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Protein recovery from mechanically deboned turkey residue by enzymic hydrolysis
Process Biochemistry, Vol. 31, No. 6, pp. 605-616, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00 + 0.00 ELSEVIER
0032-9592(95)00101-8
Protein Recovery from Mechanically Deboned Turkey Residue by Enzymic Hydrolysis L. G. Fonkwe & R. K. Singh* Purdue University, Food Science Department, Smith Hall, West Lafayette, IN 47907-1160, USA (Received 30 August 1995; revised manuscript received and accepted 19 November 1995)
Enzymic hydrolysis was used to recover a potentially edible high protein hydrolysate from mechanically deboned turkey residue (MDTR), a turkey processing waste. The hydrolysis process reduced the weight of the original MDTR by 51% on a dry weight basis, and recovered 46% of the MDTR proteins. The hydrolysate contained 78% protein, 4"6% ash and only 5.7% fat. The proteins consisted mainly of amino acids and small peptides with molecular masses lower than 6"5 kD due to a high degree of hydrolysis (65- 70%), and were very soluble in water over a wide pH range. It had a yellowish colour, was rich in calcium, phosphorus, potassium and magnesium and had a large monolayer value, but had low viscosity and low emulsion capacity at p H values of 4, 7and 10. Its buffering capacity was at a minimum between pH 5 and 7.
INTRODUCTION
produced in the US in 1991. 6 It is estimated that about 169000 tons of MDTR were produced during turkey deboning (private communication). Thus a large volume of deboner waste, containing valuable proteins, was generated for disposal. MDTR has been shown to contain approximately 51% moisture, 19% protein, 9% fat and 15% ash. 7 Similar figures have been reported for chicken bone residue by other researchers. 2'5 The protein content of MDTR includes approximately 20% of salt- and alkali-soluble proteins which can be recovered and used for human consumption. The MDTR proteins also include approximately 40% collagen from tendons and bones 2 which, although not salt- or alkali-soluble, can be enzymically hydrolyzed. Yields reported for the recovery of proteins, using alkali extraction, from poultry deboner residue have been low. A yield of 12%, based on the total amount of proteins in MDTR, in a
Mechanical deboning is a unit operation in the poultry processing industry. During this process, pressure is used to separate meat from a slurry of ground bones and meat inside a pressure chamber in a mechanical deboner. The separated ground meat is currently being used in the manufacture of comminuted poultry products such as patties, bologna and sausages. 1'2 The waste material leaving the deboner is a bony residue. In the case of turkey processing, the residue is known as mechanically deboned turkey residue (MDTR). This residue still contains a valuable amount of protein and has been recognized as a valuable source of proteins for human food and is currently being used in the manufacture of animal feed. 2-5 Approximately 2415000 tons of turkey were *To whom correspondence should be addressed. 605
606
L. G. Fonkwe, R. K. Singh
laboratory process has been reported. 7 The method involved the recovery of proteins from mechanically deboned turkey residue by extraction with an alkaline sodium chloride solution and precipitation with polysaccharides, such as carrageenan, carboxymethyl cellulose and chitosan. Protein yields of 9-16% in pilot scale extractions and 6-15% in laboratory experiments from chicken bone residue have also been reported. 5 Thus, it is important to investigate alternative processes that could lead to higher protein yields from poultry deboner residue. Enzymic hydrolysis is potentially an effective method for the recovery of proteins from MDTR. Enzymic hydrolysis of MDTR has some advantages over the alkaline extraction method of recovering proteins from MDTR. First, enzymic hydrolysis has the advantage that both the collagenous and non-collagenous proteins (as well as any other proteins in the MDTR such as blood and bone marrow) are hydrolyzed, unlike the alkaline extraction of proteins in which the major proteins extracted are non-collagenous proteins. This would lead to a higher protein yield. Second, this method of protein recovery also has the potential of reducing the volume of waste (wastewater and collagen/bone slurry) generated by a large amount compared to the alkaline recovery methods. Furthermore, the enzymic hydrolysis process is simple, efficient and involves mild alkaline conditions that would not destroy the proteins recovered by racemization and/or other chemical reactions. 8-1° A limitation to enzymic hydrolysis of proteins is the fact that a bitter hydrolysate may be formed. 11 However, the latter author also indicated that animal protein hydrolysates generally do not have a bitter taste. Some studies have been carried out utilizing enzymic hydrolysis in the recovery of proteins from other poultry processing wastes. 12-~4 Treating chicken meat with an enzyme having a high endopeptidase activity could produce a bitter hydrolysate while the same treatment with an enzyme having both endo- and exopeptidase activity would produce a hydrolysate with no bitter taste. ~2 Protein hydrolysates have been produced from broiler chicken heads, using alcalase and neutrase, and reported to have good organolpetic and physicochemical properties. 12,13 A protein hydrolysate has also been prepared
from the bone residue resulting from the mechanical deboning of chickens, using a Polish enzyme preparation (proteopol BP-S). la The reported yield was 23-24% of nitrogenous compounds, and the hydrolysate had excellent solubility, good emulsifying capacity and emulsion stability, and a molecular weight less than 40 kD. The main objectives of this investigation were: (I) to investigate the production of an enzymic hydrolysate from MDTR using crude papain, and (2) to characterize the hydrolysate produced in terms of its physical, chemical and functional properties. MATERIALS AND METHODS Hydrolysis conditions
Five hundred grams of MDTR (19% protein) were added to 1 litre of water held at 60°C in a water bath. The MDTR/water mixture was allowed to reach a temperature of 60°C and crude protein [activity of 1.7 units/mg solid, purchased from Sigma Chem. Co. (St Louis, MO) and partially dissolved in warm water (60°C)] was added to the mixture. The ratio of MDTR to enzyme (w/w) was 250 g MDTR/g papain. Preliminary experiments had shown that these were excellent conditions for the enzymic hydrolysis. The mixture was continuously stirred with a mechanical stirrer and broth samples were collected at 5, 15, 30, 45, 60, 90 and 120 min of hydrolysis. At the end of the hydrolysis, all the hydrolysates collected were filtered through a metal screen, then through multiple layers of cheesecloth, and centrifuged at 3500g for 10min in a bench-top centrifuge (International Equipment Co., Needham Hts, MA) to remove all solid particles and fat. The protein hydrolysates were heated to about 95°C for at least 15 min to inactivate the enzyme, then allowed to cool to room temperature (20-22°C). Any fat layer on the surface of the liquid hydrolysate was siphoned off. The bulk of the hydrolysate was then freeze-dried. The powder formed was analyzed for protein, fat and ash contents using the approved standard methods. 15 All the hydrolysate samples collected were analyzed for protein content using the Kjeldahl and bicinchoninic acid methods. The bony residue also was weighed, dried and analyzed for moisture, protein, fat and ash con-
Protein recoveryfrom mechanicalS,deboned turkeyresidue tent, using approved standard methods. 15 This was done to determine any possible uses for this final waste material. The bony residue was then discarded. Determination of the degree of hydrolysis The degree of hydrolysis (DH) of the protein hydrolysates was determined using the trichloroacetic acid m e t h o d . 16 The method is based on the ratio of 10% trichloroacetic acid (TCA) soluble protein in the hydrolysate compared to the total amount of protein in the sample. After hydrolysis, 40 ml of the protein hydrolysate were mixed with 40 ml of 20% TCA (making a total TCA concentration of 10%). The mixture was stirred for 5 min, then centrifuged for 15min at 16000g in a Beckman centrifuge (Model J2-21, Beckman Instrument Inc., Palo Alto, CA). The supernatant was collected and freeze-dried. The amount of protein in the freeze-dried hydrolysate and in the freeze-dried TCA-soluble hydrolysate were determined by the Kjeldahl method. The degree of hydrolysis was calculated using eqn (1). 10% TCA-soluble protein in sample DHx 100. total protein in sample
(1) General characterization Proximate analysis Proximate analysis on protein powders was carried out using the approved standard methods. 15 Protein analysis on solids was carried out using the Kjeldahl method. Fat analysis was performed using the Soxhlet method, and ash analysis was carried out gravimetrically by heating the sample at 550°C in a muffle furnace for 24 h. However, protein analysis in solution was carried out using the bicinchoninic acid (BCA) method using bovine serum albumin as the reference protein. 17 This is because the Kjeldahl method is a time-consuming method while the BCA method takes a very short time to perform. Thus, the BCA method is preferable in situations in which large numbers of protein determinations are to be carried out. The relationship between the two methods needs to be established on the same sample so that results from the BCA method can be con-
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verted to those from the Kjeldahl method (the official method). 18'19 This is because the two methods measure different quantities and will give different readings for the same sample. The Kjeldahl method measures the total amount of nitrogen in a sample and converts this into the total amount of protein. The BCA method quantifies the number of peptides and proteins in solution which have at least three amino acids (two peptide bonds). Thus the Kjeldahl method of protein estimation will give a higher value than the BCA method.
