MMaatteerriiaallss aanndd MMeetthhooddss

III. MATERIALS AND METHODS

Increasing awareness and consciousness of mother for better growth and health of

their children signifies the need for developing weaning food which could supplement the

balanced nutrition and calories besides imparting health benefits. In order to meet the

growing demand of the modern society the investigation pertaining to formulation of

functional weaning food has been ventured. Weaning food was formulated by the

application of functional food ingredients such as probiotics and whey protein

hydrolysate along with the admixture of malted ragi, malted wheat, malted green gram,

refined vegetable oil, sugar and other ingredients. The materials used in the investigation

and methods adopted in developing weaning food are presented in this chapter.

3.1 Materials

3.1.1 Ingredients

The ingredients used in the investigation namely ragi, wheat, green gram, sugar,

refined sunflower oil, multivitamins, corn starch, sugar and Daber honey were procured

from the local market.

3.1.2 Whey protein concentrate

Whey Protein Concentrate (WPC) (PROCON 3700 WPC 70) was procured from

Mahaan Protein Ltd, New Delhi.

3.1.3 Enzymes

Neutrase enzyme 0.8 L, was procured from Novozymes, South Asia Pvt Ltd. The

enzymes Trypsin and Papain were obtained from Sisco Research Laboratories, Mumbai.

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3.1.4 Probiotic culture

Freeze dried probiotic cultures namely Bifidobacterium bifidum NCDC 232 was

obtained from National Dairy Research Institute Bangalore. Lactobacillus acidophilus

NCFM LYO 10 D was procured from Danisco (India) Private Ltd, Mumbai. These

cultures were sub cultured and maintained at the post graduate laboratory of Dairy

Technology Department.

3.1.5 Microbiological Media

3.1.5.1 Rogosa SL Agar

Rogosa SL agar M130-500G was procured from HiMedia, laboratories Mumbai

to enumerate Lactobacillus acidophilus NCFM LYO 10D.

3.1.5.2 Bifidobacteria Agar

Bifidobacteria agar M1396-500G was procured from HiMedia to enumerate

Bifidobacterium bifidum NCDC 232.

3.1.6. Packaging material

Poly Ethylene Terephthalate (PET) and High Density Poly Ethylene (HDPE) were

procured from Shakthi packaging company, Bangalore.

3.1.7 Experimental animals

Twenty one days old male albino rats (Wister strains) were procured from

Department of Livestock Production and Management, Veterinary College, KVAFSU,

Bangalore for bioassay studies.

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3.1.8 Chemicals and reagents

The chemicals and reagents used were of analytical grade. For all analytical

purpose freshly prepared reagents were used.

3.1.9 Glassware

The “Borosil” make glassware was used for chemical and microbiological

analysis of weaning food. Prior to use all the glasswares were thoroughly washed, air

dried and sterilized in a hot air oven at 160-180oC for 2 h for microbiological study.

3.1.10 Utensils

Stainless steel utensils were used to handle the ingredients in the formulation of

weaning food. All the utensils and accessories were thoroughly washed with suitable

detergent and rinsed with tap water before use.

3.1.11 Equipments

3.1.11.1 Spray dryer

Pilot scale Anhydro spray dryer of 7 kg of water evaporation capacity per hour

(Make: Anhydro, Copenhagen Denmark) was used for spray drying of whey protein

hydrolysates and malted functional weaning food.

3.1.11.2 Fluid bed dryer

GMP model fluid bed dryer of two kg wet material capacity per batch, fabricated

and supplied by Milk-Tech Engineers, Bangalore was used to dry malted functional

weaning food.

SPRAY DRIER FLUID BED DRIER

PLATE:1- Equipments used for drying of Functional Weaning Food

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3.2 Methods

3.2.1 Initial processing of the ingredients

The ingredients used in the formulation of functional weaning food were

preprocessed by following standard protocol before used in the formulation.

3.2.1.1 Preparation of malted ragi flour

Ragi malt flour was prepared as per the procedure described by Swamy, (2003)

with slight modification. A good quality ragi (Eleusine coracana) variety Indaf-9 was

cleaned and soaked in clean water for 16 h followed by draining out the water. Further, it

was held for germination for 36 h in wet cloth and sprinkling with water at regular

interval. After germination, it was sun dried for 8-9 h to terminate germination followed

by devegetation to remove root lets. Further, it was roasted for 15 min to improve the

flavour. Then conditioning was carried out by spraying water to get 7 per cent moisture

and mixed well followed by grinding to fine powder using attrition mill. The powdered

ragi malt was sieved using 60 mm mesh sieve and stored in clean air tight container until

used.

