Study of Red Sorghum (Sorghum Bicolor) Fermentation By Saccharomyces Cerevisiae Strain MTCC 170 and Its Effect on Total Polyphenolic Compound

Study of Red Sorghum (Sorghum Bicolor) Fermentation By Saccharomyces Cerevisiae Strain MTCC 170 and Its Effect on Total Polyphenolic Compound

Amesh V. Pawar*, Vishal R. Parate, Mohammad I. Talib

Department of Food Technology, University Institute of Chemical Technology Jalgaon 425001, Country- India

Abstract– The main objective of this work was to study the yeast fermentation and its effect on polyphenolic compound present in red sorghum (Sorghum bicolor (L.) monech) at different temperature ranges and also at different concentration of mash of red sorghum. During the study it was observed that the total polyphenolic compounds in fermentation broth were slightly increased up to 24 hrs. and rapidly increased till the end of 72 hrs. The changes occur in polyphenolic compound was different at different temperature. The maximum changes occur at 300 C and 250 C but there was minute increase at 350 C.It was also observed that the polyphenolic compound increase in concentration of mash. The maximum biomass and ethanol percentage were observed at 15% mash concentration at 300 C and at pH 6 followed by 250C and maximum sugar was consumed at the same.

Keywords- Polyphenolic Compound, Temperature, Fermentation, Red Sorghum.

1. INTRODUCTION

   The strain MTCC170 From IMTECH, Chandigarh of Saccharomyces Cerevisiae having capacity to produce Beta fructofuranosidases (invertase) enzyme that cleave Beta- 1,4- glucosidic linkage between D- glucose and D- fructose molecules of sucrose by hydrolysis producing glucose and fructose. Beta- fructofuranosidases are intracellular enzyme. It is used fermentation of cane molasses into ethanol, in calf feed preparation and also in manufacture of inverted sugar as food for honeybees [1].

   India is semi-arid country and his main cereal crop is Sorghum (sorghum bicolor (L.) Monech). India is ranked

   second in world to production of sorghum. Other cereals like wheat (Triticum aestivum L.) and rice (Oryza sativa L.) require more water and fertilizer than sorghum [2]. Red sorghum mainly contains tannins, which are located

   primarily in testa layer of the grain. Comparatively sorghum contain higher amount polyphenolic compound. The amount of polyphenolic compound in sorghum depends on genetic factor and environmental factor. Mainly sorghum ranges white color to red color and red color contain high amount of polyphenolic compound. There is direct relationship between polyphenolic compound and antioxidant activity and antimicrobial activity [3].

   Polyphenolic compounds are very important in human diet as they have many medicinal properties such antioxidant, prevent heart diseases, anticarcinogenic. There two sources from where the polyphenolic compounds can be derived, they are phenylalanine and tyrosine [4][5][6] sorghum may contain benzoic acid phenylpropanoids, flavonoids,tannins,lignins Chemically, phenolics can be defined as substances possessing an aromatic ring bearing one or more hydroxyl groups, including their functional derivatives [7][8].Sorghum is very important grain for human consumption in Asian and African region and in U.S. it is used as source of animal feed [9] .Phenolics compound contributes to the flavor of the beer and also with implicated in nonbiological haze formation [10][11][12]. Phenolics substances in beer are present in various forms; theirolatility depends on their molecular weight Beer contains 283 mg/L of nontannin and nonflavonoid phenolics compounds [13]. Phenolics compounds of beer are classified as beneficial, harmful, or neutral in so far as their influence on beer stability and sensory properties is concerned [14].

   Some beer phenolics may also act as antioxidants or may contribute to the formation of carbonyls in beer. It has been found that ferulic acid markedly increases the formation of carbonyls in beer aging under high and low air conditions. Moreover, ferulic acid and quercetin have been found to be active promoters of diacetyl formation in aging beer [15].

   Some 67 different phenolics compounds have been identified in beer. Simple phenolics, aromatic carboxylic- and phenol carboxylic acids,hydroxycoumarins,catechins, leucoanthocyanidins,anthocyanidins,flavonols,flavonones,f lavones,prenylated flavonoids and Phenolicglycosides are included in this list [16][17][18][19].

   The objective of the present investigation was to determine the effect of yeast fermentation on total polyphenolic compounds of red sorghum.

