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STUDIES ON THE DEVELOPMENT OF VALUE ADDED FOOD BY BARLEY WITH RHIZOPUS OLIGOSPORUS IN KOJI FERMENTATION.

Byline: Zia-ullah Khokhar - Email zia2_khokhar@hotmail.com , M. Amin Athar, Qurat-ul-Ain Syed, Shahjahan Baig, M. Nadeem, M. Gul Sher and Shahbaz Ali and Irsa Islam

ABSTRACT: The present work was aimed to study the proximate analysis and composition of fermented (Tempeh) and unfermented barley. The investigation on the influence of soaking time, boiling time, inoculum size, pH and incubation time and incubation temperature on the development of fermented barley by solid state fermentation using Rhizopus oligosporus A-4 was also studied. The percentage of moisture increased after fermentation while the ash and fat percentage decreased, after fermentation. The major chemical constituent of barley kernel is starch, the amount of which varied inversely with protein percentage. Similarly dietary fibers are of considerable importance in human food and barley contains a sizeable amount of fiber after fermentation. Further the activity of enzymes e.g. protease, amylase, and lipase in barley kernels before and after fermentation were investigated.

The results showed that the activity of protease and amylase was increased after fermentation where as the activity of phytase was found to be decreased.

1. INTRODUCTION

All fermentations such as production of beer, wine, distilled alcoholic beverages, bread, and cheese are basically are the out put of enzymatic process and depend upon the metabolism of living organisms [1-6]. These products defined as fermented food when subjected to the action of desirable biochemical actions cause significant modification to the food value of these products. The production of value added fermented food has opened a new era in food industry. The process of fermentation results in the decrease of crude fat, carbohydrates, phytic acid, in soluble fiber with increasing several vitamins.

Fermented food may be defined as those foods, which have been subjected to action of microorganisms, or enzymes so that desirable bio-chemical changes cause significant modification of the food. The importance of fermented food is indicated by world annual production estimate of 700 million hectoliters of beer, 12 million tones of cheese and 1 million ton of mushrooms. Other groups of fermented foods include bread, yoghurt, soy sauce, fish sauce, pepperoni, Kim chi and gari. The terms solid substrate fermentation or biotechnology are new but the art of food fermentation is old [6].

Historically Egyptians, Samarians, Babylonians and Assyrians knew about the use of barley to produce alcoholic beverages; a cuneiform Inscription on a Babylonian brick from 2800 B.C gave a recipe for the production of barley wine a type of alcohol [7]. Today, South East and East Asia are main regions of its production. Differences in the incidence of cancer in Asian and American cultures may be due to fundamental differences in diet. American consumes a typical Western diet consisting of high-fat foods while in Asian especially India and Pakistan cultures typically consume a low-fat diet. Consumption of soybean products, such as tofu, miso and tempeh as well as low-fat foods in common Asian cultures. Among the Japanese, the incidence of cancer is less for Japanese living in Japan compared to Japanese who immigrated to Hawaii from Japan. That difference may reflect differences in diet.

Dietary and lifestyle changes are well-established ways to help prevent disease. The United States Department of Agriculture (USDA) developed the Food Guide Pyramid in 1992. The Food Guide Pyramid provides a suggested number of servings for each division or category of food. The quality and quantity of fermented food and content of water-soluble vitamins is enhanced [8]. Fermentation results in a lower proportion of dry matter in the food and the concentration of vitamins, minerals and protein appear to increase when measured on dry weight basis.

Barley is second most abundant cereal grain. Barley is grown around the world and has been used for food and beer making since ancient times. The barley composition varies among varieties, consisting of 49.66 % starch, 14.28 % fiber and 9.22 % crude protein. The amylopectin content of barley starch is about 74-78 %, leaving about 22-26% amylase. The consumption of barley decreases total serum lipid and LDL cholesterol levels, and thus, decreases the risk of cardiovascular disease . Barley consists of large quantities of phytate [9], which is a major storage form of phosphorus [10]. Phytate has negative influence on the bioavailability of essential minerals, phytate complexes and thereby inhibits absorption by converting them into soluble minerals [11-12]. Lower metabolic responses may elicit by the consumption of barley products and induce a great feeling of Satiety compared with which wheat bread [13] and could therefore reduces the risk of type two diabetes [14].

