Indian Patents. 224338:"COMPOSITION COMPRISING BIOENHANCERS FOR INHIBITION OF BROWNING OF WHOLE WHEAT"

Title of Invention	"COMPOSITION COMPRISING BIOENHANCERS FOR INHIBITION OF BROWNING OF WHOLE WHEAT"
Abstract	The invention discloses a composition comprising a blend of bioenhancers for inhibition of browning of whole wheat by a process of phenolic substrate modification. The blend of bioenhancers comprises pentosanases, proteolytic enzymes, redox enzymes, reducing agents, acidulants, stabilizers, surfactants and fillers.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION [See section 10, Rule 13] COMPOSITION COMPRISING BIOENHANCERS FOR INHIBITION OF BROWNING OF WHOLE WHEAT WE, ADVANCED ENZYME TECHNOLOGIES LIMITED, A COMPANY ORGANISED AND EXISTING UNDER THE LAWS OF INDIA WHOSE ADDRESS IS SUN MAGNECTICA, 5TH FLOOR NEAR LIC SERVICE ROAD, LOUIS WADI THANE-400 604, MAHARASHTRA, INDIA. THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE Technical Field of Invention: The present invention relates to the use of scientifically blended bioenhancers with browning inhibitors to irreversibly modify the phenolic substrate, which in turn inactivates poly phenol oxidase in whole wheat. The resultant product has controlled browning effect than that compared to natural whole wheat dough. The present invention attributes technologically and economically to the life of common man through the improvement and benefits brought about herein. Background of Invention and Prior Art: Browning of foods during processing and storage, especially during manufacture of meat, fish, fruit and vegetable products decreases the sensory properties of products due to associated changes in colour, flavour and softening, besides nutritional properties. Therefore, its control is essential to preserve the quality of the food. Appearance, flavour, texture and nutritional value are four attributes considered by consumers when making food choices. Appearance, which is significantly impacted by colour, is one of the first attributes used by consumers in evaluating food quality. Colour may be influenced by naturally occurring pigments such as chlorophylls, carotenoids and anthocyanins in food, or by pigments resulting from both enzymatic and non-enzymatic reactions. Enzymatic browning is a significant problem in a number of important commodities, specially fruits such as apricots, apples, pears, peaches, bananas and grapes; vegetables such as potatoes, mushrooms ,lettuce and sea food. This discoloration limits the shelf life as well as the acceptance of the product. The major enzymatic browning is caused by poly phenol oxidase enzyme present inherently in the fruits, vegelabJes, cereaJs etc. Mechanism of browning due to poly phenol oxidase can be explained as follows: Enzymatic browning is the discoloration that results when monophenolic compounds of plants or shellfish, in the presence of atmospheric oxygen and poly phenol oxidase (PPO), are hydroxylated to ortho phenols, and the latter are oxidized to ortho quinines. The quinine condenses and reacts nonenzymatically with other phenolic compounds to produce dark brown, black or red pigment of intermediate structure. 2 Many approaches have been done in fruits but not in whole wheat. The present invention is related to the prevention of enzymatic browning in whole wheat dough by using a blend of bioenhancers. Whole wheat is nutritious than white wheat. This is because whole wheat retains bran layer containing iron (non-haem), zinc, copper, thiamin, riboflavin and niacin which are water solubles. Whole wheat aleuronic layer of cells contains valuable nutrients like vitamins B complex, vitamin E and minerals. The present invention is related to prevent the enzymatic browning in chapatti, breads, rotis, nans etc made with whole wheat dough. The whole wheat retains the nutritional value with improved visual appearance. The main basis of the present invention is to improve the organoleptic properties of whole wheat products and to retain its nutritional value. Abstract of EP0903083 discloses process for inhibiting enzymatic browning and maintaining textural quality of fresh peeled potatoes, where a process is disclosed for inhibiting enzymatic browning in raw, peeled potatoes comprising dipping the potatoes in a solution of heated organic acids (45-65°C), followed by treatment in a weakly basic solution to neutralize the potato surface and treatment with reducing agents, and followed by storage in modified atmosphere packaging. The process tends to delay the onset of enzymatic browning and, once browning has begun, limits the extent of enzymatic browning. Abstract of US6020018 discloses inhibition of enzymatic browning of raw fruit and/or vegetable juice. The juice is treated with at least one sulfated polysaccharide in an amount sufficient to inhibit browning. A promoter may also be present, said promoter is selected from the group consisting of chelating agents, acidulents, or mixtures thereof. Abstract of W08911227 describes inhibition of enzymatic browning, a process for inhibiting oxidative darkening of foods by treating the food with a protease effective to inhibit oxidative darkening of the food, a composition for inhibiting oxidative darkening and a kit for preparing the composition. Abstract of US 4,814,192 discloses process for preserving raw fruits and vegetables, including juices, comprising treating the products with ascorbic acid-2-phosphate esters and ascorbyl-6-fatty acid esters, individually or in combination. The treatments may be applied in an aqueous carrier and may further comprise other browning inhibitors, polyphenol oxidase inhibitors, emulsifying agents, dispersing agents and complexing agents. Treatments tend to delay or prevent the onset of enzymatic browning or, once browning has begun, to limit the extent of enzymatic browning. Object of the Invention: The main object of present invention relates to the use of bioenhancers with browning inhibitor in the whole wheat to inactivate the poly phenol oxidase and thereby to reduce the undesirable