Antioxidant and functional properties of low molecular weight peptides

A. Gnanamani*, S.T K Raja, T. Thiruselvi, V. Kavitha

Microbiology Division
Central Leather Research Institute
(CSIR, New Delhi)
Adyar, Chennai 20
Tamil Nadu, India

*Author for correspondence

Dr. A. Gnanamani
Microbiology Division
Central Leather Research Institute
Adyar, Chennai 20
Tamil Nadu, India.
Fax No. +91-44-24912150
Phone.No.. +91-44-24404955
Email: gnanamani3@gmail.com

1
Abstract

The present study demonstrates preparation and functional characterization of low

molecular weight peptides (LMwP) obtained from controlled enzymatic hydrolysis of

macromolecular protein, type I collagen. Type I collagen extracted from bovine skin was

hydrolyzed using commercial protease (100:1 w/w) and the low molecular weight

peptides are separated by following the standard procedures and characterized

accordingly. Degree of hydrolysis was calculated with respect to time. LMwP obtained

upon 40% of degree of hydrolysis exhibited molecular weight < 5 Kda as assessed using

Tricine-SDS-PAGE (16%) analysis. With regard to functional characterization, LMwP

exhibit >85% solubility, 85% water absorption capacity, appreciable emulsification

activity and with 49% increase in foam expansion property. The antioxidant profile of

LMwP revealed 99% radical scavenging activity at 95 µg concentration. The results of

the study add value to the utilization of collagen hydrolysates.

Keywords: Type I collagen, collagen hydrolysate, low molecular weight peptides,

Functional foods, antioxidant activity

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 Introduction

In the current scenario of living system, we indented to expose to number of

contaminants, which induces oxidative stress and associated health problems [1].

Fortunately, oxidative stress induced by physical, chemical and environmental agents is

well managed by the innate antioxidant systems. However, whenever there is an

imbalance in the antioxidant level and the stress induced complications, antioxidants are

supplied in the form of food as well as food additives [2]. Nevertheless, it has always

been a debate on when and how these antioxidants are transformed to prooxidant and vice

versa in the human system. Externally provided antioxidant in the form natural materials

is highly preferable, because of the deleterious effects reported in the case of synthetic

antioxidants (BHA, BHT) [3]. Nature has immense antioxidant systems in the form of

fruits, vegetables, greens, meat and including proteins; and intake of these natural sources

alleviates the problems associated with oxidative stress [1].

Hence, most of the food manufacturing industries incorporated the necessary

antioxidants in different forms during manufacturing processes. Several proteins like

alumin, metallothioneins, transferrins and ferritins and protein hydrolysates or food-

derived peptides like opioid, capelin -round scad muscle, skin of sole and squid and sea

cucumber (Stichopus japonicas) were profiled for their antioxidative properties [4-6].

Bioactive peptides of natural proteins are recognized as ‘Functional Foods’ or

‘Nutraceuticals’ [7]. Collagen (natural protein) and the denatured collagen (gelatin) are

extensively utilized in food industries as food additives. However, all the available

reports emphasize and describe the functional properties of gelatin hydrolysates and only

meager reports are on native protein (collagen) hydrolysates. Since, the functional

3
properties of natural protein hydrolysates and the denatured protein hydrolysates differ

widely, it is of high demand for the natural protein hydrolysates. Amongst the various

types of collagen, the most predominate Type I collagen (commonly called as skin

collagen) is always the suitable choice for research. Furthermore, skin collagen is a

mixture of Type I & III, elastin, laminin, etc., and since no reports are available

specifically on native collagen hydrolysate of Type I family, in the present study we

made an attempt to prepare the collagen hydrolysates of Type I collagen and evaluated

the bioactive as well as functional properties. In brief, the experimental study includes

process of extraction of acid soluble Type I collagen (ASC) from the skin followed by

hydrolyzing the extracted collagen by proteases and separating the Low Molecular

Weight peptides (LMwP). Experimenting the antioxidant property through radical

scavenging effect on DPPH free radical, inhibition of linoleic acid autoxidation, reducing

power, and metal chelating assay. In addition, functional properties including solubility,

water absorption, foaming and emulsifying activities were assessed, which makes study

more beneficial in the field of nutrition and medicine.