The recent research make realized the fact that the phytate content of barley could be minimized by 97% during the process of fermentation with Rhizopus Oligusporus [9]. Charlotte Eklund-Jonsson [15] aimed at evaluating the tempeh fermentation process as a means to develop nutritionally improved vegetarian products from barley and oats.

Although for many centuries tempeh has been produced in Indonesia but the tempeh mould was identified firstly by Dutch scientist Prinsen Geerlings in 1895. Soya bean tempe is known for its bioactivity in reducing the severity of diarrhoea in piglets. This bioactivity is caused by an inhibition of the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells [16]. Rhizopus oligosporus dominates among the different moulds found in tempeh. There are three species group of Rhizopus oligosporus (Mucorales, Mucoraceae, and Zygomycota). R.oryzae group, R.stolonifer group and R.microsporous group [17]. Latter group species are dominating due to the properties such as (i) Rapid growth at high temperature (30-42oC) (ii) inability to ferment sucrose (iii) High proteolytic and lippolytic activities and (iv) Production of strong antioxidants [18-19].

Rhizopus oligosporus is the most species in tempeh fermentation [20]. Rhizopus oligosporus is considered as a domesticated form of Rhizopus microsporus, which can produce toxic secondary metabolites such as Rhizoxin and Rhizonins A and B [21]. Rhizopus oligosporus produces enzymes such as carbohydrates (e.g. Polygalacturonase, xylanase, small quantities of a-D-galactosidase, etc).Lipases, Proteases and phytases [22]. In contrast Rhizopus oligosporus did not produces a-galactosidase and consequently can not degrade flatulence-causing compounds such as stachyose and raffinose [23].

The objective of this study was to optimize the various parameters e.g. soaking time, boiling time, pH, incubation time, and incubation temperature on barley fermentation with Rhizopus oligosporus and to determine the proximate composition of unfermented and fermented barley and estimation of the activity of Protease, amylase and Phytase.

2. MATERIAL AND METHOD

2.1 Organisms

The culture of Rhizopus oligosporous A-4 was obtained from Microbiology Lab, Food and Biotechnology Research center, PCSIR Lab complex Lahore, Pakistan. The culture was maintained on Potato dextrose agar slants for 5 to 7 days at 35oC.

2.2 Collection of sample

Barley used for experiments were obtained from the local market and they were sorted out to remove any impurities and dirt.

2.3 Growth medium

Potato dextrose agar (PDA oxide) was used for the development of Rhizopus oligosporous. The composition of PDA in g/L is as: Potato extracts 4.0gm (extract from 200g potato), D (+) glucose 20gm, Agar 15gm. The PDA slants were prepared for the growth of Rhizopus oligosporous A-4. The 5.6 pH was maintained with 1N HCl/NaOH before sterilization at 121C0 for 15min. The Chloramphenicol was used as antibiotic before autoclaving. The culture was regularly revived after a weak for getting newly 4 to 7 days old strains.

2.4 Buffers

Phosphate buffer 0.05M

K2HPO4 (Base) = 8.709g/L KH2PO4 (acid) = 6.8045g/L

Citrate buffer 0.05M

Citric acid=4.803g/500ml Tri sodium citrate = 7.35g/500ml

Glycine-NaOH Buffer: was prepared by adding 50ml of 0.1M Glycine and 32ml of 0.1M NaOH, final

Volume was made up to 200ml with distill water.

2.5 Solution and reagents

(i) 0.1 mM Maltose solution

The solution was prepared by mixing 3.6gm of maltose in 100ml distilled water.

(ii) Casein Solution

This solution was prepared in glycine-NaOH buffer different percentages of casein in grams were Dissolved in Glycine-NaOH buffer, Tee value of casein in grams were 0.5, 1.0, 1.5, 2.5, 3.0, 3.5 and 4.0 g. The pH of the whole solutions was maintained at 10 by using 0.1M NaOH and stored at 4oC.