browning. The other object of present invention relates to the fact that the nutritional value of the whole wheat flour before and after treatment with formulation of bioenhancers with browning inhibitor is retained without any lowering or deterioration. Summary of the Invention: The object of the invention is to provide a process for modification of phenolic substrates as bioenhancers by inhibiting poly phenol oxidase activity in the whole wheat. The bioenhancer comprises of specific pentosanases which act on pentosans like pectin, xylan and such like, proteolytic enzymes selected from neutral proteases, papain and such like, redox enzymes such as catalase, peroxidase and such like in combination with reducing agents such as cysteine, glutathione and ascorbic acid with chelating agents such as phosphates, EDTA and such like with some acidulants like fumaric acid, phosphoric acid and citric acid with some stabilizers. Phenolic substrate modification by bioenhancers in whole wheat formulation comprises of bioenhancers such as pentosanases - 0.1-5%, proteolytic enzyme -0.1-2%, redox enzymes - 0.1-3%, reducing agents such as L-cysteine, ascorbic acid 0.5-6%, stabilizers such as sodium and calcium salts at 7-10% with acidulants such as fumaric acid and maleic acid at 10- 20%, thus phenolic substrate modification by the use of bioenhancers in the range of 0.1 - 0.4 % reduces the rate of browning in the whole wheat flour preparation, yet retaining its nutritional value. Detailed Description: The present invention claims the composition which consists of blend of bioenhancers like specific pentosanases which act on pentosans like pectin, xylan, cellulose and proteolytic enzymes like neutral proteases, plant protease (papain, bromalein, ficin) and redox enzymes such as catalase, gluconase oxidase and lipoxidase. The bioenhancers are formulated in conjunction with browning inhibiting components like reducing agents namely cysteine, glutathione and ascorbic acid, chelating agents such as phosphates and EDTA. The rate of action of bioenhancers and browning inhibitors can be enhanced with acidulants like fumaric acid, phosphoric acid, ascorbic and citric acid which stabilize the whole browning inhibition in conjunction with calcium, sodium salts and emulsifying agents such as polysorbates. The present invention discloses the range of composition of bioenhancer such as pentosanase - 0.1 - 5%, proteolytic enzyme - 0.1- 2%, redox enzymes 0.1- 3%, reducing agents 0.5-6%, stabilizers such as sodium and calcium salts at 7-10% with acidulants 10- 20% and white dextrin, defatted soya flour as inert filler: q.s. (1) Bioenhancer The mechanism of bioenhancers can be exploited for the control of undesirable enzyme activities to inhibit browning effect. 1. Substrate and/or product modification other than the target enzymes 2. Direct inactivation of the target enzyme. 3. Inactivation by secondary reactions of highly reactive products. (i)Pentosanase: It helps in selective inhibition of browning by irreversible modification of phenolic substrates. Due to distorted phenolic structure of substrate, the active site on the poly phenol oxidase cannot come in contact with the substrates to give 100% reaction thus helping in reducing the browning effect. (ii)Proteolytic enzymes: The plant proteases like ficin, papain, and bromelain are sulphydryl enzymes of broad specificity, which are very effective browning inhibitors. This inhibitory effect is thought to be due to either binding or hydrolysis at specific sites necessary for poly phenol oxidase activity. (iii)Redox enzymes: 5 These bioenhancers are capable of methylating the 3 rd position of 3, 4 dihydroxy aromatic compounds. It was observed to cause irreversible modification of phenolic substrates, thus preventing them from serving as substrates for poly phenol oxidase, thus preventing browning reaction. (2) Reducing agents Reducing agents play a role in the prevention of enzymatic browning either by reducing O-quinones of the phenolic substrates to colorless diphenols, or by reacting irreversibly with O-quinones to form stable colorless products. (i)Ascorbic acid is a strong reducing compound, which is acidic in nature, forms neutral salts with bases and is highly water soluble. Polyphenol oxidase inhibition by ascorbic acid has been attributed to the reduction of enzymatically formed O-quinones to their precursor diphenols. Ascorbic acid is irreversibly oxidized to dehydroascorbic acid during the reduction process, thus preventing browning reaction to the desirable extent. (ii)Cysteine is an effective inhibitor of enzymatic browning. It is reported to be more effective than bisulphites. Cysteine forms intermediate complex with quinine, which serves as competitive inhibitor of polyphenol oxidase enzyme. (3) Acidulants Acidulants are generally applied in order to maintain the acidity for optimum catalytic activity of an enzyme. Acidulants such as citric acid, malic acid and phosphoric acid are capable of lowering acidity of the system, thus rendering poly phenol oxidase inactive. (4) Chelators Enzymes generally posses metal ions at their active sites. Removal of these ions by chelating agents can therefore render enzymes inactive. Chelating agents like sodium salt of ethylene diamine tetra acetic acid, sodium tripolyphosphate with pro-oxidative agents such as copper and iron ions, act through an unshared pair of electrons in their molecular structures which captures the metal ion present in poly phenol oxidase and reduces the browning action. 