Enzymatic Hydrolysis of Recovered Protein from Frozen Small Croaker and Functional Properties of Its Hydrolysates

Authors

  • Yeung Joon Choi,

    1. Authors Y.J. Choi, Hur, and B.D. Choi are with Div. of Marine Life Science/Inst. of Marine Industry, Gyeongsang Natl. Univ., 445, Inpyeong-dong, Tongyeong, Korea, 650-160. Author Konno is with Lab. of Food Biochemistry, Faculty of Fisheries, Hokkaido Univ., Hokkaido 041, Japan. Author Park is with Seafood Lab. and Dept. of Food Science and Technology, 2001 Marine Drive #253, Astoria, OR 97103, U.S.A. Direct inquiries to author Y.J. Choi (E-mail: yjchoi@gnu.ac.kr).
    Search for more papers by this author
  • Sungik Hur,

    1. Authors Y.J. Choi, Hur, and B.D. Choi are with Div. of Marine Life Science/Inst. of Marine Industry, Gyeongsang Natl. Univ., 445, Inpyeong-dong, Tongyeong, Korea, 650-160. Author Konno is with Lab. of Food Biochemistry, Faculty of Fisheries, Hokkaido Univ., Hokkaido 041, Japan. Author Park is with Seafood Lab. and Dept. of Food Science and Technology, 2001 Marine Drive #253, Astoria, OR 97103, U.S.A. Direct inquiries to author Y.J. Choi (E-mail: yjchoi@gnu.ac.kr).
    Search for more papers by this author
  • Byeong-Dae Choi,

    1. Authors Y.J. Choi, Hur, and B.D. Choi are with Div. of Marine Life Science/Inst. of Marine Industry, Gyeongsang Natl. Univ., 445, Inpyeong-dong, Tongyeong, Korea, 650-160. Author Konno is with Lab. of Food Biochemistry, Faculty of Fisheries, Hokkaido Univ., Hokkaido 041, Japan. Author Park is with Seafood Lab. and Dept. of Food Science and Technology, 2001 Marine Drive #253, Astoria, OR 97103, U.S.A. Direct inquiries to author Y.J. Choi (E-mail: yjchoi@gnu.ac.kr).
    Search for more papers by this author
  • Kunihiko Konno,

    1. Authors Y.J. Choi, Hur, and B.D. Choi are with Div. of Marine Life Science/Inst. of Marine Industry, Gyeongsang Natl. Univ., 445, Inpyeong-dong, Tongyeong, Korea, 650-160. Author Konno is with Lab. of Food Biochemistry, Faculty of Fisheries, Hokkaido Univ., Hokkaido 041, Japan. Author Park is with Seafood Lab. and Dept. of Food Science and Technology, 2001 Marine Drive #253, Astoria, OR 97103, U.S.A. Direct inquiries to author Y.J. Choi (E-mail: yjchoi@gnu.ac.kr).
    Search for more papers by this author
  • Jae W. Park

    1. Authors Y.J. Choi, Hur, and B.D. Choi are with Div. of Marine Life Science/Inst. of Marine Industry, Gyeongsang Natl. Univ., 445, Inpyeong-dong, Tongyeong, Korea, 650-160. Author Konno is with Lab. of Food Biochemistry, Faculty of Fisheries, Hokkaido Univ., Hokkaido 041, Japan. Author Park is with Seafood Lab. and Dept. of Food Science and Technology, 2001 Marine Drive #253, Astoria, OR 97103, U.S.A. Direct inquiries to author Y.J. Choi (E-mail: yjchoi@gnu.ac.kr).
    Search for more papers by this author

Abstract

ABSTRACT:  Fish protein isolate were recovered from frozen small croaker using pH shift. The partial enzymatic hydrolysates were fractionated as soluble and insoluble parts. They were dried using the drum dryer and their functional properties were examined. The total nitrogen content of the enzymatic hydrolysates ranged from 12.9% to 13.7%. The degree of hydrolysis of precipitates was 18.2% and 12.2% for croaker hydrolysates treated with Protamex 1.5 MG (Bacilllus protease complex) and Flavourzyme 500 MG (endoproteases and exoproteases, Aspergillus oryzae), respectively. The TCA supernatant, after centrifugation of hydrolysates, contained numerous peptides ranging from 100 to 4000 daltons. The solubility of the supernatants was higher than that of the precipitates at 0% to 3% NaCl and pH 2 to 10. The precipitate of Flavourzyme- and Protamex-treated hydrolysates showed a high emulsion activity index value compared to egg white and bovine plasma protein. In addition, the highest emulsion stability was observed for Protamex-treated precipitate hydrolysates. Emulsion stability of Protamex-treated precipitate hydrolysates was comparable to those of protein additives (egg white, bovine plasma protein, and soy protein concentrate). Water and fat binding capacity of precipitates were higher than those of supernatant. The results indicate that precipitate hydrolysate from undersized croaker can be used in processed muscle foods as a functional and nutritional ingredient.

