iT

MATERIALS
AND
METHODS

\ i
I. MATERIALS

A. CHEMICALS

All the chemicals used were of analytical grade. The special

reagents used were as follows; heat-stable a-amylase (Sigma),

pancreatin, pepsin (Loba-Chemie), amyloglucosidase (Hi Media),

glucose oxidase/peroxidase diagnostic kit (Qualigens

Diagnostics, Glaxo), Autozyme kit for creatinine (Accurex

Biomedical Pvt. Ltd., Mumbai), Urea kit (Nicholas Piramal,

Mumbai), Uric acid kit (Aspen Laboratories, New Delhi), kit for

serum proteins (CDR Diagnostics, Hyderabad), Kit for SGOT and

SGPT (Euro Diagnostic Systems Pvt. Ltd., Chennai) and kit for

serum alkaline p h o s p h a t a s e (Agappe Diagnostics, Kerala).

1. FOODS

All raw food grains for the formulation of enteral foods

such as rice {Oryza sativa), ragi {Eleusine coracana), barley

{Hordeum vulgare) and green gram [Vigna radiata) were procured

in bulk from the open market. Other ingredients such as oats,

soya flour, soya oil, safflower oil, coconut oil, sugar and milk

powder were also purchased from the local market. The food

grains were cleaned of extraneous particles a n d stored at room

temperature :: 28°C in plastic bins before being subjected to

various processing treatments.

86
 Commercially available oatmeal (Bagrry's brand), defatted

soya flour (Proflo, Ruchi Soya Industries Ltd.), powdered sugar,

Ruchi b r a n d of soya oil. Parachute brand coconut oil, Saffola

brand of safflower and Dhara brand of g r o u n d n u t oils were

procured from the local market. Sagar b r a n d of skim milk

powder (SMP) and Amul Whole milk powder (WMP) were also

procured from the local market for addition to the food as a

source of milk in the formulated enteral food.

2 . NUTRACEUTICALS

All essential vitamins, minerals and other micronutrients

were procured as pharmaceutical formulations from the market.

All the necessary vitamins and minerals were added to the food

as per recommended daily allowances with appropriate overages.

These were of Indian/British or United States Pharmacopoeia

grade and were first constituted into a vitamin-mineral mix and

then added to food as required.

a. PROBIOTICS

A probiotic in the form of L. acidophilus spores was also

added to the food. Spores of Lactobacillus were p u r c h a s e d in the

form of the preparation Sporolac.

87
B. METHODS

1. SURVEY OF HOSPITALISED PATIENTS

Initially, selected hospitals in Mysore city having ICUs and

care facilities for the critically ill viz., Holdsworth Memorial

Hospital, BGS Apollo Hospital, Vikram Hospital, Bharath

Hospital and Institute of Oncology, Basappa Memorial Hospital

and J S S Hospital were visited to observe the ICUs and their

functioning. A nutritional need-assessment was conducted on

patients admitted to these u n i t s i.e. surgical ICUs, non-surgical

ICUs as well as on other critically ill patients to collect

information on the type and extent of nutritional support

provided. A schedule (Annexure I) was prepared for the purpose

of collecting this information, both from the patients or their

caregivers and the attending doctors and dietitians. A total of

100 patients were assessed.

This set of information was used as a base for identifying

the category of patients and their specific needs for formulating

condition-specific enteral foods.

2 . PROCESSING OF F O O D INGREDIENTS

The food grains cleaned and stored as described earlier

were processed after being checked again for contamination and

malted a s per the method described later. The oatmeal was

88
lightly toasted over a flame at z. 80+5°C, milled and passed

through 60 mesh sieve and stored in plastic bins for further

processing The defatted soya flour was also toasted at z 80+5°C

and stored till further use. This heat treatment is reported to

help in improving the nutritional quality of soya flour by

inhibiting certain anti-nutrient factors that may be present

(176).

a. Malting of cereals and legumes

Cereals and legumes selected for their nutritional quality

viz. ragi (Eleusine coracana), rice [Oryza sativa), barley [Hordeum

vulgare) a n d greengram [Vigna radiata) were malted as given

below.

i) Rice
The raw paddy were cleaned and washed to remove any

c o n t a m i n a n t s and steeped in excess a m o u n t of clean, potable

water for 24h. The water was changed after a b o u t 10 h o u r s and

the grains mixed thoroughly to help in swelling and to dissipate

any heat produced. The water that was added also contained

H2O2 at a l%v/v level to inhibit the growth of micro organisms

during germination. The steeped grains were then spread in a

double layer of moistened j u t e cloth. The thickness of the grain

bed was maintained at about 1 inch and another double layer of

wet j u t e cloth was used to cover it. Paddy was germinated for 3

89
days (~72h) at room temperature (28±3°C) in ambient conditions

for the duration. The grains were intermittently sprayed with

water and mixed/turned to maintain moisture as well as

temperature by dissipating heat produced during germination

and also to avoid clumping of germinated grains.