6 (5) Stabilizers: Salts of sodium and calcium ions stabilize the composition of browning inhibitor containing bioenhancers. According to the present invention, bioenhancers were prepared by fermentation using controlled conditions with the help of pressure, adjustments in temperature and using suitable fermentation medium referring to the environment. The fermentation process is carried out by using the fermentation substrate and the carbohydrate source that is metabolized by the fermenting microorganism(s). The fermentation media includes fermentation substrate and other raw materials used in the fermentation process. In the present invention, the fermentation media can bring out liquefaction and saccharification processes or other desired processes prior to or simultaneously with fermentation. Schematic representation of process 7 Conventional process Schematic representation of process Bioenhancer Treatment Observe every 1/2 hour till 4-6 hours Observations and results: Example 1: Lab trial results for the market wheat variety lokawan: i) Lokawan variety Effect of treatment of bioenhancer on Lokawan variety Observations for 6 consecutive days are as follows: Day 1 Observations 8 Hunter Lab Colorimeter 0th Hour Observations (1st ay) Day 2 Observations 9 Hunter Lab Colorimeter after 24 hours Observations (2nd Day) Day 5 Observations 11 Hunter Lab Colorimeter after 96 hours Observations (5 th Day) Day 6 Observations Hunter Lab Colorimeter after 120 hours observations (6th Day) Graphical representation of reduction in ppo content: Effect of ppo on lokawan wheat variety with bioenhancer (refer to graph no 1) 12 Example 2: Effect of treatment of bioenhancer on Sarbathy variety Observations for alternate days are as follows: Hunter lab colorimeter observations for 0th hour Day 1 observations Colour measurement of whole wheat (before grinding)by hunter lab colorimeter 13 Colour measurement of whole wheat dough by hunter lab colorimeter Treated wheat (0.3%) 58.12 6.06 17.99 Treated wheat (0.4%) 58.82 5.92 18.33 L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) Loss on Drying for 24 hour Resudual Enzyme Level after 24 hours Samples PPO content in the whole wheat flour after 24 Hour observations Note: There are inherent enzymes like protease and amylase present in the whole wheat. The added Bioenhancer is not contributing any additional protease and amylase activity to the final wheat flour and thus maintaining the original rheological properties of the wheat flour. Hunter Lab Colorimeter Observations After 72 Hour Day 3 Observations Colour measurement of whole wheat Dough by hunter lab colorimeter L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) 15 PPO content in the whole wheat for 72 Hour observations Loss on Drying for 72 Hour Residual Enzyme Level for 72 Hour Samples Note: There are inherent enzymes like protease and amylase present in the whole wheat. The added Bioenhancer is not contributing any additional protease and amylase activity to the final wheat flour and thus maintaining the original rheological properties of the wheat flour. Hunter Lab Colorimeter Observations for 120 Hour Day 5 Observations 16 Colour measurement of whole wheat Dough by hunter lab colorimeter L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) Loss on Drying for 120 hour PPO content in the whole wheat for 120 Hour observations 17 Residual Enzyme Level for 120 Hour samples Note: There are inherent enzymes like protease and amylase present in the whole wheat. The added Bioenhancer is not contributing any additional protease and amylase activity to the final wheat flour and thus maintaining the original rheological properties of the wheat flour. Graphical representaion of redution in ppo content Effect of ppo on sharbhati wheat variety with bioenhancer (refer to graph no 2) Example 3: Wheat variety: WH 147 Effect of treatment of bioenhancer on WH 147 variety Observations are as follows: Colour measurement of whole wheat before grinding bv hunter lab colorimeter L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) 18 Colour measurement of whole wheat Dough bv hunter lab colorimeter sample control dough 54.32 6.31 17.12 Treated dough 58.06 5.22 15.71 L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) Graphical representation of reduction in ppo content Effect of ppo on WH 147 wheat variety with bioenhancer after 24 hours treatment (refer to graph no 3) Example 4: Wheat variety: sure Effect of treatment of bioenhancer on sure variety Observations are as follows Colour measurement of whole wheat before grinding bv hunter Lab colorimeter L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) 19 Colour measurement of whole wheat Dough bv hunter Lab colorimeter L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue) Graphical representation of reduction in ppo content Effect of ppo on sure wheat variety with bioenhancer after 24 hours treatment (refer to graph no 4) Overall benefits of using bioenhancer in whole wheat atta are as follows: • inhibits the poly phenol oxidase activity • improves the colour of dough • increases the water absorption of the flour • Makes chapatti, rotis softer for longer time with improved shelf life • Improves digestibility of the final product Amount of polyphenol content in different varieties of whole wheat Graphical representation is as follows (refer to graph no 5) Comparison of ppo content in original and treated varieties of wheat (refer to graph no 7) 20 Analysis of Wheat Flour The analysis of wheat flour treated and untreated wheat flour for various parameters such as moisture, ash, gluten, protein etc for all four varieties are as follows: Moisture analysis of control and treated wheat flour (refer to Graph no 8) Wheat variety Control % moisture Treated % moisture Lokawan 13.1 13.1 Sarbathy 12.99 12.99 WH147 13.45 13.45 Sure 12.98 12.98 Gluten analysis of control and treated flour (refer to Graph no 9) Wheat variety Control % gluten Treated % gluten Lokawan 9.1 9.1 Sarbathy 8.9 8.98 WH147 9 8.99 Sure 9.12 9.11 Protein analysis of control and treated wheat flour (refer to Graph no 10) Wheat variety Control % protein Treated % protein Lokawan 9.2 9.2 Sarbathy 9.2 9.23 WH147 8.9 8.99 Sure 7.68 7.66 Ash analysis of control and treated wheat flour (refer to Graph no 11) Wheat variety Control % Ash Treated % ash Lokawan 1.2 1.2 Sarbathy 1 1 WH147 1.1 1.1 Sure 1.23 1.23 Nutritional report for control and treated wheat flour 21 Nutritional report of Lokawan flour before and after treatment Sample Thiamine content in mg/100 g of flour Whole Lokawan wheat flour untreated 0.592 