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Extraction and characterization of gelatin from bovine heart
Author’s Accepted Manuscript Extraction and Characterization of Gelatin from Bovine Heart Bimol C. Roy, Chamali Das, Hui Hong, Mirko Betti, Heather L. Bruce www.elsevier.com/locate/sdj
PII: DOI: Reference:
S2212-4292(17)30219-5 https://doi.org/10.1016/j.fbio.2017.09.004 FBIO228
To appear in: Food Bioscience Received date: 16 May 2017 Revised date: 28 September 2017 Accepted date: 29 September 2017 Cite this article as: Bimol C. Roy, Chamali Das, Hui Hong, Mirko Betti and Heather L. Bruce, Extraction and Characterization of Gelatin from Bovine Heart, Food Bioscience, https://doi.org/10.1016/j.fbio.2017.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Extraction and Characterization of Gelatin from Bovine Heart
Bimol C. Roy*, Chamali Das, Hui Hong, Mirko Betti and Heather L. Bruce Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
*
Correspondence: Bimol C. Roy, Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, Alberta, Canada T6G 2P5 e-mail: [email protected] Fax: 1- (780) 492-4265 Phone: Cell: 1-(780)-908-8403; Office- 1-(780)-492-7282
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ABSTRACT The objective of this study was to investigate the effects of initial heat extraction time and enzyme concentration during subsequent pepsin digestion on gelatin quality from bovine heart (BH) connective tissue (CT). BH gelatin was extracted from isolated BH CT at 80°C for 4 or 6 hrs. Gelatin remaining in the CT residue was extracted with pepsin at either 100 or 200 mg pepsin/g CT to examine the yield and properties of enzyme-extracted gelatin. BH gelatins extracted at 80°C showed very good gel strength (241-269 g) compared to that subsequently extracted with pepsin (54-96 g) and gelatin yield increased with the length of heat extraction (3%) although gel strength was reduced. Heat-extracted BH gelatin regardless of duration of heat extraction contained α1 and α2 chain as main components with some degradation peptides. The frequency sweep test of BH gelatins showed independency in a wide range of frequency (1-196 hertz) regardless of whether extracted by heat or pepsin. Heat-extracted BH gelatins exhibited the highest storage modulus values (1165-1245 pa). BH gelatins extracted with the lowest concentration of pepsin showed a higher mean storage modulus value than gelatin extracted with the highest pepsin concentration. The present study is the first to demonstrate that gelatin extracted from BH collagen using heat and pepsin has characteristics comparable to other sources of gelatin, making it suitable for many applications, and confirms that the use of pepsin increases BH gelatin yield with a concomitant reduction in gelatin quality.
Keywords Gelatin; Bovine heart; Gel strength; Amino acids; ATR-FTIR
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1. Introduction Gelatin is a fibrous protein with thermo-reversible forms derived from its parent molecule collagen which comprises about 25 to 35% of total body protein (Vijayaraghavan et al., 2010). Commercial gelatin is mainly produced from bovine or porcine skin and bones (Ward and Courts, 1977). Recently, aquatic (Cheow et al., 2007) and poultry sources (Sarbon et al., 2013) have received considerable attention for gelatin production as these species are unencumbered by religious restrictions. Gelatins from mammalian sources are preferred, however, to aquatic animal sources because of their high gel strength, desirable gelling and melting temperatures, suitable viscosity (Kittiphattanabawon et al., 2010), and lack of fishy odor or allergens (Haug and Draget, 2011). Also, the low availability and increased prices of raw materials limit the production of gelatin from fish and poultry sources (Schrieber and Gareis, 2007) and reduce their economic competitiveness compared to mammalian gelatins (Haug and Draget, 2011). In Canada, bovine hearts (BH) are rendered along with other carcass by-products or treated as a meat with low market value. It is well known that collagen is the major component of heart, and heart valves are comprised of approximately 50% collagen on a dry matter basis. Bovine heart valves contain primarily type I and III collagens, and therefore contain more hydroxyproline than skin (Bashey et al., 1972) but there is little information about BH gelatin. Use of BH as a source of gelatin may be limited due to a perception of lack of gelatin quality or yield. Commercially, mammalian gelatins are extracted from animal tissues at low temperatures for the best gelling properties followed by subsequent extractions at progressively increasing temperatures (Schrieber and Gareis, 2007). Enzymes are also used to increase final yield, and this usually results in reduced gelling quality (Muyonga et al., 2004a). Therefore, the objective of this study was to investigate the effects of initial heat extraction time and pepsin concentration during subsequent enzymatic digestion on gelatin quality from bovine heart (BH) connective tissue (CT). 2. Materials and methods 2.1. Collection of BH
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Thirty-two BH were collected fresh from an abattoir, transported to the laboratory in ice and frozen at -20°C until used. All measurements were performed in triplicate with the exception of gel strength, which were performed in duplicate.
2.2. Isolation of connective tissue (CT) from BH Frozen BH was thawed and external adhering fat was removed, cut into small cubes. An aliquot of BH was retained for proximate analysis and the remainder was used for CT isolation. BH cubes were blended in deionised (DI) water in a Waring™ laboratory blender two speeds, commercial (Fisher Scientific, Cat No. 1450919, Edmonton, Canada). The homogenate was filtered through a metal sieve (1 mm2) and the material remaining on sieve was deemed CT. CT was suspended in 0.5M NaOH solution with constant stirring for 30 min and centrifuged at 5000 x g for 10 min. The sediment was collected, washed in DI water and the pH was adjusted to neutral using HCl. The resulting was centrifuged at 5000 x g for 10 min and the sediment was collected and treated with 10% butyl alcohol (Sigma Aldrich, Catalog W217816) to remove fat. The defatted CT was then washed with DI water and blotted dry with filter paper. 2.3. Gelatin extraction Defatted wet CT from 32 BH was combined and then divided into 15 × 100 g portions randomly and treated as presented in Figure 1. 2.3.1. Extraction with heat Wet CT was suspended in DI water (1:10 w/v) and gelatin was extracted at 80°C for either 4 (R4) or 6 (R6) hrs. After extraction, the CT suspension was cooled and filtered through cheese cloth. Filtered CT residue was retained for further gelatin extraction with pepsin. The filtrate (soluble gelatin) was collected and dialyzed against DI water (Spectra/Por 4 dialysis tubing, 12– 14K MWCO, Spectrum Laboratories Inc., Canada) until the conductivity was below 50 μSiemens. The dialysed gelatin was then frozen at -80°C, lyophilized, and weighed for yield calculation. 2.3.2. Extraction with pepsin 4
The CT residue was suspended in 0.5M acetic acid with pepsin (Sigma Aldrich, St. Louis, USA. Catalog P7125) and gelatin was extracted as illustrated in Figure 1 and processed as described for heat extracted gelatin. 2.4. Yield of gelatin The yield of gelatin was calculated as follows: Yield (%)
Weight of lyophilized gelatin (g) =
Weight of lyophilized CT of BH (g) / Weight of raw or DM of BH meat (g)
×
100
2.5. Proximate composition Proximate analyses were performed on lyophilized BH meat and bovine heart gelatin (BHG) (AOAC International (2000) moisture (950.46), ash (920.153), and fat (960.39)). Crude protein was measured using a TruSpec carbon/nitrogen determinator (Leco Corp., St. Joseph, USA). Nitrogen conversion factors 6.25 and 5.55 used for BH meat and lyophilized BHG, respectively.
2.6. Determination of gel strength The gel strength (British Standard Institution, 1975) of BHG was estimated using a 6.67% gelatin solution prepared by dissolving 7.50 g lyophilized gelatin in 105 mL DI water in a standard Bloom jar. Gelatin was allowed to absorb water for 3 hrs, then dissolved at 45°C, cooled to room temperature and then matured at 10°C for 17 hrs. Gel strength was measured using a TA-XT2 texture analyzer (Stable Micro Systems, Surrey, UK) with a 2 kN load cell equipped with a 1.27 cm diameter flat faced cylindrical Teflon plunger at 0.5 mm/sec speed with a penetration distance of 4 mm.
2.7. Dynamic viscoelastic properties Dynamic viscoelastic properties of 6.67% gelatin solution were measured (Physica MCR 301 rheometer, Anton Paar GmbH) according to the method of Roy et al. (2017) under oscillation using 25 mm parallel plate geometry with a gap of 1 mm between plates. The analysis was run under a constant strain of 5%, a constant frequency of 1 hertz, with the temperature decreasing from 45 to 10°C and then increased to 45°C with a heating/cooling rate of 2°C/min. The storage 5
modulus (G′, Pa) and loss modulus (G″, Pa) were represented as a function of temperature. The gelling point was evaluated by the sharp increase of G′ during the cooling process and the melting point was determined by sharp decrease of G′ during the subsequent heating process (Figure 2).
2.8. Color Color of 6.67% BHG gels was measured (Konica Minolta Chroma Meter CR-410, Folio Instruments Inc., Kitchener, Ontario) and expressed as L* (lightness), a* (redness/greenness), b* (yellowness/blueness), hue and chroma. The colorimeter used illuminant D65 and was calibrated prior to measurements using a standard white calibration plate provided by the manufacturer.
2.9. Determination of pH of gelatin solution The pH of 2% BHG solution was measured according to the British Standard Institution (1975) method with a glass electrode (Orion 2 Star™, ThermoFisher Scientific, Calgary, Canada) connected to a pH meter (Star Plus Meter, ThermoFisher Scientific, Calgary, Canada) standardized with pH 4.0, 7.0 and 10.0 standard buffers.
2.10. Transmittance Percent transmittance of 2% gelatin solution was measured at 620 nm using an Evolution™ 60S UV-Visible Spectrophotometer (Thermo Scientific, Calgary, Canada) as described by Roy et al. (2017).
