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Production and characterization of gelatinous protein extracts from turkey deboner residue
Process Biochemist~., Vol. 32, No. 4, pp. 309-318, 1997 Copyright © 1997 Elsevier Science Ltd Printed in Great Britain. All rights reserved 01132-9592/97 $17.00 + 0.00 ELSEVIER
PII:
S0032-9592
(96)00087-8
Production and characterization of gelatinous protein extracts from turkey deboner residue* Linus G. Fonkwe and Rakesh K. Singh Food Science Department, Purdue University, Smith Hall, West Lafayette, IN 479(17-11611, USA (Received 9 August 1996: accepted 27 August 1996)
Abstract
Mechanically deboned turkey residue (MDTR) is a turkey processing waste with a high collagen content. The objectives of this investigation were to produce gelatinous protein extracts from MDTR by heating at 55, 70 and 85°C following an acid pre-treatment method and then to characterize them using physicochemical methods. The three extractions recovered 15.5% of total MDTR proteins and reduced the original weight of the MDTR by 55%. All the gelatinous protein extracts were rich in protein and the amino acids proline, glycine and alanine. The samples had higher mineral contents than a commercial gelatin sample, and exhibited similar molecular weight patterns. However, they had different colours, lower viscosities and gel strength than the commercial gelatin sample. The gelatinous protein extracts prepared at 55 and 70°C had better buffering capacities and higher gel strengths than those produced at 85°C. © 1997 Elsevier Science Ltd. All rights reserved
Keywords:deboner residue, processing waste, gelatinous protein, physicochemical properties.
related to the recovery of sarcoplasmic and myofibrillar proteins from poultry bone residue, as well as the functional and nutritional qualities of these proteins [3, 5-9]. However, the production of gelatinous protein extracts from MDTR has not been investigated. The vertebrate collagen (or tropocollagen) is considered to be a cross-striated polymer with three helical polypeptide chains held together and stabilized by crosslinks between adjacent chains [10, 11]. Some of these crosslinks between the collagen subunits are thought to be hydrogen bonds. Upon heating in water, collagen can be broken down to its constituent subunits resulting in gelatin. Low heating temperatures produce gelatin with a low yield; high heating temperatures may lead to the formation of low grade gelatin and glue, but with higher protein yields. There are two major processing methods for the production of gelatin. In the acid method, the collagenous raw material, usually pork bones, is soaked in acid for about 24 h. The soaked material is washed to remove excess acid and heated at different temperatures to produce type A gelatin. In another method, the raw material, usually cattle hide, is soaked in lime for about 2 weeks, then washed and heated at different temperatures to produce type B gelatin [10, 12, 13]. Thus, the objectives of this investigation were: 1. to produce gelatinous protein extracts from MDTR by
Introduction
Mechanically deboned turkey residue (MDTR) is a waste material from the mechanical deboning of turkey frames in the poultry processing industry. During the mechanical deboning process, a ground slurry of meat and bones is put into a mechanical deboner wherein pressure is used to separate meat and soft tissue from the rest of the bony material [1]. It is estimated that 169000 tons of MDTR were produced in 1991 (private communication) from approximately 2415000 tons of turkey produced [2]. The MDTR contains approximately 20% protein (wet weight basis) and can therefore be a valuable source of animal protein. Most of the bone residue is currently being used mainly in the manufacture of animal feed [3-6]. MDTR has a high content of bones and connective tissues. The major protein found in these tissues is collagen, a precursor of the protein gelatin, which has wide applications in the food and pharmaceutical industries. It has been estimated that MDTR contains between 30 and 40% of its proteins in the form of collagen [5]. Several researchers have investigated various aspects *Approved as Paper No. 14732 of Purdue University Agricultural Research Programs. 309
310
L. G. Fonkwe, R. K. Singh
heating at three different temperatures, using the acid soak method for production of type A gelatin; and 2. to characterize the gelatinous protein extracts produced using physicochemical methods and compare some of their properties to those of a commercial gelatin sample. Materials and Methods
Mechanically deboned turkey residue was obtained from Farbest Foods Inc. (Huntingburg, IN). The sample, which was received frozen, was cut into blocks each weighing approximately 300 g and stored frozen at - 2 0 ° C until needed. Prior to use, the frozen sample was thawed overnight at 8-10°C in a refrigerator. All chemicals and supplies used during experimentation were of reagent grade and purchased from commercial suppliers.
Gelatin production Salt and alkali-soluble proteins were extracted from M D T R using an alkaline solution following a procedure described in the literature [8]. In this extraction, a 1% sodium chloride solution was used at room temperature. The pH of the extraction slurry was maintained between 10.5 and 10.7 pH units for 30 min. The slurry formed was then filtered through multiple layers of cheese-cloth to obtain a protein solution and a wet bony residue. The wet bony residue was then subjected to an acid pretreatment similar to that used in the manufacture of type A gelatin as described in the literature [10, 11, 13]. The residue from the alkali extraction process was soaked in 5% hydrochloric acid for 24 h and washed in running tap water for approximately 15 min to remove acid, excess soluble proteins and fat. The acid-soaked residue was then combined with three times ~ts weight of ordinary tap water in a steel pot and heated at 55°C for 5 h in a water bath. After 5 h of heating, the residue was allowed to cool to room temperature and its weight was recorded. The volume of the broth obtained was also noted. The broth obtained was concentrated in a vacuum concentrator (Rotavapor, Buchi, Switzerland) with a water bath temperature of 50°C and was then dried in a vacuum oven. The gelatinous protein extract recovered was weighed and labelled as 'GEL55'. The residue from the extraction process at 55°C was combined with three times its weight of tap water in a steel pot and heated at 70°C for another 5 h. The final volume of the broth as well as the weight of the cooled residue produced were noted. The broth formed was collected, concentrated in a vacuum evaporator and dried in a vacuum oven under the same conditions as GEL55. The gelatinous protein extract recovered was weighed and labelled as 'GEL70'. The residue from the aforementioned procedure was
combined with three times its weight of tap water in a steel pot and heated at 85°C for 5 h. The weight of the cooled residue and the volume of the resultant broth also were noted. The broth formed was collected, concentrated in a vacuum evaporator and dried in a vacuum oven as described earlier. The gelatinous protein extract recovered was weighed and labelled as 'GEL85'. The final residue was then discarded. All the gelatinous protein extracts collected were analysed for moisture, protein, fat and ash contents using standard methods [14].
Mineral analysis Mineral analysis on the samples was carried out using inductively coupled plasma/atomic emission spectrophotometry (ICP/AES). Samples were digested with 70% nitric acid and hydrogen peroxide and mineral analyses were carried out on a Perkin Elmer Plasma 400 ICP/AES instrument (Norwalk, CT) with a model AS 90 autosampler.
Amino acid analysis Amino acid analysis was performed in the Chemistry Department, Purdue University, West Lafayette, IN, using an Amino Prep Hydrolyser (AP100 Savant) for sample hydrolysis, and a High Performance Beckman 7300 Analyser for amino acid analysis.
Amino acid score The amino acid score (AAS) was determined using the method recommended by the Joint F A O / W H O expert consultation [15, 16]. The AAS of each of the nine essential amino acids (histidine, isoleucine, leucine, lysine, methionine + cystine, phenylalanine + tyrosine, threonine, tryptophan and valine) was calculated using eqn (1). The amino acid requirements for pre-school age children were used to calculate the AAS. AAS -
mg essential amino acid in lgsample essential amino acid required per g
(1)
The lowest ratio was considered to be the chemical score (CS) of the protein.
Essential amino acid index (EAA D The EAAI was determined using a formula described in the literature [17]. The AAS of each of the essential amino acids was calculated. The EAAI was calculated as the ninth root of the product of the AAS for the nine essential amino acids using eqn (2): EAAI = 9 ~ / A A S I x A A S 2 x . . . x A A S 9
(2)
where AAS,-AAS9 represent the amino acid scores for the nine essential amino acids.
Gelatinous protein extractsfrom turkey deboner residue
311
Physicochemical Properties
Sorption isotherms
Colour of gelatin dispersions
Samples were placed in pre-weighed aluminum dishes and weighed. The dishes containing the samples were then placed in evacuated air-tight dessicators containing saturated solutions of lithium chloride, potassium acetate, magnesium chloride, potassium carbonate, magnesium nitrate, sodium nitrite, sodium chloride, potassium chloride, or 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 2 weeks, based on preliminary investigations which showed that 2weeks was enough time for the samples to attain equilibrium with their environments. After 2 weeks, the samples were weighed again and the moisture contents (MC) of the samples were calculated using eqn (4):
A 1% (w/v) dispersion of each protein sample in water was prepared. The colour of each dispersion was determined on the L-a-b scale using a Hunter colorimeter (Model D25-PC2, Hunter Associates 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.
