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REVIEW OF LITERATURE

Idli is a fermented breakfast food widely consumed in southern India. It is

prepared from a wet ground mixture of rice and black gram dhal and it is

famous for its soft, spongy texture, desirable sour taste and characteristic

aroma. Cereal grains are considered to be one of the most important sources

of dietary proteins, carbohydrates, vitamins, minerals and fibre for people all

over the world. Fermentation may be the most simple and economical way

of improving the nutritional value, sensory properties and functional

qualities of food. The indigenous fermented food products produced from

different cereal substrates (sometimes mixed with other pulses) fermented

by lactic acid bacteria and yeast are included. Traditional fermented foods

prepared from most common types of cereals (such as rice, wheat, corn or

sorghum) are well known in many parts of the world.

Several aspects such as methods of idli preparation, effect of raw materials,

effect of temperature and biochemical changes also plays an important role

in deciding the body and textural properties of idli. Therefore, the present

investigation was evaluated the suitability of whey protein concentrate

(WPC) in preparing idli and its effect on some physico-chemical and

sensory characteristics of idli. The relevant information has been reviewed

under the following broad heading:

2.1 Production and properties of idli

2.2 Production and properties of whey protein concentrate (WPC)

2.3 Microbiological quality of idli

2.4 Health benefits of whey protein concentrate (WPC)

2.5 Uses of whey protein concentrate (WPC)

2.6 Nutritional aspects of whey protein concentrate (WPC)

2.7 Cereal based fermented foods

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Production and properties of idli

2.1.1 Idli

Desikachar et al., (1993) stated that an increase in non-protein nitrogen and

a decrease in reducing sugars have been observed during fermentation of idli

batters, the batters are usually prepared by soaking rice (Oryza sativum) and

decuticled black gram (Phaseolus mungo) dhal in water, grinding them

separately, mixing and allowing the mixture to ferment overnight. Both

titratable acidity and the volume of the batter increase as a result of

fermentation and have been used as criteria for judging the progress of

fermentation. A temperature range of 25-30˚C has been found to be optimal

for the fermentation. That both yeasts and bacteria participate in the

fermentation has been shown using penicillin G and chlortetracylin as

selective inhibitors. Acid and gas production have been found to be mostly

dependent on the growth of microbes belonging to the bacterial group.

Radhakrishnamurthy et al., (1993) suggested that black gram has been

reported to play a major role in idli fermentation as a source of micro-

organisms and as a fermenting substrate.

Veen et al., (1993) observed that idli prepared by fermenting a mixture of

soaked and milled parboiled rice and dehulled black gram (Phaseolus

mungo), no appreciable increase in methionine was found after 24 hours of

fermentation, when idli would normally be steamed. The PER and

digestibility in rats were the same as of the unfermented mixture. The

riboflavin content was decreased because the presence of Streptococcus

faecalis in the fermented batter. The presence of pharmacological active

amines such as tyramin was expected but they were not detected.

Reddy and Salunkhe (1993) reported that parboiled rice and black gram

dhal in various proportions are soaked and wet ground separately with added

water to yield a batter of desired consistency. A small quantity of salt is

added and allowed for fermentation overnight during which time

23

Leuconostoc mesenteroides and Streptococcus faecalis, naturally present on

the grains/ legumes/ utensils grow rapidly out- numbering the initial

contaminants and dominating the fermentation. The organisms produce

lactic acid and carbon dioxide, which make the batter anaerobic and leavens

the product.

Reddy et al., (1993) suggested that idli, a popular fermented breakfast food

consumed in the Indian subcontinent is made mainly from rice and black

gram. It is very popular because of its textural and sensory attributes.

Sathe and Salunke (1993) explored the potential use of white beans (Great

Northern) in idli production instead of black gram. It was reported that white

beans could be successfully substituted for black gram in the production of

idli. During the fermentation insignificant protein hydrolysis and gradual

reduction in total sugars were reported.

Juliano and Sakuraj (1993) suggested that parboiled rice is better suited

than raw rice for producing idli, i.e. it is soft without becoming sticky.

Kaw et al., (1993) revealed that the microbiological quality of the

fermenting batters was not affected by the amylase content of the rice used.

However, it was found that among the sensory quality attributes evaluated,

cohesiveness was found to be correlated with the amylase content of rice

used. Filipino and Indian panels evaluated the quality of the idlis in almost

the same part of the scale except for flavour where in the former seemed to

notice the sour off flavour which were not detected by the latter. Probably,

Indians consider this sour off flavour inherent of idli.

Khetarpaul and Chauhan (1994) stated that natural as well as single,

mixed and sequential pure culture (S. diastaticus, S. cerevisiae, L. brevis and

L. fermentum) fermentations of pearl millet flour for 72 hrs lowered pH and

raised titratable acidity. The fermentation either decreased or did not change

the protein content of pearl millet flour. Natural fermentation increased

whereas pure culture fermentation decreased the fat content. Ash content did

24

not change. Natural fermentation at 20˚C and 25 ˚C increased whereas at

30˚C it decreased the thiamine content of the pearl millet flour. Yeast

fermentation raised the level of thiamine two-to three-fold, while lactobacilli

fermentation lowered it significantly.

Swanson and Swanson (1994) investigated the protein quality in idli

produced using black and white beans and rice. They also concluded that

fermentation does not improve protein quality in idli.

Joseph and Swanson (1994) observed that a fermented steamed idli

prepared from beans (Phaseolus vulgaris) and rice. Feed Efficiency Ratio

(FER), Protein Efficiency Ratio (PER) and relative PER of fermented ‗idli‘

diets were significantly smaller (p 0.05) than the FER, PER and rPER of

unfermented ‗idli‘ diets. The true Digestibility Coefficient (DC) and Net

Protein Utilization (NPU) of fermented ‗idli‘ diets were significantly smaller

(p 0.05) than the DC and NPU of unfermented ‗idli‘ diets. Biological value

(BV) of fermented and unfermented ‗idli‘ diets were similar to the BV of a

casein control diet. They further reported that fermentation does not improve

the protein quality of ‗idli‘ prepared from beans and rice.

Yadav and Khetarpaul (1994) observed that the indigenous fermentation

of coarsely ground dehulled black gram dhal slurry at 25, 30 and 35 ˚C for

12 and 18 h reduced the levels of Phytic acid and polyphenols significantly

(P < 0.05). The unfermented legume batter had high amounts of phytic acid

(1000 mg / 100 g) and polyphenols (998 mg / 100 g) and these were reduced

to almost half in the product fermented at 35 ˚C for 18 hrs. In vitro

digestibility of starch and protein improved significantly (P < 0.05) with

increases in the temperatures and period of fermentation. A significant (P <

0.01) and negative correlation found between the in vitro digestibility and

the anti- nutrient further strengthens.

Murthy et al., (1994) has developed an automatic idli making unit that

produces 1200 idlis per hour. The unit consists of automatic idli batter

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depositor, a special idli pan conveyor, steam chamber and idli scooping

system.

Joseph (1994) suggested that fermented foods prepared from cereals and

legumes are an important part of the human diet in Southeast Asia and parts

of East Africa. The popularity of legume based fermented foods is due to

desirable changes including texture and organoleptic characteristics.

Improvement in digestibility and enhancement of keeping quality, partial or

complete elimination of anti-nutritional factors or natural toxins, increased

nutritive value, and reduced cooking time.

Murthy and Rao (1997) observed that the thermal diffusivity (α) of idli

batter was determined experimentally assuming infinite slab geometry under

transient heat transfer conditions. The value α of batter determined using

batch (1.38 × 10 -7 m2 s -1) and continuous (1.1 × 10 -7 m2 s -1) idli steaming

units were compared with the value obtained using Riedel‘s equation (1.42 ×

10 -7 m2 s -1) and Martens equation (1.1 × 10 -7 m2 s -1).

Nagaraju and Manohar (2000) observed that idli fermentation was carried

out in the conventional way in a batter having rice to black gram in the ratio

of 2:1, 3:1 and 4:1 at room temperature. The rheology of the product was

assessed using a Brookfield viscometer having disc spindles. Yield stress

values were in the range of 13- 43 Pa and reached a maximum value at 7 hrs

of fermentation. Flow behaviour includes were in the range 0.287- 0.605.

Flow behaviour indices at 23 hrs were significantly lower than those at Oh.

Consistency index values, at any fermentation time, increased as the rice to

black gram ratio increased. Mean particle size ranged from 500 to 600 µm

and there was no definite trend noticed with respect to time of fermentation

and rice to black gram ratio. There was a steep change in volume increase

after 4 h fermentation.

Agarwal et al., (2000) reported that the predominant fermentation

microflora comprises lactic acid bacteria and yeast and causes an

26

improvement in the nutritional, textural and flavour characteristics of the

final product. The flavour profile of idli batter prepared with initial levels of

2 × 104 c.f.u. g -1 of Candida versatilis CFR 505 and 2 × 101 c.f.u. g -1 of

Pediococcus pentosaceus CFR 2123 in 500 g idli batter, packed in polyester

polylaminate pouches and stored at 30 ± 2 ˚C was periodically analysed by

GC-MS. The desirable flavour compounds such as ketones, diols and acids

were found to be present upto 8 days of storage, whereas undesirable

flavours like sulphurous and oxazolidone compounds, ethanone and thiazole

appeared in the batter subsequent to 6 days of storage. They further observed

that the flavour profile of traditional fermented foods can be a reliable

qualitative and quantitative parameter.

Teniola and Odunfa (2001) suggested that idli is a low calorie, starchy and

nutritious food, which is consumed as breakfast or snack. Steamed idli

contains about 3.4% protein, 20.3% carbohydrate and 70% moisture.

Nisha et al., (2005) studied that the stabilization of the idli batter at room

temperature (28- 30 ˚C) and refrigerated storage (4- 8 ˚C) by using various

hydrocolloids and some surface active agents. The batter was evaluated in

terms of percentage decrease in volume and percent whey separation. While

hydrocolloids gave good stabilization, surface active agents failed to

stabilize the batter although they reduced whey separation. Among the

various hydrocolloids. 0.1% guar gave best batter stabilization and idlis

made there from after 10 days of room temperature and 30 days of

refrigerated storage of batter were found to be of acceptable quality.

Chandini et al., (2005) studied that the effect of varietal differences and

polishing of rice on quality parameters of idli. Two varieties of raw rice,

―Jaya‖ and ―Minilong,‖ and one variety of parboiled rice ―Ponni‖ with two

degrees of polishing (high and low) were selected. Emulsification capacity

ranged from 102 to 110 mL/ 100 g and foam capacities at different pH range

were similar. Rice with a lesser degree of polishing fermented better with

higher batter volume and microbial count, lesser shear value and gave softer

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idlis. Sensory analysis revealed that idlis prepared with low-polish rice

scored significantly lower for appearance and color quality compared with

products prepared with high-polish rice. Further concluded that the quality

characteristics of idli were influenced by the variety of rice and the degree of

polishing, but the two types of black gram used, whole and split, had no

effect.

Steinkraus (2005) suggested that the traditional fermented foods contain

high nutritive value and developed a diversity of flavours, aromas and

textures in food substrates.

Sharma and Ali (2006) suggested that replacing rice with kodo in idli had

no deleterious effect on the nutritive value but enhancement of protein, fat,

calcium, phosphorus and fibre. This kodo idli is found to be acceptable,

palatable and nutritious.

Lyer and Ananthanarayan (2008) studied that the fermentation time of the

batter varies from 14 to 24 hrs with overnight fermentation being the most

frequent time interval. Reduction in the fermentation time of the idli batter is

of great commercial significance for large scale idli production and this can

be potentially achieved by addition of enzymes externally. They further

concluded that the possibility of expediting the idli batter fermentation

process by adding an exogenous source of α- amylase enzyme. 5, 15 and 25

U per 100 g batter of amylase were added to the idli batter which was

allowed to ferment. Different parameters were monitored and sensory

attributes were also studied and compared with that of the control set. The

fermentation time was reduced from a conventional 14 h to 8 h and the

sensory attributes of the final product were also successfully maintained.

Susheelamma et al., (2007) studied that greater increase in hydration

capacity of black gram dhal was observed during 5-15 min of soaking, a

gradual increase up to 90 min. Apparent viscosity of batter showed 100%

increase up to 90 min and less than 20% increase beyond 120 min and shear-

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thinning nature of batter indicated that they were pseudo- plastic in nature.

Yield stress and consistency index showed greater changes after 90 min.

Compared with the addition of native rice flour, incorporation of flaked rice

flour and expanded rice flour into black gram batter significantly increased

the cold paste viscosity of the batter, probably because of their greater

water- holding capacity, compared with that from native rice.

Reddy et al., (2007) suggested that the increase of methionine content

during idli fermentation by which path way methionine is synthesized and

identification and isolation of microorganisms responsible for methionine

production or synthesis.

Balasubramanian and Viswanathan (2007) studied that the blend ratio

(66% parboiled rice (Oryza sativa): 33% decorticated black gram

(Phaseolus Mungo Roxb) was fermented for 12 hrs and the batter was steam

cooked for 10 min. Texture profile analysis (TPA) test was performed for

idli, making cylinder samples (13.5 mm diameter, 10 mm long) of idli. In

Pearson correlation matrix, majority of the parameters were positively

correlated at p<0.01 and p<0.05. The firmness value positively correlated

with gumminess and chewiness, which depicts the soft nature of idli.

Resilience is not correlated with other textural parameters. From principle

component analysis (PCA), the first and second principle component,

describe 42.5 and 27.2% of the variance, respectively in the TPA

parameters. The first principle component is highly positively correlated

with gumminess, chewiness and cohesiveness. The second principle

component is positively correlated with firmness and negatively correlated

with springiness. Resilience contributed very weakly to both these principle

components. Based on the results of PCA, the firmness is the prime factor to

illustrate idli texture followed by chewiness, gumminess, cohesiveness and

springiness.

Varnashree et al., (2008) observed that idli prepared by ragi (R), ragi flour

(RF), parboiled rice (PR), black gram dhal (B) and black gram dhal flour

29

(BF) were processed differently and used in different ratios to prepare four

variations of idli [RPRB (1.5:1.5:1), RB (3:1), RFB (3:1) and RFBF (3:1)].

The degree of gelatinization was found to be higher in idlis prepared with

whole ragi and black gram dhal. The water absorption capacity of black

gram dhal was higher and hence soft textured idlis were obtained. They

further reported that whole ragi could be used to replace rice in the

preparation of idli which enhances the nutritional quality without

considerable effect on the quality parameters of idli.

Kanchana et al., (2008) reported that idli was dried by microwave drying

(MD), vacuum assisted MD and hot air drying. Moisture, bulk density, water

activity (aw) and instrumental colour value. Instrumental texture quality was

also studied in the fresh and rehydrated idli. Moisture content of dehydrated

idli ranged between 6.7 and 9.4% and the water activity (aw) was between

0.166 and 0.441. Low power density and low temperature dehydrated idli

was acceptable. Rehydration of idli was better in hot air oven drying

followed by vacuum assisted MD and domestic MD.