Mineral analysis Mineral analysis was carried out on freeze-dried samples using the Inductively Coupled Plasma/ Atomic Emission Spectrophotometry (ICP/ AES) method in the Food Science Dept., Purdue University. Samples were digested with 70% nitric acid and hydrogen peroxide and mineral analysis were carried out on a Perkin Elmer Plasma 400 ICP/AES instrument (Norwalk, CT) with a model AS 90 auto sampler. Functional properties Water-holding capacity and protein solubility The procedure for the determination of waterholding capacity (WHC) was a recommended method in the literature with modification.TM A sample of the protein hydrolysate powder was mixed with water in the ratio 1:20 (w :v). The pH of the hydrolysate solutions was then adjusted to pH 2, 4, 5, 5.5, 6, 8 and 10 using hydrochloric acid and/or sodium hydroxide solutions and stirred for 30min. The pH of hydrolysate solutions were checked and corrected to the desired pH value every 5 min during stirring until it remained constant. The solutions were then centrifuged at 16000g for 15 min. The volume of each resultant supernatant was measured using a graduated cylinder, and used to determine WHC using eqn (2): WHC=
volume of water absorbed (ml) x 100. (2) weight of sample (g)
The protein solubility index (or nitrogen solubility index, NSI) was calculated by comparing the amount of protein in the supernatant (converted to Kjeldahl units) to the amount of protein in the original sample, using eqn (3).
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NSI-
L. G. Fonkwe, R. K. Singh
amount of protein in solution x 100%. amount of protein in the originalsample
noticeable change in the sound of the homogenizer, using the equation volume of canola oil used (ml)=duration of
(3) The amount of protein in solution was determined using the BCA method while the amount of protein in the freeze-dried hydrolysate was determined using the Kjeldahl method. The protein content from the BCA assay was then converted into protein estimates in the Kjeldahl unit using a predetermined scale.
Fat binding capacity The fat binding capacity (FBC) was measured using a method described in the literature 2° with modification. One gram of the sample was mixed with 20 ml of Crisco puritan canola oil (Procter & Gamble, Cincinnati, OH) and stirred for 10 min. The mixture was then centrifuged at 16000g for 15min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Palo Alto, CA) and the volume of the unabsorbed oil was measured using a graduated cylinder. The FBC was calculated using eqn (4) FBC=
amount of fat absorbed (ml) weight of sample (g)
process × rate of oil addition. The emulsion capacity (EC) was calculated as the amount of oil emulsified per gram of protein, using eqn (5). EC-
Emulsion capacity The procedure utilized in the measurement of the emulsion capacity (EC) was a method described in the literature 18 with modification. A 1% hydrolysate solution was prepared by dissolving the hydrolysate in a 0.5 M sodium chloride solution. The hydrolysate solution (50 ml) was poured into a 600 ml plastic beaker. The protein solution was homogenized with a polytron homogenizer at 1700 rpm for 15 s prior to the start of oil addition to the protein solution. Crisco puritan canola oil (Procter & Gamble, Cincinnati, OH) was added to the protein solution at a rate of 1.2ml/s using a Masterflex pump (model 7016-52, Cole Parmer, Chicago, IL) with continuous homogenization until a steady change in the sound of the homogenizer was noticed. The duration of the oil addition process was measured with a stop watch and used to calculate the volume of canola oil delivered to the protein solution that caused the increase in viscosity leading to a
wt of protein in sample (g)
(5) Physicochemical properties Colour of the hydrolysate solution The colour of the hydrolysate solution was determined on the L-a-b scale using a Hunter colorimeter (Model D25-PC2, Hunter Associated Laboratories, Reston, VA). A HunterLab standard white plate (No. C2-30636) was used to standardize the colorimeter prior to use. Three parameters, hue, chroma and AE, were calculated from the L-a-b readings obtained using eqns (6)-(8):
x 100%. (4)
volume of canola oil used (ml)
Hue=tan-1 (a/b )
(6)
Chroma = (a 2 + b 2)1/2
(7)
AE = (AL z + Aa 2 + Ab 2),/2
(8)
and where AL, Aa and Ab are differences in the measured L-a-b values between any two solutions.
Viscosity of the hydrolysate solutions Solutions of the protein hydrolysate were prepared, in concentrations ranging from 1 to 5% (w/v), in volumetric flasks using the dry hydrolysate powders. Each solution then was centrifuged at 16000g for 15 min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Palo Alto, CA) and the supernatant was collected. The viscosity of each solution was measured using an Oswald capillary viscometer, and the relationship between protein concentration and relative viscosity was determined. Sorption isotherm Hydrolysate powder was placed in pre-weighed aluminum dishes and weighed. The dishes containing the samples were then placed in
Protein recovery from mechanicalty deboned turkey residue
evacuated air tight dessicators containing saturated solutions of lithium chloride, potassium acetate, magnesium chloride, potassium carbonate, magnesium nitrate, sodium nitrite, sodium chloride, potassium chloride, and potassium nitrate in water. These solutions had equilibrium relative humidities of 12, 23, 33, 44, 55, 64, 76, 85 and 93%, respectively. The samples were held in the dessicators for two weeks, based on preliminary investigations which showed that two weeks were enough time for the samples to attain equilibrium with their environments. After two weeks, the samples were weighed again and the moisture contents (MC) of the samples were calculated using eqn (9): ME-
mass of water in sample mass of dry sample
x 100.
(9)
Determination of titration curve A 1% (w/v) solution of the protein hydrolysate in water was prepared. The solution was cooled to room temperature using cold tap water. The protein solution (25 ml) was measured into an Erlenmeyer flask and its pH lowered to approximately pH 2.0 using a 3 M hydrochloric acid solution. The protein solution then was titrated using a 0-01 M sodium hydroxide solution until its pH rose to approximately pH 12. Buffering capacity The buffering capacity (BC) of the protein hydrolysate was calculated from the titration curve data using eqn (10), 21'22 for each pH change of 0.5 units.
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determined using a QTest texture analyzer (QTest, a division of Sintech Inc., Cary, NC). Immediately prior to measuring the gel strength, the gel was carefully removed from the 10 ml beaker and placed on a platform. The gel strength was determined as the maximum amount of force required to crush the gel (diameter of 21 mm and height of 30 ram) to either the break point or to 75% deformation using a 5 kgf (10 lbf) load cell. RESULTS AND DISCUSSION Hydrolysis parameters Two curves showing the rate of production of proteins from MDTR using papain are shown in Fig. 1. The amount of protein in solution increased significantly ( P < 0.05) during the first 45 rain of hydrolysis and then levelled off at about 20 mg/ml based on the BCA method of protein quantification (40 mg/ml by the Kjeldahl method). However, the amount of protein in the hydrolysate did not increase significantly
55 50 45 40 -6 35
i
~ 3o ' ~ 25
BC=
meq titrant mass of protein (g) x ApH
(10)
Gel formation The procedure for gel formation was obtained from the literature 18 with modification. A 20% (w/v) hydrolysate solution was prepared by adding the sample to deionized distilled water, mixing and holding in a volumetric flask. The hydrolysate solution was heated to 100°C for 30 min, the volume adjusted, and the solution was poured into 10 ml beakers and allowed to cool to room temperature (20-22°C). The solution then was held at 8-100C in a refrigerator overnight. The strength of the gel formed was
~ 20
4
-'*-- BCA - 4 - - Kjeldahl
10
~So
ids
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Duration of hydrolysis (rain)
Fig. 1. Hydrolysis curves for the recovery of proteins from mechanically deboned turkey residue (MDTR) by enzymic hydrolysis using papain (BCA=bicinchoninic acid method; Kjeldahl=Kjeldahl method of protein quantification). The substrate to enzyme ratio was 250 g MDTR/g papain. Each point is the mean of triplicate determinations and bars represent a 95% confidence level.