Gel eleetrophoresis Gel electrophoresis was carried out on a 12% nongradient S D S - P A G E gel using an established method [18]. Samples were prepared as follows: 50mg of sample were extracted with 0.5 ml of 5% sodium dodecylsuiphate (SDS) for 30min at room temperature (20-22°C), with occasional shaking in a centrifuge tube. The tubes were centrifuged at 16000g for 5 min and the supernatant was collected. The supernatant was mixed with S D S - P A G E sample buffer (100ml supernatant with 50ml SDS-PAGE sample buffer) containing 2%/~-mercaptoethanok and heat denatured in boiling water for 5 min. After gel preparation, 50 Id of each treated sample and 20 Id of protein molecular weight standards (Sigma Chemical Co., St louis, M e ) were applied to the gel and electrophoresed overnight using a voltage of 60 V. The gel formed was stained with Coomassie blue stain.
Viscosity of the gelatinous dispersions Dispersions of various concentrations (0-25-1.0%, w/v) of the gelatinous protein extracts in water were prepared. Each dispersion was then centrifuged at 16000g for 15 min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Pale Alto, CA) and the supernatant was collected. The viscosity of each dispersion was measured in an Oswald capillary viscometer, and the relationship between protein concentration and relative viscosity was determined using the modified Einstein equation for dilute solutions [19, 20] as shown in eqn (3): qr = 1 +qC
(3)
where q~ is the relative viscosity, q is the intrinsic viscosity and c is the concentration of the protein solution. Therefore, a plot of the relative viscosity versus the concentration of each dispersion would give a straight line with a slope representing the intrinsic viscosity of the dispersion. From this relationship, the intrinsic viscosities (the viscosities at zero concentration) of the protein samples were estimated.
MC=
mass of water in sample mass of dry samplc
x 100%
(4)
Gel .formation and gel strength The procedure for gel formation was a method adapted from the literature [11]. Dispersions of the protein extracts in hot tap water were made in beakers and heated to about 70°C until the samples were completely dissolved. The solutions were then poured into l(I ml beakers and allowed to cool to room temperature (2(l-22°C). The solutions then wcrc held at 8-10°C in a refrigerator overnight. The strength of the gels formed was determined using a QTest texture analyser (QTest, Division of Sintech Inc., Cary, NC). Immediately prior to measuring gel strength, the gel was carefully removed from the 10ml 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 mm) to either the break-point or to 75% deformation using a 5 kgf load cell.
Determination of titration curves For each protein sample, a 1% (w/v) dispersion in hot water was prepared. The dispersion was cooled to room temperature using cold tap water (20°C). The protein dispersion (25 ml) was measured into an Erlcnmeyer 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 increased to approximately 12.
Buffering capacity The buffering capacity (BC) of each sample was calculated from the titration curve data using an
L. G. Fonkwe, R. K. Singh
312
amount of protein in the extract increasing with an increase in extraction temperature, as shown in Table 2. This phenomenon was not unexpected since the production of gelatin from collagen is essentially a 'melting' process in which heat is used to break down the triple helix structure of collagen (Mr>300000) to produce smaller gelatin molecules. Thus, as the heating temperature was increased, the rate of collagen breakdown increased and more gelatinous proteins were formed. There was no significant difference (p<0.05) in the amount of fat in the first two gelatin extracts, but the amount of fat in GEL85 was significantly lower. Table 2 also indicates that the amount of ash in the gelatin samples progressively decreased with increase in extraction temperature.
established equation from the literature [21, 22]. The equation is shown as eqn (5), for each pH change of 0-5 units.
Bc=
meq titrant mass of protein (g) × A pH
(5)
Results and Discussion
Gelatin yield All the broths extracted were very dilute as shown in Table 1. Table 1 also shows the yields from the sequential extractions. The yields were low and increased with an increase in extraction temperature. The overall process recovered approximately 15.5% of the total M D T R proteins. The final residue, following the extraction of gelatinous proteins, contained 18% protein on a dry weight basis. This represented approximately 45% of the original M D T R (dry weight basis). This final residue can still be rendered and used as animal feed or as a fertilizer.
Mineral analysis The mineral contents in the gelatin extracts are shown in Table 3. The concentration of sodium and potassium ions decreased with increase in extraction temperature. These concentrations were considerably higher than those present in a commercial gelatin sample, SANOFI (Sanofi Bio-lndustries, Waukesha, WI). There were larger amounts of magnesium, phosphorus and calcium in GEL55 compared to any other protein extract. However, the concentrations of these ions in all three protein extracts were also considerably higher than those in the commercial gelatin sample. Among the microelements measured, only zinc and
Proximate analysis All the gelatinous protein powders produced in this investigation were high in protein content with the
Table 1. Yields" of gelatinous protein extracts from acid-soaked mechanically deboned turkey residue (MDTR), following alkali extraction of proteins by extracting sequentially at three temperatures
% Solids Protein yield (g/kg acid-soaked residue) Protein yield (g/kg MDTR) Protein yield (% MDTR proteins)
55°C
Extraction temperature 70°C
85°C
0"49±0-0 4"00±0"3 3"00±0"2 1"5
0"7±0"0 6"0±0"6 4"4±0-5 2-0
2"68±0-0 31"00±7-0 23"60±1"5 12"0
"All yields are the means of three determinations.
Table 2. Approximate composition~ of mechanically deboned turkey residue (MDTR) and gelatinous protein extracts recovered from the MDTR Proteinh MDTR GEL55 GEL70 GEL85
(%)
(%)
Fat (%)
Ash (%)
50'9 (0"8) 10"0 (0"2) 9.3 (0'1) 9"6 (0"2)
20"0 (0"1) 45-3 (0"3) 59"2 (0"2) 79-2 (0"0)
6"3 (0"4) 10-8 (0"6) 12"0 (0-5) 1"4 (0-1)
16-9 (0"5) 15"7 (0-7) 7"8 (0.4) 5"3 (0-1)
aEach value is a mean of three determinations with S.D. in parentheses. hProtein conversion factor was 5'55, except for the MDTR where the protein conversion factor was 6"25. GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C.
Gelatinous protein extracts from turkey dehoner residue
313
Table 3. Mineral composition (ppm) of mechanically deboned turkey residue (MDTR) and gelatinous protein extracts recovered from the MDTR
MDTR GEL55 GEL70 GEL85 SANOFI
Na
NK
NP
Mg
Ca
Mo
Zn
B
Mn
Fe
Cu
4720 73 172 6009 1908 783
1930 794 687 155 18
75 535 27 959 3701 6954 321
2563 3678 23894 1368 105
155 784 35 948 4288 4966 199
0 0 0 124 0
145 77 45 36 0
I 0 0 7 (I
3 5 0 4 (~
30 164 126 397 107
2 0 0 4 0
GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction al 70°C. SANOFI = calf skin gelatin.
iron w e r e f o u n d in large a m o u n t s in G E L 5 5 a n d G E L 7 0 . H o w e v e r , G E L 8 5 had small a m o u n t s o f m o l y b d e n u m , zinc and iron, as well as small a m o u n t s o f b o r o n , m a n g a n e s e a n d c o p p e r . A m o n g these s a m e m i c r o e l e m e n t s , only iron c o u l d be d e t e c t e d in the c o m mercial gelatin sample, as shown in T a b l e 3. T h e higher m i n e r a l c o n t e n t of the gelatins p r o d u c e d from M D T R c o m p a r e d to the c o m m e r c i a l gelatin can be a t t r i b u t e d to the raw m a t e r i a l s used in their p r o d u c tion. T h e c o m m e r c i a l gelatin was m a d e f r o m calf skin [13] while the g e l a t i n o u s p r o t e i n extracts w e r e m a d e from M D T R which c o n t a i n s a lot o f b o n e s rich in m i n e r a l content.
Amino acid analysis T a b l e 4 shows the a m i n o acid c o m p o s i t i o n o f the t h r e e g e l a t i n o u s p r o t e i n extracts ( G E L 5 5 , G E L 7 0 , G E L 8 5 )
along with the M D T R from which they were p r o d u c e d . T h e results indicate that the M D T R was rich in p r o line, gtycine a n d alanine, the p r e d o m i n a n t a m i n o acids in collagen. T h e a m o u n t s o f these t h r e e a m i n o acids in M D T R greatly e x c e e d e d the a m o u n t s f o u n d in mechanically d e b o n e d m e a t in the l i t e r a t u r e [23]. T a b l e 4 also indicates that these a m i n o acids were the p r e d o m i nant a m i n o acids in the t h r e e g e l a t i n o u s p r o t e i n extracts. T h e c o n c e n t r a t i o n s of these t h r e e a m i n o acids in the p r o t e i n extracts e x c e e d e d the c o n c e n t r a t i o n s in the M D T R . In fact, except for the c o n t e n t s o f cysteine a n d arginine, the c o n c e n t r a t i o n s of all o t h e r a m i n o acids were lower in the p r o t e i n extracts than in the MDTR. T h e r e was no cysteine in G E L 7 0 a n d G E L 8 5 a l t h o u g h t h e r e was an a c c u m u l a t i o n in G E L 5 5 . T h e t h r e e g e l a t i n o u s p r o t e i n extracts had low essential a m i n o acid c o n t e n t s o f 19-3, 15.1 and 15.2%, respec-
Table 4. Amino acid composition (g/100 g sample) of freeze-dried mechanically deboned turkey residue (MDTR) and gelatinous protein extracts produced from the MDTR
Aspartic Threonine '' Serine Glutamic Proline Glycine Alanine Cysteine;' Valine 'l Methionine " Isoleucine" Leucine " Tyrosine ~' Phenylalanine '' Histidine" Lysine ~' Arginine Tryptophan ~'b
MDTR
GEL55
GEL70
GEL85
7"08 2.95 3"24 10"00 10.91 28-69 11-63 0.44 3'44 1.22 2.35 4-49 0"97 2-20 1"10 3"80 5"32 -
5"85 2"31 2.68 8'94 11.31 33"55 12.98 1.04 3"21 0-97 1-73 3-80 0"38 1.77 0"71 3-42 5.26 _
5"82 2" 10 2.57 8.66 13-87 35-14 12-99 0-00 2-24 0-89 1.36 2.89 0.43 1.65 0.58 2.99 5.74 _
5"83 2-05 2.65 8.57 13.18 36.33 12.59 0.00 2"20 0.91 1.41 2.96 0-46 1-67 0-60 2.93 5'57 _
~'Essential amino acids. bTryptophan content not determined. GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C.
