolia oil to protect cowpea seeds has been reported as an eco-friendly and an economically viable method [50]. Biodiesel due to its environmentally safe, non-toxic, biodegradable and renewable nature, as against the conventional diesel, has been regarded as the fuel of future. For use as biodiesel, fatty acid methyl esters of various seed oils, both edible and non-edible, have been found suitable. The edible sources mainly include soybean, rapeseed, sunflower and safflower [12, 72, 73]. Few attempts have been made for producing biodiesel from non-edible sources like greases, tallow, lard, jatropha (Jatropha curcas), neem (Azadirachta indica), castor (Ricinus communis), karanj (Pongamia pinnata), mahua (Madhuca indica) and kokkam (Garcinia indica) [74–78]. 123 180 Waste Biomass Valor (2012) 3:175–189 Table 4 Composition of mahua oil seed Components [3] [9] [18] [4] [12] [56] [49] Mahua seed cake Mahua seed cake Mahua seed cake Mahua seed cake Mahua seed cake Defatted flour 19.1 19.1 24.39 15.38 24 10.39 1 7.8 5.52 Mahua seed cake Defatted flour Protein (N 9 6.25) (%) 16.9 29.4 Oil (%) 51.5 1.1 3.2 8.6 8.4 Fibre (%) Carbohydrates (%) 22 16.3 55–56 42.8 51.3 Ash (%) 3.4 6 6.3 6.8 9.2 Saponins (%) 2.5 9.8 4.6 5.1 5.1 Tannins (%) 0.5 1 Moisture (%) 8 64.36 4.63 34 10.85 6 5.89 10.4 1.2 8.7 6.5 12 Phosphorus as P2O5 (%) 0.78 Volatile matter (%) Fixed carbon (%) 68.3 18.4 Lignin (%) 20.21 Cellulose (%) 60.42 Hemicellulose (%) 16.21 Bulk density (g/cc) 0.3482 Organic matter (%) 90.8 Sapogenols (%) 95.37 1.6 1.6 CaO (%) 0.49 K2O (%) 2.93 Table 5 Composition and properties of mahua seed oil [3] [18] Refractive index 1.4795 1.452–1.462 Specific gravity 0.915 Saponification value 196 187–197 Iodine value 80.2 55–70 Peroxide value (meq/kg) 24 24.5 1 1.0–3.0 [12] [1] [16] [80] Physico-chemical property Cetane number (CN) Unsaponifiable matter (%) 1.456 198.34–202.78 190 52.09–68.59 59 58.0–51.58 Acid value (mg KOH/g of oil) 16 Fatty acids composition (%) Palmitic Stearic 17.8 14 24.5 22.7 11.65–61.5 19.11–32.16 17.8 14 16–28.2 20–25.1 16–28.3 20–25.2 Oleic 46.3 37 32.91–48.65 46.3 41–51 41–52 Linoleic 17.9 14.3 9.36–15.42 17.9 8.9–13.7 8.9–13.8 Linolenic 1.7 Arachidic 0.9 0–3.3 0–3.4 Total unsaturated 65.9 46.66–60.65 Total saturated 32.7 39.33–52.75 Mahua with its huge oil potential offers a great scope for biodiesel production. There are at least four ways in which oils and fats can be converted into biodiesel, namely, 123 transesterification, blending, micro emulsions and pyrolysis—transesterification being the most commonly used method [79, 80]. Mahua oil methyl esters (MOME) have Waste Biomass Valor (2012) 3:175–189 181 Table 6 Properties of mahua oil biodiesel (MOB) Property [17] MO [16] MOB B20 MO [16] [17] [47] MOB MO MOB Standards Diesel USA ASTM Europe EN Flash point (°C) 212 129 103 212 129 232 208 68 [130 [120 Kinematic viscosity @ 40°C 28.58 5.1 4.04 27.63 4.85 24.58 3.98 2.6 1.9–6.0 3.5–5.0 Copper strip corrosion 1.5 1 1 Cloud point (°C) 17 4 -3 18 9 Density at 15°C (kg/m3) 897 876 838 915 883 960 880 850 Calorific value (MJ/kg) 35.61 36.91 41.75 35.61 36.91 36 37 42 14 5 51 15 6 7 51 Pour point (°C) Cetane number 1 -6 860–900 [47 [51 Acid value 38 0.41 0.35 \0.8 \0.5 Water content (%) 1.6 0.04 0.02 \0.03 \0.05 \0.02 Ash content (%) 0.9 0.01 0.01 Carbon residue (%) 3.7 0.2 0.17 been prepared by transesterification using potassium hydroxide (KOH) as a catalyst [17]. The fuel properties of mahua oil biodiesel (MOB) are closely related to diesel and confirm to the ASTM standards. From the engine test analysis, it has been observed that the MOB, B5 and B20 blend results in lower CO, HC and smoke emissions as compared to diesel. Hence MOB or blends of MOB and diesel (B5 or B20) can be used as a substitute for diesel in diesel engines used