After germination, the grains were dried in an oven at

about 65+5°C. The grains were kilned at about 75-80±5°C till the

grains developed the 'malt aroma'. The h u s k of the malted rice

was removed j u s t before milling using a roller sheller. The grains

were later tempered with 5-7% of water and allowed to stand for

10 min. before being milled in a commercial plate mill.

Subsequently the rice malt flour was sieved through 100

mesh sieve (BS) to obtain a uniform particle size of ISOp. The

tempering of grains carried out prior to milling prevented its

pulverization and facilitated the removal of most of the bran

from the flour a s overtails at this stage.

ii) Ragi

The finger millet was steeped in excess a m o u n t of clean,

potable water for about 24h. The water was changed after about

10 h o u r s and the grains mixed thoroughly to help in steeping

and to dissipate heat produced. The water contained H2O2 at a

90
l%v/v level to inhibit the growth of micro organisms during

germination. The steeped grains were then spread on a double

layer of moistened jute cloth. The thickness of the grain bed was

maintained at about 1 inch and another double layer of wet j u t e

cloth was used to cover it. Ragi was allowed to germinate for 2

days (~48h) at room temperature {28±3°C) in ambient conditions.

The grains were intermittently sprayed with water and

m i x e d / t u r n e d to maintain moisture a s well a s temperature by

dissipating heat produced during germination and also to avoid

clumping of germinated grains.

After germination, the grains were dried in an oven at

about 65±5°C. The grains were kilned at about 70-75±5°C till the

grains developed the 'malt aroma'. The rootlets were removed

manually by abrasion and the derooted grains were later

tempered with 5-7% of water and allowed to stand for 10 min.

before being milled in a commercial plate mill.

Subsequently the ragi malt flour were sieved through 100

mesh sieve to obtain a uniform particle size of 150|a.

Hi) Barley

The barley grains were also steeped in excess a m o u n t of

clean, potable water for about 24h. The water was changed after

91
about 10 h o u r s and the grains mixed thoroughly to help in

steeping and to dissipate heat produced. The water contained

H2O2 at a l%v/v level to inhibit the growth of micro organisms

during germination. The steeped grains were then spread on a

double layer of moistened j u t e cloth. The thickness of the grain

bed was maintained at about 1 inch and another double layer of

wet j u t e cloth was used to cover it. Barley was allowed to

germinate for 3 days (~72h) at room t e m p e r a t u r e (28±3°C) in

ambient conditions. The grains were intermittently sprayed with

water and mixed/turned to maintain moisture as well as

temperature by dissipating heat produced during germination

and to avoid clumping of the grains.

After germination, the grains were dried in a n oven at

about 65+5°C. The grains were kilned at about 75-80±5°C till the

grains developed the 'malt aroma'. The rootlets were removed

manually by abrasion and the derooted grains were later

tempered with 5-7% of water and allowed to s t a n d for 10 min.

before being milled in a commercial plate mill.

Subsequently the barley malt flour was sieved through 100

mesh sieve to obtain a uniform particle size of 150)LI.

92
iv) Green gram

The previously cleaned green gram steeped in excess

a m o u n t of clean, potable water for about 24h. The water was

changed after about 10 h o u r s and the grains mixed thoroughly

to help in steeping and to dissipate heat produced. The water

contained H2O2 at a l%v/v level to inhibit the growth of micro

organisms during germination. The steeped grains were then

spread on a double layer of moistened j u t e cloth. The thickness

of the grain bed was maintained at about 1 inch and another

double layer of wet jute cloth was used to cover it. Green gram

was allowed to germinate for 2 days (~48h) at room temperature

(28±3°C) in ambient conditions. The grains were intermittently

sprayed with water and m i x e d / t u r n e d to maintain moisture as

well as temperature by dissipating heat produced during

germination a n d also to avoid clumping of germinated grains.

After germination, the grains were dried in a n oven at

about 65±5°C. The grains were kilned at about 70-75±5°C till the

grains developed the 'malt aroma'.

The rootlets were removed manually by abrasion and the

h u s k of the green gram is also removed during derooting. The

derooted grains were later milled in a commercial plate mill.

93
Subsequently the green gram malt flour were sieved through 100

mesh sieve to obtain a uniform particle size of 150|LI.

3 . ENTERAL F O O D FORMULATION

A total of 4 formulations were prepared a s follows (Table

14).

a. General formulation

The flour from the malted grains viz., rice, barley, ragi and

green gram, lightly roasted soya flour, oat flour, milk powder,

oil, sugar along with the vitamin-mineral mix were dry-blended

to prepare the general enteral food formulation and coded as

GEF.

The general formulation was suitably modified to contain

conditionally essential nutrients and designed for cancer

patients, HIV/AIDS patients and diabetics.

b. Formulation specific for cancer patients

The cancer-specific enteral food (CSEF) contained higher

a m o u n t s of soya flour (10%) and milk powder (18%) compared to

the base formulation (7 and 15% respectively). It also contained

coconut oil at a level of 1%, a source of medium chain

triglycerides (MCTs).

