9. NUTRITIONAL AND NUTRACEUTICAL EVALUATION

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1 9. NUTRITIONAL AND NUTRACEUTICAL EVALUATION Wild mushrooms are becoming more important in our diet due to their nutritional value in view of the high protein and low fat/energy contents (Diéz and Alvarez, 21; Agahar-Murugkar and Subbulakshmi, 25; Barros et al., 27 c). In the present study the proximate composition of wild edible species of termitophilous and lepiotoid mushrooms for the presence of nutrients and nutraceutics have been undertaken. The evaluation of nutrient composition included the determination of proteins, fats, ash, fibers, energy value, carbohydrates, heavy metals and mineral elements. The evaluation of nutraceutical composition included the determination of phenolic compounds, flavonoids, carotenoids, alkaloids and vitamins. Besides, experiments were also performed to screen and evaluate the lignocellulolytic enzyme production by the mushroom mycelium. These aspects have been worked out following standard techniques listed under Material and Methods. The results of the chemical composition and estimated energetic value (expressed on dry weight basis) of the wild edible mushrooms have been given in tables 4 to Nutritional studies Proteins: The wild mushroom species are reported to contain higher contents of protein and amino acids (Kurtzman, 1975) usually around 2-3% by dry weight which is quite useful for vegetarians or anyone looking to increase the protein content in their diet. Mushrooms due to high quantity and quality of proteins have been recognized by FAO as the food contributing to the protein nutrition of the countries depending largely on cereals. During present investigations the total proteins were estimated by AOAC (199) method through Kjeldhal apparatus. Amongst the worked out species maximum protein content has been found in T. medius (46.2%) followed 274

2 by T. badius (44%) while T. striatus (12.95%) contained lowest amount of protein per 1 gram of dry sample. The nutrient profile, in general, revealed that termitophilous mushrooms are rich source of protein (Table 4) and the values obtained are comparable to vegetables like Soybean (36.5%) in this regard. In comparison in lepiotoid mushrooms the net protein percentage evaluated ranged between %, which is slightly higher than T. striatus (12.95%) but much less in comparison to rest of the termitophilous mushrooms evaluated during the present investigations (Table 4 & 6). Fibers: Mushrooms are a valuable source of dietary fiber and important for the digestive system. Analysis of mushrooms confirms the presence of relatively large amounts of fiber varying from 3-32 % (Chang and Hayes, 1978). Presently crude fibres were determined by acid and alkali digestion methods. Crude fibers in dried samples were found to be maximum in T. mammiformis (8%) followed by T. medius (7.5%) and minimum percentage of these were documented in T. badius (2.5%) and Macrolepiota rhacodes (2.5%) (Table 4 & 6). Crude Fat: Like proteins and carbohydrates, mushrooms possess a wide range of fat content varying from 1.2 to 2.6% (Adriano and Cruz, 1933). Presently, crude fat was determined by extraction through Soxhlet apparatus using petroleum ether. It was found to be maximum in M. procera (3.4%) followed by T. mammiformis (3.3%) while other mushrooms contain considerably low percentage of crude fat and in T. heimii the amount of crude fat is lowest (1.65%) per 1 g of dry sample (Table 4 & 6). Total Ash: For ash content, 5-1 g of sample was ignited in silica dish up to 525 C for constant weight. Presently, Ash content was maximum in T. microcarpus (15.6% 275

3 Percentage in dry sample) followed by T. striatus (12.13%) and lowest amount of ash was recorded in M. procera (1.93%). Table 4. Proximate chemical composition (g/1 g) and energetic value (kj/1 g) of wild Termitomyces species (On dry weight basis) (mean ± SD; n = 3) Sr. No. Protein Moisture Crude fat Fiber Ash Carbohydrates Energy (kj/1 g) 1 T. microcarpus T. radicatus T. mammiformis T. medius T. badius T. striatus T. heimii CD (p.5%) Proteins Moisture Crude fat Fibers Ash Carbohydrates Fig. 16: Proximate analysis of seven Termitomyces species Carbohydrates: Carbohydrates constitute the greatest fraction of the mushroom dry matter. Carbohydrate percentage was found to be maximum in M. rhacodes (68.19%) 276