Introduction

Croaker (Pennahia argentata) is found in abundance in the West Sea of Korea, but its commercial utilization has been limited due to its small size. This small croaker is often converted into low-grade surimi and used as a binder for fried seafood cakes (twighin ahmook) in Korea. Improving the value of this underutilized species as a food protein source is, therefore, desirable.

The application of enzyme technology to convert fish processing wastes or underutilized species into protein concentrates for food ingredients has become a considerable interest. Hydrolysis can be used to improve or modify the physicochemical, functional, and/or sensory properties of native proteins without losing nutritional value (Kristinsson and Rasco 2000a). Enzymatic hydrolysis of proteins, especially partial hydrolysis, is often employed to improve the functionalities of food proteins (Althouse and others 1995; Kristinsson and Rasco 2000b; Kuipers and others 2005). The functionalities of food proteins vary according to the protein source, the proteases used (Sugiyama and others 1991), the degree of hydrolysis (Kristinsson and Rasco 2000a), the pH and ionic strength of buffer (Doucet and others 2003), and heat treatment (Feng and Xiong 2003). Protein hydrolysate characteristics that are often related to emulsion properties are the degree of hydrolysis (DH) and the apparent molecular weight distribution (van der Ven and others 2001). The solubility is an important measure for liquid foods, while emulsion and water adsorption is important for semi-solid foods. To widen the application of protein hydrolysate in various foods, we thought that hydrolysates must be fractionated into soluble and insoluble parts after the process of enzymatic hydrolysis before determining their functionalities. Most published studies cover the biochemical properties of the hydrolysate itself without fractionation.

Enzymatic hydrolysis of fish has been applied to various species with noticeable benefits: Pacific whiting solid wastes (Benjakul and Morrissey 1997), conger eel scrap (Kang and others 2002), Atlantic salmon (Kristinsson and Rasco 2000a), cod (Gilmartin and Jervis 2002), and sharp-toothed eel (Cho and Ahn 2002). Fish sauce fermentation was also accelerated by enzymatic hydrolysis (Lee and others 1988; Kim and others 1999, 2002). Based on the need to upgrade underutilized small croaker and available technology, we hypothesized that the use of commercial enzymes for partial hydrolysis of croaker, with our additional efforts in fractionation, could add a significant value as a functional and nutritional food ingredient.

The objectives of this study were to recover fish protein isolates from frozen small croaker, to fractionate soluble and insoluble hydrolysates after the process of enzymatic hydrolysis by commercial proteases, and to evaluate functionalities for soluble and insoluble fractions. Functionalities of hydrolysates were also compared with those of known protein additives.

Materials and Methods

Fish and enzyme

Frozen whole small croakers (Pennahia argentata, body weight, 125 ± 36 g; body length, 18.2 ± 2.4 cm) preserved at −20 °C were purchased from a local market in March 2004 (Tongyeong, South Korea). Fish were thawed in the cold room (4 to 7 °C) over 12 h. After removing head, viscera including kidney and bladder, and fins, the fish were washed with cold tap water and ground 3 times with a meat grinder (5 mm plate hole diameter; M-12S, Fuji Eng, Gyeonggido, South Korea). Minced muscle, including bones and blood, was subjected to protein recovery using the extraction of fish muscle protein.

The food-grade proteases, Alcalase (endopeptidase, Bacillus licheniformis), Neutrase 0.8 L (endoprotease, Bacillus amyloliquefaciens), Protamex 1.5 MG (Bacillus protease complex), and Flavourzyme 500 MG (endoprotease and exopeptidase, Aspergillus oryzae), were provided by Biosis (Busan, South Korea) (Table 1). According to our preliminary study, the Vmax and Km values were 322.6 uM Tyr/min/mg and 10.2 mg for Alcalase, 384.6 uM Tyr/min/mg and 15.7 mg for Neutrase, 476.2 uM Tyr/min/mg and 43.1 mg for Protamex, and 91.7 uM/Tyr/min/mg and 5.1 mg for Flavourzyme against the recovered protein as substrate. We selected Protamex with the highest Km value and Flavourzyme with the lowest Km value for hydrolysis of the recovered protein.