Sridevi et al., (2010) studied that idli batter samples were prepared using

lactic starter cultures like Pediococcus pentosaceus (Pp), Enterococcus

faecium MTCC 5153 (Ef), Ent. Faecium (IB2 Ef - IB2), individually, along

with the yeast culture, Candida versatilis (Cv). Idli batter prepared using Ef

and Ef- IB2 cultures gave better results, when evaluated for the rise in batter

volume (80 ml), level of CO2 production (23.8%), titratable acidity 2.4-3.5%

(lactic acid) and pH 4.3-4.4. Storage stability of batter made with selected

starter cultures was determined by analyzing the idlis prepared using the

batter stored for 1 and 5 days for texture, nutrient composition and sensory

quality. They further reported that the idlis of different combination of

cultures, whereas these results are better than that of the idlis made using

naturally fermented idli batter.

Nazni and Shalini (2010) stated that the pearl millet can be used in idli

preparation instead of rice. Replacing rice with pearl millet had good impact

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on the nutritive value by increasing the protein, fat, fibre, calcium and iron

content in the developed idlis. Thus pearl millet idli is found to be

acceptable, palatable and nutritious.

Rekha and Vijayalakshmi (2011) studied that to reduce the natural

fermentation period of ‗idli‘ from the conventional 14 h to 10 h by adding

underutilized okara for the preparation of ‗idli‘. Black gram was partially

substituted with soy residue okara in the ratio of (1:1). After 14 h of natural

fermentation, the pH and total acidity of control ‗idli‘ batter was 4.51 and

0.64% and that of okara fortified ‗idli‘ batter was 4.53 and 0.43%. The

amount of CO2 released by the control and okara fortified batter was 19.7%

and 33.6%. The viable count of yeast and mould, lactics and mesophilic

bacteria in control and okara batter increased with time reaching 9.00 &

10.34, 8.66 & 7.69 and 8.65 & 9.47 log10 cfu/g respectively at the end of 10

hrs of natural fermentation. Okara fortified ‗idli‘ was soft and spongy

compared to control ‗idli‘.

Ghosh and Chattopadhyay (2011) stated that idli batter is prepared by

soaking polished parboiled rice and decorticated black gram for 4 h at 30 ± 1

˚C in water. The soaked mass was ground using a grinder with adequate

amount of water. The blend ratios of 2:1, 3:1 and 4:1 (w/w) batter were

allowed for fermentation for different periods with the addition of 2% (w/w)

of salt. The rheology of the product was assessed using a Brookfield

Viscometer having disc spindles. Shear stress values were in the range of

0.22 and 4 Pa and reached a maximum value at 7 h of fermentation. The

density, pH and percentage total acidity of batter during fermentation for

different blend ratios ranged between 0.93 and 0.59 gm cm-3, 4.21 and 5.9

and 0.44 and 0.91% respectively. During fermentation, maximum

production of riboflavin and thiamine were found to be 0.76 mg/100 gm and

0.73 mg/ 100 in 3:1 blend ratio of idli batter and the folic acid content was

found to be at a maximum of 0.75 mg/ 100 gm of idli batter after 10 h of

31

fermentation. Digestibility in terms of amino N2 content was analysed by

formal titration.

Aachary et al., (2011) studied that the use of Xylooligosaccharides (XOS)

as a prebiotic in idli, a cereal/ legume based fermented cake and its effect on

texture, fermentation and sensory characteristics was investigated. Idli batter

was fermented with different concentrations of XOS (0, 0.2, 0.4 and 0.6%

w/v) for 4 – 18 hrs conventionally. The addition of XOS markedly increased

lactic acid bacteria number (9.88 ± 0.08 log cfu g -1) which resulted in rapid

reduction in pH (4.61 ± 0.03) and specific gravity after 6 h of fermentation

when compared to conventional batter fermentation for 18 h without XOS

(9.46 ± 0.06 log cfu g -1). Instrumental (colour and texture) and sensory

evaluation indicated that the optimum conditions were 0.4% XOS and 6 h

fermentation. Idlis with XOS had higher moisture content and a softer

texture. Addition of XOS benefits both fermentation and idli quality.

2.1.2 Semolina

Erbas et al., (2004) observed that moisture adsorption isotherms of

semolina (from hard wheat) and farina (from soft wheat) were determined at

20, 35, 50 and 60 ˚C using the isopiestic method. The adsorbed moisture

content significantly affected product type, temperature and water activity.

Moisture up-take accelerated about 0.75 water activity. Henderson, Halsey

and GAB equations were found to be the most suitable model to describe the

isothermal water sorption of semolina and farina at 0.1-0.9 water activity

range. Monolayer moisture content (m0) for two products were calculated

from BET and GAB equations. The m0 values of both models decreased with

increasing temperature. Adsorption isosteric heat (Qs) decreased, the

maximum heat of adsorption was obtained in the moisture content 6-7% and

was higher for semolina (15.2 kJ / mol) than farina (14.34 kJ / mol). The Qs

value quickly decreased with increase in moisture content to approximately

15% and then was plateau on axis of moisture content. Semolina and farina

must be storage below 75% relative humidity at 20 ˚C prevents caking and

32

deterioration because moisture sorption acceleration was increased after

0.75aw. The moisture content of the products in this storage conditions could

be approximately 12.5%.

2.1.3 Functional properties of semolina

Reddy and Yenagi (1997) observed that the semolina yield of dicoccum

wheat varieties is comparable to durum and traditional products of dicoccum

wheat have better taste, flavour and suited for preparation of Godihuggi,

Gulladaki laddu and roasted madeli.

Reddy et al., (1998) observed that Dicoccum (Triticum dicoccum) is a class

of wheat grown in India and represents a small percentage of total wheat

production. It is a quality wheat and also known as ‗Jave‘ wheat and used

for preparation of semolina for use in various Indian traditional products.

Patil et al., (2003) reported that swelling power and percent solubility of

semolina of different grades of dicoccum wheat was lower than durum and

bread wheat varieties. Dense products of ‗very coarse‘ and ‗coarse‘ semolina

of dicoccum varieties are highly acceptable and are more nutritious. It was

also found that ‗DWR-2006‘ durum variety and high swelling and solubility

index with good pasting characters.

Ranhotra (2006) suggested that wide varieties of products are prepared

from milled fractions of wheat and is well documented that soft wheat is

used for preparation of cakes, biscuits and pastry. Hard wheat is used for

preparation of bread and chapatti and durum wheat is used for preparation of

dense breakfast and pasta products.

2.2 Production and properties of whey protein concentrate (WPC)

Whey protein concentrate (WPC), a co- product of cheese making and

casein manufacture, represent a rich and heterogeneous mixture of proteins

with a broad range of nutritional and functional properties. Functional

properties of whey proteins in foods include solubility, dispersibility, heat

33

stability, formation (gels and edible films) and surface activity (emulsions

and foams).

Berlin et al., (1973) studied that measurements of the heat of fusion of free

water in concentrated solutions of purified whey proteins showed that .5 g

water/g whey protein would not freeze at –40 C. This water was defined as

bound. Total bound water in protein solutions containing lactose and salts

varied between .5 and 1.2 g water/g solids, with unfreezable water

increasing as the concentrations of lactose and salts were increased. Bound

water values observed with several whey protein products agreed with

values computed from data both for high and low molecular weight fractions

of these products. Thermal denaturation did not cause significant changes in

water binding.

Forsum (1974) reported that nutritional evaluation of whey protein

concentrates and whey protein fractions was performed by Protein

Efficiency Ratio and Net Protein Utilization assays as well as by

calculations of chemical scores. The whey protein concentrates were

industrially produced by gel filtration and ultrafiltration and were

fractionated further by large-scale gel filtration. Nutritional values of the two

concentrates were similar and high. Whey protein fractions containing α

lactalbumin had high Protein Efficiency Ratio and Net Protein Utilization

values while fractions containing β-lactoglobulin had high Net Protein

Utilization values but only moderate Protein Efficiency Ratio values.

Mcdonough et al., (1974) stated that protein concentrates were susceptible

to heat; normal pasteurization temperatures resulted in approximately 20%

denaturation. Whey protein exhibited excellent water retention. Addition of

1.5% protein to skim milk followed by heating formed a custard-like gel

with sufficient body to stand alone without leakage. Approximately twice as

much egg albumin was required to achieve comparable results. Whipping

properties were very good when butterfat content was less than 2%.

34

Excellent stable whips could be produced by a combination of heat and pH

adjustment.

Forsum et al., (1974) observed that large-scale fractionation of whey

protein concentrates was performed by pre-paratory gel filtration. A

prototype of a stacked column, KS 450, packed with Sephadex® G-75 and

equilibrated with .1 M phosphate buffer pH 6.3 was utilized. Both an

ultrafiltered and a gel-filtered whey protein concentrate were fractionated

into three different fractions, the protein composition of which was

evaluated by electrophoresis, amino acid analyses, and casein estimations.

The nitrogen distribution between the different fractions was also analyzed.

By large-scale gel filtration, β-lactoglobulin of high purity and preparations

rich in α-lactalbumin could be obtained in large quantities.

Mcdonough et al., (1976) stated that WPC were prepared from cheese whey

by ultrafiltration and were evaluated as a milk extender. Ash values

generally were lowered by ultrafiltration averaging 10.5% for cottage cheese

whey and 7.5% for its corresponding concentrate. Feeding trials of rats

indicated bioavailability of the dried concentrates was higher than that of

both casein and skim-milk powder. Addition of dried concentrate to nonfat

dry milk as a 40% blend raised the protein efficiency ratio of the nonfat dry

milk from 2.51 to 2.83. Sensory evaluation indicated that up to 40%

concentrate from sweet whey or 20% from acid whey can be blended with

skim milk without adversely affecting organoleptic quality.

Jost et al., (1976) observed that the casein digestion test indicated that

neutral or alkaline proteases were absent. Hemoglobin digestion at acidic pH

indicates trace amounts of acidic proteases, most likely derived from the

rennet. No changes were detected by gel filtration on Sephadex G-75 and by

polyacrylamide gel electrophoresis with sodium dodecyl sulphate, in the

protein pattern upon aging wheys for a few days. Whey proteins were

incubated at pH 6.2 and 4.5 with high concentrations of commercial rennet.

α-Lactalbumin and β-Lactoglobulin were not affected. In contrast, at pH 4.5

35

serum albumin was degraded extensively, and the H-chain of the

immunoglobulins underwent limited digestion.

Hidalgo and Gamper (1977) reported that rennet whey protein concentrates

have excellent nutritional properties, but their use in fluid food systems is

impaired by the poor heat stability of the protein. Heating whey protein

concentrated solutions at neutral pH caused up to 70% loses in solubility. In

the absence of added calcium, protein coagulation occurred near the iso-

electric zone whereas in the presence of .03 M calcium chloride, similar

protein coagulation occurred in the whole pH range (pH 2 to pH 12). Tryptic

hydrolysis of the protein increased the heat stability of whey protein

concentrates considerably.

Modler and Emmons (1977) stated that WPC prepared by heating at pH

2.5 to 3.0 had a minimum solubility of 78%. This was reduced to 51% when

the pH during heating was 3.5. Although addition of iron to whole whey

increased protein recovery, solubility was reduced. Concentrating whey

prior to heating greatly increased protein recovery but substantially reduced

solubility. The viscosity of spray-dried samples of acid-heat WPC

reconstituted to 33% solids ranged from 4,000 to 36,800 centipoise while

commercial samples had viscosities of 400 to 1,840 centipoise.

Experimental samples of WPC gelled at protein concentrations of 2, 4, 6,

and 8% heating at 95 C for 20 min. All experimental samples had excellent

color stability while commercial samples darkened upon heating.

Thompson and Reyes (1980) reported that heat coagulated cheese WPC

was modified by reaction with succinic anhydride at pH 8 followed by

isoelectric precipitation, neutralization, and freeze drying. All functional

properties except whippability were improved. Succinylated WPC dispersed

readily as highly swollen particles with high viscosity. It has high

emulsifying capacity and can form stable emulsions at 1% usage. Gel

filtration indicates extensive unfolding of the protein after succinylation.

36

Succinylation increased the protein content of the WPC with little loss in

biological value and yield.

Phillips (1981) stated that proteins are among the most widely used natural

emulsifiers because they can decrease the interfacial tension and form a

protective layer around oil/ fat globules in emulsions.

Cheftel and Lorient (1982) stated that whey protein concentrate (WPC)

represent an important and valuable source of ingredients due to their

effective nutritional, sensory and functional properties such as water

absorption, gel formation, emulsification and foaming.

Chan (1983) reported that accelerated storage tests of WPC were carried out

to characterize changes of the proteins that may affect its nutritional quality

or functional properites. After 42 days at 37°C and 75% relative humidity,

pH 4.6 soluble protein decreased only slightly (14% loss), and there were no

significant changes of sulfhydryl or disulfide content, Hydroxyethylfurfural

increased dramatically (17 to 192 μmoles/100 WPC). ―Free‖ lactose, and

total, dinitrobenzenesulfonate-available and pepsin-pancreatin-digestible

lysine contents also decreased (17, 34, 57, and 72% losses).

Melachouris (1984) observed that the technical aspects are whey source,

whey pre-treatment, ultrafiltration membrane performance, characterization

of liquid process streams, characterization of whey protein concentrates,

application of whey protein concentrates (WPC) and effluent stream

utilization. Functional whey protein concentrates (WPC) produced under

strict quality control conditions could find a growing market and could be

used as key ingredients in development of new food products.

Dewit and Klarenbeek (1984) observed that mild heat treatments up to

60°C may affect reversibly the solubility and foaming properties of whey

proteins. Conformational changes, as reflected by differential scanning

calorimetry and observed above 60°C for α-lactalbumin and near 80 and

140°C for β-lactoglobulin, however, exert more serious effects on the

37

functional properties of whey proteins. Modifications of cystine-residues in

the polypeptide chain are detected by amino acid analysis upon heat

treatments above 100°C under identical heating conditions as used for

differential scanning calorimetry.

Mangino (1984) reported that many of the desirable attributes of foods may

be directly or indirectly related to the functionality of their protein

components. In which food proteins interact with other food components as

well as with themselves determines their functionality. The protein structure

relate to physical requirements necessary for water binding, gelation,

emulsification, and foam formation. Factors that affect these functional

properties are related to changes of protein structure. The nature of

interactions required for optimal functionality are related to conditions that

alter protein structure in such a way as to encourage occurrence of these

interactions.

Kester and Richardson (1984) stated that Modification of whey proteins to

enhance or alter their functional properties may increase food applications.

Whey protein modification can be accomplished by chemical, enzymatic, or

physical techniques.

Schmidt et al., (1984) reported that WPC preparations, prepared by

electrodialysis, ultrafiltration, reverse osmosis, gel filtration, and reagent

complexation, are highly variable in their composition and functionality.

Factors affecting the functional properties of WPC include: whey source and

composition, cheese or casein manufacturing conditions, heat treatment

conditions, fractionation and isolation conditions, storage conditions, overall

sanitation conditions, and techniques used for functionality evaluation.

Process modifications such as selective heat treatment, selective

demineralization or ion exchange, and preteolytic enzyme hydrolysis may be

used to alter these functional properties for a desired use application.

