WO2017034390A2

WO2017034390A2 - Bioactive swiftlet nest supplement

Info

Publication number: WO2017034390A2
Application number: PCT/MY2016/050050
Authority: WO
Inventors: Abdul SALAM BABJI
Original assignee: Universiti Kebangsaan Malaysia
Priority date: 2015-08-24
Filing date: 2016-08-23
Publication date: 2017-03-02
Also published as: WO2017034390A3; MY202071A

Abstract

Present invention discloses a natural food supplement, obtained from EBN, which comprises bioactive glycopeptides, produced by selective enzymatic hydrolysis of low dosage irradiated micro-particulates of EBN. The natural food supplement has significant antihypertensive and antioxidative (anti-aging) activities. Present invention also discloses a process for the preparation of natural food supplement. The invention is aimed to develop food product from edible bird nest (EBN) which is focused on the effects of micro-particulates, irradiation and enzymatic hydrolysis processing steps undertaken to explore the 'bioactive' peptides and glycopeptides in relation to the functional properties. This bioactive swiftlet nest supplement is a superior product as compared to traditional raw swiftlet nest in terms of natural antihypertensive and antioxidant activities, and is also safe, nutritious and convenient alternative of synthetic bioactive supplement sources.

Description

BIOACTIVE SWIFTLET NEST SUPPLEMENT FIELD OF THE INVENTION

[0001] The present invention relates generally, to a natural food supplement, and more particularly, relates to a bioactive swiftlet nest supplement which has significant antihypertensive and antioxidative activities. The present invention also describes a process for preparation of bioactive swiftlet nest supplement.

BACKGROUND OF THE INVENTION

[0002] Hypertension is a global health problem and continues to rise at an alarming trend. Over time it is a major risk factor for hypertensive heart disease, coronary artery disease, stroke, aortic aneurysm, peripheral artery disease, and chronic kidney diseases.

[0003] A large number of diseases such as cardiovascular disease, cancer and aging etc. are caused by the excess of oxygen free radicals. During pathological conditions or due to external factors, human body produces large number of active oxygen with reduced capability of clean-up, repair, and body protection, which results in to free radicals imbalance. This excessive reactive oxygen will attack the cell membrane, unsaturated fatty acids, proteins, amino acids, nucleic acids, etc., and destroy the cell structure and function, causing damage to the body, giving rise to a series of diseases (such as cardiovascular disease, cancer, and accelerate the body aging, etc.).

[0004] Antioxidants can help the body to maintain and balance of oxygen free radicals, thereby preventing the occurrence of diseases and maintain the body's health. It plays an important role in neutralization of harmful peroxides and free radicles.

[0005] Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases. Edible bird nest (EBN) hydrolysate is considered as a potential dietary source of natural antihypertensive and antioxidants with functional biological activities. Specific health and beauty care benefits have been long known with EBN. It is believed that EBN have health enhancing effects such as anti-aging, growth promoting and immuno-enhancing properties.

[0006] EBN is a dried glutinous secretion from the salivary glands of several different swiftlet species. It is widely consumed as health food due to its high beneficial effects to human health and has been considered to be one of the most precious food items by the Chinese for thousands of years. The glycoproteins, a major component of edible bird nest (EBN) has unique properties related to claims of health, vigour, beauty and cure for all illnesses. [0007] In nutrition and cosmetics industries, bird's nest nutritional formulation extraction method is relatively very simple, mainly in the rough way of cooking. Protein bioavailability of macromolecules or glycoproteins is generally low in nutrition and cosmetics industries.

[0008] The present invention addresses the need to develop a food supplement with high bioactive glycopep tides, which acts as a potential dietary source of natural antihypertensive and antioxidants properties. There is also a need to develop a preparation process for the natural food supplement.

[0009] As glycoprotein is the major components of EBN, research and food product development on EBN will be focused on the effects of micro-particulates, irradiation and enzymatic hydrolysis processing steps undertaken to explore the 'bioactive' glycopeptides in relation to the functional properties. Therefore, it would be desirable to provide a natural antihypertensive and antioxidant digestible supplement that is safe, nutritious and convenient as an alternative of synthetic bioactive supplement sources.