L. G. Fonkwe, R. K. Singh
314
tively (tryptophan content not included). Without considering their tryptophan content, the gelatinous protein extracts from M D T R had very low CS values of 0.34, 0.31 and 0.32 for GEL55, GEL70 and GEL85, respectively, and essential amino acid indices of 0.62, 0.49 and 0.49. These values indicate that the gelatinous protein extracts from M D T R were of low nutritional quality.
Physicochemical Properties Colour of 1% gelatin dispersions The Hunter L-a-b values for the gelatin samples are shown in Table 5. The lightness (or L-values) for GEL55 and GEL70 were similar to one another and higher than the L-value for GEL85 and the commercial gelatin sample. The hue values show that the protein solutions had a yellowish colour, based on Francis and Clydesdale's relationship between a/b ratio, hue angle and colour [24]. The chroma of the gelatin solutions indicated that the yellow colour was not pure. AE was used to compare the colours of the four samples. The h E values for all comparisons (not shown) were large, indicating that the colours of the gelatinous protein solutions were different from one another and from the colour of the commercial gelatin solution. The colour measurements indicated that the gelatinous proteins produced from M D T R were more yellowish in colour than the commercial gelatin sample. This difference in colour arises from the source of the raw material for gelatin production. The commercial gelatin was produced from calf skin which does not contain bony material. Gelatin made from bony material has been shown to have a more intense yellow colour than gelatin made from non-bony material [13].
Gel electrophoresis Figure 1 shows a photograph of the S D S - P A G E gel for the gelatinous protein extracts produced from M D T R as well as a commercially produced gelatin
sample. The gelatinous protein extracts (lanes b, c and e) had few bands with molecular masses less than 31 kDa, although GEL85 had more of these bands than the other two protein extracts. Most of the protein bands in these extracts had molecular masses that were greater than 45 kDa. The bands in GEL70 were more intense than those in GEL55 indicating that the concentrations of the larger molecular mass proteins were higher in GEL70 than in GEL55. Similarly, the commercial gelatin sample (lane d) showed few bands with molecular masses below 31 kDa, and the bulk of its proteins appeared as bands with molecular masses above 45 kDa. The molecular mass of gelatin has been estimated to be approximately 100 kDa, but smaller gelatin molecules have also been found with molecular masses around 70 kDa resulting from peptide bond hydrolysis [11-13]. Figure 2 shows that the gelatinous proteins had some proteins with molecular masses smaller than 70 kDa. GEL85 (lane d) contained more low molecular weight protein bands than any of the other samples. This may indicate a deterioration in protein quality due to the high heating temperature used in recovering this extract.
Viscosity of dilute gelatin dispersions The relationship between the relative viscosity and protein concentration (up to a concentration of 1%) for GEL55 and GEL70 can be represented by eqns (6) and (7), respectively: q~ = 1.016+0.334c
(6)
r/r = 1"169+0"720c
(7)
From these equations, the intrinsic viscosities of GEL55 and GEL70 were determined to be 0.334 and 0.720 dl/g, respectively. The higher viscosity of GEL70 can be related to the molecular mass distribution discussed in the preceding section. It appears that GEL70 contained a higher concentration of high molecular mass proteins with masses larger than 45 kDa, hence a higher intrinsic viscosity. Values for the intrinsic viscosities of many proteins can be found in the literature
Table 5. Hunter L-a-b values and the calculated hue and chroma for three gelatinous protein extracts produced from mechanically deboned turkey residue Hunter values
Hue
GEL55 GEL70 GEL85 SANOFI
50-7 56.9 32"2 8.9
Chroma -
1.1 2-3 1.4 1.9
L
a
b
2.0 5"0 - 2"7 - 1.0
- 28"8 - 24.7 27"4 62"2
2.3 5"5 3"0 2.1
GEL55 = first gelatinous protein extract produced by heating at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract at 85°C, following extraction at 70°C. SANOFI = calf skin gelatin.
Gelatinous protein extractsfrom turkey deboner residue [19]. Some examples include values of 0.036, 0.039, 2.34, 0.69 and 0.88 dl/g for haemoglobin (M~ 68000), lactate dehydrogenase (Mr 138000), myosin (Mr 226000), gelatin (Mr 383 000) and gelatin (Mr 320000), respectively. The literature values for the gelatins are different from the values obtained for the protein extracts in this investigation because the former were pure proteins (narrow molecular mass range) while the samples analysed here had a wider molecular mass range.
60'
-~ 50'
40
:= 30
Sorption &otherms The sorption isotherms of all the gelatinous samples are shown in Fig. 2. They all looked similar and were sigmoidal in shape with three distinct regions, which is typical for the sorption isotherms of most foods [25, 26]. The monolayer values of the gelatinous proteins can
315
P E
20
o 10
0
!
,
,
I
I
I
0.1
0.2
0.3
0.4
0.5
0.6
TGEL8~ 0.7
I).8
09
Water activity
Fig. 2. Sorption isotherms of gelatinous extracts produced from mechanically deboned turkey residue following alkaline extraction of muscle proteins. Each point is the mean of duplicate determinations and the bars represent a 95% confidence interval. GEL 55 = first gelatinous extract produced at 55°C; GEL 70 = second gelatinous extract produced at 70°C, following extraction at 55°C; GEL 85 = third gelatinous extract produced at 85°C following, extraction at 55 and 70°C.
200,000
I:°88
be calculated from the sorption isotherms using the BET equation (eqn (8)): a
45,000
m(1-a)
-
rnjc
c-I +
mlc
a
(8)
where, a = w a t e r activity, m = moisture content, m l = monolayer value and c = constant. A BET plot for the first portion of the sorption isotherms for the gelatin extracts is shown in Fig. 3. The BET monolayer values were determined to be 7.50, 7.67 and 8.90g water per gram of extract for GEL55, GEL70 and GEL85, respectively. These values are high compared to values for other proteins in the literature. For example, the monolayer values for casein and soy protein are 5.47 and 5.8, respectively [25]. These values may be higher because the gelatinous protein extracts had high salt contents which also absorbed moisture.
31,000
21,000
14,400 6,500 I
1 -
~
~
:
~
~
~ a b c d e Fig. 1. Photograph of an SDS-PAGE gel for the gelatinous protein extracts produced from mechanically deboned turkey residue and a commercial gelatin sample. Lane a = molecular weight marker; lane b = gelatinous extract produced at 55°C (GEL 55); lane c = second gelatinous extract produced at 70°C, following extraction at 55°C (GEL 70); lane d = commercial gelatin sample (SANOFI); lane e = gelatinous extract produced at 85°C, following extraction at 55 and 70°C (GEL 85).
Gel forming capacity and gel strength All the gelatinous protein extracts formed gels as expected. Table 6 shows the gel strengths of the various protein extracts produced from MDTR and those of a commercial gelatin sample at various concentrations. The gels prepared from the commercial gelatin sample were significantly stronger (p<0.05) than those made from the gels produced in this investigation at each concentration. GEL85 formed the
316
L. G. Fonkwe, R. K. Singh
weakest gels at each concentration and did not form a solid gel at the lowest concentration (2.4%) investigated. Although the commercial gelatin sample demonstrated a linear increase in gel strength with increase in protein concentration, the gelatinous protein extracts did not show the same trend. There was an increase in gel strength with increase in protein concentration, but the relationship was non-linear. Titration curves and buffering capacity
The titration curve for GEL55 is displayed in Fig. 4 and the buffering capacities of the three gelatinous protein extracts are displayed in Fig. 5.