3.2.1.2 Preparation of malted wheat flour

The whole malted wheat flour was prepared as per the procedure of Taragopal et

al., (1982). Good quality wheat (Triticum aestivum) variety GWR-162 was cleaned and

soaked in clean water for 12 h. Then water was drained out and kept for germination for

36 h at room temperature covered with wet cloth, followed by sun drying till the grain

becomes dry completely. Dried wheat was devegetated to remove rootlets and roasted for

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15 min. The germinated dried wheat was conditioned to have 4 per cent moisture with

clean water and left for few minutes before grinding by attrition mill to obtain malted

whole wheat flour. The flour thus obtained was sieved using 60 mm mesh sieve and

stored in cleaned airtight container until used.

3.2.1.3 Preparation of malted green gram flour

The green gram malt was prepared as per the procedure outlined by Malleshi

(1995). A good quality green gram variety PB-1 procured from the local market was

cleaned and soaked in clean water for 12 h. Further, water was drained and kept for

germination for 24 h at room temperature covering with wet cloth, followed by sun

drying till the grains become crisp. Sprouted dried green gram was devegetated and

dehusked to obtain malted dhal. The dhal was roasted for 15 min followed by grinding to

get fine powder using attrition mill. The flour thus obtained was sieved using 60 mm

mesh sieve and stored in clean airtight container until used.

3.2.1.4 Preparation of carrot and tomato juice

Good quality carrot procured from the local market was cleaned and properly

washed with potable water. Carrot were cut in to small pieces and grinded in wet grinder

followed by filtering and squeezing to get juice. The resultant juice was used for blending

with the whey medium. Similarly fresh tomatoes procured from local market were

washed with tap water. The washed tomatoes were cut in to small pieces and grinded in

wet grinder, the juice obtained was filtered to remove fibre material. Resultant juice was

used for blending with whey medium for propagating probiotics.

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3.2.2 Propagation of probiotic culture

The probiotic culture was propagated in whey medium enriched with different

prebiotics at various levels in order to obtain higher viable count.

3.2.2.1 Growth and maintenance of probiotic cultures in whey medium

Probiotic cultures namely and Lactobacillus acidophilus NCFM LYO 10D (La-N)

and Bifidobacterium bifidum NCDC 232 (Bb-N) were inoculated separately to the

sterilized cheese whey medium at 0.5, 1.0, 1.5 and 2.0 per cent levels and incubated at

37oC for a period of 24 h. The growth of probiotics as measured in terms of pH, acidity

and viable counts was monitored at a regular interval of 3 h.

3.2.2.2 Effect of prebiotics on the growth of probiotics

Sterilized whey medium was fortified with various prebiotic namely honey, blend

of carrot and tomato juice (1:1) and whey protein hydrolysate. The fortified whey

medium was inoculated with probiotic culture and incubated at 37oC for a known period

to study the effect of prebiotics.

3.2.2.2.1 Effect of honey on the growth of probiotics

Cheese whey medium was added with honey at 1, 2 and 3 per cent levels and

subjected to sterilization. Sterilized medium was inoculated separately with Bb-N and La-

N at their optimum level and incubated for an optimum period at 37oC as per the results

obtained in the para 3.2.2.1. The effect of honey on the activity of probiotics was

measured in terms of pH, acidity and viable counts to adjudge the optimum level of

honey to be added.

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3.2.2.2.2 Effect of carrot and tomato juice blend on the activity of probiotics

Cheese whey medium was added with an optimum level of honey and further

added with 3, 4 and 5 per cent levels of carrot and tomato juice blend (1:1) and subjected

to sterilization. Sterilized medium was inoculated separately with Bb-N and La-N at their

optimum concentration and incubated at 37oC. At a regular interval, the growth of

probiotics in terms of pH, acidity and viable counts was monitored to select an

appropriate level of carrot and tomato juice to be blended.

3.2.2.2.3 Effect of hydrolyzed whey protein concentrate on the activity of probiotics

Cheese whey medium was added with an optimum level of honey and a blend of

carrot and tomato juice. Whey medium was further added with 0.5, 1.0, 1.5 and 2.0 per

cent levels of Whey Protein Hydrolysate (WPH) and subjected to sterilization. Sterilized

whey medium was inoculated with Bb-N and La-N separately at their optimum level and

incubated at 37oC. At a regular interval, the growth of probiotics was monitored in terms

of pH, acidity and viable counts to elicit the effect of whey protein hydrolysate on the

growth of probiotics.

