Optimization of enzymatic hydrolysis conditions on the degree of hydrolysis of edible bird ’ s nest using Alcalase ® and nutritional composition of the hydrolysate

This study aimed to determine the optimum enzymatic hydrolysis conditions for edible bird’s nest (EBN) using Alcalase in order to achieve maximum degree of hydrolysis (DH). Four parameters namely temperature (45-65°C), pH (7.5-9.5), Alcalase to substrate concentration (1-2%) and hydrolysis time (60-180 mins) were employed at three levels using a face-centered central composite design (CCD) by using RSM. The optimum conditions for hydrolysis of EBN were at the temperature of 64.99°C, Alcalase concentration of 2%, hydrolysis time of 179.55 min and pH of 9.46. The suggested model was a 2-factor interaction (2FI) model. Under the optimum conditions, the predicted value for DH (37.90%) was close to the experimental data (37.92%). EBN hydrolysate comprised of 67.63±0.78% protein, 2.62±0.37% ash, 0.61±0.30% fat and 29.14±0.11 carbohydrate. EBN contained all essential amino acids except for tryptophan (which was not quantifiable using the method in this study), and their levels were higher than the suggested profile of essential amino acid requirements for adults, except for methionine. EBN hydrolysate also contained 34.04% hydrophobic amino acid.


Introduction
Edible bird's nest (EBN) is made from the saliva of swiftlet species, Aerodromus fuciphagus.Swiftlets may stay in natural caves or man-made swiftlet house.EBN has become an important cuisine and pharmacy since the beginning of Tang (618-907 AD) dynasty as it is high in medical values with properties of anti-aging, anti-cancer, immune-enhancing, dissolving phlegm, suppressing cough and others (Ma and Liu, 2012).
Traditionally in Chinese culture, EBN drink is a type of delicate food prepared by double boiling the EBN with rock sugar.However, double boiled EBN has low solubility.Thus, enzymatic protein hydrolysis may help to increase EBN solubility and thus maximize its functionality, digestibility and bioactivity.EBN hydrolysate may offer more convenient and marketable value-added ingredients for food and nutraceutical application.Nowadays, EBN is marketed as raw cleaned nest, ready to drink soup, health supplement and cosmeceutical products.
Protein hydrolysis is commonly used to modify physicochemical and bioactivity properties of EBN.Marcone (2005) reported that glycoprotein is the main component in EBN.Guo et al. (2006) have found that the enzymatic hydrolysis of EBN by Pancreatin F hydrolyzed glycoprotein to glycopeptides that have the ability to inhibit influenza virus.Matsukawa et al. (2011) reported that hydrolysis of EBN by Pancreatin F consisted of water-soluble compound like chondroitin glycosaminoglycans (GAG) which is the main component in the bone that can prevent osteoporosis.Epidermal growth factors are also present in EBN and EBN treated with HCl (Kong et al., 1987;Rashed and Nazaimoon, 2010).Furthermore, hydrolysis of EBN using Alcalase® has resulted in angiotensin-I converting enzyme (ACE)inhibitory activity (Amiza et al., 2014;Babji et al., 2018), while enzymatic hydrolysis of EBN has also resulted in antioxidative activity (Ghassem et al., 2017;Babji et al., 2018).
Enzymatic hydrolysis by commercial exogenous food-grade enzymes is more favourable due to better control in terms of properties of the hydrolysates such as the length of the peptide and resulted in desirable and consistent products.The four main factors that affect the enzymatic hydrolysis process are types of enzyme, pH, temperature and time (Bhaskar and Mahendrakar, 2008).In addition, pH and temperature are mainly depending on the type of enzyme as different enzymes have their own range of pH and temperature where they are active.
According to Ovissipour, Abedian, Motamedzadegan et al. (2009), hydrolysis time and enzyme concentration will affect the degree of hydrolysis (DH) where higher DH will result in longer hydrolysis time.Thus, optimization of enzymatic hydrolysis of EBN protein is important to control the enzymatic hydrolysis process and as a basis to understand the structure-function relationship in EBN.Alcalase® is one of the most effective commercial enzymes to prepare food protein hydrolysis (Shahidi et al., 1995).Previous study has been reported on the optimization of enzymatic hydrolysis of various foods including dark muscle tuna (Saidi et al., 2013); palm kernel cake protein (Ng et al., 2013); visceral waste proteins of Catla catla (Bhaskar et al., 2008); pine seed protein (Wang et al., 2011); and fish soluble concentrate (Nilsang et al., 2005).It will be interesting to know how hydrolysis of glycoprotein in EBN differs from other food proteins.
Response Surface Methodology (RSM) is a collection of statistical and mathematical technique used in the study of optimization in order to solve multivariate problem, graphically represent as response surface (Raissi and Farsani, 2009).Based on the experimental data, RSM may be used to fit and predict a mathematical model to explain the relationship between the experimental variables and its response as well as to determine the optimum condition for a specific response.
Thus, this study aimed to optimize the enzymatic protein hydrolysis conditions on the DH of EBN using Alcalase®.Besides, the proximate analysis and amino acid composition of EBN hydrolysate prepared under optimum condition were determined as well.

