Nutrients , Nutraceuticals and Bioactive Properties of Multi-Whole Grain Mix for Drink and Porridge

Whole grains are reported to be rich in nutrients, nutraceuticals and have number of health beneficial effects. A convenient multi-whole grain mix for the preparation of a drink or porridge was formulated by using cereals, millets, pulses and nuts. Particle size was mostly of 180-250 microns (52%). Amylograph characteristics like GT, PV, HPV, CPV were 82 ° C, 285BU, 310BU, and 605BU, respectively were ideal for drink. The mix was found to be rich in carbohydrate, protein, fibre and calorie. The 100g of the mix had nutraceuticals like carotenoids (290 g), -tocopherol (4.6mg), tocopherol (1.5mg), and polyphenols-soluble, bound and total (94,132 and 226mg GA Eq.). Bioactive properties like vitamin E activity, free radical scavenging activity, total antioxidant activity and starch digestibility were 2.6i.u., 153mg catechin.Eq./100g, 17mg Tocopherol equivalent and 61.8%. Mix was sensorily acceptable in the form of drink and porridge and can be used as an ideal nutritious food for all age group.


INTRODUCTION
Whole grains contain all parts of the grain viz., the endosperm, germ, and bran.Whole grains are rich in nutrients and phytochemicals with known health benefits.As reviewed by [1], whole grains have high concentrations of dietary fibre, resistant starch, and oligosaccharides.They are also rich in antioxidants including trace minerals and phenolic compounds and these compounds have been linked to disease prevention.Other protective compounds in whole grains include phytate, phyto-oestrogens such as lignan, plant stanols and sterols, and vitamins and minerals.Epidemiological studies find that whole-grain intake is protective against cancer, CVD, diabetes, and obesity.Whole grain feeding studies in human subjects also report improvements in biomarkers such as weight loss, blood-lipid improvement and antioxidant protection.
The major cereals and millets consumed as whole grains in India are wheat, sorghum, Pearl millet, finger millet and to some extent brown rice.These grains are the major source of nutrients in diets contributing to around 7.3-11.6% protein, 1.3-5 % fat, 60-72 % carbohydrate, and 328 -361 kcal of the daily energy intake [2].
The foods which we commonly consume also contains tocopherols, carotenoids, phenolics and *Address corresponding to this author at the Department of Grain Science and Technology, Central Food Technological Research Institute (Council of Scientific and Industrial Research), Mysore -570 020, Karnataka, India; Tel: +91 821 251 0843; Fax: +91 821 251 7233; E-mail: jayadeep@cftri.res.inflavanoids, which serve as a good source of natural antioxidants [3,4,5] and are reported to have health beneficial effects [6].Vitamin E is a fat-soluble vitamin and includes both tocopherols and tocotrienols [7].It has many biological functions especially as antioxidant that stops the production of reactive oxygen species [8], neuroprotective [9], inhibition of platelet aggregation [10], control of gene expression etc [11].
Carotenoids are important in human nutrition and health.They are valuable as antioxidants [12], in the prevention of atherosclerosis in the maintenance of immune function [13], in the health of eyes [14] and some are precursors of vitamin A. Presence of carotenoids was reported in grains such as maize, wheat and sorghum [15,16].
Cereals and legumes contain a wide range of phenolics and act as good source of natural antioxidants [17].Presence of antioxidative phenolics have been reported in millets also [18,19].Flavonoids like tannin and anthocyanins also have antioxidative potential [20].All these indicate the bioactive potential of whole grains.However, number of palatable products from individual whole grains and multiple whole grains are few.Reports on the nature of lipid soluble as well as other polyphenolic antioxidants of the multi-whole grain products are also limited.
Reports on multigrain weaning foods based on polished rice, malted legumes and millets are also reported [21,22].However, drink or porridge mixes from multi-whole grains are scanty.Hence a convenient multi-whole grain mix was formulated by selecting nutrients and nutraceuticals rich cereals, millets and other ingredients, and optimization of conditions for development of flavor and appropriate texture.Main objective was to assess the quality of the formulated product with respect to physico-chemical, viscographic parameters, nutraceuticals and bioactive properties, and also the sensory quality of the cooked product for use as drink or porridge.

