Efficacy of Multigrain Bread in - Vivo

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ORIGINAL ARTICLE Efficacy of Multigrain Bread in - Vivo

K. Jothilakshmi 1*, G.G. Kavitha Shree 1, G. Gurumeenakshi 2 and M.R. Duraisamy 3

Assistant Professor 1, KVK, Dharmapuri, Assistant Professor 2, PHTC, AEC and RI, Assistant Professor 3, KVK, Needamangalam, TNAU, Tamil Nadu, India.

Abstract The increasing interest in healthy foods causes a change in the

buying behavior of the consumer. When watching the baker’s shop

window, more browner and whole-wheat bread with a healthy image can be seen. As millets are nutritive foods, when it is mixed with whole wheat flour for preparation of bread is satisfies the need of consumers. Hence, the multigrain mixes were used for the preparation of breads with an improved the nutrient content. High fibre bread containing wheat, millet, pulse and *Corresponding Author: purified dietary fiber namely inulin has been standardized. The standardized wheat based multigrain bread contained kodo millet, bengal K. Jothilakshmi gram, horse gram and inulin (fructo-oligosacharride). The rheological

properties, nutrient content, cellular structure and sensory characteristics Email: [email protected] such as colour and appearance, flavour, texture, taste and over all acceptability of the multigrain bread was evaluated and highly accepted. Received: 07/07/2016 The most acceptable proportion was used for animal study to assess the hypoglycemic and hypolipidemic effects. The study was conducted on Accepted: 02/09/2016 Wistar rats. The effect of wheat based multigrain bread on the glucose levels in diabetic induced rats after a feeding trial of 28 days was studied. There was a statistically significant reduction in the blood glucose level at the end of four weeks. The initial glucose levels reduced from 204.16 ±

3.57 to 121.16 ± 3.4 mg/dL which is a 40.65 per cent reduction. The HDL

level was found to be 22.2 ± 0.68, LDL 22.08 ± 0.95 mg/dL and atherogenic index was 2.18 ± 1.13 respectively at the end of the study. Further, from the study it was found that the multigrain mixes can be used to develop bakery, extruded, convenience foods which will provide both therapeutic and nutraceutical benefits.

Keywords : Kodo millet, Bengal gram, Inulin, Animal study.

1. Introduction Life style disease is associated with the way a Coarse cereals provide viable alternatives to person or group of people live in a careless, ignorant diversify sources of health components in foods. manner. The onset of these life style diseases is Obviously, the benefits are highest in whole grain insidious, they take years to develop and once cereal consumption. Although millets are nutritionally encountered are not easily cured and do not lend superior to cereals, their utilization in the country is not themselves to quick fix by medicines alone and require widespread. They are mostly used in the preparation of dietary intervention. Formulation of functional food traditional dishes. One possible way of extending their with the inclusion of whole grains, legumes, raw or less utilization could be by blending them with wheat flour processed food particles retain all the fibre and can after suitable processing. According to Dykes and prevent and treat such diseases (Kulkarni, 2009). Rooney (2007), whole grain foods are a unique source Millets are very important foods owing to their of dietary fibre, in the plant cell wall that constitutes nutritional composition and high satiety value. High the complex matrices, within which several bioactive dietary fibre and the complex nature of carbohydrates compounds co-exist. In addition to non starch have immense health implications such as lowering polysaccharides of plant cell walls, whole grain foods blood cholesterol, triglyceride and sugar levels and thus are rich in vitamins, minerals and other compounds that have a potential role in the life style disorders alone and in combination are likely to have significant (Chimnad, 2009). The term "whole grain" refers to health benefits (Marquart, 2002). flour that is made from all parts of the wheat grain