Whole Lokawan wheat flour treated with 0.3% bioenhancer 0.593 Whole Lokawan wheat flour treated with 0.4% bioenhancer 0.592 Nutritional report of WH 147 flour before and after treatment Sample Thiamine content in mg/100 g of flour Whole WH 147 wheat flour untreated 0.511 Whole WH 147 wheat flour treated with 0.3% bioenhancer 0.512 Whole WH 147 wheat flour treated with 0.4% bioenhancer 0.511 Nutritional report of Sarbathy flour before and after treatment Sample Thiamine content in mg/100 g of flour Whole Sarbathy wheat flour untreated 0.536 Whole Sarbathy wheat flour treated with 0.3% bioenhancer 0.536 Whole Sarbathy wheat flour treated with 0.4% bioenhancer 0.536 Nutritional report of Sure flour before and after treatment Sample Thiamine content in ppm Whole Sure wheat flour untreated 0.521 Whole Sure wheat flour treated with 0.3% bioenhancer 0.522 Whole Sure wheat flour treated with 0.4% bioenhancer 0.522 Sensory evaluation of chapattis prepared from treated and untreated whole wheat flour. Chapatti preparation: Chapattis are prepared from treated and control wheat flour. Sensory evaluation Each chapatti samples was evaluated by a panel of judges for various sensory attributes color, flavour, taste, texture, chewing ability, folding ability and appearance. Results and Discussion: The sensory evaluations of the chapattis prepared from different flour are mentioned below. The total pooled scores obtained by control and treated flour of chapattis for colour, flavour, taste, texture, chewing ability; folding ability and appearance were 34.47 and 46.65 respectively. These results revealed that chapattis prepared from treated whole wheat flour were ranked at the top than that of control. However, on the basis of scores assigned by the panel of trained judges to different sensory attributes, it is obvious that chapattis prepared from treated flour showed the better quality characteristics than that of control in the above mentioned properties. Pooled data for sensory evaluation of chapattis Sample Colour Flavour Taste Texture Chewing ability Folding ability Total score of acceptability Untreated wheat flour (control) 6.33 5.16 4.66 4.66 3.83 6.00 34.47 Treated wheat flour 6.50 6.66 7.00 6.83 6.33 7.00 46.65 Where the value of score represents 1= Very Poor; 7= Excellent. Pooled data for sensory evaluation of chapattis prepared from control and treated flour (refer to Graph no 12) Organoleptic properties of chapatti prepared from the wheat treated with bioenhancer Observations: Sensory evaluation of dough and chapatti Poly phenol oxidase activity determination was done as per method specified in "Enzyme and related biochemicals" by Worthington™ Amylase Method (Skbu/ Gm): Reagents: Sodium acetate buffer, dilute iodine solution, buffered starch solution. Enzyme solution: Estimate quantity of enzyme necessary to be in range and dissolve in water. Procedure - Pipette 5.0 ml of dilute solution of iodine into series of test tubes. Pipette 10 ml of buffered starch substrate solution into a test tube. Equilibrate at 30°c for 15 min. at zero time add 5 ml of appropriate enzyme dilution into equilibrated buffered substrate solution and start the stop watch as zero time. After 15 min of reaction time, Pipette one of the hydrolysis material into 5 ml dilute iodine solution. Mix the tube and compare against color comparator. Near the end point the comparison should be made at 30 seconds interval. Calculation: One unit of SKB activity is defined as that amount of enzyme to dextrinize 0.1 gm of soluble starch per 60 minutes under the condition of assay. SKBU/GM: 0.2 X 60 X DILUTION FACTOR 0.1 X Time X5 Protease (PC/GM) was determined referring to Food Chemical Codex I. Thiamine was estimated referring to Analytical Methods of Vitamins. Determination of moisture, ash, gluten was as per aoac standard methods. Determination of proteins was carried out referring to Pearson's Composition and Analysis of Foods. We claim: 1. A composition comprising a blend of bioenhancers which comprises pentosanases in an amount of 0.1 to 5%; proteolytic enzymes in an amount of 0.1% to 2%; redox enzymes in the range of 0.1% to 3%; formulated in conjunction with reducing agents, acidulants, stabilizers; surfactants and fillers, wherein said composition synergistically inhibits the browning of whole wheat specifically using bioenhancer treatment process, thereby modifying the phenolic substrate. 2. The composition as claimed in claim 1 wherein the said pentosanases are selected from cellulase, beta glucanase, mannanase, arabinase in the range of 15 u/gm to 60u/gm. 3. The composition as claimed in claim 1 wherein the said proteolytic enzyme is selected from papain, bromelain and ficin in the range of 125 pcu/gm to 550 pcu/gm. 4. The composition as claimed in claim 1 wherein said redox enzyme is selected from catalase, gluconase oxidase and lipoxidase in the range of 1050u/gm to 1750 u/gm. 5. The composition as claimed in claim 1 wherein said reducing agent is selected from L-cysteine and ascorbic acid in the range of 0.5 to 6% by weight. 6. The composition as claimed in claim 1 wherein said acidulant is selected from maleic acid and fumaric acid in the range of 10 to 20% by weight. 7. The composition as claimed in claim 1 wherein said stabilizer is selected from sodium and calcium salts in the range of 7 to 10% by weight. 8. The process as claimed in claim 1, for inhibiting browning of whole wheat comprises the following steps: a. Adding formulation of bioenhancer to whole wheat and soaking overnight, b. Grinding in mill to obtain 40-60 mesh size, c. Making dough with 60-61% water and closing it with lid and d. Observing every half hour for four to six hours. 9. A composition comprising a blend of bioenhancers wherein said composition as claimed in claim 1, inhibits browning of whole wheat by a process of modification of phenolic substrate as exemplified substantially in the foregoing examples 1-4. Dated this 1st day of July, 2005. FOR ADVANCED ENZYME TECHNOLOGIES LIMITED By their Agent (SAIMA SAGHIR ANSARI) KRISHNA & SAURASTRI