38

Matthews (1984) stated that whey protein properties that have been

exploited commercially include: molecular size differences (ultrafiltration,

gel filtration), insolubility of protein at high temperature, charge

characteristics demineralization, protein removal by ion exchange),

aggregation by polyphosphates, and crystallization of lactose. Numerous

other isolation procedures have been investigated. Chemical, physical, and

functional characteristics vary according to method of manufacture. Capital

costs for most of these processes are high. As yields are characteristically

low, careful economic analysis is necessary.

Harper (1984) reported that the procedures for development of model food

systems for evaluation of functionality of whey protein concentrates as an

alternative to traditional functionality testing in simple systems. The

approach is illustrated for two model systems, a coffee whitener and a

whipped topping. Results indicate factors that must be considered in model

development. In the coffee whitener, protein type must be considered, and

the same model may not be applicable to different types of proteins.

Kilara (1985) stated that the enzymatic hydrolysis of proteins includes:

enzyme specificity, extent of protein denaturation, substrate/ enzyme ratio,

pH, ionic strength, temperature and presence or absence of inhibitory

substances.

Mulvihill and Donovan (1987) stated that whey protein isolate (WPI) is

widely used because of its unique nutritional, physico- chemical and a

functional property of WPI is their ability to form gels. Gelation is

considered to be a result of partial unfolding followed by aggregation.

Garrett et al., (1988) reported that the thermal coagulation of unfractionated

whey proteins was inhibited by various sugars. The diasaccharides, sucrose

and lactose were most effective and the amino sugar, glucosamine, least

effective in this respect. Ultraviolet absorption and light- scattering

measurements on the thermal denaturation and coagulation of both

39

unfractionated and individual whey proteins (α- lactalbumin, β-

lactoglobulin and bovin serum albumin) showed that sucrose promotes the

denaturation of these proteins but inhibits their subsequent coagulation.

Hsu and Fennema (1989) observed that various functional properties

(protein solubility, foam stability, emulsifying capability) and development

of browning of dry WPC containing 52% protein were monitored during 6

month at temperatures ranging from −40 to 40°C and water activities

ranging from .15 to .41. To achieve good retention of the initial attributes of

WPC during 6-mo storage, the temperature should be no higher than 20°C

and the water activity should not exceed about .2.

Sienkiewicz and Riedel (1990) studied that the water binding capacity of

WPC is influenced by protein concentration, mineral content and the extent

of heating during manufacture.

Patel and Kilara (1990) studied that the concentrates were prepared from

cheese whey obtained from skim milk, whole milk, and buttermilk-enriched

skim milk. In comparison with the other whey protein concentrates,

concentrates prepared from skim milk whey had lower surface

hydrophobicity and concentrates prepared from buttermilk-enriched skim

milk whey had lower solubility. Whey protein concentrates prepared from

whole milk whey had poor foaming and emulsifying properties. In general,

free fat and bound fat were negatively related with foaming and emulsifying

properties, whereas, ash, calcium, and denaturation enthalpy were positively

related with foaming and emulsifying properties.

Patel et al., (1990) observed that the samples were prepared from three milk

viz. skim milk, whole milk, and skim milk enriched with buttermilk. The

concentrates from skim milk were lower in all fat components and higher in

proteins, except for the membrane-associated protein. The buttermilk-

enriched samples had the most membrane-associated components. The

concentrates from whole milk and buttermilk-enriched, skim milk were

40

similar in protein composition, except for membrane-associated protein. The

whole milk samples had the highest concentrations of total and free fat

components. Lactose content and mineral composition were similar for the

three types of concentrates. Thermal properties and denaturation kinetics

were examined by differential scanning calorimetry.

Wit (1990) suggested that the functional properties of whey proteins are

often impaired by inevitable heat treatments during processing of whey

protein products and preservation of food products. The effect of heat

treatments between 70 and 90°C is analyzed in terms of the kinetics of whey

protein unfolding and aggregation. The results reveal that thermal

denaturation of whey proteins during industrial heating processes is

predictable. An important determinant for functional properties is the salt

composition of whey protein products during heat treatments at temperatures

above 75°C.

Renner and Salam (1991) suggested that whey proteins have higher protein

efficiency ratio, net protein utilization and biological value compared to

casein and any other food proteins. These proteins are considered as the best

quality protein.

Morr and Ha (1991) studied that the chemistry of off- flavour formation in

WPC products in comparison to milk and whey by maillard reaction, lipid

oxidation and riboflavin decomposition during storage and suggests whey

pre-treatment processing technology to improve the flavour stability of

WPC.

Geoffrey and Geoffrey (1991) reported that seasonal changes generally

included a reaction in the α- lactalbumin content of WPC manufactured

during the final three months of lactation, concomitant with a rise in the

level of β- lactoglobumin. These changes contrassed with an increase in the

casein content of WPC prepared during the first 4 month of lactation.

41

Seasonal variation in the relative proportion of the major protein constituents

of WPC has important implications for the dairy industry.

Nagendra et al., (1991) stated that the decrease in the scores above 40%

levels of replacement could be attributed to the formation of large grains

because of incorporation of WPC.

Jayaprakasha (1992) stated that fresh chedder cheese whey was subjected

to fractionation by ultrafiltration plant (Permionics Ltd. Baroda) possessing

polysulphone membrane with 1.8 m2 effective surface area at a temperature

of 50 ˚C and pressure of 100 psi until the retentate reached to a TS content

of 9.0% by processing whey to a volume reduction of about 80 to 85%.

Geoffrey and et al., (1992) reported that transition energy associated with

protein denaturation was reduced for WPC prepared in the final 3 mo of

lactation. This decrease paralleled a similar decrease in α- lactalbumin

content and surface hydrophobicity of the WPC. Other seasonal changes in

the functional properties of WPC prepared in the letter half of lactation. Heat

induced gel firmness was generally higher for WPC manufactured in the

final 3 mo of the lactation cycle, on observation that paralled an increased

proportion of β- lactoglobulin in WPC prepared at that time.

Gauthier et al., (1993) suggested that enzymatic hydrolysis of whey

proteins has the potential to improve solubility, emulsifying and foaming

properties.

Barbut and Foegeding (1993) stated that protein unfolding and aggregation

are particularly sensitive to pH and ionic strength. At low ionic strength

gelation is prevented by electrostatic repulsion between molecules. Addition

of ions after pre- heating of protein dispersions induces gelation.

Kawachi et al., (1993) suggested that whey protein gelation at ambient

temperatures has potential applications in the food industry. It can be used in

42

the production of desserts, dressing, spreads, bakery products, pressed ham

and surimi.

Donald et al., (1993) reported that concentrating milk by ultrafiltration

shortened coagulation time and increased gel firmness. UHT processed milk

did not coagulate when rennet was added. Its 3 × concentrated counterpart

did coagulate although only a weak gel was formed. UHT heating caused the

casein micelles to increase in size with additional protein material adhering

to their surface, especially in the 3 × skim milk heated to 140 ˚C. This

diffuse layer of material around casein micelles was not observed in 3 ×

whole milk. It is suggested that this denatured protein is adsorbed onto the

fat water interfaces during homogenization.

Althouse et al., (1995) suggested that the functional properties of whey

proteins concentrate (WPC) for their effective utilization in the food

products.

Banerjee and Chan (1995) stated the the functional properties of whey

protein concentrate films were compared with those of the films derived

from sodium caseinate, potassium caseinate, calcium caseinate, and whey

protein isolate. Water vapor permeability of simple whey protein concentrate

film was lower than that for films of sodium caseinate, potassium caseinate,

and whey protein isolate. Composite whey protein concentrate film had the

lowest water vapor permeability of all the milk protein films. The ultimate

tensile strengths of simple whey protein concentrate films were similar to

those of caseinate films. Whey protein concentrate films had good water

vapor barrier and mechanical properties that were comparable with those of

films from other commercial milk proteins.

Jayaprakasha et al., (1995) stated that technology for the conservation of

whey solids in the form of whey protein concentrate (WPC) is the best way

to redeem these solids.

43

Turgeon et al., (1996) studied that whey protein concentrate (WPC), a heat-

treated WPC (90 °C, pH 2.5, 10 min) and peptidic fractions obtained by

ultrafiltration of their tryptic and chymotryptic hydrolysates were

incorporated in a salad dressing formulation at 0.5, 1.0 or 1.5% (w/w)

protein. Peptidic fractions obtained from tryptic hydrolysates produced the

most stable salad dressings (over 6 months at the 1.0% and 1.5% protein

level) with rheological properties similar to a commercial mayonnaise. The

most important factors for emulsion stability were the incorporation level

and the nature of peptides.

Regester et al., (1996) stated that it is estimated that 30% of the world‘s

milk production is utilized for cheese preparation, which generates nearly

83,030 million kg of whey.

Vaghela and Kilara (1996) stated that the freeze- dried WPC containing 35

and 75% protein and varying amounts of residual lipids, were manufactured

by pretreating whey with calcium chloride and heat. These and commercial

WPC were subjected to proximate analysis, resulted in WPC that had

significantly lower total lipids and a lower lipids to protein ratio. The

commercial WPC had ratios of lipid to protein that were significantly higher

than all experimental WPC. The pre-treatment significantly increased the

proportions of phospholipid and monoacylglycerol and decreased the

proportion of triacylglycerol.

Mleko and Achremowicz (1996) suggested that the most important

functional properties of whey protein isolates, as a food component is their

ability to form gels. Dispersions of heated whey proteins can form gels when

salt is added before or after heating.

Milena and Douglas (1996) reported that skim milk was heated at

temperature in the range 75-90 ˚C at pH values of 6.8, 6.2 and 5.8. The

amounts of α- lactalbumin and β- lactoglobulin which interacted with the

casein micelles during heat treatment. Both α- lactalbumin and β-

44

lactoglobulin appeared to interact similarly with casein micelles at

temperatures up to 85 ˚C. The amount of whey protein complexed with

micelles increased with time, reaching plateau values that, at the highest

temperature, were comparable with the quantity present in the original skim

milk. In general, faster reaction of the whey proteins with the micelles was

found at lower pH and higher temperatures.

Kinekawa and Kitabatake (1996) reported that β-Lactoglobulin was

purified from WPC by a combination of pepsin treatment and membrane

filtration. Porcine pepsin was added to whey protein (1:200, wt/wt), and the

mixture was then incubated at pH 2.0 and 37°C for 60 min. The protein

fraction was collected by ammonium sulfate precipitation, and the

precipitate was either dialyzed against water using a dialysis membrane (20-

kDa pore size) of filtered using an UF membrane (30-kDa pore size). The β-

LG did not differ from standard β-LG as measured by chromatography.

Hsu and Kolbe (1996) reported that WPC containing 33 or 72% protein

were evaluated as functional ingredients to improve the textural properties of

surimi seafoods made from Pacific whiting. The development of least cost

formulations of these products using nonlinear programming techniques was

used to evaluate the economic viability of WPC. WPC ingredients were

promising because they remained economically competitive with potato

starch and beef plasma protein, which are commonly used as ingredients in

whiting surimi seafoods. Factors affecting the amount of WPC in the least

cost formulations.

Mleko (1997) stated that whey protein gels find a wide range of applications

in forming texture of a new product or mimic the texture of an existing one.

Elofsson et al., (1997) studied that the cold- gelling whey protein powder

was found to consist of large, micron-sized, drying-induced, weak

aggregates consisting of primary disulfidebridged aggregates of 20 – 30 nm

in diameter. In dilute solutions under conditions of large repulsive forces

45

(low ionic strength and pH far from the isoelectric point) the large

aggregates dissolved slowly over many hours. In less good solvents, larger

aggregates remained or were formed.

Nielsen (1997) observed that partial hydrolysis of protein generally

increases the number of polar groups and hydrophobicity, decreases the

molecular weight, alters the globule structure of proteins and exposes

previously buried hydrophobic regions. These changes will alter their

emulsifying properties.

Casper et al., (1999) studied that whey protein concentrates were prepared

from two caprine and one ovine specialty cheese wheys by ultrafiltration-

diafiltration and freeze-drying processes. The WPC were compared with a

bovine WPC prepared by same method. Ovine WPC showed better foam

overrun, foam stability, and gel strength than did bovine and caprine WPC

and both caprine WPC showed better gel strength than did bovine WPC.

Caprine WPC produced from rennet whey, showed better emulsifying

capability at low pH than did both bovine and ovine WPC. Caprine WPC

produced from direct-acidified whey, had less emulsification capability than

bovine WPC produced from rennet whey.

Mleko and foegeding (1999) used two- stage process to obtain whey

protein gels. The first stage was performed at pH 8.0, when whey protein

molecules get polymerized by disulphide bonds and the second stage was

carried out at pH 6.0-7.0, which favour noncovalent bonds.

Jayaprakasha and Brueckner (1999) stated that the production of cheese

nearly 50% of the milk solids are lost through whey, resulting in colossal

losses of nutritious solids. Whey solids are known to carry excellent

nutritional and functional properties.

Keogh and Kennedy (1999) reported that the increasing of homogenisation

pressure, reduced the fat globule diameter and increasing the number of

homogenisation passess reduced the diameter of the largest globules.

46

Increasing fat and salts reduced fat globule diameter stability after 2

homogenisation passes but the reduction in stability was less after 4 passes.

Increasing the lactose: whey protein concentrate (WPC) ratio reduced free

fat and fat globule aggregation after powder reconstitution, but not the

surface fat. The higher level of fat increased the surface fat on powder

particles and the level of oxidation during storage.

Hayes and Nielsen (2000) observed that the plasmin activity in whey

protein products may cause breakdown of food proteins to have desirable or

undesirable effects on food quality. Acid whey protein products had

significantly higher plasmin concentrations then sweet whey. Plasmin

activities associated with acid and sweet whey protein products were both

significant affected by the growth of Pseudomonas fluorescens M 3/6. The

interaction effect between bacterial growth and whey type on plasmin

activity was not significant. Plasmin activity in the reconstituted commercial

WPC (i.e., sweet and acid) varied considerably (16.3 to 330 µg/ g of

protein), but was significantly lower (2.1 to 4.4 µg/ g) of protein in whey

isolates.

Jayaprakasha (2000) suggested that the most promising way could be

inclusion of WPC in traditional food products, as these traditional foods

occupy a very important place in Indian dietary. Several food formulations

have been developed utilizing WPC which impart better physico- chemical

and functional properties, besides improving the nutritional profile of such

products.

Brandsma and Rizvi (2001) stated that optimal cheese manufacturing

parameters were determined to be 80–100 μL rennet kg–1 MF cheesemilk,

coagulation temperature of 32–36 °C, and post-coagulation curd cutting time

of 15 min. As compared with ultrafiltration (UF) retentate cheese

manufacture, cheese made from MF retentates has potential for improved

textural and functional qualities, along with recovery of highly functional

whey proteins (WP) from permeate.

47

Mleko et al., (2002) observed that whey protein gels were obtained at

ambient temperatures from no salt heated dispersions with calcium ions as

an inducing agent. Double heating of whey proteins resulted probably in

extensive unfolding of whey proteins and subsequent formation of polymers/

aggregates. Further increase of calcium concentration caused probably

higher aggregation, which influenced the balance between inter and intra-

molecular forces and affected rheological properties.

Mleko (2002) studied that whey protein polymers/ aggregates were obtained

in a two-stage heating process. Sample were mixed for 5 min at 20,000 rpm

and rheological properties were compared with non sheared dispersions.