Journal of Ready to Eat Food | July-September, 2016 | Volume 03 | Issue 03 | Pages 28-33 © 2016 Jakraya Jothilakshmi et al... Efficacy of Multigrain Bread in - Vivo kernel, the bran, germ and endosperm. The bran and vanaspathi were purchased and stored in airtight plastic germ are the most nutritious parts of the wheat grain. containers till used. They contain vitamin B1, B2, B3, vitamin E, folic acid, calcium, phosphorus, zinc, copper, iron and dietary 2.1 Rheological Characteristics fiber. During the refinement process, the majority of Alveograph (Alveolink NG, Alveo CH- Chopin, these nutrients get lost. Eating a diet full of whole ICC-no171- no 50) used to study the rheological grains can reduce your risk for diabetes and heart properties of multigrain mixes to found the suitability disease. It takes the body longer to absorb whole grain of the bakery products. products. The slower absorption rate prevents sharp rises in sugar and insulin levels. Dietary fiber is a 2.2 For Baking crucial part of any weight loss regime. Soluble fiber Baking oven (Memmert model 854, Schwabach, slows down the digestive process, allowing the food to West Germany) with thermostatic adjustment with a spend more time in the digestive tract and increasing temperature range of 0°C-250°C was used for baking the amount of nutrients absorbed into the body. Fiber the bread. also creates a feeling of fullness, making less likely to over indulge. Fiber can also reduce cholesterol levels in 2.3 Analysis of Chemical Constituents the blood. Multigrain bread has a ton of dietary fiber, Hot air oven used to estimate the moisture as the majority of the fiber is found in the germ. On the content of the bread, Kel Plus used to estimate the other hand, whole grain means the bread or baked good protein content of the bread, Sox Plus used to estimate includes the nutritious bran and germ of the grain. So the fat content of the bread, Fibra Plus used to estimate the product contains the dietary fiber, magnesium, the crude fibre content of the bread, Spectronic 20 vitamin B6 and vitamin E and other important phyto- (Bosch and Lamb, USA) used to estimate the reducing nutrients. sugar, total sugar, calcium, iron, phosphorus and inulin Multigrain bread can be an important part of a content of the bread. healthy diet and an excellent way to eat more whole grains. Understanding the name is simple-"multigrain"- 2.4 Experimental Animals indicates the loaf should contain several different types Wistar albino rats weighing 180-220 g were of grains, these grains include kodo millet, bengal obtained from Thiruvananthapuram Medical College, gram, horse gram and inulin (fructo-oligosacharride). Thiruvananthapuram, India and the study was Inulin is resistant to digestion in the upper conducted in K.M. College of Pharmacy, Madurai. The gastrointestinal tract and it reaches the large intestine experiments were approved by the institutional animal essentially intact, where it is fermented by colonic ethics committee (Appendix VII). The wistar albino bacteria causing an increase in fecal biomass, rats were housed in large spacious cages and they were production of short- chain fatty acids and decrease in fed with multigrain mixes puffed snack food and access ceco-colonic pH. Hence, it is classified as a soluble to water ad libitum . The animals were acclimatized to dietary fiber (Murphy, 2001). In recent times, fortified the standard environmental condition of temperature foods with purified dietary fibre or the direct addition (22°C±5°C) and humidity (55±5%) and 12 hrs light of fibre rich source to improve the nutritional quality of and dark cycles throughout the experimental period. food product are encouraged owing to health benefits Diabetes mellitus was induced in wistar albino associated with dietary fibre. In this context, inulin is a rats by single intra-peritoneal injection of freshly potential ingredient that can be used in a functional prepared solution of Alloxan monohydrate (150 mg/kg food product development which meets the needs of BW) in physiological saline after overnight fasting for the food industry, public health professionals and 12 hrs. individuals. With this background the present Alloxan is commonly used to produce diabetes investigation on “Standardization of fructo- mellitus in experimental animals due to its ability to oligosacharride (inulin) incorporated multigrain mixes destroy the β-cells of pancreas possibly by generating and assessment of its hypolipidemic and hypoglycemic the excess reactive oxygen species such as H 2O2, O 2 effect” was taken up and HO-. The development of hyperglycemia in rats is confirmed by plasma glucose estimation on 72 h post 2. Materials and Methods alloxan injection. The rats with fasting plasma glucose Whole wheat ( Triticum aestivum ), kodo millet level of >150 mg/dL were used for this experiment. (Pasplum scrobiculatum ), bengalgram (Cicer arietinum) , horse gram ( Dolichos biflorus) , inulin, 2.5 Processing of Multigrain Bread Mixes sugar, dry yeast ( Saccharomyces cerevicea ) and