Raw material
EBN was purchased from a swiftlet farm in Gong Badak, Terengganu.The raw material purchased was in raw uncleaned form.The raw uncleaned EBN was stored in a plastic container at room temperature until further use.The food grade commercial enzyme used for in this study was Alcalase ® 2.4 L in liquid form (2.4 AU/g), purchased from Novo Industry (Denmark).All other chemical reagents used were of analytical grades.

Preparation of raw material
Prior to protein hydrolysis, the raw uncleaned bird's nest was cleaned by soaking them in water until the nest cement is softened and the tightly bound laminae partially loosen.After that, it was rinsed many times with water to remove its contaminants (guano, cracked eggs, dust) and feathers.Next, the small feather and fine plumage were removed manually by using fine tips tweezers and with the aid of magnifying lens.The cleaned bird's nest was then kept in a container and stored in freezer (-20 o C) until further use.

Determination of proximate analysis of EBN sample
The raw cleaned EBN and the lyophilized EBN hydrolysate prepared under optimum condition were subjected to proximate analysis according to AOAC method (AOAC, 2000).Prior to optimization study, protein content of raw cleaned EBN is needed to calculate the mass of EBN, water and enzyme needed for each run of enzymatic hydrolysis (Kristinsson and Rasco, 2000).

Experimental design for optimization study
There are four independent variables involved in this optimization study.The independent variables are temperature (°C), enzyme to substrate concentration (E/ S, %v/w), hydrolysis time (mins) and pH was employed at equidistant level (-1, 0 and +1) as shown in Table 1.The ranges of the independent variables used were chosen based on the previous study on optimization of enzymatic hydrolysis from silver catfish (Pangasius sp.) frame by using Alcalase ® (Amiza et al., 2011).Degree of hydrolysis (DH) was set as a dependent factor or response variable in this study.
The experimental design used in this study was a face-centered central composite design.A total of 30 runs of protein hydrolysis of EBN using various combinations of independent variables were automatically generated by Design Expert software (Version 6.0.10)(Stat Ease Inc.).There were 6 center points set in this experimental design.After each run, the resulting hydrolysate was freeze-dried.Then, the lyophilized powder was subjected to determination of DH.The data on DH was further analyzed using Design Expert software (Version 6.0) (Stat Ease Inc.).

Enzymatic hydrolysis of EBN
Firstly, the frozen cleaned raw EBN was thawed.Then, the mass of the raw material and water was calculated as described by Kristinsson and Rasco (2000).Enzymatic hydrolysis of EBN was employed as described by Amiza et al. (2014).For each run, the EBN sample was added to distilled water and homogenized using homogenizer (IKA ® T18 basic, IKA Works Inc, USA) at 10,000 rpm for 2 mins.Prior to enzymatic hydrolysis, the homogenized EBN was boiled for half an hour in a water bath (TE-10D, Techne ® , UK) and allowed to cool to room temperature afterward.After cooling, the temperature and the pH of the solution were adjusted (amount of pH added included in the mass of FULL PAPER water needed for hydrolysis), before the addition of enzyme.After that, an Alcalase ® enzyme solution was added to the homogenize EBN and the hydrolysis process was carried out using water bath shaker (Shaker bath-903, Protech ® , UK) at the specific temperature and time as designated by Response Surface Methodology.After the hydrolysis process was completed, the enzyme was inactivated at 85°C for 20 mins in the water bath.The resultant mixture was freeze-dried, and the lyophilized hydrolysate sample was crushed into powder and kept in an airtight container until further analysis.