3.2.2.2.4 Media preparation for enumerating Bifidobacerium bifidum NCDC232 and

Lactobacillus acidophilus NCFM LYO 10D

The stock solution was prepared by dissolving 3.4 g of Potassium dihydrogen

phosphate in 100 ml of distilled water and pH was adjusted to 7.0 with 0.5N NaoH.

1.25ml of stock phosphate buffer solution was taken and the volume was made up to

1000ml with distilled water and distributed in 9ml or 99ml quantities in tubes or bottles

and sterilized by autoclaving at 121oC/15 min.

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49.3 g of Bifidobacteria agar was suspended in 1000 ml distilled water, mixed

well and heated till it was boiling to dissolve the medium completely and filling in to

flasks followed by sterilization by autoclaving for 15 lb pressure at 121 oC for 15 min,

cooled to 50oC and used for plating. The plates were incubated in anaerobic condition for

72-96 h in an anaerobic jar adopting candle method to enumerate Bfidobactrium bifidum

NCDC 232.

75 grams Rogosa SL agar media was suspended in 1000 ml distilled water and

heated to boiling to dissolve the medium completely. Then 1.32 ml glacial acetic acid

was added and mixed thoroughly and distributed into culture tubes or flasks and heated to

90-100o C for 2 to 3 min followed by cooling to 40o C for direct inoculation. The plates

were incubated an anaerobic condition at 37oC for 72-96 h in an anaerobic jar adopting

candle method to enumerate Lactobacillus acidophilus NCFM LYO 10D.

3.2.3 Process optimization of hydrolysis

In order to select an appropriate enzyme for enzymatic hydrolysis of WPC, spray

dried WPC was reconstituted to have 12 per cent protein and heated to 80oC for 5 min.

The reconstituted medium was inoculated separately with Neutrase, papain and trypsin

enzymes at 1:25 enzyme to substrate ratio (E: S) and incubated for a period of 180 h at

their respective optimum pH and temperature. The effect of enzyme on degree of

hydrolysis was monitored at a regular interval of 30 minutes by measuring change in pH.

An enzyme which resulted in a maximum hydrolysis with minimum time was used

further in the investigation.

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3.2.4 Optimization of protein concentration in substrate and enzyme to substrate

ratio

Spray dried WPC-70 was reconstituted to 12, 15, 18 and 21 per cent total solids

using potable water and adjusted to optimum pH. Neutrase enzyme was added to the

reconstituted samples at various enzymes to substrate ratio (1:25, 1:50, 1:75 and 1:100).

In vitro digestion was carried out for a period of 150 min in a water bath maintained at 40

oC. Subsequently, the enzyme activity was terminated by heating the samples to 85oC for

10 min. The extent of hydrolysis was estimated. The samples which resulted in maximum

hydrolysis with minimum enzyme concentration was selected for further studies.

3.2.5 Preparation of spray dried whey protein hydrolysate powder

Spray dried WPC was reconstituted to have15 per cent protein. The reconstituted

WPC was added with Neutrase enzyme at the enzyme substrate ratio of 1:50. The in-vitro

digestion was carried out by incubating at 40oC for a period of 150 min. The resultant

hydrolysate was spray dried and further used for formulation of weaning food.

3.2.6 Formulation of weaning food

The functional malted weaning food was formulated by using malted ragiflour,

malted wheat flour, malted green gram flour, hydrolyzed WPC, sugar, oil and

multivitamins besides supplementation with probiotics.

3.2.6.1 Optimization of ragi malt and wheat malt levels in weaning food

Based on the desired nutritional requirements profile of the weaning food base

blend containing 30 parts of cereals/ millets, 38 parts pulses, 24 parts sugar and 8 parts of

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oil was prepared as per the procedure outlined by Swamy (2003). In the portion of 30

parts of cereals/ millets base, wheat malt was replaced with ragi malt at 25, 50, 75 and

100 per cent levels. The blends were subjected to chemical and sensory evaluation studies

after cooking to make into porridge in order to select the right levels of ragi malt that

could be incorporated in the weaning food.

3.2.6.2 Optimization of level of replacement of green gram with WPC and WPH

The optimized weaning food with respect to malted ragi was further tried with

WPC and WPH for replacing malted green gram. The malted green gram powder used in

the study was replaced with WPC and hydrolysed WPH at various levels (0, 10, 20, 30 &

40 %). The blends were reconstituted to 25 per cent TS and cooked and made into

porridge. The resultant gruel was subjected to chemical and sensory evaluation attribute

studies in order to select the right level of replacement of green gram with WPC and

WPH.