Cleaned, dehusked and undamaged whole flour for preparation of bread is satisfies the need wheat, kodo millet, bengal gram and horse gram were of consumers. Hence the multigrain mixes were sun dried for six to eight hours. The dried and roasted used for the preparation of breads with an (80 oC for 8-10 minutes) ingredients were milled into improved the nutrient content. flour in a commercial flour mill, cooled and stored in airtight containers at room temperature. Table 2: Ingredients used for Bread Preparation

Ingredients Weight 2.5.1 Standardization of Multigrain Bread Mix Multigrain mixes (g) 100 Wheat, kodo millet, bengal gram and horse Sugar (g) 24 gram were the grains chosen and standardized for multi Shortening (g) 8 grain bread, 30 per cent millets and 30 per cent pulses Yeast (g) 1.5 and 15 per cent inulin with 40 per cent of wheat flour Salt (g) 1.5 containing bread were highly acceptable. The Water (ml) 60 standardized mixes were evaluated the rheological characteristics to found the suitability of the bread preparation and given in table 1. 2.5.3 Analysis of Chemical Constituents of Standardized Multigrain Bread The chemical constituents of the multigrain Table 1: Standardized multigrain bread mixes bread were analyzed. The parameters like moisture, Ingredients (g) T1 T2 T1I T2I reducing sugar, total sugar, starch, protein, fat, crude Wheat flour 40 40 40 40 fibre, insoluble, soluble and total dietary fibre, inulin, Kodo millet flour 30 30 22.5 22.5 ash, iron, calcium, phosphorus and alcoholic acidity Bengal gram flour 30 - 22.5 - were analyzed as per the methods of AACC (2000). Horse gram flour - 30 - 22.5 Inulin - - 15 15 2.5.4 Sensory Evaluation of Multi Grain Bread Sensory evaluation was conducted as per the 2.5.2 Rheological Characteristics of the Multigrain method described by Watts et al . (1989). A panel of ten Bread Mixes trained judges and evaluated the multigrain bread. The The rheological characteristics such as organoleptic evaluation sessions were conducted one alveograph of multigrain bread mixes were analyzed by hour before lunch under adequate conditions of using alveograph. Alveograph was used to determine temperature, humidity and illumination. The panelists the resistance, tenacity, extensibility and baking were asked to score the colour, appearance, flavour, strength of the flour blends as per the standard method. texture, taste and overall acceptability of the above For the constant hydration, alveograph always use 50 bread on a scale of 9 to 1 hedonic scale. per cent hydration (250 g flour, 125 g of water-salt solution). 2.6 Animal Experimental Protocol a) P - Stands for maximum pressure that was In the experiment, a total of 36 rats (30 diabetic withstood and shows how strong the dough rats and six normal rats) were used. Diabetes was was (Tenacity). induced three days before starting the experiment. The b) L- Stands for the height of the bubble that was rats were divided into 6 groups (n=6 rats/ group) after achieved, measured from where the slops of inducing diabetes. In the experiment, six rats were used the bubble started to the top of the bubble. in each group This shows how flexible the dough was Treatment Protocol: (Extensibility). a) Group I: (Control) consist of normal rats. c) P/L - for the ratio of P to L b) Group II: Control (diabetic control) given 150 d) G- Stands for the square root of the volume of mg/Kg of BW, Alloxan monohydrate through air in the bubble that was possible before the intra-peritoneal to induce diabetes. bubble burst. This shows how flexible the Treatment Groups: Experiments I to VI were run for 28 dough was (Swelling index). days. e) W - Stands for the energy needed to make the a) Group III: Diabetic rat given only multigrain bubble as big as it could go before it burst and mix extruded snack food (T 1). indicate the strength of the flour (baking b) Group IV: Diabetic rat given only multigrain strength). mix with inulin extruded snack food (T 1I). Preparation of Multigrain Bread As millets are c) Group V: Diabetic rat given only multigrain nutritive foods, when it is mixed with whole wheat mix extruded snack food (T 2).