2. MATERIALS AND METHOD

   Strain

   Saccharomyces Cerevisiae MTCC 170, IMTECH, Chandigarh.

   Raw material

   Red Sorghum was procured from local market of Jalgaon.

   Malting

   Malting involves germination, steeping and limiting growth of seed. During malting the enzymes are generated these are -Amylase, – Amylase, -Glucosidase and Peroxidases [20] .Amylase catalyses random hydrolysis of starch at (1, 4) linkage Amylase activity in sorghum malt is 25 to 183 U/g depending on sorghum variety [21].Beta amylase catalyses the hydrolysis of penultimate (l, 4) glycosidic bond at non- reducing end of polysaccharides causing the release of maltose. Amylase activity in sorghum malt is 11 to 41 SDU/g [Sorghum Diastatic Unit/g][22]. Glucosidase in germinating grains catalyses hydrolysis of terminal, non-reducing a-(l, 4) glucosidic linkages in both oligosaccharides and glucans yielding glucose [23]. Peroxidase catalyses the reductive destruction of hydrogen peroxide and invariably contributes to the defense system of living organism against peroxidation of unsaturated lipids involving oxygen radicals40. Lipid peroxidation is undesirable in malting and brewing and result in the production of hydroperoxides and their decomposition products, aldehydes[24].Lipase (triacylglycerol acyhydrolase) catalyses the hydrolysis of triacylglycerides to free fatty acids and Glycerol [25].Sorghum grains contain detectable lipase activity which vary slightly during 24 h steeping period at 30°C and

   increases during germination to about 4-fold after 96 h.

   However, lipase activity varies among different sorghum cultivars [26].

   Mashing

   Mashing in conventional brewing is basically by two methods, viz., decoction and infusion processes. During mashing water soluble substances dissolve, enzymes hydrolyse gelatinised starch and solubilised proteins and to a lesser extent other higher molecular weight substances essential for the type and character of beer, and finally dissolved substances are separated. Hydrolyses of substances involve enzymes such as amylases, proteases, peptidases, transglucosidases and phosphorylases which are regulated by factors like temperature, pH, time and concentration of the wort. Mashing extracts about 80% of the dry matter from the malt while cold water extracts about 15% [27]. Mashing of sorghum malt at 65°C and 70°C for 30 min each, at second and third stages respectively, of

   three stage decoction process, provides wort with complete hydrolysis [28].

   Fermentation

   Alcohol Fermentation is a process where different types of species of yeast are used to produce ethanol. The most economical species is Saccharomyces Cerevisiae which gives desirable amount of ethanol. The fermentation process is affected by various factors such as temperature, pH, sugar concentration, incubation period and media composition [1].

   Determination of Alcohol

   The alcohol content was calculated by potassium dichromate method. General principle includes that the potassium dichromate is yellowish color solution reacts with alcohol in presence of sulphuric acid and forms green color complex due to reductionof potassium dichromate and it is directly proportional to the content of alcohol. The absorbance is measured at 575 nm [29].

   Determination of sugar

   Sugar can be determined by phenol sulphuric acid method. Principal of this method is the carbohydrate means disaccharides polysaccharides and simple sugar reacts with strong acid and heat to generate furan derivatives that condensed to phenol which is yellow golden color complex which directly proportional to concentration of total sugar. The absorbance is measured at 550 nm [30].

   Determination of biomass

   Biomass produced during fermentation can be calculated by simple filtration method [31]. Simply filter the sample only for five minute at room temperature otherwise it will affect on the concentration of volatile component. Place the filter paper to hot air oven for drying for overnight. Next day simply weight the paper [32].

   Determination of total polyphenolic compound Folin- Denis Assay

   The Folin-Denis assay is the most widely used procedure for quantification of total phenolics in plant materials and beverages. Reduction of phosphomolybdic- phosphotungstic acid (Folin-Denis) reagent to a blue- colored complex in an alkaline solution occurs in the presence of phenolics compounds [33]. Folin-Ciocalteu reagent is not specific and detects all phenolics groups found in extracts including those found in the extractable proteins. A disadvantage of this assay is the interference of reducing substances such as ascorbic acid with the determinations. The total phenolics are assayed colorimetrically as modified [34] [35]. 2.5 mL of 10-fold diluted Folin-Ciocalteu reagent, 2 mL of a 7.5% solution of sodium carbonate, and 0.5 mL of phenolics solution are mixed well. The absorbance is measured at 765 nm after a 15-min heating at 45°C; a mixture of water and reagents is used as a blank. The content of phenolics is expressed as Gallic acid or catechin equivalent.