Table 1—.  Biochemical properties of proteases according to the manufacturer and our measurement.
ProteaseManufacturer (Biosis, Busan, Korea)aOur measurementb
Declared activityOptimumOptimumKinetics
pHTemp,°CpHTemp,°CVmax uM Tyr/min/mgKm, mg
  1. aActivity and optimum conditions were measured against hemoglobin as substrate.

  2. bOptimum condition and kinetic parameters were measured against the fish protein isolate as substrate.

Alcalase 0.6 L0.6 AU/g850 to 608.065322.610.2
Neutrase 0.5 L0.5 AU/g7 to 1040 to 509.060384.615.7
Protamex 1.5 MG1.5 AU/g7 to 8509.560476.243.1
Flavourzyme 500 MG500 LAPU/g5.5 to 7.550 to 557.555   91.74 5.1

Egg white powder (EW; EW 54822, Prineff, Cameron, Wis., U.S.A.), bovine plasma protein (BPP; Proliant, Ames, Iowa, U.S.A.), soy protein concentrate (SPC; Promine, Solae, St. Louis, Mo., U.S.A.), and whey protein concentrate (WPC; Nutrilac 7723, Arla Foods Ingredients, Union, N.J., U.S.A.) were used to compare the functional properties of hydrolysates from the recovered fish protein.

Extraction of muscle protein

Croaker mince, including bone, was homogenized at a 1: 2 (w/v) ratio with cold (<5 °C) distilled water at 8000 rpm for 1 min using a homogenizer (Ika 25T basic, IKA Works Inc., Wilmington, N.C., U.S.A.). The homogenization continued with 7 vol of cold distilled water further to disperse the muscle proteins. The homogenates were then filtered through 2 layers of moth-proof nylon mesh (0.3 × 0.1 mm) to remove connective tissues, skin, and bones. The suspension was adjusted to pH 5.5 using 6 N HCl, and then kept in the cold room (4 °C) for 30 min. The precipitated fish proteins were recovered using 2 layers of cheesecloth. After measuring protein concentration of the filtered paste (Lowry and others 1951), fish protein isolate (FPI) was homogenized and diluted to 25 mg/mL with cold distilled water, and adjusted to pH 7.0 using 7.5 N NaOH solution. The FPI suspension was used as the substrate for the proteases.

Preparation of hydrolysate

The ratio of Protamex or Flavourzyme to FPI suspension was 1: 100 (w/w) or 1: 125 (w/w), respectively. The mixture of enzyme and substrate at pH 7.0 was incubated at 25 °C for 2 h using temperature circulator (Multitemp-III, Pharmacia-Biotech & Geburder Haake GmbH, Germany) while stirring with a digital dispenser (MS-280D, Tops, Seoul, South Korea). The supernatant, after centrifugation (SUPPRA 22K Plus, Hanil Science Industrial Co. Ltd., Incheon, South Korea) at 5000 ×g for 15 min, was evaporated under vacuum below 50 °C (N-1, EYELA Co., Tokyo, Japan) up to 25 Brix with reflectometer (N2, ATAGO Co., Tokyo, Japan). The precipitate obtained after centrifugation was used without further treatments. The concentrated supernatant and precipitates were separately dried at 121 °C in a drum dryer (YSCSDT-1, Youngnam Machinery Ind. Co., Busan, South Korea) and then pulverized using a food processor at room temperature (Kitchen Aid, 350 Watt, St. Joseph, Mich., U.S.A.).

Moisture, nitrogen, ash, and the degree of hydrolysis (DH)

Moisture content was measured using oven drying at 105 °C until a constant weight was obtained (AOAC 1984). Total nitrogen by Kjeldahl and ash contents were determined according to the respective AOAC methods (1984). The degree of hydrolysis (DH) was assayed as the TCA soluble index. Trichloroacetic acid (TCA) soluble index was determined according to the method of Hoyle and Merritt (1994) with slight modifications. Powdered supernatant and precipitate, 0.5 g each, were mixed in 10 mL of distilled water using a bio homogenizer (M133/1281-0, Biospec products Inc., Bartlesville, Okla., U.S.A.). TCA solution (15%) was then added until a final concentration of 5% (w/v) was obtained. The mixtures were kept at room temperature for 30 min and centrifuged at 3000 ×g for 15 min to remove insoluble particles. The TCA soluble index was calculated as:

image

The amino nitrogen content in the supernatant was assayed with TNBS (2,4,6-trinitrobenzenesulfonic acid, P2297, Sigma Chemical Co., St. Louis, Mo., U.S.A.) according to the method by Nielsen and others (2001) with modification. Into the test tubes containing 0.125 mL of hydrolysate, 1 mL of 0.2125 M sodium phosphate (pH 8.2), and 0.5 mL of 0.01% TNBS were added. The mixture was then incubated at 50 °C for 60 min before adding 1 mL of 0.1 M sodium sulfite solution to stop the reaction. The absorbance was measured at 420 nm using a UV-Vis spectrophotometer (Heλios, Unicam Limited, Cambridge, U.K.). Free amino acid and nitrogen contents were calculated according to a standard curve calibrated using L-leucine and the nitrogen content of L-leucine.

Molecular weight distribution of hydrolysates

The molecular weight distribution of hydrolysates was investigated by gel filtration on a Superdex 30 prep grade gel column (1.6 × 60 cm) (GE Healthcare, Seoul, Korea). Cytochrome c (C7120, MW, 12400 daltons), aprotinin (A1153, MW, 6500 daltons), vitamin B12 (C3607, MW 1344 daltons), and carnosine (C9625, MW, 226 daltons) produced in Sigma Chemical Co. were used as standards. Two milliliters of Protamex-treated hydrolysate (5.88 mg protein) and Flavouryzyme-treated hydrolysate (4.52 mg protein) were injected and eluted with an elution buffer (0.05 M sodium phosphate, pH 7.0) containing 0.1 M NaCl. The elution was carried out at a rate of 1 mL/min using a UV detector set at 206 nm with a recovery volume of 3 mL/tube. For molecular weight distribution of the precipitate, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was run according to the method of Laemmli (1970) using a 5% stacking gel and a 12% separating gel. To prepare samples for SDS-PAGE, the precipitate was suspended in 1% (w/v) SDS solution, followed by incubation at 80 °C for 30 min to dissolve proteins. Centrifugation (3000 ×g, 15 min) was conducted to remove undissolved parts and further to prevent the streaking of protein bands in the stacking gel. Twenty micrograms of protein, which were determined by the method of Lowry and others (1951) using bovine serum albumin as a standard, were applied to the gel. Gels were stained in 0.1% (w/v) Coomassie brilliant blue R-250 and destained in a solution of methanol, acetic acid, and distilled water (50: 7: 43, v/v/v). A wide range marker standard mixtures (M 4038, Sigma Chemical Co.) used for the determination of molecular weight contained the following standard proteins: myosin of rabbit muscle (205 kDa), β-galactosidase of Escherichia coli (116 kDa), phosphorylase b of rabbit muscle (97 kDa), fructose-6-phosphate kinase of rabbit muscle (84 kDa), bovine serum albumin (66 kDa), glutamic dehydrogenase of bovine liver (55 kDa), ovalbumin of chicken egg (45 kDa), glyceraldehydes-3-phosphate dehydrogenase of rabbit muscle (36 kDa), carbonic anhydrase of bovine erythrocytes (29 kDa), trypsinogen of bovine pancreas (24 kDa), trypsin inhibitor of soybean (20 kDa), α-lactoalbumin of bovine milk (14.2 kDa), and aprotinin of bovine lung (6.5 kDa).

Solubility

Fifty milligrams of hydrolysate were dispersed in 5 mL of buffer at various pH (pH 2.0, 0.1, M HCl-KCl; pH 3.0 to 6.0, 0.1 M citrate-phosphate; pH 7.0 to 8.0, 0.1 M Tris-HCl; pH 9.0 to 10.0, 0.1 M glycine-NaOH), and homogenized at 8000 rpm for 1 min within 30 min. The dispersion was centrifuged at 3000 ×g for 15 min. The supernatant was recovered and then the amount of protein in the supernatant was determined by the method of Lowry and others (1951). Solubility was calculated as the percentage ratio of protein in the supernatant to total protein in the initial sample. The effect of ionic strength on solubility was determined at different NaCl concentrations (0% to 3% at pH 7.0).