L. G. Fonkwe, R. K. Singh
610
(P < 0.05) after about 45 min of hydrolysis. Similarly, the amount of total solids in solution did not increase significantly (P<0.05) after about 30 min of hydrolysis, as shown in Fig. 1 also. Therefore, it appears that the process was complete within approximately 30-45 min of hydrolysis. The DH of the final protein hydrolysate was between 65 and 70% indicating a high degree of hydrolysis. The DH values on the final protein hydrolysate can be used to estimate the number-average chain length of its proteins (amino acids and peptides) using eqn (11): 11 100 PCL--x NSI DH%
(11)
where PCL=average protein chain length, and NSI=nitrogen solubility index. Thus, the protein hydrolysates had an average PCL of approximately 1.3-1.4 amino acid residues, based on an NSI of 90% at 22°C and pH 7 (data shown under NSI discussion) for the calculation of PCL. The solubilities of the individual amino acids in the hydrolysate is included in this calculation, making the PCL value lower than that for the smallest peptide possible (PCL > 2). Thus there may have been a large amount of individual amino acids in the hydrolysate. Taking an average molecular weight of 120 kD for amino acids, the numberaverage molecular mass (/f/n) of the peptides in the hydrolysate would be between 155 and 166 kD. One way of comparing the BCA method and the Kjeldahl method for protein quantification during enzymic hydrolysis of proteins is to plot the ratio of the Kjeldahl protein value to the BCA protein value versus the duration of hydrolysis. In this report, the ratio of the amount of protein in a given volume of hydrolysate estimated by the Kjeldahl method divided by the amount of protein in an identical volume of hydrolysate estimated by the BCA method has been termed RATIO. RATIO can be written in the form of eqn (12): RATIO
protein content determined by Kjeldahl method protein
content
determined
by BCA
method
(12) A plot of RATIO versus duration of hydro-
lysis is shown in Fig. 2. The RATIO was observed to increase with the duration of hydrolysis during the first 45 min of hydrolysis. Beyond this duration, the RATIO was found to remain constant at approximately 2.0. This can be explained by the fact that during the first 45 min of hydrolysis, the enzyme (papain), which has broad specificity, cleaved the protein chains at different points along their lengths leading to the formation of peptides with different lengths and individual amino acids. While the BCA method only quantified those peptides with at least three amino acid residues, the Kjeldahl method quantified total nitrogen in the hydrolysate. In the beginning of hydrolysis, the concentration of small peptides (with less than three amino acids) and amino acids was low in proportion to the amount of total nitrogen (protein), hence a low denominator in eqn (12). As hydrolysis progressed during the first 45 min, however, the enzyme was hydrolyzing the longer chain polypeptides and the protein
2.1
1.9
o 1.8
1.7
1.6
1.5
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20
30
40
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60
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120
Duration of extraction (min)
Fig. 2. Plot of RATIO (ratio of protein content determined by the Kjeldahl method to the protein content determined by the bicinchoninicacid method) during the enzymic hydrolysisof mechanicallydeboned turkey residue (MDTR) using papain. The MDTR/papain (w/w) ratio was 250 g MDTR/g papain. Each point is the mean of duplicate determinations and bars represent a 95% confidenceinterval.
Protein recoveryfrom mechanicallydeboned turkey residue
substrate into smaller peptides. This led to an increase in the concentration of amino acids and dipeptides in the hydrolysate, which cannot be quantified by the BCA method. This caused a decrease in the magnitude of the numerator and an increase in the denominator in eqn (12), hence an increase in the value of RATIO. After approximately 45 min of hydrolysis, the substrate and long chain polypeptides were exhausted, hence RATIO remained constant as the numerator and denominator in eqn (12) became constant. From this analysis, it appears that the hydrolysis process was complete after approximately 45 min of hydrolysis. The magnitude of RATIO at the end of hydrolysis was approximately 2.0. This indicated that the protein hydrolysate contained approximately 50% of its proteins in the form of amino acids and dipeptides. The remaining proteins were longer chain polypeptides. However, the molecular weight distribution of these polypeptides was not determined. Analysis by gel electrophoresis (results not shown) indicated that none of the polypeptides in the protein hydrolysate had a molecular mass greater than 6-5 kD. The protein hydrolysate prepared from MDTR using papain had a final pH ranging from pH 7.11 to 7-24, and a relative density of approximately 1-03. Thus, it was a dilute protein solution but with a high protein content. The hydrolysate had a mean protein content of about 40 mg/ml (40 g/litre) as estimated by the Kjeldahl method. This protein content was significantly higher (P<0.05) than the amount of total protein nitrogen recommended in poultry broth, 100 ppm or 100 mg/litre. 23 Yield The hydrolysis of MDTR produced, on the average, approximately l13g of freeze-dried hydrolysate per kilogram MDTR. With a protein content of approximately 78% (Kjeldahl method), the hydrolysate contained 88 g of protein, representing 8.8% of the MDTR on a wet weight basis and 46% of total MDTR proteins. Very little visible flesh material was left on the bones after hydrolysis of MDTR with papain for more than 45 min. The bones in the MDTR following hydrolysis constituted about 40% of the original weight of MDTR on a wet weight basis (51% on a dry weight basis). The protein content of these bones represented
611
approximately 54% of total MDTR protiens. Therefore, these bones could be ground and used as animal feed or as fertilizer. Enzymic hydrolysis of MDTR has advantages over the alkali extraction process. First, enzymic hydrolysis reduced the original weight of MDTR by approximately 60% (51% on a dry weight basis). Second, only a small amount of water was used in the hydrolysis process [MDTR:water (w/v) ratio of 1:2] and less energy would be required for drying if a hydrolysate powder were needed. Third, enzyme inactivation required heating the hydrolysate at 90°C for 15 min, a heating time-temperature combination that may be too harsh for all types of bacteria associated with poultry. This is evident if one considers the fact that L. monocytogenes (strains Scott A and V7), one of the more heat resistant nonspore forming pathogens in poultry, has D-values of 0.48 and 0.19min, respectively, at 65°C in chicken gravy. 24 Thus if properly handled, the enzymic hydrolysate may be ready for use as a protein supplement or high protein drink or meal. General characterization Proximate analysis
The freeze-dried protein hydrolysate had a high protein content of approximately 78% and a moisture content of 9.1%. It was low in fat and ash content (5.7 and 4.6%, respectively). Mineral analysis
The freeze-dried hydrolysate had high contents of sodium (7951 ppm), potassium (7001 ppm), phosphorus (1286 ppm), calcium (2121 ppm) and magnesium (673 ppm). It also contained zinc (14 ppm) and iron (72 ppm). Thus, a thermally processed hydrolysate meal or drink would be a valuable source of these minerals. For example, a 1% solution of the hydrolysate in water would contain approximately 1750 mg potassium, 322 mg phosphorus, 500 mg calcium, and 168 mg magnesium per cup (250 ml) compared to 381 mg potassium, 235 mg phosphorus, 300 mg of calcium and 34 mg magnesium per cup of 1% low fat m i l k y Functional properties Protein solubility The solubility profile of the protein hydrolysate (measured as nitrogen solubility index, NSI) is shown in Fig. 3. The protein hydrolysate was
L. G. Fonkwe, R. K. Singh
612
and medical f o o d s . 29 The small sizes of the hydrolysate proteins (less than 6.5 kD) makes them weakly immunogenic, thus suitable for use in hypoallergenic diets. Most of the protein hydrolysates being used currently in these areas have been prepared from soy protein and casein. During the determination of protein solubility on the freeze-dried protein hydrolysate, the hydrolysate did not absorb much water. The water holding capacity was only approximately 0.5 ml water per gram protein hydrolysate.
100'
95
.o 1
.=_
~6
85
~
8o
Emulsion capacity and fat binding capacity .~_ Z
75'
70
~
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I 3
4
5
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10
pH of protein solution
Fig. 3. Solubilityprofile of a protein hydrolysate produced from mechanically deboned turkey residue (MDTR) using papain. The ratio of MDTR to papain was 250 g MDTR/g papain. Each point is the mean of triplicate determinationsand bars represent a 95% confidence interval. very soluble at all pH values and also in a 2% sodium chloride solution, with NSI values greater than 90%. The hydrolysate was significantly more soluble in 2% sodium chloride solution (NSI of 95%) than in water adjusted at pH 2-10 (P<0.05). Comparison of means was carried out using the Duncan Multiple range test. The excellent solubility of the protein hydrolysate can be attributed to the small sizes of its polypeptide chains and the increased hydrophilicity of these polypeptides. Determination of the approximate sizes of the peptides in the hydrolysate using 12% SDS-PAGE showed that they had molecular masses lower than 6.5 kD. The increased hydrophilicity is due to their larger charge to size ratios compared to longer proteins such as those found in non-hydrolyzed proteins from a similar source. The solubility profile suggests that the protein hydrolysate can be used in food applications where their high solubility in a wide range of pH is a useful p r o p e r t y . 11 Some of the areas of potential applications include sports nutrition,26 weight control diets, hypoallergenic f o o d s 27'28
The mean emulsion capacity (EC) of the protein hydrolysate at pH 4, 7 and 10 were 175.2, 172.1 and 183.7 ml oil per gram of hydrolysate, respectively. Comparison of these means using Duncan's multiple range test showed that these values were not significantly different (P< 0.05) from one another. These low EC values can be attributed to the high degree of hydrolysis (DH), hence the small size of the peptides in the hydrolysate.28 The DH for the production of the hydrolysate was between 65 and 70%, indicating that the mean protein chain length of the peptides in the hydrolysate was approximately 1.4 amino acid residues. The emulsion activity of a pancreatic casein hydrolysate was reported to decrease linearly with DH and the final hydrolysate (DH 67%) to have a reduced emulsion activity.32 Other reports indicate that there may be an optimum molecular size for a protein to have good emulsion properties. 33'34 It also has been reported that although small peptides can migrate to and adsorb more rapidly at the oil/water interfaces in emulsions, they are not efficient at lowering the interfacial tension and they can also be easily displaced by larger proteins. 28 In addition to its low EC, the protein hydrolysate was a poor fat binder, holding approximately 1.0 ml oil per gram.
Physicochemicai properties Colour of protein hydrolysate The protein hydrolysate solution (prior to drying) had L-a-b values of 48.0, -2-1 and 17, respectively. This gave a hue of - 7 . 0 indicating that the solution had a yellowish colour, based on Francis and Clydesdale's relationship between the a/b ratio, angle and colour. 3° The
Protein recoveryfrom mechanically deboned turkey residue
chroma of the hydrolysate solution was low (17.1), indicating that the colour of the solution was not pure.