2.11. Differential scanning calorimetric (DSC) measurements Thermal properties of gelatin were measured using a multi-cell micro differential scanning calorimeter (CSC 4100 MC-DSC, TA Instruments). Approximately 500 mg of 6.67% gelatin were weighed into a stainless steel ampoule and scanned from 20 to 60°C at a heating rate of 1°C/min with a blank ampoule as reference. The total energy required for denaturing gelatin, the helix-coil transition temperature (Tm) and the enthalpy change (∆H) were derived by integrating the area under the endotherm peak (MC-DSC RUN 2.6.9 software). 6
2.12. Emulsifying properties The emulsion stability index (ESI) and the emulsion activity index (EAI) of gelatin were determined as described by Roy et al. (2017). Gelatin solutions of 0.5, 1 and 2% were prepared and 8 ml of each were mixed with 2 ml of sunflower oil and homogenized for 1 min at 24200 rpm. Fifty µl of emulsion were aspirated at 0 and 30 min after homogenization, diluted with 5 ml of 0.1% sodium dodecyl sulphate (SDS) solution, and then mixed thoroughly for 10 sec by vortex. The absorbance was measured at 500 nm against a 0.1% SDS solution. EAI and ESI were calculated asEAI (m2 / g)
=
2 × 2.303 × A500 × DF C × φ × 104
A0 × ΔT ΔA
ESI (min) =
Where A500 = absorbance at 500 nm, DF= dilution factor (100), C= protein concentration (g / ml) before emulsification, φ = oil volume fraction (v/v) of the emulsion (i. e., the volume of emulsion droplets divided by the total volume of emulsion, φ = 0.2), and A30 and A0 the absorbance after 30 min and time zero, respectively, where ΔA = A30 – A0 and ΔT = 30 min. 2.13. Foaming properties Foam expansion (FE) and foam stability (FS) of gelatin solutions were determined by following the method of Roy et al. (2017). Gelatin solutions of 0.5, 1 and 2% were prepared and 25 ml of each were homogenized (Ultra-Turrax Ika T-18 Homogenizer, Cole-Parmer, Montreal, Canada) to incorporate air for 1 min at 20000 rpm. Total volume was measured at 0, 30 and 60 min after homogenization. The FE and FS were calculated by the following formula: (VT - V0) FE (%) =
V0
(Vt - V0) × 100
FS (%) =
V0
× 100
Where, VT is the total volume after homogenization (ml); V0 is the volume before homogenization; and Vt is the total volume after storage at room temperature for 30 and 60 min. Foam stability was calculated as the volume of foam remaining after 30 and 60 min.
2.14. Water holding capacity and fat binding capacity
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Water holding capacity (WHC) and fat binding capacity (FBC) of BHG were measured as described by Roy et al. (2017). Briefly, 0.25g BHG was dissolved in 25 ml of DI water or sunflower oil, respectively, and allowed to stand for 1 hr with agitation every 15 min. The residue was collected by centrifugation at 4500 x g for 20 min at 4°C. The supernatant was discarded and the tubes were allowed to drain at a 45° angle for 30 min. The WHC and FBC of BHG were calculated by following equation: WHC or FBC (%) =
Weight of the contents of the tube after draining (g) Weight of the lyophilised gelatin (g)
× 100
2.15. Molecular weight determination Gelatin molecular weight (MW) was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Gelatin (5 mg/ml) MW was examined in non-reduced and reduced (β-mercaptoethanol) states. Samples were heated at 95°C for 5 min and then centrifuged at 5000 x g for 5 min prior to electrophoresis. Gelatin samples (10 µL) and MW markers (Bio-Rad Laboratories Inc.) were loaded onto precast 4 to 20% gradient commercial gels (Bio-Rad Laboratories Inc.). Gels were run at 170V (Power Pack BasicTM, Bio- Rad Laboratories Inc.), then stained with Coomassie Brilliant Blue R250 and destained in a mixture of DI water, methanol and acetic acid (50:40:10, v/v/v).
2.16. Amino Acid Composition Gelatin solutions were hydrolysed for 1 hr at 160°C in a vacuum-sealed vial with 6M HCl containing 0.1% phenol. Hydrolysates were labeled with AccQ-Tag Ultra Derivatization Kit (Waters, Milford, MA) according to the manufacturer protocol and then analyzed by a HPLC system (Agilent 1200 Series) using the AccQ-Tag C18 column (3.9 × 150 mm, Waters) with detection at 254 nm. Norleucine (Sigma-Aldrich Inc, Edmonton, Canada) served as the internal standard and hydroxyproline (BioVision Incorporated, California, USA) as the external standard. 2.17. Infrared spectroscopy measurements Attenuated total reflectance-Fourier transform infrared (ATR–FTIR) spectra of lyophilised -1
BHG were recorded at 4 cm resolution as described by Muyounga et al. (2004b). Spectra were 8
recorded from 400 to 4000 cm
-1
at room temperature and signals were collected with
accumulation of 32 scans per spectrum. A background spectrum was provided by a clean empty cell. Analysis of spectral data was performed using the OMNIC 7.3 data collection software program (Thermo Electron Corporation).
2.18. Statistical analysis The characteristics of heat extracted BHG were examined using PROC GLM in the Statistical Analysis System (SAS, SAS Institute Inc., Cary, USA) with a one-way analysis of variance where the sole source of variation was heating duration (4 or 6 hrs). For pepsin-extracted BHG, the effect of pepsin concentration and duration of heating were again determined using a oneway analysis of variance with prior heating time and pepsin concentration (4hrs-100 mg/mL, 4hrs-200 mg, 6hrs-100 mg, 6hrs-200 mg/mL) as fixed sources of variation. Mean differences between treatments were determined using Tukey’s Honestly Significant Difference at P < 0.05.
3. Results and discussion
3.1. Proximate composition
BH meat contained a high proportion of moisture followed by crude protein, fat and trace amounts of ash, with protein concentration increasing, and fat and moisture decreasing in the extracted gelatin (Tables 1 and 2). There was no difference in proximate composition due to heat or pepsin treatment (Tables 1 and 2) and recommended maximum ash, moisture and fat contents of 2.5% (Jones, 1977), 15% (GME, 2008), and 2% (GMIA, 2012), respectively, were observed. The low fat content of BHG indicated that the method of de-fatting in this study was effective. The protein content of bovine gelatin ranges from 81.75 to 90.22% (Sarbon et al., 2013), and that of the extracted BHG was comparable. Pepsin-extracted BHG showed numerically higher protein content when compared to those obtained with heat treatment and this agreed with the report of Lassoued et al. (2014).
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3.2. Yield
The yields of extracted BHG are shown in Tables 1 and 2, respectively. For gelatin extracted with heat, yield increased significantly (P = 0.0267) as the extraction period increased from 4 to 6 hrs, and agreed with the results of Kittiphattanabawon et al. (2010). Prolongation of heat extraction of gelatin provides additional time to hydrolyze intra- and inter-molecular bonds and disassociate the γ-chains into α- or β-chains or to hydrolyze the α-chains into peptides and increase gelatin yield (Wong, 1989). Gelatin yield from duck feet was shown to be 3.65-5.75% by Abedinia et al. (2017) and 0.75-3.31% by Park et al. (2013), while that of chicken feet was 1.72-5.33% (Lim et al., 2001) on a wet weight basis, values which are similar to or comparable with those observed in the present study. The yield of gelatin depends on species, collagen content, degree of cross linking in the raw materials, and method of extraction (Widyasari and Rawdkuen, 2014). Pepsin acts on the collagen telopeptides and disrupts inter-molecular crosslinks thus releasing additional gelatin (Nalinanon et al., 2008). The pepsin-digested CT residues previously heated at 80°C yielded 7 to 11 times more gelatin than the prior heat extractions, demonstrating that total gelatin yield was increased by pepsin after the initial heat extraction of gelatin. Doubling the pepsin concentration increased the gelatin yield of CT previously heated at 80°C for 4 hrs (R4) but not that heated for 6 hrs (R6) (P = 0.0004). This indicated that additional gelatin could be recovered from the 4 hrs heat-extracted CT by increasing the pepsin, but not after 6 hrs at 80°C.
3.3. Gelatin quality An additional 2 hrs of heat-extraction during the initial extraction of gelatin decreased a* values and increased transmittance, but there was no effect of pepsin concentration on these measurements (Tables 1 and 2). Gelatin that is light-colored or translucent is best for incorporation into food products because it rarely interferes with the main color of the product. The reduction in a* values and increase in transmittance value of the heat-extracted BHG coincided with increased extraction duration (Ockerman and Hansen, 1999), suggesting denaturation of an endogenous chromophore or solubilisation of hydrophobic aggregates
10
(Montero et al., 2002), but this was not investigated further. In general, color does not affect the functional properties of gelatin (Cheow et al., 2007). Frequency sweep tests showed that all extracted BHG did not exhibit frequency dependence (supplementary Figure 1). At 10°C, both heat and pepsin-extracted BHG formed gels indicating that they were capable of network formation. The G′ values were higher than the G″ values [not shown] throughout the frequency range deployed confirming the elastic nature of the gelatin at 10°C. Heat extracted BHG gels showed numerically higher G′ values than pepsin extracted BHG gels (Tables 1 and 2), which indicated heat extracted BHG gels were likely more stable and stronger than pepsin extracted gels (Sarbon et al., 2013) because an increased G′ value is indicative of an enhanced ability of a gelatin molecule to refold into a triple-helix. The decrease in gel strength with the additional 2 hrs heating approached significance (P = 0.0924, Table 1), suggesting that gel strength tended to decrease as extraction time increased, which agreed with the results of Babin and Dickinson (2001). Also, gel strength decreased with increased pepsin concentration regardless of the prior duration of heat extraction (P = 0.0040, Table 2). The mean gelling and melting temperature of BHG are presented in Tables 1 and 2, and illustrated in Figure 2. Sharp increases in G′ values during cooling indicated rapid junction zone formation in the protein network (Saha and Bhattacharya, 2010). The gelling temperature of BHG did not differ between R4 and R6, which indicated that the length of gelatin extraction had no influence on gelling temperature, but melting temperature was highest in R4. Also, for the pepsin-extracted gelatin, gelling temperature was highest in gelatin extracted from CT residue previously exposed to 80°C for 4 hrs using the 100 mg pepsin (R4-100) concentration while melting temperature was highest for the gelatin extracted using 100 mg pepsin regardless of the previous gelatin extraction period at 80°C (R4-100 and R6-100) (Table 2). In general, high gelling and melting temperatures of gelatin indicate that the gelatin is of good quality and can be used in many applications (Tabarestani et al., 2010). A high melting temperature is desirable for gelatin because it indicates that the gel condition is maintained for a long time in the mouth, thus providing the best mouth feel (Sinthusamran et al., 2014).