in transportation as well as in the agriculture sector [16]. It has also been observed that the fuel properties of mahua biodiesel are similar to those of karanja and jatropha biodiesel except the viscosity which can be suitably modified by transesterification [16]. Table 6 lists the various properties of mahua oil biodiesel (MOB) as reported by various authors. Even though mahua oil is used for consumption, some authors have reported its toxicological nature. The incidence of aflatoxin contamination in mahua seeds was several-fold higher than permissible limits of imposed regulatory guidelines by FAO, WHO, European and Asian countries [67, 81]. It was observed that inclusion of alkaline-refined edible grade mahua oil (from Madhuca latifolia) at a rate of 10% in the diet of weanling albino rats for 14 weeks reduced their reproductive performance in the second generation. Histological studies showed bilateral testicular atrophy with degenerative changes in the seminiferous tubules [1]. Mahua Seed Cake (MC) After the extraction of oil from mahua seed, a major portion (60–70%) of raw material is left over as the seed cake. Mahua seed meal/cake is a good source of nitrogen and proteins [18]. Small quantities of the cake are sometimes fed to cattle without any apparent ill effects. It has also been used as manure either alone or in mixture with other \0.02 \0.3 cakes and ammonium sulphate. The cake goes mainly as a low-grade fertilizer [82]. It is also used along with shikakai (Acacia concinnca) as a hair wash in south India [9, 18, 25, 54]. The foaming agent of mahua seed cake makes the cake suitable in preparation of detergent, shampoo and toiletry [18]. Active carbon with good bleaching properties has been prepared from the husks of mahua seeds [9]. At an optimum condition (pH: 2; temperature: 30°C; dosage: 6 g/l; shaking speed: 150 rpm), cake has a potential to absorb Congo red dye for lower concentration range up to 75 mg/l [56]. The defatted mahua seed flour has an oil absorption capacity of 4.5 ml/g flour i.e. twice that of water; which makes it suitable for bakery products. High emulsification capacity of flour makes it advantageous for preparation of sausages and other meat products [3]. Mahua seed cake is applied to lawns and golf greens, as saponins present in it have a specific action against earthworms. The cake may be used in the formulation of moulding powder composition. It possesses insecticidal, piscicidal and pesticidal properties [9, 12, 24, 25, 29]. Mahua cake independently or in combination with a chemical nematicide (Carbofuran3G) has been reported for the management of Pratylenchus delattrei in crossandra under glass house conditions. Application of cake against nematodes (Meloidogyne incognita) has shown to improve plant growth [55]. Mahua also inhibited the mycelial growth (by 64.16%) and spore germination (by 71.41%) of Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice [83]. The residum or cake left after the expression of the oil has also been employed as an emetic [23]. Toxicity of Mahua Seed Cake (MC) The use of mahua seed cake in food or livestock feeds is limited by the presence of bitter and toxic components, viz. the saponins [46]. Saponins are a diverse group of low molecular-weight 123 182 Waste Biomass Valor (2012) 3:175–189 Fig. 1 Aglycones present in mahua saponins [1] secondary plant metabolites that are widely distributed in the plant kingdom. The chemical structure of saponins consists of an aglycone of either steroidal or a triterpenoid nature and one or more sugar chains (glycosides). Saponins can form stable foam in aqueous solutions, hence the name ‘‘saponin’’ from the Latin word for soap (sapo) [84]. In Madhuca, the triterpenoids mostly occur in the seeds up to a concentration of