Emulsifying properties

Emulsifying properties were measured using emulsifying activity index (EAI) and the emulsifying stability index (ESI) according to the method of Pearce and Kinsella (1978) with slight modification. One hundred twenty-five milligrams of powdered hydrolysate were dissolved in 20 mM sodium phosphate solution (pH 7.0) containing 0.6 M NaCl and the final volume was adjusted to 25 mL. Then 6.7 mL of corn oil were added to 25 mL of hydrolysate solution, after which the mixture was homogenized at 8000 rpm at room temperature for 15 s with a bio homogenizer (M 133/1281-0, Biospec Products Inc.). Seventeen microliters of the emulsion were then diluted with 5 mL of 0.1% SDS solution. The absorbance of the diluted emulsion was determined at 500 nm with the UV-Visible spectrophotometer. The oil volume fraction (Φ) of the emulsion was measured using the method of Pearce and Kinsella (1978). The EAI was calculated using the following equation:

image

where A500 is the absorbance at 500 nm, Φ is the volume fraction of the oil phase, c is the concentration of hydrolysate in the aqueous solution before the emulsion was made, and L is the path length of the cuvette.

The emulsion stability index (ESI) was determined by separation between the oil- and water-phase during storage of the emulsion in a measuring cylinder at refrigeration temperature, and was expressed as the percent change of the cream layer compared to the total emulsion.

Fat and water absorption capacity

Fat and water absorption capacity was assayed in triplicate (Shahidi and others 1995). One gram of hydrolysate was transferred into an accurately weighed 50 mL Felcon tube (35-2070, Becton Dickinson Co, N.J., U.S.A.). Soybean oil (20 mL) was added and the mixture was thoroughly mixed in a bio homogenizer at 8000 rpm for 30 s. The sample was kept at room temperature for 20 min, followed by centrifugation (3000 ×g, 10 min). Free oil was decanted, and the adsorption of the sample was determined from the weight difference. For the measurement of water absorption capacity, water was used instead of soybean oil. Water absorption capacity was measured from the same sample used for fat adsorption. Fat and water absorption were expressed in terms of combined weight (g) of fat and water adsorbed by 1 g of hydrolysates or the nonmuscle proteins, respectively.

Statistical analysis

All experiments were repeated at least in triplicate. Data were analyzed for the degree of variation and significance of difference based on analysis of variance (ANOVA), using the JMP statistics package (SAS Inst. Inc, Cary, N.C., U.S.A.). Tukey's pair-wise comparison test was used to determine statistical difference (P < 0.05) between treatment means.

Results and Discussion

Chemical composition and DH of hydrolysates

Total nitrogen content of the supernatants from Flavourzyme-treated hydrolysates was lower than the precipitate (P < 0.05). However, the total nitrogen content of our sample (partial hydrolysates from croaker) was higher than from the nonmuscle protein additives (P < 0.05). The moisture and crude protein (total nitrogen × 6.25) contents of hydrolysate were ranged at 3.6% to 5.2% and 80.7% to 85.9%, respectively (Table 2).

Table 2—.  Moisture, total nitrogen, ash content, and the degree of hydrolysis (DH) from small croaker and nonmuscle proteins.
Hydrolysates Moisture, %Total nitrogen (g/100 g) (Protein, g/100 g)Ash, %DHA
  1. Data are means (± SD) of 3 replicates.

  2. The same letters in a column are not significantly different (P > 0.05).

  3. ADegree of hydrolysis was not analyzed for nonmuscle protein additives.

  4. Values in the parentheses represent crude protein (nitrogen × 6.25).

ProtamexPrecipitate4.4 ± 0.7c13.7 ± 0.1a4.7 ± 0.2cd18.2
(85.9 ± 0.6)A  
Supernatant4.4 ± 0.7c13.7 ± 0.1a8.4 ± 0.4b46.7
(85.4 ± 0.6)   
FlavourzymePrecipitate3.6 ± 0.0c13.4 ± 0.1a3.7 ± 0.1de12.2
(83.9 ± 0.6)   
Supernatant 5.2 ± 0.3bc12.9 ± 0.1b8.4 ± 0.3b42.0
(80.7 ± 0.9)   
Egg white powder6.0 ± 0.2b12.8 ± 0.0b 5.2 ± 0.4c  -
(79.9 ± 0.2)   
Whey protein concentrate 5.2 ± 0.5bc11.5 ± 0.2c2.5 ± 0.5e-
(72.1 ± 1.4)   
Bovine plasma protein7.9 ± 0.2a10.2 ± 0.1d11.4 ± 0.2a  -
(63.7 ± 0.3)   
Soy protein concentrate3.8 ± 0.3c  9.9 ± 0.0d8.8 ± 0.2b-
(61.6 ± 0.1)   

The major component of fish protein hydrolysate evaluated from fish scrap of 5 marine species by enzymatic treatment was a mixture of peptides of various lengths, which accounted for 82.3% to 85.8% of total nitrogenous compounds (Khan and others 2003). Similarly, Benjakul and Morrissey (1997) reported that the protein content of freeze-dried hydrolysates from Pacific whiting solid wastes was 82.3%. Ash content of croaker hydrolysate in this study ranged from 3.7% to 8.4%. The ash content of the supernatant was higher than that of the precipitate (P < 0.05) regardless of enzymes, indicating that the high ash content in the supernatants was probably caused by the creation of sodium chloride during the adjustment of pH.