Preparation of RTC Multi-Whole Grain Mix
Whole grain mix was prepared by roasting the grains individually, mixing in suitable proportion, pulverization and size reduction (Figure 1).

Analysis of Particle Size Distribution
The particle size distribution of the multi-whole grain mix were studied by shaking 100 g of sample for 10 min in a set of Jayant Standard Test Sieves (Jayant Scientific Industries, Mumbai, India) ranging from mesh no.28 to 100 or openings of 600 to 150 μm, in a sieve rack fitted in a Rotap sieve shaker.Sieved fractions were weighed.

Colour
Colour values of the RTC multi-whole grain mix was determined by using Hunter lab scan XE model (M/S Hunter associate laboratory Inc., Reston-V.A., USA) with a view angle of 2 °. Colour values of the sample was determined by the Hunter system L, a, b values.

Bulk Density (BD), and Sedimentation Volume (SV)
The bulk density (BD) of the RTC multi-whole grain mix was determined according to the method of [23].Sedimentation volume of the RTC multi-whole grain mix was determined according to the method of [24].Two gram of fine multi-whole grain mix was taken in a 25 ml stoppered cylinder and 25 ml 0.05N HCl was added and the contents were shaken vigorously.Thereafter, 2 drops of amyl alcohol was added and the contents were shaken gently.The cylinder was placed undisturbed for about 4 h.Sedimentation volume was recorded at the end of 4h and percentage increase in volume was reported.

Pasting Characteristics and Apparent Viscosity
The pasting characteristics of multi-whole grain mix at 10% concentration was studied with a Brabender Viscograph, Type VSK 4 (Duisburg, FRG) fitted with a 700 cmg sensitivity cartridge using the procedure described in [25].Viscosity of cooked multi-whole grain drink was measured at 10% concentration by using Brookfield DV -II + Pro (Brookfield Engineering Laboratories, INC, MA USA), at room temperature using disc spindle measuring system in a Synchroelectric viscometer.Apparent viscosity was measured as a relation between torque and spindle speed rotation.Measurement was carried out at 100 rpm using spindle number 2 depending on the measurement range of sample viscosity.

Proximate Analyses
The moisture content of the RTC multi-whole grain mix was determined by drying at 130° C for 2 h as per [26].The micro Kjeldhal method was employed to determine the total nitrogen and the protein content (Nx6.25)[27].Fat was estimated by extraction with petroleum ether (60 -80° C), with a soxhlet apparatus and ash content was determined as per [27].Carbohydrate was calculated by difference method as 100 -(moisture+protein+ash+fat).Dietary fibre was estimated using the rapid enzymatic assay of [28].

Determination of Total Carotenoids
Total carotenoid was determined by the procedure of [29].Five gram of sample was mixed with about 50 ml of acetone and ground with pestle and mortar.The extract was filtered and the extraction repeated till sample becomes colourless.The extracts were pooled and mixed with 50 ml petroleum ether and 400 ml distilled water in a separating funnel.The petroleum ether layer was separated and washed 2-3 times with water, dried with anhydrous sodium sulphate and made up to 100 ml with petroleum ether.The absorbance was measured at 452 nm and the total carotene content was calculated based on the molar extinction co-efficient of -carotene.

Characterization of Carotenoids by HPLC
The solvent used for extraction of total carotenoid was evaporated using nitrogen at 40-50° C in a water bath and the residue was dissolved immediately in a known volume of methanol and stored at -20° C until analysis.The carotenoids were fractionated on C18 (250 4.6 mm, 5 μm) column using HPLC system [Shimadzu HPLC with SCL-10A system controller, LC 10AT pump, and SPD, 10A UV-visible detector] and isocratic solvent system containing acetonitrile, chloroform, isopropanol and water (78: 16: 3.5: 2.5 v/v) set at a flow rate of 1 ml/min and the detector was set at 452 nm.Linear response of carotenoid was in the range 0.5-10 ng [30].