Determination of degree of hydrolysis
Determination of DH was carried out using trichloroacetic acid (TCA) method (Hoyle and Merritt, 1994).Total nitrogen content was determined by analyzing 0.5 g of freeze-dried EBN powder using Kjeldahl method.As for the determination of 10% TCA soluble nitrogen, 0.5 g of freeze-dried EBN powder was added to 10 mL of distilled water.Then, 10 mL of 20% (w/v) TCA was mixed with the sample.The sample was left to stand for 30 mins for precipitation.Later, the sample was centrifuged (High speed centrifuges model 1580R, Gyrozen Co., Ltd, Korea) at 4000 rpm for 15 mins.The resulting supernatant was filtered and analyzed using Kjeldahl method (AOAC, 2000).DH is defined as the percentage ratio between the number of peptide bonds cleaved and the total number of peptide bonds in the sample (Adler-Nissen, 1979).DH was computed as follows:

Verification for optimum condition
After optimization, the suggested solution for the optimum condition was obtained.From the optimum condition, enzymatic hydrolysis of the suggested condition was carried out in quadruplicate.Then, the degree of hydrolysis of lyophilized hydrolysate resulted from the hydrolysis were determined.The predicted value given by Response Surface Methodology (RSM) was then compared with the experimental value obtained using one sample t-test.

Determination of amino acid composition
Amino acid composition of the lyophilized EBN hydrolysate was analyzed using amino acid analyzer (L-8800 Hitachi) (Guo et al., 2007).Two types of hydrolysis were carried out i.e. 6N HCl and performic acid.Only 17 types of amino acids are quantifiable using this method, excluding tryptophan.There is a limitation of the acid hydrolysis method, whereby cysteine, methionine and tryptophan could not be accurately quantified due to partial oxidation of cysteine and methionine, and complete destruction of tryptophan.Apart from that, deamidation of asparagine and glutamine to aspartic acid and glutamic acid, respectively, will lead to a mixture quantification for asparagine with aspartic acid and glutamine with glutamic acid.To quantify tryptophan, alkaline hydrolysis is needed, and it is not carried out in this study.

Data analysis
All data was stated as mean ± standard deviation.For optimization study, the experimental run and data analysis were carried out by using the Design Expert software Version 6.0.10 (Stat Ease Inc., USA).Analysis of variance of the RSM model was carried out using Design Expert software (version 10.0.7) (StatEase Inc., USA).The experimental data from the experiments were fitted into an empirical second-order polynomial using steepest accent method according to the following equation: Here, Y is the response variable.β 0 is the offset term.βi , βii, and βij are the linear, quadratic, and interaction regression coefficient variables, respectively.While Xi

Experimental data for optimization study
Response surface methodology was used to optimize the protein hydrolysis of EBN using Alcalase ® .The independent variables used in this study were temperature, pH, hydrolysis time and Alcalase ® concentration.Degree of hydrolysis (DH) was set as an independent variable in order to measure the cleavage of the peptide bond in the EBN.DH of EBN ranged from 14.27-37.89%.The highest DH (37.89%) was obtained at the temperature of 65°C, pH of 9.5, hydrolysis time of 60 mins and enzyme concentration of 2%.Meanwhile, the lowest DH (14.27%) was obtained at 45°C, pH of 7.5, hydrolysis time of 60 mins and enzyme concentration of 1% The range of DH obtained in this study is in similar range to those of EBN hydrolysis using Protamex® (13.79-33.26%)(Amiza et al., 2019) and visceral waste protein of beluga sturgeon (Huso huso) hydrolysis using Alcalase® (11.28-30.87%)(Ovissopour et al., 2009b).However, it was higher compared to those of Grass carp skin hydrolysis which ranged from 1-15.2% (Wasswa et al., 2008) and lower compared to salmon skin hydrolysate which ranged from 43.98-74.54%(See et al., 2011).The difference in DH obtained between these studies could be due to the difference in protein source used, the type of enzyme used as well as the different range of parameters applied during enzymatic hydrolysis that may promote proteolysis.Different protein source will have different types of protein and amino acid composition which may lead to different proteolytic susceptibility, thus resulting in different DH.Different enzymes will work differently during proteolysis depending on their preferred amino acid to be cleaved during proteolysis.For example, okara protein hydrolysates produced by Alcalase® and Flavourzyme™ gave DH values of 33.6% and 5.8%, respectively, indicating that Alcalase® cleaved more peptide bonds than Flavourzyme™ (Sbroggio et al., 2016).However, Bamdad et al. (2011) reported that Flavourzyme hydrolysis (100 LAPU/g protein) of hordein was relatively more extensive than the hydrolysis by Alcalase (0.24 AU/g protein), which resulted in lower DH values.Thus, in order to maximize the DH, we need to optimize the enzymatic hydrolysis condition for a particular protein source.