1. Materials and Methods

2.1 Materials and Reagents

Bovine skin obtained from local slaughtering house was stored at

4degreesCelsius. Chemicals required for antioxidant assay including 1, 1-diphenyl-2-

picrylhydrazyl (DPPH), Linoleic acid, Iron (II) chloride, ferrozine (3-(2-pyridyl)-5,6-

bis(4-phenyl-sulfonic acid)-1,2,4-triazine) and Tricine were obtained from Sigma Aldrich

Chemical co. Protease and all other chemicals used were of analytical grade and

commercially available.

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2.2 Extraction of Type I Acid Soluble Collagen from bovine Skin

Bovine skin obtained from slaughterhouse was subjected to hair and flesh

removal by conventional methods and the resultant collagen matrix was further subjected

to extraction using acetic acid and sodium chloride precipitation method as shown in flow

chart (Fig. 1). The extracted acid soluble collagen (ASC) was lyophilized and stored at

4ºC. The molecular profile of ASC was examined using 8% SDS- PAGE analysis [8].

2.3 Preparation of Low Molecular Weight Peptides (LMwP).

ASC dissolved in 50 mL of 1mmol L-1 tris buffer (pH 9.0) treated with

commercial protease at 100:1 (Substrate: Enzyme) weight ratio respectively. At

scheduled time intervals the reaction was terminated by placing the samples in a water

bath at 90ºC for 5 min with occasional agitation. Samples were then cooled and

centrifuged at 4000rpm for 10 min and the resultant supernatant was lyophilized and

stored at less than 4 ºC.

Degree of enzymatic hydrolysis (DH) of collagen was assessed by measuring the

increasing amount of primary amines released in the samples according to the modified

ninhydrin colorimetric method summarized by Moore and Stein [9]. ASC (4mg)

completely hydrolyzed using 6 mol L-1 HCl for 16 h at 110 ºC served as standard and

compared with the free amino group of LMwP received at different intervals.

2.3 Molecular and Functional characterization of LMwP

2.3.1 Molecular profile of LMwP

It is well known that peptides below 20 kDa cannot be resolved in Glycine- SDS

PAGE. Thus in the present study we followed Tricine-SDS PAGE as summarized by

Schagger [10]. Acrylamide solution and the cross linker concentrations used for the

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preparation of gel were designated as T and C respectively. In the present study,

acrylamide solution consisting of 49.5% T and 6% C were used to prepare 16%

Resolving gel and 4% stacking gel. The length of the separating and stacking gel were of

6 and 2 cm respectively, with a gel thickness of 1 mm. Initially, electrophoresis was

performed at a constant voltage of 60 V for stacking and 120 V for separation. Followed

by electrophoresis, the gels were fixed in 50% methanol, 10% acetic acid for 1hr before

staining in 0.025% Coomassie brilliant blue G in 10% acetic acid for 24 h and destained

using 10% acetic acid.

2.3.2 Amino acid analysis of LMwP

The lyophilized LMwP (10mg) was hydrolyzed with HCl (6 N) at 110 ºC for 24

h. After hydrolysis, the samples were vacuum dried and neutralized and dissolved in

column buffer, dilution ratio of 1:100 (Sample: Buffer). Reversed Phase High

performance liquid chromatography (RP-HPLC) analysis was performed, after

precolumn derivatization with o-phthaldialdehyde (OPA).

2.4 Antioxidant activity of LMwP

2.4.1 Radical scavenging activity of LMwP

LMwP received from the above summarized experiments of 10, 20, 30 & 40%

degree of hydrolysis were subjected to assessment on radical scavenging effect using

DPPH free radical under room temperature in dark condition. LMwP samples were

dissolved in distilled water and the final concentration of 1mg/ml was used for the study.

To 100µl of samples added 900µl of 99.5% ethanol and 125µl of 0.02% of DPPH in

99.5% of ethanol. After the scavenging period of one hour, the decrease in absorbance

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was measured at 517nm [11]. Tests were performed in triplicate and the scavenging effect

was calculated as follows:

Radical Scavenging Activity (RSA) % = (A – B)/B × 100

where, A, B are absorbance of control and sample respectively. The absorbance of water

(500µl), ethanol (500µl) and DPPH (125µl) where taken as control and sample without

DPPH where taken as blank. The standard consists of α – tocopherol (Vitamin E) with

final concentration of 10mg/ml in absolute alcohol. Sample which shows maximum

percentage of RSA was taken in varying concentration to calculate the IC50 values and

compared to the standard. The IC50 represents the content of LMwP responsible for

inhibition of 50% radical activity of DPPH.