94
 Table 14. Proportion of ingredients in the
formulated enteral foods

Pro port ion (%)

Ingredient General Modified for Modified Modified
enteral cancer for for
food patients diabetics HIV/AIDS
Malted rice 10 10 10 12
Malted ragi 20 20 18 20
Malted barley 20 20 14 20
Malted green gram 10 10 10 8
Oatmeal 5 - 7 -
Soya flour 7 10 10 8
Skim milk powder - - 18 -
Whole Milk powder 15 18 - 20
Coconut oil - 1 - 1
Soya oil 2 2 2 2
Safflower oil 3 2 3 2
G r o u n d n u t oil 2 1 2 1
Sugar 3 3 3 3
Vitamin-mineral 3 3 3 3
mix

95
c. Formulation specific for HIV/AIDS patients

The HIV/AIDS-specific enteral food (HSEF) was formulated

with higher a m o u n t of rice malt for its hypoallergenic properties.

It also contained higher a m o u n t of milk powder to deliver good

quality protein required in cachexia or wasting t h a t often

accompanies this condition.

d. Formulation specific for diabetic patients

For the diabetes-specific enteral food (DSEF), the general

formula was modified to provide lower amounts of freely

available glucose and more a m o u n t of dietary fibre. Skimmed

milk powder was added and the a m o u n t of oils was adjusted to

deliver a mixture of m o n o u n s a t u r a t e d and polyunsaturated fatty

acids (MUFA and PUFA).

The different enteral formulations were packed in

polypropylene pouches recommended for foods and stored at

room temperature (28±3°C) till use.

4 . PHYSICO-CHEMICAL CHARACTERISTICS

a. Proximate c o m p o s i t i o n

Standard AOAC approved methods (200) were used for

determining the proximate composition of all the raw and

96
processed food samples. All estimations described here were

carried out in triplicate and the data pooled for average values.

i) Moisture

An accurately weighed sample (z. 5mg) of food was spread

over a petriplate and dried overnight (about 16h) in an oven at

50°C. it was covered and cooled in a dessicator and weighed.

Sample was dried again for a further 2h a n d reweighed till

constant weight was obtained for two readings.

ii) Protein

The total nitrogen in dried enteral food was determined by

micro-Kjeldahl procedure. A factor of 6.25 was used to convert

the nitrogen value obtained into protein content.

Hi) Crude fat

Crude fat was estimated in the food by solvent extraction

in a Soxhlet a p p a r a t u s for 14-16h with petroleum ether solvent

(b.p. 60-80°)

iv) Total ash

Ash was determined by drying the food samples overnight

in an oven at 100°C to give a dry weight of 2g, charring on a hot

plate for I h and then ashing in a muffle furnace at 550°C for 4-

97
5h. The weight was recorded after cooling. The difference in

weight gives an estimation of ash content.

v) Carbohydrate by difference

The total carbohydrate content by difference was estimated

by subtracting the a m o u n t of crude fat, protein and a s h from the

sample on dry basis.

b. Carbohydrate profile

i) Sugars

The reducing and total sugar content was estimated by

titrimetric determination using the Shaffer and Somogyi micro

method (201). 2.5g sample was transferred to a 250ml beaker.

50ml water was added and boiled. After cooling, it was

transferred to 250 ml volumetric flask. 2ml lead acetate was

added and the solution was allowed to s t a n d fore 10 min. the

excess lead was precipitated out with 2ml of 22% potassium

oxalate solution, it was mad up to volume and filtered.

Reducing sugar

Five ml of the filtrate was pipetted into a test tube. 5 ml of

Shaffer-Somogyi reagent was added and mixed well. The test

tube was capped with a funnel and kept in a boiling water bath

for 15 min. after cooling for 4 min u n d e r r u n n i n g water, the

funnels were removed and 2ml of iodide oxalate solution was

98
added from the side along with 3ml of 2N sulphuric acid. This

was mixed thoroughly to ensure complete dissolving of the

cuprous oxide and was allowed to stand for 5 min in cold water.

The solution was then titrated with 0.005M thiosulphate

solution u s i n g starch indicator.

Total s u g a r s

For estimation of total sugars, 25 ml of the filtrate was

taken in a 50 ml volumetric flask along with 5ml of hydrochloric

acid (1 + 1). This was allowed to stand for 24h at room

temperature and then neutralised with 2N sodium hydroxide.

The solution was made u p to volume with water. An aliquot was

taken and the total invert sugar was determined as in reducing

sugar.

Reducing sugar (%) = mg dextrose x volume made u p x 100

5 X weight of sample x 1000

ii) Glucose

Glucose was estimated in all foods using a glucose

oxidase/peroxidase diagnostic kit (Qualigens Diagnostics, Glaxo)

(202) by treating the unknown solution with the working

solution and incubating the assay mixture for 15min at 37°C.