Viscosity of the protein hydrolysate The relative viscosity of the protein hydrolysate solution increased with increase in the concentration of the hydrolysate. The relationship between the relative viscosity and the hydrolysate concentration (1-5%) can be expressed using eqn (13): r/r = 0"07C +
0.941,
(13)
where r/r is the relative viscosity of the protein hydrolysate solution (cps) and c is the concentration (g/dl) of the protein solution. The relationship was a linear one with a coefficient of determination of 96.4%.
Sorption isotherm The sorption isotherm of the protein is shown in Fig. 4. The curve is typical of the sorption
613
isotherm of most proteins and other biological materials. The monolayer value of the protein hydrolysate can be calculated from the sorption isotherms using the BET equation, shown in eqn (14). a
1
m(1-a)
-
mlc
c-1 -~
mlc
.a,
(14)
where a=water activity, m=moisture content, ml=monolayer value and c=constant. A BET plot for the first portion of the sorption isotherm is shown in Fig. 5. The relationship was a linear one with a coefficient of determination of 98.9%. The BET monolayer value for the hydrolysate was determined to be 8.22 g water per gram hydrolysate. This value is high compared to values for other proteins in the literature, probably because of the high salt content in the hydrolysate. For example, the monolayer values for casein and soy protein are 5.47 and 5.8g water/gram protein, respectively.31
90
0.045
80 0.04 70 0.035
0.03
~ 50
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E 0.025
40
~
30
0.02
20 0.015 I0 0.01 0
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0.8
m
0.9
Water activity (%)
Fig. 4. Sorption isotherm of a protein hydrolysate produced from mechanically deboned turkey residue using papain. Each data point is the mean of duplicate determinations and bars represent a 95% confidence interval.
0.1
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0.25
0.3
0.35
Water activity
Fig. 5. Plot for the determination of the BET monolayer value of a protein hydrolysate produced from mechanically deboned turkey residue and based on eqn (14). Each data point is the mean of duplicate determinations and bars represent a 95% confidence interval.
614
L. G. Fonkwe, R. K. Singh
Titration curve and buffer capacity Figure 6 shows the titration curve of the protein hydrolysate (1% solution) during titration with a 0.01 M sodium hydroxide solution. The buffering capacity (BC) of the protein hydrolysate decreased rapidly from pH 2-5 to 5, remained fairly constant between pH 5 and 7 and then rose between pH 7 and 11. At low pH values (pH<5), the solution was positively charged since both the amino and carboxyl groups on the amino acids and peptides in the hydrolysate were protonated. The amino acids and peptides existed in the form shown in eqn (15a). As alkali was added, the carboxyl and amino groups on the amino acids and peptides were being deprotonated, the amino acids and peptides acquired some negative charges and existed in the form of zwitterions (eqn 15(b)). This deprotonation caused the rapid decrease in BC between pH 2.5 and 5.5. Between pH 5-5 and 7, BC was at a minimum as most of the amino acids and peptides were becoming negatively charged. Beyond pH 7, all the carboxyl
and amino groups had been deprotonated, the amino acids and peptides were negatively charged and existed in the form shown in eqn (15c). +
H3N - RCH - COOH
(15 a)
H 3 N - R C H - COO
(15b)
HzN-RCH-CO0
05c)
Theoretically, BC was not supposed to increase after pH 7 since less protons were left on the amino acids and peptdies to neutralize the added alkali. The increase in BC seen in Fig. 7, however, resulted from the fact that as the solution became more alkaline, more alkali was needed to increase the pH of the solution. This increase in the amount of alkali added to obtain the required change in pH caused the
1.6 101112. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
f 1.4 ¸
9' 1.2 ¸ 8
.~ 7 6 0.8 0.6'
4 t~ 3'
0.4' 2 0.2' 0 0
20
40
60
80
100
120
140
160
180
200
mL 0.01M NaOH added per 25 mL hydrolysate solution
Fig. 6. Titration curve for the titration of a 1% solution of a protein hydrolysate, recovered from mechanically deboned turkey residue, with 0.01 M sodium hydroxide solution. Each point is the mean of triplicate determinations and bars represent a 95% confidence interval.
0 2
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Fig. 7. Buffering capacity of a protein hydrolysate produced from mechanically deboned turkey residue. Each point is the mean of duplicate determinations and bars represent a 95% confidence interval.
Protein recovery from mechanically deboned turkey residue
phantom increase in BC between pH 7 and 12 observed in Fig. 7. Gel formation The protein hydrolysate solution (20% w/v) did not form a gel upon heating and cooling. This may be due to the small sizes (few amino acid residues) of the peptides present in the hydrolysate. These peptides may not be long enough to form the bonds required to form a strong matrix present in protein-based gels.
SUMMARY AND CONCLUSIONS
Enzymic hydrolysis was shown to be an effective method of recovering potentially edible proteins from mechanically deboned turkey residue, a poultry processing waste. The protein recovery yield was high and the amount of the original residue was reduced by more than half. This could translate to higher income for the poultry industry as the cost of waste disposal would be reduced. More income would also be generated through the sale of protein hydrolysate produced. The hydrolysate produced has potential to be used in a wide range of applications due to its excellent solubility in water over a wide pH range: diet foods, high protein sports drinks, hypoallergenic baby food, etc. At the present time, this market is dominated by hydrolysates from caein and soya. The hydrolysate could also be used as a source of calcium, phosphorus, magnesium and potassium in human nutrition.
REFERENCES 1. Stadelman, W. J., Olson, V. M., Shemwell, G. A. & Pasch, S., Egg and Poultry-Meat Processing. Ellis and Horwood, Chichester, UK, 1988. 2. Kijowski, J. & Niewiarowicz, A., A method of protein extraction from chicken bone residue and the chemical and electrophoretic characteristics of the extract. J. Food Technol., 20 (1985) 43-9. 3. Wallace, M. J. D. & Froning, G. W., Protein quality determination of bone residue from mechanically deboned chicken meat. Poultry Sci., 48 (1979) 333-6. 4. Young, L. L., Composition and properties of an animal protein isolate prepared from bone residue. J. Food Sci., 41 (1976) 606-8. 5. McCurdy, S. M., Jelen, P., Fedec, P. & Wood, D. F., Laboratory and pilot scale recovery of protein from mechanically separated chicken residue. J. Food Sci., 51 (1986) 742-7, 753.
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6. USDA, Agricultural Statistics. United States Department of Agriculture, Washington, DC, 1992. 7. Fonkwe, L. G. & Singh, R. K., Protein recovery from mechanically deboned turkey residue. Trans. ASAE, 37 (1994) 527-34. 8. Lawrence, R. A. & Jelen, P., Formation of lysinoalanine in alkaline extracts of chicken proteins. J. Food Prot., 45 (1982) 923-4. 9. Masters, P. M. & Friedman, M., Amino acid racemization in alkali-treated food proteins - - chemistry, toxicity, and nutritional consequences. In Chemical Deterioration of Food Proteins, ed. J. R. Whitaker & M. Fujimaki. American Chemical Society Symposium. Series, Am. Chem. Soc., Washington, DC, 1980. 10. Whitaker, J. R., Changes occurring in proteins in alkaline solution. In Chemical Deterioration of Food Proteins, ed. J. R. Whitaker & M. Fujimaki. American Chemical Society Symposium. Series, Am. Chem. Soc., Washington, DC, 1980. 11. Adler-Nissen, J., Enzymic Hydrolysis of Food Proteins. Elsevier, New York, 1986. 12. Fik, M. & Surowka, K., Preparation and properties of protein concentrate from broiler chicken heads. J. Sci. Food Agric., 37 (1986) 445-54. 13. Surowka, K. & Fik, M., Studies on the recovery of proteinaceous substances from chicken heads. I. An application of neutrase to the production of protein hydrolysate. Int. J. Food Sci. Technol., 27 (1992) 9-20. 14. Kijowski, J., Stangierski, J. & Lesnierowski, G., Enzymic protein hydrolysate from bone residues from mechanical deboning of chickens. Przemysl-Spozywczy, 46 (1992) 149-52. 15. AOAC, Official Methods of Analysis, 15th edn Association of Official Analytical Chemists, Washington, DC, 1990. 16. Hoyle, N. T. & Merritt, J. H., Quality of fish protein hydrolysates from herring (Clupea harengus). J. Food Sci., 59 (1994) 76-9. 17. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C., Measurement of protein using bicinchoninic acid. Anal. Biochem., 150 (1985) 76-85. 18. Regenstein, J. M. & Regenstein, C. E., Food Protein Chemistry. Academic Press, New York, 1984. 19. Chang, S. K. C., Protein analysis. In Introduction to the Chemical Analysis of Foods, ed. S. S. Nielsen. Jones and Bartlett, Boston, 1994, Chapt. 14, pp. 207-20. 20. Ozimek, G., Poznanski, S. & Cichon, R., Influence of selected factors on functional properties of textured milk proteins. J. Food Technol., (1981) 575-85. 21. Morr, C. V., Swenson, P. E. & Richter, R. L., Functional properties of whey protein concentrates. J. Food Sci., 38 (1973) 324. 22. Coffmann, C. W. & Garcia, V. V., Functional properties and amino acid content of a protein isolate from mung bean flour. J. Food Technol., 12 (1977) 473-84. 23. Binsted, R. & Devey, J. D., Legal aspects: meat soups; cream soups; vegetable soups; clear soups; broths; miscellaneous. In Soup Manufacture: Canning, Dehydration and Quick-Freezing, 3rd edn. Food Trade Press, London, 1970., pp. 132-3. 24. Huang, I. D., Yousef, A. E., Marth, E. H. & Mathews, M. E., Thermal inactivation of listeria monocytogenes in chicken gravey. J. Food Prot., 55 (1992) 492-6. 25. Whitney, E. N. & Rolfes, S. R., Understanding Nutrition. West, Minneapolis, MN, 1993.