The degree of hydrolysis (DH) for supernatants of Protamex and Flavourzyme-treated hydrolysates was 46.7% and 42.0%, respectively (Table 2). The DH of minced eel hydrolyzed with Protamex and Flavourzyme for 2 h were 65% and 45%, respectively, as determined by TCA soluble index (Cho and Ahn 2002). The DH of hydrolysates with Alcalase ranged from 26.8% in herring press cake to 44.7% in raw herring (Hoyle and Merritt 1994). The difference of DH in hydrolysates was probably due to the protease concentration and the state of substrates (that is, mince, cake, or hydrolysates) during the hydrolysis reaction. The results suggested that hydrolysates obtained from the supernatants contained more soluble and lower molecular weight nitrogen compounds as compared to the precipitates.

Molecular weight range of hydrolysates

The precipitates of Protamex- and Flavourzyme-treated hydrolysates contained more bands with higher molecular weight compared to the supernatants (Figure 1). The precipitate of Flavourzyme-treated hydrolysate showed a wide range of molecular weights (29 to 205 kDa). The high density of FP (precipitate of Flavorzyme-treated hydrolysate) proteins between 100 and about 200 kDa were probably the function of Flavourzyme itself. We previously observed a similar pattern in Flavourzyme-treated precipitate from Jack mackerel (not shown). Salmon hydrolysates, treated with various alkaline proteases, showed the maximum peptide size at about 20 kDa and the peptide profile was strongly enzyme dependent (Kristinsson and Rasco 2000b).

Figure 1—.

SDS-PAGE pattern of recovered protein and hydrolysates. M, wide range marker proteins; UN, unhydrolyzed protein; PP, precipitate of Protamex hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; FS, supernatant of Flavourzyme hydrolysate.

To investigate peptides with low molecular weights below 10 kDa, the supernatants were fractionated on a Superdex 30 prep grade column. The molecular weight and distribution of peptides varied between Protamex- and Flavourzyme-treated hydrolysates (Figure 2). The main peaks of peptide in supernatants were 4000, 800, and 100 daltons for both treatments. However, the supernatants from Flavourzyme-treated hydrolysate produced more peptides in smaller molecular size compared to those from Protamex-treated hydrolysate. It was probably due to the Flavourzyme, which is a mixture of endo- and exo-proteases making smaller fragments, while Protamex is an endo-protease.

Figure 2—.

Gel chromatogram of the supernatant from hydrolysates treated by Protamex (A) and Flavourzyme (B).

Solubility

The solubility of the supernatants was higher than that of the precipitates. Protein solubility of the supernatants from Flavourzyme-treated hydrolysates was higher than that of Protamex-treated hydrolysates. The solubility of supernatants from hydrolysates was not affected by NaCl concentration (Figure 3) probably due to a reduction in the molecular weight and an increase in the number of polar groups by enzymatic hydrolysis. However, the precipitates from the 2 enzyme treatments showed a steady and gradual increase as the salt content increased, except for Protamex-treated precipitates at 3% (approximately 0.52 M) salt. We were not sure if this reduction was something to do with salting-out effect in relation to this specific enzyme.

Figure 3—.

Effect of NaCl on the solubility of croaker hydrolysate (top) and nonmuscle protein (bottom) at pH 7.0. FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, precipitate of Protamex hydrolysate; WPC, whey protein concentrate; BPP, bovine plasma protein; SPC, soy protein concentrate; EW, egg white.

The NaCl dependence of precipitate solubility probably indicates the presence of undegraded myofibrillar protein residues. The higher solubility of the supernatant was, therefore, due to the higher DH. The supernatants contained more small fragments compared to precipitates. The solubility of the precipitates increased as the DH increased. This is mainly due to a reduction in the molecular weight and an increase in the number of polar groups. The effect of DH on protein solubility also depended on protein structure (Nielsen 1997).