The absorbance against blank was measured at 510 nm.

Hi) Starch profile

To m e a s u r e the different fractions of starch viz., rapidly

digestible starch (RDS), slowly digestible starch (SDS) and

99
resistant starch (RS) fractions, the method described by Englyst

et al. (203) was adopted.

The various categories of starch were measured after

incubation of the formulated foods with invertase, pancreatic

amylase and amyloglucosidase at 37°C in capped conical flask in

a shaking water bath. A value for rapidly available glucose (RAG)

was obtained a s the glucose released after 20 min. (G20). A

second m e a s u r e m e n t (G120) was obtained as the glucose released

after a further 100 min incubation. A third m e a s u r e m e n t for

total glucose (TG) was obtained on further treatment with 7M

potassium hydroxide at 0°C, followed by complete enzymatic

hydrolysis with amyloglucosidase.

nm"^
. y 1/ L ^ S

Resistant starch was measured as the starch that

remained unhydrolysed after 120min incubation. Free glucose

(FG) was also determined by treating the sample with acetate

buffer and placing the tube in water bath at 100°C for SOmin to

obtain values for RDS and TS. To obtain values for RDS and TS,

correcting RAG and TG for FG is necessary. Hence, FG including

the glucose released from sucrose would have to be measured.

Here, SDS was computed as G120-RAG. This method allows for

TG and G120 without the necessity to correct for FG, since this is

included in both m e a s u r e m e n t s .

100
 The values for different starch fractions of TS, RDS, SDS

and RS were obtained by combining the values of G20, G120, FG

and TG as follows:

TS = (TG - FG) X 0.9

RDS = (G120 - FG) X 0.9
SDS = (G120 - G20) X 0.9
RS = TS - (RDS + SDS) or
(TG - G120) X 0.9

The procedure used for estimating the starch fractions is

summarised in Figure 14.

iv) Dietary fibre

The method described by Asp et al. (204) was used to

measure total dietary fibre as the s u m of water-insoluble and

water-soluble fractions based on digestion of food samples with

enaymes. The enzymatic hydrolysis of starch an protein are

carried out in three steps: gelatinization in the presence of a

heat stable a-amylase (lOOmg, 100°C, 15 min, pH 6.0),

incubation with pepsin (lOOmg, 40°C, 60 min, pHl.5) and

incubation with pancreatin (lOOmg, 140°C, 60 min, pH 6.8).

Insoluble dietary fibre was recovered by filtration with celite as

filter aid. Soluble dietary fibre was precipitated from the filter

with four volumes of 9 5 % ethanol and recovered by filtration.

Schematic description of the method is given in Figure 15.

101
 SAMPLE 2 X STANDARD BLANK
0.8-4.0g (@ 700-900 mg dry matter) 25 ml gljcose std. 25 ml acetate buffer

Take 50 ml polypropylene tubes Take 50 ml polypropylene tubes

Add SOmg guar gum

Add 25 ml acetate buffer (sample only)

I
Add 25 ml acetate buffer

30 min. in boiling water bath Equilibrate at 37°C in water bath

Add 5 ml of enzyme solution (atfixedintervals)

— After 20 min.
After 120 min.
Cool to room temperature Vortex Mix well

Take 1ml in 2ml 66% ethanol 30 min. in boiling water bath

I Vortexl Mix well

Take 0.5ml in 2ml 66% ethanol
Centrifuge 5min. @ 1500g Add 10 ml 7M KOH
< ' »
Dilute 1ml in 5ml water Dilute 1ml in 20ml water
I Centrifuge 5mln. @ ISOOg

(Sample) (Standard) 30 min. in Ice water

L J
Estimate Free Glucose Estimate G20 Estimate G120

Take 1 ml In 10 ml of 0.5M acetic add

;
Take 0.2ml dil. Amyloglucosidase (50AGU/ml)
Vortex I Mix well

30 min. at 70°C

10 min. in boiling water bath

Cool to room temperature

Dilute with 40 ml water

I
Centrifuge 5min. @ ISOOg
I
Estimate Totai Glucose

Figure 14. Schematic flowchart of starch e s t i m a t i o n

(Source: Englyst (203)

102
 Gelatinization and •C-amy lase
i n c u b a t i o n , pH 6 . 0 , 15 m i n , 100 '-C

^
Pepsin incubation , pH 1.5 , 1 h , JtO°C

^^
Pancreatin incubation, pH 6 8 , 1 h, 40° c]

< N
Total f i b r e determination
I n s o l u b l e and s o l u b l e I

Filtration 'Z2 Precipitation with 4 v o l .

ethanol

/ \
Washing Precipitation
with alcohol with 4 vol.
and acetone ethanol

i
Filtration
Drying Filtration
(insoluble
fibre)
i i
Washing w i t h Washing w i t h a l c o h o l andl
a l c o h o l and acetone
acetone

i i
Drying Drying total fibre)
(soluble fibre)

1 1
I
C o r r e c t i o n for indigest b l e p r o t e i n and ash

Figure 15. Schematic representation of Fibre estimation

Source: Asp (204)

103
v) Degree of gelatinisation

Degree of gelatinisation was determined by the method of

Wootton et al. (205).