Full Text

FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
& THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]
COMPOSITION COMPRISING BIOENHANCERS FOR INHIBITION OF BROWNING OF WHOLE WHEAT
WE, ADVANCED ENZYME TECHNOLOGIES LIMITED, A COMPANY ORGANISED AND EXISTING UNDER THE LAWS OF INDIA WHOSE ADDRESS IS SUN MAGNECTICA, 5TH FLOOR NEAR LIC SERVICE ROAD, LOUIS WADI THANE-400 604, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION
PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE

Technical Field of Invention:
The present invention relates to the use of scientifically blended bioenhancers with browning inhibitors to irreversibly modify the phenolic substrate, which in turn inactivates poly phenol oxidase in whole wheat. The resultant product has controlled browning effect than that compared to natural whole wheat dough. The present invention attributes technologically and economically to the life of common man through the improvement and benefits brought about herein.
Background of Invention and Prior Art:
Browning of foods during processing and storage, especially during manufacture of meat, fish, fruit and vegetable products decreases the sensory properties of products due to associated changes in colour, flavour and softening, besides nutritional properties. Therefore, its control is essential to preserve the quality of the food. Appearance, flavour, texture and nutritional value are four attributes considered by consumers when making food choices. Appearance, which is significantly impacted by colour, is one of the first attributes used by consumers in evaluating food quality. Colour may be influenced by naturally occurring pigments such as chlorophylls, carotenoids and anthocyanins in food, or by pigments resulting from both enzymatic and non-enzymatic reactions.
Enzymatic browning is a significant problem in a number of important commodities, specially fruits such as apricots, apples, pears, peaches, bananas and grapes; vegetables such as potatoes, mushrooms ,lettuce and sea food. This discoloration limits the shelf life as well as the acceptance of the product.
The major enzymatic browning is caused by poly phenol oxidase enzyme present inherently in the fruits, vegelabJes, cereaJs etc. Mechanism of browning due to poly phenol oxidase can be explained as follows:
Enzymatic browning is the discoloration that results when monophenolic compounds of plants or shellfish, in the presence of atmospheric oxygen and poly phenol oxidase (PPO), are hydroxylated to ortho phenols, and the latter are oxidized to ortho quinines. The quinine condenses and reacts nonenzymatically with other phenolic compounds to produce dark brown, black or red pigment of intermediate structure.
2

Many approaches have been done in fruits but not in whole wheat. The present invention is related to the prevention of enzymatic browning in whole wheat dough by using a blend of bioenhancers.
Whole wheat is nutritious than white wheat. This is because whole wheat retains bran layer containing iron (non-haem), zinc, copper, thiamin, riboflavin and niacin which are water solubles. Whole wheat aleuronic layer of cells contains valuable nutrients like vitamins B complex, vitamin E and minerals.
The present invention is related to prevent the enzymatic browning in chapatti, breads, rotis, nans etc made with whole wheat dough. The whole wheat retains the nutritional value with improved visual appearance. The main basis of the present invention is to improve the organoleptic properties of whole wheat products and to retain its nutritional value.
Abstract of EP0903083 discloses process for inhibiting enzymatic browning and maintaining textural quality of fresh peeled potatoes, where a process is disclosed for inhibiting enzymatic browning in raw, peeled potatoes comprising dipping the potatoes in a solution of heated organic acids (45-65°C), followed by treatment in a weakly basic solution to neutralize the potato surface and treatment with reducing agents, and followed by storage in modified atmosphere packaging. The process tends to delay the onset of enzymatic browning and, once browning has begun, limits the extent of enzymatic browning.
Abstract of US6020018 discloses inhibition of enzymatic browning of raw fruit and/or vegetable juice. The juice is treated with at least one sulfated polysaccharide in an amount sufficient to inhibit browning. A promoter may also be present, said promoter is selected from the group consisting of chelating agents, acidulents, or mixtures thereof.
Abstract of W08911227 describes inhibition of enzymatic browning, a process for inhibiting oxidative darkening of foods by treating the food with a protease effective to inhibit oxidative darkening of the food, a composition for inhibiting oxidative darkening and a kit for preparing the composition.

Abstract of US 4,814,192 discloses process for preserving raw fruits and vegetables, including juices, comprising treating the products with ascorbic acid-2-phosphate esters and ascorbyl-6-fatty acid esters, individually or in combination. The treatments may be applied in an aqueous carrier and may further comprise other browning inhibitors, polyphenol oxidase inhibitors, emulsifying agents, dispersing agents and complexing agents. Treatments tend to delay or prevent the onset of enzymatic browning or, once browning has begun, to limit the extent of enzymatic browning.
Object of the Invention:
The main object of present invention relates to the use of bioenhancers with browning inhibitor in the whole wheat to inactivate the poly phenol oxidase and thereby to reduce the undesirable browning.
The other object of present invention relates to the fact that the nutritional value of the whole wheat flour before and after treatment with formulation of bioenhancers with browning inhibitor is retained without any lowering or deterioration.
Summary of the Invention:
The object of the invention is to provide a process for modification of phenolic substrates as bioenhancers by inhibiting poly phenol oxidase activity in the whole wheat.
The bioenhancer comprises of specific pentosanases which act on pentosans like pectin, xylan and such like, proteolytic enzymes selected from neutral proteases, papain and such like, redox enzymes such as catalase, peroxidase and such like in combination with reducing agents such as cysteine, glutathione and ascorbic acid with chelating agents such as phosphates, EDTA and such like with some acidulants like fumaric acid, phosphoric acid and citric acid with some stabilizers.
Phenolic substrate modification by bioenhancers in whole wheat formulation comprises of bioenhancers such as pentosanases - 0.1-5%, proteolytic enzyme -0.1-2%, redox enzymes - 0.1-3%, reducing agents such as L-cysteine, ascorbic acid 0.5-6%, stabilizers such as sodium and calcium salts at 7-10% with acidulants such as fumaric acid and maleic acid at 10- 20%, thus phenolic substrate modification by the use of bioenhancers in the range of 0.1 - 0.4 % reduces the rate of browning in the whole wheat flour preparation, yet retaining its nutritional value.