The solubilities of BPP and EW were higher than those of SPC and WPC. The solubilities of BPP and EW were not affected by NaCl concentration, while those of SPC and WPC decreased as the salt concentration increased to 3%. In another study (Oshodi and Ojokan 1997), the solubility of bovine plasma protein concentrate (BPPC) was slightly higher in 1% NaCl compared to 0% and 5%, but was not significantly different between 0% and 5%. These results, overall, are in agreement with the findings of this study with regard to the performance of nonmuscle protein solubility in NaCl solutions.

The solubility of the precipitates was increased gradually with increasing pH (Figure 4). However, the solubility of the supernatant was not affected by pH (P < 0.05). The results suggested that the solubility of precipitates slightly depended on the pH compared to that of supernatants. The solubility of croaker protein was very low at pH 4.5 and increased as the pH is shifted away from the isoelectric point of fish muscle protein, around pH 5.0 (Park and others 2003). The pH dependence of precipitates was probably caused by unhydrolyzed muscle protein with respect to their low DH. The solubility might be attributed to the difference in the isoelectric point of the peptides formed with different DH (Nielsen 1997). The pH influenced the charge on weakly acidic and basic side-chain groups, thus protein and hydrolysates showed low solubility at their isoelectric point (Gbogouri and others 2004)

Figure 4—.

Effect of pH on solubilities of partial hydrolysate (top) and nonmuscle protein (bottom). FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, precipitate of Protamex hydrolysate; WPC, whey protein concentrate; BPP, bovine plasma protein; SPC, soy protein concentrate; EW, egg white.

The solubility of nonmuscle proteins depended on pH. The solubility of BPP and EW at pH 7 was the highest (50% to 60%), while that of WPC and SPC was quite low (10% to 18%) (P < 0.05). According to Morr and others (1985), the solubilities of WPC, SPC, and EW at pH 7.0 were 90.4% to 93.4%, 18.2% to 19.2%, and 95.3% to 99.0%, respectively. The lower solubility observed in the current experiments could be due to the ionic strength of the buffer and/or different manufacturing processes. The solubility of EW and SPC was lowest at pH 3.0, while that of WPC were lowest at pH 4.0 (P < 0.05).

WPC solubility was affected by temperature and pH (Pelegrine and Gasparetto 2005). The lowest solubility value for WPC was obtained at pH 4.5, which was the isoelectric point of whey proteins (Pelegrine and Gasparetto 2005). The variations in protein solubility of BPP depended on pH and salt concentration. In another study (Oshodi and Ojokan 1997), the solubility curve of BPP showed a minimum at about pH 5 in the absence of salt.

Emulsifying properties

The EAI of hydrolysates and nonmuscle protein additives are shown in Figure 5. The EAI of the Flavourzyme-treated precipitates was much higher than that of the supernatants, which were comparable to the 2 protein additives (SPC and WPC) that were tested (P < 0.05). The results suggested that EAI did not correlate with solubility. The emulsion stability results, however, corresponded with the results of Nielsen (1997). The EAI of the 2 hydrolysate precipitates was much higher than EW and BPP (P < 0.05), while that of the supernatants was comparable to EW and BPP. The results might be attributed to the differences in the molecular size among the hydrolysates. The EAI of hydrolysates also depends on protease type and DH.

Figure 5—.

Emulsion activity index (EAI) of croaker hydrolysates and nonmuscle proteins at pH 7.0. FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, precipitate of Protamex hydrolysate; EW, egg white; BPP, bovine plasma protein; SPC, soy protein concentrate; WPC, whey protein concentrate.

Except for the precipitate from Protamex-treated hydrolysates, all other hydrolysates showed a lower emulsion stability index (ESI) compared to the nonmuscle protein additives after 20 min of emulsion storage (Figure 6). A reason for the low ESI could be that the peptide produced by hydrolysis was not amphiphilic enough to impart a high stability to the emulsion (Chobert and others 1988).

Figure 6—.

Emulsion stability of croaker hydrolysates and nonmuscle proteins at pH 7.0 after 20 min of storage at 4 ± 2 °C. FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, precipitate of Protamex hydrolysate; EW, egg white; BPP, bovine plasma protein; SPC, soy protein concentrate; WPC, whey protein concentrate.