d) Group VI: Diabetic rat given only multigrain 4. Discussion mix with inulin extruded snack food (T 2I). 4.1 Nutrient Content 2.7 Sample Collection Protein, fibre and calcium increased with At the start and after 28 days of feeding trial, the incorporation of millets and pulses in MGM bread blood glucose level and body weight were measured. while the fat and starch content decreased when Then blood was collected retro-orbitally from the eye compared to control bread. The reducing sugar content under light ether anesthesia using capillary tubes. was found to be the lowest in 22.5 per cent of millets, Blood was collected in fresh vials containing EDTA as 22.5 per cent pulses and 15 per cent of inulin anticoagulant agent and serum was separated in a T8 incorporated mixes when compared to 30 per cent of electric centrifuge at 2000 rpm for two minutes. Then millets and 30 per cent of pulse mixes. The values were serum samples were used for various biochemical tests T2 (1.03 g) compared to inulin added mixes such as T 2I (Al- shamaony et al ., 1994). (0.91 g). The starch content was maximum in T0 (50.2 g) and the values decreased with the level of 3. Results incorporation of millets and pulses. Hence, this multigrain bread is highly suitable for diabetic patient. 3.1 Rheological Characteristics of Multigrain It could be inferred from the values that as the Bread Mixes incorporation of pulses increased there was an increase The multigrain mixes with and without inulin in the protein content of the multigrain bread compared when compared to control exhibited differences in the to control. Accordingly the bread that were rheological characteristics as measured by Alveograph. incorporated with horse gram (T 2) had the highest protein content of 15.15 g per cent. As 15 per cent of 3.2 Influence of Multigrain Mixes Dough on the inulin replaced the millet and pulse in the multigrain Viscoelastic Properties mixes, the protein content was lower in these mixes Tenacity (P) of the MGM was compared to when compared to those that were not incorporated control. The tenacity value was found to be higher for with inulin. Among the treatments T 2 (4.3 g) had the highest fibre content followed by T 2I (3.7%), T 1 (3.2%) T2I (86 mm) which was closer to the control (92 mm). and T 1I (2.8 %). The dietary fibre (insoluble, soluble The L (extensibility) values of the MGM from T 1 to T 2I were in the range of 80 to 84 mm. The extensibility of and total) was significantly higher for the multigrain the multigrain mixes with and without inulin did not bread compared to wheat bread. It was found that T 2I vary to a great extent and the values were found to be had the highest soluble and TDF content when closer to that of the control. The P/L ratio was the compared the other multigrain bread. This is due to the maximum for the control. It was found that the value composition of T 2I which has 40 per cent wheat flour, for all MGM were lesser than that of the control. The 22.5 per cent kodo millet flour, 22.5 per cent horse 2 gram flour and 15 per cent inulin. Horse gram and baking strength (W) of T 2I (248 cm ),) and the T 2 (236 cm 2) MGMs were higher among treated mixes and inulin contributed more soluble and total dietary fibre 2 respectively. And also multigrain bread contained comparable to the control T 0 (260 cm ). Among the MGM, the inulin incorporated mixes highest mineral content when compared to wheat had higher index of swelling than the multigrain mixes bread. The bread from multigrain mixes had smaller size air cells compared to control bread because of less without inulin. The elasticity of T 2I was 40.2 per cent. From the above findings, it was found that the various gluten and more fibre content due to the whole grains. rheological characteristics the wheat flour differed The scores for colour and appearance, texture, flavour, when millets, pulses and inulin were added and found it taste and overall acceptability indicated that T 2I bread was most suitable for development for multigrain was the best among the other breads. All the bread bread. samples were found to be acceptable. Codina et al. (2010) reported that the P, L, G, W and P/L ratio of sixty samples of commercial wheat 4.2 Efficacy of Multigrain Bread flour ranged from 21-127 mm, 13-126 mm, 8.03- 25 The hypolipidemic and hypoglycemic effect of mm, 15:300 x 10-4 and 0.36- 2.70 respectively. In this multigrain bread were evaluated in wistar albino rats study, the values obtained for different parameters of for a period of 28 days. The parameters such as body multigrain mixes were within these ranges. The weight, glucose level, lipid profile, plasma insulin nutrient content and sensory characteristics of the bread haemoglobin and glycosylated haemoglobin were were developed from the multigrain mixes is given in studied. After 28 days of study there was a statistically- Table 3-4.

