Abstract: Whey protein concentrate (WPC 80) was modified by partial hydrolysis with Protamex to 5, 10, 15 and 20% degree of hydrolysis (DH). Whey protein concentrate 80 and its hydrolysates were analyzed, compared to FAO/WHO/UNU amino acid requirement pattern based on amino acid requirements of preschool-age child. The enzymatic hydrolysis of WPC 80 leads to numerous alterations in protein functional characteristics, like changes in solubility. The pH-protein solubility profiles of WPC 80 and its hydrolysates were used to determine protein solubility. A modified method of AOAC was used to determine Cysteine, Branched-Chain Amino Acids (BCAA) and Tryptophan at various degree of hydrolysis. Results showed that modified whey protein concentrate by partial hydrolysis degree at 15% were highly solubility and the global amino acids determined in an optimal content that makes them appropriate for food formulations as a source of health benefits amino acids.
INTRODUCTION
Whey protein concentrate standardized to 80% protein content (WPC 80) is the ideal protein used in a wide range of food applications has a health benefits (Farthing, 2001; Low et al., 2003; Kent et al., 2003; Hazen, 2003). Kawase et al. (2000) found that supplement fermented milk with an added whey protein concentrate would affect serum lipids and blood pressure.
Today, whey protein is often described as a nutritionally perfect protein in the sense that it contains all the essential and non-essential amino acids required by the human body. Wheys amino acid profile is closely related to the optimal physiological needs of the human body, including an abundance of sulfur-containing amino acids, all in a highly bioavailable form also features the highest percentages of Branched-Chain Amino Acids (BCAA) (Walzem and Dilland, 2002; Pacheco and Sgarbieri, 2005).
Sometimes whey protein concentrate is modified by partial hydrolysis with enzymatic treatment (Kleber et al., 2006; Bernasconi et al., 2006) that transfer part of the protein into peptides to improve some properties with enrich of product supplemented (Konrad et al., 2005). Partial hydrolysis can be done to the whey proteins product e.g., cheese using different protease and obtained product containing bioactive peptides that are free of any bitterness (Mann, 2000; Nelson et al., 2002).
In recent years, a lot of scientific interest has been focused on physiologically active peptides deriving from milk upon hydrolysis. Partial hydrolysis of whey proteins can either change or evidence the functional properties of the peptides in this industrial residue, thereby increasing their applications. The hydrolysates obtained by treatment Whey Protein Concentrate (WPC) with pancreatin and protamex are good sources of peptides with activity to stimulate glutathione synthesis (Pacheco and Sagarbieri, 2005). The degree of hydrolysis is important to point out that competition for the active site between the original substrate and the peptides.
This process can be performed to different degrees; that is, vastly different size peptides can be produced having different functional characteristics. Furthermore, hydrolysis can yield a variety of new peptides that may offer many physiological benefits for humans (Otte et al., 1997).
The aim of this investigate was to evaluate the meaningful health benefits of this modified whey protein at different degree of WPC 80 hydrolysis (DH) through the amino acid pattern (mg g-1 protein) and identify the ideal degree of hydrolyzation have unique a health benefits amino acids content.
MATERIALS AND METHODS
Whey protein concentrate (WPC 80, 80% protein based on dry weight) was obtained from Davisco Foods International, Inc., U.S. Protamex, a commercial Bacillus proteinase complex, was obtained from Novo Nordisk’s Enzyme Business (Wuxi, China). It is an endopeptidase with a broad specificity to hydrophobic amino acids.
Prior to enzymatic treatment the aqueous solution of WPC 80 was allowed to hydrate for 1 h at room temperature with gentle mixing, adjusted to pH 4.6 with 2 mol L-1 HCl and heated to 85°C for 30 min to denature WPC 80. The protein solution was then equilibrated at 50°C and the pH for Protamex hydrolysis was adjusted to 8.0 with 1 mol L-1 NaOH before addition of enzyme.