4 followed by M. procera (6.82%) while T. medius (33.3%) contained lowest amount of carbohydrates (Table 4 & 6). Moisture: Moisture content does become a limiting factor in using mushrooms as a significant contributor to the diet, although moisture content in itself may not be of any nutritional significance, however, it does considerably influence the nutritional value. Fresh mushrooms like most vegetables contain high amount of moisture content (9% on an average) which may be altered by environmental and storage conditions (Purkayastha and Chandra, 1985). The average moisture content recorded in dried samples was highest in M. dolichaula (8.8%) followed by M. procera (8.5%) and minimum in T. badius (5.7%) on dry weight basis. Table 5:-. Proximate Mineral elements (mg/1 g) of wild Termitomyces species (On dry weight basis) (mean ± SD; n = 3) Sr. No. Mineral elements (mg/1 g) of dry samples Fe Mg Cu Mn Ca Se Zn 1 T. microcarpus T. radicatus T. badius T. medius T. heimii T. striatus T. mammiformis CD (p.5%)

5 (mg/1g of Dry Weight) Ca Mg Fe Cu Mn Se Zn Fig. 17: Mineral elements of seven Termitomyces species Table 6. Proximate chemical composition (g/1 g) and energetic value (kj/1 g) of wild Macrolepiota species (On dry weight basis) (mean ± SD; n = 3) Sr. No. Proteins Moisture Crude fat Fibers Ash Carbohydrates Energy (kj/1 g) 1 M. procera M. rhacodes M. dolichaula CD (p.5%) Table. 7. Proximate Mineral elements (mg/1 g) of wild Macrolepiota species (On dry weight basis) (mean ± SD; n = 3) Sr. No Mineral elements (mg/1 g) of dry samples Fe Mg Cu Mn Ca Se Zn 1 M. procera M. rhacodes M. dolichaula CD (p.5%)

6 (mg/1g of Dry Weight) Percentge on dry weight basis Macrolepiota procera Macrolepiota rhacodes Macrolepiota dolichaula Moisture Proteins Crude fat Fibers Ash Carbohydrates Fig. 18: Proximate analysis of three Macrolepiota species Fe Ca Cu Mn Mg Se Zn -5 M. procera M. rhacodes M. dolichaula Fig. 19: Mineral elements of three Macrolepiota species Determination of mineral elements and heavy metals During the present investigation mineral estimation and heavy metal detection was done by the method of Jackson (1967) with the help of Atomic Absorption Spectrophotometer and results are documented in tables-5, 7 & 8. Out of 279

7 1 wild samples examined for estimation, maximum amount of Fe (673 mg) was recorded in T. mammiformis followed by T. radicatus (482 mg) while T. striatus (82 mg) contained lowest amount of Fe. Other seven species also possessed significant levels of the mineral element. Mg was maximum in T. medius (33 mg) followed by T. heimii (287 mg) where as minimum quantity of Mg (6 mg) was recorded in T. microcarpus. Maximum amount of Ca (24 mg) was recorded in T. medius followed by T. radicatus (19 mg) whereas minimum quantity was documented in M. dolichaula (5 mg). Cu was maximum in T. striatus (11 mg) followed by T. radicatus (9 mg/1 gm dry wt.) and T. badius (7 mg) where as minimum quantity of this element was detected in T. mammiformis (4 mg). Mn was maximum in T. medius (13 mg) followed by T. radicatus (1 mg). Minimum amount of Mn (1 mg) was recorded in M. dolichaula. Zn was maximum (.9 mg) in M. rhacodes followed by T. microcarpus (.8 mg), whereas minimum quantity (.4 mg) of Zn was recorded in T. radicatus. Maximum amount of Se (.12 mg) was recorded in T. microcarpus followed by.11 mg in T. heimii whereas minimum amount (.5 mg) was recorded in T. striatus. The determination of heavy metal concentration in the fruiting bodies of mushrooms is essential in dietary intake studies. Some of the heavy metals such as As, Cd, Ni, Cr, Pb, and Hg are toxic. Traces of some heavy metals viz. As, Pb, Ag, Hg, Cd and Cr were found to be present in some of the presently investigated samples. Out of 1 wild samples examined, highest accumulation of Hg was found in T. medius (.1 mg) followed by T. striatus (.99 mg). Other seven species also possesses significant levels of this element with T. radicatus having minimum quantity (.16 mg). Maximum amount of As was observed in T. striatus (.185 mg) followed by M. rhacodes (.74 mg) and least in T. mammiformis (.37 mg). No 28