Table 3: Nutrient Content of Multigrain Bread

Nutrients/100g T0 T1 T2 T1 I T2 I Moisture(g) 24.30 24.1 24.3 24.3 24.36 Reducing sugar(g) 1.53 1.50 1.03 1.39 0.91 Total sugar(g) 4.22 4.13 3.33 3.94 3.25 Starch(g) 50.00 49.64 46.09 48.72 45.88 Protein(g) 12.05 13.50 15.15 13.30 15.13 Fat(g) 8.30 8.29 7.92 7.80 7.50 Crude Fibre (g) 2.0 3.2 4.3 2.8 3.7 Inulin(g) 2.76 1.50 1.50 16.50 16.50 Ash (g) 1.7 1.5 1.6 1.6 1.7 Iron (mg) 4.00 4.04 4.07 4.00 4.01 Calcium (mg) 38 85 106 81 101 Phosphorus(mg) 297 294 290 290 284

Table 4: Sensory Characteristics of Multigrain Bread

Treatments Colour and Flavour Texture Taste Overall Appearance Acceptability T0 9.0 9.0 9.0 9.0 9.0 T1 8.6 8.5 8.6 8.5 8.5 T2 8.8 8.8 8.7 8.7 8.7 T1I 8.7 8.7 8.8 8.8 8.7 T2I 8.8 8.8 8.9 8.9 8.8 significant increase in the body weight of the rat in a positive effect on insulin levels. Rats given inulin normal (G 1) and treatment groups (G 3-G6) while the containing multigrain bread had lower HbA1C toxic control (G 2 which were the rats with diabetes but indicating a hypoglycemic effect due to the inclusion of not given any treatment) showed a decrease in weight. inulin. Presence of kodo millet, horse gram and bengal The average weight gain in each group ranged from 19 gram appeared to have an appreciable hypoglycemic g (T 1) to 36g (T 2I) after 28 days of study period. effect on the diabetic rats. The initial blood glucose level of the normal The results proved that the inulin incorporated (G 1) and toxic control (G 2) rats was 71.5±5.67 and multigrain bread exerted a better hypoglycemic and 148.66±5.48 mg/dL blood respectively. There was a hypolipidemic effect when compared to the multigrain statistically significant reduction in the blood glucose at bread without inulin. Of the inulin included multigrain the end of four weeks feeding trials. The maximum bread T2I samples exhibited the highest effect which reduction in blood glucose was found in G 6 rats, fed was ev ident from the increase in body weight, reduction with T2I sample where initial glucose levels reduced in blood glucose level, total cholesterol, triglycerides from 204.16 ± 3.57 to 121.16 ± 3.4 mg/dL (a reduction and glycosylated Hb levels of the animals. of 40.65 %). The HDL level of Group VI was found to be 22.2±0.68, LDL 22.08±0.95 and AI 2.18±1.13 5. Conclusion mg/dl. The results show that T 2I (G 6) exhibited more, Today’s consumers hold high standards for the hypolipidemic effect followed by T 1I (G 4). The foods they consume. They demand foods that taste multigrain bread with inulin had more hypolipidemic great, fat and calorie reduced, and they are interested in effect than the multigrain mixes without inulin and the foods that provide added health benefits. It is also differences were statistically significant. expected that these foods will be convenient and The plasma insulin level in normal control rats affordable. The desire of consumers to look good and was 25.21±0.54 μu/ml. Plasma insulin level of G 4 and stay healthy in a fast-paced environment is becoming G6 were close to that of G 1. The G 2 rats which were more difficult to fulfil. Quick fixes and shortcuts are diabetic induced, and with no treatment given, had the attractive to the consumer, whether they refer to food least plasma insulin of 11.83±0.19 μu /ml. Except G 2 preparation, weight loss or disease prevention. group the plasma insulin values for all the groups were Consumers are also more informed and more aware of found to be higher indicating that the feeding trials had the links between diet and health than ever before.

Consequently they are looking for foods to provide body, beyond adequate nutritional effects, in a way multiple benefits as well as good taste. This speaks of a which is relevant to either the state of well being and strong focus on disease prevention and indicates that health or the reduction of the risk of a disease and the time is right for optimizing health. By and large inulin can be regarded as functional food and the functional foods can be defined as the food that developed multigrain bread exert a therapeutic and beneficially affect one or more target functions in the nutraceutical effect.

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