0.045 0.04 0.0351
During the titration of the gelatinous protein solutions (1% concentration) with a 0"01M sodium hydroxide solution, a phenolphthalein end-point (a change in colour of the solution to the first sign of a permanent purple colour) of the titration was observed. The end-point of the titration occurred at pH values of about 8-6, 8.7 and 8.7 for GEL55, GEL70 and GEL85, respectively. Since the colour change of phenolphthalein occurs close to the isoionic point of the protein solution during titration, it can be approximated that the isoionic point of the gelatins lies between pH 8 and 9. This falls in the same region as the isoionic point of type A gelatins in the literature (7.0-9.0) [11-13]. Figure 5 shows that the buffering capacity of GEL55 was highest between pH 3 and 4 and between pH 6 and 8. The buffering capacity curve for GEL70 was similar to the curve for GEL55 except that the buffering capacity was lower in magnitude. Maximum buffering occurred between pH 4 and 5 and between pH 6 and 8. The buffering capacity of GEL85 was much lower than those of the previous two samples.
0.03T Summary and Conclusions
? 0025| 0.02~ 0.015~
°°'T 0"0050T I 0.1 0.15
l ! I 0.2 0.5 Wateractivity
0.3
0.35
Fig. 3. Plot for the determination of the monolayer values of gelatinous extracts produced from mechanically deboned turkey residue based on eqn (8) in the text. Each point is the mean of duplicate determinations and the bars represent a 95% confidence interval. GEL 55 = first gelatinous protein extract produced at 55°C; GEL 70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C; GEL 85 = third gelatinous protein extract produced at 85°C following, extraction at 55 and 70°C.
Three gelatinous protein extracts were produced from M D T R at 55, 70 and 85°C. They all exhibited some properties that were similar to those of a commercial gelatin sample, such as a high molecular weight profile and good gel forming capacity. However, the gelatinous protein extracts produced at 55 and 70°C exhibited properties that were superior to those of the extract produced at 85°C. The mineral contents of these gelatinous extracts were very much higher than those of a commercial gelatin, and their colours were very different from the colour of a commercial gelatin dispersion. The gels formed by the gelatinous protein extracts were significantly softer than gels formed by a commercial gelatin sample. Gelatins of different gel strengths (bloom) are being used in different concentrations in the food industry. For example, gelatin is used at concentrations of 0"2-9% in the confectionery industry [13]. Soft gels
Table 6. Gel strength ~ of gels prepared using gelatinous protein extracts produced from mechanically deboned turkey residue and a commercial gelatin sample. Each reading is a mean of duplicate runs Peak load (g) Concentration (%) 2-4 5.0 7-14
GEL55
GEL70
GEL85
SANOFI
140-94 _+53 (271-54 _+33) h 842.44 _ 170
229-40 + 49 652-72 + 79 831-84 _+64
no gel 141.14 + 0 605.05 _+55
302.28 _ 47 1113.36 + 142 1706-57 + 85
~Measured using a 5 kg load cell on a Qtest texture analyser and mean of two observations. hConcentration was 3"75% of GEL55. GEL55 = third gelatinous protein extract produced at 55°C; GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C; GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C; SANOFI-- calf-skin gelatin acquired from Sanofi Bio-Industries Inc. (Rousselot 250, Lot # :0-5680), Waukesha, WI.
Gelatinous protein extractsfrom turkey deboner residue
12
317
as in the manufacture of glue in the glue manufacturing industry.
...........................................................................................................................................................................
11' 10'
Acknowledgements 9
This project was supported by a grant from the Commissioner of Agriculture and Rural Development, Indiana, USA. The raw material (mechanically deboned turkey residue) for this project was provided by Farbest Food Inc., Huntingburg, IN. Sanofi BioIndustries Inc., Waukesha, WI, provided the commercial gelatin sample (calf skin gelatin Rousselot 250, Lot # :0-5680) used in this project.
a 7 .o ~ 5
3'
References
1
0
I 10
I 20
I 30
I 40
I 50
I 60
: 70
: 80
: 90
100
m l 0.01M N a O H a d d e d p e r 25 m l p r o t e i n s o l u t i o n
Fig. 4. Titration curve for the titration of a 1% solution of the first gelatinous protein extract (GEL55), recovered from mechanically deboned turkey residue at 55°C, with a 0-01 M sodium hydroxide solution. Each point is the mean of triplicate determinations and the bars represent a 95% confidence interval.
such as the ones produced from MDTR could potentially be used in sauces and soups and as binders and coatings in the meat industry, as well as in the preparation of Kosher dishes. These gels could also be used in the pharmaceutical industry for coating tablets, as well
1.4
........................................................................................................................................................................
'~-GELSS~ ~GEL70 [
1.2'
N
g
(I.8
~ o.6 a~ 0.4" ..=
0.2"
0
: I
' 2
. . . . . . . . . 3 4 5 6 7 8 9 pH of protein solution
10
: I1
12 J
-0.2
Fig. 5. Buffering capacities of the three gelatinous protein extracts (GEL55, GEL70 and GEL85) produced from mechanically deboned turkey residue at 55, 70 and 85°C, respectively. Each point is the mean of triplicate determinations and the bars represent a 95% confidence interval.
1. Stadelman, W. J., Olson, V. M., Shemwell, G. A. and Pasch, S., Egg and Poultry-Meat Processing. Ellis and Horwood, Chichester, UK, 1988. 2. USDA, Agricultural Statistics. United States Department of Agriculture, Washington DC, 1992. 3. Young, L. L., Composition and properties of an animal protein isolate prepared from bone residue. Journal of Food Science, 1976, 41, 6[)6-608. 4. Wallace, M. J. D. and Froning, G. W., Protein quality determination of bone residue from mechanically deboned chicken meat. Poultry Science, 1979, 58, 333-336. 5. Kijowski, J. and Niewiarowicz, A., A method of protein extraction from chicken bone residue and the chemical and electrophoretic characteristics of the extract. Journal of Food Technology, 1985, 20, 43 -49. 6. McCurdy, S. M., Jelen, P., Fedec, P. and Wood, D. F., Laboratory and pilot scale recovery of protein from mechanically separated chicken residue. Journal of Food Science, 1986, 51,742-747. 7. Chi, S. P. and Chen, T. C., Selected chemical and physical characteristics of chicken broth from broiler deboning by-products. Journal of Food Process and Preservation, 1994, 17, 407-420. 8. Fonkwe, L. G. and Singh, R. K., Protein recovery from mechanically deboned turkey residue. Trans. ASAE, 1994, 37, 527-534. 9. Fonkwe, L. G. and Singh, R. K., Protein recovery from mechanically deboned turkey residue by enzymic hydrolysis. Process Biochemist~', 1996, 31, 605-616. 10. Gustavson, K. H,, The Chemistry and Reactivity of Collagen. Academic Press Inc. Publishers, New York, 1956. 11. Glicksman, M., Gelatin. In Gum Technology in the Food Industry. Academic Press Inc., New York, 1969. 12. Courts, A., Properties and uses of gelatin. In Applied Protein Chemistry, ed. R. A. Grant. Applied Science Publishers Ltd, London, 1980. 13. Anonymous, Technical Brochure - - Gelatin. Sanofi Bio-Industries, Waukesha, WI, 1992. 14. AOAC, Official Methods of Analysis, 15th edn.
318
15.
16.
17. 18.
19.
20.
L. G. Fonkwe, R. K. Singh Association of Official Analytical Chemists, Washington DC, 1990. Joint FAO/WHO Expert Consultation, Protein quality evaluation, FAO Food and Nutrition Paper No. 51. Food and Agriculture Organization of the United Nations, Rome, Italy, 1991. Joint FAO/WHO/UNU Expert Consultation, Energy and protein requirements, WHO Technical Report, Serial No. 724. World Health Organization, Geneva, Switzerland, 1985. Rasco, B. A., Protein quality tests. In Introduction to the Chemical Analysis of Foods, ed. S. S. Nielsen. Jones and Bartlett Publishers, Boston, 1994. Weber, K. and Osborn, M. J., The reliability of molecular weight determinations by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Journal of Biological Chemistry, 1969, 244, 4406. Rha, C. and Pradipasena, R., Viscosity of proteins. In Functional Properties of Food Macromolecules, eds J. R. Mitchell and D. A. Ledward. Elsevier Applied Science Publishers, New York, 1986. Tanglertpaibul, T. and Rao, M. A., Intrinsic viscosity of tomato serum as affected by methods of
21. 22.
23.
24. 25. 26.
determination and methods of processing concentrates. Journal of Food Science, 1987, 52, 1642-1645. Morr, C. V., Swenson, P. E. and Richter, R. L., Functional properties of whey protein concentrates. Journal of Food Science, 1973, 38, 324. Coffmann, C. W. and Garcia, V. V., Functional properties and amino acid content of a protein isolate from mung bean flour. Journal of Food Technology, 1977, 12, 473-484. Babji, A. S., Froning, G. W. and Satterlee, L. D., Protein nutritional quality of mechanically deboned poultry meat as predicted by the C-PER assay. Journal of Food Science, 1980, 45, 441-443. Francis, F. J. and Clydesdale, F. M., Food Colorimetry: Theory and Applications. The AVI Publishing Co. Inc., Westport, CT, 1975. Chou, D. H. and Morr, C. V., Protein-water interactions and functional properties. Journal of the American Oil Chemists Society, 1979, 56, 53A-62A. Kinsella, J. E., Functional properties of soy proteins. Journal of the American Oil Chemists Society, 1979, 56, 242-258.