SUMMARY OF THE INVENTION

[0010] The present invention recognizes and addresses various disadvantages and drawbacks of traditional raw swiftlet nest. The natural food supplement and its preparation process has been devised in the light of above mentioned circumstances and aims to solve the above mentioned problems. [0011] In accordance with one aspect of the invention, disclosed is a natural EBN food supplement which comprises bioactive glycopeptides, associated with significant antihypertensive and antioxidative (anti-aging) activities.

[0012] The bioactive glycopeptides is produced by selective enzymatic hydrolysis of low dosage irradiated micro-particulates of EBN.

[0013] In accordance with another aspect of the invention, disclosed is a process for the preparation of natural food supplement, obtained from EBN, having characteristics bioactive glycopeptides. The process comprising the steps of:

a) Production of EBN micro-particulate powder with size of 710, 300 and 38mm.

b) Production of irradiated EBN micro-particulate powder at doses of 0.0, 1.0, 2.0, 5.0, 7.5, 10.0, 20.0 and 30.0 kGy using cobalt-60 irradiator (220 Gammacell ® Excel) at a rate of 2.17 kGy.h^"1. c) Determination of the effect of gamma irradiation on nutritional and microbiological quality of the EBN micro-particulate powder. Analysis have been undertaken were proximate analysis (AO AC, 2003), amino acid profiling, microbiological analysis.

d) Production of EBN hydrolysate from irradiated EBN micro-particulate powder via enzymatic hydrolysis process.

e) Determination of the bioactivity (antihypertensive and antioxidant) of EBN hydrolysate. f) Production of Bioactive Swiftlet Nest capsule.

Accordingly, objects of the present invention are listed below:

• It is the main object of the present invention to provide a natural food supplement which has significant antihypertensive and antioxidative (anti-aging) activities.

• It is another object of the present invention to provide bioactive glycoproteins which have high protein digestibility and solubility.

• It is still another object of the present invention to provide natural food supplement, whose daily consumption of 4.3 gram provide the benefits of an ti -hypertensive (maintenance of good health) and antioxidant (anti-aging).

• It is further another object of the present invention to develop a process which is focused on selective enzymatic hydrolysis of low dosage irradiated micro-particulates of edible bird nest (EBN). • It is another object of the present invention to provide natural food supplement which is nutritious, safe to consume, digestible and convenient as an alternative of synthetic bioactive supplement sources. [0014] The foregoing and other objects and advantages of the invention will be apparent upon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] The accompanying drawings, which are incorporated into, and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

Figure 1 (a) and (b) is a Schematic illustration of comparison of antioxidant activities between edible bird nest, chicken and fish soup.

Figure 2 shows an angiotensin converting enzyme inhibitory (ACE-1) activities of edible bird nest, chicken and fish soup.

Figure 3 shows a DPPH inhibition for EBN samples with different sizes in comparison with BHA, BHT, and BHA + BHT

Figure 4 shows a flow chart, illustrating the steps involved in the formation of bioactive capsule processing.

Table 1 shows the degree of hydrolysis (DH), protein solubility, peptide content and antioxidant activity of EBN at different time of hydrolysis. Table 2 shows the antioxidant activity of EBN hydrolysates in different time of hydrolysis at concentration of 5mg mL^"1

Table 3 shows the percentage of angiotensin converting enzyme (ACE) inhibitory activity and IC50 value for different hydrolysis time of sample fraction of edible bird nest hydrolysate.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The following detailed description of the present invention provides specific details of certain embodiments of the invention illustrated in the drawings to provide a thorough understanding of those embodiments. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. It should be recognized, however, that the present invention can be reflected in additional embodiments and the invention may be practiced without some of the details in the following description.