Extraction of Sample for the Vitamin E Analysis, Antioxidant Property and Polyphenol Analysis
The multi-whole grain mix (0.4 g) was extracted with 4ml of methanol for one hour with occasional stirring.The extract centrifuged at 3000 rpm and the supernatant was filtered, and then stored at -20°C until used for the analysis of Vitamin E, antioxidant property and soluble polyphenols [31].Further for extracting the bound polyphenols, the residue was extracted and supernatant collected as above with 1% HCl methanol reagent [32].

Estimation of Polyphenols
Known quantity of methanolic extract of sample was mixed with Folin Ciocalteus reagent and sodium carbonate, volume adjusted with water, kept in dark for 30min, centrifuged and supernatant read for OD at 760 nm.Ferulic acid was used as standard [33].

Characterization of Vitamin E by HPLC
Vitamin E (tocopherols and tocotrienols) content of multi-whole grain mix was quantified by the method of [30], as explained by [5].Reverse phase HPLC (CBM-10A Shimadzu system with RF10AXL fluorescent detector, LC10AT pump) was used and the chromatograms were recorded and processed by LC-10A class software.The extracts were separated on Merck Purospher Star C18 column (4.6X250 mm, 5 mm); (Merck, Darmstadt, Germany) using a gradient solvent system consisting of acetonitrile, methanol, isopropanol and aqueous acetic acid [45:40:5:10] as solvent A and acetonitrile, methanol and isopropanol (25:70:5) as solvent B. The Fluorescence detector was set at excitation and emission wavelengths of 298 and 328 nm, respectively.Standards of both tocopherol and tocotrienol exhibited a linear response in the range as follows; 4-45 ng, 3-55 ng, 0.4-5ng.

In-Vitro Starch Digestibility
In -vitro starch digestibility of multi-whole grain mix was estimated according to the method described by [34].The glucose in the digested sample was estimated by DNS method [35].

Total Antioxidant Activity
The total antioxidant activities of the multi-whole grain mix was quantified using the phosphomolybdenum reagent [36].An aliquot of methanolic extract of sample (20 μL) mixed with 1230 μl of the reagent in a microtube.The tubes were capped, shaken well and incubated at 90°C for 90 min in water bath and the absorbance was measured at 695 m against a reagent blank.Results were calculated and expressed as -tocopherol equivalents (TE) per gram using the molar extinction coefficient of -tocopherol.Linearity of reaction was found to be in the range 2 10 -4 to 2 10 -5 moles.

Free Radical Scavenging Activity
Methanol soluble extract of the sample was mixed with DPPH reagent, kept in dark for 30 min at room temperature and read for OD at 517nm.DPPH reagent was used as blank and percentage reduction was noted.Catechin was used as the standard and the catechin equivalent was calculated from the linearity curve of percentage reduction against the quantity [37].

Preparation of Drink/Porridge from the Ready to Cook Multi-Whole Grain Mix
Drink was prepared by mixing 10 g sample and 25 gm in the case of porridge, in a pan with 100 ml of water, 60 ml of milk, 2.5 g sugar.This slurry was cooked for 3-4 min in a thick vessel in low flame with continuous stirring.Cooking time was fixed based on sensory acceptability.