2.6 Proximate analysis of barley

Proximate composition of a food sample determines the total proteins, fats, fiber, carbohydrates, ash, and moisture content as the percentage composition of the product. The samples were analyzed by standard procedure (AOAC, 2005) [24]. All determinations were carried in triplicate. Samples were ground in laboratory hammer mill in to a meal on which the proximate analysis was done. All samples were combined and taken their mean average.

Ash of the Tempe sample and unfermented barley was calculated according to the standard method (AOAC, 2005). Moisture content of barley sample was determined by gravimetric method [25]. The classical method for fat determination is based on continuous extraction performed on dried samples of food in a Soxhlet extractor. Total crude protein was estimated by Kjeldahl method (Nx6.25). Proteins and other organic materials in the sample were converted to ammonium sulphate by digesting the sample with sulphuric acid in the presence of catalyst. Crude fiber is the loss of ignition of dried residue remaining after digestion of the samples with H2SO4 and sodium hydroxide under specific conditions (AOAC method, 2005)

2.7 Fermentation of Barely grain

The soaked barely was boiled for various times and drained the extra boiling water, dried in sterilized Petri plates in oven at 60oc for 20 to 30 min to remove the moisture. The plastic bags were taken and made small pores with sterilized needle. The dried barely grain were inoculated with prepared spores suspension used as inoculums under sterilized conditions near the flame. Inoculum was mixed well with the help of sterilized loop and put into the plastic bags. Sealed the mouth of plastic bags. Incubated the inoculated barely grains at different temperature for different time.

2.8 Protease Activity

The protease activity in the fermented barley was estimated by the method of kuntz, (1947) [26].

"One unit enzyme activity was defined as the amount of enzyme that releases 1 ug of tyrosine per ml per min under the above assay conditions. Specific enzyme activity was expressed as units/mg of protein.

2.9 Amylase Activity

Amylase activity in the fermented barley was estimated by the method of Berg Meyer, (1983) [27].

0.25ml of substrate (soluble starch 1% w/v in 0.1M phosphate buffer) and add 150 ul of enzyme solution, incubate at 37oC for 30 min, add 400 ul of DNS to stop the reaction then boil the reaction mixture at 95oC for 15min, cool it at room temperature then add 8ml of deionized water and read the absorbance at 489nm. From the standard curve, concentration per unit absorbance (standard factor) was calculated. Standard factor of curve was 7520.91.

2.10 Phytase activity:

The phytase activity in fermented barley was estimated by the method of Heinonen and Lathi, [28].

Take 0.2 ml of extracted enzyme from fermented barely in washed and rinsed test tubes. And incubate at 37oC for 15 min. added 0.2ml of substrate (1% phytic acid) and incubate at 37oC for 15 min. Add 0.4ml of 15% TCA and centrifuge at 10000rpm for 10min at 4oC. Collect the fraction and take 0.2ml of supernatant with 1.8ml of deionized water .Add 2ml of fresh color reagent to each tube and mix well and placed at 50 oC for 15 min.

Enzyme activity was measured by following formula.

Enzyme activity (mg/ml) = mean absorbance at 280 nm x average standard factor.

3. RESULTS AND DISCUSSION

A Study was conducted to investigate the proximate composition of fermented and unfermented barley under the influence of fungal mold Rhizopus oligosporus A-4, optimization of fermentation conditions. The activity of enzymes e.g. protease, amylase and phytase was also investigated.

3.1 Proximate composition of unfermented barley

The results of proximate composition of unfermented barley are given in table-1. All the experiments were conducted in triplicate. The average composition of Pakistani varity of barley cereal was found to be 62.68% Carbohydrate, 12.00 % Crude fiber, 10.50 % Protein, 9.94% Moisture, 2.15% Ash and 2.11% Crude fat. Proximate composition may vary in different verities of barley and climatic conditions in which barley grow.