Degree of gelatinisation is defined as the ratio of

gelatinised starch to total starch in a product. Two grams of

sample after crude defatting by Soxhlet extraction with

petroleum ether (b.p. 40-60°C) was taken for the estimation. The

sample was suspended in 100 ml water, centrifuged and

duplicate aliquots (1ml) diluted with 10ml water were treated

with iodine solution (0.1ml). The absorbance of this sample at

600nm was measured in a spectrophotometer against a reagent

blank (Ai). A further suspension of the product (2g) in water

(95ml) was prepared as described above. To this suspension was

added a q u e o u s potassium hydroxide (lOM, 5ml) and the mixture

was allowed to stand for 5min with gentle agitation. The alkaline

suspension was then centrifuged, duplicate aliquots (1ml) were

treated with 1ml hydrochloric acid (0.5N) and diluted with water

to lOml. These samples were treated with 0.1ml iodine solution

and their absorbance was measured as before (A2). Degree of

gelatinisation is computed by the formula shown below:

Degree of gelatinisation = Ai_x 100

A2

104
c. Amino acid profile

The amino acid content of the enteral food formulations

were analysed using an Amino acid analyser. For amino acid

analysis, 200 to 250 mg of food samples were hydrolysed with 5

ml of 6 N HCl (redistilled) at 110°C for 24 h o u r s . The hydrolysate

was filtered (Whatman No. 2) into a 50-ml flask and lyophilized

in a freeze dryer. The dried sample was subsequently dissolved

in 3 ml of water and again lyophilized. This was repeated three

times to remove traces of HCl. The final dried sample was

dissolved in about 2.0 ml of water.

An aliquot based on the total amino acids of the sample

was taken (5 ml of 2.5 mmol), determined a s alanine, and dried

in a vacuum. The sample was redried using 25 ml of

ethanol:triethylamine:water (2:2:1) and 20 ml of derivatizing

solution, ethanol:triethylamine:water: phenylisothio-cyanate

(7:1:1:1). The sample was incubated at room t e m p e r a t u r e for 25

minutes, a n d then excess reagent was removed by drying under

vacuum. It was then analysed by reverse-phase high-

performance liquid chromatography (RP-HPLC) in a n amino acid

column (Water Associate System, Column Pico Tag) with sodium

acetate buffer and acetonitrile. Amino acids were detected at 254

n m by analysis with a Shimadzu CR4A Chromatopac. Amino

acids were calculated from the peak areas as follows: Amino

105
acids (pmol) = (Area of unknown amino acid/Area of standard

amino acid) x 312.5 (or 156.2 for cysteine).

i) Chemical score

Chemical score is based on the a m o u n t of the most

limiting amino acid present in the test protein relative to the

a m o u n t of t h a t amino acid in the reference protein (egg). This

was calculated using the following formula (206).

essential amino acid total essential amino

Chemical = in test protein (g) X acids in egg (g) X 100
score essential amino acid total essential amino
in egg (g) acids in test protein (g)

ii) Essential amino acid index

Essential amino acid index was calculated according to the

method of Oser (207) taking into account the ratio of essential

amino acids in the test protein relative to their respective

a m o u n t s in whole egg protein.

Hi) Predicted Biological value

The predicted biological value was calculated according to

Oser's method (207) using the following formula:

Biological Value = 1.09 (essential amino acid index - 11.7)

106
d. Functional properties

i) Bulk density

The method of Wang and Kinsella (208) was used to

determine bulk density. 5g of the foods were weighed into 15ml

graduated t u b e s and packed tightly with a plunger. Final volume

was noted and expressed as g/ml.

ii) Fat absorption capacity

2g of the foods was added to 12.5 ml of refined groundnut

oil in a 5ml graduated centrifuge tube. The contents were stirred

every 15 min. for 30 min. after which, the tube was centrifuged

for 25 min. at 3200 rpm and the volume of free oil was read. Fat

absorption is expressed as the a m o u n t of oil bound by Ig of

flour and also by Ig of protein (208).

Hi) Water absorption capacity

To 2.5g of the food, 15ml of water was added and the

material was suspended in water by mixing with a thin glass

rod, taking care to see that no food adheres to the sides of the

tube. The suspension was then centrifuged at 1600 rpm for 25

min. the s u p e r n a t a n t was discarded and the tube was kept in a

hot-air oven maintained at 50°C for 25 min. It was then kept in

a dessicator and subsequently weighed (208).