Sensory Evaluation
A trained panel was employed for carrying out sensory evaluation of multi-whole grain drink/porridge prepared from the mix were analysed for sensory quality acceptance by following the method of quantitative descriptive analysis (QDA).The scorecard consisted of 15 cm scale where in 1.25 cm was anchored as 'Low' and 13.75 cm as 'High' [38].The sensory attributes such as appearance of buff colour, consistency, toasted cereal aroma, pulsy aroma, milk like aroma, cardamom aroma, bland and sweet taste and overall acceptability were analysed.Evaluations were carried out in sensory booths under white fluorescent light, air conditioned at 20±2 °C with relative humidity 50±5 %.The drink was served to panelists in cups and porridge in porcelain plates coded with 3-digit random numbers to minimize bias.

Statistical Analysis
Results are presented as mean + SD (standard deviation) of three independent determinations [39].

Physical Properties of the Mix
Particle size varied from 600 microns to <150 microns (Table 1).Coarse particles were very less 0.36 %.Mix contains mostly 180-250 micron size particles (52 %).This make the quality of the cooked product smoother with better mouth feel.Endosperm hardness, amount of pericarp remaining after decortication, flour particle size distribution, etc. in sorghum is reported to affect the quality of porridge.Isolated sorghum starch (<45 microns) was of equal or better quality and contained less soluble solids than to prepared from sorghum flours containing particles <250 microns or <425 microns [40].
In the Hunter colour measuring system L value indicating the brightness was found to be 68.5 for the mix.Lower 'a' value indicated that reddish tint is less in the mix even though red rice and fingel millet were used.'b' value of 14.7 indicate the yellow tinge since the mix was prepared using maize, wheat and finger millet which are rich in carotenoids.Percentage change (~257 %) in sedimentation value for the mix shows the high degree of partial gelatinization of the starches present in various grains which has undergone the process of roasting and consequent higher water absorption capacity.Bulk density of the mix was 0.45 kg/L indicating the fluffy nature of the mix.

Viscographic Parameters
The various parameters measured in the Brabender Viscograph are shown in (Table 2).The mix at 10 %  Values are mean ± standard deviation of three independent determinations.concentration on cooking had gelatinization temperature (82°C), indicating that the granules in this mix loses their birefringence at an early temperature due to the pre-gelatinization occurring at the roasting step of the processing.Peak viscosity (PV) is the viscosity where the starch granules reach highest swelling while cooking.It was observed that the mix showed least peak viscosity of about 285 BU, indicating that the various grains in the mix had undergone high degree partial gelatinization, which means grains had achieved almost cooked nature, while processing.Hot paste viscosity (HPV) is the viscosity registered at the end of cooking.It was observed that in the mix the HPV was 310 BU, and it was an indication that the swollen granules did not change or did not break, indicating that the mix was behaving like cross linked starch, as cross linked starch will not break down while cooking [41].Break down value was negative, further indicating the behavior of cross linked type starch.The cold paste viscosity (CPV) value was 605 B U. Correspondingly the set back was less indicating the fact that the mix had undergone lesser retrogradation.Total set back value was low; hence the quantity of linear molecules precipitation was less in the mix.Where as PV, HPV and CPV were 570, 410 and 980 BU, respectively, in finger millet flour which is traditionally used for drink or porridge purpose which will have thick slurry [42].
Viscosity analysis by digital brook field viscometer of the cooked mix at 10% concentration showed the least viscosity (141.6 cps) at 45 °C indicating the semi crystalline nature of the starch granules.