Gels prepared from pre sheared dispersions at pH 7.0 and 6.5 characterized

higher storage moduli and lower phase angels. Pre shearing of the

dispersions obtained at pH 6.0 resulted in a weaker gel, probably because of

higher aggregation. Non sheared gels had a higher optical density then pre

sheared gels, which suggests formation of a gel composed of larger

aggregates.

Desrumaux and Marcand (2002) reported that the emulsifier used was

whey protein concentrate (1.5%). The properties of the emulsions were

characterized by laser light scattering (droplet size distribution) and coaxial

cylinders rheometry (rheological behaviour). The protein adsorption fraction

was obtained by a spectrophotometric method using bicinchoninic acid

reagent. No change was revealed by polyacrylamide gel electrophoresis of

the whey protein within the pressure range studied. Microdifferential

scanning calorimetry scans indicated that the changes of the structural and

textural properties may be because of changes in the protein conformation.

Yoshida et al., (2002) stated that edible films, using whey protein as the

structural matrix, were tested for water vapour diffusion properties. Whey

protein films were prepared by dispersing 6.5% whey protein concentrate

(WPC) in distilled water with pH kept at 7.0. Glycerol was the plasticizer

agent. Film slabs (13.5 × 3.5 cm) were put in a chamber at 25 °C and 75%

48

relative humidity, being held in vertical planes for different periods of time.

The mass gain was determined throughout the experiment. Moisture

adsorption by milk whey protein films is well described by a linear diffusion

equation model.

Spiegel and Huss (2002) studied that the effects of pH-value and a

reduction in calcium content on the kinetics of whey protein denaturation

and the aggregation behaviour, under shear in a scraped surface heat

exchanger. Aggregates which are produced under shear between pH 4 and

5.5 reveal a small particle size (<5 μm) irrespective of the lactose content

and the heating temperature. At a reduced calcium concentration the heat-

and shear-treatment resulted in a gritty structure with large rubber-like

particles. These are not to be taken as primary whey protein aggregates but

as fragments of a fine-stranded gel.

Totosaus et al., (2002) reported that protein gelation has been traditionally

achieved by heating, but some physical and chemical processes form protein

gels in an analogous way to heat-induction. A physical means, besides heat,

is high pressure. Chemical means are acidification, enzymatic cross-linking,

and use of salts and urea, causing modifications in protein–protein and

protein–medium interactions. The characteristics of each gel are different

and dependent upon factors like protein concentration, degree of

denaturation caused by pH, temperature, ionic strength and/or pressure.

Downey (2002) stated that freezing and thawing have been shown to

adversely affect the centrifugal drip loss and maximum resistance to

penetration of cooked, puréed vegetables (potatoes, carrots and turnips).

Amelioration of these effects has been investigated through the addition of

cryoprotectants (xanthan gum, guar gum, pectin, carrageenan, sodium

caseinate, WPC). In general, gums (xanthan and guar) proved most effective

in reducing drip losses although carrageenan and pectin exhibited some

ability in this regard. Dairy powders produced no effect on drip loss but did

alter maximum resistance values after thawing.

49

Foegeding et al., (2002) suggested that whey protein ingredients are used

for a variety of functional applications in the food industry. Each application

requires one or several functional properties such as gelation, thermal

stability, foam formation or emulsification. Whey protein ingredients can be

designed for enhanced functional properties by altering the protein and non-

protein composition, and/or modifying the proteins. Modifications of whey

proteins based on enzymatic hydrolysis or heat-induced polymerization have

a broad potential for designing functionality for specific applications.

Briczinski and Robert (2002) observed that whey was ultrafiltered and

diafiltered to remove lactose and salt, freeze- dried and milled to a powder.

Unfermented hydrolyzed and unhydrolyzed whey controls were processed in

the same manner. The EPS-WPC ingredients contained approximately 72%

protein and 6% EPS but they exhibited low protein solubility (65%, pH 7.0,

pH 3.0).

Yoshida et al., (2003) stated that the macroscopic aspects of moisture

transmission in whey protein films were determined by measuring water

vapour adsorption. A theoretical model was constructed in which two kinds

of water vapour fluxes were considered: one originating from diffusion,

whilst the other was a flux due to the gravitation drift of moisture. The

comparison of theoretical and experimental results showed that only the

diffusion process was present.

Bailey (2003) stated that the milk protein concentrates are used in the United

States in many different products, including the starter culture of cheese, or

in non standard cheeses such as baker's cheese, ricotta, Feta and Hispanic

cheese, processed cheese foods, and nutritional products. One of the difficult

aspects of trying to assess the impact of MPC imports on the US dairy

industry is to quantify the protein content of these imports. The protein

content of MPC imports typically ranges from 40 to 88%.

50

Piyasena and Chambers (2003) studied that the influence of whey protein

dispersions (WPDs) on syneresis of renneted curd. Curd produced from milk

with added WPDs contained less protein and less fat than that produced

from raw milk. These findings reveal the importance of substrate pH in

combination with homogenization of added protein dispersions from whey

when utilized by cheese producers to optimize cheese yield and

composition.

Bals and Kulozik (2003) observed that the effect of the thermal

denaturation of whey proteins on the formation, stability and structure of

their respective foams. A membrane foaming apparatus, which is a very

gentle foaming method was used to produce the foams. It was shown that the

denaturation of the β-lactoglobulin, the main component in whey protein

isolate, strongly improves the foam stability. At a denaturation degree

>70%, it is possible to reduce drainage to a large extent. The image analysis

demonstrated that higher levels of denaturation of the proteins and thus

higher viscosities of the protein solution produced coarser foam textures

with larger bubbles. The incorporation of the bubbles was more difficult

when the viscosity of the continuous phase was high.

Miralles et al., (2003) capillary electrophoresis (CE) was used to determine

the whey protein to total protein ratio in raw and UHT milk samples with

different degrees of proteolysis caused by storage. In UHT milks, the

overestimation of the whey protein to total protein ratio took place after 30

or 60 d of storage. However, the ratios αS1-CN/β-CN and αs1-CN/κ-CN

permitted detection of the samples of raw or UHT milk with degraded

proteins.

Pasin and Miller (2004) suggested that whey protein concentrate (WPC)

are dairy ingredients that are highly nutritious with a protein efficiency ratio

of 3.6 and a protein digestibility corrected amino acid score of 1.14 as

against 1.5 and 0.25 for wheat proteins.

51

Wroblewska et al., (2004) reported that commercial whey protein

concentrate (WPC) was hydrolysed with either Alcalase 2.4 FG (Novo

Nordisk), or papain (Sigma) (in one-step process) or with two enzymes (in

two-step process) to determine the changes in the immunoreactivity of α-

lactalbumin and β-lactoglobulin. The ‗two-step‘ process was observed to be

the most effective however allergenic epitopes were still present, as it was

found by enzyme- linked immunosorbent assay (ELISA) with anti-α-la and

anti-β-lg antibodies. The addition of papain as the second enzyme in the

hydrolysis process contributed to the improvement of the sensory properties

of WPC hydrolysate as compared with the Alcalase hydrolysate. Alcalase-

papain partially hydrolysated WPC can be found a promising base for

production of the tolerogenic formula.

Resch et al., (2004) studied that the freeze-dried and spray-dried derivatized

WPC powders, along with polysaccharide thickeners, were reconstituted in

water and evaluated by using a range of rheological studies. The effects of

temperature, concentration, and shear on viscosity as well as the mechanical

spectra were assessed to characterize the ability of the powders to function

in food systems. Rheological characterization revealed the modified

derivatization procedure yielded an ingredient having the same cold-set

thickening and gelling ability as the original derivatized powder. The

modified whey proteins were also able to achieve, at higher usage levels,

textural properties similar to several polysaccharide thickeners.

Christiausen et al., (2004) observed on the stability of WPC and β-LG

dressings, while hydrolysate dressings showed reduced stability, except for

the combination of low pH (4.0), high protein (4%) and fat content (30%).

This dressing was highly stable at high processing temperature. The

microstructure of WPC dressing showed homogenous, non-aggregated

structure in contrast to hydrolysate and β-LG dressing, which showed highly

ordered, aggregated structure which supports the rheological measurements

for gel formation.

52

Veith and Reynolds (2004) observed that process for the production of a

whey protein concentrate (WPC) with high gel strength and water-holding

capacity from cheese whey. To maintain whey protein solubility, it is

necessary to minimize heat exposure of the whey during pre-treatment and

processing. The presence of the caseinomacropeptide (CMP) in the WPC

was found to be detrimental to gel strength and water-holding capacity. All

of the commercial WPC that produced high-strength gels exhibited ionic

compositions that were consistent with acidic processing to remove divalent

cations with subsequent neutralization with sodium hydroxide.

Zafer et al., (2005) observed that the fermentation of whey by

Kluyveromyces marxianus strain MTCC 1288 using varying lactose

concentrations at constant temperature and pH. The increase in substrate

concentration up to a certain limit was accompanied by an increase in

ethanol formation. An increase in lactose concentration to 100 g L−1 led to a

drastic decrease in product formation and substrate utilization. The

maximum ethanol yield was obtained with an initial lactose concentration of

50 g L−1. A method of batch kinetics was utilized to formulate a

mathematical model using substrate and product inhibition constants.

Reskin et al., (2006) reported that fat globule aggregation and adsorbed

protein content in whipped frozen emulsions were determined after

application of thawing, dilution or centrifugation. Micrographs indicated that

in aerated products, partial replacement of native whey proteins by pre-

denatured whey proteins or casein introduced (i) more homogeneity in air

bubble size, (ii) more attachment of fat globules to their air serum interface,

(iii) fat globules in the continuous matrix that were in closer contact with

each other. These differences in the microstructures of whipped frozen

emulsions were attributed to differing surface heterogeneity of adsorbed

protein particles of fat globule interfaces.

Anema et al., (2006) result revealed that the effect of storage time and

temperature on the solubility of milk protein concentrate (MPC85) using

53

solubility tests, gel electrophoresis and mass spectrometry. It was found that,

at a given temperature, the solubility of MPC decreased exponentially with

time and a master curve was obtained using a temperature–time

superposition. Gel electrophoresis indicated that the insoluble proteins were

the caseins, whereas the whey proteins remained soluble. Mass spectrometry

showed that, with storage time, the casein was lactosylated. In the light of

these measurements, it is speculated that the insolubility of the MPC could

have been due to cross-linking of the proteins at the surface of the MPC

powder.

Roman and Sgarbieri (2006) reported that the hydrophilic and surfactant

properties of casein concentrates made by different processes such as

isoelectric precipitation and neutralization (commercial casein, CC)

coagulation by rennet (casein clots, COC) and microfiltration/diafiltration

(casein micelles, CM). Water absorption capacity (WAC), water solubility

(WS) and water-holding capacity (WHC) were highest for CM and lowest

for COC. Solubility was higher in water for both CM and COC. Foaming

capacity was better for CM than for CC. Foam stability was low for both

CM and CC but it was high for CM and for CC in the absence of salt.

Emulsifying capacity was higher for CC. Stability of emulsion was high for

CC at pH 4.0 and for CM at pH 7.0.

Onwulata et al., (2006) reported that WPI pastes (60% solids) were

extruded in a twin-screw extruder at 100°C with 4 pH-adjusted water

streams: acidic (pH 2.0 ± 0.2) and alkaline (pH 12.4 ± 0.4) streams from 2 N

HCl and 2 N NaOH. Acidic (pH 2.5 ± 0.2) and alkaline (pH 11.5 ± 0.4)

electrolyzed water streams; these were compared with WPI extruded with

deionized water. Alkaline conditions increased insolubility caused yellowing

and increased pasting properties significantly. Acidic conditions increased

solubility and decreased WPI pasting properties. Subtle structural changes

occurred under acidic conditions, but were more pronounced under alkaline

conditions.

54

Sinha et al., (2007) results revealed that the functional and nutritional

properties of enzymatically hydrolyzed WPC and to formulate a beverage

mix. The water absorption capacity of WPC was 10 ml/100 g and increased

in enzyme treated samples from 16 to 34 ml/100 g with increase in the time

of hydrolysis. Emulsion capacity (45 ml of oil/g of control WPC) showed a

decreasing trend with increasing time of hydrolysis. The gel filtration pattern

of enzyme treated samples increase in low molecular weight fractions. The

content of methionine in samples treated with enzymes is higher, compared

to the control. The in vitro protein digestibility of untreated WPC was 25%

and increased in all treated samples to varying degrees (69–70%).

Formulated beverage had 52% protein, 10% fat and 6.6% ash. There were

no significant differences in the sensory attributes of formulated and

commercial beverage.

Dangaran and Krochta (2007) stated that WPI films plasticised with

sucrose were stored in 53% relative humidity for up to 60 days. The oxygen

permeability, tensile properties and gloss of the films were measured.

Changes in properties were compared with changes in WPI films plasticised

with glycerol (no crystallisation) or plasticised with sucrose (crystallisation)

plus a crystallisation inhibitor. The inhibitors hindered sucrose

crystallisation, and the desired film properties were maintained for a longer

period of time. Raffinose was the more effective inhibitor, maintaining the

film flexibility and barrier properties for over 28 days and maintaining gloss

at almost 90% of the initial value for 60 days of storage.

Cheison et al., (2007) observed that whey protein isolate (WPI) was

hydrolyzed to whey protein hydrolysates (WPH) of degree of hydrolysis

equal to 15% using Protease N ‗Amano‘ G (IUB 3.4.24.28) in a batch

reactor at 55 °C and pH 7.0 according to the pH-stat procedure. Ash was

removed by adsorbing WPH onto macroporous adsorption resins (MAR).

Kim et al., (2007) examined the effects of enzymes on the production and

antigenicity of native and heated WPC hydrolysates. Native and heated (10

55

min at 100˚C) WPC (2% protein solution) were incubated at 50˚C for 30, 60,

90 and 120 min with 0.1, 0.5 and 1% pepsin and then with 0.1, 0.5 and 1%

trypsin on a protein equivalent basis. Results suggested that incubation of

heated WPC with 1% pepsin and then with 1% trypsin was the most

effective for producing low antigenic hydrolysates by WPC hydrolysis and

obtaining low molecular weight small peptides.

Herceg et al., (2007) observed that the interactions between whey proteins

(whey protein isolate (WPI), whey protein concentrate (WPC) and β-

lactoglobulin) and carbohydrates (glucose, sucrose, starch and inulin) on

some physical and functional properties of whey proteins and carbohydrates

suspensions (10% dry matter (w/v)). Carbohydrate addition in model

suspensions of whey proteins resulted in significantly enhanced foam

stability of protein suspensions (FSI; MFS). Model systems were also

analyzed for emulsion activity index (EAI) and emulsion stability index

(ESI) by the turbidometric technique. EAI and ESI values increased

significantly in model suspensions prepared with WPI and β-lactoglobulin in

combination with mono and disaccharides.

Tosi et al., (2007) studied that the influence of thermal treatment on foaming

properties of sweet whey solutions. The tests were carried out on solutions

of cheese sweet whey powder by a factorial experiment design of two

variables, namely treatment temperature and time, at three levels, 75, 80 and

85 °C, and 300, 750 and 1200 s. Selected responses were foam volume,

liquid volume in foam, foam forming power and foam stability. The applied

thermal treatments modified foam properties in such a way that the foam

volume of thermally treated samples was always higher than that of the non-

treated samples, with the exception of that treated at 85 °C for 750 s.