Endogenous enzymatic hydrolysates were found to produce the most stable emulsions at 10% and 15% DH (Kristinsson and Rasco 2000a). The EAI and ESI decreased as DH increased. It was assumed that the hydrolysates had less surface hydrophobicity with increasing DH. The relative ESI of hydrolysates and nonmuscle protein additives were unchanged until storage time extended to 100 h (data not shown). A protein used in an emulsified product must be sufficient in quantity to give complete coverage of the interface. Displacement of adsorbed protein from the interface by a low molecular weight amphiphile may lead to rapid flocculation as the steric stabilization layer is lost (Hall 1996). Emulsion instability due to coalescence was related to apparent molecular weight distribution of hydrolysates. A relative high amount of peptide larger than 2 kDa positively influenced emulsion stability (van der Ven and others 2001). Emulsion obtained with small peptides usually showed significant coalescence (Walstra and van Vliet 2008). It was assumed that the high ESI in the precipitates was caused by high molecular weight distribution compared to supernatants. Flavouzyme is a fungal protease and peptidase complex, and can produce more the lower molecular weight from recovered protein of croaker. The high ESI of Protamex-treated precipitates is probably due to difference of fragment composition and net charge.

Fat and water absorption capacity

The fat and water absorption capacity (FAC and WAC) of hydrolysates and nonmuscle additives were evaluated for possible use as a functional ingredient for meat products. The precipitate from Flavourzyme-treated hydrolysates had a significantly higher FAC compared to the other hydrolysates and nonmuscle proteins (P < 0.05) (Figure 7). BPP was equaled to the 2 supernatants from the hydrolysates (FS and PS). The FAC of the supernatants did not show a significant difference between Flavourzyme- and Protamex-treated hydrolysates (P < 0.05). The FAC of the Flavourzyme- and Protamex-treated precipitates with low DH was higher than that of the Flavourzyme- and Protamex-treated supernatants with high DH. The results suggested that FAC depended on DH and the molecular size of the peptide composition of the hydrolysates. The protein functionality is affected by composition, conformation and homogeneity as intrinsic factors. Protein composition and conformation have significant effects on water holding capacity and fat holding capacity. The amount of water is affected by factors such as protein type, concentration, number of exposed polar groups, pH, salts, and temperature (Barbut 1996).

Figure 7—.

Fat absorption capacities of croaker hydrolysates and nonmuscle proteins. FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, precipitate of Protamex hydrolysate; EW, egg white; BPP, bovine plasma protein; SPC, soy protein concentrate; WPC, whey protein concentrate.

Nonhydrolyzed salmon protein had FAC significantly higher than its hydrolysates (Gbogouri and others 2004). In addition, the hydrolysates at 5% DH had a significantly higher fat absorption than 10% and 15% DH due to the larger peptide sizes (Kristinsson and Rasco 2000a).

WAC showed 7.2 ± 0.4 g/g for WPC and 6.5 ± 0.2 g/g for SPC, followed by the precipitate from Flavourzyme-treated hydrolysates (3.9 ± 0.1 g/g) and the precipitate from Protamex-treated hydrolysate (3.4 ± 0.0 g/g) (Figure 8). WAC of supernatant from Flavourzyme- and Protamex-treated hydrolysates ranged from 0.3 ± 0.0 g/g to 0.5 ± 0.0 g/g. Enzyme treatment with different proteases affected WAC, but WAC was unaffected by DH (Kristinsson and Rasco 2000b). The difference of WAC between fractions of hydrolysates was probably due to the difference in the molecular size of the hydrolysates.

Figure 8—.

Water absorption capacities of croaker hydrolysates and nonmuscle proteins. FS, supernatant of Flavourzyme hydrolysate; PS, supernatant of Protamex hydrolysate; FP, precipitate of Flavourzyme hydrolysate; PP, residue of Protamex hydrolysate; EW, egg white; BPP, bovine plasma protein; SPC, soy protein concentrate; WPC, whey protein concentrate.

Conclusions

The higher solubility of hydrolysates was obtained from the supernatants, indicating the dependence of solubility on the degree of hydrolysis. The major peptides of the supernatant ranged from 100 to 4000 daltons. The EAI, ESI, FAC, and WAC were higher in the precipitate than in the supernatant. Croaker hydrolysates were superior to nonmuscle protein additives for FAC, and were comparable to those for EAI. Extended hydrolysis was thought to contribute to high solubility, while limited hydrolysis could improve EAI, ESI, FAC, and WAC. Based on the comparison with nonmuscle protein additives, the precipitate from enzyme-treated hydrolysates from underutilized small croaker could be used as a functional ingredient.

Acknowledgments

This study was financially supported by the Ministry of Commerce, Industry, and Energy (MOCIE) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Regional Innovation.

Ancillary