[0017] In accordance with one aspect of the invention, is a natural bioactive bird nest EBN food supplement whose properties of antihypertensive and antioxidative activities are explored extensively. [0018] In accordance with another aspect of the invention, is a process for the preparation of natural bioactive EBN food supplement which involves selective enzymatic hydrolysis of low dosage irradiated micro-particulates of bioactive EBN which have resulted in highly bioactive glycopeptides with significant antihypertensive and antioxidative (antiaging) activities. [0019] Micro-particulate size reduction was applied to the raw edible bird nest (EBN) as a purpose to increase the surface area per unit volume of sample, thus increase the solubility and bioactivity. If this part is not carried out, edible bird nest (EBN) sample will less soluble and the bioactivity will be low. [0020] The effectiveness of bioactivity in food systems depends on the concentration and solubility factor. According to Wu and Nancollas (1998), smaller particle size contributes to a greater surface area per unit volume, thus helps to increase the solubility. Solubility of antioxidants will determine whether the soluble antioxidant in the lipid phase or water and may affect the overall antioxidant activity (Naveena et al., 2013). By reducing the particle size of sample, the surface area per volume will increase which also increase the solubility, thus give high potential to bioactivity. Micro- particulate size reduction indirectly will increase the antihypertensive and antioxidative activity of edible bird nest (EBN).

[0021] Referring to Figure 1 (a), (b), and Figure 2 are a Schematic illustration of comparison of antioxidant and antihypertensive activities between edible bird nests (EBN), chicken and fish soup. Both the figure shows that edible bird nest (EBN) has the highest antioxidant and antihypertensive properties.

[0022] Besides that, gamma irradiation was done to the micro-particulate EBN sample to reduce the microbial count to accepted level for safety purpose. If this step is not applied the safety of the product will be questioned.

[0023] Gamma irradiation is a safe alternative method of reducing the microbial load in EBN sample. Even though conventional drying method will reduce the water content and cause death of microorganisms, small number of vegetative microorganisms, yeasts and fungi, yet there remains a possibility of bacterial spores, microscopic fungi and yeasts with high resistance to survive after the drying process (Miteva et al., 2008).

[0024] Gamma irradiation with radioactive elements (Cobalt-60) is widely used in medical and pharmaceutical products as the sterilization purpose to inhibit the microbial growth.

[0025] In the present invention, study was carried out to determine the effect of gamma irradiation on the microbiological quality and amino acid profile of edible bird nest (EBN) powder. As shown in Table 1, eight doses of irradiation were chosen, namely: 0.0, 1.0, 2.0, 5.0, 7.5, 10.0, 20.0 and 30.0 kGy. Total plate count (TPC) of edible bird nest (EBN) samples was reduced to < 100 CFU.g^" ¹ at 20.0 kGy. Irradiation dose of 5.0 kGy was required to reduce the coliform and Staphylococcus aureus count to < 100 CFU.g^"1 and reduced the yeast and mould count to < 1000 CFU.g^"1. While for Escherichia coli 1.0 kGy was sufficient enough to reduce the count to < 100 CFU.g^"1. Salmonella spp. was not detected in all the samples. Twenty kGy was the minimum irradiation dose to sterilize the edible bird nest (EBN) powder samples most effectively. [0026] The pH value of the samples were in the range of 8.34-8.90, while water activity (a_w) of the samples were in the range of 0.621-0.674. Microbiological quality changes of irradiated samples were caused by the gamma irradiation alone.

Table 1 : Microbiological Count in irradiated (low dosage) Edible bird nest (EBN)

[0027] Gamma irradiation at doses as high as 10.0 and 20.0 kGy did not give significant changes on amino acid profile of Edible bird nest (EBN). Gamma irradiation improved the microbiological quality of Edible bird nest (EBN) without affecting its amino acid profile as shown in Table 2.

Table 2: Amino Acid Composition (raw EBN versus different irradiation doses)

Amino Acid Irradiation dose (kGy)

0 10 20

Non-essential amino acid

Aspartic acid 5.35 + 4.62^a 5.32 + 0.05^a 5.03 + 0.49^a

Serine 5.19 + 0.24^a 5.19 + 0.17^a 4.95 + 0.50^a

Glutamic acid 4.20 + 0.18^a 4.18 + 0.25^a 3.87 + 0.36^a

Glycine 2.23 + 0.11^a 2.20 + 0.07^a 2.11 + 0.20^a

Alanine 1.95 + 0.06^a 2.18 + 0.07^a 2.02 + 0.25^a

Proline 7.65 + 3.97^a 7.42 + 2.57^a 4.10 + 0.18^a

Tyrosine 2.71 + 0.33^a 3.09 + 0.03^a 2.98 + 0.39^a

Cysteine 1.34 + 0.02^a 1.43 + 0.06^a 1.46 + 0.01^a

Essential amino acid

Arginine 3.62 + 0.37^a 3.62 + 0.21^a 3.53 + 0.50^a

Histidine 2.09 + 0.19^a 2.11 + 0.07^a 2.05 + 0.24^a Threonine 3.91 + 0.17^a 3.78 + 0.16^a 3.61 + 0.38^a