Stability also behaved in the same way, except the sample treated at 75 °C

for 300 s.

Wang et al., (2007) assessed the film-forming abilities of six types of

proteins, as well as six types of polysaccharides at various concentrations

56

(proteins: 0–16%; polysaccharides: 0–4%) and heating temperatures (60–

80 °C). Biopolymer films evaluated included: sodium caseinate (SC), whey

protein isolate (WPI), gelatine (G); caboxymethyl cellulose (CMC), sodium

alginate (SA) and potato starch. Film-forming conditions were achieved

using SC and G (4% and 8%), WPI (8% and 12%), PS, CMC (2% and 3%)

or SA (1% and 1.5%) solutions heated to 80 °C in combination with 50%

(w/w) glycerol. Films manufactured from 1.5% SA, 8% G and 3% CMC had

the highest tensile strength (24.88 MPa); flexibility (89.69%) and puncture

resistance (22.66 N). SC, WPI and G-based films were more resistant to

solvent than SA, CMC and PS.

Zhong and Jin (2008) revealed that the WPI and WPC powders and a 10%

(wt/vol) WPI solution were treated with supercritical carbon dioxide

(scCO2). The WPI solution was treated at 40°C and 10 MPa for 1 h whereas

WPI and WPC powders were treated with scCO2 at 65°C and 10 or 30 MPa

for 1 h. The improvement in gelling properties was more significant for the

scCO2-treated WPC. In addition, the scCO2-processed WPI and WPC

powders appeared to be fine and free-flowing in contrast to the clumps in the

unprocessed samples. The results suggest that functionalities of whey

proteins can be improved by scCO2 treatment to produce novel ingredients.

Wang et al., (2008) stated that response surface methodology (RSM) was

used to investigate pH and corn oil (CO) effects on the properties of films

formed from WPI. Test films were evaluated for tensile strength (TS),

puncture strength (PT), percentage elongation at break point (E), water

vapour permeability (WVP) and oxygen permeability (OP). When WPI

solution pH increased, film TS generally decreased with CO addition. E

values increased dramatically with increasing levels of CO when pH for

WPI solutions were >8.5. WPI solutions possessing high pH values

produced WPI films with the highest PT values. WVP had a quadratic

relationship with pH and CO addition. OP had an inversely linear

57

relationship with increasing pH and a quadratic relationship with CO

addition.

Michael et al., (2008) suggested that the WPC higher than WPI for milky,

sweet, and caramel flavors. Instrumental analysis showed that WPC

products had a greater number of volatiles than WPI products. Sensory

results indicated that the flavor of WPC and WPI was not affected by

instantizing, ion exchange, or bleaching; alternatively, instrumental results

indicated slight differences in numbers of volatiles identified for each

aforementioned process.

Kucukcetin (2008) studied that different heat treatments (95 ˚C/ 256 s, 110

˚C/ 180 s and 130 ˚C/ 80 s) were applied to the yoghurt milk with the CN to

WP ratio of 1.5:1, 2:1, 3:1 and 4:1. Physical properties, including graininess

and roughness of stirred yoghurt were determined during storage at 4 ˚C for

15 days. Visual roughness number of grains, perimeter of grains, storage

modulus and yield stress decreased, when heating temperature or CN to WP

ratio increased.

Lim et al., (2008) studied that use of high hydrostatic pressure (HHP) to

improve functional properties of fresh WPC, compared with functional

properties of reconstituted commercial whey protein concentrate 35 (WPC

35) powder. Additionally, HHP-WPC treated at 300 MPa for 15 min

acquired larger overrun than commercial WPC 35. The HHP treatment of

300 MPa for 0 min did not improve foam stability of WPC. However, WPC

treated at 300 or 400 MPa for 15 min and 600 MPa for 0 min exhibited

significantly greater foam stability than commercial WPC 35. The HHP

treatment was beneficial to enhance overrun and foam stability of WPC.

Kresic et al., (2008) revealed that the effects of three emerging

technologies: high pressure (HP: 500 MPa, 10 min), ultrasound (US:

20 kHz, 15 min) and tribomechanical activation (TA: 40000 rpm) on

flowing behaviour and thermophysical properties of WPI and WPC. HP and

58

US were carried out on 10% (w/w) model dispersions while for TA samples

were in powdered form. Pressurization caused significant decrease in

solubility of WPC and WPI, while both samples treated with US and TA

exhibited significantly better solubility compared to control. Apparent

viscosity data described with power law equation (r2 = 0.97–0.99)

significantly increased after all treatments while HP caused the most

intensive changes in rheological behaviour.

Padiernos et al., (2009) evaluate the foaming properties of selected low-fat

whipping cream formulations containing whey protein concentrate (WPC)

that did or did not undergo high hydrostatic pressure (HHP) treatment.

Whipping cream containing untreated WPC and HHP-treated WPC resulted

in greater overrun and foam stability than the control whipping cream

without WPC. High hydrostatic pressure-treated WPC can improve the

foaming properties of low-fat whipping cream.

Guyomarch et al., (2009) revealed that the aggregates were formed by

heating mixtures of whey protein isolate (WPI) and pure κ-casein or sodium

caseinate at pH 7 and 0.1 M NaCl. The aggregates were characterized by

static and dynamic light scattering and size exclusion chromatography. After

extensive heat-treatment at 80 °C for 24 h, almost all whey proteins and κ-

casein formed mixed aggregates. At a given WPI concentration the size of

the aggregates decreased with increasing κ-casein or sodium caseinate

concentration, but the overall self-similar structure of the aggregates was the

same. The presence of κ-casein or caseinate therefore inhibited growth of the

heat-induced whey protein aggregates.

Sugiarto et al., (2009) studied that the binding of iron (ferrous sulphate) to

two commercial milk protein products, sodium caseinate and WPI dissolved

in 50 mM HEPES buffer was examined as a function of pH and iron

concentration. Sodium caseinate had more sites (n=4) than WPI (n=8) for

binding iron and the affinity of caseinate to bind iron was also higher than

that of WPI. These differences were attributed to the presence of clusters of

59

phosphoserine residues in casein molecules. The amount of iron bound to

sodium caseinate was found to be independent of pH in the range 5.5 – 7.0

whereas acidification (pH range 7.0 – 3.0) caused a marked decrease in the

amount of iron bound to WPI.

Kuhn et al., (2010) stated that cold-set whey protein isolate (WPI) gels

formed by sodium or calcium chloride diffusion through dialysis membranes

were evaluated by mechanical properties, water-holding capacity and

microscopy. The increase of WPI concentration led to a decrease of porosity

of the gels and to an increase of hardness, elasticity and water-holding

capacity for both systems (CaCl2 and NaCl). WPI gels formed by calcium

chloride addition were harder, more elastic and opaque, but less deformable

and with decreased ability to hold water in relation to sodium gels.

Gauche et al., (2010) reported that at temperatures higher than 85 ˚C the

apparent viscosity measurements of whey protein solutions with

transglutaminase were significantly higher than those of the control samples.

DSC analysis showed that thermal denaturation occurred at temperatures

close to 82 ˚C and the enzymatic reaction was enhanced at higher

temperature. The gel point of whey proteins decreased with transglutaminase

addition. This decrease became greater as a function of reaction time due to

the formation of high weight protein polymers catalyzed by

transglutaminase, which was also observed in the turbidity analysis.

Chai et al., (2010) reported that effect of the free and the pre-encapsulated

calcium ions on the physical properties of the WPI film were studied for

improving calcium content in the edible films. At pH 8, the film-forming

process was hindered by serious protein aggregation and gelation caused by

0.5% (w/w) free calcium ions added in an 8% WPI solution. If the calcium

ions were pre-encapsulated in the protein microparticles (contained 17%

Ca2+) using spray drying method, and then added in the film-forming

solution prepared using the same protein, the calcium content could be

60

doubled (1%, w/w) without significant effects on the physical properties of

the film.

Evans et al., (2010) studied that identify and compare the composition,

flavor, and volatile components of 80% serum protein concentrates (SPC)

and whey protein concentrates (WPC). Each pair of 80% SPC and WPC was

manufactured from the same lot of milk. Consumer acceptance testing of

acidified 6% protein beverages made with 80% SPC and WPC produced in

the pilot plant and with WPC from commercial sources was conducted. The

SPC was lower in fat and had a higher pH than the WPC produced in the

pilot plant or commercial WPC. The pilot-plant WPC had higher

concentrations of lipid oxidation products compared with SPC, which may

be related to the higher fat content of WPC. There was a large difference in

appearance between 80% SPC and WPC: solutions of SPC were clear and

those of WPC were opaque.

Kelly et al., (2010) observed that the effects of protein concentration on

astringency and interactions between whey and salivary proteins. Changes in

astringency with protein concentration depended on pH. At pH 3.5,

astringency significantly increased with protein concentration from 0.25 to

4% (wt/wt) and then remained constant from 4 to 13% (wt/wt). Furthermore,

saliva flow rates increased with increasing protein concentrations. Maximum

turbidity of whey protein–saliva mixtures was observed between pH 4.6 and

5.2. Both sensory evaluation and in vitro study of interactions between β-LG

and saliva indicate that astringency of whey proteins is a complex process

determined by the extent of aggregation occurring in the mouth, which

depends on the whey protein beverage pH and buffering capacity in addition

to saliva flow rate.

Hiller and Lorenzen (2010) revealed that milk proteins were modified by

Maillard reaction with glucose, lactose, pectin and dextran and analysed for

changes in molar mass distribution and functional properties. Further

concluded that oligomeric (20,000–200,000 g/mol) and polymeric

61

(>200,000 g/mol) Maillard reaction products with heterogeneous functional

property. Compared to untreated milk proteins, milk protein/saccharide

Maillard products formed highly viscous solutions and performed increased

antioxidant capacity. Improved heat stability and increased overrun for milk

protein/pectin and milk protein/dextran products.

Mimouni et al., (2010) studied that a sample preparation method for

scanning electron microscopy analysis of rehydrated milk protein

concentrate (MPC) powder particles and used to characterize the time course

of dissolution and the effects of prior storage on the dissolution process. The

results show that a combination of different types of interactions (e.g.,

bridges, direct contact) between casein micelles results in a porous, gel-like

structure that restrains the dispersion of individual micelles into the

surrounding liquid phase without preventing water penetration and

solubilization of nonmicellar components. During storage of the powder,

increased interactions occur between and within micelles, leading to

compaction of micelles and the formation of a monolayer skin of casein

micelles packed close together, the combination of which are proposed to be

responsible for the slow dissolution of stored MPC powders.

Singh (2011) stated that milk proteins usually exert several interdependent

functional properties simultaneously in each food application. The

functional properties of proteins vary with pH, temperature, ionic strength,

and concentration of calcium and other polyvalent ions, sugars, and

hydrocolloids, as well as with processing treatments. In addition, the

processes used in the manufacture of milk protein products can modify the

native structures of proteins, which can lead to further protein–protein

interactions, consequently affecting the protein functionality.

Foegeding et al., (2011) suggested that the significant progress in the

utilization of whey protein has been made in the past 30 years and the future

growth of whey utilization is expected to be led by the industry‘s increasing

62

focus on nutritional products, particularly in the dietary, sports and clinical

segments of the market.

Huang et al., (2011) reported that the use of whey protein isolate (WPI)

edible coatings to improve the rehydration behaviour of freeze-dried (FD)

strawberry pieces. First, the optimal ratio sample volume of coating solution

was optimised by determining the rehydration ratio, bulk density and

nutritional quality of the samples. Second, the effect of changing the pH and

the variation in temperature–time to denaturate WPI on rehydration

characteristics was also evaluated. The rehydration ratio of strawberry pieces

decreased with increasing the denaturation temperature and time, while it

increased with increasing pH of the coating solution.

Nicorescu et al., (2011) studied to compare the effect of thermal treatments

on the foaming properties of whey protein isolate (WPI) and egg white

proteins (EWP): EWP was pasteurized in dry state from 1 to 5 days and

from 60 °C to 80 °C, while WPI was heat-treated between 80 °C and 100 °C

under dynamic conditions using a tubular heat exchanger. WPI exhibited a

higher foamability than EWP. For WPI, heat treatment induced a slight

decrease of overrun when temperature was above 90 °C. Further concluded

that the dry heat treatment of EWP provided softer foams, despite more rigid

than the WPI-based foams, whereas dynamically heat-treated WPI gave

firmer foams than native proteins.

Hemar et al., (2011) observed that fluorescence spectroscopy was used to

investigate the interaction between resveratrol and whey proteins. The whey

proteins examined were lactoferrin, holo-lactoferrin, apo-lactoferrin, whey

protein isolate (WPI) and the β-lactoglobulin- and α-lactalbumin-rich

fractions of WPI. In all the systems studied, it was found that resveratrol

interacted with the whey proteins to form a 1:1 complex. The binding

constant, Ks, for the protein–resveratrol complex for all the proteins

examined varied from 1.7 × 104 to 1.2 × 105 m−1.

63

Kizeminski et al., (2011) studied that the effect of whey protein addition on

structural properties of stirred yoghurt systems at different protein and fat

content was studied using laser diffraction spectroscopy, rheology and

confocal laser scanning microscopy (CLSM). The composition of heated

milk systems affected micro- and macroscopic properties of yoghurt gels.

Particle size increased as a function of increasing whey protein content and

decreased as a function of increased fat level.

Listiyani et al., (2011) stated that the hydrogen peroxide (HP) bleached

WPC 34% displayed higher cardboard flavor and had higher volatile lipid

oxidation products than benzoyl peroxide (BP) bleached or control WPC34.

Benzoyl peroxide-bleached WPC34 had higher benzoic acid (BA)

concentrations than unbleached and HP bleached WPC34 and BA

concentrations were also higher in BP-bleached WPC80 compared with

unbleached and HP-bleached WPC80, with smaller differences than those

observed in WPC34. Benzoic acid extraction from permeate showed that

WPC80 permeate contained more BA than did WPC34 permeate. Benzoyl

peroxide is more effective in color removal of whey and results in fewer

flavor side effects compared with HP and residual BA is decreased by

ultrafiltration and diafiltration.

Yang and foegeding (2011) explain the macroscopic foaming properties of

egg white protein (EWP) and whey protein isolate (WPI). Foam properties

were altered by adding different amounts of sucrose (4.27–63.6 g/100 mL).

Addition of sucrose decreased the initial bubble size, corresponding to

higher foam stability and lower air phase fraction. EWP foams were

composed of smaller bubbles and lower air phase fractions than WPI foams.

Increased sucrose concentration caused a decreased liquid drainage rate due

to a higher continuous phase viscosity and smaller bubble sizes. WPI foams

had faster rates for liquid drainage and bubble coarsening than EWP foams.

Campbell et al., (2011) stated that the effect of annatto color and starter

culture on the flavor and functionality of whey protein concentrate (WPC).

64

Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of

milk, annatto with 3% wt/vol norbixin content) was manufactured using a

mesophilic lactic starter culture or by addition of lactic acid and rennet.