107
e. Storage Study

The formulated enteral food were packed in polypropylene

pouches, numbered suitably and stored at room temperature (28° ±

2°C) for shelf study. All the samples were stored for a total period of

60 days and were evaluated twice for quality and sensory evaluation

and microbiological analysis (i.e. at the end of 30 and 60 days).

Moisture, peroxide value and free fatty acids were evaluated by the

methods of AOAC (209).

f) Sensory evaluation of enteral foods

The acceptability of formulated enteral foods was assessed by

sensory evaluation. A 10-point quality rating scale for attributes

such as colour, aroma, taste, consistency and overall acceptability

was used. The samples were coded and given to 12 semi-trained

panellists for evaluation. Along with the sample, water was provided

to remove any traces of previous sample from the oral cavity. The

evaluation was conducted for the fresh and stored (30 and 60 days)

samples.

g) Microbiological analysis of Enteral Foods

The formulated enteral foods were subjected to microbial

analysis immediately after the formulation and at the end of each

storage period viz 30 and 60 days respectively. Each of these samples

108
was enumerated for Coliform, yeast, moulds and spores (mesophils).

A total plate count was also conducted.

Total plate count, coliforms, yeast and moulds are spores

(mesophiles and thermophiles) were enumerated in the samples. lOg

of sample was weighted aseptically and transferred to sterilized

pestle and mortar. 90ml of diluent i.e. quarter strength ringers

solution was added and macerated. One ml of the sample thus

prepared was transferred to sterilized petriplates for different

parameters in duplicates.

1. Total plate count - 1 ml from lO-i dilution was transferred to

sterilized petriplates in duplicates. Dextrose tryptone agar

(DTA) was poured and after solidifying, plates were incubated

at 37°C for 48 hours.

2. Coliforms - 1 ml from IQi dilution was transferred to sterilized

petriplates in duplicates. Violet Red Bile Agar (VRBA) was

poured and after solidifying plates were incubated at 37°C for

24 hours.

i) Yeast and Moulds

One ml from lO-i dilution was transferred to sterilized

petriplates in duplicates. Acidified Potato Dextrose Agar (PDA) was

109
poured and after solidifying, plates were incubated at 30°C for 5

days.

ii) Spores

About 10 ml of sample from 10"^ dilution was taken in

sterilized empty test tubes and was kept in boiling water bath for 15

minutes. Then the tubes were cooled and from this sample were

plated.

5. CLINICAL STUDIES

H u m a n clinical studies were conducted in a multi-centric

study involving hospitals in Mysore and Gujarat (Table 15). The

study involved four groups of subjects. Group I - Head and Neck

cancer patients, Group II- Critically ill patients on enteral food,

Group III - Critically ill patients with diabetes and Group IV -

HIV/AIDS patients. Each group had ten subjects being fed the

formulated enteral food and ten on control diet, except the last

group which h a d 3 patients each.

a. Pilot study

An initial pilot study was conducted on 3 to 5 patients

from each group. This study was conducted to a s s e s s the

acceptability, efficacy and safety of the designed enteral food.

110
 Table 15. Details of subjects s e l e c t e d for clinical studies
Cases
(Men, Women)^
Hospital
General Head &
Diabetes
Critically Neck HIV/AIDS
Mellitus
ill cancer
17 8
BHIO, Mysore
(lOM, 7W) (5M, 3W)
Vikram 8
Hospital,
Mysore (5M, 3W) (2M, 2W)

J S S Hospital, 8
Mysore (4M, 4W) (3M, 3W)
Sri Krishna 5 5 7
Hospital,
Gujarat (2M, 3W) (3M, 2W) (2M, 3W) (4M, 3W)

Total 21 22 23
*Figures in p a r e n t h e s e s indicate n u m b e r of Men and Women
respectively

111
 wiPAf^CH^y^L seer
The GEF was tested on a few surgical and non-surg

patients from the general ICU and feedback collected. The CSEF

was tested on patients with cancer who could be fed orally as

well as by Ryle's tube. This was done to get patient feedback on

the oral acceptability of the food since the food is designed to be

bland for patients able to ingest food orally. The DSEF was

tested in critically ill patients with Type 2 diabetes to ensure the

acceptability and safety of the food by observation of their

glycaemic control to prevent further complications in their

condition.

Feedback was elicited from the patients, caregivers,

attending doctors and dieticians during the pilot study. Based

on this, the mode of feeding, feeding regimen and protocol for

the study was finalised. The final clinical study was conducted

taking a larger sample.

b. S e l e c t i o n of subjects

A total of 73 subjects were selected and randomly assigned

to the experimental or control group. A few more subjects in

each group were enrolled to account for patients who dropped

out midway for treatment reasons or mortality. Informed consent

was secured from the patients or their legal guardians on

enrolment in the study.

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 The age group of the subjects ranged from 18 to 55 years

and it was attempted to enrol cases with similar medical

histories for the experimental and control groups for validity.