PII:
S0032-9592
(96)00087-8
Production and characterization of gelatinous protein extracts from turkey deboner residue* Linus G. Fonkwe and Rakesh K. Singh Food Science Department, Purdue University, Smith Hall, West Lafayette, IN 479(17-11611, USA (Received 9 August 1996: accepted 27 August 1996)
Abstract
Mechanically deboned turkey residue (MDTR) is a turkey processing waste with a high collagen content. The objectives of this investigation were to produce gelatinous protein extracts from MDTR by heating at 55, 70 and 85°C following an acid pre-treatment method and then to characterize them using physicochemical methods. The three extractions recovered 15.5% of total MDTR proteins and reduced the original weight of the MDTR by 55%. All the gelatinous protein extracts were rich in protein and the amino acids proline, glycine and alanine. The samples had higher mineral contents than a commercial gelatin sample, and exhibited similar molecular weight patterns. However, they had different colours, lower viscosities and gel strength than the commercial gelatin sample. The gelatinous protein extracts prepared at 55 and 70°C had better buffering capacities and higher gel strengths than those produced at 85°C. © 1997 Elsevier Science Ltd. All rights reserved
Keywords:deboner residue, processing waste, gelatinous protein, physicochemical properties.
related to the recovery of sarcoplasmic and myofibrillar proteins from poultry bone residue, as well as the functional and nutritional qualities of these proteins [3, 5-9]. However, the production of gelatinous protein extracts from MDTR has not been investigated. The vertebrate collagen (or tropocollagen) is considered to be a cross-striated polymer with three helical polypeptide chains held together and stabilized by crosslinks between adjacent chains [10, 11]. Some of these crosslinks between the collagen subunits are thought to be hydrogen bonds. Upon heating in water, collagen can be broken down to its constituent subunits resulting in gelatin. Low heating temperatures produce gelatin with a low yield; high heating temperatures may lead to the formation of low grade gelatin and glue, but with higher protein yields. There are two major processing methods for the production of gelatin. In the acid method, the collagenous raw material, usually pork bones, is soaked in acid for about 24 h. The soaked material is washed to remove excess acid and heated at different temperatures to produce type A gelatin. In another method, the raw material, usually cattle hide, is soaked in lime for about 2 weeks, then washed and heated at different temperatures to produce type B gelatin [10, 12, 13]. Thus, the objectives of this investigation were: 1. to produce gelatinous protein extracts from MDTR by
Introduction
Mechanically deboned turkey residue (MDTR) is a waste material from the mechanical deboning of turkey frames in the poultry processing industry. During the mechanical deboning process, a ground slurry of meat and bones is put into a mechanical deboner wherein pressure is used to separate meat and soft tissue from the rest of the bony material [1]. It is estimated that 169000 tons of MDTR were produced in 1991 (private communication) from approximately 2415000 tons of turkey produced [2]. The MDTR contains approximately 20% protein (wet weight basis) and can therefore be a valuable source of animal protein. Most of the bone residue is currently being used mainly in the manufacture of animal feed [3-6]. MDTR has a high content of bones and connective tissues. The major protein found in these tissues is collagen, a precursor of the protein gelatin, which has wide applications in the food and pharmaceutical industries. It has been estimated that MDTR contains between 30 and 40% of its proteins in the form of collagen [5]. Several researchers have investigated various aspects *Approved as Paper No. 14732 of Purdue University Agricultural Research Programs. 309
310
L. G. Fonkwe, R. K. Singh
heating at three different temperatures, using the acid soak method for production of type A gelatin; and 2. to characterize the gelatinous protein extracts produced using physicochemical methods and compare some of their properties to those of a commercial gelatin sample. Materials and Methods
Mechanically deboned turkey residue was obtained from Farbest Foods Inc. (Huntingburg, IN). The sample, which was received frozen, was cut into blocks each weighing approximately 300 g and stored frozen at - 2 0 ° C until needed. Prior to use, the frozen sample was thawed overnight at 8-10°C in a refrigerator. All chemicals and supplies used during experimentation were of reagent grade and purchased from commercial suppliers.
Gelatin production Salt and alkali-soluble proteins were extracted from M D T R using an alkaline solution following a procedure described in the literature [8]. In this extraction, a 1% sodium chloride solution was used at room temperature. The pH of the extraction slurry was maintained between 10.5 and 10.7 pH units for 30 min. The slurry formed was then filtered through multiple layers of cheese-cloth to obtain a protein solution and a wet bony residue. The wet bony residue was then subjected to an acid pretreatment similar to that used in the manufacture of type A gelatin as described in the literature [10, 11, 13]. The residue from the alkali extraction process was soaked in 5% hydrochloric acid for 24 h and washed in running tap water for approximately 15 min to remove acid, excess soluble proteins and fat. The acid-soaked residue was then combined with three times ~ts weight of ordinary tap water in a steel pot and heated at 55°C for 5 h in a water bath. After 5 h of heating, the residue was allowed to cool to room temperature and its weight was recorded. The volume of the broth obtained was also noted. The broth obtained was concentrated in a vacuum concentrator (Rotavapor, Buchi, Switzerland) with a water bath temperature of 50°C and was then dried in a vacuum oven. The gelatinous protein extract recovered was weighed and labelled as 'GEL55'. The residue from the extraction process at 55°C was combined with three times its weight of tap water in a steel pot and heated at 70°C for another 5 h. The final volume of the broth as well as the weight of the cooled residue produced were noted. The broth formed was collected, concentrated in a vacuum evaporator and dried in a vacuum oven under the same conditions as GEL55. The gelatinous protein extract recovered was weighed and labelled as 'GEL70'. The residue from the aforementioned procedure was
combined with three times its weight of tap water in a steel pot and heated at 85°C for 5 h. The weight of the cooled residue and the volume of the resultant broth also were noted. The broth formed was collected, concentrated in a vacuum evaporator and dried in a vacuum oven as described earlier. The gelatinous protein extract recovered was weighed and labelled as 'GEL85'. The final residue was then discarded. All the gelatinous protein extracts collected were analysed for moisture, protein, fat and ash contents using standard methods [14].
Mineral analysis Mineral analysis on the samples was carried out using inductively coupled plasma/atomic emission spectrophotometry (ICP/AES). Samples were digested with 70% nitric acid and hydrogen peroxide and mineral analyses were carried out on a Perkin Elmer Plasma 400 ICP/AES instrument (Norwalk, CT) with a model AS 90 autosampler.
Amino acid analysis Amino acid analysis was performed in the Chemistry Department, Purdue University, West Lafayette, IN, using an Amino Prep Hydrolyser (AP100 Savant) for sample hydrolysis, and a High Performance Beckman 7300 Analyser for amino acid analysis.
Amino acid score The amino acid score (AAS) was determined using the method recommended by the Joint F A O / W H O expert consultation [15, 16]. The AAS of each of the nine essential amino acids (histidine, isoleucine, leucine, lysine, methionine + cystine, phenylalanine + tyrosine, threonine, tryptophan and valine) was calculated using eqn (1). The amino acid requirements for pre-school age children were used to calculate the AAS. AAS -
mg essential amino acid in lgsample essential amino acid required per g
(1)
The lowest ratio was considered to be the chemical score (CS) of the protein.
Essential amino acid index (EAA D The EAAI was determined using a formula described in the literature [17]. The AAS of each of the essential amino acids was calculated. The EAAI was calculated as the ninth root of the product of the AAS for the nine essential amino acids using eqn (2): EAAI = 9 ~ / A A S I x A A S 2 x . . . x A A S 9
(2)
where AAS,-AAS9 represent the amino acid scores for the nine essential amino acids.
Gelatinous protein extractsfrom turkey deboner residue
311
Physicochemical Properties
Sorption isotherms
Colour of gelatin dispersions
Samples were placed in pre-weighed aluminum dishes and weighed. The dishes containing the samples were then placed in evacuated air-tight dessicators containing saturated solutions of lithium chloride, potassium acetate, magnesium chloride, potassium carbonate, magnesium nitrate, sodium nitrite, sodium chloride, potassium chloride, or 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 2 weeks, based on preliminary investigations which showed that 2weeks was enough time for the samples to attain equilibrium with their environments. After 2 weeks, the samples were weighed again and the moisture contents (MC) of the samples were calculated using eqn (4):
A 1% (w/v) dispersion of each protein sample in water was prepared. The colour of each dispersion was determined on the L-a-b scale using a Hunter colorimeter (Model D25-PC2, Hunter Associates 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.
Gel eleetrophoresis Gel electrophoresis was carried out on a 12% nongradient S D S - P A G E gel using an established method [18]. Samples were prepared as follows: 50mg of sample were extracted with 0.5 ml of 5% sodium dodecylsuiphate (SDS) for 30min at room temperature (20-22°C), with occasional shaking in a centrifuge tube. The tubes were centrifuged at 16000g for 5 min and the supernatant was collected. The supernatant was mixed with S D S - P A G E sample buffer (100ml supernatant with 50ml SDS-PAGE sample buffer) containing 2%/~-mercaptoethanok and heat denatured in boiling water for 5 min. After gel preparation, 50 Id of each treated sample and 20 Id of protein molecular weight standards (Sigma Chemical Co., St louis, M e ) were applied to the gel and electrophoresed overnight using a voltage of 60 V. The gel formed was stained with Coomassie blue stain.