The barley composition varies among varieties, consisting of 49.66 % starch, 14.28 % fiber and 9.22 % crude protein [29].Table-1

3.2 Effect of various Process parameters on the fermentation of barley

The soaking time is to be considerable importance, as it influenced the consistency of the barley kernels to a large extent. The soaking time was varied from 24, 26, 28 and 30 hours in distilled water as given in table 2. The soaking time after 28 hours of soaking was found to be suitable for the production of fermented whole grain cereal product. This result is closer to the work of winaro and Reddy (1986) [30], he reported that in Indonesia Soya beans undergo a natural microbial acidification during the soaking process. During this period of 12-48 hours at approximately 280C was found to be best. Partial cooking or boiling of the cereal raw material is also known to play a vital role in the production of fermented food. Therefore boiling time varied from 15, 20, 25 and 30 minutes as given in table-2. The combination of soaking and boiling is most favorable for the growth of Rhizopus oligosporus A-4.

A longer boiling time did not give the same elasticity of the kernel as did the combination of soaking and boiling. For longer boiling the gelatinization of starch content occurred and the cake formation deteriorated. The boiling time of 25 minutes was found to be suitable for the production of fermented whole grain cereal product. Mulyowidaro et al [31] reported that mild boiling (15 min at 95oC) of soaked and biologically acidified beans results in sufficient survival of a mixed flora of lactic acid bacteria and bacillus spp. Nout and Rombouts [22] denoted that the strong proteolytic Bacillus spoilage took place in beans which had been heated for 60 min at 95oC or 15 min at 121oC. In the latter case a near pure culture of Bacillus survived as spores and grows without competition .

The inoculum size of Rrhizopus oligosporus A-4 strongly influences the fermentation Process. The 100g barley after 24, 26, 28 and 30hrs soaking in distilled water was treated with 0.5, 0.75, 1.0 and 1.5 ml of inoculum comprising of Rhizopus oligusporus, respectively. The results are given in table-2. The spore suspension was prepared by streaking a slant with inoculum needle into 10ml of sterilized distilled water. The fermentation was carried out at 25 to 40 0C for 24 to 48hrs.The best growth of Rhisopus oligosporous was observed with 1.0 ml inoculum size as evident from better cake formation. At 0.5ml to 0.7 ml inoculum size of fungus grew slowly and the barely with dense mycelia was not observed. At high inoculum size the fungus grew rapidly and the dense cake formation was uneven. These results are in good agreement with the work reported by Olsson et al [32].

According to them Rhizopus oligosporus was inoculated at approximately 104 spores/g moist substrate, a barley cake with dense mycelia growth was obtained after 20 hours. With inoculation approximately 102 spores/g moist barley. thus fungus grew more slowly and a barley cake with dense mycelial growth was not obtained until after 28 hours to 32 hours. This slow growth may increase the risk of contamination with pathogens. When Rhizopus oligosporus was inoculated at approximately 106 spores/g moist barley, the time for obtaining dense mycelial growth was shortened to 15 hrs to 20 hours. However, the growth was uneven, probably due to oxygen limitation in the center. Nout and Kiers [33] have also been reported the similar results for temph fermentation of soy bean.

The pH has a strong influence on the growth of mycelia and on better cake quality. The results showed in table-2.The initial pH was in the range of 2.5 to 4.0 and final in the range of 3.0 to 4.5The results were similar to that of Reu et al [34] determined that during fermentation the pH value of barley tempeh only increased slightly from pH 4.6 to pH 4.9. Reu et al [34] also reported that germination in aqueous suspension was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0.05 %( w/v incubation medium), at pH 4.0. When soybean was soaked in presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium but not in Tempe. However the results are also nearly similar to Eriksson et al [35].

The incubation time 30 to 36 hours was preferred. It was found that during this time period the fungus growth was very good, and the fungus mycelia evenly distributed in the entire product. The incubation time and temperature was found to be considerable importance. For the production of a fermented cereal product, and in particular a fermented whole grain barley based product of tempeh type. The best mycelial growth was reported at 30oC to 35oC (Table-2). The results of Steinkraus et al [19] are in good agreement. They reported that at 300C the mycelial growth was shown. Berg, et al [36] advocated optimum conditions for the germination of Rhizopus oligosporus were at 350C.