Nutritional Composition
Moisture content of the mix was 8 %.Carbohydrate, determined by difference was 71 %, as this mix contained maize, wheat, barley as major grains which are rich sources of carbohydrates.Protein content was 10.5 % due to the presence of pulses and nuts.Lipid content was high (8.6 %), this could be due to the presence of maize, ground nut and cashew nut which are rich sources of lipid.Ash content was 1.92 % and calorie of the mix was 402 kcal.Insoluble dietary fibre was high (11.7 %) because of the presence of seed coat of the grains as mix was prepared from whole grains.Soluble fibre was 1 % and total dietary fibre was high 12.7 % (Table 3).Investigations on whole grains of rice suggest that content of total dietary fibre is only 4.96-8.8% [43], where as that in polished rice is still lower at 0.22 to 1.25 % [44].The use of whole wheat flour instead of refined flour significantly improved the nutritional profile of flour tortillas [45].There are different functional foods developed in different countries like USA, Japan, EU from whole grains considering its nutritional potential [46].Effect of replacement of whole wheat flour with multigrain blend, increased the protein, fat, dietary fibre and mineral contents of north Indian parotta, [47].Multi-whole grain mix also contributes significantly to RDA of protein (22 %), fibre (51 %) and calorie (18 %).This indicates that multigrain composition is nutritionally superior to refined grains.Whole grain and refined wheat flours showed distinct metabolic profiles in rats due to difference in the content of health beneficial components [48].Whole grains and dietary fibre continue to win honors in preventing various diseases [49].Values are mean ± standard deviation of three independent determinations.

Total Carotenoids and its Charecterization
The total carotenoids content in the mix was high 290 g/100g.This could be due to the presence of whole grains like maize, wheat and Finger mullet in the mix.This value was comparable with the carotenoid content of the proso millet (366 g/100g) [5], but significantly higher than the carotenoids content of wheat 150-200 g/100g, sorghum 180-230 g/100g, finger millet 199 g/100g and little millet 78 g/100g [16,5].Whole multi-grain products will provide the good proportion of carotenoids.However, the presence of ßcarotene could not be detected by HPLC analysis (Figure 3), unlike in maize which is reported to contain about 15.7 g/100g [50].Even then, contribution of other carotenoid types like xanthophylls (lutein and astaxanthin) detected in the mix should not be overlooked.These are present in a wide variety of fruits and vegetables and also in grains [51,49].Lutein and zeaxanthin are repoted to prevent eye diseases also [52].

Vitamin E Characterization
The characterization of vitamin E in multi-whole grain mix was carried out by reverse phase HPLC (Figure 4).Vitamin E in the mix was found to be in the form of tocopherols as the major component and tocotrienols as minor component.The content of total tocopherols was 6.1 mg/100g.Gama tocopherol in the mix was higher (4.6 mg/100g) followed by alpha (1.5 mg/100g).Tocotrienols are not quantified since the contents are only in traces.Vitamin E component in other cereals like wheat also is tocopherol where as the major component in rice is tocotrienols [53].Rice and wheat flours contain only 0.8 mg and 1.23 mg/100g total Vitamin E, respectively, whereas the vitamin E content in refined little millet is 1.3 mg and whole grain finger millet flour is 4.1 mg/100g [5].
Vitamin E mainly exists as alpha, gamma and delta isomers.Among those, -Tocopherol is an important lipid-soluble antioxidant and it protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction [54,8].Other forms of tocopherols like gama also have antioxidant properties and their own unique properties [7].Both tocopherols and tocotrienols have antioxidant potential against cholesterol oxidation [55].Since vitamin E has a number of health beneficial effects the multi-whole grain mix could serve as a good source of these to the consumers.

Polyphenols
The soluble, bound and total polyphenolic contents in multi-whole grain mix were 94, 132 and 226 mg GA Eq./100g, respectively.Main source of polyphenols in the mix is from finger mullet which is reported to contain 450mg/100g soluble and 900 mg/100g bound polyphenols [56], sorghum and maize [57].Content in the mix is better than the refined grains were it is very low, 6 mg/100g, in the case of polished rice due to the loss of bran layers during polishing [58].

Vitamin E Activity
The biological activity of vitamin E in animals is defined by its influence on symptoms of deficiency, including neuropathy, fetal death, or myopathy (muscle disease), and is dependent upon distinct regulatory processes.Vitamin E activity indicates the estimated biopotency of vitamin E and that in i.u. for alpha tocopherol 1.0; gamma tocopherol 0.25; and delta tocopherol 0.01 [59].Vitamin E activity was 1.9 i.u./100 g in whole grain mix where as it is low in refined cereal and millet flours based on the vitamin E composition.