2.4.2 Assay of reducing power

The reducing power of LMwP at different degree of hydrolysis (10, 20, 30 &

40%) was determined according to the procedure followed by Oyaiza [12]. To 0.5ml of

sample solution mixed with 2.5 ml of 0.2 mol L-1 phosphate buffer (pH 6.6) and 2.5 ml of

10 g kg-1 potassium ferricyanide. The mixture was incubated at 50º C for 20 min. Then

2.5 ml of 100 g kg-1of trichloroacetic acid was added to the mixture, followed by

centrifugation at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed

with 2.5 ml of distilled water and 2.5 ml of 1 g kg-1 of ferric chloride and the increase in

absorbance was measured at 700nm.

2.4.3 Metal-chelating activity

The metal chelating activity of LMwP was determined according to Decker &

Welch [13]. One milliliter of sample solution was mixed with 3.7 ml of distilled water.

The mixture was then reacted with 0.1 ml of 2 mmol L-1 FeCl2 and 0.2 ml of 5 mmol L-1

7
ferrozine for 20 min at room temperature and the absorbance was read at 562 nm. The

control tube receives distilled water instead of the sample. Percentage of chelating

activity was calculated using the following equation;

Metal Chelating Activity (%) = [{(Abs) control – (Abs) sample} / (Abs) control] × 100

2.4.4 Measurement of Lipid Peroxidation in Linoleic Acid Model System.

Inhibitory effect of auto oxidation of linoleic acid by LMwP was determined by

following procedure given by Osawa & Namiki [14]. LMwP at 1.3mg dissolved in 10ml

of 50 mmol L-1 phosphate buffer (pH 7) and added to a solution containing 0.13 mL of

linoleic acid and 10 mL of 99.5 % ethanol. With distilled water the total volume adjusted

to 25 mL. The mixture was then incubated for 5 days in dark at 40 ºC. The degree of

oxidation was evaluated by measuring the absorbance of ferric thiocyanate values at

500nm.

2.5 Functional characterization of LMwP

2.5.1 Solubility nature of LMwP

Solubility of LMwP was determined by dispersing 200 mg of LMwP in 20 ml of

deionized water at different pHs adjusted to 2- 12 with 1mol L-1 HCl and 1 mol L-1

NaOH. The reaction mixture was stirred at room temperature for 30 min and centrifuged

at 10000 rpm for 15 min. Protein concentration (mg) in the supernatant (m2) was

determined by biuret method. And total protein content in the sample (m1) was

determined after solubilization of the sample in 0.5 mol L-1 NaOH separately and the

solubility of LMwP was calculated as described below:

Solubility (%) = (m2/m1) × 100

2.5.2 Determination of Water Absorption property

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 One gram of LMwP of 10 and 40% degree of hydrolysis was evenly spreaded

individually in the pre-weighed petridish and the total weight (g) was measured (m1). The

petridish was placed in a humidity chamber. Increase in weight measured at 4 h interval

(m2) and the water absorption capacity of the sample was calculated according to the

following equation:

Water absorption (%) = {(m2 – m1) / Sample quantity} × 100

2.5.3 Determination of Foaming Properties

Foam expansion and stability of LMwP was carried out for both 10 and 40%

hydrolyzed sample according to the procedure given by Shahidi, Xiao-Quing &

Synowiecki [15]. Different concentration of LMwP (0.4, 0.8 and 1.2%) in 50 ml of water

was taken and homogenized in 100 ml cylinder at a speed of 16,000 rpm using

homogenizer for 1 min. Followed by homogenation the volume of the sample was

measured at 0, 0.5, 1, 5 10, 20 and 30 min intervals. Foam capacity was expressed as

foam expansion at 0 min, calculated accordingly as

Foam expansion (%) = (A – B)/B × 100

Where, A is volume after whipping (ml) and B is volume before whipping (ml). Foam

stability was calculated from the following equation.

Foam stability (%) = (A – B)/B × 100

where, A is the volume at 0.5, 1, 5 10, 20 and 30 min after whipping(ml) and B is the

volume before whipping (ml). All determinations were means of at least three

measurements.