Reuetal [34]supported that inoculation of beans with Rhizopus oligosporus at various temperatures followed by incubation at 30oC resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 hours lag phase was found between initial bean temperatures of 25 and 37oC.

3.3 Characterization of cake

The barley cake production by its fermentation with Rhizopus oligosporus A-4 resulted in the production of very faint neutral and pleasant taste and flavor. The product has a light neutral color, as it was evident from the comparison of given fermented and unfermented Barley cakes in table-2. The quality of fermented barley makes it easy to add a desired taste, flavor or color to the invented product and the hygienic stability of the product was evident from its resistance to the contaminants. The product was stored at +8oC for 2 months. Table-2

3.4 Moisture, ash, carbohydrate, Protein, crude Fat, crude Fiber content during barley fermentation with Rhizopus oligosporus

The nutritional value of cereals are numerous, they can be a valuable source of energy. Energy is required for all metabolic processes. The energy cereals come from the nutrient supply of protein, fat and carbohydrate. The results of moisture, ash, carbohydrate, protein, crude fat and crude fiber are shown in table-3. The fat contents and carbohydrate percentage decreases after fermentation. Protein level was seen to be high. Deu Reu [34] gave approximately similar kinds of report. However carbohydrate contents reduced to 51.20% and 50.82 from 62.8%. Mulyowidaro et al [31] also gave similar report. The percentage of ash varied in each experiment at incubation temperature of 250C to 400C, from 0.9 to 1% after fermentation wherease the percentage of ash before fermentation was 1.21 to 1.05 respectively, so the ash contents were decreased after fermentation. Sahana Perveen et al [37] gave similar kind of report.

As the percentage of moisture varied from 10 to 10.8 at incubation temperature range of 25 0C to 40 0C, the results of N.G. Edwinoliver [38] showed the similar data for moisture analysis. Table-3

3.5 Estimation of the activity of Protease, Amylase and

Phytase enzymes in fermented barley

Protease is an important enzyme produced through fermentation by Rhizopus oligosporus A-4. It is considered worth wide to investigate the release of acid Protease from Rhizopus oligosporus during fermentation which might closely concern with the improvement of nutritional protein in Tempe. Acid protease released from Rhizopus oligosporus after 26 hours to 30 hours soaking at 25 to 400C, were studied. Acid Protease activity at pH 3.0 was greater than at pH 2.5, 3.5, 4.0. Simultaneously, no acid-Protease was detected not only in raw barley but also in pretreated barley prepared before inoculation. Moreover, it could be noticed that pH of enzyme extraction obtained from Tempe was in the pH stability range of acid-Protease from Rhizopus oligosporus. After 24 hours of fermentation at 280C,the mold propagate slowest when compared with that at 300C and 350C and caused to slightly release of acid Protease in Tempe.

In addition, the release at 300C was significantly higher than at 350C (table-4), Although the abundant and fastest growth of Rhizopus oligosporus A-4 was observed at 350C but the higher heat resulting from active growth was accumulated than at 350C and infact, resulted in increasing the incubation temperature and affecting the enzyme production. This is the reason why the release of acid-Protease was reduced when incubation temperature increased from 30 to 350C. Nevertheless, it can be concluded that there was a strong effect of incubation temperature on the release of acid-Protease in Tempeh after 26 hours fermentation. The highest acid-Protease activity was released with fermentation on at 300C for 26 hours. In addition, it can be noticed that the release of acid-Protease was dropped after 30 hours fermentation (Table-4) .Although many enzymes are often destructed by protease produced in the same culture.

Wang and Heseltine have reported that no degradation product of self-digestion acid-Protease isolated from culture filtrate of Rhizopus oligosporus could be detected when the enzyme preparation was incubated at 26 hours for 300C.Anyway ,Wang and Hesseltine have found that the inactivation of protease was explained as being caused by an alkaline shift in pH Acid-Protease produced by Rhizopus oligosporus are very unstable as pH approaches to a greater level. This was the reason why acid-protease was reduced in Tempeh after 30 hours. Consequently, it can be concluded that there was a significant effect of fermentation time on the release of acid-protease in Tempeh when incubated at 300C. The above mentioned results discussion is approximately similar to that of Waraw, Krusong et.a l[39].