   Yield

   Yield (YPS) was calculated by using following formula

   YPS

   = Product produced (ethanol ) substrate consumed (sugar )

   Substrate Conversion Ratio

   Substrate Conversion Ratio (SCR) was calculated by using below formula

   SCR= Initial Sugar Residual sugar

   Initial sugar

3. RESULT AND DISCUSSION

   Effect of yeast fermentation at 250C and 24 hrs. on sugar, ethanol, polyphenolic compound and biomass

   Table 1. Effect of Yeast Fermentation at 250C and 24 Hrs. On Sugar, Ethanol, Polyphenolic Compound and Biomass Values are Mean of Triplicate.

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound Biomass
   (%)	(gm)	(gm)	(%)	Yps SCR	(µgm/ml) (gm)
   5	3.5159	2.1718	0.5728	0.4261 0.3822	1.0733 0.3645
   10	7.0318	5.3618	0.7451	0.4461 0.2374	1.2992 0.2501
   15	10.5477	8.066	1.087	0.4369 0.2358	1.4247 0.4482
   20	14.0636	12.4705	0.7078	0.4442 0.1132	1.8686 0.2540
   25	17.5795	16.9037	0.2851	0.4218 0.0384	1.9546 0.0411

   Table no. 1 showing that the maximum sugar was consumed; maximum alcohol production and high biomass were produced at 15% concentration. Maximum yield was at 10% mash concentration and maximum substrate conversion was at 5% mash concentration. The maximum polyphenolic compounds are observed at 25% mash concentration.

   2.5

   Total polyphenolic compound µgm/ml

   2

   1.5

   1

   0.5

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   Red sorghum mash concentration %

   10 15 20 25

   Residual sugar(gm)

   5

   Initial sugar(gm)

   20

   18

   16

   14

   12

   10

   8

   6

   4

   2

   0

   Sugar %

   Total polyphenolic compound µgm/ml

   Figure 1. Effect of fermentation at 250C and time 24 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate.

   Fig. no. 1 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration.

   Figure 3. Effect of fermentation at 250C and time 24 hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method. Values are mean of triplicate

   Fig. no. 3 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash concentration.

   1.2

   1

   Alcohol %

   0.8

   0.6

   0.4

   0.2

   0

   5 10 15 20 25

   Alcohol%

   0.5

   0.45

   0.4

   0.35

   Biomass gm

   0.3

   0.25

   0.2

   0.15

   0.1

   0.05

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   Biomass(gm)

   Red sorghum mash concentration %

   Figure 2. Effect of fermentation at 250C and time 24 hrs. on alcohol, determined by potassium dichromate method. Values are mean of triplicate

   Fig. no. 2 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   Figure 4. Effect of fermentation at 250C and time 24 hrs. on biomass, determined by filtration method. Values are mean of triplicate

   Fig. no. 4 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

   Effect of yeast fermentation at 250C and 48 hrs. on sugar, ethanol, polyphenolic compound and biomass

   Table 2. Effect of Yeast Fermentation at 250C and 48 Hrs. on Sugar, Ethanol, Polyphenolic Compound and Biomass Values are Mean of Triplicate

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound	Biomass
   (%)	(gm)	(gm)	(%)	Yps	SCR (µgm/ml)	(gm)
   5	3.5159	1.3868	0.9372	0.4401	0.6055 1.0905	0.3503
   10	7.0318	1.2444	2.5823	0.4461	0.823 1.3851	0.5728
   15	10.5477	0.7926	4.5281	0.4641	0.9248 1.8034	1.5189
   20	14.0636	8.355	2.4935	0.4367	0.4059 2.0465	0.5614
   25	17.5795	15.5964	0.8725	0.4399	0.1128 2.2131	0.2380

   Table no. 2 showing that the maximum sugar was consumed, maximum alcohol production, high biomass was production and Maximum yield was at 15% mash concentration and maximum substrate conversion was at 15% mash concentration. The maximum polyphenolic compounds are observed at 25% mash concentration but the maximum changes in total polyphenolic compound were observed at 15% mash concentration.