Preparation of Hydrolysates and Degree of Hydrolysis
Preparation of Hydrolysates
Whey Protein Concentrate (WPC 80) was reconstituted at 50°C in distilled
water to give a starting protein concentration of 5% (w/v). Prior to enzymatic
treatment the aqueous solution of WPC 80 was allowed to hydrate for 1 h at room
temperature with gentle mixing, adjusted to pH 4.6 with 2 mol L-1
HCl and heated to 85°C for 30 min to denature WPC 80. The WPC solution was
then equilibrated at 50°C and initially the pH adjusted to 8 using 0.5 N
NaOH to avoid any coagulation during enzymatic hydrolysis of protein to improve
digestibility (Sindayikengera and Xia, 2006). Controlled
enzymatic hydrolysis whey protein concentrate process by heating the reaction
mixture at 90°C for 10 min.
Proteolytic enzyme (1.5 AU g-1) was added at a rate of 0.40 AU per 1 g of WPC 80. Partial hydrolysis of WPC solution was carried out at 50°C under appropriate conditions. The degree of hydrolysis (DH) was monitored using the pH-stat technique Adler-Nissen (1986). During hydrolysis, samples were withdrawn after different times (min) and the enzymes were inactivated by heating the reaction mixture for 10 min at 90°C. The supernatants were taken as WPC hydrolysates and the precipitates were discarded. The WPC hydrolysates were stored at -20°C for subsequent estimation of degree of hydrolysis (DH).
Calculation of the Degree of Hydrolysis (DH) Using the Ph-Stat Technique
The hydrolysis was carried out using the pH-stat method described by Adler-Nissen
(1986) and the DH (%) was calculated from the volume and the normality of
alkali used to maintain constant pH 8.
Sodium hydroxide (0.5 N) was utilized to monitor the consumption of a titrating, necessary to control the system pH during batch hydrolysis assays carried out. It calculated the number of peptide bonds hydrolyzed by the enzyme.
Where:
| DH | = | Degree of hydrolysis |
| B | = | The base consumption (mL) |
| Nb | = | Base normality |
| α | = | The average degree of dissociation of a-NH groups (the values presented by Adler-Nissen (1986)) |
| MP | = | The mass of protein (g) |
The modified whey protein at 5, 10, 15 and 20% DH were under investigation.
Determination Amino Acid Composition
A modified method of AOAC 982.30a (AOAC, 1990) was
used to determine Met-Cys and Branched-Chain Amino Acids (BCAA), except tryptophan.
Sixty milligrams of freeze-dried sample were hydrolyzed with 8 mL of 6 mol L-1
HCl under vacuum at 110°C for 24 h. After cooling, the hydrolysate was washed
with distilled water, filtered (Whatman No. 2) and dried at 60°C (also under
vacuum) in a rotary evaporator. The dried sample was then dissolved in 0.01
mol L-1 HCl. The amino acids in the hydrolysate were separated and
quantified by injecting 50 μL into a Hitachi 835-50 amino acid analyzer
equipped with a 2.6 x150 mm2 ion exchange column coated with resin
2619#. The column temperature was 53°C. Sodium citrate buffers
(pH 3.3, 4.3 and 6.3) were used as eluents with a flow rate of 0.225 mL min-1.
The light absorbance of the amino acids was detected with a 166 Detector (Beckman
Instruments) at 570 nm and the amino acids were quantified by comparing them
with amino acid profiles from external amino acid standard.
Determination of Tryptophan
Tryptophan was estimated by the ninhydrin method of Pintér-Szakács
and Molnán-Perl (1990). One gram of sample was introduced into a 25 mL
polypylene test tube with caps and then 10 mL of 0.075 mol L-1 NaOH
was added and mixed until there were no lumps. The dispersion was shaken for
30 min and centrifuged at 5000 r min-1 for 10 min and the supernate
was transferred to a clean test tube. To 0.5 mL of supernate, 5 mL of ninhydrin
reagent (1.0 g of ninhydrin in 100 mL mixture of 37% HCl and 96% HCOOH at a
ratio of 2:3) was added and then solution was incubated at 35°C for 2 h
and then cooled to room temperature after which the volume was made up to 10
mL with diethyl ether, thoroughly mixed with a Vortex mixer, filtrated and the
clear filtrate was read at 380 nm. A standard tryptophan curve was prepared
using 0~100 μg tryptophan. From the standard graph, the concentration of
tryptophan was calculated and expressed as g/100 g protein. Measurement the
pH-protein solubility profiles of WPC 80 and its hydrolysates (Protein solubility)
(PS).