Detailed Description:
The present invention claims the composition which consists of blend of bioenhancers like specific pentosanases which act on pentosans like pectin, xylan, cellulose and proteolytic enzymes like neutral proteases, plant protease (papain, bromalein, ficin) and redox enzymes such as catalase, gluconase oxidase and lipoxidase. The bioenhancers are formulated in conjunction with browning inhibiting components like reducing agents namely cysteine, glutathione and ascorbic acid, chelating agents such as phosphates and EDTA. The rate of action of bioenhancers and browning inhibitors can be enhanced with acidulants like fumaric acid, phosphoric acid, ascorbic and citric acid which stabilize the whole browning inhibition in conjunction with calcium, sodium salts and emulsifying agents such as polysorbates.
The present invention discloses the range of composition of bioenhancer such as
pentosanase - 0.1 - 5%, proteolytic enzyme - 0.1- 2%, redox enzymes 0.1- 3%,
reducing agents 0.5-6%, stabilizers such as sodium and calcium salts at 7-10% with
acidulants 10- 20% and white dextrin, defatted soya flour as inert filler: q.s.
(1) Bioenhancer
The mechanism of bioenhancers can be exploited for the control of undesirable
enzyme activities to inhibit browning effect.
1. Substrate and/or product modification other than the target enzymes
2. Direct inactivation of the target enzyme.
3. Inactivation by secondary reactions of highly reactive products. (i)Pentosanase:
It helps in selective inhibition of browning by irreversible modification of phenolic substrates. Due to distorted phenolic structure of substrate, the active site on the poly phenol oxidase cannot come in contact with the substrates to give 100% reaction thus helping in reducing the browning effect. (ii)Proteolytic enzymes:
The plant proteases like ficin, papain, and bromelain are sulphydryl enzymes of broad specificity, which are very effective browning inhibitors. This inhibitory effect is thought to be due to either binding or hydrolysis at specific sites necessary for poly phenol oxidase activity. (iii)Redox enzymes:
5

These bioenhancers are capable of methylating the 3 rd position of 3, 4 dihydroxy aromatic compounds. It was observed to cause irreversible modification of phenolic substrates, thus preventing them from serving as substrates for poly phenol oxidase, thus preventing browning reaction.
(2) Reducing agents
Reducing agents play a role in the prevention of enzymatic browning either by reducing O-quinones of the phenolic substrates to colorless diphenols, or by reacting irreversibly with O-quinones to form stable colorless products. (i)Ascorbic acid is a strong reducing compound, which is acidic in nature, forms neutral salts with bases and is highly water soluble. Polyphenol oxidase inhibition by ascorbic acid has been attributed to the reduction of enzymatically formed O-quinones to their precursor diphenols. Ascorbic acid is irreversibly oxidized to dehydroascorbic acid during the reduction process, thus preventing browning reaction to the desirable extent.
(ii)Cysteine is an effective inhibitor of enzymatic browning. It is reported to be more effective than bisulphites. Cysteine forms intermediate complex with quinine, which serves as competitive inhibitor of polyphenol oxidase enzyme.
(3) Acidulants
Acidulants are generally applied in order to maintain the acidity for optimum catalytic activity of an enzyme. Acidulants such as citric acid, malic acid and phosphoric acid are capable of lowering acidity of the system, thus rendering poly phenol oxidase inactive.
(4) Chelators
Enzymes generally posses metal ions at their active sites. Removal of these ions by chelating agents can therefore render enzymes inactive.
Chelating agents like sodium salt of ethylene diamine tetra acetic acid, sodium tripolyphosphate with pro-oxidative agents such as copper and iron ions, act through an unshared pair of electrons in their molecular structures which captures the metal ion present in poly phenol oxidase and reduces the browning action.
6

(5) Stabilizers:
Salts of sodium and calcium ions stabilize the composition of browning inhibitor
containing bioenhancers.
According to the present invention, bioenhancers were prepared by fermentation using controlled conditions with the help of pressure, adjustments in temperature and using suitable fermentation medium referring to the environment. The fermentation process is carried out by using the fermentation substrate and the carbohydrate source that is metabolized by the fermenting microorganism(s). The fermentation media includes fermentation substrate and other raw materials used in the fermentation process. In the present invention, the fermentation media can bring out liquefaction and saccharification processes or other desired processes prior to or simultaneously with fermentation.
Schematic representation of process
7
Conventional process

Schematic representation of process Bioenhancer Treatment

Observe every 1/2 hour till 4-6 hours Observations and results: Example 1: Lab trial results for the market wheat variety lokawan:
i) Lokawan variety
Effect of treatment of bioenhancer on Lokawan variety Observations for 6 consecutive days are as follows:
Day 1 Observations

8
Hunter Lab Colorimeter 0th Hour Observations (1st ay)

Day 2 Observations
9
Hunter Lab Colorimeter after 24 hours Observations (2nd Day)

Day 5 Observations
11
Hunter Lab Colorimeter after 96 hours Observations (5 th Day)

Day 6 Observations
Hunter Lab Colorimeter after 120 hours observations (6th Day)

Graphical representation of reduction in ppo content: Effect of ppo on lokawan wheat variety with bioenhancer (refer to graph no 1)
12
Example 2: Effect of treatment of bioenhancer on Sarbathy variety Observations for alternate days are as follows:
Hunter lab colorimeter observations for 0th hour
Day 1 observations
Colour measurement of whole wheat (before grinding)by hunter lab colorimeter