Pasteurized fat-separated whey was then ultrafiltered and spray dried into

WPC. WPC manufactured from whey with starter culture compared with

WPC from rennet-set whey. The WPC with annatto had higher

concentrations of p-xylene, diacetyl, pentanal, and decanal compared with

WPC without annatto. Results suggest that annatto has a no effect on whey

protein flavor, but that the starter culture has a large influence on the

oxidative stability of whey.

Whitson et al., (2011) studied that the effects of holding time of liquid

retentate on flavor of spray-dried whey proteins: Cheddar whey protein

isolate (WPI) and Mozzarella 80% whey protein concentrate (WPC80).

Liquid WPC80 and WPI retentate were manufactured and stored at 3°C.

After 0, 6, 12, 24, and 48 h, the product was spray-dried (2 kg) and the

remaining retentate held until the next time point. Powders were stored at

21°C and evaluated every 4 mo throughout 12 mo of storage. Sensory

results, lipid oxidation products (hexanal, heptanal, octanal) and sulfur

degradation products (dimethyl disulfide, dimethyl trisulfide) increased in

spray-dried products with increased liquid retentate storage time, whereas

diacetyl decreased. Shelf stability was decreased in spray-dried products

from longer retentate storage times.

Ye (2011) revealed that the emulsifying properties of milk protein

concentrates (MPC) and stabilities of emulsions formed with MPCs by

examining emulsions formation, adsorption behaviours of proteins and

emulsion microstructures. Compared with emulsions formed with higher

calcium MPCs at a given protein concentration, emulsions formed with low

calcium MPCs were finer, the total surface protein concentration was lower

and the protein composition on the surface of the emulsion droplets was

altered. In low-calcium-MPC-stabilized emulsions, the stability of the

65

emulsions decreased with an increase in the emulsion size at low protein

concentrations.

Zhu and Damodaran (2012) observed that the extent of partitioning of

annatto between protein, milk fat globule membrane (MFGM), and aqueous

(serum) phases of cheese whey. The MFGM was separated from Cheddar

cheese whey and quantitative analysis of the distribution of annatto in the

fat-free whey protein isolate (WPI), the MFGM fractions, and the serum

phase revealed that annatto was not bound to the protein fraction. The results

showed that a colorless WPI or whey protein concentrate could be produced

from Cheddar cheese whey by separation of MFGM from the whey,

followed by diafiltration.

Ramos et al., (2012) reported that the effectiveness of antimicrobial edible

coatings to wrap cheeses, throughout 60 d of storage, as an alternative to

commercial nonedible coatings. Coatings were prepared using whey protein

isolate, glycerol, guar gum, sunflower oil, and together with several

combinations of antimicrobial compounds—natamycin and lactic acid,

natamycin and chitooligosaccharides (COS), and natamycin, lactic acid, and

COS. Application of coating on cheese decreased water loss (~10%, wt/wt),

hardness, and color change; however, salt and fat contents were not

significantly affected. The antimicrobial edible coating containing

natamycin and lactic acid was the best in sensory terms.

Hussain et al., (2012) reported that the 5% (wt/vol) whey protein isolate

(WPI) dispersion (pH 6.5) with different concentrations of NaCl was

submitted to dynamic heat treatment. The gelation temperature was also

influenced by ionic strength and an increase in denaturation temperature and

thermal stability was also observed by using differential scanning

calorimetry. Results demonstrated the strong interaction between ionic

strength and dynamic thermal treatment on protein functional properties and

their careful adjustment could enable the food industry to effectively use

WPI as a gelling agent.

66

Jervis et al., (2012) stated that compare two commercially approved

bleaching agents, benzoyl peroxide (BP) and hydrogen peroxide (HP), and

their effects on the flavor and functionality of WPC 80%. Colored and

uncolored liquid wheys were bleached with BP or HP, and then ultrafiltered,

diafiltered, and spray-dried; WPC80 from unbleached coloured and

uncoloured Cheddar whey were manufactured as controls. The HP-bleached

WPC80 were higher in lipid oxidation compounds and had higher fatty and

cardboard flavors compared with the other unbleached and BP-bleached

WPC80. The WPC80 bleached with BP had lower norbixin concentrations

compared with WPC80 bleached with HP. Whey bleached with HP

treatments had more soluble protein after 10 min of heating at 90°C at pH

4.6 and pH 7 than the no-bleach and BP treatments, regardless of additional

colour. Overall, HP bleaching caused more lipid oxidation products and

subsequent off-flavors compared with BP bleaching. However, heat stability

of WPC80 was enhanced by HP bleaching compared with control or BP-

bleached WPC80.

Dissanayake et al., (2012) reported that the effect of microparticulation at

low pH on the functionality of heat-denatured whey proteins (WP). Spray-

dried microparticulated WP (MWP) powders were produced from 7%

(wt/wt) WP dispersions at pH 3, acidified with citric or lactic acid and

microfluidized with or without heat denaturation. Microparticulated WP

exhibited enhanced heat stability as indicated by thermograms, along with

better emulsifying properties compared with those produced at neutral pH.

However, MWP powders created weaker heat-induced gels at neutral pH

compared with controls. However, they created comparatively strong cold

acid-set gels. At low pH a combination of heat and high hydrodynamic

pressure produced WP micro-aggregates with improved colloidal stability

that affects other functionalities.

67

2.3 Microbiological quality of idli

The microbiological studies were carried out to assess the standard of

cleanliness during production, packaging, transportation and storage and for

evaluating the shelf life of idli. The market samples of idli analysed at

Department of Food Technology, Tirupathi, Andhra Pradesh (Suneetha et

al., 2011) were heavily contaminated with variety of organisms.

Mukherjee et al., (1995) stated that the micro-organisms responsible for the

characteristic changes in the batter were isolated and identified. Although

there is a sequential change in the bacterial flora, the predominant micro-

organisms responsible for souring as well as for gas production, was found

to be Leuconostoc mesenteroides. In the later stages of fermentation, growth

of Streptococcus faecalis and still later of Pediococcs cerevisiae becomes

significant. The fermentation of idli demonstrates a leavening action caused

by the activity of the hetrofermentative lactic acid bacterium, L.

mesenteroides. As far as is known, this is the first record of a leavening

action produced exclusively by the activity of a lactic acid bacterium.

Kaw (1995) observed that batter volume showed an increase up to 24 hr of

fermentation period and non- amylose variety IR29 showed the maximum

expansion. Microbial counts in idli batter which were low initially increased

steeply with the advancement in fermentation time. Maximum counts of

Lctobacilli and coliforms were obtained after 12 h and those of yeasts and

molds after 24 h fermentation. Sensory evaluation revealed that idlis made

from IR29, the waxy variety of rice were unacceptable and significantly

different from the amylase type of rice varieties. While no significant

differences between the rice varieties were observed for aroma, density, off-

flavour and fermented flavour, the differences in cohesiveness, coarseness

and general acceptability were significant. Highly positive correlations were

revealed between pH and reducing sugars, batter volume and total acidity,

total acidity and non- protein nitrogen and highly negative correlations of

pH with total acidity and non protein nitrogen. Yeasts and molds had highly

68

significant and positive association with batter volume, non- protein nitrogen

and total acidity. Lactobacilli revealed significantly negative correlations

with reducing sugars and pH.

Soni et al., (1996) reported that bacteria alone or in combination with yeasts

were found to be responsible for the fermentation of dosa. Leuconostoc

mesenteroides, Streptococcus faecalis, Lactobacillus fermentum and

Bacillus amyloliquefaciens were the predominant bacteria responsible for

souring and leavening of dosa batter. Yeasts whenever present, belonged

mainly to Saccharomyces cerevisiae, Debaryomyces hansenii and

Trichosporon beigelli. They produced flavour, enzymes and helped in the

saccharification of starch. Both bacteria and yeasts were contributed by the

ingredients Oryza sativa and Phaseolus mungo. The prevalence of bacteria

and yeasts was affected by seasonal variations but bacteria always

dominated the overall microbial load.

Soni and Sandhu (1998) stated that idli prepared from a fermented batter

containing both rice (Oryza sativa) and black gram (Phaseolus mungo).

Analysis of 35 different samples of idli batter, both commercial and

laboratory prepared, revealed the occurrence of bacteria belonging to six

species in the range of 106 – 109 / g, 68% of the samples were also positive

for yeasts, ranging up to 106 / g and yielding six types. Laboratory studies on

ways to improve the nutritional and organoleptic properties of idli identified

changes in fermentation conditions that led to appreciable increases in the

microbial cell counts, total acids volume, soluble solids, reducing sugars,

non- protein nitrogen, free amino acids, amylases, proteinases and water

soluble vitamins, including B2, B2, B12, and C. Novel idli batters, prepared

by replacing the conventional black gram with other legumes, revealed

comparable changes but with differences in the levels of some biochemical

constituents. Incubation of idli batters at 28 ˚C with initial pH around 4.5

and supplemented with sucrose (1- 2%) proved to be favourable for

improving the nutritional constituents and organoleptic characteristics.

69

Jama and Varadaraj (1999) stated that the inherent viable bacterial

populations of mesophilic aerobes and lactics in idli batter increased in their

numbers with time at 35 ˚C reaching numbers in the range of 13 to 15 log10

CFU g -1. Simultaneously, the pH level decreased from 6.2 to 4.4. Strains of

Bacillus cereus F 4810, Escherichia coli D 21 and Staphylococcus aureus

FRI 722 (foodborne pathogens) introduced into the idli batter at an initial

level of 4.3 log10 CFU g -1 was able to survive and grow well in an initial

period of 6 h. However, the strain of S. aureus showed a constant increase in

its numbers reaching 9.3 log10 CFU g -1 in 12 h. The addition of plantaricin

LP84, a bacteriocin produced by Lactobacillus plantarum NCIM 2084 to idli

batter at 1% (v/w) level was able to retard the growth of the inoculated

cultures during fermentation.

Roy et al., (2007) examined six different type samples of legume-based

popular fermented foods viz. amriti, dhokla, dosa, idli, papad and wadi

purchased from retail outlets in West Bengal. These market samples carried

higher micro-organisms counts than laboratory sample. In this study the

result indicated that these foods were manufactured using poor quality

starting materials, processed under unhygienic conditions or/ and

temperature abused during transportation and storage.

Lyer et al., (2011) observed that idli batter was used as a source for isolation

of lactic acid bacteria (LAB). A total of 15 LAB strains were isolated on the

basis of their gram nature and catalase activity. Of these, one lactobacilli

strain and one lactococci strain which showed antimicrobial activity were

identified using biochemical characterization, sugar utilization and

molecular sequencing. The microbes, labelled as IB-1 (Lactobacillus

plantarum) and IB-2 (Lactococcus lactis) were tested for their in vitro

tolerance to bile salts, resistance to low pH values and acidifying activity.

Both the strains showed good viability (IB-58.11%, IB-60.84%) when

exposed to high bile salt concentration (2%) and acidic pH of ≤ pH 3.0 (IB1-

88.13%, IB2-89.85%).

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Hussain and Uddin (2011) studied that the total microbial load in

germinated wheat and mungbean seed flour for preparing of weaning foods.

The plates were incubated at (35 ± 1) °C for 48h, and the numbers of

bacterial colonies and of yeast and mould colonies are counted. The results

show that the total viable counts were ranged from 1.7 x 102 cfu/g -9.3 x

102 for wheat seed flour cfu/g, , and1.7 x 102 - 6.0 x 102 cfu/g for the

mungbean seed flour. It was concluded that flour was prepared at 33°C for

60 hour was safe in sense of microbiological purity and can be used for

weaning food preparation.

2.4 Health benefits of whey protein concentrate (WPC)

Renner (1992) suggested that a high quality protein with significant

quantities of essential amino acids such as lysine, methionine and

tryptophan, WPC can be used for supplementing plant proteins in

developing high protein foods and other special dietetic foods.

Pihlanto and Korhonen (2003) suggested that significant amounts of

essential amino acids, whey proteins possess excellent biological active

peptide sequences that promote good health.

Vermeirssen et al., (2003) stated that in vitro gastrointestinal digestion was

the predominant factor controlling the formation of angiotensin-I-converting

enzyme (ACE) inhibitory activity, hence indicating its importance in the

bioavailability of ACE inhibitory peptides.

Xu (2009) evaluated the effects of whey protein on osteoblasts. The whey

protein was added to the culture medium at concentrations of 0.02 and

0.1 mg/mL. In vitro, whey protein stimulated the proliferation and

differentiation of osteoblasts cultured in different concentrations of whey

protein. The levels of osteocalcin and insulin-like growth factor-I in the

culture medium also increased. Real-time reverse transcription-PCR results

showed that the mRNA expression of osteoprotegerin (OPG) and receptor

activator of nuclear factor-κ B ligand (RANKL) increased in the cells in a

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dose-dependent manner, and when the results were expressed as

OPG/RANKL ratio, a significant increase could be seen in the 0.1 mg/mL

whey protein group. Results showed that the active component in the whey

protein plays an important role in bone formation and a potential therapeutic

role in osteoporosis by activating osteoblasts.

2.5 Uses of whey protein concentrate (WPC)

Whey protein concentrate (WPC) by virtue of their high protein content and

functionality are increasingly used in a great number of food products, for

replacing traditional additives like milk powder, egg albumin based foods

and is now finding applications in infant and dietetic foods, ice cream and

frozen beverages, meat products, pasta and Indian traditional products like

khoa.

Vitti (1991) reported that the composition and functional properties of whey

protein concentrate (WPC) are such that it can be used to replace egg partly

in bread and cakes.

Thompson and Reniers (1992) were determined the effects on quality and

acceptance on succinylated whey concentrate was substituted for sodium

caseinate in the formulation of coffee whitener and for 20% of the egg yolk

in salad dressing. Succinylated whey concentrate has emulsification

properties suitable to these products. A coffee whitener was obtained with

higher viscosity, greater stability, lower whitening power, and greater

acceptability than the control sample made with sodium caseinate. Similarly,

a more stable salad dressing with a higher viscosity and no significant

difference in acceptability was produced.

Thompson et al., (1993) revealed that the effect on quality of substituting

succinylated cheese whey protein concentrate for nonfat dry milk in ice

cream and instant pudding. The use of succinylated whey protein

concentrate in ice cream increased viscosity and resistance to melting and

reduced freezing time and overrun. In the absence of stabilizers and

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emulsifiers, trends were the same and ice cream remained stable.

Incorporating succinylated whey protein concentrate into instant pudding

increased hydration and retarded rate of firming during storage.

Modler et al., (1993) studied that the eighteen skim milk yogurts, prepared

from all combinations of six protein types (three casein- and three whey-

based products) and three protein concentrations (.05, 1.0, and 1.5% added

protein). Addition of increasing amounts of protein increased gel firmness

and decreased syneresis. The casein-based proteins, particularly sodium

caseinate, produced yogurts that were generally inferior to gelatin for

smoothness and appearance. WPC at 1.0 and 1.5% of protein addition,

produced yogurts generally superior to casein-based products for both

appearance and smoothness.

Rajorhia et al., (1990) incorporated 10 and 18% WPC (27.14% TS) solids

in buffalo milk for the manufacture of khoa. Greater amount of WPC

produced bigger grains in khoa, which is a desirable property for preparing

kalakand.