   20

   18

   16

   14

   Sugar %

   12

   10

   8

   6

   4

   2

   0

   5 10 15 20 25

   Initial sugar(gm) Residual sugar(gm)

   5

   4.5

   4

   3.5

   Alcohol %

   3

   2.5

   2

   1.5

   1

   0.5

   0

   5 10 15 20 25

   <>Alcohol%

   Red sorghum mash concntration % Red sorghum mash concentration %

   Figure 5 Effect of fermentation at 250C and time 48 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate

   Figure 6 Effect of fermentation at 250C and time 48 hrs. on alcohol, determined by potassium dichromate method. Values are mean of triplicate

   Fig. no. 5 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration

   Fig. no. 6 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   2.5

   2

   Total polyphenolic compound µgm/ml

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   1.5

   1

   0.5

   0

   0.2

   0

   Biomass(gm)

   1.6

   1.4

   1.2

   1

   0.8

   0.6

   0.4

   Total polyphenolic compound µgm/ml

   Biomass gm

   Figure 7 Effect of fermentation at 250C and time 48 hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method. Values are mean of triplicate

   Fig. no. 7 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash concentration. But as compared to fig. no. 3 the maximum effect on polyphenolic compound was at 15% mash concentration.

   Figure 8 Effect of fermentation at 250C and time 48 hrs. on biomass, determined by filtration method. Values are mean of triplicate

   Fig. no. 8 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

   Effect of yeast fermentation at 300C and 24 hrs. on sugar, ethanol, polyphenolic compound and biomass.

   Table 3 Effect of yeast fermentation at 300C and 24 hrs. on sugar, ethanol, polyphenolic compound and biomass Values Are Mean Of Triplicate

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound Biomass
   (%)	(gm)	(gm)	% Yps	SCR	(µgm/ml) (gm)
   5	3.5159	1.9053	0.7075 0.4392	0.458	1.1188 0.1196
   10	7.0318	4.8749	0.968 0.4487	0.3067	1.3336 0.3119
   15	10.5477	7.6612	1.2758 0.4419	0.2736	1.3791 0.5375
   20	14.0636	11.7036	1.0751 0.4555	0.1678	1.8498 0.267
   25	17.5795	16.8267	0.3312 0.4399	0.0428	1.8701 0.0453

   Table no. 3 showing that the maximum sugar was consumed; maximum alcohol production and high biomass were produced at 15% concentration. Maximum yield was at 20% mash concentration and maximum substrate conversion was at 5% mash concentration. The maximum polyphenolic compounds were observed at 25% mash concentration.

   20

   18

   16

   14

   12

   10

   8

   6

   4

   2

   0

   Initial sugar(gm)

   Residual sugar(gm)

   2

   1.8

   1.6

   1.4

   1.2

   1

   0.8

   0.6

   0.4

   0.2

   0

   Total polyphenolic compound µgm/ml

   Figure 9 Effect of fermentation at 300c and time 24 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate.

   Fig. no. 9 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   Sugar gm

   Total polyphenolic compound µgm/ml

   Figure 11 Effect of fermentation at 300c and time 24 hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method. Values are mean of triplicate.

   Fig. no. 11 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash concentration.

   1.4

   1.2

   Alcohol %

   1

   0.8

   0.6

   0.4

   0.2

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   Biomass(gm)

   0.6

   0.5

   0.4

   0.3

   0.2

   Biomass gm

   Alcohol%

   0.1

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   Figure 10 Effect of fermentation at 300c and time 24 hrs. on Alcohol, determined by potassium dichromate method. Values are mean of triplicate

   Fig. no. 10 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   Figure 12 Effect of fermentation at 300c and time 24 hrs. on biomass, determined by Filtration method. Values are mean of triplicate.

   Fig. no. 12 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

   Effect of yeast fermentation at 300C and 48 hrs. on sugar, ethanol, polyphenolic compound and biomass.