Exclusion criteria were set by the attending doctors based

on medical grounds of existing treatment, medications being

given or disease profile. Overtly obese patients were excluded

from the study to decrease confounding variables t h a t might be

introduced since obesity can alter disease progression and

treatment modalities in cancer as well as diabetic subjects.

A questionnaire was developed around the following

information needs of the study - personal history, demographic

information including socio-economic s t a t u s , their health and

medical histories, present medical condition, treatment,

nutritional s t a t u s , dietary pattern, food frequency intake of

enteral food, and m e a s u r e m e n t s of outcome m e a s u r e s .

Patients who were sent to the general ward were only

selected for the investigations as the protocol for patients at the

special wards was not suitable for measuring the outcome of the

study.

The study was designed such t h a t the patients could be

approached both, at the ICU as well as the wards. At the ICU,

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the data was collected from secondary sources, viz., hospital

charts. As the patients recovered and were moved to the wards,

the relevant data such as anthropometry and other feedback was

collected from the patients themselves.

c. Preparation of enteral food for feeding

The enteral foods were prepared by mixing the formulated

food (25% solids w/v) in cold/warm water. The mixture was

stirred to prepare a uniform slurry. This slurry was then heated

in a water bath for about 20 minutes to facilitate enzymatic

hydrolysis of starch characterised by thinning of the slurry. This

was followed by a final cooking by bringing the slurry to a boil

over a low flame with constant stirring. The slurry was then

cooled a n d fed to the patients as required. If required to be

stored for more t h a n 6 hours, the slurry was refrigerated.

d. Mode of feeding

The patients who were able to ingest food orally were also

fed the formulated enteral foods orally. This included patients

before surgery and patients recovering from t r e a t m e n t who did

not require Ryle's tube placement. However, in perioperative

patients, patients in the ICU and patients who were unable to

ingest food orally, enteral feeding was carried out using Ryle's

tube having bore of 12-15 FG. The preferred mode of tube

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placement was nasogastric since this method is easiest to

initiate and shown to have the least complications.

e. Feeding regimen

A modified equation for estimating the daily energy

requirement a s shown was used to compute dietary intake.

Energy (Kcal) Males = [(789 x BSA) + 137]* + 50%[*]

Females = [(544 x BSA) + 414]* + 50%[*]

Protein (g) = 1.5g/kg body weight.

The formulated foods were prepared fresh each day and

refrigerated if required till consumption. The feeding was either

oral or by Ryle's tube and amounted to 2 to 3-hourly feeds of

200-350ml depending on requirement. The control and

experimental groups were given isocaloric, diets with similar

protein contents and the hospital-blenderised food for the

control patients was modified in terms of calories and protein to

this effect.

f. Outcome indicators

The outcome indicators assessed included anthropometric

indices as well as routine biochemical and haematological tests.

A specially designed protocol was used for the a s s e s s m e n t of

outcome of the study (Annexure I).

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i) Anthropometry

Somatic status of the patients was assessed by

anthropometric m e a s u r e m e n t s which included, height, weight,

mid upper arm circumference (MUAC) and skinfold thickness

(SFT). These were measured by standard procedures as

described by Jelliffe (210)

ii) Biochemical indices

Serum creatinine

Serum creatinine was estimated by the alkaline picrate

method (211). The estimation was carried out using the

Autozyme kit manufactured by Accurex Biomedical Pvt. Ltd.,

Mumbai.

Urea

Urea was estimated in serum by the Bertehlot method

which uses the enzyme urease to hydrolyse urea in a

colourimetric reaction (212). A kit manufactured by Nicholas

Piramal, Mumbai was used for the samples.

Uric acid

Uric acid in serum was estimated by the TBHBA method

(213). A kit containing the required reagents manufactured by

Aspen Laboratories, New Delhi was used for the estimation.

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Serum proteins

Total protein - Total protein was estimated colourimetrically by

precipitating the proteins as a coloured complex in the presence

of copper reagent in an alkaline medium.

Albumin - Albumin was also estimated colourimetrically using

bromo cresol green (BCG) reagent at 4.2 pH. Globulin is

calculated by subtracting the a m o u n t of albumin from total

proteins. A kit containing the requisite reagents manufactured

by CDR Diagnostics, Hyderabad was used for estimating both,

total protein a s well as albumin.

Electrolytes

The readings of the electrolytes Calcium, Sodium,

Potassium and Chloride in serum were recorded from the

hospital medical records as they were conducted regularly on a

routine basis.

Liver function tests

SCOT, SGPT - The methods given by the International

Federation of Clinical Chemistry (IFCC) were used to estimate

SGOT and SGPT in serum (214). The colourimetric estimations

were done using diagnostic kits available from Euro Diagnostic

Systems Pvt. Ltd., Chennai.

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5. Alkaline p h o s p h a t a s e - Serum alkaline p h o s p h a t a s e was

colourimetrically estimated in serum using a diagnostic kit

provided by Agappe Diagnostics, Kerala (215).