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In the present study, the gelling and melting temperatures and gel strength differences between pepsin and heat extracted BHG appeared to be due to differences in peptide MW distribution (Nalinanon et al., 2008). In general, fragmentation of collagen α-chains during gelatin extraction is associated with lowered gel strength (Karim and Bhat, 2009). The SDSPAGE (Figure 3) showed that BHG extracted at 80°C contained longer peptides with fewer low MW peptides than the gelatin subsequently derived using pepsin. Gel strength in the present experiment appeared to be solely dependent upon the proportions of α-chains and β-components in the gelatin (Johnston-Banks, 1990). According to Gilsenan and Ross-Murphy (2000), high MW gelatins showed high melting temperatures. It might be that the low MW gelatins require additional cross-links per unit of volume to form a gel (See et al., 2015). The postulation that the MW of gelatin determined melting temperature is supported by the DSC results, which indicated that prolonged extraction of gelatin at 80°C decreased denaturation end temperature (Table 1). Also, for the pepsin-extracted gelatins, MW was highest for the gelatin extracted using 100 mg of pepsin (R4-100), coincident with this gelatin also having the highest denaturation onset temperature and greatest enthalpy of denaturation (Table 2). The gelatin extracted using 200 mg pepsin on the CT residue previously extracted for gelatin at 80°C for 6 hrs (R6-200) had decreased mean denaturation onset temperature and enthalpy but increased mean denaturation end temperature (Table 2). These results indicated that BHG extracted at 80°C for 6 hrs had lower denaturation temperatures, suggesting fewer or less complex bonds than that extracted for 4 hrs (Usha and Ramasami, 2004). Previous studies have shown that decreased thermal properties of gelatin were mainly related with decreased concentrations of proline, hydroxyproline, and glycine (Gilsenan and Ross-Murphy, 2000). In the present study, however, there were no differences in amino acid concentrations between the gelatins extracted at 80°C for either 4 or 6 hrs (Table 3), although gelatin extracted using pepsin after extraction of the heat-soluble gelatin showed differences in serine, glycine, threonine, isoleucine, and leucine concentrations, with decreased polar basic amino acid concentrations associated with the highest quality pepsin-extracted gelatin (R4-100) (Table 4). The result of this study clearly supported that pepsin-extracted BHG with its short peptide chain lengths did not form as strong a gel as the gelatin extracted at 80°C, most likely because of the lack of interjunction zones (Intarasirisawat et al., 2007). Pepsin increased the yield of gelatin at the expense 12
of gel strength but low gel strength gelatin can be used as a wine, beer or fruit juice clarifying agent (Lassoud et al., 2014). Water holding capacities and FBC of heat- and pepsin-extracted BHG are presented in Tables 1 and 2, respectively. Duration of gelatin extraction at 80°C did not affect WHC. In pepsin extracted BHG, however, WHC was not affected by the pepsin concentration used to extract additional gelatin from CT previously extracted at 80°C for 4 hrs, but was significantly decreased when 200 mg of pepsin were used to extract gelatin from CT residue previously extracted for gelatin at 80°C for 6 hrs (P = 0.04) (Table 2). The WHC of gelatin has been shown to be mainly related to its hydrophilic amino acids and hydroxyproline content (Ninan et al., 2014) although such a relationship was not observed in this study. Fat binding capacity was also unaffected by the duration of gelatin extraction at 80°C or pepsin concentration during enzymatic gelatin extraction regardless of the duration of prior gelatin extraction at 80°C. Increased concentrations of exposed hydrophobic residues and tyrosine, leucine, valine and isoleucine have been related to an increased FBC (Ninan et al., 2014). The amounts of non-polar side chains available to bind the oil hydrocarbon side chains have positively influence gelatin FBC (Nurul and Sarbon, 2015). In this study, the non-polar amino acids content did not differ between BHG extracted at 80°C or that extracted using pepsin, indicating little influence of extraction process on FBC.
3.4. Other Gelatin Properties The ESI and EAI of the heat- and pepsin- extracted BHG are presented in supplementary Tables 1 and 2, respectively. The ESI or EAI of pepsin-extracted BHG did not differ among gelatins at any concentrations, indicating that pepsin concentration had little effect on these measures. The BHG extracted with heat for 4 hrs had a significantly lower ESI than that extracted for 6 hrs (P = 0.0455) at 2% gelatin concentration. The amphoteric nature of gelatin with its peptidal hydrophobic zones allows it to act as an emulsifier (Koli et al., 2012). This result disagreed with the results of other researchers who observed that ESI decreased as the concentration of gelatin increased (Jridi et al., 2013) although contradictions exist (Kaewruang et al., 2013). Surh et al. (2006) reported that ESI is higher in gelatin with high MW peptides than in that with low MW peptides. Additionally, the EAI of BHG extracted with either heat or pepsin 13
did not show any difference due to heat or pepsin treatment, although the EAI in all BHG numerically increased as the gelatin concentration decreased which agreed with other reports (Ahmed and Benjakul, 2011). It might be that, at low concentrations, the protein in gelatin is highly soluble and rapidly migrates to the surface of the fat droplets. Generally, in the dispersing phase, highly soluble protein increases the efficiency of emulsification (Sikorski, 2001). The FE and FS of heat- and pepsin-extracted BHG are presented in supplementary Tables 1 and 2, respectively. The FE and FS at different concentrations of BHG were unaffected by duration of extraction at 80°C, but FS was highest for gelatin extracted at 100 mg pepsin on CT residue previously extracted at 80°C for 4 hrs (R4-100) and lowest for gelatin from CT residue previously extracted at 80°C regardless of duration of heat-extraction or pepsin concentration used when tested at 2% gelatin concentration after 60 min (P = 0.012). The reduced FE and FS may be due to aggregation of gelatin proteins, as aggregation hinders the interactions between protein and water necessary for foam formation (Kinsella, 1977). Increased content of hydrophobic amino acids in gelatin is related to increase foaming capacity (Jeloulli et al., 2011).
3.5. Infrared spectroscopy measurements A typical FTIR spectrum of BH gelatin (Figure 4) showed low intensities of Amides A and I followed by Amide II bands and only negligible intensities of Amide III band. The Amide A band is associated with stretching vibrations of the N-H group (Kong and Yu, 2007) coupled with hydrogen bonds, and was observed in the present study at 3302-3305 in R4, R6 and R4-100 and 3283-3298 cm -1 in R4-200, R6-100 and R6-200. The Amide A band of R4-200, R6-100 and R6-200 shifted to a lower wavenumber than BH gelatin of R4, R6 and R4-100, which indicated that hydrogen bonding between N-H groups of shorter peptide fragments is occurring (Ahmad and Benjakul, 2011). The Amide I band at 1635 cm-1 indicates the characteristic coil structure of gelatin and appeared at the wavenumbers 1635-1632 cm-1 for all BHG, and may be due to C=O stretch vibration along with N-H bending (Kong and Yu, 2007). The Amide II band was observed at the wavenumbers 1527-1535 cm-1 in BHG and most likely resulted from the combination of a C-N stretch and N-H deformation in the gelatin peptides (Bandekar, 1992). The Amide III band of BHG was observed at 1231-1232 cm-1 and is usually associated with the loss 14
of the triple-helical structure and transformation to a random coil when the collagen converts to gelatin (Muyounga et al., 2004b).
4. Conclusion Bovine heart, a low valued organ from beef, was shown in this study to be a promising source of high quality gelatin. Yield of gelatin with heat extraction for 4 or 6 hrs was low, but yield increased as the duration of heat extraction increased, although gel strength tended to decrease. The use of pepsin to extract gelatin from BH regardless of concentration dramatically increased gelatin yield but produced gelatin with low gel strength and compromised functional properties. The results of this study indicated that heat extracted gelatin had amino acid composition, MW distribution, viscoelastic properties and functional properties comparable with other sources of gelatins. FTIR data supported the conclusion that pepsin extractions may contain more intermolecular cross-links and low MW peptides which compromised gelatin quality. Therefore, bovine heart can be considered an alternative source of raw material for gelatin extraction. Acknowledgements This research was supported financially by the Alberta Livestock and Meat Agency. Conflict of interests There are no conflicts of interest to report. References Abedinia, A., Ariffin, F., Huda, N., & Nafchi, A. M. (2017). Extraction and characterization of gelatin from the feet of Pekin duck (Anas platyrhynchos domestica) as affected by acid, alkaline, and enzyme pretreatment. International Journal of Biological Macromolecules, 98, 586-594. Ahmad, M., & Benjakul, S. (2011). Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pre-treatment and extraction time. Food Hydrocolloids, 25, 381-388. 15
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Tabarestani, H. S., Maghsoudlou, Y., Motamedzadegan, A., & Mahoonak, A. R. S. (2010). Optimization of physico-chemical properties of gelatin extracted from fish skin of rainbow trout (Oncorhynchus mykiss). Bioresource Technology, 101, 6207-6214. Usha, R., & Ramasami, T. (2004). The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity. Thermochimica Acta, 409, 201-206. Vijayaraghavan, R., Thompson, B. C., Macfarlane, D. R., Kumar, R., Surianarayanan, M., Aishwarya, S., & Sehgal, P. K. (2010). Biocompatibility of choline salts as crosslinking agents for collagen based biomaterials. Chemical Communications, 46, 294-296. Ward, A. G., & Courts, A. (1977). The science and technology of gelatin. New York: Academic Press. Widyasari, R., & Rawdkuen, S. (2014). Extraction and characterization of gelatin from chicken feet by acid and ultrasound assisted extraction. Food Applied Bioscience Journal, 2, 85-97. Wong, D. W. S. (1989). Mechanism and theory in food chemistry. New York: Van Nostrand Reinhold Company Inc.