around 100 g/kg as for example in M. butyracea [49] and M. longifolia [3, 85]. Species of Madhuca contain pentacyclic triterpenoid saponins based on an oxygenated oleanolic acid skeleton. The known Madhuca saponins contain protobassic acid (2b, 6b, 28-trihydroxyoleanolic acid), 16a-hydroxyprotobassic acid (2b, 6b, 16a, 28 tetrahydroxyoleanoic acid), or their 2-oxo derivatives as aglycones (Fig. 1), with sugar residues attached in most case to both C-3 and C-28 to form bidesmidic saponins [85–87]. New saponins are continuously 123 being isolated and their structures determined [86–90]. However, structural details proposed for some of the isolated saponins are not reliable, and it is therefore difficult to conclude about the exact number of known Madhuca saponins. Structures shown in Table 7 can be regarded as representative examples rather than as a comprehensive list [1]. Many authors have reported the toxic effects of saponins [91, 92]. Rats fed with Madhuca latifolia meal, containing 5–6% saponin, at the level of 10–12% died in a month [93]. The oral LD50 in mice of crude Madhuca saponins (exact botanical source not given) was about 1.0 g/kg body weight. In mice and rats, Madhuca saponins caused local gastrointestinal toxicity as well as liver and kidney toxicity. At lower doses, Madhuca saponins can cause feed refusal and starvation with reduced body weight gain and increased mortality. Results from studies on Madhuca seed cakes, which contain saponins, on ruminants indicate that Waste Biomass Valor (2012) 3:175–189 183 Table 7 Saponins isolated from Madhuca species [1] Madhuca species Saponin Structure References M. longifolia Madlongiside A 6b,23-dihydroxy-2-oxooleanoic acid 28-O-a-L-arabinopyranoside [87] M. longifolia Madlongiside B 3-O-b-D-glucopyranosyl 6b,23-dihydroxy-2-oxooleanoic acid 28-O-a-Larabinopyranoside [87] M. longifolia Madlongiside C Protobassic acid acid 28-O-a-L-arabinopyranoside [87] M. longifolia Madlongiside D Protobassic acid 28-O-a-L-rhamnopyranosyl(1-2)-O-a-L-arabinopyranoside [87] M. longifolia M. butyracea Mi-saponin A 3-O-b-D-glucopyranosyl protobassic acid 28-O-b-D-rhamnopyranosyl(1-3)b-D-xylopyranosyl(1-4)-a-L-arabinopyranoside [86, 87, 90] M. butyracea Butyroside A 3-O-b-D-glucopyranosyl protobassic acid 28-O-b-D-apiofuranosyl(1-3)-b-Dxylopyranosyl(1-4)-a-L-rhamnopyranosyl(1-2)-a-L-arabinopyranoside [90] M. butyracea Butyroside B 3-O-b-D-glucopyranosyl 16a-hydroxyprotobassic acid 28-O-b-Dapiofuranosyl(1-3)-b-D-xylopyranosyl(1-4)-a-L-rhamnopyranosyl(1-2)-a-Larabinopyranoside [90] M. butyracea Butyroside C 3-O-b-D-glucuronopyranosyl protobassic acid 28-O-b-D-rhamnopyranosyl(13)-b-D-xylopyranosyl(1-4)-a-L-rhamnopyranosyl(1-2)-a-Larabinopyranoside [86] M. butyracea Butyroside D 3-O-b-D-glucuronopyranosyl 16a-hydroxyprotobassic acid 28-O-b-Dapiofuranosyl(1-3)-b-D-xylopyranosyl(1-4)-a-L-rhamnopyranosyl(1-2)-a-Larabinopyranoside [86] M. butyracea 16a-hydroxy Misaponin A 3-O-b-D-glucopyranosyl 16a-hydroxyprotobassic acid 28-O-b-Drhamnopyranosyl(1-3)-b-D-xylopyranosyl(1-4)-a-L-arabinopyranoside [90] they are more tolerant to Madhuca saponins than monogastric animals and can tolerate inclusion levels of up to a maximum of 20% of the total diet [1]. Except for piscicidal effect of Madhuca saponins by water exposure in guppy fish, no toxicity studies after dietary exposure have been identified in fish [1]. The defatted meal, though contains a good amount of protein, is not edible due to the presence of saponins which are toxic [94]. Thus, detoxification of mahua seed flour is necessary before its extensive utilization in food or feed products. Table 8 Different methods for saponin removal from mahua seed cake (MC) Treatment Detoxification of Mahua Seed Cake A number of processes have been examined for reducing