8 As was documented in T. microcarpus, T. heimii, M. procera and M. dolichaula. Cd was found to be maximum in T. microcarpus (.48 mg) and least amount of this metal was recorded in M. rhacodes (.14 mg). Cr, Ag and Pb were found to be absent in all these samples. Maximum permissible concentrations and approximately permissible levels of harmful concentration of heavy metals in food stuff have been depicted in table 8. Results showed that the amount of heavy metals in lepiotoid and termitophilous mushrooms evaluated is well within the range of permissible limits for human consumption (FAO/WHO, 1976). Table. 8: Heavy elements (mg/1 g) of Termitomyces and Macrolepiota species (On dry weight basis) (ND = Not detected) Sr. No. Heavy metals (mg/1 g) of dry samples As Cr Hg Pb Cd Ag 1 T. microcarpus ND ND.94 ND.488 ND 2 T. radicatus ND ND.16 ND.22 ND 3 T. badius.2 ND.96 ND.45 ND 4 T. medius.1 ND.1 ND.39 ND 5 T. heimii ND ND.18 ND.4 ND 6 T. striatus.185 ND.99 ND.17 ND 7 T. mammiformis.37 ND.43 ND.27 ND 8 M. procera ND ND.87 ND.19 ND 9 M. dolichaula ND ND.62 ND.19 ND 1 M. rhacodes.74 ND.69 ND.14 ND 11 Permissible limit in individual food article 1 mg/ kg 5.3 mg/kg 1 mg/kg 2.5 mg/kg 1.5 mg/kg 2.5 mg/kg 281

9 (mg/1g of Dry Weight) As Cr Hg Pb Cd Ag Fig. 11: Histogram showing heavy elements of Termitomyces and Macrolepiota species. 9.3 Neutraceutical studies Evaluation for the presence of phenolics, flavonoids, carotenoids and alkaloids in the wild edible lepiotoid and termitophilous mushrooms was done by employing standard biochemical techniques Evaluation for Phenolic compounds:- Phenolic compounds in mushrooms are known to contribute largely to antioxidant potential and are suggested to be major bioactive compounds for health benefits associated with inhibition of atherosclerosis and cancer (Cheung and Cheung, 25). In the last few years, an increasing interest in the consumption of mushrooms has arisen, due to their elevated polyphenol concentration, which correlates with their elevated antioxidant activity. Total phenolic content of the edible termitophilous and lepiotoid mushrooms was determined colorimetrically using Folin - phenol method. Maximum amount of phenolic content was documented in T. microcarpus (25.85 mg/g) followed by T. mammiformis (22.49 mg/g) and minimum amount of phenolic content was evaluated in M. dolichaula (

10 mg/g). Out of the examined species, all species of Termitomyces contained substantial amount of phenolics. (Table 9) Flavonoids: - Flavonoids are also polyphenolic compounds that are ubiquitous in nature. The quantity of flavonoids ranged from 1.36 mg/g in M. rhacodes to 2.2 mg/g in T. microcarpus Carotenoids: - The β-carotene content was found in very low amount in different species ranging from.11 μg/g in T. heimii to.5 μg/g in T. badius. Lycopene content ranged from.3 μg/g in T. heimii to.27 μg/g in T. striatus. Table 9:- Phenolic compounds (mg/g), Flavonoids (mg/g), β-carotene (μg/g), Lycopene (μg/g) and Alkaloids (mg/g) in wild Termitomyces and Macrolepiota species (On dry weight basis) (mean ± SD; n = 3). Sr. No. Phenolic compounds (mg/g) Flavonoids (mg/g) β-carotene (μg/g) Lycopene (μg/g) Alkaloids (mg/g) 1 T. microcarpus T. badius T. medius T. striatus T. heimii T. mammiformis T. radicatus M. dolichaula M. procera M. rhacodes CD at P (.5%)

11 mg/g (Phenolic, flavonoids & alakaloids) μg/g ( β carotene & lycopene) on dry weight Phenolics ( mg/g) Flavonoids ( mg/g) β-carotene (μg/g) Lycopene (μg/g) Alkaloids (mg/g) Fig. 111: Bioactive compounds of 1 wild species of Termitomyces & Macrolepiota Alkaloids:- Alkaloids are a group of naturally occurring chemical compounds with neutral and even weakly acidic properties which finds use for therapeutic and recreational purposes, in many synthetic and semi-synthetic drugs to enhance or change the primary effect of the drug and reduce unwanted side-effects. M. dolichaula contained.13 mg/g of alkaloids which was maximum in comparison to all other evaluated species of Macrolepiota and Termitomyces. 9.4 Vitamins Wild edible termitophilous and lepiotoid mushrooms were evaluated for the amount of vitamin A, vitamin B-complex and vitamin C, The results have been expressed in mg/1 g for all vitamins in table-1. All the studied mushrooms possessed significant amount of vitamins (Fig. 112). The maximum content of vitamin A in the analyzed mushrooms was observed in Lepiota humei (.16 mg/1 g) followed by T. heimii (.12 mg/1 g) and minimum quantity was documented in T. reticulatus.1 mg/1 g. 284