Urinary creatinine

A 24-hr urine sample using thymol as preservative was

used for the estimation of 24-hr urinary creatinine by alkaline

picrate (211). This estimation was also carried out using the

Autozyme kit manufactured by Accurex Biomedical Pvt. Ltd.,

Mumbai.

6. COMPUTATION

Laboratory values were computed and expressed as

mean+SD. Biochemical and haematological indices were

classified into percentiles of standard.

The information collected during the clinical studies was

consolidated in terms of each subject a n d computed for the

whole group using descriptive analysis. Study information was

analysed by calculating the number, percentage distribution and

frequency c o u n t s .

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 Heights and weights were classified into percentiles of

s t a n d a r d (187, AU). Body m a s s index was computed using

weight a n d height using the formula below.

Body m a s s index = Weight (kg)

Height^ (cm)

The MUAC and SFT were also classified into percentiles of

standard while the MUAMC was computed as per the standard

equation shown below.

MUAMC = MUAC - 7r(SFT)

For computing the nutrient intake of the subjects, the

foods consumed by each subject were grouped u n d e r specific

food grouping as per the ICMR pattern, i.e. cereals, pulses,

vegetables, milk and milk products, sugar and jaggery, fats and

oils and compared with a desirable dietary pattern (DDP). DDP

was computed based on RDI for adults with ideal body weight to

give 1800-2100 Kcal and 50-60g protein per day. From the

grouped food intake data, energy along with other n u t r i e n t s -

carbohydrate, protein, fat, iron, calcium, retinol, thiamine,

riboflavin, niacin and vitamin C were calculated using the food

composition tables given by ICMR (216) for Indian foods.

Recommended dietary intake (RDI) was derived for the

subjects using ICMR recommended dietary allowance. The

119
computation of RDI and nutrient intake along with DDP is

described below.

1. Food consumed by each person was converted to its raw weights,

i.e., breaking u p the menu items into its basic raw ingredients

and assigning them to different food groups like cereals, pulses,

vegetables, milk and milk products, meat, eggs (animal foods

other than milk), fruits, fats and oils and sugar and jaggery.

2. Translating the data on food intake (total amounts consumed raw

under each group) into energy and nutrients - carbohydrate,

protein, fat and other micro-nutrients were calculated using food

composition tables.

3. Nutrient intake of patients was compared against recommended

dietary intake (RDI) for ensuring appropriateness of intake for

energy and protein for each person which was derived based on

the ICMR, RDA, for age, sex and activity. Resting metabolic energy

rate (RMR) predictable for each patient computed using

WHO/FAO Equations.

4. The nutrient intake data of farm women was also used to derive

single value nutrient allowances per 1000 kcal to measure the

nutritional adequacy of the diets relative to single value nutrient.

Single value nutrient allowances per 1000 kcal were calculated

based on ICMR-RDA.

This analysis served both to evaluate the nutrient to energy

ratio of foods consumed and to arrive at a nutritional quality index

120
(INQ or NQI) . The equations for the derivations in percentage

nutrient adequacy and INQ are as follows:

PNA = Actual nutrient intake x 100

Recommended dietary intake

INQ = Actual nutrient intake per 1000 kcal

Recommended dietary intake per 1000 kcal

5. The RDI of the individual was used to derive a desirable dietary

pattern (DDP) by translating the RDI into foods in amounts

desirable to ensure nutritional adequacy in terms of

macronutrients. The DDP was derived as given below based on

the RDA for adults with respect to energy and protein:

a) Identifying the source of carbohydrates - major (cereals and

pulses) and minor (sugar, fruits and vegetables).

b) Calculating the amount of sugar as equivalent to 3 to 4

teaspoons (i.e., used for 2 cups of coffee/tea) per day -

approximately 20 - 30 g.

c) Approximately an amount of 70 g of total carbohydrates was

derived from sugar, fruits and vegetables (30+10+30)

respectively. This amount was subtracted from the total

carbohydrates.

d) The amount of cereals and pulses was calculated on the basis

of the remainder total carbohydrates in the ratio of 7:1.

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 e) The amount of proteins from cereals and pulses (as per the

amount fixed in the earlier step) was calculated and the

remainder was made up with the milk and milk products (in a

vegetarian diet at least 25 % of the recommended protein has

to be from the animal sources amounting to about 200 to 250

ml of milk in a diet containing 50 g of protein).

f) The total amount of fat was derived by summing up the total

fat (invisible) content of the foods already listed.

g) The total fat in the DDP was derived by subtracting the

invisible fat from the total. The resulting amount was provided

in the form of fats and oils (visible).

7 . S T A T I S T I C A L ANALYSIS

The d a t a was analysed for statistical significance using

appropriate statistical tests viz. Chi-square, paired t-test, f-

value. Graphical representation was achieved using computer

programmes (SPSS 10.0 and MS Excel 2003).

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