Analysis of variance (ANOVA) for degree of hydrolysis
ANOVA table is used to summarize the test for significance of regression models, test for significance for individual model coefficient and test for lack of fit (Kohli and Singh, 2011).In this study, Design Expert software has suggested two-factor interaction (2FI) model for the EBN hydrolysis using Alcalase®.Then, the model reduction was performed to further improve the model.The model reduction was carried out by excluding all insignificant model terms (i.e.model terms with p value>0.05).Table 2 shows the ANOVA table of Response Surface Reduced 2FI model for degree hydrolysis.It is important to examine the fitted model to ensure adequate approximation is provided to the true system.Table 2 shows the model F-value of 21.56, which implies that the model is significant.There is only a 0.01% chance that a 'Model F-Value' could occur due to noise.The "p-value" for "Lack of Fit" of 1.42 implies that the "Lack of Fit" is not significant relative to the pure error.There is a 37.44 % chance that a "Lack of Fit-F value" could occur due to noise.The non-significant "Lack of Fit" is good in optimization study because it shows how well the model fits well with the experimental data.The coefficient of determination (R 2 ) of 0.8179 was in good agreement with the adjusted R 2 of 0.7800.The ANOVA table shows that all linear model terms (pH, temperature, enzyme concentration, hydrolysis time) and one interaction model term (AD, which was interaction between temperature and pH) affected the DH significantly.Based on the sequential model sum of squares, the appropriate models were selected based on the highestorder polynomial where the additional terms were significant.The experimental result in this study was evaluated and the model equation for DH of EBN as a function of four independent variables was generated.In this study, the model suggested for enzymatic protein hydrolysis of EBN is a two-factor interaction (2FI) model.However, most of the previous studies suggested quadratic models to predict the DH of an enzymatic protein hydrolysis.For example, optimization of DH from EBN using Protamex® (Amiza et al., 2019), visceral waste protein of beluga sturgeon Huso huso (Ovissipour, Taghiof, Motamedzadegan et al., 2009), grass carp (Ctenopharyngodon idella) skin (Wasswa et al., 2007), silver catfish (Pangasius sp.) frame (Amiza et al., 2011), Salmon (Salmo salar) skin (See et al., 2011), tuna dark muscle by-product (Saidi et al., 2013) and threadfin bream (Nemipterus japonicus) (Normah et al., 2005).The difference in the model between this study and the above-mentioned protein sources could be due to the same reasons as mentioned previously for differences in DH.

Response surface plot and effect of factor for degree of hydrolysis
Figure 1 shows the contour plots that represented a function of two factors which are temperature and pH and the other factors such as enzyme concentration and time was hold at optimum level.The figure shows that the DH increased with an increased in temperature and pH.In this study, pH and temperature were maximum at its maximum points.This study was in an agreement with the hydrolysis of Grass carp skin using Alcalase® (Wasswa et al., 2008) where an increase of DH during was achieved by increasing the temperature to 60°C and pH 9.Besides that, enzymatic hydrolysis of silver catfish (Pangasius sp.) frame (Amiza et al., 2011) also found that DH was increased with pH.The optimum pH of silver catfish (Pangasius sp.) frame was pH 9.5.
In this study, the optimum condition for EBN in terms of DH was highest at pH at 9.5.This is because Alcalase ® showed a broad range of activity in alkaline pH.The optimum pH range for Alcalase ® to function is ranged from pH 6 to pH 10 ( Aspmo et al., 2005).pH can affect both the substrate and enzyme by changing the distribution and confirmation of the molecules at very acidic or alkaline condition.Enzyme tends to undergo irreversible denaturation and loss of stability (Benjakul and Morrisey, 1997).A sharp decrease in Alcalase ® activity at pH 11.5 was reported by Benjakul and Morrisey (1997).
Besides that, the protein present in EBN was glycoprotein that is a combination of carbohydrate and protein.According to Roussel et al. (1988), glycoprotein found in EBN contain hundreds of carbohydrate chains attached to the peptide by O-glycosidic linkages between N-acetylgalactosamine and a hydroxylated amino acid made from serine and threonine.The O-glycosidic linkages are alkaline labile (Shylaja and Seshadri, 1989).This may explain why in alkaline condition (pH 9.5), EBN gave the highest DH.The breakage of Oglycosidic linkages enhanced the protein hydrolysis by Alcalase® enzyme.Furthermore, the optimum condition for EBN in terms of DH was highest at the temperature of 65°C.Benjakul and Morrisey (1997) have found that above 70°C , enzyme activity decrease due to thermal irreversible denaturation of enzyme.This finding is in an agreement with Baek and Cadwallader (1995) whereby they also reported that the optimum temperature of Alcalase ® on crayfish processing by-product was at the temperature of FULL PAPER Figure 1.Response surface plot of degree of hydrolysis (%) of EBN hydrolysate as a function of temperature (°C) and pH during enzymatic hydrolysis of EBN using Alcalase® 70°C.Thus, the range of temperature and pH should be increased in order to get the curvature ideally within the midpoint of the two factors.