Figure 1. Schematic diagram of gelatin extraction from bovine heart Figure 2. Temperature sweep test of the extracted BH gelatins. The changes of storage modulus (G′, Pa) as a function of temperature in (a) cooling (from 45 °C to 10 °C) and (b) subsequent heating (from 10 °C to 45 °C) ramp Figure 3. SDS-PAGE pattern of gelatin extracted from the bovine heart (BH) with temperature and pepsin enzyme. Lane 1 & 15: Standard, protein concentration was 5 mg/ml; Lane 2: BH meat; Lane 3: R4-100, native; Lane 4: R4-100, reduced; Lane 5: R6-200, native; Lane 6: R6-200, reduced; Lane 7: R6-100, native; Lane 8: R6-100, reduced; Lane 9: R4-200, native; Lane 10: R4200, reduced; Lane 11: R6, native; Lane 12: R6, reduced; Lane 13: R4, native; Lane 14: R4, reduced and 10 µl of each sample was loaded Figure 4. The ATR-FTIR spectra of extracted gelatin from bovine heart with temperature and pepsin enzyme
20
Table 1. Proximate composition of bovine heart meat and bovine heart gelatin, and gelatin yield, gel strength, viscoelastic and other physical properties of gelatin extracted at 80°C for 4 (R4) or 6 (R6) hrs Treatments Standard Error R4 R6 of the Mean 3 3 15.21 15.04 0.54
Properties n Crude protein (%) Bovine heart meat
Bovine heart gelatin
0.8404
Crude fat (%)
2.98
2.87
0.24
0.7618
Ash (%)
0.91
0.90
0.03
0.7514
Moisture (%)
77.16
77.07
0.23
0.8011
Crude protein (%)
85.97
88.89
1.35
0.1104
Crude fat (%)
0.91
1.03
0.15
0.6113
Ash (%)
0.16
0.16
0.02
0.8581
13.03
12.91
0.79
0.9182
b
10.69
a
0.72
0.0267
Moisture (%) on lyophilised CT of BH Yield (%)
P value
7.19
on wet basis of heart meat
0.50
0.93
0.20
0.2045
on dry basis of heart meat
2.19
4.06
0.89
0.2113
268.84
241.21
8.87
0.0924
25.67
24.30
0.45
0.1015
a
b
0.18
0.0396
1161.66
1245.00
175.08
0.1052
31.06
21.22
3.69
0.1323
L*
55.46
53.94
1.71
0.5636
a*
0.36a
-0.36b
0.12
0.0164
b*
6.12
6.95
0.42
0.2384
Chroma
6.13
6.97
0.42
0.2324
Hue
93.40
93.06
1.09
0.8485
pH
5.95
6.10
0.06
0.1972
b
7.70
a
0.61
0.0098
Gel strength (g) Gelling temperature (°C) Melting temperature (°C) Storage modulus (Gʹ , Pa) at 10°C of cooling ramp Loss modulus (Gʺ , Pa) at 10°C of cooling ramp
33.43
32.65
Transmittance
3.67
Denaturation onset temperature (°C)
27.91
27.06
0.45
0.2664
Denaturation peak temperature (°C)
36.28
35.04
0.42
0.1066
a
b
0.63
0.0405
67.98
0.7189
Denaturation end temperature (°C)
45.61
42.91
Enthalpy (kJ/ mol)
717.06
754.21
21
Water holding capacity (%)
1015.97
938.98
46.16
0.3037
782.94
883.93
86.57
0.4558
Fat binding capacity (%) a, b
Means within a row with different letters are significantly different between with the mentioned P values
Table 2. Proximate composition, gelatin yield, gel strength, viscoelastic and other physical properties of gelatin extracted with subsequent pepsin treatment from bovine heart connective tissue residue Treatments 4 hrs heat extraction 6 hrs heat extraction 100 mg 200 mg 100 mg 200 mg Properties P value pepsin pepsin pepsin pepsin R4-100 R4-200 R6-100 R6-200 n
5
Crude protein (%)
4
3
3
92.85 (0.60)
92.66 (0.69)
92.88 (0.69)
0.9960
Crude fat (%)
0.35 (0.06)
0.54 (0.06)
0.63 (0.07)
0.58 (0.07)
0.0685
Ash (%)
0.42 (0.02)
0.45 (0.02)
0.44 (0.04)
0.47 (0.03)
0.6731
Moisture (%)
7.12 (0.64)
5.58(0.72)
6.21(1.02)
6.48 (0.83)
0.4970
on lyophilised CT of BH
65.60 (2.0)b
79.57 (2.24)a
77.82 (2.59)a
on wet basis of heart meat
2.91 (0.40)
4.13 (0.46)
4.00 (0.53)
4.29 (0.53)
0.1733
on dry basis of heart meat
12.74 (1.85)
18.13 (2.07)
17.51 (2.39)
18.76 (2.39)
0.1840
96.10 (6.27)a
62.40 (7.01)bc
90.67 (8.10)ab
54.19 (8.10)c
0.0040
18.51 (0.35)
a
b
b
13.96 (0.45)
c
<0.0001
28.66 (0.43)
a
24.28 (0.55)
c
0.0005
b
0.0023
Yield (%)
92.80 (0.53)
Gel strength (g)
Gelling temperature (°C) Melting temperature (°C) Storage modulus, Gʹ (Pa) at 10°C of
16.18 (0.39) 26.48 (0.48)
bc
17.80 (0.45) 27.45 (0.55)
ab
85.33 (2.59)a
603.25 (64.79)a
215.50 (74.81)b
325.83 (74.81)ab
15.94 (1.39)a
8.49 (1.61)b
11.45 (1.61)ab
4.55 (1.61)b
0.0029
L*
47.46 (0.60)
48.12 (0.68)
47.59 (0.78)
46.25 (0.78)
0.4245
a*
0.95 (0.09)
1.09 (0.10)
1.22 (0.11)
1.19 (0.11)
0.3081
b*
6.26 (0.41)
7.46 (0.48)
6.43 (0.53)
7.42 (0.53)
0.1942
Chroma
6.34 (0.40)
7.54 (0.45)
6.54 (0.52)
7.52 (0.52)
0.1887
Hue
81.17 (0.95)
81.60 (1.06)
79.02 (1.23)
80.75 (1.23)
0.4597
pH
6.15 (0.05)
6.09 (0.63)
6.11 (0.07)
6.03 (0.07)
0.6159
Transmittance (%)
5.40 (1.02)
4.90 (1.14)
6.86 (1.32)
6.40 (1.32)
0.6657
Denaturation onset temp. (°C)
28.11 (0.53)a
26.44 (0.59)ab
25.21 (0.69)b
24.95 (0.69)b
0.0122
Denaturation peak temp. (°C)
31.81 (0.35)
30.35 (0.39)
30.70 (0.46)
30.37 (0.46)
0.0620
38.00 (0.71)ab
37.07 (0.80)b
38.34 (0.92)ab
41.04 (0.92)a
0.0458
cooling ramp
Loss modulus, Gʺ (Pa) at 10°C of
62.33 (74.81)
0.00 04
cooling ramp
Denaturation end temp. (°C)
22
Enthalpy (kJ/mol) Water holding Capacity (%) Fat binding Capacity (%) a,b,c
1198.69 (92.52)a 925.98 (60.14)ab 4373.12 (158.37)
979.84 (103.44)ab 843.81 (67.24)ab 4351.09 (177.07)
945.14 (119.44)ab 1045.27 (77.64)a 4290.69 (204.46)
671.82 (119.44)b 684.71 (77.64)b 4168.35 (204.46)
Means within a row with different letters are significantly different with the mentioned P values
Standard error of mean in parentheses
23
0.0350 0.0400 0.8709
Table 3. Amino acids (% mole) composition of gelatin extracted at 80°C for 4 or 6 hrs from connective tissue isolated from Bovine heart Amino acids (% mole) n Hydroxyproline
Treatments R4 R6 3 10.03
Standard Error of the Mean
P value
3 9.78
0.10
0.1743
Aspartic acid
6.49
6.64
0.04
0.0682
Serine
3.63
3.61
0.06
0.8214
Glutamic acid
7.52
7.13
0.19
0.2337
Glycine
31.38
31.66
0.15
0.2596
Histidine
0.78
0.84
0.05
0.5173
Arginine
5.43
5.44
0.02
0.6213
Threonine
2.14
2.11
0.04
0.7521
Alanine
10.26
10.27
0.09
0.9064
Proline
11.23
11.19
0.07
0.7310
Cysteine
ND
ND
Tyrosine
0.53
0.57
0.05
0.6037
Valine
2.19
2.23
0.03
0.5823
Methionine
0.50
0.55
0.02
0.2510
Lysine
2.58
2.51
0.03
0.2741
Isoleucine
1.42
1.44
0.04
0.8559
Leucine
2.60
2.60
0.01
0.9051
Phenylalanine
1.34
1.38
0.03
0.5592
Hydroxyproline + Proline
21.26
20.96
0.14
0.2119
Polar acidic
35.01
35.27
0.12
0.2074
Polar basic
8.99
9.00
0.10
0.9837
Polar uncharged
20.76
20.48
0.21
0.4000
Non-polar hydrophobic
40.06
39.79
0.12
0.1880
ND = not detected
24
Table 4. Amino acids (% mole) composition of gelatin extracted with subsequent pepsin treatment from Bovine heart connective tissue residue Treatments Amino acids (% mole)
n Hydroxyproline Aspartic acid Serine Glutamic acid Glycine Histidine Arginine Threonine Alanine Proline Cysteine Tyrosine Valine Methionine Lysine Isoleucine Leucine Phenylalanine Hydroxyproline + proline Polar acidic Polar basic Polar uncharged Non-polar hydrophobic a,b
4 hrs heat extraction 100 mg 200 mg pepsin pepsin R4-100 R4-200 5 4 7.83 (0.20) 8.69 (0.22) 5.63 (0.12) 6.16 (0.14) b 3.27 (0.04) 3.75 (0.05)a 6.03 (0.26) 6.56 (0.30) a 30.68 (0.14) 29.71 (0.16)b 0.82 (0.12) 0.60 (0.13) 4.00 (0.14) 4.49 (0.16) b 1.84 (0.05) 2.33 (0.06)a 13.25 (0.21) 12.36 (0.24) 10.98 (0.04) 10.85 (0.05) ND ND 0.70 (0.02) 0.72 (0.03) 5.07 (0.18) 4.30 (0.20) 0.47 (0.03) 0.50 (0.03) 2.28 (0.05) 2.38 (0.05) ab 1.94 (0.03) 1.87 (0.04)ab 3.35 (0.06)a 2.97 (0.07)b 1.78 (0.05) 1.69 (0.06)
6 hrs heat extraction 100 mg 200 mg pepsin pepsin R6-100 R6-200 3 3 8.54 (0.26) 8.35 (0.26) 5.68 (0.16) 5.85 (0.16) a 3.55 (0.06) 3.74 (0.06)a 6.18 (0.34) 6.03 (0.34) a 30.49 (0.18) 30.22 (0.18)a 0.93 (0.16) 0.94 (0.16) 4.43 (0.18) 4.35 (0.18) a 2.16 (0.07) 2.26 (0.07)a 12.48 (0.28) 12.46 (0.28) 10.83 (0.06) 10.81 (0.06) ND ND 0.76 (0.03) 0.81 (0.03) 4.45 (0.24) 4.46 (0.24) 0.56 (0.04) 0.55 (0.04) 2.34 (0.06) 2.35 (0.06) b 1.79 (0.05) 2.02 (0.05)a 3.08 (0.08)ab 3.04 (0.08)ab 1.70 (0.07) 1.70 (0.07)
18.81 (0.21)
19.55 (0.23)
19.37 (0.27)
19.17 (0.27)
0.1832
33.95 (0.16) 7.80 (0.19)b 25.18 (0.35) 38.40 (0.24)
33.47 (0.17) 8.70 (0.21)a 23.82 (0.40) 38.69 (0.27)
34.05 (0.20) 8.39 (0.25)ab 24.05 (0.46) 38.65 (0.31)
33.97 (0.20) 8.64 (0.25)ab 23.90 (0.46) 38.45 (0.31)
0.1639 0.0402 0.0879 0.8460
Means within a row with different letters are significantly different at the mentioned P value
ND = not detected Standard error of mean in parentheses
25
P value
0.0779 0.0844 0.0001 0.5671 0.0066 0.3749 0.1776 0.0008 0.0649 0.1527 0.1994 0.0737 0.3336 0.6756 0.0403 0.0110 0.6858
PII: DOI: Reference:
S2212-4292(17)30219-5 https://doi.org/10.1016/j.fbio.2017.09.004 FBIO228
To appear in: Food Bioscience Received date: 16 May 2017 Revised date: 28 September 2017 Accepted date: 29 September 2017 Cite this article as: Bimol C. Roy, Chamali Das, Hui Hong, Mirko Betti and Heather L. Bruce, Extraction and Characterization of Gelatin from Bovine Heart, Food Bioscience, https://doi.org/10.1016/j.fbio.2017.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Extraction and Characterization of Gelatin from Bovine Heart
Bimol C. Roy*, Chamali Das, Hui Hong, Mirko Betti and Heather L. Bruce Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
*