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26. Frokjaer, S., Use of hydrolysates for protein supplementation. Food Technol., 48 (1994) 68-71. 27. Cordle, C. T., Control of food allergies using protein hydrolysates. Food Technol., 48 (1994) 72-6. 28. Mahmoud, M. I., Physicochemical and functional properties of protein hydrolysates in nutritional products. Food Technol., 48 (1994) 89-95. 29. Schmidl, M. K., Taylor, S. L. & Nordlee, J. A., Use of hydrolysate-based products in special medical diets. Food Technol., 48 (1994) 77-80, 85. 30. Francis, F. J. & Clydesdale, F. M., Food Colorimetry: Theory and Applications. AVI, Westport, CT, 1975. 31. Chou, D. H. & Morr, C. V., Protein-water inter-
actions and functional properties. J. Am. Oil Chemists' Soc., 56 (1979) 53A-62A. 32. Mahmoud, M. I., Malone, W. T. & Cordle, C. T., Enzymatic hydrolysis of casein: effect of degree of hydrolysis on antigenicity and physical properties. J. Food Sci., 57 (1992) 1223-9. 33. Adler-Nissen, J., Enzymatic hydrolysis of soy protein for nutritional fortification of low pH food. Ann. Nutr. Alim., 32 (1978) 205-16. 34. Lee, S. W., Shimizu, M., Kaminogawa, S. & Yamauchi, K., Emulsifying properties of peptides obtained from the hydrolysis of fl-casein. Agric. Biol. Chem., 51 (1987) 161-6.
0032-9592(95)00101-8
Protein Recovery from Mechanically Deboned Turkey Residue by Enzymic Hydrolysis L. G. Fonkwe & R. K. Singh* Purdue University, Food Science Department, Smith Hall, West Lafayette, IN 47907-1160, USA (Received 30 August 1995; revised manuscript received and accepted 19 November 1995)
Enzymic hydrolysis was used to recover a potentially edible high protein hydrolysate from mechanically deboned turkey residue (MDTR), a turkey processing waste. The hydrolysis process reduced the weight of the original MDTR by 51% on a dry weight basis, and recovered 46% of the MDTR proteins. The hydrolysate contained 78% protein, 4"6% ash and only 5.7% fat. The proteins consisted mainly of amino acids and small peptides with molecular masses lower than 6"5 kD due to a high degree of hydrolysis (65- 70%), and were very soluble in water over a wide pH range. It had a yellowish colour, was rich in calcium, phosphorus, potassium and magnesium and had a large monolayer value, but had low viscosity and low emulsion capacity at p H values of 4, 7and 10. Its buffering capacity was at a minimum between pH 5 and 7.
INTRODUCTION
produced in the US in 1991. 6 It is estimated that about 169000 tons of MDTR were produced during turkey deboning (private communication). Thus a large volume of deboner waste, containing valuable proteins, was generated for disposal. MDTR has been shown to contain approximately 51% moisture, 19% protein, 9% fat and 15% ash. 7 Similar figures have been reported for chicken bone residue by other researchers. 2'5 The protein content of MDTR includes approximately 20% of salt- and alkali-soluble proteins which can be recovered and used for human consumption. The MDTR proteins also include approximately 40% collagen from tendons and bones 2 which, although not salt- or alkali-soluble, can be enzymically hydrolyzed. Yields reported for the recovery of proteins, using alkali extraction, from poultry deboner residue have been low. A yield of 12%, based on the total amount of proteins in MDTR, in a
Mechanical deboning is a unit operation in the poultry processing industry. During this process, pressure is used to separate meat from a slurry of ground bones and meat inside a pressure chamber in a mechanical deboner. The separated ground meat is currently being used in the manufacture of comminuted poultry products such as patties, bologna and sausages. 1'2 The waste material leaving the deboner is a bony residue. In the case of turkey processing, the residue is known as mechanically deboned turkey residue (MDTR). This residue still contains a valuable amount of protein and has been recognized as a valuable source of proteins for human food and is currently being used in the manufacture of animal feed. 2-5 Approximately 2415000 tons of turkey were *To whom correspondence should be addressed. 605
606
L. G. Fonkwe, R. K. Singh
laboratory process has been reported. 7 The method involved the recovery of proteins from mechanically deboned turkey residue by extraction with an alkaline sodium chloride solution and precipitation with polysaccharides, such as carrageenan, carboxymethyl cellulose and chitosan. Protein yields of 9-16% in pilot scale extractions and 6-15% in laboratory experiments from chicken bone residue have also been reported. 5 Thus, it is important to investigate alternative processes that could lead to higher protein yields from poultry deboner residue. Enzymic hydrolysis is potentially an effective method for the recovery of proteins from MDTR. Enzymic hydrolysis of MDTR has some advantages over the alkaline extraction method of recovering proteins from MDTR. First, enzymic hydrolysis has the advantage that both the collagenous and non-collagenous proteins (as well as any other proteins in the MDTR such as blood and bone marrow) are hydrolyzed, unlike the alkaline extraction of proteins in which the major proteins extracted are non-collagenous proteins. This would lead to a higher protein yield. Second, this method of protein recovery also has the potential of reducing the volume of waste (wastewater and collagen/bone slurry) generated by a large amount compared to the alkaline recovery methods. Furthermore, the enzymic hydrolysis process is simple, efficient and involves mild alkaline conditions that would not destroy the proteins recovered by racemization and/or other chemical reactions. 8-1° A limitation to enzymic hydrolysis of proteins is the fact that a bitter hydrolysate may be formed. 11 However, the latter author also indicated that animal protein hydrolysates generally do not have a bitter taste. Some studies have been carried out utilizing enzymic hydrolysis in the recovery of proteins from other poultry processing wastes. 12-~4 Treating chicken meat with an enzyme having a high endopeptidase activity could produce a bitter hydrolysate while the same treatment with an enzyme having both endo- and exopeptidase activity would produce a hydrolysate with no bitter taste. ~2 Protein hydrolysates have been produced from broiler chicken heads, using alcalase and neutrase, and reported to have good organolpetic and physicochemical properties. 12,13 A protein hydrolysate has also been prepared
from the bone residue resulting from the mechanical deboning of chickens, using a Polish enzyme preparation (proteopol BP-S). la The reported yield was 23-24% of nitrogenous compounds, and the hydrolysate had excellent solubility, good emulsifying capacity and emulsion stability, and a molecular weight less than 40 kD. The main objectives of this investigation were: (I) to investigate the production of an enzymic hydrolysate from MDTR using crude papain, and (2) to characterize the hydrolysate produced in terms of its physical, chemical and functional properties. MATERIALS AND METHODS Hydrolysis conditions
Five hundred grams of MDTR (19% protein) were added to 1 litre of water held at 60°C in a water bath. The MDTR/water mixture was allowed to reach a temperature of 60°C and crude protein [activity of 1.7 units/mg solid, purchased from Sigma Chem. Co. (St Louis, MO) and partially dissolved in warm water (60°C)] was added to the mixture. The ratio of MDTR to enzyme (w/w) was 250 g MDTR/g papain. Preliminary experiments had shown that these were excellent conditions for the enzymic hydrolysis. The mixture was continuously stirred with a mechanical stirrer and broth samples were collected at 5, 15, 30, 45, 60, 90 and 120 min of hydrolysis. At the end of the hydrolysis, all the hydrolysates collected were filtered through a metal screen, then through multiple layers of cheesecloth, and centrifuged at 3500g for 10min in a bench-top centrifuge (International Equipment Co., Needham Hts, MA) to remove all solid particles and fat. The protein hydrolysates were heated to about 95°C for at least 15 min to inactivate the enzyme, then allowed to cool to room temperature (20-22°C). Any fat layer on the surface of the liquid hydrolysate was siphoned off. The bulk of the hydrolysate was then freeze-dried. The powder formed was analyzed for protein, fat and ash contents using the approved standard methods. 15 All the hydrolysate samples collected were analyzed for protein content using the Kjeldahl and bicinchoninic acid methods. The bony residue also was weighed, dried and analyzed for moisture, protein, fat and ash con-
Protein recoveryfrom mechanicalS,deboned turkeyresidue tent, using approved standard methods. 15 This was done to determine any possible uses for this final waste material. The bony residue was then discarded. Determination of the degree of hydrolysis The degree of hydrolysis (DH) of the protein hydrolysates was determined using the trichloroacetic acid m e t h o d . 16 The method is based on the ratio of 10% trichloroacetic acid (TCA) soluble protein in the hydrolysate compared to the total amount of protein in the sample. After hydrolysis, 40 ml of the protein hydrolysate were mixed with 40 ml of 20% TCA (making a total TCA concentration of 10%). The mixture was stirred for 5 min, then centrifuged for 15min at 16000g in a Beckman centrifuge (Model J2-21, Beckman Instrument Inc., Palo Alto, CA). The supernatant was collected and freeze-dried. The amount of protein in the freeze-dried hydrolysate and in the freeze-dried TCA-soluble hydrolysate were determined by the Kjeldahl method. The degree of hydrolysis was calculated using eqn (1). 10% TCA-soluble protein in sample DHx 100. total protein in sample
(1) General characterization Proximate analysis Proximate analysis on protein powders was carried out using the approved standard methods. 15 Protein analysis on solids was carried out using the Kjeldahl method. Fat analysis was performed using the Soxhlet method, and ash analysis was carried out gravimetrically by heating the sample at 550°C in a muffle furnace for 24 h. However, protein analysis in solution was carried out using the bicinchoninic acid (BCA) method using bovine serum albumin as the reference protein. 17 This is because the Kjeldahl method is a time-consuming method while the BCA method takes a very short time to perform. Thus, the BCA method is preferable in situations in which large numbers of protein determinations are to be carried out. The relationship between the two methods needs to be established on the same sample so that results from the BCA method can be con-
607
verted to those from the Kjeldahl method (the official method). 18'19 This is because the two methods measure different quantities and will give different readings for the same sample. The Kjeldahl method measures the total amount of nitrogen in a sample and converts this into the total amount of protein. The BCA method quantifies the number of peptides and proteins in solution which have at least three amino acids (two peptide bonds). Thus the Kjeldahl method of protein estimation will give a higher value than the BCA method.