2.5.4 Emulsifying Properties

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 The emulsifying activity index (EAI) and the emulsion stability index (ESI) of

LMwP determined according to the method of Pearce and Kinsella, [16] using soybean

oil (1 ml) and LMwP (both 10 and 40 % degree of hydrolyzed samples) solution (3 ml) at

different concentrations (0.1, 0.5 and 1%) were mixed and homogenized at a speed of

18,000 rpm for 1 min using homogenizer. Aliquots of the emulsion (100 µl) was taken

from the bottom of the container at 0 and 10 min after homogenization and diluted to 10

ml with 0.3% SDS solution (1:200). The absorbance of the diluted solution was measured

at 500 nm. The absorbance measured immediately 0min (A0) and 10 min (A10) and the

emulsifying activity index (EAI) was calculated as shown below:

EAI (mg2/g) = (2×2.303×dil×A) / (C × 10000 × Φ× l)

where, dil is the dilution factor (200); A is the absorbance at 500 nm; c is the protein

concentration (g/ ml ); Φ is the disperse phase volume fraction (0.22); and l is the optical

path (0.01 m).

The emulsion stability index (ESI) was calculated from the following equation:

ESI(min) = [Δt/(A0-A10)] × A0

Where ∆t is time difference (10 min), A0 and A10 are absorbance at 0 and 10 min

All determinations were means of at least three measurements.

2. Results and Discussion

3.1 Extraction and molecular characterization of ASC

The collagen matrix obtained after the removal of hair and flesh was subjected to

extraction of collagen. We found, pre-swelling of collagen matrix with 0.5M sodium

acetate prior to acetic acid extraction, enhances the total yield of collagen extracted. Pre

swelling in sodium acetate make the collagen matrix more porous and loose structure

10
caused by charge repulsion and enhances the penetration and interaction of acetic acid

with extra cellular matrix. With regard to the molecular profile analysis of extracted

ASC using SDS-PAGE analysis, Fig. 2 illustrate one β band (200 kDa) and two α bands

(116 kDa for α1 and α2), which are the unfolding polypeptide chains of the triple helix

(2[α1 (I)][α2(I)]). Molecular profile study confirms the extracted collagen belongs to

Type I family [17].

3.2 Preparation and Molecular characterization of LMwP

3.2.1 Preparation and characterization of LMwP

With protease treatment of type I collagen we received hydrolysate sample of 10,

20, 30 and 40 % degree of hydrolysis (Fig. 3a). In order to resolve the molecular size of

hydrolyzed peptides, Tricine SDS-PAGE (16%) was performed. As shown in Fig. 3b the

band patterns observed in electrophoretogram increases as the degree of hydrolysis

increased. Interestingly, peptide of molecular weight less than 5 kDa was dominant in

the samples of 40% degree of hydrolysis.

3.2.2 Amino acid analysis of LMwP

In general, composition of amino acid residues decides the functional and

bioactive properties of peptides. The aminogram of LMwP, expressed as weight

percentage was displayed in Table 1. We found percentage of Glycine was

comparatively higher in LMwP due to high glycine content of collagen. And the weight

percentage, increases in the following order, viz., hydroxyproline, proline, alanine and

glycine. Proline and hydroxyproline contributes 24 % of total amino acid concentration

and suggests the non-covalent bonding of pyrrolidone ring provides more thermal

stability to the native collagen [18]. In addition, the predominating hydrophobic residues

11
in the form of alanine and proline and the hydrophilic residues in the form of glutamic

and aspartic acid found responsible for lipid interactions and high water absorption

capacity respectively for LMwP as described in the following sections.

3.3 Bioactive characterization of LMwP

3.3.1 Antioxidant property of LMwP

It is well known that the nature and the type of protein, molecular mass, and

structure and process conditions were the highly influential factors on the bioactive and

functional properties of peptides. The antioxidant activity of LMwP at different degree of

hydrolysis was tested for their ability to scavenge the DPPH free radical and α-

tocopherol was used as a positive control. In the presence of ethanol, DPPH radical

exhibit maximum absorbance at 517nm. Since degree of hydrolysis play a major role on

the characteristics of LMwP, we found, at 20% degree of hydrolysis LMwP scavenge,

99% of DPPH (Fig. 4a). Further increase in the degree of hydrolysis reduces the activity.