Correspondence: Bimol C. Roy, Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, Alberta, Canada T6G 2P5 e-mail: [email protected] Fax: 1- (780) 492-4265 Phone: Cell: 1-(780)-908-8403; Office- 1-(780)-492-7282
1
ABSTRACT The objective of this study was to investigate the effects of initial heat extraction time and enzyme concentration during subsequent pepsin digestion on gelatin quality from bovine heart (BH) connective tissue (CT). BH gelatin was extracted from isolated BH CT at 80°C for 4 or 6 hrs. Gelatin remaining in the CT residue was extracted with pepsin at either 100 or 200 mg pepsin/g CT to examine the yield and properties of enzyme-extracted gelatin. BH gelatins extracted at 80°C showed very good gel strength (241-269 g) compared to that subsequently extracted with pepsin (54-96 g) and gelatin yield increased with the length of heat extraction (3%) although gel strength was reduced. Heat-extracted BH gelatin regardless of duration of heat extraction contained α1 and α2 chain as main components with some degradation peptides. The frequency sweep test of BH gelatins showed independency in a wide range of frequency (1-196 hertz) regardless of whether extracted by heat or pepsin. Heat-extracted BH gelatins exhibited the highest storage modulus values (1165-1245 pa). BH gelatins extracted with the lowest concentration of pepsin showed a higher mean storage modulus value than gelatin extracted with the highest pepsin concentration. The present study is the first to demonstrate that gelatin extracted from BH collagen using heat and pepsin has characteristics comparable to other sources of gelatin, making it suitable for many applications, and confirms that the use of pepsin increases BH gelatin yield with a concomitant reduction in gelatin quality.
Keywords Gelatin; Bovine heart; Gel strength; Amino acids; ATR-FTIR
2
1. Introduction Gelatin is a fibrous protein with thermo-reversible forms derived from its parent molecule collagen which comprises about 25 to 35% of total body protein (Vijayaraghavan et al., 2010). Commercial gelatin is mainly produced from bovine or porcine skin and bones (Ward and Courts, 1977). Recently, aquatic (Cheow et al., 2007) and poultry sources (Sarbon et al., 2013) have received considerable attention for gelatin production as these species are unencumbered by religious restrictions. Gelatins from mammalian sources are preferred, however, to aquatic animal sources because of their high gel strength, desirable gelling and melting temperatures, suitable viscosity (Kittiphattanabawon et al., 2010), and lack of fishy odor or allergens (Haug and Draget, 2011). Also, the low availability and increased prices of raw materials limit the production of gelatin from fish and poultry sources (Schrieber and Gareis, 2007) and reduce their economic competitiveness compared to mammalian gelatins (Haug and Draget, 2011). In Canada, bovine hearts (BH) are rendered along with other carcass by-products or treated as a meat with low market value. It is well known that collagen is the major component of heart, and heart valves are comprised of approximately 50% collagen on a dry matter basis. Bovine heart valves contain primarily type I and III collagens, and therefore contain more hydroxyproline than skin (Bashey et al., 1972) but there is little information about BH gelatin. Use of BH as a source of gelatin may be limited due to a perception of lack of gelatin quality or yield. Commercially, mammalian gelatins are extracted from animal tissues at low temperatures for the best gelling properties followed by subsequent extractions at progressively increasing temperatures (Schrieber and Gareis, 2007). Enzymes are also used to increase final yield, and this usually results in reduced gelling quality (Muyonga et al., 2004a). Therefore, the objective of this study was to investigate the effects of initial heat extraction time and pepsin concentration during subsequent enzymatic digestion on gelatin quality from bovine heart (BH) connective tissue (CT). 2. Materials and methods 2.1. Collection of BH
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Thirty-two BH were collected fresh from an abattoir, transported to the laboratory in ice and frozen at -20°C until used. All measurements were performed in triplicate with the exception of gel strength, which were performed in duplicate.
2.2. Isolation of connective tissue (CT) from BH Frozen BH was thawed and external adhering fat was removed, cut into small cubes. An aliquot of BH was retained for proximate analysis and the remainder was used for CT isolation. BH cubes were blended in deionised (DI) water in a Waring™ laboratory blender two speeds, commercial (Fisher Scientific, Cat No. 1450919, Edmonton, Canada). The homogenate was filtered through a metal sieve (1 mm2) and the material remaining on sieve was deemed CT. CT was suspended in 0.5M NaOH solution with constant stirring for 30 min and centrifuged at 5000 x g for 10 min. The sediment was collected, washed in DI water and the pH was adjusted to neutral using HCl. The resulting was centrifuged at 5000 x g for 10 min and the sediment was collected and treated with 10% butyl alcohol (Sigma Aldrich, Catalog W217816) to remove fat. The defatted CT was then washed with DI water and blotted dry with filter paper. 2.3. Gelatin extraction Defatted wet CT from 32 BH was combined and then divided into 15 × 100 g portions randomly and treated as presented in Figure 1. 2.3.1. Extraction with heat Wet CT was suspended in DI water (1:10 w/v) and gelatin was extracted at 80°C for either 4 (R4) or 6 (R6) hrs. After extraction, the CT suspension was cooled and filtered through cheese cloth. Filtered CT residue was retained for further gelatin extraction with pepsin. The filtrate (soluble gelatin) was collected and dialyzed against DI water (Spectra/Por 4 dialysis tubing, 12– 14K MWCO, Spectrum Laboratories Inc., Canada) until the conductivity was below 50 μSiemens. The dialysed gelatin was then frozen at -80°C, lyophilized, and weighed for yield calculation. 2.3.2. Extraction with pepsin 4
The CT residue was suspended in 0.5M acetic acid with pepsin (Sigma Aldrich, St. Louis, USA. Catalog P7125) and gelatin was extracted as illustrated in Figure 1 and processed as described for heat extracted gelatin. 2.4. Yield of gelatin The yield of gelatin was calculated as follows: Yield (%)
Weight of lyophilized gelatin (g) =
Weight of lyophilized CT of BH (g) / Weight of raw or DM of BH meat (g)
×
100
2.5. Proximate composition Proximate analyses were performed on lyophilized BH meat and bovine heart gelatin (BHG) (AOAC International (2000) moisture (950.46), ash (920.153), and fat (960.39)). Crude protein was measured using a TruSpec carbon/nitrogen determinator (Leco Corp., St. Joseph, USA). Nitrogen conversion factors 6.25 and 5.55 used for BH meat and lyophilized BHG, respectively.
2.6. Determination of gel strength The gel strength (British Standard Institution, 1975) of BHG was estimated using a 6.67% gelatin solution prepared by dissolving 7.50 g lyophilized gelatin in 105 mL DI water in a standard Bloom jar. Gelatin was allowed to absorb water for 3 hrs, then dissolved at 45°C, cooled to room temperature and then matured at 10°C for 17 hrs. Gel strength was measured using a TA-XT2 texture analyzer (Stable Micro Systems, Surrey, UK) with a 2 kN load cell equipped with a 1.27 cm diameter flat faced cylindrical Teflon plunger at 0.5 mm/sec speed with a penetration distance of 4 mm.
2.7. Dynamic viscoelastic properties Dynamic viscoelastic properties of 6.67% gelatin solution were measured (Physica MCR 301 rheometer, Anton Paar GmbH) according to the method of Roy et al. (2017) under oscillation using 25 mm parallel plate geometry with a gap of 1 mm between plates. The analysis was run under a constant strain of 5%, a constant frequency of 1 hertz, with the temperature decreasing from 45 to 10°C and then increased to 45°C with a heating/cooling rate of 2°C/min. The storage 5
modulus (G′, Pa) and loss modulus (G″, Pa) were represented as a function of temperature. The gelling point was evaluated by the sharp increase of G′ during the cooling process and the melting point was determined by sharp decrease of G′ during the subsequent heating process (Figure 2).
2.8. Color Color of 6.67% BHG gels was measured (Konica Minolta Chroma Meter CR-410, Folio Instruments Inc., Kitchener, Ontario) and expressed as L* (lightness), a* (redness/greenness), b* (yellowness/blueness), hue and chroma. The colorimeter used illuminant D65 and was calibrated prior to measurements using a standard white calibration plate provided by the manufacturer.
2.9. Determination of pH of gelatin solution The pH of 2% BHG solution was measured according to the British Standard Institution (1975) method with a glass electrode (Orion 2 Star™, ThermoFisher Scientific, Calgary, Canada) connected to a pH meter (Star Plus Meter, ThermoFisher Scientific, Calgary, Canada) standardized with pH 4.0, 7.0 and 10.0 standard buffers.