Mineral analysis Mineral analysis was carried out on freeze-dried samples using the Inductively Coupled Plasma/ Atomic Emission Spectrophotometry (ICP/ AES) method in the Food Science Dept., Purdue University. Samples were digested with 70% nitric acid and hydrogen peroxide and mineral analysis were carried out on a Perkin Elmer Plasma 400 ICP/AES instrument (Norwalk, CT) with a model AS 90 auto sampler. Functional properties Water-holding capacity and protein solubility The procedure for the determination of waterholding capacity (WHC) was a recommended method in the literature with modification.TM A sample of the protein hydrolysate powder was mixed with water in the ratio 1:20 (w :v). The pH of the hydrolysate solutions was then adjusted to pH 2, 4, 5, 5.5, 6, 8 and 10 using hydrochloric acid and/or sodium hydroxide solutions and stirred for 30min. The pH of hydrolysate solutions were checked and corrected to the desired pH value every 5 min during stirring until it remained constant. The solutions were then centrifuged at 16000g for 15 min. The volume of each resultant supernatant was measured using a graduated cylinder, and used to determine WHC using eqn (2): WHC=
volume of water absorbed (ml) x 100. (2) weight of sample (g)
The protein solubility index (or nitrogen solubility index, NSI) was calculated by comparing the amount of protein in the supernatant (converted to Kjeldahl units) to the amount of protein in the original sample, using eqn (3).
608
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L. G. Fonkwe, R. K. Singh
amount of protein in solution x 100%. amount of protein in the originalsample
noticeable change in the sound of the homogenizer, using the equation volume of canola oil used (ml)=duration of
(3) The amount of protein in solution was determined using the BCA method while the amount of protein in the freeze-dried hydrolysate was determined using the Kjeldahl method. The protein content from the BCA assay was then converted into protein estimates in the Kjeldahl unit using a predetermined scale.
Fat binding capacity The fat binding capacity (FBC) was measured using a method described in the literature 2° with modification. One gram of the sample was mixed with 20 ml of Crisco puritan canola oil (Procter & Gamble, Cincinnati, OH) and stirred for 10 min. The mixture was then centrifuged at 16000g for 15min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Palo Alto, CA) and the volume of the unabsorbed oil was measured using a graduated cylinder. The FBC was calculated using eqn (4) FBC=
amount of fat absorbed (ml) weight of sample (g)
process × rate of oil addition. The emulsion capacity (EC) was calculated as the amount of oil emulsified per gram of protein, using eqn (5). EC-
Emulsion capacity The procedure utilized in the measurement of the emulsion capacity (EC) was a method described in the literature 18 with modification. A 1% hydrolysate solution was prepared by dissolving the hydrolysate in a 0.5 M sodium chloride solution. The hydrolysate solution (50 ml) was poured into a 600 ml plastic beaker. The protein solution was homogenized with a polytron homogenizer at 1700 rpm for 15 s prior to the start of oil addition to the protein solution. Crisco puritan canola oil (Procter & Gamble, Cincinnati, OH) was added to the protein solution at a rate of 1.2ml/s using a Masterflex pump (model 7016-52, Cole Parmer, Chicago, IL) with continuous homogenization until a steady change in the sound of the homogenizer was noticed. The duration of the oil addition process was measured with a stop watch and used to calculate the volume of canola oil delivered to the protein solution that caused the increase in viscosity leading to a
wt of protein in sample (g)
(5) Physicochemical properties Colour of the hydrolysate solution The colour of the hydrolysate solution was determined on the L-a-b scale using a Hunter colorimeter (Model D25-PC2, Hunter Associated Laboratories, Reston, VA). A HunterLab standard white plate (No. C2-30636) was used to standardize the colorimeter prior to use. Three parameters, hue, chroma and AE, were calculated from the L-a-b readings obtained using eqns (6)-(8):
x 100%. (4)
volume of canola oil used (ml)
Hue=tan-1 (a/b )
(6)
Chroma = (a 2 + b 2)1/2
(7)
AE = (AL z + Aa 2 + Ab 2),/2
(8)
and where AL, Aa and Ab are differences in the measured L-a-b values between any two solutions.
Viscosity of the hydrolysate solutions Solutions of the protein hydrolysate were prepared, in concentrations ranging from 1 to 5% (w/v), in volumetric flasks using the dry hydrolysate powders. Each solution then was centrifuged at 16000g for 15 min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Palo Alto, CA) and the supernatant was collected. The viscosity of each solution was measured using an Oswald capillary viscometer, and the relationship between protein concentration and relative viscosity was determined. Sorption isotherm Hydrolysate powder was placed in pre-weighed aluminum dishes and weighed. The dishes containing the samples were then placed in
Protein recovery from mechanicalty deboned turkey residue
evacuated air tight dessicators containing saturated solutions of lithium chloride, potassium acetate, magnesium chloride, potassium carbonate, magnesium nitrate, sodium nitrite, sodium chloride, potassium chloride, and potassium nitrate in water. These solutions had equilibrium relative humidities of 12, 23, 33, 44, 55, 64, 76, 85 and 93%, respectively. The samples were held in the dessicators for two weeks, based on preliminary investigations which showed that two weeks were enough time for the samples to attain equilibrium with their environments. After two weeks, the samples were weighed again and the moisture contents (MC) of the samples were calculated using eqn (9): ME-
mass of water in sample mass of dry sample
x 100.
(9)
Determination of titration curve A 1% (w/v) solution of the protein hydrolysate in water was prepared. The solution was cooled to room temperature using cold tap water. The protein solution (25 ml) was measured into an Erlenmeyer flask and its pH lowered to approximately pH 2.0 using a 3 M hydrochloric acid solution. The protein solution then was titrated using a 0-01 M sodium hydroxide solution until its pH rose to approximately pH 12. Buffering capacity The buffering capacity (BC) of the protein hydrolysate was calculated from the titration curve data using eqn (10), 21'22 for each pH change of 0.5 units.
609
determined using a QTest texture analyzer (QTest, a division of Sintech Inc., Cary, NC). Immediately prior to measuring the gel strength, the gel was carefully removed from the 10 ml beaker and placed on a platform. The gel strength was determined as the maximum amount of force required to crush the gel (diameter of 21 mm and height of 30 ram) to either the break point or to 75% deformation using a 5 kgf (10 lbf) load cell. RESULTS AND DISCUSSION Hydrolysis parameters Two curves showing the rate of production of proteins from MDTR using papain are shown in Fig. 1. The amount of protein in solution increased significantly ( P < 0.05) during the first 45 rain of hydrolysis and then levelled off at about 20 mg/ml based on the BCA method of protein quantification (40 mg/ml by the Kjeldahl method). However, the amount of protein in the hydrolysate did not increase significantly
55 50 45 40 -6 35
i
~ 3o ' ~ 25
BC=
meq titrant mass of protein (g) x ApH
(10)
Gel formation The procedure for gel formation was obtained from the literature 18 with modification. A 20% (w/v) hydrolysate solution was prepared by adding the sample to deionized distilled water, mixing and holding in a volumetric flask. The hydrolysate solution was heated to 100°C for 30 min, the volume adjusted, and the solution was poured into 10 ml beakers and allowed to cool to room temperature (20-22°C). The solution then was held at 8-100C in a refrigerator overnight. The strength of the gel formed was
~ 20
4
-'*-- BCA - 4 - - Kjeldahl
10
~So
ids
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Duration of hydrolysis (rain)
Fig. 1. Hydrolysis curves for the recovery of proteins from mechanically deboned turkey residue (MDTR) by enzymic hydrolysis using papain (BCA=bicinchoninic acid method; Kjeldahl=Kjeldahl method of protein quantification). The substrate to enzyme ratio was 250 g MDTR/g papain. Each point is the mean of triplicate determinations and bars represent a 95% confidence level.