13
Colour measurement of whole wheat dough by hunter lab colorimeter

Treated wheat (0.3%) 58.12 6.06 17.99
Treated wheat (0.4%) 58.82 5.92 18.33
L equals 0-100 (0=black and 100=white)
a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue)

Loss on Drying for 24 hour

Resudual Enzyme Level after 24 hours Samples

PPO content in the whole wheat flour after 24 Hour observations
Note:
There are inherent enzymes like protease and amylase present in the whole wheat. The added Bioenhancer is not contributing any additional protease and amylase activity to the final wheat flour and thus maintaining the original rheological properties of the wheat flour.
Hunter Lab Colorimeter Observations After 72 Hour

Day 3 Observations
Colour measurement of whole wheat Dough by hunter lab colorimeter

L equals 0-100 (0=black and 100=white)

a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue)
15
PPO content in the whole wheat for 72 Hour observations

Loss on Drying for 72 Hour
Residual Enzyme Level for 72 Hour Samples
Note:
There are inherent enzymes like protease and amylase present in the whole wheat. The added Bioenhancer is not contributing any additional protease and amylase activity to the final wheat flour and thus maintaining the original rheological properties of the wheat flour.
Hunter Lab Colorimeter Observations for 120 Hour Day 5 Observations
16
Colour measurement of whole wheat Dough by hunter lab colorimeter

L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue)
Loss on Drying for 120 hour

PPO content in the whole wheat for 120 Hour observations

17
Residual Enzyme Level for 120 Hour samples

Note:
There are inherent enzymes like protease and amylase present in the whole wheat.
The added Bioenhancer is not contributing any additional protease and amylase
activity to the final wheat flour and thus maintaining the original rheological
properties of the wheat flour.
Graphical representaion of redution in ppo content Effect of ppo on sharbhati wheat variety with bioenhancer (refer to graph no 2)
Example 3: Wheat variety: WH 147
Effect of treatment of bioenhancer on WH 147 variety
Observations are as follows:
Colour measurement of whole wheat before grinding bv hunter lab colorimeter

L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green)

b equals yellow to blue (+ = yellow and - = blue)
18
Colour measurement of whole wheat Dough bv hunter lab colorimeter

sample
control dough 54.32 6.31 17.12
Treated dough 58.06 5.22 15.71
L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green)
b equals yellow to blue (+ = yellow and - = blue)
Graphical representation of reduction in ppo content
Effect of ppo on WH 147 wheat variety with bioenhancer after 24 hours treatment
(refer to graph no 3)
Example 4: Wheat variety: sure
Effect of treatment of bioenhancer on sure variety Observations are as follows
Colour measurement of whole wheat before grinding bv hunter Lab colorimeter

L equals 0-100 (0=black and 100=white)

a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue)
19
Colour measurement of whole wheat Dough bv hunter Lab colorimeter

L equals 0-100 (0=black and 100=white) a equals red to green (+ = red and - = green) b equals yellow to blue (+ = yellow and - = blue)
Graphical representation of reduction in ppo content
Effect of ppo on sure wheat variety with bioenhancer after 24 hours treatment
(refer to graph no 4)
Overall benefits of using bioenhancer in whole wheat atta are as follows:
• inhibits the poly phenol oxidase activity
• improves the colour of dough
• increases the water absorption of the flour
• Makes chapatti, rotis softer for longer time with improved shelf life
• Improves digestibility of the final product
Amount of polyphenol content in different varieties of whole wheat Graphical representation is as follows (refer to graph no 5)

Comparison of ppo content in original and treated varieties of wheat (refer to graph no 7)
20

Analysis of Wheat Flour
The analysis of wheat flour treated and untreated wheat flour for various parameters
such as moisture, ash, gluten, protein etc for all four varieties are as follows:
Moisture analysis of control and treated wheat flour (refer to Graph no 8)

Wheat variety Control % moisture Treated % moisture
Lokawan 13.1 13.1
Sarbathy 12.99 12.99
WH147 13.45 13.45
Sure 12.98 12.98
Gluten analysis of control and treated flour (refer to Graph no 9)

Wheat variety Control % gluten Treated % gluten
Lokawan 9.1 9.1
Sarbathy 8.9 8.98
WH147 9 8.99
Sure 9.12 9.11
Protein analysis of control and treated wheat flour (refer to Graph no 10)

Wheat variety Control % protein Treated % protein
Lokawan 9.2 9.2
Sarbathy 9.2 9.23
WH147 8.9 8.99
Sure 7.68 7.66
Ash analysis of control and treated wheat flour (refer to Graph no 11)

Wheat variety Control % Ash Treated % ash
Lokawan 1.2 1.2
Sarbathy 1 1
WH147 1.1 1.1
Sure 1.23 1.23
Nutritional report for control and treated wheat flour
21

Nutritional report of Lokawan flour before and after treatment

Sample Thiamine content in mg/100 g of flour
Whole Lokawan wheat flour untreated 0.592
Whole Lokawan wheat flour treated with 0.3% bioenhancer 0.593
Whole Lokawan wheat flour treated with 0.4% bioenhancer 0.592
Nutritional report of WH 147 flour before and after treatment

Sample Thiamine content in mg/100 g of flour
Whole WH 147 wheat flour untreated 0.511
Whole WH 147 wheat flour treated with 0.3% bioenhancer 0.512
Whole WH 147 wheat flour treated with 0.4% bioenhancer 0.511
Nutritional report of Sarbathy flour before and after treatment

Sample Thiamine content in mg/100 g of flour
Whole Sarbathy wheat flour untreated 0.536
Whole Sarbathy wheat flour treated with 0.3% bioenhancer 0.536
Whole Sarbathy wheat flour treated with 0.4% bioenhancer 0.536
Nutritional report of Sure flour before and after treatment

Sample Thiamine content in ppm
Whole Sure wheat flour untreated 0.521
Whole Sure wheat flour treated with 0.3% bioenhancer 0.522
Whole Sure wheat flour treated with 0.4% bioenhancer 0.522
Sensory evaluation of chapattis prepared from treated and untreated whole wheat flour.
Chapatti preparation:
Chapattis are prepared from treated and control wheat flour.