Amylases are the enzymes which hydrolyse starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units. Each application of a -amylase requires unique properties with respect to specificity,

Table-1 Proximate composition of unfermented barley

Sr no.###Moisture%###Ash%###Carbohydrate###Protein###Crude fat###Crude fiber

###%###%###%###%

1###10.1###2.21###63.10###10.88###2.13###11.95

2###9.97###2.11###62.88###10.47###2.17###12.10

3###9.84###2.13###62.06###10.15###2.03###11.95

Mean

value###9.94###2.15###62.68###10.50###2.11###12.00

Table-2, optimization of various process parameters

Sr. No###Soaking Time(hr)###BoilingTime(min)###InoculumsSize(ml)###Initial pH###Incubation Time (hr)###Incubation Temp (0C)###Cake formation

1###24###15###0.5###2.5###24###25###_

2###24###15###0.5###2.5###30###25###_

3###26###20###0.75###3.0###30###30###+

4###26###20###0.75###3.0###36###30###++

5###28###25###1.0###3.5###36###35###+++

6###28###25###1.0###3.5###42###35###++

7###30###30###1.5###4.0###42###40###+

8###30###30###1.5###4.0###48###40###_

Where as; +++ =Excellent, ++ =very good, +###= moderate, - = poor

Table-3 Percentage of Ash, Moisture, Total protein, crude fat, fiber and Carbohydrates in fermented and unfermented barley.

Activity of Protease, Amylase and Phytase in fermented and unfermented barley

Sr.No.###Fermented###Parameters###Ash%###Moisture%###Total Protein%###Crude Fat%###Fiber%###Carbohydrate%###

###Soakig (h)###Boiling(min)###Fermented###Unfermented###Inoculum size (ml) Incubation temperature (oC) Incubation time(h)###Fermented Un-fermented###Fermented Un-fermented###Fermented Un-fermented###Fermented Un-fermented*###Fermented Un- fermented

1###2###15###10###10.5###0.5###25###24###0.9###1.21###9###8.5###1###1.3###2###0.9###65###77

###4

2###2###15###10.2###10.6###0.5###25###30###1###1.22###9.4###8.6###0.9###1.5###2.5###0.8###64###78

###4

3###2###20###10.1###10.1###0.75###30###30###0.8###1.23###9.9###8.9###0.9###1.23###2.1###0.8###63###77.5

###6

4###2###20###10###10.3###0.75###30###36###0.9###1.24###10###8.1###0.8###1.21###2.3###0.7###65###78

###6

5###2###25###10.5###10.2###1###35###36###1###1.1###10.2###9.1###0.8###1.12###2###1###64###76.6

###8

6###2###25###10.2###10.1###1###35###42###1.1###1.8###9.7###8.9###0.9###1.14###1.98###0.9###66###76.1

###8

7###3###30###10###10.3###1.5###40###42###1.2###1.05###9.5###9###1###1.31###2.01###0.9###65###76

###0

8###3###30###10.8###10.2###1.5###40###48###1###1.06###9.8###8.8###0.8###1.3###2###0.8###64###76.5

###0

Table 4-Activity of Protease, Amylase and Phytase in fermented and unfermented barley

Sr. No.###Fermentation Parameters###Protease###Amylase###Phytase

###Soaking(h) Boiling (min) Inoculum size (ml) Incubation temperature (oC) Incubation time(h)###Proteolytic Activity (mg/ml)###Control###Unit Activity (ug/ml)###Control###Unit Activity (ug/ml)###Control

1###24###15###0.5###25###24###302###289###179.03###43.466###24.5931###57.008

2###24###15###0.5###25###30###310###290###232.424###43.399###24.8104###56.005

3###26###20###0.75###30###30###325###300###871.59###62.669###22.44315###54.5497

4###26###20###0.75###30###36###320###298###232.724###62.596###19.04094###54.4497

5###28###25###1###35###36###358###300###179.251###61.669###19.612###55.235

6###28###25###1###35###42###389###280###276.943###61.659###21.556###55.23

7###30###30###1.5###40###42###310###285###1190.446###78.661###25.216###55.807

8###30###30###1.5###40###48###310###295###134.16###78.66###24.12996###55.8

(i) Protease activity in fermented and unfermented barley Average Standard factor=1233