   Table 4 Effect of yeast fermentation at 300C and 48 hrs. on sugar, ethanol, polyphenolic compound and biomass the values are mean of triplicate

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound	Biomass
   (%)	(gm)	(gm)	(%)	Yps	SCR (µgm/ml)	(gm)
   5	3.5159	0.9093	1.1684	0.4482	0.7413 1.2168	0.3663
   10	7.0318	0.5256	2.9558	0.4543	0.9252 1.5324	0.6351
   15	10.5477	0.4243	4.8891	0.4829	0.9597 1.7029	1.6495
   20	14.0636	5.7663	3.6379	0.4384	0.5899 2.342	0.6283
   25	17.5795	14.4677	1.2143	0.3902	0.177 2.3626	0.2648

   6

   5

   4

   3

   2

   Initial sugar(gm)

   Residual sugar(gm)

   Sugar %

   Alcohol %

   Table no. 4 showing that the maximum sugar was consumed, maximum alcohol production and high biomass was produced at 15% concentration. Maximum yield and maximum substrate conversion was also at 15% mash concentration. The maximum polyphenolic compounds were observed at 25% mash concentration but maximum changes in polyphenolic compound was observed at 15% mash concentration.

   20

   18

   16

   14

   12

   10

   8

   6

   4

   2

   0

   Alcohol%

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   1

   0

   Figure 13 Effect of fermentation at 300 C and time 48 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate.

   Fig. no. 13 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration.

   Figure 14 Effect of fermentation at 300C and time 48 hrs. on alcohol, determined by potassium dichromate method. Values are mean of triplicate.

   Fig. no. 14 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   2.5

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   0.5

   Total polyphenolic compound µgm/ml

   1

   1.5

   2

   Total polyphenolic compound µgm/ml

   Figure 15 Effect of fermentation at 300c and time 48 hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method. Values are mean of triplicate.

   Fig. no. 15 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash

   concentration. But as compared to fig. no.11 the maximum effect on polyphenolic compound was at 15% mash concentration.

   1.8

   1.6

   1.4

   1.2

   1

   0.8

   0.6

   0.4

   0.2

   0

   Biomass(gm)

   5 10 15 20 25

   Red sorghum mash concentration %

   Biomass gm

   Figure 16 Effect of fermentation at 300c and time 48 hrs. on biomass, determined by Filtration method. Values are mean of triplicate.

   Fig. no.16 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

   Effect of yeast fermentation at 350C and 24 hrs. on sugar, ethanol, polyphenolic compound and biomass

   Table 5 Effect of yeast fermentation at 350C and 24 hrs. on sugar, ethanol, polyphenolic compound and biomass the values are mean of triplicate

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound Biomass
   (%)	(gm)	(gm)	(%) Yps	SCR	(µgm/ml) (gm)
   5	3.5159	2.7652	0.3094 0.4121	0.2135	1.1352 0.0142
   10	7.0318	6.4445	0.3225 0.4543	0.0835	1.2743 0.0165
   15	10.5477	9.275	0.5292 0.4158	0.1206	1.331 0.0334
   20	14.0636	13.3227	0.3247 0.4382	0.0526	1.5787 0.0154
   25	17.5795	16.9156	0.2961 0.446	0.0377	1.7287 0.0061

   Table no. 5 showing that the maximum sugar was consumed; maximum alcohol production and high biomass were produced at 15% concentration. Maximum yield was at 10% mash concentration and maximum substrate conversion was at 5% mash concentration. The maximum polyphenolic compounds were observed at 25% mash concentration.

   5 10 15 20 25

   Red sorghum concentration %

   6

   4

   2

   0

   Initial sugar(gm)

   Residual sugar(gm)

   20

   18

   16

   14

   12

   10

   8

   Sugar %

   Total polyphenolic compound µgm/ml

   Figure 17 Effect of fermentation at 350C and time 24 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate

   Fig. no. 17 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration.

   2

   1.8

   1.6

   1.4

   1.2

   1

   0.8

   0.6

   0.4

   0.2

   0

   Total polyphenolic compound µgm/ml

   5 10 15 20 25

   Red sorghum mash concentration %

   Figure 19 Effect of fermentation at 350C and time 24 hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method. Values are mean of triplicate.

   Fig. no. 19 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash concentration.