L. G. Fonkwe, R. K. Singh
610
(P < 0.05) after about 45 min of hydrolysis. Similarly, the amount of total solids in solution did not increase significantly (P<0.05) after about 30 min of hydrolysis, as shown in Fig. 1 also. Therefore, it appears that the process was complete within approximately 30-45 min of hydrolysis. The DH of the final protein hydrolysate was between 65 and 70% indicating a high degree of hydrolysis. The DH values on the final protein hydrolysate can be used to estimate the number-average chain length of its proteins (amino acids and peptides) using eqn (11): 11 100 PCL--x NSI DH%
(11)
where PCL=average protein chain length, and NSI=nitrogen solubility index. Thus, the protein hydrolysates had an average PCL of approximately 1.3-1.4 amino acid residues, based on an NSI of 90% at 22°C and pH 7 (data shown under NSI discussion) for the calculation of PCL. The solubilities of the individual amino acids in the hydrolysate is included in this calculation, making the PCL value lower than that for the smallest peptide possible (PCL > 2). Thus there may have been a large amount of individual amino acids in the hydrolysate. Taking an average molecular weight of 120 kD for amino acids, the numberaverage molecular mass (/f/n) of the peptides in the hydrolysate would be between 155 and 166 kD. One way of comparing the BCA method and the Kjeldahl method for protein quantification during enzymic hydrolysis of proteins is to plot the ratio of the Kjeldahl protein value to the BCA protein value versus the duration of hydrolysis. In this report, the ratio of the amount of protein in a given volume of hydrolysate estimated by the Kjeldahl method divided by the amount of protein in an identical volume of hydrolysate estimated by the BCA method has been termed RATIO. RATIO can be written in the form of eqn (12): RATIO
protein content determined by Kjeldahl method protein
content
determined
by BCA
method
(12) A plot of RATIO versus duration of hydro-
lysis is shown in Fig. 2. The RATIO was observed to increase with the duration of hydrolysis during the first 45 min of hydrolysis. Beyond this duration, the RATIO was found to remain constant at approximately 2.0. This can be explained by the fact that during the first 45 min of hydrolysis, the enzyme (papain), which has broad specificity, cleaved the protein chains at different points along their lengths leading to the formation of peptides with different lengths and individual amino acids. While the BCA method only quantified those peptides with at least three amino acid residues, the Kjeldahl method quantified total nitrogen in the hydrolysate. In the beginning of hydrolysis, the concentration of small peptides (with less than three amino acids) and amino acids was low in proportion to the amount of total nitrogen (protein), hence a low denominator in eqn (12). As hydrolysis progressed during the first 45 min, however, the enzyme was hydrolyzing the longer chain polypeptides and the protein
2.1
1.9
o 1.8
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Duration of extraction (min)
Fig. 2. Plot of RATIO (ratio of protein content determined by the Kjeldahl method to the protein content determined by the bicinchoninicacid method) during the enzymic hydrolysisof mechanicallydeboned turkey residue (MDTR) using papain. The MDTR/papain (w/w) ratio was 250 g MDTR/g papain. Each point is the mean of duplicate determinations and bars represent a 95% confidenceinterval.
Protein recoveryfrom mechanicallydeboned turkey residue
substrate into smaller peptides. This led to an increase in the concentration of amino acids and dipeptides in the hydrolysate, which cannot be quantified by the BCA method. This caused a decrease in the magnitude of the numerator and an increase in the denominator in eqn (12), hence an increase in the value of RATIO. After approximately 45 min of hydrolysis, the substrate and long chain polypeptides were exhausted, hence RATIO remained constant as the numerator and denominator in eqn (12) became constant. From this analysis, it appears that the hydrolysis process was complete after approximately 45 min of hydrolysis. The magnitude of RATIO at the end of hydrolysis was approximately 2.0. This indicated that the protein hydrolysate contained approximately 50% of its proteins in the form of amino acids and dipeptides. The remaining proteins were longer chain polypeptides. However, the molecular weight distribution of these polypeptides was not determined. Analysis by gel electrophoresis (results not shown) indicated that none of the polypeptides in the protein hydrolysate had a molecular mass greater than 6-5 kD. The protein hydrolysate prepared from MDTR using papain had a final pH ranging from pH 7.11 to 7-24, and a relative density of approximately 1-03. Thus, it was a dilute protein solution but with a high protein content. The hydrolysate had a mean protein content of about 40 mg/ml (40 g/litre) as estimated by the Kjeldahl method. This protein content was significantly higher (P<0.05) than the amount of total protein nitrogen recommended in poultry broth, 100 ppm or 100 mg/litre. 23 Yield The hydrolysis of MDTR produced, on the average, approximately l13g of freeze-dried hydrolysate per kilogram MDTR. With a protein content of approximately 78% (Kjeldahl method), the hydrolysate contained 88 g of protein, representing 8.8% of the MDTR on a wet weight basis and 46% of total MDTR proteins. Very little visible flesh material was left on the bones after hydrolysis of MDTR with papain for more than 45 min. The bones in the MDTR following hydrolysis constituted about 40% of the original weight of MDTR on a wet weight basis (51% on a dry weight basis). The protein content of these bones represented
611
approximately 54% of total MDTR protiens. Therefore, these bones could be ground and used as animal feed or as fertilizer. Enzymic hydrolysis of MDTR has advantages over the alkali extraction process. First, enzymic hydrolysis reduced the original weight of MDTR by approximately 60% (51% on a dry weight basis). Second, only a small amount of water was used in the hydrolysis process [MDTR:water (w/v) ratio of 1:2] and less energy would be required for drying if a hydrolysate powder were needed. Third, enzyme inactivation required heating the hydrolysate at 90°C for 15 min, a heating time-temperature combination that may be too harsh for all types of bacteria associated with poultry. This is evident if one considers the fact that L. monocytogenes (strains Scott A and V7), one of the more heat resistant nonspore forming pathogens in poultry, has D-values of 0.48 and 0.19min, respectively, at 65°C in chicken gravy. 24 Thus if properly handled, the enzymic hydrolysate may be ready for use as a protein supplement or high protein drink or meal. General characterization Proximate analysis
The freeze-dried protein hydrolysate had a high protein content of approximately 78% and a moisture content of 9.1%. It was low in fat and ash content (5.7 and 4.6%, respectively). Mineral analysis
The freeze-dried hydrolysate had high contents of sodium (7951 ppm), potassium (7001 ppm), phosphorus (1286 ppm), calcium (2121 ppm) and magnesium (673 ppm). It also contained zinc (14 ppm) and iron (72 ppm). Thus, a thermally processed hydrolysate meal or drink would be a valuable source of these minerals. For example, a 1% solution of the hydrolysate in water would contain approximately 1750 mg potassium, 322 mg phosphorus, 500 mg calcium, and 168 mg magnesium per cup (250 ml) compared to 381 mg potassium, 235 mg phosphorus, 300 mg of calcium and 34 mg magnesium per cup of 1% low fat m i l k y Functional properties Protein solubility The solubility profile of the protein hydrolysate (measured as nitrogen solubility index, NSI) is shown in Fig. 3. The protein hydrolysate was
L. G. Fonkwe, R. K. Singh
612
and medical f o o d s . 29 The small sizes of the hydrolysate proteins (less than 6.5 kD) makes them weakly immunogenic, thus suitable for use in hypoallergenic diets. Most of the protein hydrolysates being used currently in these areas have been prepared from soy protein and casein. During the determination of protein solubility on the freeze-dried protein hydrolysate, the hydrolysate did not absorb much water. The water holding capacity was only approximately 0.5 ml water per gram protein hydrolysate.