(ii) Amylase activity in fermented and unfermented barley Average Standard factor=7520.91

(iii) Phytase activity in fermented and unfermented barley.Average Standard factor=285.90

Each experiment was conducted in triplicate

stability, temperature and pH dependence. It was observed that maximum production of enzyme by Rhizopus oligosporus A-4 occurred when cell population reached the peak. The maximum enzyme activity was at 42 hours (Table-4). The amylase synthesis by Rhizopus oligosporus A-4 has been correlated to the presence or absence of different nitrogen sources and various amino acids in the growth medium. A decrease in cell growth and enzyme production was observed when glucose was added to the fermentation. Among the physical parameters the pH of the growth medium plays an important role. The maximum yield of amylase occurred at pH 5. Enzyme synthesis occurred at temperature 350C.The influence of the temperature on the amylase production is related to the growth of organism. A wide range of temperature (35-800C) has been reported for the optimum growth and a-amylase production by Bajpai et al [40].However the results are nearly similar to Asgher et al [41].

Phytase [myo-inositol (1,2,3,4,5,6) hexakisphosphate], a phytate-specific phosphatase, is already used as a supplement in diets monogastric animals to improve phosphate utilization This class of enzyme has also been found increasingly interesting for use in processing and manufacturing of food for human consumption ,particularly because the decline in food phytate result in enhancement of mineral bioavailability.Various food processing and preparation methods result in a reduction in the phytate content of the processed material. Regarding enzymatic phytate dephosphorylation during food processing and preparation, adjustment of optimal conditions during food processing for the native plant or microbial phytases has to be distinguished from the addition of exogenous one. Phytate hydrolysis during, for example, germination, soaking, cooking and fermentation is a result of the phytate-degrading activity naturally present in plants and microorganisms.

The temperature and pH value are the major factors determining enzyme activity, favorable properties for phytase that should be used in food processing are high phytate-degrading capability even at room temperature, acceptable heat resistance and a high activity over a broad pH range. According to pH optimum phytase could be classified in to acid phytases with pH from 3.5-6.0. The majority of the so far characterized phytases showed maximal phytate-grading activity in the acid pH range. So the maximum activity was shown at pH 3.5. Enzyme activity increases with temperature up to maximal temperature. A further increase in temperature results in a heat-induced denaturation of the enzymes. Depending on the enzyme source, optimal temperature for phytate hydrolysis varies from 35 to 400C. As the fermentation time increases the enzyme activity changed so the time influenced the activity of enzyme at 400C for 42 h was maximum. This discussion is very similar with the description of Ralf Greiner et al [42].

CONCLUSION

From the results obtained in this work, it can be inferred that Pakistani barley cereal is rich of Carbohydrate (62.68%), Crude fiber (12.00 %), Protein (10.50 %), Moisture (9.94%), Ash (2.15%) and Crude fat (2.11%). After fermentation the nutritional value of barley tempeh is improved i.e. the percentage of moisture is increased while the ash and fat percentage decreased and is effective diet for cancer patients. The activity of protease and amylase is increased after fermentation where as the activity of phytase is decreased. The development of Koji fermentation technology in Pakistan is essential for improvement of food and production of well trained and skilled manpower.

ACKNOWLEDGMENTS

Author acknowledges the financial support from the HEC, Govt. of Pakistan under the INDEGINOUS 5000 FELLOWSHIP PROGRAM, PIN No. 074-0697-Ps4-383. Auther is also thankful to Education Department, Govt of the Punjab for grant of study leave with full pay to complete the Ph.D.

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Institute of Biochemistry and Biotechnology, Punjab University, Lahore, Pakistan

Food and Biotechnology Research Center PCSIR Laboratories Complex, Ferozpur Road Lahore, 54600 Pakistan

Department of Chemistry, Government Islamia Post Graduate College Gujranwala, Pakistan
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