the saponin content in mahua seed cake. Jakhmola et al. [85] reported that simple water treatment of Madhuca cake to reduce the saponins content appears to improve the utilization potential of the press cake as an animal feed. The detoxification has been achieved in a number of different ways, including ureaammoniation, soaking in cold or hot water, salt solutions, etc. [3, 49, 95–98]. Table 8 lists the various solvents/methods tried for the detoxification of mahua cake. Saponins removal (%) Sapogenols removal (%) Tanins removal (%) 31.77 References Ethanol 86.46 – Single stage ethanol extraction 55 – – [49] [95] Double stage ethanol extraction Cold water 90 87.1 – 99.4 – – [49] [4] Hot water 85.7 98.6 – [4] 0.25% NaOH 61.9 98.5 – [4] – [4] 0.5% NaOH 54.6 98.3 Water 65.44 – 35.71 [96] Ammonium dihydrogen phosphate buffer (pH 6) 70.8 – 61.21 [96] Ammonium ferrous sulphate 88.39 – 52.87 [96] Ammonium acetate 60.23 – 39.05 [96] Triammonium citrate 50.66 – 61.21 [96] Diammonium hydrogen citrate 45.24 – 52.87 [96] Diammonium hydrogen carbonate 68.9 – 48.12 [96] Ammonium ferric sulphate 54.834 – 39.05 [96] 123 184 Experiments with Mahua Seed Cake (MC) From above, we find that almost every part of mahua is utilized in one way or another. Most importantly, mahua flowers and oil have extensive usage. After the extraction of the oil, much of the material is left as the seed cake, which though being rich in nutrients is not much utilized because of its toxic saponins. Since the cake is a rich source of sugars and proteins, attempts were made for its effective utilization. A simple water treatment was given to detoxify the cake. The raw cake and the detoxified cake so obtained were checked for the production of biogas and the growth of Pleurotus mushrooms. Materials and Methods Characterization of Cake Waste Biomass Valor (2012) 3:175–189 up was designed in a specific manner so as to collect the biogas by water displacement method and periodically withdraw the slurry samples from the digester for analysis. The total solids in each set up were, however, maintained at 10% and the working volume of each digester at 5 l. The experiment was run for a period of 70 days in a room with its average temperature maintained at 35 ± 2°C with the help of a room heater thermostat. Each treatment was set up in triplicate and repeated twice. To each set, an inoculum from a running biogas plant was added [99]. The quantity of biogas produced in different treatments was measured by the water displacement method daily. Gas samples from each set up were collected weekly and analyzed for their methane and carbon dioxide content using Gas Chromatography (Agilent Technologies 7890A GC system). The total solids and combustible solids in the slurries obtained were determined as above. The cake used in experiments was procured from Pratapgarh area in U.P., India and characterized by NISCAIR, CSIR, India. The toxic saponins present in the cake were extracted following the protocol of Lalitha et al. [88] and their %age content in the cake was determined. Moisture content and total solids (TS) were determined by heating the cake at 60°C for 24 h and then at 103°C for about 3 h using a hot air oven. These oven dried samples were further heated to 550 ± 5°C temperature for about 5 h in a muffle furnace to determine the total combustible solids [99]. Carbon and nitrogen contents were estimated using CHN analyzer (CHNOS Elementar, Vario EL III model) and the crude protetin was obtained by multiplying the nitrogen content by a factor of 6.25 [99]. Total soluble sugars were determined by the anthrone method and oil content by using hexane as the extracting solvent and heating at 60–65°C in a soxhlet apparatus [99]. A simple treatment was given to detoxify the cake as follows. The