100'
95
.o 1
.=_
~6
85
~
8o
Emulsion capacity and fat binding capacity .~_ Z
75'
70
~
I
1
2
I 3
4
5
6
7
8
9
10
pH of protein solution
Fig. 3. Solubilityprofile of a protein hydrolysate produced from mechanically deboned turkey residue (MDTR) using papain. The ratio of MDTR to papain was 250 g MDTR/g papain. Each point is the mean of triplicate determinationsand bars represent a 95% confidence interval. very soluble at all pH values and also in a 2% sodium chloride solution, with NSI values greater than 90%. The hydrolysate was significantly more soluble in 2% sodium chloride solution (NSI of 95%) than in water adjusted at pH 2-10 (P<0.05). Comparison of means was carried out using the Duncan Multiple range test. The excellent solubility of the protein hydrolysate can be attributed to the small sizes of its polypeptide chains and the increased hydrophilicity of these polypeptides. Determination of the approximate sizes of the peptides in the hydrolysate using 12% SDS-PAGE showed that they had molecular masses lower than 6.5 kD. The increased hydrophilicity is due to their larger charge to size ratios compared to longer proteins such as those found in non-hydrolyzed proteins from a similar source. The solubility profile suggests that the protein hydrolysate can be used in food applications where their high solubility in a wide range of pH is a useful p r o p e r t y . 11 Some of the areas of potential applications include sports nutrition,26 weight control diets, hypoallergenic f o o d s 27'28
The mean emulsion capacity (EC) of the protein hydrolysate at pH 4, 7 and 10 were 175.2, 172.1 and 183.7 ml oil per gram of hydrolysate, respectively. Comparison of these means using Duncan's multiple range test showed that these values were not significantly different (P< 0.05) from one another. These low EC values can be attributed to the high degree of hydrolysis (DH), hence the small size of the peptides in the hydrolysate.28 The DH for the production of the hydrolysate was between 65 and 70%, indicating that the mean protein chain length of the peptides in the hydrolysate was approximately 1.4 amino acid residues. The emulsion activity of a pancreatic casein hydrolysate was reported to decrease linearly with DH and the final hydrolysate (DH 67%) to have a reduced emulsion activity.32 Other reports indicate that there may be an optimum molecular size for a protein to have good emulsion properties. 33'34 It also has been reported that although small peptides can migrate to and adsorb more rapidly at the oil/water interfaces in emulsions, they are not efficient at lowering the interfacial tension and they can also be easily displaced by larger proteins. 28 In addition to its low EC, the protein hydrolysate was a poor fat binder, holding approximately 1.0 ml oil per gram.
Physicochemicai properties Colour of protein hydrolysate The protein hydrolysate solution (prior to drying) had L-a-b values of 48.0, -2-1 and 17, respectively. This gave a hue of - 7 . 0 indicating that the solution had a yellowish colour, based on Francis and Clydesdale's relationship between the a/b ratio, angle and colour. 3° The
Protein recoveryfrom mechanically deboned turkey residue
chroma of the hydrolysate solution was low (17.1), indicating that the colour of the solution was not pure.
Viscosity of the protein hydrolysate The relative viscosity of the protein hydrolysate solution increased with increase in the concentration of the hydrolysate. The relationship between the relative viscosity and the hydrolysate concentration (1-5%) can be expressed using eqn (13): r/r = 0"07C +
0.941,
(13)
where r/r is the relative viscosity of the protein hydrolysate solution (cps) and c is the concentration (g/dl) of the protein solution. The relationship was a linear one with a coefficient of determination of 96.4%.
Sorption isotherm The sorption isotherm of the protein is shown in Fig. 4. The curve is typical of the sorption
613
isotherm of most proteins and other biological materials. The monolayer value of the protein hydrolysate can be calculated from the sorption isotherms using the BET equation, shown in eqn (14). a
1
m(1-a)
-
mlc
c-1 -~
mlc
.a,
(14)
where a=water activity, m=moisture content, ml=monolayer value and c=constant. A BET plot for the first portion of the sorption isotherm is shown in Fig. 5. The relationship was a linear one with a coefficient of determination of 98.9%. The BET monolayer value for the hydrolysate was determined to be 8.22 g water per gram hydrolysate. This value is high compared to values for other proteins in the literature, probably because of the high salt content in the hydrolysate. For example, the monolayer values for casein and soy protein are 5.47 and 5.8g water/gram protein, respectively.31
90
0.045
80 0.04 70 0.035
0.03
~ 50
'
E 0.025
40
~
30
0.02
20 0.015 I0 0.01 0
I
0
0.1
0.2
i
0.3
0,4
i
0.5
0.6
0.7
0.8
m
0.9
Water activity (%)
Fig. 4. Sorption isotherm of a protein hydrolysate produced from mechanically deboned turkey residue using papain. Each data point is the mean of duplicate determinations and bars represent a 95% confidence interval.
0.1
I
I
I
I
0.15
0.2
0.25
0.3
0.35
Water activity
Fig. 5. Plot for the determination of the BET monolayer value of a protein hydrolysate produced from mechanically deboned turkey residue and based on eqn (14). Each data point is the mean of duplicate determinations and bars represent a 95% confidence interval.
614
L. G. Fonkwe, R. K. Singh
Titration curve and buffer capacity Figure 6 shows the titration curve of the protein hydrolysate (1% solution) during titration with a 0.01 M sodium hydroxide solution. The buffering capacity (BC) of the protein hydrolysate decreased rapidly from pH 2-5 to 5, remained fairly constant between pH 5 and 7 and then rose between pH 7 and 11. At low pH values (pH<5), the solution was positively charged since both the amino and carboxyl groups on the amino acids and peptides in the hydrolysate were protonated. The amino acids and peptides existed in the form shown in eqn (15a). As alkali was added, the carboxyl and amino groups on the amino acids and peptides were being deprotonated, the amino acids and peptides acquired some negative charges and existed in the form of zwitterions (eqn 15(b)). This deprotonation caused the rapid decrease in BC between pH 2.5 and 5.5. Between pH 5-5 and 7, BC was at a minimum as most of the amino acids and peptides were becoming negatively charged. Beyond pH 7, all the carboxyl
and amino groups had been deprotonated, the amino acids and peptides were negatively charged and existed in the form shown in eqn (15c). +
H3N - RCH - COOH
(15 a)
H 3 N - R C H - COO
(15b)
HzN-RCH-CO0
05c)
Theoretically, BC was not supposed to increase after pH 7 since less protons were left on the amino acids and peptdies to neutralize the added alkali. The increase in BC seen in Fig. 7, however, resulted from the fact that as the solution became more alkaline, more alkali was needed to increase the pH of the solution. This increase in the amount of alkali added to obtain the required change in pH caused the
1.6 101112. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
f 1.4 ¸
9' 1.2 ¸ 8
.~ 7 6 0.8 0.6'
4 t~ 3'
0.4' 2 0.2' 0 0
20
40
60
80
100
120
140
160
180
200
mL 0.01M NaOH added per 25 mL hydrolysate solution
Fig. 6. Titration curve for the titration of a 1% solution of a protein hydrolysate, recovered from mechanically deboned turkey residue, with 0.01 M sodium hydroxide solution. Each point is the mean of triplicate determinations and bars represent a 95% confidence interval.
0 2
I
I
I
I
I
I
I
I
I
3
4
5
6
7
8
9
10
II
pH of hydrolysate solution
Fig. 7. Buffering capacity of a protein hydrolysate produced from mechanically deboned turkey residue. Each point is the mean of duplicate determinations and bars represent a 95% confidence interval.
Protein recovery from mechanically deboned turkey residue
phantom increase in BC between pH 7 and 12 observed in Fig. 7. Gel formation The protein hydrolysate solution (20% w/v) did not form a gel upon heating and cooling. This may be due to the small sizes (few amino acid residues) of the peptides present in the hydrolysate. These peptides may not be long enough to form the bonds required to form a strong matrix present in protein-based gels.
SUMMARY AND CONCLUSIONS
Enzymic hydrolysis was shown to be an effective method of recovering potentially edible proteins from mechanically deboned turkey residue, a poultry processing waste. The protein recovery yield was high and the amount of the original residue was reduced by more than half. This could translate to higher income for the poultry industry as the cost of waste disposal would be reduced. More income would also be generated through the sale of protein hydrolysate produced. The hydrolysate produced has potential to be used in a wide range of applications due to its excellent solubility in water over a wide pH range: diet foods, high protein sports drinks, hypoallergenic baby food, etc. At the present time, this market is dominated by hydrolysates from caein and soya. The hydrolysate could also be used as a source of calcium, phosphorus, magnesium and potassium in human nutrition.
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26. Frokjaer, S., Use of hydrolysates for protein supplementation. Food Technol., 48 (1994) 68-71. 27. Cordle, C. T., Control of food allergies using protein hydrolysates. Food Technol., 48 (1994) 72-6. 28. Mahmoud, M. I., Physicochemical and functional properties of protein hydrolysates in nutritional products. Food Technol., 48 (1994) 89-95. 29. Schmidl, M. K., Taylor, S. L. & Nordlee, J. A., Use of hydrolysate-based products in special medical diets. Food Technol., 48 (1994) 77-80, 85. 30. Francis, F. J. & Clydesdale, F. M., Food Colorimetry: Theory and Applications. AVI, Westport, CT, 1975. 31. Chou, D. H. & Morr, C. V., Protein-water inter-
actions and functional properties. J. Am. Oil Chemists' Soc., 56 (1979) 53A-62A. 32. Mahmoud, M. I., Malone, W. T. & Cordle, C. T., Enzymatic hydrolysis of casein: effect of degree of hydrolysis on antigenicity and physical properties. J. Food Sci., 57 (1992) 1223-9. 33. Adler-Nissen, J., Enzymatic hydrolysis of soy protein for nutritional fortification of low pH food. Ann. Nutr. Alim., 32 (1978) 205-16. 34. Lee, S. W., Shimizu, M., Kaminogawa, S. & Yamauchi, K., Emulsifying properties of peptides obtained from the hydrolysis of fl-casein. Agric. Biol. Chem., 51 (1987) 161-6.