2.10. Transmittance Percent transmittance of 2% gelatin solution was measured at 620 nm using an Evolution™ 60S UV-Visible Spectrophotometer (Thermo Scientific, Calgary, Canada) as described by Roy et al. (2017).
2.11. Differential scanning calorimetric (DSC) measurements Thermal properties of gelatin were measured using a multi-cell micro differential scanning calorimeter (CSC 4100 MC-DSC, TA Instruments). Approximately 500 mg of 6.67% gelatin were weighed into a stainless steel ampoule and scanned from 20 to 60°C at a heating rate of 1°C/min with a blank ampoule as reference. The total energy required for denaturing gelatin, the helix-coil transition temperature (Tm) and the enthalpy change (∆H) were derived by integrating the area under the endotherm peak (MC-DSC RUN 2.6.9 software). 6
2.12. Emulsifying properties The emulsion stability index (ESI) and the emulsion activity index (EAI) of gelatin were determined as described by Roy et al. (2017). Gelatin solutions of 0.5, 1 and 2% were prepared and 8 ml of each were mixed with 2 ml of sunflower oil and homogenized for 1 min at 24200 rpm. Fifty µl of emulsion were aspirated at 0 and 30 min after homogenization, diluted with 5 ml of 0.1% sodium dodecyl sulphate (SDS) solution, and then mixed thoroughly for 10 sec by vortex. The absorbance was measured at 500 nm against a 0.1% SDS solution. EAI and ESI were calculated asEAI (m2 / g)
=
2 × 2.303 × A500 × DF C × φ × 104
A0 × ΔT ΔA
ESI (min) =
Where A500 = absorbance at 500 nm, DF= dilution factor (100), C= protein concentration (g / ml) before emulsification, φ = oil volume fraction (v/v) of the emulsion (i. e., the volume of emulsion droplets divided by the total volume of emulsion, φ = 0.2), and A30 and A0 the absorbance after 30 min and time zero, respectively, where ΔA = A30 – A0 and ΔT = 30 min. 2.13. Foaming properties Foam expansion (FE) and foam stability (FS) of gelatin solutions were determined by following the method of Roy et al. (2017). Gelatin solutions of 0.5, 1 and 2% were prepared and 25 ml of each were homogenized (Ultra-Turrax Ika T-18 Homogenizer, Cole-Parmer, Montreal, Canada) to incorporate air for 1 min at 20000 rpm. Total volume was measured at 0, 30 and 60 min after homogenization. The FE and FS were calculated by the following formula: (VT - V0) FE (%) =
V0
(Vt - V0) × 100
FS (%) =
V0
× 100
Where, VT is the total volume after homogenization (ml); V0 is the volume before homogenization; and Vt is the total volume after storage at room temperature for 30 and 60 min. Foam stability was calculated as the volume of foam remaining after 30 and 60 min.
2.14. Water holding capacity and fat binding capacity
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Water holding capacity (WHC) and fat binding capacity (FBC) of BHG were measured as described by Roy et al. (2017). Briefly, 0.25g BHG was dissolved in 25 ml of DI water or sunflower oil, respectively, and allowed to stand for 1 hr with agitation every 15 min. The residue was collected by centrifugation at 4500 x g for 20 min at 4°C. The supernatant was discarded and the tubes were allowed to drain at a 45° angle for 30 min. The WHC and FBC of BHG were calculated by following equation: WHC or FBC (%) =
Weight of the contents of the tube after draining (g) Weight of the lyophilised gelatin (g)
× 100
2.15. Molecular weight determination Gelatin molecular weight (MW) was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Gelatin (5 mg/ml) MW was examined in non-reduced and reduced (β-mercaptoethanol) states. Samples were heated at 95°C for 5 min and then centrifuged at 5000 x g for 5 min prior to electrophoresis. Gelatin samples (10 µL) and MW markers (Bio-Rad Laboratories Inc.) were loaded onto precast 4 to 20% gradient commercial gels (Bio-Rad Laboratories Inc.). Gels were run at 170V (Power Pack BasicTM, Bio- Rad Laboratories Inc.), then stained with Coomassie Brilliant Blue R250 and destained in a mixture of DI water, methanol and acetic acid (50:40:10, v/v/v).
2.16. Amino Acid Composition Gelatin solutions were hydrolysed for 1 hr at 160°C in a vacuum-sealed vial with 6M HCl containing 0.1% phenol. Hydrolysates were labeled with AccQ-Tag Ultra Derivatization Kit (Waters, Milford, MA) according to the manufacturer protocol and then analyzed by a HPLC system (Agilent 1200 Series) using the AccQ-Tag C18 column (3.9 × 150 mm, Waters) with detection at 254 nm. Norleucine (Sigma-Aldrich Inc, Edmonton, Canada) served as the internal standard and hydroxyproline (BioVision Incorporated, California, USA) as the external standard. 2.17. Infrared spectroscopy measurements Attenuated total reflectance-Fourier transform infrared (ATR–FTIR) spectra of lyophilised -1
BHG were recorded at 4 cm resolution as described by Muyounga et al. (2004b). Spectra were 8
recorded from 400 to 4000 cm
-1
at room temperature and signals were collected with
accumulation of 32 scans per spectrum. A background spectrum was provided by a clean empty cell. Analysis of spectral data was performed using the OMNIC 7.3 data collection software program (Thermo Electron Corporation).
2.18. Statistical analysis The characteristics of heat extracted BHG were examined using PROC GLM in the Statistical Analysis System (SAS, SAS Institute Inc., Cary, USA) with a one-way analysis of variance where the sole source of variation was heating duration (4 or 6 hrs). For pepsin-extracted BHG, the effect of pepsin concentration and duration of heating were again determined using a oneway analysis of variance with prior heating time and pepsin concentration (4hrs-100 mg/mL, 4hrs-200 mg, 6hrs-100 mg, 6hrs-200 mg/mL) as fixed sources of variation. Mean differences between treatments were determined using Tukey’s Honestly Significant Difference at P < 0.05.
3. Results and discussion
3.1. Proximate composition
BH meat contained a high proportion of moisture followed by crude protein, fat and trace amounts of ash, with protein concentration increasing, and fat and moisture decreasing in the extracted gelatin (Tables 1 and 2). There was no difference in proximate composition due to heat or pepsin treatment (Tables 1 and 2) and recommended maximum ash, moisture and fat contents of 2.5% (Jones, 1977), 15% (GME, 2008), and 2% (GMIA, 2012), respectively, were observed. The low fat content of BHG indicated that the method of de-fatting in this study was effective. The protein content of bovine gelatin ranges from 81.75 to 90.22% (Sarbon et al., 2013), and that of the extracted BHG was comparable. Pepsin-extracted BHG showed numerically higher protein content when compared to those obtained with heat treatment and this agreed with the report of Lassoued et al. (2014).
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3.2. Yield
The yields of extracted BHG are shown in Tables 1 and 2, respectively. For gelatin extracted with heat, yield increased significantly (P = 0.0267) as the extraction period increased from 4 to 6 hrs, and agreed with the results of Kittiphattanabawon et al. (2010). Prolongation of heat extraction of gelatin provides additional time to hydrolyze intra- and inter-molecular bonds and disassociate the γ-chains into α- or β-chains or to hydrolyze the α-chains into peptides and increase gelatin yield (Wong, 1989). Gelatin yield from duck feet was shown to be 3.65-5.75% by Abedinia et al. (2017) and 0.75-3.31% by Park et al. (2013), while that of chicken feet was 1.72-5.33% (Lim et al., 2001) on a wet weight basis, values which are similar to or comparable with those observed in the present study. The yield of gelatin depends on species, collagen content, degree of cross linking in the raw materials, and method of extraction (Widyasari and Rawdkuen, 2014). Pepsin acts on the collagen telopeptides and disrupts inter-molecular crosslinks thus releasing additional gelatin (Nalinanon et al., 2008). The pepsin-digested CT residues previously heated at 80°C yielded 7 to 11 times more gelatin than the prior heat extractions, demonstrating that total gelatin yield was increased by pepsin after the initial heat extraction of gelatin. Doubling the pepsin concentration increased the gelatin yield of CT previously heated at 80°C for 4 hrs (R4) but not that heated for 6 hrs (R6) (P = 0.0004). This indicated that additional gelatin could be recovered from the 4 hrs heat-extracted CT by increasing the pepsin, but not after 6 hrs at 80°C.
3.3. Gelatin quality An additional 2 hrs of heat-extraction during the initial extraction of gelatin decreased a* values and increased transmittance, but there was no effect of pepsin concentration on these measurements (Tables 1 and 2). Gelatin that is light-colored or translucent is best for incorporation into food products because it rarely interferes with the main color of the product. The reduction in a* values and increase in transmittance value of the heat-extracted BHG coincided with increased extraction duration (Ockerman and Hansen, 1999), suggesting denaturation of an endogenous chromophore or solubilisation of hydrophobic aggregates
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(Montero et al., 2002), but this was not investigated further. In general, color does not affect the functional properties of gelatin (Cheow et al., 2007). Frequency sweep tests showed that all extracted BHG did not exhibit frequency dependence (supplementary Figure 1). At 10°C, both heat and pepsin-extracted BHG formed gels indicating that they were capable of network formation. The G′ values were higher than the G″ values [not shown] throughout the frequency range deployed confirming the elastic nature of the gelatin at 10°C. Heat extracted BHG gels showed numerically higher G′ values than pepsin extracted BHG gels (Tables 1 and 2), which indicated heat extracted BHG gels were likely more stable and stronger than pepsin extracted gels (Sarbon et al., 2013) because an increased G′ value is indicative of an enhanced ability of a gelatin molecule to refold into a triple-helix. The decrease in gel strength with the additional 2 hrs heating approached significance (P = 0.0924, Table 1), suggesting that gel strength tended to decrease as extraction time increased, which agreed with the results of Babin and Dickinson (2001). Also, gel strength decreased with increased pepsin concentration regardless of the prior duration of heat extraction (P = 0.0040, Table 2). The mean gelling and melting temperature of BHG are presented in Tables 1 and 2, and illustrated in Figure 2. Sharp increases in G′ values during cooling indicated rapid junction zone formation in the protein network (Saha and Bhattacharya, 2010). The gelling temperature of BHG did not differ between R4 and R6, which indicated that the length of gelatin extraction had no influence on gelling temperature, but melting temperature was highest in R4. Also, for the pepsin-extracted gelatin, gelling temperature was highest in gelatin extracted from CT residue previously exposed to 80°C for 4 hrs using the 100 mg pepsin (R4-100) concentration while melting temperature was highest for the gelatin extracted using 100 mg pepsin regardless of the previous gelatin extraction period at 80°C (R4-100 and R6-100) (Table 2). In general, high gelling and melting temperatures of gelatin indicate that the gelatin is of good quality and can be used in many applications (Tabarestani et al., 2010). A high melting temperature is desirable for gelatin because it indicates that the gel condition is maintained for a long time in the mouth, thus providing the best mouth feel (Sinthusamran et al., 2014).