   0.04

   0.035

   0.6

   0.5

   0.4

   0.3

   0.2

   0.03

   Biomass gm

   0.025

   0.02

   Alcohol %

   0.015

   0.01

   Alcohol%

   0.005

   0.1

   0

   0

   Biomass(gm)

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   Figure 18 Effect of fermentation at 350C and time 24 hrs. on Alcohol, determined by potassium dichromate method. Values are mean of triplicate

   Fig. no. 18 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   Figure 20 Effect of fermentation at 350C and time 24 hrs. on biomass, determined by filtration method. Values are mean of triplicate.

   Fig. no.20 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

   Effect of yeast fermentation at 350C and 48 hrs. on sugar, ethanol, polyphenolic compound and biomass

   Table 6 Effect of yeast fermentation at 350C and 48 hrs. on sugar, ethanol, polyphenolic compound and biomass the values are mean of triplicate

   Mash conc.	Initial sugar	Residual sugar	Alcohol		Total polyphenolic compound Biomass
   (%)	(gm)	(gm)	(%)	Yps SCR	(µgm/ml) (gm)
   5	3.5159	2.6941	0.3338	0.4061 0.2337	1.1644 0.0267
   10	7.0318	6.1207	0.4028	0.4421 0.0911	1.2881 0.0238
   15	10.5477	9.1781	0.5799	0.4234 0.1298	1.3516 0.1078
   20	14.0636	13.2004	0.379	0.439 0.0653	1.5999 0.0193
   25	17.5795	16.5758	0.323	0.3218 0.057	1.7536 0.0033

   20

   18

   16

   14

   12

   10

   8

   6

   4

   2

   0

   Sugar %

   Alcohol %

   Table no. 6 showing that the maximum sugar was consumed; maximum alcohol production and high biomass was produced at 15% concentration. Maximum yield was at 10% mash concentration and maximum substrate conversion was at 5% mash concentration. The maximum polyphenolic compounds were observed at 25% mash concentration but maximum changes in total polyphenolic compound was observed at 15% mash concentration

   0.7

   0.6

   0.5

   0.4

   0.3

   Alcohol%

   0.2

   0.1

   0

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   Initial sugar(gm)

   Residual sugar(gm)

   Figure 21 Effect of fermentation at 350 C and time 48 hrs. on sugar, determined by phenol sulphuric method. Values are mean of triplicate.

   Fig. no. 21 showing that the maximum sugar was consumed at 15% mash concentration and minimum at 25% mash concentration.

   Figure 22 Effect of fermentation at 350C and time 24 hrs. on Alcohol, determined by potassium dichromate method.

   Values are mean of triplicate.

   Fig. no. 22 showing that the maximum alcohol was produced at 15% mash concentration and minimum at 25% mash concentration.

   2

   1.8

   1.6

   1.4

   1.2

   1

   0.8

   0.6

   0.4

   0.2

   0

   Total polyphenolic compound µgm/ml

   0.12

   0.1

   0.08

   0.06

   0.04

   Biomass(gm)

   5 10 15 20 25

   Red sorghum mash concentration %

   5 10 15 20 25

   Red sorghum mash concentration %

   Total polyphenolic compound µgm/ml

   Figure 23 Effect of fermentation at 350C and time 48hrs. on total polyphenolic compound, determined by Folin-Ciocalteu method.

   Values are mean of triplicate.

   Fig. no. 23 showing that the maximum total polyphenolic compound found at 25% mash concentration and minimum at 5% mash concentration. But as compared to fig. no. 19 the maximum effect on polyphenolic compound was at 15% mash concentration.

   0.02

   0

   Biomass gm

   Figure 24 Effect of fermentation at 350C and time 48 hrs. on biomass, determined by filtration method. Values are mean of triplicate.

   Fig. no.24 showing that the maximum biomass was produced at 15% mash concentration and minimum at 25% mash concentration.

4. CONCLUSION

   From the above study it was conclude that maximum sugar consumption, alcohol production, biomass production was occurred at 300C and 15% mash concentration. Maximum changes in total polyphenolic compound were observed at 300C temperature and 15% mash concentration. The same results were followed by fermentation. At 250C the consumption of sugar and production of alcohol and biomass was decreased as compared to 300C.At 350C there was no adequate consumption of sugar and production of alcohol and biomass.

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