Optimization of degree of hydrolysis
For optimization purposes, the importance of all four independent variables in this study was set at 3 or (+ + +).While the response variable of this study (DH) was considered the most importance by setting the goal to the maximum and the "Importance" has been set as 5 or (+ + + + +).
After this limitation was set, the optimized test was evaluated by Design Expert Software version 6.0 and the 10 best solutions of optimization conditions for further evaluation were shown in Table 3.The desirability of the 10 optimization conditions was closed to 1.The desirability function brings the meaning of "goodness" of a specific setting.If the desirability is close to 1, this indicates a desirable set of various response (Vining and Kowalski, 2011).Solution No. 1 was selected, giving the optimum condition at the temperature of 64.99°C, Alcalase® concentration of 2%, hydrolysis time of 179.55 min and pH of 9.46.The optimum conditions obtained in this study is in agreement with that of protein enzymatic hydrolysis of blood cockles whereby the optimum were at temperature of 65°C, enzyme concentration of Alcalase® at 2%, hydrolysis time at 180 mins and pH at 9.5 (Amiza and Masitah, 2012).However, it is quite different compared to that of EBN hydrolysis using Protamex® (Amiza et al., 2019) which were at the temperature of 59.9 o C, pH of 6.3, enzyme concentration of 2% and hydrolysis time of 5.4 hr (or 324 mins).This could be due to the different enzymes used to hydrolyze EBN, whereby Alcalase is active in alkaline region (6.5-8.5) and Protamex® is active in neutral region (pH 5.5-7.5).The only similarity between the optimum condition in these two EBN studies is the optimum enzyme concentration at 2%.The optimum condition for EBN hydrolysis using Alcalase® is at a shorter time, but higher pH and temperature as compared to Protamex®.

Verification of optimum conditions
By using the optimum condition at the temperature of 64.99°C, Alcalase® concentration of 2%, hydrolysis time of 179.55 min and pH of 9.46., the predicted value of the response variable (DH) was 37.90%.To validate the model, four replicates of EBN hydrolysis were carried out under optimum condition as suggested by this study.A one sample t-test showed that there was no significant difference between the experimental value (37.92%) and the predicted value (37.90%) (p<0.05).This shows that the equation fitted well with the experimental data in this study.