Protein Solubility (PS) was determined in duplicate by the method of Bera and Mukherjee (1989). Two hundred milligrams of proteins were dispersed in 10 mL of deionized water. The pH of suspensions was adjusted to different levels (2.0 to 8.0) by using 1 mol L-1 HCl or 1 mol L-1 NaOH. The suspensions were stirred at room temperature for 30 min and then centrifuged at 10000x g for 30 min (Kika Ultra Turrax T18 basic, Germany). Protein contents in supernates were determined by Kjeldahl method (Ceirwyn, 1995). The percentage of protein solubility in each suspension was calculated by the ratio of protein in the supernate to protein in 200 mg sample.
RESULTS
Enzymatic protein hydrolysis is the degradation of proteins into peptides and/or amino acids by proteolytic enzymes. During protein hydrolysis amide bonds are cleaved and, after addition of a water molecule, peptides and/or free amino acids are released. The most commonly used parameter describing the result of a hydrolysis process is the degree of hydrolysis (DH), used as an indicator of the extent of hydrolysis.
Solubility
The whey protein solubility was measured in the pH range of 2 to 8 (the
pH-protein solubility profiles of WPC 80 and its hydrolysates; (Fig.
1). Results showed that WPC 80 and its hydrolysate had minimum solubility
at pH 4.0-5.0. Solubility in the pI range increased from 75.0 to 77.2, 79.0,
81.1 and 86.1% for WPC 80 and its hydrolysates at 5, 10, 15 and 20% DH, respectively.
At pH away isoelectric point (pI), the number of ionizable groups increased specially in which related to the peptides formed from the hydrolysate that improved the solubility. The WPC 80 and its hydrolysates had the highest solubility values at the both acidity pH range between 2.0 and 3.0 and alkaline pH range between 7.0 and 8.0. The results showed also that changes in solubility with DH were small at 5 and 10% DH and became more noticeable up to 15 and 20% DH for WPC 80.
The Degree of Hydrolysis and the Health Benefits Amino Acid Content
Results showed that WPC 80 and its hydrolysates were the high content of
sulfur-containing amino acids (Met and Cys), so WPC 80 had 79.0 mg g-1
protein and at 5, 10, 15 and 20% DH had 40.2, 43.1, 41.4 and 42.0 mg g-1
protein, respectively as shown in (Fig. 2) whereas FAO/WHO/UNU
recommended requirements of preschool-age child were 25.0 mg g-1
protein.
| Fig. 1: | Protein solubility of WPC 80 and its hydrosylate |
The three Branched-Chain Amino Acids (BCAA) are Leucine, Isoleucine and Valine. Leucine in WPC 80 had 106.0 mg g-1 protein and at 5, 10. 15 and 20% DH had 104.0, 109.0, 107.4 and 105.2 mg g-1 protein respectively whereas the optimal physiological needs of the human body are 66.0 mg g-1 protein as recommended by FAO/WHO/UNU (Fig. 3-5). It is obvious that Leucine less sensitive to dehydrolyzation. Isoleucine in WPC 80 had 48.6 mg g-1 protein and at 5, 10. 15 and 20% DH had 53.0, 52.0, 52.5 and 48.2 mg g-1 protein, respectively whereas the optimal physiological needs of the human body are 28.0 mg g-1 protein. Valine in WPC 80 had 17.5 mg g-1 protein and at 5, 10. 15 and 20% DH had 52.5, 50.0, 53.4 and 48.2 mg g-1 protein, respectively whereas the optimal physiological needs of the human body are 35.0 mg g-1 protein.
The nutritional quality of the protein used in the formulation could be extrapolated to the expected tryptophan content. Tryptophan content in WPC 80 was 17.0 mg g-1 protein and the requirement pattern is 11 mg g-1 protein. With increasing the DH 5, 10, 15 and 20% in WPC 80, Trp. content increased or kept the around the same concentrate as in non-hydrolyzed WPC 80.
| Fig. 2: | Met and Syst content in WPC 80 and its hydrosylate compared to FAO/WHO/UNU reference standard |
| Fig. 3: | Leucinecontent in WPC 80 and its hydrosylate compared to FAO/WHO/UNU reference standard |
| Fig. 4: | Isoleucine content in WPC 80 and its hydrosylate compared to FAO/WHO/UNU reference standard |
| Fig. 5: | Valine content in WPC 80 and its hydrosylate compared to FAO/WHO/UNU reference standard |
| Fig. 6: | Tryptophane content in WPC 80 and its hydrosylate compared to FAO/WHO/UNU reference standard |
As gradually increase DH, Trp. content was 21.3, 18.0, 19.0 and 17.0 mg g-1 protein, respectively (Fig. 6).