Viscosity of the gelatinous dispersions Dispersions of various concentrations (0-25-1.0%, w/v) of the gelatinous protein extracts in water were prepared. Each dispersion was then centrifuged at 16000g for 15 min in a Beckman centrifuge (Beckman Instrument Inc., Model J2-21, Pale Alto, CA) and the supernatant was collected. The viscosity of each dispersion was measured in an Oswald capillary viscometer, and the relationship between protein concentration and relative viscosity was determined using the modified Einstein equation for dilute solutions [19, 20] as shown in eqn (3): qr = 1 +qC
(3)
where q~ is the relative viscosity, q is the intrinsic viscosity and c is the concentration of the protein solution. Therefore, a plot of the relative viscosity versus the concentration of each dispersion would give a straight line with a slope representing the intrinsic viscosity of the dispersion. From this relationship, the intrinsic viscosities (the viscosities at zero concentration) of the protein samples were estimated.
MC=
mass of water in sample mass of dry samplc
x 100%
(4)
Gel .formation and gel strength The procedure for gel formation was a method adapted from the literature [11]. Dispersions of the protein extracts in hot tap water were made in beakers and heated to about 70°C until the samples were completely dissolved. The solutions were then poured into l(I ml beakers and allowed to cool to room temperature (2(l-22°C). The solutions then wcrc held at 8-10°C in a refrigerator overnight. The strength of the gels formed was determined using a QTest texture analyser (QTest, Division of Sintech Inc., Cary, NC). Immediately prior to measuring gel strength, the gel was carefully removed from the 10ml 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 mm) to either the break-point or to 75% deformation using a 5 kgf load cell.
Determination of titration curves For each protein sample, a 1% (w/v) dispersion in hot water was prepared. The dispersion was cooled to room temperature using cold tap water (20°C). The protein dispersion (25 ml) was measured into an Erlcnmeyer 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 increased to approximately 12.
Buffering capacity The buffering capacity (BC) of each sample was calculated from the titration curve data using an
L. G. Fonkwe, R. K. Singh
312
amount of protein in the extract increasing with an increase in extraction temperature, as shown in Table 2. This phenomenon was not unexpected since the production of gelatin from collagen is essentially a 'melting' process in which heat is used to break down the triple helix structure of collagen (Mr>300000) to produce smaller gelatin molecules. Thus, as the heating temperature was increased, the rate of collagen breakdown increased and more gelatinous proteins were formed. There was no significant difference (p<0.05) in the amount of fat in the first two gelatin extracts, but the amount of fat in GEL85 was significantly lower. Table 2 also indicates that the amount of ash in the gelatin samples progressively decreased with increase in extraction temperature.
established equation from the literature [21, 22]. The equation is shown as eqn (5), for each pH change of 0-5 units.
Bc=
meq titrant mass of protein (g) × A pH
(5)
Results and Discussion
Gelatin yield All the broths extracted were very dilute as shown in Table 1. Table 1 also shows the yields from the sequential extractions. The yields were low and increased with an increase in extraction temperature. The overall process recovered approximately 15.5% of the total M D T R proteins. The final residue, following the extraction of gelatinous proteins, contained 18% protein on a dry weight basis. This represented approximately 45% of the original M D T R (dry weight basis). This final residue can still be rendered and used as animal feed or as a fertilizer.
Mineral analysis The mineral contents in the gelatin extracts are shown in Table 3. The concentration of sodium and potassium ions decreased with increase in extraction temperature. These concentrations were considerably higher than those present in a commercial gelatin sample, SANOFI (Sanofi Bio-lndustries, Waukesha, WI). There were larger amounts of magnesium, phosphorus and calcium in GEL55 compared to any other protein extract. However, the concentrations of these ions in all three protein extracts were also considerably higher than those in the commercial gelatin sample. Among the microelements measured, only zinc and
Proximate analysis All the gelatinous protein powders produced in this investigation were high in protein content with the
Table 1. Yields" of gelatinous protein extracts from acid-soaked mechanically deboned turkey residue (MDTR), following alkali extraction of proteins by extracting sequentially at three temperatures
% Solids Protein yield (g/kg acid-soaked residue) Protein yield (g/kg MDTR) Protein yield (% MDTR proteins)
55°C
Extraction temperature 70°C
85°C
0"49±0-0 4"00±0"3 3"00±0"2 1"5
0"7±0"0 6"0±0"6 4"4±0-5 2-0
2"68±0-0 31"00±7-0 23"60±1"5 12"0
"All yields are the means of three determinations.
Table 2. Approximate composition~ of mechanically deboned turkey residue (MDTR) and gelatinous protein extracts recovered from the MDTR Proteinh MDTR GEL55 GEL70 GEL85
(%)
(%)
Fat (%)
Ash (%)
50'9 (0"8) 10"0 (0"2) 9.3 (0'1) 9"6 (0"2)
20"0 (0"1) 45-3 (0"3) 59"2 (0"2) 79-2 (0"0)
6"3 (0"4) 10-8 (0"6) 12"0 (0-5) 1"4 (0-1)
16-9 (0"5) 15"7 (0-7) 7"8 (0.4) 5"3 (0-1)
aEach value is a mean of three determinations with S.D. in parentheses. hProtein conversion factor was 5'55, except for the MDTR where the protein conversion factor was 6"25. GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C.
Gelatinous protein extracts from turkey dehoner residue
313
Table 3. Mineral composition (ppm) of mechanically deboned turkey residue (MDTR) and gelatinous protein extracts recovered from the MDTR
MDTR GEL55 GEL70 GEL85 SANOFI
Na
NK
NP
Mg
Ca
Mo
Zn
B
Mn
Fe
Cu
4720 73 172 6009 1908 783
1930 794 687 155 18
75 535 27 959 3701 6954 321
2563 3678 23894 1368 105
155 784 35 948 4288 4966 199
0 0 0 124 0
145 77 45 36 0
I 0 0 7 (I
3 5 0 4 (~
30 164 126 397 107
2 0 0 4 0
GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction al 70°C. SANOFI = calf skin gelatin.
iron w e r e f o u n d in large a m o u n t s in G E L 5 5 a n d G E L 7 0 . H o w e v e r , G E L 8 5 had small a m o u n t s o f m o l y b d e n u m , zinc and iron, as well as small a m o u n t s o f b o r o n , m a n g a n e s e a n d c o p p e r . A m o n g these s a m e m i c r o e l e m e n t s , only iron c o u l d be d e t e c t e d in the c o m mercial gelatin sample, as shown in T a b l e 3. T h e higher m i n e r a l c o n t e n t of the gelatins p r o d u c e d from M D T R c o m p a r e d to the c o m m e r c i a l gelatin can be a t t r i b u t e d to the raw m a t e r i a l s used in their p r o d u c tion. T h e c o m m e r c i a l gelatin was m a d e f r o m calf skin [13] while the g e l a t i n o u s p r o t e i n extracts w e r e m a d e from M D T R which c o n t a i n s a lot o f b o n e s rich in m i n e r a l content.
Amino acid analysis T a b l e 4 shows the a m i n o acid c o m p o s i t i o n o f the t h r e e g e l a t i n o u s p r o t e i n extracts ( G E L 5 5 , G E L 7 0 , G E L 8 5 )
along with the M D T R from which they were p r o d u c e d . T h e results indicate that the M D T R was rich in p r o line, gtycine a n d alanine, the p r e d o m i n a n t a m i n o acids in collagen. T h e a m o u n t s o f these t h r e e a m i n o acids in M D T R greatly e x c e e d e d the a m o u n t s f o u n d in mechanically d e b o n e d m e a t in the l i t e r a t u r e [23]. T a b l e 4 also indicates that these a m i n o acids were the p r e d o m i nant a m i n o acids in the t h r e e g e l a t i n o u s p r o t e i n extracts. T h e c o n c e n t r a t i o n s of these t h r e e a m i n o acids in the p r o t e i n extracts e x c e e d e d the c o n c e n t r a t i o n s in the M D T R . In fact, except for the c o n t e n t s o f cysteine a n d arginine, the c o n c e n t r a t i o n s of all o t h e r a m i n o acids were lower in the p r o t e i n extracts than in the MDTR. T h e r e was no cysteine in G E L 7 0 a n d G E L 8 5 a l t h o u g h t h e r e was an a c c u m u l a t i o n in G E L 5 5 . T h e t h r e e g e l a t i n o u s p r o t e i n extracts had low essential a m i n o acid c o n t e n t s o f 19-3, 15.1 and 15.2%, respec-
Table 4. Amino acid composition (g/100 g sample) of freeze-dried mechanically deboned turkey residue (MDTR) and gelatinous protein extracts produced from the MDTR
Aspartic Threonine '' Serine Glutamic Proline Glycine Alanine Cysteine;' Valine 'l Methionine " Isoleucine" Leucine " Tyrosine ~' Phenylalanine '' Histidine" Lysine ~' Arginine Tryptophan ~'b
MDTR
GEL55
GEL70
GEL85
7"08 2.95 3"24 10"00 10.91 28-69 11-63 0.44 3'44 1.22 2.35 4-49 0"97 2-20 1"10 3"80 5"32 -
5"85 2"31 2.68 8'94 11.31 33"55 12.98 1.04 3"21 0-97 1-73 3-80 0"38 1.77 0"71 3-42 5.26 _
5"82 2" 10 2.57 8.66 13-87 35-14 12-99 0-00 2-24 0-89 1.36 2.89 0.43 1.65 0.58 2.99 5.74 _
5"83 2-05 2.65 8.57 13.18 36.33 12.59 0.00 2"20 0.91 1.41 2.96 0-46 1-67 0-60 2.93 5'57 _
~'Essential amino acids. bTryptophan content not determined. GEL55 = first gelatinous protein extract produced at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C.