Mccane and Widdowson (1991) suggested that whey protein concentrate

(WPC) contains 70- 75% protein and is being used to enrich soft drinks and

other beverages, infant foods, ice cream and yoghurt.

Gruetzmacher and Bradley (1991) examined to use as a replacement for

sodium caseinate in spray-dried coffee whiteners. Fifty-two spray-dried

coffee whitener formulations were prepared using demineralized acid whey

protein concentrate and compared with standard commercial whitener

formulations. Optimal stability and functionality were obtained at 1.5% acid

whey protein. A dipotassium phosphate to protein ratio of 1.0 yielded good

stability. Demineralized acid whey protein was an acceptable replacement

for sodium caseinate in spray-dried coffee whiteners and can replace sodium

caseinate at a 1:2 ratio.

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Hofi et al., (1993) suggested that whey protein concentrate (WPC) have

been successfully used in frozen desserts for years because they bind water

and form weak networks that mimic the structure of full fat ice cream.

Patel et al., (1993) studied that the addition of 5% WPC solids to cow milk

improved the flavour, body and texture and colour of khoa prepared. WPC

incorporated cow milk khoa compared well with the traditional buffalo milk

khoa.

Brandsma and Rizvi (2001) studied that Low-moisture, part-skim (LMPS)

Mozzarella cheeses were made from highly concentrated skim milk

microfiltration (MF) retentate and butteroil. Differing combinations of

rennet concentration, coagulation temperature and post-coagulation curd

cutting time were used, with comparisons made of the rheological and

functional characteristics of cheeses during ageing. Rennet concentration

was the only experimental factor to significantly affect MFM rheological

and functional development.

Dewani and Jayaprakasha (2002) studied that whey solids (in the form of

WPC) can be effectively used in the preparation of khoa and khoa based

sweets such as peda and gulabjamun by properly inducing selective heat

treatment, optimizing levels of pre-concentration and blending whey protein

concentrate (WPC) and milk in appropriate proportions. Further peda and

gulabjamun were prepared by the standard methods. With the increase in

levels of WPC in the admixture, yield, acidity, hydroxyl methyl furfural

(HMF) and penetration values increased correspondingly whereas fat,

protein, lactose, moisture and ash content decreased. Sensory attributes of

peda increased upto 40% and 50% WPC levels. Similarly gulabjamun with

30% WPC level was found to be on compare with the control.

Kumar and Sangwan (2002) observed that whey protein concentrate (WPC

15% w/v) were hydrolysed with trypsin, α- chymotrypsin and papain

enzymes separately and influence of enzymatic hydrolysis on emulsifying

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properties. The emulsifying activity index (EAI) and emulsifying capacity

(EC) increased up to certain level of hydrolysis and decreased on future

hydrolysis at pH 7.0. In case of trypsin and papin, the maximum ESI and EC

was found after 2 h of hydrolysis while it was after 1 h in α- chymotrypsin

hydrolysed sample.

Mleko and Gustaw (2002) studied that dairy desserts with starch, k-

carrageenan and total milk proteins were prepared and their rheological

behaviour was compared with desserts obtained by substitution of milk

protein by whey proteins. As whey protein desserts were held at 90 ˚C for 5

min, an increase in apparent viscosity was observed. In comparison to whey

proteins, total milk proteins produced desserts with lower apparent viscosity.

Holding at 90 ˚C did not increase of apparent viscosity and there was

smaller increase in apparent viscosity as the samples were cooled.

Dewani and Jayaprakasha (2002) reported that replacement of MSNF up

to 30% with WPC resulted in increased overall acceptability scores of

gulabjamun. A mechanized semi-continuous system has been developed for

the manufacture of gulabjamun from khoa at commercial scale.

Aryana et al., (2002) observed that the functionality of various

combinations of egg white (EW), whey protein concentrate (WPC) and

bovine serum albumin (BSA) and compare the microstructures of their gels.

The combination of WPC with EW or BSA resulted in a synergistic effect

for thermostability (TS) and foam stability (FS) and an additive effect for oil

holding capacity (OHC), water holding capacity (WHC), foam density (FD),

emulsifying activity index (EAI), gel stress and strain. On the contrary, an

antagonistic effect was observed for FA. If a multifunctional combination

was to be picked, it would be a 1 : 1 ratio of WPC and BSA as it had the

highest number of attributes with synergistic effects.

Singh et al., (2003) studied that egg was replaced with WPC at levels of 0 to

100%. Replacement of egg with WPC up to 50% did not result in significant

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differences in physical properties of cake, thereafter differences were

significant except for the cells of the cake. For the optimization of WPC,

levels of 4, 6, 8, 10 g were used. With the increase in WPC from 4 to 10 g,

volume and weight of cakes increased significantly. But no changes in other

physical characteristic in cake. The cake made with WPC 6 g/ 100 g flour

was observed to have maximum score for overall acceptability.

Tripathy et al., (2003) studied that ragi (Finger millet) based products were

formulated utilizing whey protein concentrate (WPC) to enhance their

nutritional profile. WPC was used at 10 to 40% to replace ragi flour and the

products such as ragi malt and ragi dosa were prepared. The results revealed

that ragi malt and ragi dosa were best accepted at 30% level of WPC

supplement and had a protein content of 14.8 and 14.2% for ragi malt and

ragi dosa then compared to control.

Pawar et al., (2003) developed to enrich the nutritional quality of beverage

by adding whey protein concentrate (WPC) at 2, 3 and 4% levels. The

sensorial quality score of all the parameters reported comparatively higher in

case of beverage containing 3% WPC over that of other beverages. The

storage study of formulated WPC beverage reported 40 and 15 days as

active storage period at refrigerated (4 + 1°C) and room temperature (25 +

1°C) storage conditions without significant changes in nutritional and

sensory quality parameters. The unit cost of production of beverage

(300mL) worth of Rs. 7/- was observed to be comparatively higher because

of small scale production.

Gunasekaran (2003) observed that hydrogels made from WPC are pH-

sensitive with a minimum swelling ratio near the isoelectric point (pI) of

WPs (~5.1). These hydrogels are suitable for controlled drug release. The

swelling and release behavior of the WPC hydrogels can be controlled by

coating them with layers of calcium alginate. Beta-lactoglobulin (BLG), the

primary WP, can be used to prepare nanoparticles of about 60 nm average

diameter using desolvation method. The stability of the particles was

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investigated by degradation experiments at neutral and acidic conditions

with and without proteolytic enzymes.

Conforti and Lupano (2004) reported that the effects of honey, lemon

juice, and two different whey protein concentrates (WPC) on the structural

and functional properties of biscuits, were analyzed. The presence of WPC

with a high protein content produced a decrease in the firmness and

consistency and an increase in the cohesiveness of dough. Honey increased

the adhesiveness of dough, mainly in samples with the WPC of lower

protein content and lemon juice, and tended to decrease dough relaxation

time. The fracture stress of biscuits decreased with the incorporation of

WPC. Also, honey increased the red undertone and yellowness of biscuits

and decreased their lightness; however, the addition of lemon juice reduced

these effects.

Singh and Nath (2004) reported that protein enrichment of bael fruit

beverages was prepared by using partially denatured WPC complexed with

acidic polysaccharides i.e carboxymethyl cellulose (CMC) and pectin. A

beverages base with 25% bael fruit pulp, 16‘ brix and pH 3.9 was found

optimum and was fortified with 1.75, 2.75 and 3.75% level of WPC

polysaccharide complex. The products with 1.75% protein level of pectin

WPC complex and 1.75 and 2.75% protein level of CMC - WPC complex

were rated superior then other combinations. The beverages with 1.75%

whey protein level of CMC WPC complex scored maximum for all sensory

attributes.

Rai and Jayaprakasha (2004) reported that the spray dried sweet cream

butter milk (SCBM) was blended with spray dried WPC at various

properties along with other ingredients. Gulabjamun were prepared from the

mix developed by the various admixtures and compared with the control

samples. The results revealed that SCBM which had undergone a heat

treatment of 95 ˚C/ 20 min was highly suitable and instant gulabjamun mix

could be successfully prepared from an admixture of spray dried WPC and

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SCBM at 30:70 level without jeopardizing any of the functional and sensory

characteristics of the resulted gulabjamun.

Keskinler et al., (2004) observed that soluble whey proteins (WPs),

adsorbed on yeast cells, were recovered by a crossflow microfiltration (MF)

technique using a cellulose nitrate membrane with a pore size of 0.45 μm.

This technique not only provides for the recovery of protein but also may

give rise to the direct use of yeast cells, which are rich in protein, in the

baking industry. Whey protein absorbed by yeast cells can be used to

produce nutritionally rich products in areas where yeasts have been already

used.

Dewani and Jayaprakasha (2004) reported that replacement of milk solids-

not-fat (MSNF) up to 40% with WPC improved all the sensory attributes of

plain peda. They also applied RO process for pre-concentration of milk as an

intermediate step in the production of plain peda. It was concluded that such

product was nutritionally better than the conventionally made peda.

Salve et al., (2005) prepare low fat paneer from buffalo milk added with

Whey Protein Concentrate (WPC) at different levels. Paneer was prepared

from buffalo milk standardized to 6% fat and at lower fat levels viz, 5%, 4%

& 3%. Results revealed that the quality attributes of paneer differed

significantly with lowering of fat from 6% to 3% except appearance. The

sensory scores for body & texture and overall acceptability of paneer made

from buffalo milk with 4% fat and control paneer were comparable. Paneer

made from milk with 6% fat recorded highest yield as well as recovery of fat

and total solids. The product with 5% fat had almost similar recovery of

solids but with slightly low in yield. Incorporation of WPC at different

levels significantly influenced the sensory quality of low fat paneer. WPC @

2% was found most effective as compared to other levels for improving the

quality attributes of low fat paneer.

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Steinkraus (2005) suggested that soybean, green gram and Bengal gram can

bande substituted for black gram. Wheat or maize can be substituted for the

rice to yield Indian dhokla.

Alvarez et al., (2005) studied that two milk protein concentrates (MPC, 56

and 85%) as substitutes for 20 and 50% of the protein content in ice cream

mix. The basic mix formula had 12% fat, 11% non fat milk solids, 15%

sweetener and 0.3% stabilizer/emulsifier blend. Protein levels remained

constant, and total solids were compensated for in MPC mixes by the

addition of polydextrose. MPC formulations had higher mix viscosity, larger

amount of fat destabilization, narrower ice melting curves, and greater shape

retention compared with the control. MPC did not offer significant

modifications of ice cream physical properties on a constant protein basis

when substituted for up to 50% of the protein supplied by non fat dry milk.

Milk protein concentrates may offer ice cream manufacturers an alternative

source of milk solids non-fat, especially in mixes reduced in lactose or fat,

where higher milk solids non fat are needed to compensate other losses of

total solids.

Herrero and Requena (2006) stated that yoghurt was manufactured from

goat's milk and supplemented with 30 g L−1 of whey protein concentrate

(WPC). The addition of WPC to goat's milk enhanced the textural

characteristics of yoghurt. These advantageous attributes included increased

firmness, hardness and adhesiveness. These attributes were quantitatively

similar to those obtained from yoghurt made from cow's milk. In addition,

the textural properties were maintained constant throughout the shelf-life of

the product.

Prabha (2006) developed a technology for the production of dietetic burfi

using alternative ingredients, viz. whey protein concentrate (WPC), sorbitol,

maltodextrin and sucralose and optimized the ingredients using Response

Surface Methodology (RSM). The product was found to be highly

acceptable by the consumers.

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Gupta (2006) suggested that dairy ingredients are preferred ingredients due

to their functional suparmacy and good flavour, colour and nutritional

profile.

Perez (2006) studied that the effect of antioxidant and content alone or in

combination with edible coatings for fresh cut apples. Edible composite

coatings were prepared from WPC and beeswax (BW). Ascorbic acid at 0.5

and 1% content, cysteine at 0.1, 0.3 and 0.5% content and 4- hexylresorcinol

at 0.005 and 0.02% were incorporated in the formulations as antioxidants.

Result revealed that incorporation of the antioxidant to the coating reduced

browning compared to the use of the antioxidant alone.

Sodini et al., (2006) reported that two conditions of whey processing, pH

and heat treatment, affect the physical properties of stirred yoghurts fortified

to 45g protein kg -1 with whey protein concentrates (WPC). Cheddar whey

was heated at pH 6.4 or pH 5.8 at 72˚C for 15 s, eventually heated further at

82 or 88˚C for 78 s, ultra filtered and spray dried. Resulting WPC contained

38% protein, the denaturation level of the whey protein was 10 – 53%.

There were significant differences in physical properties of WPC fortified

yoghurts, water holding capacity ranged from 33% to 46% and elastic

modules ranged from 63 to 145 Pa depending on whey processing. WPC

with low denaturation level produced yoghurts with high elastic modules

and water holding capacity. Minimizing the heat treatment during whey

processing maximized the functional properties of WPC to be used in

yoghurt.

Patocka et al., (2006) examined that the addition of WPI up to 10%

decreased the apparent viscosity of a commercial yoghurt drink. The original

viscosity was restored at addition of 15% WPI. In buttermilk, minimum

viscosity was observed after addition of 6% and original value was restored

at 12%. Addition of the WPI was investigated either before fermentation

(BF) or after fermentation (AF) of the heated unfortified yoghurt milk.

Addition of WPI to commercial stirred yoghurt decreased G‘, while G‖ was

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affected only marginally. BF yoghurts behaved similarly to the commercial

yoghurt while AF yoghurt exhibited complete breakdown of the system.

Pinto et al., (2007) studied that low fat ice cream (LFIC) was prepared

containing WPC as a fat replacer. Addition of WPC in the LFIC is increased

in protein content and did not show any significant influence on pH and

acidity. It increased viscosity and whipping ability of the mixes and

increased in overrun, melting resistance and decreased firmness of hardened

product. The ice cream containing 1.25% WPC recorded better sensory

attributes compared to all the other samples. Addition of WPC above 1.25%

level resulted in a foamy, frothy, fluffy, slightly slimy/ sticky product.

Narender et al., (2007) studied that biscuits prepared from the blends

containing 10, 20 and 30% of whey protein concentrate (WPC). The protein

and ash contents of WPC containing biscuits were significantly higher then

the control. Blending of refined wheat flour with WPC up to 30% did not

have any adverse effect on the sensory quality of protein enriched biscuits.

The cutting and compression strengths of the 30% WPC incorporated

biscuits were significantly higher then the control. These protein enriched

biscuits can be stored for 60 days at ambient temperature (30- 35 ˚C).

Pinto et al., (2007) studied that blend of cheddar cheese comprising of 66%

of 2- 3 months old and 34% of 4- 5 months old cheddar cheese were used to

prepare processed cheese spread. Cheese solids were partially replaced by

WPC and solids at different levels viz. 1.5, 3.0 and 4.5 percent.

Incorporation of WPC resulted in a significant improvement in body and

texture score of spread particularly at 3.0 and 4.5% level. However, addition

of WPC at higher levels imparted a milder flavour to the product. Processed

cheese spread with good melt ability, desired characteristics with improved

spread ability can be prepared by using dried WPC at levels up to 4.5% of

cheese solids.