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In the present study, the gelling and melting temperatures and gel strength differences between pepsin and heat extracted BHG appeared to be due to differences in peptide MW distribution (Nalinanon et al., 2008). In general, fragmentation of collagen α-chains during gelatin extraction is associated with lowered gel strength (Karim and Bhat, 2009). The SDSPAGE (Figure 3) showed that BHG extracted at 80°C contained longer peptides with fewer low MW peptides than the gelatin subsequently derived using pepsin. Gel strength in the present experiment appeared to be solely dependent upon the proportions of α-chains and β-components in the gelatin (Johnston-Banks, 1990). According to Gilsenan and Ross-Murphy (2000), high MW gelatins showed high melting temperatures. It might be that the low MW gelatins require additional cross-links per unit of volume to form a gel (See et al., 2015). The postulation that the MW of gelatin determined melting temperature is supported by the DSC results, which indicated that prolonged extraction of gelatin at 80°C decreased denaturation end temperature (Table 1). Also, for the pepsin-extracted gelatins, MW was highest for the gelatin extracted using 100 mg of pepsin (R4-100), coincident with this gelatin also having the highest denaturation onset temperature and greatest enthalpy of denaturation (Table 2). The gelatin extracted using 200 mg pepsin on the CT residue previously extracted for gelatin at 80°C for 6 hrs (R6-200) had decreased mean denaturation onset temperature and enthalpy but increased mean denaturation end temperature (Table 2). These results indicated that BHG extracted at 80°C for 6 hrs had lower denaturation temperatures, suggesting fewer or less complex bonds than that extracted for 4 hrs (Usha and Ramasami, 2004). Previous studies have shown that decreased thermal properties of gelatin were mainly related with decreased concentrations of proline, hydroxyproline, and glycine (Gilsenan and Ross-Murphy, 2000). In the present study, however, there were no differences in amino acid concentrations between the gelatins extracted at 80°C for either 4 or 6 hrs (Table 3), although gelatin extracted using pepsin after extraction of the heat-soluble gelatin showed differences in serine, glycine, threonine, isoleucine, and leucine concentrations, with decreased polar basic amino acid concentrations associated with the highest quality pepsin-extracted gelatin (R4-100) (Table 4). The result of this study clearly supported that pepsin-extracted BHG with its short peptide chain lengths did not form as strong a gel as the gelatin extracted at 80°C, most likely because of the lack of interjunction zones (Intarasirisawat et al., 2007). Pepsin increased the yield of gelatin at the expense 12
of gel strength but low gel strength gelatin can be used as a wine, beer or fruit juice clarifying agent (Lassoud et al., 2014). Water holding capacities and FBC of heat- and pepsin-extracted BHG are presented in Tables 1 and 2, respectively. Duration of gelatin extraction at 80°C did not affect WHC. In pepsin extracted BHG, however, WHC was not affected by the pepsin concentration used to extract additional gelatin from CT previously extracted at 80°C for 4 hrs, but was significantly decreased when 200 mg of pepsin were used to extract gelatin from CT residue previously extracted for gelatin at 80°C for 6 hrs (P = 0.04) (Table 2). The WHC of gelatin has been shown to be mainly related to its hydrophilic amino acids and hydroxyproline content (Ninan et al., 2014) although such a relationship was not observed in this study. Fat binding capacity was also unaffected by the duration of gelatin extraction at 80°C or pepsin concentration during enzymatic gelatin extraction regardless of the duration of prior gelatin extraction at 80°C. Increased concentrations of exposed hydrophobic residues and tyrosine, leucine, valine and isoleucine have been related to an increased FBC (Ninan et al., 2014). The amounts of non-polar side chains available to bind the oil hydrocarbon side chains have positively influence gelatin FBC (Nurul and Sarbon, 2015). In this study, the non-polar amino acids content did not differ between BHG extracted at 80°C or that extracted using pepsin, indicating little influence of extraction process on FBC.
3.4. Other Gelatin Properties The ESI and EAI of the heat- and pepsin- extracted BHG are presented in supplementary Tables 1 and 2, respectively. The ESI or EAI of pepsin-extracted BHG did not differ among gelatins at any concentrations, indicating that pepsin concentration had little effect on these measures. The BHG extracted with heat for 4 hrs had a significantly lower ESI than that extracted for 6 hrs (P = 0.0455) at 2% gelatin concentration. The amphoteric nature of gelatin with its peptidal hydrophobic zones allows it to act as an emulsifier (Koli et al., 2012). This result disagreed with the results of other researchers who observed that ESI decreased as the concentration of gelatin increased (Jridi et al., 2013) although contradictions exist (Kaewruang et al., 2013). Surh et al. (2006) reported that ESI is higher in gelatin with high MW peptides than in that with low MW peptides. Additionally, the EAI of BHG extracted with either heat or pepsin 13
did not show any difference due to heat or pepsin treatment, although the EAI in all BHG numerically increased as the gelatin concentration decreased which agreed with other reports (Ahmed and Benjakul, 2011). It might be that, at low concentrations, the protein in gelatin is highly soluble and rapidly migrates to the surface of the fat droplets. Generally, in the dispersing phase, highly soluble protein increases the efficiency of emulsification (Sikorski, 2001). The FE and FS of heat- and pepsin-extracted BHG are presented in supplementary Tables 1 and 2, respectively. The FE and FS at different concentrations of BHG were unaffected by duration of extraction at 80°C, but FS was highest for gelatin extracted at 100 mg pepsin on CT residue previously extracted at 80°C for 4 hrs (R4-100) and lowest for gelatin from CT residue previously extracted at 80°C regardless of duration of heat-extraction or pepsin concentration used when tested at 2% gelatin concentration after 60 min (P = 0.012). The reduced FE and FS may be due to aggregation of gelatin proteins, as aggregation hinders the interactions between protein and water necessary for foam formation (Kinsella, 1977). Increased content of hydrophobic amino acids in gelatin is related to increase foaming capacity (Jeloulli et al., 2011).
3.5. Infrared spectroscopy measurements A typical FTIR spectrum of BH gelatin (Figure 4) showed low intensities of Amides A and I followed by Amide II bands and only negligible intensities of Amide III band. The Amide A band is associated with stretching vibrations of the N-H group (Kong and Yu, 2007) coupled with hydrogen bonds, and was observed in the present study at 3302-3305 in R4, R6 and R4-100 and 3283-3298 cm -1 in R4-200, R6-100 and R6-200. The Amide A band of R4-200, R6-100 and R6-200 shifted to a lower wavenumber than BH gelatin of R4, R6 and R4-100, which indicated that hydrogen bonding between N-H groups of shorter peptide fragments is occurring (Ahmad and Benjakul, 2011). The Amide I band at 1635 cm-1 indicates the characteristic coil structure of gelatin and appeared at the wavenumbers 1635-1632 cm-1 for all BHG, and may be due to C=O stretch vibration along with N-H bending (Kong and Yu, 2007). The Amide II band was observed at the wavenumbers 1527-1535 cm-1 in BHG and most likely resulted from the combination of a C-N stretch and N-H deformation in the gelatin peptides (Bandekar, 1992). The Amide III band of BHG was observed at 1231-1232 cm-1 and is usually associated with the loss 14
of the triple-helical structure and transformation to a random coil when the collagen converts to gelatin (Muyounga et al., 2004b).