L. G. Fonkwe, R. K. Singh
314
tively (tryptophan content not included). Without considering their tryptophan content, the gelatinous protein extracts from M D T R had very low CS values of 0.34, 0.31 and 0.32 for GEL55, GEL70 and GEL85, respectively, and essential amino acid indices of 0.62, 0.49 and 0.49. These values indicate that the gelatinous protein extracts from M D T R were of low nutritional quality.
Physicochemical Properties Colour of 1% gelatin dispersions The Hunter L-a-b values for the gelatin samples are shown in Table 5. The lightness (or L-values) for GEL55 and GEL70 were similar to one another and higher than the L-value for GEL85 and the commercial gelatin sample. The hue values show that the protein solutions had a yellowish colour, based on Francis and Clydesdale's relationship between a/b ratio, hue angle and colour [24]. The chroma of the gelatin solutions indicated that the yellow colour was not pure. AE was used to compare the colours of the four samples. The h E values for all comparisons (not shown) were large, indicating that the colours of the gelatinous protein solutions were different from one another and from the colour of the commercial gelatin solution. The colour measurements indicated that the gelatinous proteins produced from M D T R were more yellowish in colour than the commercial gelatin sample. This difference in colour arises from the source of the raw material for gelatin production. The commercial gelatin was produced from calf skin which does not contain bony material. Gelatin made from bony material has been shown to have a more intense yellow colour than gelatin made from non-bony material [13].
Gel electrophoresis Figure 1 shows a photograph of the S D S - P A G E gel for the gelatinous protein extracts produced from M D T R as well as a commercially produced gelatin
sample. The gelatinous protein extracts (lanes b, c and e) had few bands with molecular masses less than 31 kDa, although GEL85 had more of these bands than the other two protein extracts. Most of the protein bands in these extracts had molecular masses that were greater than 45 kDa. The bands in GEL70 were more intense than those in GEL55 indicating that the concentrations of the larger molecular mass proteins were higher in GEL70 than in GEL55. Similarly, the commercial gelatin sample (lane d) showed few bands with molecular masses below 31 kDa, and the bulk of its proteins appeared as bands with molecular masses above 45 kDa. The molecular mass of gelatin has been estimated to be approximately 100 kDa, but smaller gelatin molecules have also been found with molecular masses around 70 kDa resulting from peptide bond hydrolysis [11-13]. Figure 2 shows that the gelatinous proteins had some proteins with molecular masses smaller than 70 kDa. GEL85 (lane d) contained more low molecular weight protein bands than any of the other samples. This may indicate a deterioration in protein quality due to the high heating temperature used in recovering this extract.
Viscosity of dilute gelatin dispersions The relationship between the relative viscosity and protein concentration (up to a concentration of 1%) for GEL55 and GEL70 can be represented by eqns (6) and (7), respectively: q~ = 1.016+0.334c
(6)
r/r = 1"169+0"720c
(7)
From these equations, the intrinsic viscosities of GEL55 and GEL70 were determined to be 0.334 and 0.720 dl/g, respectively. The higher viscosity of GEL70 can be related to the molecular mass distribution discussed in the preceding section. It appears that GEL70 contained a higher concentration of high molecular mass proteins with masses larger than 45 kDa, hence a higher intrinsic viscosity. Values for the intrinsic viscosities of many proteins can be found in the literature
Table 5. Hunter L-a-b values and the calculated hue and chroma for three gelatinous protein extracts produced from mechanically deboned turkey residue Hunter values
Hue
GEL55 GEL70 GEL85 SANOFI
50-7 56.9 32"2 8.9
Chroma -
1.1 2-3 1.4 1.9
L
a
b
2.0 5"0 - 2"7 - 1.0
- 28"8 - 24.7 27"4 62"2
2.3 5"5 3"0 2.1
GEL55 = first gelatinous protein extract produced by heating at 55°C. GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C. GEL85 = third gelatinous protein extract at 85°C, following extraction at 70°C. SANOFI = calf skin gelatin.
Gelatinous protein extractsfrom turkey deboner residue [19]. Some examples include values of 0.036, 0.039, 2.34, 0.69 and 0.88 dl/g for haemoglobin (M~ 68000), lactate dehydrogenase (Mr 138000), myosin (Mr 226000), gelatin (Mr 383 000) and gelatin (Mr 320000), respectively. The literature values for the gelatins are different from the values obtained for the protein extracts in this investigation because the former were pure proteins (narrow molecular mass range) while the samples analysed here had a wider molecular mass range.
60'
-~ 50'
40
:= 30
Sorption &otherms The sorption isotherms of all the gelatinous samples are shown in Fig. 2. They all looked similar and were sigmoidal in shape with three distinct regions, which is typical for the sorption isotherms of most foods [25, 26]. The monolayer values of the gelatinous proteins can
315
P E
20
o 10
0
!
,
,
I
I
I
0.1
0.2
0.3
0.4
0.5
0.6
TGEL8~ 0.7
I).8
09
Water activity
Fig. 2. Sorption isotherms of gelatinous extracts produced from mechanically deboned turkey residue following alkaline extraction of muscle proteins. Each point is the mean of duplicate determinations and the bars represent a 95% confidence interval. GEL 55 = first gelatinous extract produced at 55°C; GEL 70 = second gelatinous extract produced at 70°C, following extraction at 55°C; GEL 85 = third gelatinous extract produced at 85°C following, extraction at 55 and 70°C.
200,000
I:°88
be calculated from the sorption isotherms using the BET equation (eqn (8)): a
45,000
m(1-a)
-
rnjc
c-I +
mlc
a
(8)
where, a = w a t e r activity, m = moisture content, m l = monolayer value and c = constant. A BET plot for the first portion of the sorption isotherms for the gelatin extracts is shown in Fig. 3. The BET monolayer values were determined to be 7.50, 7.67 and 8.90g water per gram of extract for GEL55, GEL70 and GEL85, respectively. These values are high compared to values for other proteins in the literature. For example, the monolayer values for casein and soy protein are 5.47 and 5.8, respectively [25]. These values may be higher because the gelatinous protein extracts had high salt contents which also absorbed moisture.
31,000
21,000
14,400 6,500 I
1 -
~
~
:
~
~
~ a b c d e Fig. 1. Photograph of an SDS-PAGE gel for the gelatinous protein extracts produced from mechanically deboned turkey residue and a commercial gelatin sample. Lane a = molecular weight marker; lane b = gelatinous extract produced at 55°C (GEL 55); lane c = second gelatinous extract produced at 70°C, following extraction at 55°C (GEL 70); lane d = commercial gelatin sample (SANOFI); lane e = gelatinous extract produced at 85°C, following extraction at 55 and 70°C (GEL 85).
Gel forming capacity and gel strength All the gelatinous protein extracts formed gels as expected. Table 6 shows the gel strengths of the various protein extracts produced from MDTR and those of a commercial gelatin sample at various concentrations. The gels prepared from the commercial gelatin sample were significantly stronger (p<0.05) than those made from the gels produced in this investigation at each concentration. GEL85 formed the
316
L. G. Fonkwe, R. K. Singh
weakest gels at each concentration and did not form a solid gel at the lowest concentration (2.4%) investigated. Although the commercial gelatin sample demonstrated a linear increase in gel strength with increase in protein concentration, the gelatinous protein extracts did not show the same trend. There was an increase in gel strength with increase in protein concentration, but the relationship was non-linear. Titration curves and buffering capacity
The titration curve for GEL55 is displayed in Fig. 4 and the buffering capacities of the three gelatinous protein extracts are displayed in Fig. 5.
0.045 0.04 0.0351
During the titration of the gelatinous protein solutions (1% concentration) with a 0"01M sodium hydroxide solution, a phenolphthalein end-point (a change in colour of the solution to the first sign of a permanent purple colour) of the titration was observed. The end-point of the titration occurred at pH values of about 8-6, 8.7 and 8.7 for GEL55, GEL70 and GEL85, respectively. Since the colour change of phenolphthalein occurs close to the isoionic point of the protein solution during titration, it can be approximated that the isoionic point of the gelatins lies between pH 8 and 9. This falls in the same region as the isoionic point of type A gelatins in the literature (7.0-9.0) [11-13]. Figure 5 shows that the buffering capacity of GEL55 was highest between pH 3 and 4 and between pH 6 and 8. The buffering capacity curve for GEL70 was similar to the curve for GEL55 except that the buffering capacity was lower in magnitude. Maximum buffering occurred between pH 4 and 5 and between pH 6 and 8. The buffering capacity of GEL85 was much lower than those of the previous two samples.