Valine 6.67 + 0.57^a 6.58 + 0.23^a 6.26 + 0.63^a

Methionine 0.32 + 0.05^a 0.35 + 0.01^a 0.33 + 0.01^a

Lysine 2.31 + 0.13^a 2.38 + 0.09^a 2.25 + 0.29^a

Isoleucine 2.07 + 0.18^a 2.01 + 0.09^a 1.94 + 0.20^a

Leucine 4.01 + 0.34^a 3.95 + 0.18^a 3.82 + 0.41^a

Phenylalanine 3.74 + 0.34^a 3.71 + 0.16^a 3.58 + 0.40^a

Tryphtophan 0.58 + 0.02^a 0.61 + 0.10^a 0.67 + 0.07^a

Total non-essential amino acid 30.62 31.01 26.52

Total essential amino acid 29.32 29.10 28.04

Total Protein (%) 52.19 55.56 54.85

[0028] Many of the proteins that occur naturally in raw food materials exert their physiological action either directly or upon enzymatic hydrolysis in-vitro or in-vivo (Korhonen & Pihlanto, 2006).

[0029] Enzymatic hydrolysis is a method to produce specific bioactive protein fragments that have positive impacts on body functions, or conditions and may ultimately influence health (especially act as antihypertensive and antioxidative activity). These beneficial health effects may be attributed to numerous known peptides sequences. The activity is based on their inherent amino acid composition or sequences and peptides are known to reveal multifunctional properties (FitzGerald & Meisel, 2003). Without activity of enzyme, hydrolysis still can be done but the rate and effectiveness of hydrolysis will be lower.

[0030] In the present invention, enzymatic hydrolysis was applied to the EBN sample to produce specific bioactive protein fragments that have positive impacts on body functions, or conditions and may ultimately influence health.

[0031] Referring to Table 3 shows the degree of hydrolysis (DH), protein solubility, and peptide content of EBN at different time of hydrolysis. Table 3. Degree of hydrolysis (DH), protein solubility, peptide content and antioxidant activity of EBN at different time of hydrolysis.

Table 1 showed the degree of hydrolysis (DH), protein solubility and peptide content of EBN hydrolysate at different time of hydrolysis. Increasing the time of hydrolysis (h) to 1.5h increased the DH, protein solubility and peptide content to 82.7%, 104.1mg/g and 86.68mg/g respectively. Degree of hydrolysis indicated the percentage of peptides breakdown and it is usually used as a parameter to control the level of proteolysis (Adler-Nissen, 1979). According to the result, after an initial rapid phase of hydrolysis up to 1.5h, the rate of hydrolysis tend to decrease and entered a stationary phase. At this point, increasing the time of hydrolysis did not result in higher degree of hydrolysis because the concentration of peptide bonds available for hydrolysis may be the limiting factor. Montecalvo et al. (1984) reported that increasing of DH also caused increase in cleavage of peptide bonds which then increases the peptides solubility. The high value at 1.5 h of hydrolysis indicates that more peptide bonds were broken down by the alcalase enzyme at that duration.

Referring to Table 4 shows the antioxidant activity of EBN hydrolysates in different time of hydrolysis at concentration of 5mg mL^"1

Table 4. Antioxidant activity of EBN hydrolysates in different time of hydrolysis at concentration of 5mg mL^"1

Antioxidant activity

Sample (h) DPPH (%) ABTS (%) FRAP

0.0 27.17 + 0.20^f 29.30 + 0.6^d 0.27 + 0.03^e

0.5 41.72 + 1.2^e 51.65 + 0.Γ 0.31 + 0.04^d 1.0 42.45 + 0.3^d 52.01 + 0.6^C 0.45 + 0.02^b

1.5 59.45 + 0.8^a 69.45 + 0.2^a 0.46 + 0.01^b

2.0 56.29 + 0.8^b 69.28 + 0.5^a 0.57 + 0.02^a

3.0 55.90 +0.2^C 60.60 + 0.8^b 0.44 + 0.04^c

4.0 55.74 + 1.2^C 60.24 + 0.3^b 0.45 + 0.01^b

Each data represents average value ± standard deviation of triplicate replication, n=3.