Proximate analysis of EBN and protein hydrolysate
Proximate analysis was carried out on soaked cleaned raw EBN and lyophilized EBN hydrolysate prepared under optimum condition.The EBN hydrolysate was prepared using the hydrolysis conditions that gave the highest DH based on experimental data at the temperature 64.99°C, enzyme concentration of Alcalase® at 2%, hydrolysis time at 179.55 min and pH at 9.46.Table 4 shows the proximate analysis of the soaked cleaned raw EBN and freeze-dried EBN hydrolysate (dry basis).
The lyophilized EBN hydrolysate has lower moisture content (3.85%) as compared to the moisture contained in soaked raw EBN (91.26%).The huge differences in moisture content are due to uncleaned raw EBN was soaked in water up to 12 hours to expand so that the nest cement is softened, and tightly bound laminae were partially loosen.During soaking, the raw EBN absorbed water and result in high moisture content.According to Severin and Xia (2005), the differences in moisture content may arise due to the varying efficiency of freezedrying and storage conditions.For a fair comparison, both EBN samples should be converted to dry basis (Table 4).In dry basis, protein gave the highest composition in both cleaned raw EBN (70.55%) and lyophilized hydrolysate powder (67.63%).According to Ma and Liu (2012) and Marcone (2005), the protein content of dry EBN (in wet basis) was in the range of 42% to 63%.This shows that the protein content of EBN samples in this study was slightly higher compared to the previous study by Ma and Liu (2012) and Marcone (2005).The difference in protein content could be due to differences in the source of EBN, cleaning extent and moisture content between the samples.
It was found that the ash content in the cleaned EBN was higher than EBN hydrolysate which was 11.59% and 2.52%, respectively.The ash content in EBN hydrolysate was lower than raw EBN.The ash content in EBN hydrolysate was in reasonable agreement with Ma and Liu (2012) that was in the range of 2.1% to 7.3%, but it was a bit higher for raw EBN.The pH of cooked EBN was around pH 7. Thus, NaOH was added to adjust the pH to alkaline range prior to enzymatic hydrolysis.Severin and Xia (2005) and Benjakul and Morrissey (1997) have reported that the addition of NaOH in conjunction with the adjustment of pH before enzymatic hydrolysis caused an increased in ash content.However, in this study, the increase in ash content due to NaOH addition was not observed.This loss of mineral/ash in FULL PAPER raw materials could be due to leaching of mineral from soaked cleaned raw EBN during rinsing and storage.
The fat content (dry basis) in soaked clean raw EBN (5.43%) was higher compared to EBN hydrolysate (0.62%).Previous studies on EBN has reported that the fat content in EBN was in the range of 0.14-1.28%(Marcone, 2005;Ma and Liu, 2012).This shows that the fat content in cleaned raw EBN was quite high compared to the previous study.However, the fat content in the EBN hydrolysate is consistent with the previous study.This discrepancy could be due to loss of some fat from the raw EBN during storage, heat treatment, hydrolysis and freeze-drying process.Heating treatment carried out before enzymatic hydrolysis (cooking for half an hour) and after enzymatic hydrolysis (85 o C for 15 mins) may help to remove fat from raw materials (Bhaskar et al., 2008).Low-fat content is desirable because it will increase the stability of the hydrolysate towards lipid oxidation (Ovissipour, Taghiof, Motamedzadegan et al., 2009).
Lastly, the carbohydrate content of raw EBN (12.43%) was lower compared to EBN hydrolysate (29.14%).The values are consistent with the previous study by Ma and Liu (2012) and Marcone (2005), whereby the carbohydrate content of EBN ranged from 10.63% to 27.26%.Since carbohydrate content is calculated by the difference method, it is directly affected by other proximate composition of the EBN.