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Khillari et al., (2007) studied that incorporation of WPC replacing 20% fat

in ice cream mix (ICM) does not reduce in overrun and melting quality of

ice cream. Though, viscosity of ICM with increase in fat substitution by

WPC, the protein content of ice cream increased substantially but the

variations in carbohydrate and ash contents were negligible. Addition of

WPC substituting 20 to 40% fat is recommended for getting acceptable

quality low fat ice cream.

Prabhasankar et al., (2007) reported that effect of WPC (5%, 7.5%, 10%)

and additives on the quality of vermicelli made from Indian durum wheat.

Result revealed that with increase in WPC from 0% to 10% cooked

vermicelli weight increased from 82.5 to 88 g/25g cooking loss increased

from 6.0 to 8.4%, lightness increased (47.42 – 52.9) and yellowness

decreased (7.0 - 3.80) and shear force decreased (66 – 45g). Sensory

evaluation of vermicelli showed that addition of above 5% WPC resulted in

whitish colour vermicelli with mashy strand quality and sticky mouthfeel.

The effect of additives viz. ascorbic acid (0.01% and 0.015%), gluten (1.5%

and 3.0%) and glycerol monostearate (GSM) (0.25% and 0.5%) individually

as well as in combination on the quality of vermicelli with 5% WPC

indicated that combination of 0.01% ascorbic acid, 3% gluten and 0.5%

GSM resulted in vermicelli having lower cooking loss, creamy yellow

colour, firm, discrete strands and non-sticky mouthfeel.

Devaraju et al., (2008) reported that pasta were prepared by using finger

millet composite flour, the protein source are deffed soy flour and WPC.

Fortification of the protein content from 13.12 percent in control to 17.78

percent in pasta made from finger millet with composite flour sensory

evaluation scores indicated non significant difference among the control and

experimental products for texture.

Aziznia et al., (2008) studied that the effect of whey protein concentrate

(WPC) and gum tragacanth (GT) as fat replacers on the chemical, physical,

and microstructural properties of non fat yogurt. The WPC (7.5, 15, and

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20 g/L) and GT (0.25, 0.5, 0.75, and 1 g/L) were incorporated into the skim

milk slowly at 40 to 45°C with agitation. The yogurt mixes were pasteurized

at 90°C for 10 min, inoculated with 0.1% starter culture, and incubated at

42°C to pH 4.6, then refrigerated overnight at 5°C. A control non fat yogurt

and control full fat yogurt were prepared as described, but without addition

of WPC and GT. Increasing amount of WPC led to the increase in total

solids, total protein, acidity, and ash content, whereas GT did not affect

chemical parameters. No significant difference was observed for firmness

and syneresis of yogurt fortified with GT up to 0.5 g/L compared with

control non fat yogurt. Increasing the amount of gum above 0.5 g/L

produced softer gels with a greater tendency for syneresis than the ones

prepared without it. Addition of GT led to the coarser and more open

structure compared with control yogurt.

Lee and Vickers (2008) revealed that the acidity of whey protein solutions

was responsible for their astringency. Panelists rated acidic whey protein

and acid-only solutions for astringency and sourness. Acidic whey protein

solutions contained 6% or 1% whey protein isolate and phosphoric acid at a

pH of 3.4. Acid-only solutions were formulated to match the whey protein

solutions for either total acidity or for pH. The acid-only solutions matched

for total acidity were more astringent than the whey-containing solutions,

while those matched for pH were significantly less astringent. Sourness was

reduced by the whey proteins, most likely because of the decreased

concentration of free hydrogen ions. The astringency of acidic whey protein

solutions appears to be caused by their high acidity and not directly by the

whey proteins.

Beecher et al., (2008) studied that there are 2 types of whey protein-

containing beverages: those at neutral pH and those at low pH. Astringency

is very pronounced at low pH. Astringency is thought to be caused by

compounds in foods that bind with and precipitate salivary proteins. The pH

of the whey protein solution had a major effect on astringency. A pH 6.8

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whey protein beverage had a maximum astringency intensity of 1.2 (15-

point scale), whereas that of a pH 3.4 beverage was 8.8 (15-point scale).

Astringency decreased between pH 3.4 and 2.6, coinciding with an increase

in sourness. Decreases in astringency corresponded to decreases in protein

aggregation as observed by turbidity.

Lim et al., (2008) studied that three batches of low fat ice cream mix were

produced to contain WSU-WPC without high hydrostatic pressure (HHP),

WSU-WPC with HHP (300 MPa for 15 min), and WPC 35 without HHP.

All low fat ice cream mixes contained 10% WSU-WPC or WPC 35.

Overrun and foam stability of ice cream mixes were determined after

whipping for 15 min. The ice cream mix containing HHP-treated WSU-

WPC exhibited the greatest overrun and foam stability. Ice cream containing

HHP-treated WSU-WPC exhibited significantly greater hardness than ice

cream produced with untreated WSU-WPC or WPC 35. Improvements of

overrun and foam stability were observed when HHP-treated whey protein

was used at a concentration as low as 10% (wt/wt) in ice cream mix.

Seethalakshmi et al., (2010) stated that whey protein concentrate (WPC)

can be used as a replacer for egg in biscuit preparation. The addition of

whey protein concentrate (WPC) enhanced the nutritive value without

altering the sensory and physico chemical properties of biscuits. These

biscuits are more suitable for those do not consume egg and also for those

who need protein rich diet.

Mallasy et al., (2010) stated that supplementing pearl millet with whey

protein, samples and control were fermented in the presence of starter for

14 h. The pH, crude protein, in vitro protein digestibility (IVPD) and protein

fractions of the fermented and supplemented pearl millet were determined at

2- h intervals. Supplementation of whey protein resulted in significant

increase in protein content compared to the control. Fermentation was found

to cause a highly significant improvement in IVPD for AC, AW1 and AW2.

This would indicate an improvement in the nutritional quality of pearl millet.

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Sensory evaluation revealed higher acceptability for whey protein

supplemented formulas compared to control.

Baskaran et al., (2011) studied that the physical properties of noodles

enriched with skim milk powder, whey protein concentrate (WPC) and a

combination of skim milk powder and WPC at 5, 7.5 and 10% levels were

studied. Volume increase, weight increase and swelling ratio of the enriched

noodles were reduced at increasing levels of substitution. Total solids loss in

gruel showed increasing trend as the levels of substitution increased. It was

also found that, the loss of total solids was higher in noodles supplemented

with SMP compared to WPC and a combination of SMP and WPC.

Pintro et al., (2011) observed that whey protein ingredients were modified

to produce yoghurts with acceptable texture properties. Alteration of the

ratio of α-lactalbumin to β-lactoglobulin, heat denaturation and hydrolysis

treatments were applied to whey protein to improve their behaviour in

yoghurt formulations. Ingredients with increased proportion of α-

lactalbumin or made from partially hydrolyzed protein produced yoghurts

that closely matched the characteristics of control yoghurt. The effect of

whey protein ingredients on yoghurt rheological properties and dispersibility

was related to the concentrations of reactive thiol groups that determined the

extent of cross linking during acidification. During storage, yoghurt firmness

and viscosity increased and syneresis decreased. Yoghurt microstructure was

altered by whey protein ingredients, which significantly reduced void spaces

and increased gel matrix compactness.

Akalin et al., (2012) studied that the influence of milk protein-based

ingredients on the textural characteristics, sensory properties, and

microstructure of probiotic yogurt during a refrigerated storage period of 28

d. Milk was fortified with 2% (wt/vol) skim milk powder as control, 2%

(wt/vol) sodium calcium caseinate (SCaCN), 2% (wt/vol) whey protein

concentrate (WPC) or a blend of 1% (wt/vol) SCaCNand 1% (wt/vol) WPC.

The fortification with SCaCN improved the firmness, adhesiveness and

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higher values of viscosity in probiotic yogurts during storage. However,

WPC enhanced water-holding capacity more than the caseinate. Addition of

SCaCN resulted in a coarse, smooth, and more compact protein network;

however, WPC gave finer and bunched structures in the scanning electron

microscopy micrographs.

2.6 Nutritional aspects of whey protein concentrate (WPC)

Sandrou and Arvanitoyannis (2000) stated that low fat/ calorie products

were originally developed for diabetics and people with specific health

problems and they were considerably expensive. Now a days consumers

demand for low fat/ low calorie products have significantly risen in an

attempt to limit health problems to lose or stabilize their weight and to work

within the frame of healthier diets. The food industry has been confronted

with a new challenge in order to satisfy consumers by the development of

low fat/ calorie products with acceptable sensory characteristics and

competitive prices by preferably employing the conventional processing

equipment and in agreement with current strict legislations.

Tomar and Prasad (2002) reported that the dairy products as carries of

milk proteins and lactic acid bacteria are equipped with great potential in

prevention and cure of atherosclerosis and hypercholesterolemia.

Baker (2002) stated that there were some reductions in the incidence of

heart disease by eating low fat diets. He believed that one should increase

consumption of fruits and vegetables, olive oil, low fat dairy products and

fish oil to remain healthy and free from heart disease.

Kapoor (2007) observed that the fat intake in our diet occurs from two

sources, visible fat and invisible fat. It is easy to control the quality of visible

fat ingested. Most vegetarian foods contain intrinsically, a very low quantity

of fat except the whole milk and its products (nuts and seeds). It is easy to

separate out the milk fat and hence control the overall amount of fat eaten in

a vegetarian diet. For a vegetarian, the only source of animal fat is milk

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products. By using low fat milk and its products such as curd, cottage cheese

or paneer made from low fat milk, the vegetarian can minimize the amount

of animal fat ingested.

Madureira et al., (2010) suggested that processing of whey proteins yields

several bioactive peptides that can trigger physiological effects in the human

body: on the nervous system via their opiate and ileum-contracting

activities; on the cardiovascular system via their antithrombotic and

antihypertensive activities; on the immune system via their antimicrobial

and antiviral activities; and on the nutrition system via their digestibility and

hypocholesterolemic effects. The specific physiological effects, as well the

mechanisms by which they are achieved and the stabilities of the peptides

obtained from various whey fractions during their gastrointestinal route.

2.7 Cereal based fermented foods

Aliya and Geervani (1990) reported that as in other indigenous fermented

foods, a significant improvement in the biological value and net protein

utilisation of dhokla due to fermentation.

Chavan & Kadam (1990) suggested that the fermentation also leads to a

general improvement in the shelf life, texture, taste and aroma of the final

product. During cereal fermentations several volatile compounds are formed,

which contribute to a complex blend of flavours in the products.

Soni and Sandhu (1990) suggested that fermented foods are produced

world-wide using various manufacturing techniques, raw materials and

microorganisms. However, there are only four main fermentation processes:

alcoholic, lactic acid, acetic acid and alkali fermentation.

Purushothaman et al., (1993) reported that dhokla is also similar to idli

except that Bengal gram dhal is used instead of black gram dhal in its

preparation. A mixture of rice and chickpea flour is also used as the

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substrate for the fermentation. As in idli preparation, the fermented batter is

poured into a greased pie tin and steamed in an open steamer.

Battacharya and Bhat (1997) reported that dosa batter is prepared by

grinding wet rice and black gram separately with water. The two

suspensions are then mixed and allowed to undergo natural fermentation,

usually for 8–20 h. To make a dosa, the fermented suspension is spread in a

thin layer (of 1–5 mm thickness) on a flat heated plate, which is smeared

with a little oil or fat. A sol to gel transformation occurs during the heating

and within a few minutes, a circular, semi-soft to crisp product.

Simango (1997) stated that fermentation is one of the oldest and most

economical methods of producing and preserving food. In addition,

fermentation provides a natural way to destroy undesirable components and

enhance the nutritive value and appearance of the food and reduce the

energy required for cooking and to make a safer product.

Hirahara and Pagini (1998) suggested that the technologies for the

industrial production of fermented products from milk, fruit, vegetables,

cereals and meat are well developed and scientific work is actively carried

out all over the world.

Shukla et al., (2000) observed that biscuit along with bread form major

baked food produced in India accounting for over 30 and 50% of total

bakery products.

Shukla et al., (2000) stated that the per capita consumption of biscuit in

India is reported to be 8 kg per annum as against 15 kg per annum in

developed countries.

Manley (2001) reported that the protein fortified biscuits can be prepared

from composite flours such as wheat flour fortified with soy, cottonseed,

peanut, mustard or corn germ flour and also from vital wheat gluten and

milk powder.

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Samanth (2002) stated that India produces 3 million tonnes of bakery

products, which can be categorised into bread, biscuit and cake.

Puranik (2003) stated that bakery products are increasingly becoming

popular in India as indicated by over 2.5 fold increase in their production

during the last 2 decades.

Tyagi et al., (2007) stated that the protein fortified biscuits contain nutrients

in concentrated form useful for feeding programs at institutes such as day

care centres and schools or for emergency rations.

Riat and Sadana (2009) revealed that idli, dhokla, nan, kulcha, bread,

jalebi, bhatura, bhalla, dosa, gulgule and wadian were prepared in the

laboratory using traditional fermentation techniques. The fermented batter of

idli and dosa contained higher amount of available lysine, cystine and

methionine. After processing, maximum retention of lysine, methionine and

cystine was observed in steamed idli.

Kamble and Ghatge (2011) reported that soybased product such as

soyladoo was formulated in three different combinations with Bengal dhal

flour viz. 60:40, 50:50 and 40:60. Among these combinations was selected

and nutritionally evaluated on the basis of their storage stability. Due to

attractive colour, flavour, taste, appearance and overall acceptability of

soyladoo prepared with composition III i.e. use of soyflour 40g., with the

combination of Bengal gram dhal flour 60g. scored higher by

organoleptically. Chemical compositions like moisture, ash, crude protein,

iron, calcium zinc, carotene and vitamin B complex were found adequate in

this soyladoo.

Sharma et al., (2012) were examined the effect of blending level (0, 5, 10,

15 and 20%) of corn bran, defatted germ and gluten with wheat flour on the

physico-chemical properties, baking properties of bread, muffins and

cookies, and extrusion properties of noodles and extruded snacks prepared

from semolina. Breads from gluten blends had higher loaf volume as

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compared to bran and germ breads. Among corn byproducts, gluten cookies

were rated superior with respect to top grain. Muffins from germ blends and

gluten blends had higher acceptability scores than the bran muffins.

Blending of corn bran, defatted germ and gluten at 5 and 10% with wheat

flour resulted in satisfactory bread, cookie, and muffin score. Quality of

noodles was significantly influenced by addition of corn byproducts and

their levels. Acceptable extruded products (noodles and extruded snacks)

could be produced by blending corn byproducts with semolina up to 10%

level.

Ghosh and Chattopadhyay (2012) studied to use the method of

quantitative descriptive analysis (QDA) to describe the sensory attributes of

the fermented food products prepared with the incorporation of lactic

cultures. Panellists were evaluate to various attributes and acidity of the

fermented food products like cow milk curd and soymilk curd, idli,

sauerkraut and probiotic ice cream. Principal component analysis (PCA)

identified the six significant principal components that accounted for more

than 90% of the variance in the sensory attribute data. Overall product

quality was modelled as a function of principal components using multiple

least squares regression (R2 = 0.8). Further concluded that the utility of QDA

for identifying and measuring the fermented food product attributes that are

important for consumer acceptability.