The presence of proton donating residues in LMwP might be responsible for the

antioxidant activity of LMwP. Bo Li [19] made similar observations for procine skin

hydrolysates. According to available reports, aromatic amino acids are more responsible

for the antioxidant activity of the samples. However, we found the contribution of

phenolic residues was less than 2% (based on amino acid profile). It has been in reports

that presence of His, Met, Cys, Pro, Ala or Gly amino acids are believed to enhance the

antioxidant properties of peptides [20]. When comparing the antioxidant profile of LMwP

with standard α-tocopherol, we found, the IC50 for standard was 500 µg and the

scavenging activity takes place within 10 minutes. However, in the case of LMwP, the

IC50 value was identified as 95 µg and interestingly we observed a slow and steady

12
radical scavenging activity prolonged for more than one hour (Result not shown). Since

this kind of property was of high need for food processing industries which prevents the

deteoration of the processed food and helps for long- term storage.

3.3.2 Metal chelating and metal reducing activity of LMwP

Metal chelating property was the most important property, which decides the

bioactivity, especially the antioxidant property of peptides. In the present study, the

ferrous ion chelating ability of LMwP was shown in Fig. 4a. The maximum chelating

ability (90%) was observed with LMwP samples of 30 % degree of hydrolysis. The

carboxyl and the amino group of the branched amino acids play an important role in the

chelating metal ions and suggests peptides containing Glutamic acid, aspartic acid,

glutamine, aspartamine, lysine, histidine, phosphorylated serine and threonine are known

to bind metal ions and this property observed with LMwP suggest it can able to retard the

transition metal ions mediated oxidation of food product by chelating them [21].

With regard to metal reducing activity of LMwP, conversion of Fe3+ to Fe2+ at

different degree of hydrolysis was performed. Fig. 4a showed the maximum reducing

activity of LMwP samples of 30% degree of hydrolysis. According Wang et al [22]. To

neutralize a free radical, an electron donating reducing agent can able to donate an

electron to free radical and reduced species aquires proton from solution. Both chelating

and reducing activity of LMwP indicated the potential bioactive property.

3.3.3 Inhibition of auto oxidation of linoleic acid

Since, the free radical scavenging activity was not enough to claim LMwP as a

potential antioxidant, additional experimental analysis was required to authenticate the

said property. As discussed above, the slow and prolonged radical scavenging activity of

13
LMwP found application in food storage, however, results on the effect of LMwP on

prevention of saturation of fatty acids was also necessary to substantiate the antioxidant

property. For this purpose, LMwP tested for its preventive effect on linoleic acid auto

oxidation. Fig. 4b displayed the percentage inhibition of linoleic acid with increasing

concentration of LMwP and suggests, higher the concentration of LMwP, higher the

inhibitory activity. The inhibitory effect of LMwP was due to the chelation of proxidant

metal ions. Lipids and proteins are the main targets of oxidative reactions that affect food

quality. Formation of lipid hydroperoxides due to oxidative reaction involves the free

radical pathway [23]. Transition metal ions react very quickly with peroxides by acting as

one-electron donors to form alkoxyl radical. Transition metals, such as Fe, Cu, Co, in

foods affect both the rate of autoxidation and breakdown of hydroperoxide to volatile

compounds. The induced oxidative stress in the food product upon storage was

unavoidable and number of external factors was responsible for the induction of

oxidation, which results with spoilage of food. In order to avoid the situation external

antioxidants like BHT and BHA are employed. However, recent realization on the

deleterious effects of these compounds necessitates the need for new agents of natural

origin. Since, the results on antioxidant property of LMwP was appreciable, we suggests,

LMwP of Type I collagen will improve the quality of food effectively.

3.4 Functional characterization LMwP

3.4.1 Solubility nature of LMwP

Solubility is one of the physical factors provides information about the nature of

the product. In the present study, solubility of LMwP was tested in the pH range of 2–12.

It was observed that more than 85% solubility with pH 5. Enzymatic hydrolysis

14
potentially affects the molecular size, hydrophobicity, polarity and the ionizable groups

of protein hydrolysates. At different pH, the ionic charge equilibrium between the

hydrophobic and hydrophilic residues influences the solubility increments. Nevertheless,

in the present study, at pH 3, LMwP showed only 73 % solubility (Fig. 5a). Low

solubility of proteins was generally influenced by change in the charge or the isoelectric

pH of the peptides obtained. Solubility variations could be attributed to both net charge

of peptides that increases as pH moves away from pI and surface hydrophobicity, which

promotes the aggregation via hydrophobic interaction [24]. Furthermore, protein

solubility plays an important role in functional properties of hydrolysates such as foaming

and emulsifying properties because of rapid migration and adsorption of the peptides at

the interface are critical.