12 (mg/1g of Dry Weight) Sr. No. Table. 1 Vitamins in different species of termitophilous and lepiotoid mushrooms Vitamin A (mg/1 g) Vitamin B 1 (mg/1 g) Vitamin B 2 (mg/1 g) Vitamin C (mg/1 g) 1 T. reticulates.1±..26±.2.23± ±.1 2 T. heimii.12±.1.21±.1.25±.1.24±.1 3 T. mammiformis.11±.1.28±.1.21±.1.3±.1 4 T. radicatus.1±.2.42±.4.2±.1.96±.3 5 M. dolichaula.7±.1.75±.5.13±.2.48±.2 6 M. rhacodes.9±..8±.4.13±.3.36±.1 7 Lepiota humei.16±.1.49±.1.2±.3.18±.1 8. CD at P (.5%) Vitamin A Vitamin B1 Vitamin B2 Vitamin C Fig. 112: Histogram showing contents of various vitamins in different species of termitophilous and lepiotoid mushrooms. The thiamine (vitamin B 1 ) content ranged from.21 mg/1 g in T. heimii to.8 mg/1 g in M. rhacodes. The riboflavin (vitamin B 2 ) content was maximum in T. heimii (.25 mg/1 g) and minimum in M. rhacodes and M. dolichaula (.13 mg/1 g). Ascorbic acid (vitamin C) was 1.45 mg/1 g in T. reticulatus which was 285

13 maximum in comparison to other species while minimum amount of Vitamin C was documented in Lepiota humei (.18 mg/1 g) Enzyme Assay Lignocellulolytic enzyme production:- In this study evaluation for the production of extracellular oxidoreductases by different species using a test based on an agar medium with ABTS was done. The extracellular ABTS-oxidizing activity of the fungal strains on the modified Kirk medium has been shown in Figs. 113(A-E). The present study for the presence of enzyme was undertaken on seven cultures of wild mushrooms collected from different localities of North West India. These include Macrolepiota rhacodes, M. dolichaula, Leucocoprinus cepaestipes, Lepiota humei, Termitomyces radicatus, T. heimii and T. mammiformis. Out of these Macrolepiota rhacodes showed high ABTS-oxidizing activity while Macrolepiota dolichaula showed very low ABTS-oxidizing activity in comparison. However, the absence of extracellular ABTS-oxidizing activity does not necessarily imply the lack of capacity to produce these oxidative enzymes but could reflect a possible inhibition of their expression. As a matter of fact the oxidative enzyme system is not homogeneous and its production and properties depend on the conditions and culture media (Heinzkill and Messner, 1997). Fungal strains oxidized ABTS to dark green ABTS cation radicals (ABTS + ) indicating the production of extracellular oxidoreductases. Macrolepiota rhacodes and Lepiota humei gave positive reaction immediately after inoculation and formed dark green zone around the mycelia bit while Leucocoprinus cepaestipes showed light green zone on 3 rd 286

14 A. Lepiota humei B. M. rhacodes C. L. cepaestipes D. T. heimii E. M. dolichaula Figs. 113 (A-E): Showing extracellular ABTS oxidizing activity on agar plate 287

15 Concentration (µg/ml) Concentration µg/ml) After 5 days (µg/ml) After 1 days (µg/ml) After 15 days (µg/ml) Fig. 114: Histogram showing variation in protein concentration with respect to number of days in Wheat straw medium (WSE) After 5 days (µg/ml) After 1 days (µg/ml) After 15 days (µg/ml) -5 Fig. 115: Histogram showing variation in protein concentration with respect to number of days in Czapek medium(czp) 288

16 Laccase U/ml Laccase U/ml After 5 days (U/ml) After 1 days (U/ml) After 15 days (U/ml) Fig. 116: Graph showing variation in Laccase concentration with respect to number of days in Wheat straw medium (WSE) After 5 days (U/ml) After 1 days (U/ml) After 15 days (U/ml) -5 Fig. 117: Graph showing variation in Laccase concentration with respect to number of days in Czapek medium (CZP) 289

17 L. humei Table: 11:- Activities of lignolytic enzymes of different cultures on different media and intervals. Variety M. dolichaula M. rhacodes L. cepaestipes T. heimii T. mammiformis Medium Laccase (U/ml) 5 Days 1 Days 15 Days Protein (µg/ml) Laccase (U/ml) Protein (µg/ml) Laccase (U/ml) Protein (µg/ml) Czp. 3.±.1 17±.5 4.2± ± ±1..87±.1 WSE 2.8± ± ±.4 5.5± ± ±.2 Czp..13±. 16.8±.1.39±.3 5.2± ±.9 1.8±.2 WSE.33±. 19.9± ± ±.5 5.6±.3 2.8±.2 Czp. 11.7± ± ± ± ± ±.1 WSE 23.3± ± ± ± ± ±.2 Czp..74± ±.5 1.2±.2 2.1± ±.87.8±.8 WSE 1.85±.5 2.7± ±.1 7.5± ± ±.2 Czp..4± ±.22.63±.2 3.3±.1 1.±.33.59±.6 WSE.6±.6 22.±2..95± ±.28 1.± ±.9 Czp..5± ±.13.1±. 5.1± ± ±.5 WSE 3.44± ± ± ± ± ±.1 T. radicatus Czp..±. 17.±.5 1.3±.1 1.1± ±.2.32±.2 CD at P (.5%) WSE.23±.2 2.7± ± ±.1 2.2± ±.1 Czp WSE day after inoculation, followed by Macrolepiota dolichaula on 12 th day after inoculation while other mushroom mycelia showed very light green colour zone after 15 th day of inoculation. Laccase activity has not been observed in T. radicatus and T. mammiformis. The results obtained are depicted in table 11 and figures 113 to 117. (a) Estimation of extracellular protein:- All the cultures of termitophilous and lepiotoid mushrooms were evaluated for the occurrence of extracellular protein activity. The net extracellular protein activity was higher after the 5 th day of inoculation in M. rhacodes (23.6 µg/ml) on wheat straw extract followed by T. heimii (22.2 µg/ml) on the same substrate and least amount of extracellular protein have been detected in M. dolichaula (19.9 µg/ml) again on wheat straw medium. The 29

18 extracellular protein activity was maximum in M. rhacodes (8.54 µg/ml) followed by L. cepaestipes (7.5 µg/ml) and least amount in M. dolichaula (4.8 µg/ml) on wheat straw extract after 1 th day of inoculation. Fifteenth day of inoculation gave least extracellular protein activity which was 4.83 µg/ml in M. rhacodes followed by L. cepaestipes (3.3 µg/ml) and least amount was obtained T. mammiformis (1.2 µg/ml) on wheat straw extract, thereafter increase in number of days decreased the extracellular protein activity. From the results obtained thereoff, wheat straw substrate proved to be the best substrate for extra-cellular protein activity, while Czapek Dox agar produced less protein in general. Following maximum development after 15 th the day of incubation, extracellular protein activity started dipping depicting initiation of senescence resulting in decline in enzymatic activity. (Table - 11). (b) Estimation of extracellular Laccase activity: - On the basis of presence of colour intensity of the medium extracellular enzymatic laccase activity was further evaluated for quantitative analysis. Extracellular laccase activity was observed maximum in M. rhacodes (23.3 U/ml) followed by L. humei (2.8 U/ml) and minimum amount in M. dolichaula (.33 U/ml) on 5 th day of inoculation on wheat straw extract. Laccase activity was found to be maximum in L. humei (26.92 U/ml) followed by M. rhacodes (26.69 U/ml) and minimum in T. radicatus (1.99 U/ml) on 1 th day of inoculation on wheat straw extract. Laccase activity was found to be highest in M. rhacodes (39.25 & U/ml) and L. humei (34.66 & 4.55 U/ml), respectively on 15 th day of incubation in both Wheat straw extract and Czapek medium and least extracellular laccase activity was recorded in T. heimii (1. U/ml) under these conditions. 291

19 A. M. dolichaula B. T. heimii C. L. cepaestipes D. M. rhacodes E. T. mammiformis Figs. 118 (A-E): Different species showing degradation of CMC by the production of extracellular enzyme. 292

20 9.5.2 Evaluation of Cellulase activity:- Mushrooms have ability to digest lignocellulosic substrates. During the present study seven species were studied for the production of cellulase enzymes. Out of the seven species the mycelia of Macrolepiota dolichaula, M. rhacodes, Leucocoprinus cepaestipes, Termitomyces heimii and T. mammiformis demonstrated their ability to degrade cellulose (Figs. 118/A-E). The conclusions drawn are based on opaque clearance zones produced around the growing fungal colonies after 5-7 days of inoculation indicating degradation of CMC by the production of extracellular enzyme i.e. Cellulase or CMCase. As compared the mycelia of Termitomyces radicatus and Lepiota humei does not show any type of clear zone. 293