cake was soaked overnight in water at a dilution ratio of 1:6 (cake:solvent) sufficient as to soak the cake completely without making the mixture too thick to separate. The cake was then filtered and its saponin content was found as above. The average of three determinations was taken in all the cases and the final value was reported. Mushroom Cultivation Biogas Production Characterization of Cake For experiments on biogas production, fresh cow dung was collected from the village katwaria Sarai, Delhi, India. The raw as well as the detoxified cake were mixed in various proportions of 25, 50 and 75% with cow dung (CD) in 7 l bottles fabricated at the Biogas Lab, Centre for Rural Development and Technology (CRDT), IIT Delhi. The set Mahua seed cake was characterized as Madhuca longifolia (L.) J.F. Macbr. Var. latifolia (Roxb.) A. Chev. Syn. Madhuca indica J.F. Gmel. M. latifolia Macbr.; Bassia latifolia Roxb by NISCAIR, CSIR, India. A voucher specimen has been deposited there. Table 9 lists the composition of mahua seed cake used. 123 For experiments on mushroom cultivation, P. sajor-caju spawn was procured from Bharat Mushroom Organisation, New Delhi, India and stored at 4°C till subsequent use. Chopped wheat straw (4–5 cm) was soaked overnight with water containing formalin (100 ml formalin in 100 l water) to make it free from any contamination. The extra water was drained out. 2.5 kg wheat straw (on wet basis) was taken and raw as well as detoxified mahua seed cake were added at 1, 2, 3, 5, 10, 20, 30 and 40% proportions, as a source of nitrogen to the straw, and mixed properly. 10% spawn (on dry basis) was added to each treatment. Further, these substrate combinations were packed in polythene bags (28 cm by 20 cm) with small holes for proper aeration and allowed for spawn run at 20–25°C for 2–3 weeks. After completion of the spawn run, which was visible from the transparent polythene sheet as a white mycelial growth over the substrate, the polythene bags were cut open. Water was sprinkled regularly twice, at morning and afternoon, to develop fruit bodies. Thereafter, the mushrooms were harvested in 3–4 flushes. The total yield was reported as the fresh weight of fruit bodies in grams obtained per kg dry straw. Results and Discussion Waste Biomass Valor (2012) 3:175–189 185 Table 9 Composition of mahua seed cake (MC) Constituents (%) Mahua cake Moisture 7.81 ± 0.23 Total solids 92.18 ± 2.8 Total combustible solids 91.67 ± 1.67 Total soluble sugars 30.05 ± 1.23 Protein 19.68 ± 1.27 Fat 7.01 ± 0.7 Saponins (toxins) 16.7 ± 1.38 Carbon 44.93 ± 0.08 Nitrogen 3.15 ± 0.02 Table 10 Percentage change (? increase, -decrease) observed in biogas volume over CD for various treatments [99] Treatment % Change in biogas volume 100% CD - 25 (MC): 75 (CD) (?) 14.28 50 (MC): 50 (CD) (-) 12.7 75 (MC): 25(CD) (-) 50.34 25 (50 DMC): 75 (CD) (?) 40.13 50 (50 DMC): 50 (CD) (?) 93.36 75 (50 DMC): 25 (CD) (?) 61.22 It was found that the water treatment could reduce the saponin content by approximately 50%. The resultant cake was designated as 50% detoxified mahua cake (50 DMC). Experiments on Biogas Production The % age change in the total biogas volume obtained from various treatments over CD is given in Table 10. With pure cow dung, 196 l/kg TS of biogas could be obtained. However, with the addition of 25% raw mahua seed cake, about 14.28% increase in the production was observed. This could be because mahua seed cake is rich in sugars and proteins and might prove to be an additional nutrient source for the bacteria involved in the process of biogas production [99]. Further, addition of 50 and 75% raw mahua seed cake to CD significantly decreased the biogas volume obtained. On the other hand, 50 DMC could easily be tolerated up to a proportion of 