3.4.2 Water absorption capacity

With regard to water absorption capacity, LMwP, exhibit good water absorption

capacity, however, varied with percentage of degree of hydrolysis. Fig. 5b, illustrates,

water absorption capacity of LMwP, received for 10 and 40 % degree of hydrolysis. At

10% degree of hydrolysis LMwP showed >85% water absorption capacity, while, 40 %

DH samples exhibit only 64%. This could be due to increase in hydrolysis (>40% DH) of

collagen released peptides of <5 kDa or even as amino acids and thus exhibit the reduced

percentage of water absorption. The hydrophilic groups, carboxyl, hydroxyl, -NH2- and

carbonyl groups involved in the double hydration. In addition, it was demonstrated that

enzymatic hydrolysis exposes more ionizable polar amino acids like aspartic and

glutamic acid, hydroxyproline that are abundant in collagen. These amino acids are

capable of binding three times more water than non-ionized polar groups [25]. The high

15
water absorption capacity of LMwP suggested its application in food industries to prevent

water loss in meat, breads, cakes, cured sausages and frozen products

3.4.3 Foamability (Foam stability and foam expansion)

In general, foaming property of protein refers to its ability to form a thin tenacious

film at gas liquid interfaces so that large quantities of gas bubbles can be incorporated

and stabilized. To enable the foam to be formed, surface tension of the solution has to be

reduced. In the present study, similar to water absorption capacity, the foamability of

LMwP showed significant difference with respect to degree of hydrolysis. Hydrolysate

obtained from 10% degree of hydrolysis exhibits good foaming stability. Increase in

foam expansion percentage (from 14 to 49 %) observed with the increase concentration

of LMwP (0.1 to 3.0%.), could be due to increased solubility of LMwP as shown in Fig.

5c(i). An increase in the concentration of protein hydrolysate prepared from round scad

mince gave rise to a higher foam expansion, possibly due to an increase in the rate of

diffusion [26]. With regard to the stability of the foam (Fig. 5c (ii)), LMwP has

considerable higher percentage of Pro and Lys residues, which enhances the protein-

protein interactions via hydrogen bonds, resulting in a denser network.

With respect to foam expansion, at higher degree of hydrolysis, ie., LMwP of

40% DH, both foam expansion and stability showed drastic reduction (Fig. 5c (i & iii)).

At 0.1% LMwP concentration, only 10% foam expansion was observed and the stability

reaches zero after 20 min. This phenomenon may be attributed to the molecular size,

protein structure and hydrophobicity of the hydrolysates, which are highly dependent on

the parent protein from which they are obtained and the hydrolysis procedure followed.

The more the microscopic peptides less will be the stability [27]. Similarly, in the present

16
study LMwP of 10% degree of hydrolysis provided stable foams with appreciable

strength compared to LMwP of 40% degree of hydrolysis.

3.4.4 Emulsifying property

In general, emulsifying property is the ability of the protein/peptides to aid in the

formation and stabilization of a newly created emulsion by giving units of area of the

interface stabilized per unit weight of protein and was determined by the turbidity of the

emulsion at a wavelength of 500 nm. Emulsifying activity index (EAI) and emulsion

stability index (ESI) for LMwP of 10 & 40 % degree of hydrolysis were shown in Fig. 5d

(i & ii) and suggested EAI and ESI properties decreased with increasing degree of

hydrolysis. At lower degree of hydrolysis (10%), LMwP displayed increased and strong

EAI and ESI properties, attributed to the higher concentration of larger molecular weight

peptides or more hydrophobic peptides [28]. Furthermore, when the concentration of

LMwP (10% degree hydrolysis) increases, there was a reduction in EAI and ESI

properties which was reasoned by the protein–protein interactions resulting in a lower

protein concentration at the oil–water interface [29]. In addition, the mechanism of

emulsion formation attributed to the adsorption of peptides on the surface of freshly

formed oil droplets during homogenization and the formation of a protective membrane

that inhibits coalescence of the oil droplet [30]. The concentration dependent emulsifying

activity may be explained by adsorption kinetics. At low protein concentrations, protein

adsorption at the interface was diffusion-controlled, however, at high concentrations; the

activation energy barrier does not allow protein migration to take place in a diffusion-

dependent manner, leading to the accumulation of proteins in the aqueous phase [29].