3.2.7 Process optimization with respect to total solids in gruel and cooking of

weaning food

The formulated weaning food prepared with the admixture of wheat malt, ragi

malt green gram and WPC/WPH along with other ingredients was reconstituted with

potable water to have 20, 25, 30 and 35 per cent total solids and cooked at various

temperatures (70, 75, 80 and 85oC for 10 min). The resultant product was evaluated by

subjecting to various physical and sensory attribute studies to select the optimum level of

cooking temperature and total solids for obtaining right consistency of porridge for

consumption.

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3.2.8 Optimization of level of probiotics in weaning food

The probiotics used in the study namely La-N and Bb-N which were propagated

in enriched whey medium with prebiotics (honey, carrot and tomato juice and whey

protein hydrolysate) at the end of 15 h incubation the cell concentrate was prepared by

centrifugation of the culture. The properly grown culture containing maximum viable

counts were filled to sterilized centrifuge tubes and subjected to ultracentrifugation for

10,000 RPM for 30 min to obtain cell concentrate and decant. The cell concentrate

obtained by centrifugation was used for blending with the reconstituted and cooked

weaning blend.

Formulated weaning food was cooked at 75oC for 10 min followed by cooling to

room temperature. The probiotics containing La-N and Bb-N were incorporated

separately at different level (2.5, 5.0, 7.5 and 10 %) and subjected to sensory evaluation

to adjudge the maximum level of probiotics that could be incorporated to the porridge,

without affecting physico-chemical and sensory qualities of the weaning food.

3.2.9 Process optimization for spray drying of formulated weaning food

The formulated weaning food was adjusted to a desired level of total solids and

cooked at optimum time temperature combination. The cooked weaning porridge was

subjected to spray drying at various combinations of outlet air temperature namely 60,

65, 70 and 75oC and various feed inlet temperature viz 65, 70 and 75oC in order to assess

the effect of spray drying processing parameters on the quality of resultant powder.

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Similarly, the formulated weaning food which was cooked at right time

temperature combinations was added with an optimum level of probiotic culture and

subjected to spray drying at various combinations of outlet air temperature (60, 65, 70 &

75 oC) and feed temperature (65, 70 and 75oC) to assess the effect of drying parameters

on the viability of probiotics.

3.2.10 Standardization of fluidized bed drying

As per the result obtained in the para 3.2.8 the standardized level of culture

containing Bifidobacerium bifidum NCDC232 and Lactobacillus acidophilus NCFM LYO

10D was added to spray dried weaning food separately. And rewetted to have 15 per cent

moisture and subjected to fluid bed drying at various outlet temperatures (40, 45, 50 and

55 oC) to assess the optimum temperature of drying to retain maximum viable counts.

3.2.11 Bioassay studies for formulated weaning food

Five types of diets were selected for conducting bioassay studies. For each of

these diets, vitamin mixture was supplemented externally and corn starch was used to

adjust the total solids of the product. Formulated weaning food was subjected for

bioassay studies on albino rats.

T0: Malted weaning food

T1: Commercial weaning food

T2: Weaning food prepared by replacing green gram protein with WPC

T3: Weaning food prepared by replacing green gram protein with WPH

T4: Weaning food with WPH and probiotics

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Clinically healthy animals of weaning albino (Wister strain) rats aged 21 days

weighing about 32.20-34.50 g were randomly selected and divided in to five groups, 6

rats for each experimental group were individually housed in separate cubicles in a

metabolic cages with adequate facilities for separate feacal and urinary collection and

allowed to acclimatize to experimental condition for 3 days. The feed and water were

given ad libitum for a period of 28 days. The residual feeds were collected every day,

weighed and feed consumption was recorded. Records were kept for the weight changes

and total food intake. The gain in weight was recorded at weekly basis and evaluated for

the Protein Efficiency Ratio (PER) and Feed Efficiency Ratio (FER). Further the rats

were housed in biological cages to collect feaces and urine to assess Digestible

Coefficient (DC), Biological Value (BV) and Net Protein Utilization (NPU). The urine

from each cages were collected in small urine cups containing 0.1M H2So4 as

preservative and stored in capped bottles under refrigeration until analysed.

3.2.11.1 Bioassay parameters

The PER of the control and experimental test diets were determined as per the

procedure of IS: 7481 (1974). The nutritive value of protein was expressed as the ratio of

gain in body weight (g) to the protein consumed (g) in a specified period of time. The

feed efficiency ratio was expressed as gain in weight in grams per unit of food consumed.