50% with CD giving a maximum biogas production of 379 ± 3.6 l/kg TS, an increase of 93.36% over the control [99]. Addition of 75% 50 DMC, however, decreased the biogas production. These results suggest possibility of saponins being toxic to the bacteria involved in the biogas production process but further microbial studies are still required to validate this observation [99]. Figure 2 gives the data from these two substrates and compares the results with the biogas volume obtained from other seed cakes used in different combinations, either alone or with CD [99–105]. Methane and carbon dioxide content in all the cases varied between 50–60% and 38.5–48%, respectively. Maximum decrease of 58.60% and 71.30% in total solids and combustible solids, respectively was obtained for the same treatment, i.e., 50 (CD): 50 (50 DMC) for which the maximum biogas volume was obtained [99]. Experiments on Mushroom Cultivation Out of the several species of mushrooms being commercially available, Pleurotus species offer several advantages. They are rich in minerals, possess several medicinal properties, have low demand on resources, are easiest and least expensive to grow and can be cultivated on a variety of substrates [102]. Figure 3 lists some substrates [106] Fig. 2 Biogas volume obtained from various non-traditional substrates. Numbers in the square brackets above the histogram indicate the reference 123 186 Waste Biomass Valor (2012) 3:175–189 Fig. 3 Substrates used for cultivation of Pleurotus species. Numbers in the square brackets above the histogram indicate the reference Fig. 4 Effect of olive mill effluent and mahua seed cake (raw and detoxified) on the yield of Pleurotus mushroom species that have been used for the cultivation of different Pleurotus species [107–112]. Paddy straw and wheat straw (WS) have been reported as traditional substrates for the growth of Pleurotus species. However, the use of seed cakes as nitrogen supplements have shown 50–100% increase in the mushroom yields [106]. In the present study, experiments were also performed to check if the raw as well as detoxified mahua seed cake could support the growth of Pleurotus sajor caju mushrooms. It was observed that the addition of cake could significantly enhance the mushroom yield. Both the cakes, raw as well as detoxified, could be easily tolerated up to 10% after which the yield started decreasing. Maximum yield of 477 g/kg dry straw was obtained from the combination containing 10% 50 DMC along with WS, an 123 increase of about 128% over the control. The detoxified cake seemed to perform better than the raw cake. This was well supported by the fact that the toxic saponins, extracted from mahua seed cake (MC), when tested alone were found to inhibit the growth of Pleurotus sajor caju fungus. Figure 4 shows the trend in the total yield of Pleurotus sajor caju obtained with addition of different concentrations of raw and 50 DMC to wheat straw (WS) and compares it with the results obtained with other Pleurotus species cultivated on WS to which different concentrations of olive mill effluent were added [113–114]. As can be seen from the graph, the effluent could be suitably tolerated up to 25% as against the solid cake in our case which could be tolerated up to 10%, after which the decrease in the total yield due to their toxic nature was observed. Waste Biomass Valor (2012) 3:175–189 Conclusions From the above study, it is concluded that mahua biomass has a vast potential. After mahua flowers and oil, mahua seed cake falls next in the category due its numerous uses. Its nutrient rich composition makes it useful for various applications, which can be further enriched after the removal of its toxic saponins (detoxification). 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