(P<0.05). ^{a e} Means between time of hydrolysis are significantly different.

Hydrolysis is necessary in order to release ROS inhibitory peptides from an inactive form within the sequence of edible bird nest protein. During hydrolysis, a wide variety of smaller peptides and free amino acids is generated, depending on enzyme specificity. Antioxidant activities, as determined by DPPH, ABTS and FRAP assays of hydrolysates were shown in Table 2. EBN hydrolyzed at 1.5h (82.7% DH) showed the highest DPPH and ABTS radical-scavenging activities with value of 59.45% and 69.45% respectively. This indicated that hydrolysate produced at 1.5h have the high ability as hydrogen donor to produce non radical species. The varying scavenging radicals capabilities might be caused by the difference in chain length and amino acid composition of the peptides in the different hydrolysates. The highest FRAP reducing power activities recorded by EBN hydrolyzed at 2.0h (73.5% DH) with value of 0.57. Hydrolysis time of 2.0h produce hydrolysate with high potential of electron donor to reduce reactive species of Fe3+ to stable species of Fe2+.

Referring to Table 5 shows the angiotensin converting enzyme (ACE) inhibitory activity of EBN hydrolysate. Table 5 Angiotensin converting enzyme (ACE) inhibitory activity of EBN hydrolysate.

Hydrolysis Time Alcalase

(minutes) ACE-1 (%) IC50 value (mg/ml)

0 6.88 + 4.85^c

30 83.07 + 2.4^a 0.07

60 86.24 + 3.2^a 0.02

90 71.43 + 4.2^b 0.15

120 81.48 + 4.0^a 0.09

180 73.54^b + 5.7^b 0.18

240 71.43^b + 4.0^b 0.19 Table 3 summarizes the result obtained for the percentage of ACE inhibitory activity in the hydrolysates sample with different hydrolysis time at concentration of 2.0mg/ml. The ACE inhibitory activity is calculated by EBN protein was hydrolysed with alcalase enzyme for the extraction of ACE inhibitory peptides. Among the hydrolysates tested, Alcalase hydrolysate showed the highest ACE inhibitory activity, of 86.24% (60 minutes of hydrolysis) at 2.0mg/ml even though the highest DH was at 90 minutes. Study by Zhang et al. (2008) reported that DH value does not affect ACE inhibitory activity, but the ACE inhibitory activity is affected by the ability of the enzyme used in producing specific protein chain containing hydrophobic amino acid at the C-terminal of tripeptides. This is supported by Arihara et al. (2006) who reported that ACE inhibitory activity is affected by the enzyme specific activity in producing specific peptide chain. Several studies have reported that Alcalase hydrolysates derived from food proteins showed potent bioactivities, such as antioxidant and ACE inhibitory activities. These bioactivities are attributed to the ability of Alcalase to produce various bioactive peptides because of its endo -peptidase properties. Alcalase produces shorter peptide sequences as well as terminal amino acid sequences responsible for various health beneficial bioactivities, including ACE inhibition (Hyun & Shin 2000). Previous studies have reported that Alcalase is capable of producing bioactive peptides when it is used to hydrolyze food proteins (Li et al. 2006).

[0032] Referring to Figure 3 shows a DPPH inhibition for EBN samples with different sizes in comparison with BHA, BHT, and BHA + BHT.

Experimental Procedure

[0033] Stage 1: Literature Review

Literature review conducted by gathering information from several previous studies related to EBN and its nutritional composition. Literature review from previous studies related to the treatment of sterilization of food products using gamma irradiation, enzymatic hydrolysis, and bioactivity also be done. This level includes the listing of methods, instruments and chemicals to be used. In addition, some aspects such as the overall effectiveness and cost of the study were taken into account. [0034] Stage 2: Sample preparation, treatment and analysis

This stage involves: a) Production of EBN micro-particulate powder with size of 710, 300 and 38mm.

e) Determination of the bioactivity (antihypertensive and antioxidant) of EBN hydrolysate.

f) Production of Bioactive Swiftlet Nest capsule. [0035] Stage 3: Data Collection and Interpretation

All data and information that has been obtained from the previous analysis were recorded and documented.