DC, BV and NPU were measured as per the procedure outlined by Onweluzo and

Nwabugwu (2009).

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3.2.12 Storage studies

The formulated fluid bed dried weaning food carrying probiotics was packed in

HDPE and PET packaging material under normal and modified atmospheric packaging

conditions.

The formulated weaning food samples were subjected to MAP before storage

with the specifications as detailed in annexure-IV. The gases used in the study for MAP

are carbon dioxide and nitrogen in the ratio of 20:80, 40:60, 60:40, and 80:20. The

packaged products were stored at room temperature (30oC) for a period of 6 months. At a

regular interval of 30 days the samples were analysed for viability of probiotics and for

various chemical attribute studies in order to elicit the effect of packaging materials and

MAP.

3.3 Analytical techniques used for formulation of weaning food

3.3.1 Moisture content

The moisture content of all the ingredients and samples were estimated as AOAC

(1984).

3.3.2 Estimation of total protein

The total protein content of the dried samples of ingredients as well as final

functional weaning food was computed by estimating total nitrogen by the Micro-Jeldhal

method.

PLATE 2: Modified atmosphere packaging machine

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3.3.3 Estimation of fat

Fat content was estimated by ether extract method as per the procedure of AOAC

(1980).

3.3.4 Estimation of ash

Total ash content of the developed weaning foods was analysed as per the

procedure of AOAC (1980).

3.3.5 Estimation of crude fibre

Crude fibre of the sample was estimated by using moisture and fat-free samples

and expressed as g/ 100g or per cent of the samples used as per AOAC (1984).

3.3.6 Computation of carbohydrate

Carbohydrate content was calculated by differential method as per AOAC, 1980.

Carbohydrate (g/100g) = 100-[Protein + Fat +Fibre + Ash + Moisture].

3.3.7 Computation of energy

Energy was computed as per AOAC, 1980.

Energy (kcal) = [Protein (g) X 4] + [Carbohydrate (g) X 4] + [Fat (g) x9].

3.3.8 Determination of pH

The pH of reconstituted samples was measured using digital pH meter (Elico

make).

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3.3.9 Determination of acidity

Acidity was measured by titrating against 0.1N NaoH using phenolphthalein

indicator and expressed in terms of per cent lactic acid as per the method described in

IS:SP:18 (Part XI) 1981.

3.3.10 Spreadability

Spreadability was measured by adopting the procedure as described by

Prasannappa et al. (1972).

3.3.11 Viscosity

The viscosities of different samples at different total solids were measured using

Brook field viscometer, model D220, Make: Brookfield Engineering Laboratories DV-

11+ pro 11 commerce BLVD, Middle boro MA 02346, USA.

3.3.12 Bulk density

Bulk density estimated as per the procedure developed by Wang and Kinsella,

(1976).

3.3.13 HMF

Extent of browning was estimated by measuring 5-hydroxy methyl-2 furfural

which is the main browning compound. Browning compounds were estimated as HMF

by adopting the method specified by Sripad (1988).

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3.3.14 Peroxide value

The peroxide values were estimated as per IS: 3508-1966.

3.3.15 Free fatty acids

Free Fatty Acid (FFA) content of samples was determined as per the procedure

described by Buma (1971).

3.3.16 Sensory evaluation

Sensory evaluation of samples was carried out by a panel of 5 in house judges by

providing 9 point hedonic scale.

3.3.17 Estimation of degree of hydrolysis

Degree of hydrolysis was determined by the pH stat method as per the method of

Adler-Nissen (1986). The degree of hydrolysis (DH) was commonly measured and

monitored by the amount of base that is consumed to maintain pH during the process of

hydrolysis. Per cent degree of hydrolysis was calculated by using the following formula.

Degree of hydrolysis (DH) = B x Nb x 1/α x 1/MP x 1/htot x 100

Where

B : Base consumption in ml

Nb : Normality of the base

1/α : Average degree of dissociation of the alpha amino group (it is a constant value

depends on temperature and pH, the dissociation values for pH 7.0, 7.5, 8.0 and 8.5

are 3.0, 1.63, 1.20 and 1.06 respectively)

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MP : Mass of protein in gram

htot : Total number of peptide bonds in the protein substrate (m.eq./g of protein htot for

whey protein is 8.8),

For every 30 minutes, DH was calculated throughout 3 h of hydrolysis till the

maximum hydrolysis was attained with minimum bitterness.

3.3.18 Statistical analysis

The results were analyzed statistically for test of significance by using Statistical

Packages for Social Sciences (SPSS) version 8 software programme.