Amino acid composition in lyophilized EBN hydrolysate
Table 5 shows the amino acid composition of lyophilized EBN hydrolysate (stated in percentage).The total amount of amino acid i.e. 60.17±1.03%was quite close to the protein content of EBN hydrolysate of 65.03% (wet basis).Table 5 shows that there were 16 types of amino acids found in EBN hydrolysate using 6N HCl hydrolysis and performic acid oxidation.Thus, tryptophan is expected to be absent in the amino acid composition in this study.Marcone (2005) reported 15 types of amino acids present in EBN while Babji et al. (2018) reported 18 amino acids in EBN and EBN hydrolysate.The difference in the EBN amino acid composition in these studies is mainly due to the difference in preparation methods prior to HPLC analysis.Acid hydrolysis with HCl will result in 15 types of amino acids to be quantifiable by HPLC, because cysteine, methionine and tryptophan will be destroyed in the acid hydrolysis.Further treatment with performic acid oxidation will enable another 2 amino acids to be quantifiable i.e. cysteine and methionine.To quantify tryptophan, alkaline hydrolysis has to be carried out as well.Marcone (2005) only reported 15 types of amino acids because he only performs acid hydrolysis, while this study reported 16 types of amino acid because we performed acid hydrolysis and performic acid oxidation (cysteine supposed to be quantifiable, but not detected in our EBN hydrolysate sample).Meanwhile, Babji et al. (2018) reported 18 types of amino acid because they performed acid hydrolysis, performic acid oxidation and alkaline hydrolysis (cysteine and tryptophan were present in their EBN sample).Furthermore, the difference in amino acid composition between these studies could be due to differences in the composition of EBN obtained from different locations and in the degree of cleanliness of EBN from contaminants (such as fine FULL PAPER plumage, guano and eggshell).The habitat of swiftlets will affect the types of feed that they consume and may result in different protein content, which may indirectly affect the types of amino acid present on it (Norhayati et al., 2010).
Table 4 shows that the amino acid content in EBN hydrolysate in this study has a similar trend with that of Babji et al. (2018).This could be due to both hydrolysates were hydrolyzed using Alcalase®.As shown in Table 4, EBN hydrolysate contained 8 types of essential amino acids needed for adults except for tryptophan (which was not quantifiable using the method in this study).In fact, except for methionine, the levels of all other essential amino acid presented in EBN hydrolysate were higher than the suggested profile of essential amino acid requirements for adults (FAO/ WHO, 2007).Thus, the essential amino acid in EBN fulfilled this requirement and it can be suggested as a good source essential amino acid for nutritional value (Vidotti et al., 2003).The highest composition of essential amino acid in EBN was valine (9.11%) while the highest composition of non-essential amino acid in EBN was serine (12.19%) which is similar to the previous study reported by Marcone (2005) and Babji et al. (2018).In EBN hydrolysate, the hydrophobic amino acids are valine (9.11%), leucine (7.45%), isoleucine (3.49%), phenylalanine (5.61%), alanine (3.85%) and tyrosine (4.53%), accounted for 34.04%.Since hydrophobic amino acid is always associated with the presence of antioxidative and ACE inhibitory peptides, this indicates that EBN hydrolysate is a potential source of antioxidant and ACE inhibitory peptides to be explored.

Conclusion
In conclusion, the optimum condition for enzymatic protein hydrolysis of EBN was at the temperature of 64.99°C, Alcalase ® concentration of 2%, pH of 9.46 and hydrolysis time of 179.55 mins.The experimental DH obtained under this optimum condition (37.92%) was close to the predicted value (37.90%).The suggested model for enzymatic protein hydrolysis of EBN was twofactor interaction (2FI) model.The proximate composition of lyophilized EBN hydrolysate in dry basis consisted of 67.63±0.78%protein, 2.62±0.37%ash, 0.61±0.30%fat and 29.14±0.11carbohydrate.The EBN hydrolysate contained all essential amino acids except tryptophan (which was not quantifiable using the method in this study) and their levels were higher than the suggested profile of essential amino acid requirements for adults (FAO/WHO, 2007), except for methionine.EBN hydrolysate contained 34.04% hydrophobic amino acid.

FULL PAPER
The final equation in terms of coded factors given by the Design Expert software was: Degree of Hydrolysis = + 23.54 + 3.47 * A + 2.51* B + 2.63* C + 4.48* D + 1.52* A*D Where, A = Temperature; B = Enzyme Concentration; C = Hydrolysis time; and D = pH.For the final equation in terms of coded factors, the higher the coefficient of estimation for a particular term, the stronger the effect of the term towards DH.The positive sign of the coefficient of estimation for all factors (A, B, C and D) shows that each individual factor gave linear effect which resulted in increased DH.For linear effect, DH was most affected by pH (D)> temperature (A) > hydrolysis time (C) > enzyme concentration (B).While for AD, it also gave an increasing effect on DH.

Table 1 .
Experimental range and values of the independent variables used in the optimization of protein enzymatic hydrolysis conditions on the degree of hydrolysis of edible bird's nest and Xj are experimental factors.For the models calculated from the linear regression, analysis of variance (ANOVA) was performed and the R 2 value, residual error, pure error and lack of fit were calculated.

Table 3 .
Solutions for optimization conditions of EBN hydrolysis to obtain maximum degree of hydrolysis

Table 4 .
Proximate analysis of soaked raw edible bird's nest and lyophilized edible bird's nest hydrolysate prepared at optimum condition

Table 5 .
Amino acid composition of lyophilized edible bird's nest hydrolysate prepared at optimum condition as compared to those of other study a Values are % of amino acids expressed as mean±SD (n=2); b Suggested profile of essential amino acid requirements for adults (FAO/WHO, 2007); *Tryptophan was not analyzed in this study