17
 On the other hand, with LMwP of 40% degree of hydrolysis, emulsifying value

reduced rapidly and after 30 min the ESI value comes to zero, due to excessive hydrolysis

of proteins. To exhibit good emulsifying property, peptides with low molecular weight

should be amphiphillic [31] and this character was more important than the length of the

peptides. In general, hydrolysates are surface-active materials and promote oil-in-water

emulsion because of their hydrophilic and hydrophobic groups with their associated

charges. Since, increasing degree of hydrolysis reduces the size and amphiphilicity of low

molecular weight peptides and thus exhibits poor EAI and ESI proprties. Small peptides

migrate more rapidly and adsorb at the interface, decreasing the interface tension and

could not unfold and reorient at the interface like large peptides to stabilize emulsions

[31].

3. Conclusion

Looking into the above said experimental results, LMwP obtained through

controlled enzymatic hydrolysis of type I collagen exhibits antioxidant and other

functional properties compared to intact protein. The bioactive properties are appreciable

when compared with the natural antioxidant. Not only considering the bioactivity of

LMwP, it also holds functional characteristics, viz., non gelling, low viscosity, high

solubility in wide range of pH and low residual aromatic amino groups. Foaming,

emulsifying and water absorption properties are the added value for effective utilization

of LMwP in food processing industries. Further exploration on an use of different

digestive enzymes and their role on type I and other major collagen types is required in

addition to the role of LMwP with respect to the taste of food when used as food

additives.

18
Acknowledgments

All the authors acknowledge the Department of Biotechnology, Ministry of

Science and Technology, New Delhi, India for their financial support in the form of

project (BT/HRD/35/01/02/2008).

19
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Figure Captions

Figure. 1. Flow chart summarizes the sequential steps involved in the extraction of Acid

Soluble Collagen

Figure. 2. SDS- PAGE (8%) profile of Acid soluble collage obtained from bovine skin.

(Lane 1- Molecular weight markers, Lane 2- ASC).

Figure. 3a. Degree of hydrolysis (DH) of acid soluble collagen expressed in percentage.

Figure. 3b. Electrophoretogram of LMwP (Tricine SDS PAGE). (Lane 1- molecular

markers; Lane 2,3,4- LMwP at increasing degree of hydrolysis; Lane 5- LMwP of

40 % degree of hydrolysis). The arrow marks indicate the change in the band

patterns with reference to hydrolysis.

Figure. 4a. Antioxidant property of LMwP at different degree of hydrolysis.( radical

scavenging, metal chelating and metal reducing activity)

b. Inhibition of linoleic acid auto oxidation by LMwP.

Figure. 5a. Solubility characteristics of LMwP at different pH.

b. Water absorption (%) capacity of LMwP at 10 and 40 % Degree of hydrolysis

(DH)

c(i). Foam expansion of LMwP obtained at different degree of hydrolysis (DH) and

at varying concentration.

c(ii). Foam stability (%) of LMwP obtained after 10% degree of hydrolysis

c(iii). Foam stability (%) of LMwP obtained after 40% degree of hydrolysis

d(i). Emulsifying activity index of LMwP obtained at 10 and 40 % degree of

hydrolysis(DH) and at varying concentration.

24
 d(ii) Emulsion stability index of LMwP obtained at 10 and 40% degree of

hydrolysis at varying concentration.

Table 1 Aminoacid profile of LMwP (g Kg-1)

Amino acid Weight ( g Kg-1of LMwP)

Asp 1.4

Glu 2.06

Pro 13.5

Arg 5.69

Lys 2.32

Gly 37.7

Hyp 10.47

Ser 1.96

His 0.8

Thr 2.1

Tyr 1.4

Ala 13.9

Val 1.3

Met 1.79

Phe 0.56

Leu 1.18

Ileu 1.65

Cys ND*

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*ND- Not Detected

Fig. 1. Gnanamani et al.

26
Fig. 2. Gnanamani et al.

27
Fig. 3 a & b. Gnanamani et al

28
.

Fig. 4 a & b. Gnanamani et al.

29
30
Fig. 5 a & b. Gnanamani et al.

Fig. 5 c. Gnanamani et al.

31
Fig. 5 d. Gnanamani et al.

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33