Total Antioxidant Capacity
The total antioxidant capacity of the mix was 17mM a-Tocopherol Eq./g.Vitamin E, carotenoids, and polyphenols in the food contribute in total antioxidant capacity.Investigations in our lab also have shown that finger millets are rich in polyphenols [55] and have free radical quenching ability [60].Phenolics like ferulic acid and coumaric acid in cereals are known to express high antioxidant activity.These phytochemicals contribute to effective antioxidant potency.Other cereals like sorghum [61], wheat [62] and rice [63,64] are also reported to have antiradical properties.Polished rice contains only 3 mM a-T Eq. /g, which is much less than in the fully polished small millets [5].As investigated by [65], the antioxidant properties like TPC, DPPH radical scavenging activity, oxygen radical absorbance capacity, ferulic acid content by HPLC analysis showed the evidence on the potential health benefits to be derived from consuming whole grain products.

DPPH Free Radical Scavenging Activity
The free-radical scavenging potentials of the methanolic extract of multi-whole grain mix were analyzed by the DPPH method, and the results are shown in (Figure 5).It exhibited 33, 38, 42 and 45 % free-radical activity at the 20, 30, 40 and 50 l of methanolic extract.Free radical scavenging activity of the mix was 153 mg CAT Eq./100g and it is higher than refined grain products.The activity of the extract is attributed to its hydrogen-donating ability [66].The antioxidants are believed to intercept the free radical chain of oxidation and to donate hydrogen from the phenolic hydroxyl groups, there by forming a stable end product, which does not propagate further oxidation of the lipid [67].

In-Vitro Starch Digestibility
The starch digestibility of mix was 62 % and this medium digestibility is caused by partial gelatinization of the starches present in various grains which has undergone the process of roasting.Low digestibility in legumes is caused by the crystalline structure of starch, which protects the glucoside bonds and limits enzyme hydrolytic action [68].Once fully gelatinized, this crystalline structure is lost, leaving the molecules open for hydrolysis, which breaks the glucoside bonds, and therefore increases digestibility.Low digestibility may also be due to the presence of fibre and polyphenols in whole grains which will reduce the digestibility of starch [69,70].Relatively lower carbohydrate digestibility makes the multi-whole grain mix suitable for diabetics also.

Sensory Quality of the Product
Sensory evaluation was carried out to make sure that the mix which is rich in nutrients and nutraceuticals is acceptable to the consumers.Scores for buff colour, consistency, flavors of toasted cereals and pulse, milk, cardamom were optimum in whole-multi grain drink and porridge samples (Figure 2).Overall acceptability was 10 and 11, respectively for drink and porridge with sugar, and 7.5 and 7.7 without sugar.This indicates that the product can be used with and without sugar and can be consumed by young and adult as a source of macro and micronutrients and phytochemicals.

CONCLUSIONS
Ready to cook multi-whole grain mix is an ideal blend of cereals, millets, pulses and nuts which provide the benefits of nutrients and nutraceuticals present in different whole grains.Mix can be used for the preparation of drink or porridge with or without sugar and it was sensorily acceptable for all the age groups.Mix is rich in nutrients, nutraceuticals and bioactive properties compared to the refined grains.So this convenient, tasty multi-whole grain mix can be used as wholesome food for health and wellness.

Figure 1 :
Figure 1: Flow chart for the preparation of RTC Multi-whole grain mix.

Figure 2 :
Figure 2: Sensory profile of multi-whole grain drink and porridge.

Figure 3 :
Figure 3: HPLC chromatogram of carotenoids in the multi-whole grain mix showing astaxanthin (3.5min) and spectrum of lutein peak (4.5 min).

Figure 5 :
Figure 5: DPPH free radical scavenging activity (FRSA) of methanolic extract of multi-whole grain mix and the standard catechin.