DISCUSSION
In whey proteins, most peptide bonds are located in the interior of the protein and are not accessible for the enzyme. For these globular proteins it was postulated by Linderstrøm-Lang that reversible denaturation of the protein is needed for protein breakdown, as after denaturation more peptide bonds are exposed and at this stage the unfolded molecules are susceptible to degradation by proteolytic enzymes.
Generally, any protein has a solubility minimum at its isoelectric pH. The solubility at the isoelectric point (pI) of whey proteins concentrate 80 increases with hydrolysis, which is mainly the result of reduction in molecular weight and the increase in the number of polar groups (Chobert et al., 1988; Nielsen, 1997). Increasing the number of ionizable groups NH4 +, COO¯ in the aqueous environment was increased in hydrophilicity of the residues that follow by increasing the solubility. A high solubility of the concentrates produces a high significant biological activity (Bounous and Gold, 1991). This indicates that the proteins present are essentially in undenatured form. This functional parameter is important for application of hydrolysates in food products.
Although, Met and Cys decreased with increasing the hydrolysation, the content of these amino acids but it still above the requirements as FAO/WHO/UNU recommended. Cystine is considered to be key factors for synthesis of the glutathione (GSH), one of the major detoxifiers and antioxidants of the body. Glutathione is essential in supporting the immune system, including natural killer cells (Droege and Holm, 1997) and in the maintenance of T-lymphocytes (Gutman amd Schattini, 1998). In higher concentrations compared to the recommended dose immune function is enhanced through intracellular conversion to glutathione that supporting natural killer cells (Droege and Holm, 1997) and in the maintenance of T-lymphocytes (Gutman amd Schattini, 1998).
Isoleucine and Valine concentration were increased by increasing the partial hydrolysis compared to its concentration in WPC 80. Increasing partial hydrolysis of WPC 80 is useful for tissue growth and repair, in protein metabolism during the translation-initiation pathway of protein synthesis (Anthony et al., 2001) and synthesizing new proteins (Walzem and Dilland, 2002; Bos et al., 2000).
Tryptophan is important for the production of serotonin. Serotonin is one of the key brain chemicals involved in regulating mood. A number of studies indicate that normal mood depends in large part on adequate brain serotonin stores (Booij et al., 2005; Ruhe et al., 2007; Kaye et al., 2000). Dietary intake of L-Tryptophan directly influences the amount of serotonin in the plasma, brain and throughout the entire body.
CONCLUSIONS
In this study, it is apparent that WPC 80 enzymatic hydrolysis affected amino acid availability as indicated by some nutritional parameters such as the amino acid composition. This enzymatic hydrolysis improved the solubility and WPC 80 and its hydrolysates. The enhanced solubility of WPC hydrolysate is expecting increasing the biological activity. The undenatured conformation of whey protein was rich in cystine and with increasing DH kept cystine above the optimal physiological needs of the human body that is benefit for enhancing glutathione synthesis. Generally modified WPC 80 by partial hydrolysis was lead to increasing the concentration of Branched-Chain Amino Acids (BCAAs) that will be most useful for tissue growth and growth muscles. Tryptophan also was increased with increasing DH.
Whey protein concentrate standardized to 80% protein content (WPC 80) is the ideal protein used in a wide range of food applications. This study obvious that the optimal modified WPC 80 by partial hydrolysis was at DH 15% that is a good and makes them appropriate for food formulations or as nutritional supplements.
According to the results obtained, the content of beneficial health amino acids produced from WPC 80 hydrolysis depend on DH. It is prefer to recognize in generally the protein DH to get the optimal amino acid content benefit both this protein used as supplementation or was inside the product. Other health benefits can get from partial whey protein hydrolysates. Recent studies have described the antioxidant activity of milk protein hydrolysates and individual peptides released after hydrolysis (Pena Ramos and Xiong, 2001). The antioxidant activity has been attributed to certain amino acid sequences (Suetsuna et al., 2000).