0.03T Summary and Conclusions
? 0025| 0.02~ 0.015~
°°'T 0"0050T I 0.1 0.15
l ! I 0.2 0.5 Wateractivity
0.3
0.35
Fig. 3. Plot for the determination of the monolayer values of gelatinous extracts produced from mechanically deboned turkey residue based on eqn (8) in the text. Each point is the mean of duplicate determinations and the bars represent a 95% confidence interval. GEL 55 = first gelatinous protein extract produced at 55°C; GEL 70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C; GEL 85 = third gelatinous protein extract produced at 85°C following, extraction at 55 and 70°C.
Three gelatinous protein extracts were produced from M D T R at 55, 70 and 85°C. They all exhibited some properties that were similar to those of a commercial gelatin sample, such as a high molecular weight profile and good gel forming capacity. However, the gelatinous protein extracts produced at 55 and 70°C exhibited properties that were superior to those of the extract produced at 85°C. The mineral contents of these gelatinous extracts were very much higher than those of a commercial gelatin, and their colours were very different from the colour of a commercial gelatin dispersion. The gels formed by the gelatinous protein extracts were significantly softer than gels formed by a commercial gelatin sample. Gelatins of different gel strengths (bloom) are being used in different concentrations in the food industry. For example, gelatin is used at concentrations of 0"2-9% in the confectionery industry [13]. Soft gels
Table 6. Gel strength ~ of gels prepared using gelatinous protein extracts produced from mechanically deboned turkey residue and a commercial gelatin sample. Each reading is a mean of duplicate runs Peak load (g) Concentration (%) 2-4 5.0 7-14
GEL55
GEL70
GEL85
SANOFI
140-94 _+53 (271-54 _+33) h 842.44 _ 170
229-40 + 49 652-72 + 79 831-84 _+64
no gel 141.14 + 0 605.05 _+55
302.28 _ 47 1113.36 + 142 1706-57 + 85
~Measured using a 5 kg load cell on a Qtest texture analyser and mean of two observations. hConcentration was 3"75% of GEL55. GEL55 = third gelatinous protein extract produced at 55°C; GEL70 = second gelatinous protein extract produced at 70°C, following extraction at 55°C; GEL85 = third gelatinous protein extract produced at 85°C, following extraction at 70°C; SANOFI-- calf-skin gelatin acquired from Sanofi Bio-Industries Inc. (Rousselot 250, Lot # :0-5680), Waukesha, WI.
Gelatinous protein extractsfrom turkey deboner residue
12
317
as in the manufacture of glue in the glue manufacturing industry.
...........................................................................................................................................................................
11' 10'
Acknowledgements 9
This project was supported by a grant from the Commissioner of Agriculture and Rural Development, Indiana, USA. The raw material (mechanically deboned turkey residue) for this project was provided by Farbest Food Inc., Huntingburg, IN. Sanofi BioIndustries Inc., Waukesha, WI, provided the commercial gelatin sample (calf skin gelatin Rousselot 250, Lot # :0-5680) used in this project.
a 7 .o ~ 5
3'
References
1
0
I 10
I 20
I 30
I 40
I 50
I 60
: 70
: 80
: 90
100
m l 0.01M N a O H a d d e d p e r 25 m l p r o t e i n s o l u t i o n
Fig. 4. Titration curve for the titration of a 1% solution of the first gelatinous protein extract (GEL55), recovered from mechanically deboned turkey residue at 55°C, with a 0-01 M sodium hydroxide solution. Each point is the mean of triplicate determinations and the bars represent a 95% confidence interval.
such as the ones produced from MDTR could potentially be used in sauces and soups and as binders and coatings in the meat industry, as well as in the preparation of Kosher dishes. These gels could also be used in the pharmaceutical industry for coating tablets, as well
1.4
........................................................................................................................................................................
'~-GELSS~ ~GEL70 [
1.2'
N
g
(I.8
~ o.6 a~ 0.4" ..=
0.2"
0
: I
' 2
. . . . . . . . . 3 4 5 6 7 8 9 pH of protein solution
10
: I1
12 J
-0.2
Fig. 5. Buffering capacities of the three gelatinous protein extracts (GEL55, GEL70 and GEL85) produced from mechanically deboned turkey residue at 55, 70 and 85°C, respectively. Each point is the mean of triplicate determinations and the bars represent a 95% confidence interval.
1. Stadelman, W. J., Olson, V. M., Shemwell, G. A. and Pasch, S., Egg and Poultry-Meat Processing. Ellis and Horwood, Chichester, UK, 1988. 2. USDA, Agricultural Statistics. United States Department of Agriculture, Washington DC, 1992. 3. Young, L. L., Composition and properties of an animal protein isolate prepared from bone residue. Journal of Food Science, 1976, 41, 6[)6-608. 4. Wallace, M. J. D. and Froning, G. W., Protein quality determination of bone residue from mechanically deboned chicken meat. Poultry Science, 1979, 58, 333-336. 5. Kijowski, J. and Niewiarowicz, A., A method of protein extraction from chicken bone residue and the chemical and electrophoretic characteristics of the extract. Journal of Food Technology, 1985, 20, 43 -49. 6. McCurdy, S. M., Jelen, P., Fedec, P. and Wood, D. F., Laboratory and pilot scale recovery of protein from mechanically separated chicken residue. Journal of Food Science, 1986, 51,742-747. 7. Chi, S. P. and Chen, T. C., Selected chemical and physical characteristics of chicken broth from broiler deboning by-products. Journal of Food Process and Preservation, 1994, 17, 407-420. 8. Fonkwe, L. G. and Singh, R. K., Protein recovery from mechanically deboned turkey residue. Trans. ASAE, 1994, 37, 527-534. 9. Fonkwe, L. G. and Singh, R. K., Protein recovery from mechanically deboned turkey residue by enzymic hydrolysis. Process Biochemist~', 1996, 31, 605-616. 10. Gustavson, K. H,, The Chemistry and Reactivity of Collagen. Academic Press Inc. Publishers, New York, 1956. 11. Glicksman, M., Gelatin. In Gum Technology in the Food Industry. Academic Press Inc., New York, 1969. 12. Courts, A., Properties and uses of gelatin. In Applied Protein Chemistry, ed. R. A. Grant. Applied Science Publishers Ltd, London, 1980. 13. Anonymous, Technical Brochure - - Gelatin. Sanofi Bio-Industries, Waukesha, WI, 1992. 14. AOAC, Official Methods of Analysis, 15th edn.
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L. G. Fonkwe, R. K. Singh Association of Official Analytical Chemists, Washington DC, 1990. Joint FAO/WHO Expert Consultation, Protein quality evaluation, FAO Food and Nutrition Paper No. 51. Food and Agriculture Organization of the United Nations, Rome, Italy, 1991. Joint FAO/WHO/UNU Expert Consultation, Energy and protein requirements, WHO Technical Report, Serial No. 724. World Health Organization, Geneva, Switzerland, 1985. Rasco, B. A., Protein quality tests. In Introduction to the Chemical Analysis of Foods, ed. S. S. Nielsen. Jones and Bartlett Publishers, Boston, 1994. Weber, K. and Osborn, M. J., The reliability of molecular weight determinations by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Journal of Biological Chemistry, 1969, 244, 4406. Rha, C. and Pradipasena, R., Viscosity of proteins. In Functional Properties of Food Macromolecules, eds J. R. Mitchell and D. A. Ledward. Elsevier Applied Science Publishers, New York, 1986. Tanglertpaibul, T. and Rao, M. A., Intrinsic viscosity of tomato serum as affected by methods of
21. 22.
23.
24. 25. 26.
determination and methods of processing concentrates. Journal of Food Science, 1987, 52, 1642-1645. Morr, C. V., Swenson, P. E. and Richter, R. L., Functional properties of whey protein concentrates. Journal of Food Science, 1973, 38, 324. Coffmann, C. W. and Garcia, V. V., Functional properties and amino acid content of a protein isolate from mung bean flour. Journal of Food Technology, 1977, 12, 473-484. Babji, A. S., Froning, G. W. and Satterlee, L. D., Protein nutritional quality of mechanically deboned poultry meat as predicted by the C-PER assay. Journal of Food Science, 1980, 45, 441-443. Francis, F. J. and Clydesdale, F. M., Food Colorimetry: Theory and Applications. The AVI Publishing Co. Inc., Westport, CT, 1975. Chou, D. H. and Morr, C. V., Protein-water interactions and functional properties. Journal of the American Oil Chemists Society, 1979, 56, 53A-62A. Kinsella, J. E., Functional properties of soy proteins. Journal of the American Oil Chemists Society, 1979, 56, 242-258.