Sensory evaluation
Each chapatti samples was evaluated by a panel of judges for various sensory
attributes color, flavour, taste, texture, chewing ability, folding ability and appearance.
Results and Discussion:
The sensory evaluations of the chapattis prepared from different flour are mentioned below. The total pooled scores obtained by control and treated flour of chapattis for colour, flavour, taste, texture, chewing ability; folding ability and appearance were 34.47 and 46.65 respectively. These results revealed that chapattis prepared from treated whole wheat flour were ranked at the top than that of control. However, on the basis of scores assigned by the panel of trained judges to different sensory attributes, it is obvious that chapattis prepared from treated flour showed the better quality characteristics than that of control in the above mentioned properties.
Pooled data for sensory evaluation of chapattis

Sample Colour Flavour Taste Texture Chewing ability Folding ability Total score
of
acceptability
Untreated wheat flour (control) 6.33 5.16 4.66 4.66 3.83 6.00 34.47
Treated wheat flour 6.50 6.66 7.00 6.83 6.33 7.00 46.65
Where the value of score represents 1= Very Poor; 7= Excellent.
Pooled data for sensory evaluation of chapattis prepared from control and treated flour (refer to Graph no 12)
Organoleptic properties of chapatti prepared from the wheat treated with bioenhancer Observations:
Sensory evaluation of dough and chapatti

Poly phenol oxidase activity determination was done as per method specified in "Enzyme and related biochemicals" by Worthington™
Amylase Method (Skbu/ Gm):
Reagents: Sodium acetate buffer, dilute iodine solution, buffered starch solution. Enzyme solution: Estimate quantity of enzyme necessary to be in range and dissolve in water.
Procedure - Pipette 5.0 ml of dilute solution of iodine into series of test tubes. Pipette 10 ml of buffered starch substrate solution into a test tube. Equilibrate at 30°c for 15 min. at zero time add 5 ml of appropriate enzyme dilution into equilibrated buffered substrate solution and start the stop watch as zero time. After 15 min of reaction time, Pipette one of the hydrolysis material into 5 ml dilute iodine solution. Mix the tube and compare against color comparator. Near the end point the comparison should be made at 30 seconds interval.
Calculation: One unit of SKB activity is defined as that amount of enzyme to dextrinize 0.1 gm of soluble starch per 60 minutes under the condition of assay.
SKBU/GM: 0.2 X 60
X DILUTION FACTOR
0.1 X Time X5
Protease (PC/GM) was determined referring to Food Chemical Codex I. Thiamine was estimated referring to Analytical Methods of Vitamins. Determination of moisture, ash, gluten was as per aoac standard methods. Determination of proteins was carried out referring to Pearson's Composition and Analysis of Foods.

We claim:
1. A composition comprising a blend of bioenhancers which comprises pentosanases in an amount of 0.1 to 5%; proteolytic enzymes in an amount of 0.1% to 2%; redox enzymes in the range of 0.1% to 3%; formulated in conjunction with reducing agents, acidulants, stabilizers; surfactants and fillers, wherein said composition synergistically inhibits the browning of whole wheat specifically using bioenhancer treatment process, thereby modifying the phenolic substrate.
2. The composition as claimed in claim 1 wherein the said pentosanases are selected from cellulase, beta glucanase, mannanase, arabinase in the range of 15 u/gm to 60u/gm.
3. The composition as claimed in claim 1 wherein the said proteolytic enzyme is selected from papain, bromelain and ficin in the range of 125 pcu/gm to 550 pcu/gm.
4. The composition as claimed in claim 1 wherein said redox enzyme is selected from catalase, gluconase oxidase and lipoxidase in the range of 1050u/gm to 1750 u/gm.
5. The composition as claimed in claim 1 wherein said reducing agent is selected from L-cysteine and ascorbic acid in the range of 0.5 to 6% by weight.
6. The composition as claimed in claim 1 wherein said acidulant is selected from maleic acid and fumaric acid in the range of 10 to 20% by weight.
7. The composition as claimed in claim 1 wherein said stabilizer is selected from sodium and calcium salts in the range of 7 to 10% by weight.

8. The process as claimed in claim 1, for inhibiting browning of whole wheat
comprises the following steps:
a. Adding formulation of bioenhancer to whole wheat and soaking overnight,
b. Grinding in mill to obtain 40-60 mesh size,
c. Making dough with 60-61% water and closing it with lid and
d. Observing every half hour for four to six hours.
9. A composition comprising a blend of bioenhancers wherein said composition as
claimed in claim 1, inhibits browning of whole wheat by a process of
modification of phenolic substrate as exemplified substantially in the foregoing
examples 1-4.
Dated this 1st day of July, 2005.
FOR ADVANCED ENZYME TECHNOLOGIES LIMITED By their Agent

(SAIMA SAGHIR ANSARI) KRISHNA & SAURASTRI

"COMPOSITION COMPRISING BIOENHANCERS FOR INHIBITION OF BROWNING OF WHOLE WHEAT"

Documents:

Inventors:

PCT Conventions: