6. INVESTIGATION OF NUTRITIONAL AND NUTRACEUTICAL ASPECTS

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1 6. INVESTIGATION OF NUTRITIONAL AND NUTRACEUTICAL ASPECTS Under this part of the investigations, besides the analysis of proximate nutritional and mineral content of Lentinus squarrosulus following standard techniques, its mycelium was studied for haemagglutination and sugar specificity of its lectins so as to understand its potential as a nutraceutically and pharmaceutically relevant mushroom Investigation of nutritional aspects The nutritional aspects primarily concerns the proximate composition of mushrooms with particular reference to moisture content, proteins, amino acids, fats, carbohydrates, fiber, vitamins, minerals and number of other nutritional considerations like acid value, saponification value, ph, etc. These aspects have been worked out following standard techniques listed under Material and Methods. The results of the study are depicted in Table Nil Enery value (Kcal) 1.02 Iodine value Acid value ph 0.37 (%)Carbohydrates 3.41 (%)Crude total ash Crude fiber (%) Crude fat (%) L.squarrosulus Saponification value (%)Protein Species (%)Loss on drying at 700C Table-39: Nutritional components of L. squarrosulus per 100 gm of the dry sample 380 (a) Moisture: 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 (90 % on the average) which may be altered by changing temperature, relative humidity and cxxx

2 storage conditions (Purkayastha and Chandra, 1985). The maximum moisture content of the fresh cultivated L. squarrosulus has been determined at 87 percent. As compared in the dry sample it has been determined at %. (b) Proteins: Protein is the most critical component contributing to the nutritional value of a food (Chang and Hayes, 1978). Mushroom is a good source of proteins and amino acids (Kurtzman, 1975). Analysis of data reveals that biological values of mushroom proteins are intermediate between vegetable and animal proteins (Worgan, 1968). The degree of digestibility of mushroom proteins as reported by Lintzel (1941) varies from 72-83% and its calorific value ranges from K Cal/100gm of mushrooms. 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. Total proteins were estimated by AOAC (1990) method through Kjeldhal apparatus. In L. squarrosulus 3.41 % protein has been determined per 100 gm of the dry sample. In terms of quantity the amount of protein is not very high but is comparable to some of the vegetables like cabbage (1-5 %) as reported by Kaul (1983). (c) Crude Fat: Like proteins and carbohydrates, mushrooms possess a wide range of fat content varying from 1.2 to 20.6 % (Adriano and Cruz, 1933). Mushrooms are a low fat food as is the case in L. squarrosulus. Presently, crude fat was determined by extraction through Soxhlet apparatus using petroleum ether and was estimated at 0.37% per 100 gm of dry sample. (d) Crude Fiber: Analysis of mushrooms confirms the presence of relatively large amounts of fiber varying from 3 32 % (Chang and Hayes, 1978). The fiber content in almost all mushrooms is very high as has been documented during estimation of crude fiber in L. squarrosulus. In this mushroom 1.02 % crude fibers were determined cxxxi

3 from the 100 gm of dry sample which is substantially more than all the cultivated species of Pleurotus (Rai et al., 1988), Agaricus bisporus, Volvariella volvacea, cabbage, cauliflower and potato (Rai and Sohi, 1988). (e) Total Ash: For ash content, 5-10 gm of L. squarrosulus sample was ignited in silica dishes up to 5250C for 4-6 hrs. Substantial amount of ash (5.78 %) was estimated which is much more than recorded by Rai and Sohi (1988) in other mushrooms. (f) Carbohydrates: Carbohydrates constitute the greatest fractions of the mushroom dry matter. Total carbohydrate content of fresh mushrooms as reported by Rautavaara (1947) varies from to 76.1 % and it is mainly composed of mannitol, glycogen and hemicellulose together with smaller amounts of reducing sugars (Worgan, 1968). Presently, in case of L. squarrosulus it has been determined individually by substracting the content of other components from total dry matter. In 100 gm of dry sample of L. squarrosulus % carbohydrates has been estimated. (g) Minerals: Because of the high ash content mushrooms are quite rich in minerals. Mushrooms possess high amount of mineral constituents like phosphorus, sodium and potassium although calcium and iron are present in low quantity. Similar observation has been made in L. squarrosulus. During the present investigation macro and micro mineral estimation was done by the method of Jackson (1967) with the help of Atomic Absorption Spectrophotometer and results are documented in Table-40. Out of the four macro-minerals (Ca, Mg, Na and K) estimated, Ca (3.33 mg/100 gm) was found in highest amount followed by Na (2.392 mg/100 gm), Mg (1.200 mg/100 gm) and K ( mg/100 gm). From amongst the micro nutrients (Cu, Zn and Fe) estimated the content of Fe (6.41 mg/100 gm) was maximum followed by Zn (6.10 mg/100 gm) and Cu (0.750 mg/100 gm). cxxxii

4 Table-40: Macro and micro minerals present in L. squarrosulus Species L.squarrosulus Macro minerals (mg/100gm) Ca Mg Na K Micro minerals (mg/100gm) Cu Zn Fe As is apparent from the results obtained, L. squarrosulus possesses good amount of nutritional components because of which this species like all other edible mushrooms is good for human consumption Investigation of haemagglutination activity of extracts of mycelium for presence of lectins In the present study, L. squarrosulus mycelia has been studied for the presence of lectins by haemagglutination assay using human erythrocytes. Yeast glucose medium maintained at ph 4.0 was inoculated with homogenized inoculum aseptically and incubated at 30 ± 10C for 5, 7, 9 and 12 days. The mycelial mats obtained were harvested by filtration and washed thoroughly with double distilled water to remove the traces of media. Subsequently, the mycelium was homogenized in phosphate buffered saline (0.1 M, ph 7.2) in the ratio of 1 : 1.5 for 5 minutes in an ice bath and then ground in acidified river sand for 30 minutes. Extract was then centrifuged at 5000 rpm for 20 minutes at 4 0C and supernatant obtained was assayed for haemagglutination activity using human type A, B, AB & O erythrocytes. Lectin activity was expressed in terms of titre, defined as inverse of highest dilution causing visible haemagglutination. For this purpose 20 µl sample was 2-fold serially diluted in PBS through the wells of U bottom microtitre plate. Subsequently, 20 µl of erythrocyte suspension (2 %, v/v) was added to each well and plates were incubated at room temperature for 30 minutes. The plates were stabilized at 4 0C for 1-2 h. Mat cxxxiii

5 formation indicated the presence of haemagglutination activity, while button formation indicated the absence of haemagglutination activity (Figs.-39/A-D). Results are depicted in Table-41 in which haemagglutination activity has been expressed in terms of titre. Culture age- 5 days Culture age- 7 days Culture age- 9 days Culture age- 12 days Button Absence of haemagglutination activity Mat Presence of haemagglutination activity Fig.-39(A to D): Activity of lectins with human erythrocytes cxxxiv

6 Table-41: Haemagglutination activity of extracts from the mycelium of L. squarrosulus CULTURE AGE 6.3. LECTIN ACTIVITY (Titre) (DAYS) A B AB O Investigation of sugar specificity of the mycelial lectin Mushroom lectins display a considerable repertoire of carbohydrate specificities. The sugar specificity of lectins leads to a better understanding of the function of subcellular mechanisms in diverse fields of biological interest such as pathological markers of diseases, tissue metastasis and for controlling a variety of infections (Driessche et al., 2000). In the present study, L. squarrosulus mycelial lectin has been studied for sugar specificity. With 20 µl of appropriately diluted lectin samples, an equal volume of sugar stock solution was mixed in wells of U- bottom microtitre plate. The plate was incubated for 1 h at room temperature and then 40 µl of erythrocyte suspension (2 %, v/v)was added to each well followed by incubation for 30 minutes at room temperature. The plates were subsequently stabilized at 40C for 1 hour and observed for button or mat formation. Button formation indicated inhibitory sugar and mat formation suggested the non inhibitory sugars (Fig.-40). Results are presented in Table-42 and Fig.-40. cxxxv

7 Table-42: Sugar inhibition profile of L. squarrosulus lectin SUGAR RIBOSE RHAMNOSE RAFFINOSE XYLOSE L-FUCOSE D-MANNOSE D-ARABINOSE D-GALACTOSE D-SUCROSE DEXTROSE D-MALTOSE D-FRUCTOSE D-MANNITOL D-LACTOSE (+) : Inhibitory ; ACTIVITY (-) : Non-inhibitory Represents noninhibitory sugar Represents inhibitory sugar Fig.-40: Activity of lectins with different sugars The activity of lectins with different sugars exhibits their importance which can be utilized in applying chromatography for purification and drug targeting. Lectin present in L. squarrosulus showed non-inhibitory activity with various sugars except ribose and D-maltose as shown in Table-42. cxxxvi