[0036] Stage 4: Production of Bioactive Swiftlet Nest Capsule

Encapsulation the swiftlet hydrolysate using plant based capsules, packed and labelled in bottle for commercialization purpose.

[0037] Referring to Figure 4 shows a flow chart, illustrating the steps involved in the bioactive capsule processing. The bioactive capsule processing involves the steps of:

(a) Producing micro-particulate from EBN

(b) Sterilizing the micro-particulate of EBN

(c) Soaking the micro-particulate of EBN in distilled water (4°C for 16 hours)

(d) Double boiling the micro-particulate of EBN at temperature 100°C for 30 minutes

(e) Adjusting the pH of sample solution depending on types of enzymes used

(f) Adding enzyme to the sample solution

(g) Putting sample solution inside water-bath shaker for hydrolysis (h) Deactivating enzyme at temperature 100°C for 5 minutes

(i) Collecting supernatant after cooling and filtering the sample solution

(j) Freezing the sample in freezer for 1 day & freeze dry the sample using freeze dryer

(k) Finally dry sample is encapsulated using plant based capsules, packed and labelled in bottle for commercialization purpose.

[0038] Normally people consume EBN as soup which need cooking for 2 to 6 hours. The present invention findings reduce preparation time, while at the same time increase the nutritional value (functional peptides) and more important it is safe to consume (by low microbial count after gamma irradiation). Besides being nutritious, the product which is biotechnologically processed have high antihypertensive and antioxidant activities. Because it is in hydrolysates form, it is more digestible. All this benefits make this product superior as a natural antihypertensive and antioxidant digestible supplement that is safe, nutritious and convenient as an alternative of synthetic bioactive supplement sources.

[0039] Based on effective concentration at 50% (EC50), it is suggested that consuming about 4 to 5 gram of bioactive edible bird nest (EBN) as supplement per day will provide the benefits of antihypertensive (maintenance of good health) and antioxidant (anti-aging). This bioactive bioactive edible bird nest (EBN) supplement will be commercialized among elderly, adults in search for long quality life and patients with chronic diseases.

[0040] This invention can also be well performed by using other local resources of proteins such as MDCM, viscera, entrails from the animal industry.

Claims

CLAIMS:

1. A natural food supplement, obtained from EBN, as natural food supplement comprising bioactive glycopeptides produced by selective enzymatic hydrolysis of irradiated micro- particulates of edible bird nest (EBN).

2. The natural food supplement of claim 1 , wherein said bioactive peptides have high protein digestibility and solubility.

3. The natural food supplement of claim 1, wherein said natural food supplement has significant antihypertensive and antioxidative (anti-aging) activities.

4. The natural food supplement of claim 1 , wherein about 4 to 5 gram of bioactive EBN as supplement per day will provide the benefits of anti-hypertensive (maintenance of good health) and antioxidant (anti-aging).

5. A natural food supplement, obtained from EBN, having characteristics bioactive glycopeptides, produced by a process comprising the steps of:

a) producing edible bird nest (EBN) micro-particulate powder;

b) irradiating gamma on edible bird nest (EBN) micro-particulate powder using cobalt- 60 irradiator (220 Gammacell ® Excel) at a rate of 2.17 kGy.h^"1 ;

c) determining effect of gamma irradiation on nutritional and microbiological quality of the edible bird nest (EBN) micro-particulate powder;

d) enzymatic hydrolysis of irradiated edible bird nest (EBN) micro-particulate powder to produce edible bird nest (EBN) hydrolysate;

e) determining the bioactivity (antihypertensive and antioxidant) of edible bird nest (EBN) hydrolysate;

f) producing bioactive swiftlet nest capsule.

6. The process as claim 5, wherein edible bird nest (EBN) micro-particulate powder is in size of 710, 300 and 38mm. The process as claim 5, wherein edible bird nest (EBN) micro-particulate powder irradiated at doses 0.0, 1.0, 2.0, 5.0,

7.5, 10.0, 20.0 and 30.0 kGy.

The process as claim 5, wherein determination of the effect of gamma irradiation on edible bird nest (EBN) micro-particulate powder is performed by proximate analysis, amino acid profiling, microbiological analysis.