4. Conclusion Bovine heart, a low valued organ from beef, was shown in this study to be a promising source of high quality gelatin. Yield of gelatin with heat extraction for 4 or 6 hrs was low, but yield increased as the duration of heat extraction increased, although gel strength tended to decrease. The use of pepsin to extract gelatin from BH regardless of concentration dramatically increased gelatin yield but produced gelatin with low gel strength and compromised functional properties. The results of this study indicated that heat extracted gelatin had amino acid composition, MW distribution, viscoelastic properties and functional properties comparable with other sources of gelatins. FTIR data supported the conclusion that pepsin extractions may contain more intermolecular cross-links and low MW peptides which compromised gelatin quality. Therefore, bovine heart can be considered an alternative source of raw material for gelatin extraction. Acknowledgements This research was supported financially by the Alberta Livestock and Meat Agency. Conflict of interests There are no conflicts of interest to report. References Abedinia, A., Ariffin, F., Huda, N., & Nafchi, A. M. (2017). Extraction and characterization of gelatin from the feet of Pekin duck (Anas platyrhynchos domestica) as affected by acid, alkaline, and enzyme pretreatment. International Journal of Biological Macromolecules, 98, 586-594. Ahmad, M., & Benjakul, S. (2011). Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pre-treatment and extraction time. Food Hydrocolloids, 25, 381-388. 15
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Figure 1. Schematic diagram of gelatin extraction from bovine heart Figure 2. Temperature sweep test of the extracted BH gelatins. The changes of storage modulus (G′, Pa) as a function of temperature in (a) cooling (from 45 °C to 10 °C) and (b) subsequent heating (from 10 °C to 45 °C) ramp Figure 3. SDS-PAGE pattern of gelatin extracted from the bovine heart (BH) with temperature and pepsin enzyme. Lane 1 & 15: Standard, protein concentration was 5 mg/ml; Lane 2: BH meat; Lane 3: R4-100, native; Lane 4: R4-100, reduced; Lane 5: R6-200, native; Lane 6: R6-200, reduced; Lane 7: R6-100, native; Lane 8: R6-100, reduced; Lane 9: R4-200, native; Lane 10: R4200, reduced; Lane 11: R6, native; Lane 12: R6, reduced; Lane 13: R4, native; Lane 14: R4, reduced and 10 µl of each sample was loaded Figure 4. The ATR-FTIR spectra of extracted gelatin from bovine heart with temperature and pepsin enzyme
20
Table 1. Proximate composition of bovine heart meat and bovine heart gelatin, and gelatin yield, gel strength, viscoelastic and other physical properties of gelatin extracted at 80°C for 4 (R4) or 6 (R6) hrs Treatments Standard Error R4 R6 of the Mean 3 3 15.21 15.04 0.54
Properties n Crude protein (%) Bovine heart meat
Bovine heart gelatin
0.8404
Crude fat (%)
2.98
2.87
0.24
0.7618
Ash (%)
0.91
0.90
0.03
0.7514
Moisture (%)
77.16
77.07
0.23
0.8011
Crude protein (%)
85.97
88.89
1.35
0.1104
Crude fat (%)
0.91
1.03
0.15
0.6113
Ash (%)
0.16
0.16
0.02
0.8581
13.03
12.91
0.79
0.9182
b
10.69
a
0.72
0.0267
Moisture (%) on lyophilised CT of BH Yield (%)
P value
7.19
on wet basis of heart meat
0.50
0.93
0.20
0.2045
on dry basis of heart meat
2.19
4.06
0.89
0.2113
268.84
241.21
8.87
0.0924
25.67
24.30
0.45
0.1015
a
b
0.18
0.0396
1161.66
1245.00
175.08
0.1052
31.06
21.22
3.69
0.1323
L*
55.46
53.94
1.71
0.5636
a*
0.36a
-0.36b
0.12
0.0164
b*
6.12
6.95
0.42
0.2384
Chroma
6.13
6.97
0.42
0.2324
Hue
93.40
93.06
1.09
0.8485
pH
5.95
6.10
0.06
0.1972
b
7.70
a
0.61
0.0098
Gel strength (g) Gelling temperature (°C) Melting temperature (°C) Storage modulus (Gʹ , Pa) at 10°C of cooling ramp Loss modulus (Gʺ , Pa) at 10°C of cooling ramp
33.43
32.65
Transmittance
3.67
Denaturation onset temperature (°C)
27.91
27.06
0.45
0.2664
Denaturation peak temperature (°C)
36.28
35.04
0.42
0.1066
a
b
0.63
0.0405
67.98
0.7189
Denaturation end temperature (°C)
45.61
42.91
Enthalpy (kJ/ mol)
717.06
754.21
21
Water holding capacity (%)
1015.97
938.98
46.16
0.3037
782.94
883.93
86.57
0.4558
Fat binding capacity (%) a, b
Means within a row with different letters are significantly different between with the mentioned P values
Table 2. Proximate composition, gelatin yield, gel strength, viscoelastic and other physical properties of gelatin extracted with subsequent pepsin treatment from bovine heart connective tissue residue Treatments 4 hrs heat extraction 6 hrs heat extraction 100 mg 200 mg 100 mg 200 mg Properties P value pepsin pepsin pepsin pepsin R4-100 R4-200 R6-100 R6-200 n
5
Crude protein (%)
4
3
3
92.85 (0.60)
92.66 (0.69)
92.88 (0.69)
0.9960
Crude fat (%)
0.35 (0.06)
0.54 (0.06)
0.63 (0.07)
0.58 (0.07)
0.0685
Ash (%)
0.42 (0.02)
0.45 (0.02)
0.44 (0.04)
0.47 (0.03)
0.6731
Moisture (%)
7.12 (0.64)
5.58(0.72)
6.21(1.02)
6.48 (0.83)
0.4970
on lyophilised CT of BH
65.60 (2.0)b
79.57 (2.24)a
77.82 (2.59)a
on wet basis of heart meat
2.91 (0.40)
4.13 (0.46)
4.00 (0.53)
4.29 (0.53)
0.1733
on dry basis of heart meat
12.74 (1.85)
18.13 (2.07)
17.51 (2.39)
18.76 (2.39)
0.1840
96.10 (6.27)a
62.40 (7.01)bc
90.67 (8.10)ab
54.19 (8.10)c
0.0040
18.51 (0.35)
a
b
b
13.96 (0.45)
c
<0.0001
28.66 (0.43)
a
24.28 (0.55)
c
0.0005
b
0.0023
Yield (%)
92.80 (0.53)
Gel strength (g)
Gelling temperature (°C) Melting temperature (°C) Storage modulus, Gʹ (Pa) at 10°C of
16.18 (0.39) 26.48 (0.48)
bc
17.80 (0.45) 27.45 (0.55)
ab
85.33 (2.59)a
603.25 (64.79)a
215.50 (74.81)b
325.83 (74.81)ab
15.94 (1.39)a
8.49 (1.61)b
11.45 (1.61)ab
4.55 (1.61)b
0.0029
L*
47.46 (0.60)
48.12 (0.68)
47.59 (0.78)
46.25 (0.78)
0.4245
a*
0.95 (0.09)
1.09 (0.10)
1.22 (0.11)
1.19 (0.11)
0.3081
b*
6.26 (0.41)
7.46 (0.48)
6.43 (0.53)
7.42 (0.53)
0.1942
Chroma
6.34 (0.40)
7.54 (0.45)
6.54 (0.52)
7.52 (0.52)
0.1887
Hue
81.17 (0.95)
81.60 (1.06)
79.02 (1.23)
80.75 (1.23)
0.4597
pH
6.15 (0.05)
6.09 (0.63)
6.11 (0.07)
6.03 (0.07)
0.6159
Transmittance (%)
5.40 (1.02)
4.90 (1.14)
6.86 (1.32)
6.40 (1.32)
0.6657
Denaturation onset temp. (°C)
28.11 (0.53)a
26.44 (0.59)ab
25.21 (0.69)b
24.95 (0.69)b
0.0122
Denaturation peak temp. (°C)
31.81 (0.35)
30.35 (0.39)
30.70 (0.46)
30.37 (0.46)
0.0620
38.00 (0.71)ab
37.07 (0.80)b
38.34 (0.92)ab
41.04 (0.92)a
0.0458
cooling ramp
Loss modulus, Gʺ (Pa) at 10°C of
62.33 (74.81)
0.00 04
cooling ramp
Denaturation end temp. (°C)
22
Enthalpy (kJ/mol) Water holding Capacity (%) Fat binding Capacity (%) a,b,c
1198.69 (92.52)a 925.98 (60.14)ab 4373.12 (158.37)
979.84 (103.44)ab 843.81 (67.24)ab 4351.09 (177.07)
945.14 (119.44)ab 1045.27 (77.64)a 4290.69 (204.46)
671.82 (119.44)b 684.71 (77.64)b 4168.35 (204.46)
Means within a row with different letters are significantly different with the mentioned P values
Standard error of mean in parentheses
23
0.0350 0.0400 0.8709
Table 3. Amino acids (% mole) composition of gelatin extracted at 80°C for 4 or 6 hrs from connective tissue isolated from Bovine heart Amino acids (% mole) n Hydroxyproline
Treatments R4 R6 3 10.03
Standard Error of the Mean
P value
3 9.78
0.10
0.1743
Aspartic acid
6.49
6.64
0.04
0.0682
Serine
3.63
3.61
0.06
0.8214
Glutamic acid
7.52
7.13
0.19
0.2337
Glycine
31.38
31.66
0.15
0.2596
Histidine
0.78
0.84
0.05
0.5173
Arginine
5.43
5.44
0.02
0.6213
Threonine
2.14
2.11
0.04
0.7521
Alanine
10.26
10.27
0.09
0.9064
Proline
11.23
11.19
0.07
0.7310
Cysteine
ND
ND
Tyrosine
0.53
0.57
0.05
0.6037
Valine
2.19
2.23
0.03
0.5823
Methionine
0.50
0.55
0.02
0.2510
Lysine
2.58
2.51
0.03
0.2741
Isoleucine
1.42
1.44
0.04
0.8559
Leucine
2.60
2.60
0.01
0.9051
Phenylalanine
1.34
1.38
0.03
0.5592
Hydroxyproline + Proline
21.26
20.96
0.14
0.2119
Polar acidic
35.01
35.27
0.12
0.2074
Polar basic
8.99
9.00
0.10
0.9837
Polar uncharged
20.76
20.48
0.21
0.4000
Non-polar hydrophobic
40.06
39.79
0.12
0.1880
ND = not detected
24
Table 4. Amino acids (% mole) composition of gelatin extracted with subsequent pepsin treatment from Bovine heart connective tissue residue Treatments Amino acids (% mole)
n Hydroxyproline Aspartic acid Serine Glutamic acid Glycine Histidine Arginine Threonine Alanine Proline Cysteine Tyrosine Valine Methionine Lysine Isoleucine Leucine Phenylalanine Hydroxyproline + proline Polar acidic Polar basic Polar uncharged Non-polar hydrophobic a,b
4 hrs heat extraction 100 mg 200 mg pepsin pepsin R4-100 R4-200 5 4 7.83 (0.20) 8.69 (0.22) 5.63 (0.12) 6.16 (0.14) b 3.27 (0.04) 3.75 (0.05)a 6.03 (0.26) 6.56 (0.30) a 30.68 (0.14) 29.71 (0.16)b 0.82 (0.12) 0.60 (0.13) 4.00 (0.14) 4.49 (0.16) b 1.84 (0.05) 2.33 (0.06)a 13.25 (0.21) 12.36 (0.24) 10.98 (0.04) 10.85 (0.05) ND ND 0.70 (0.02) 0.72 (0.03) 5.07 (0.18) 4.30 (0.20) 0.47 (0.03) 0.50 (0.03) 2.28 (0.05) 2.38 (0.05) ab 1.94 (0.03) 1.87 (0.04)ab 3.35 (0.06)a 2.97 (0.07)b 1.78 (0.05) 1.69 (0.06)
6 hrs heat extraction 100 mg 200 mg pepsin pepsin R6-100 R6-200 3 3 8.54 (0.26) 8.35 (0.26) 5.68 (0.16) 5.85 (0.16) a 3.55 (0.06) 3.74 (0.06)a 6.18 (0.34) 6.03 (0.34) a 30.49 (0.18) 30.22 (0.18)a 0.93 (0.16) 0.94 (0.16) 4.43 (0.18) 4.35 (0.18) a 2.16 (0.07) 2.26 (0.07)a 12.48 (0.28) 12.46 (0.28) 10.83 (0.06) 10.81 (0.06) ND ND 0.76 (0.03) 0.81 (0.03) 4.45 (0.24) 4.46 (0.24) 0.56 (0.04) 0.55 (0.04) 2.34 (0.06) 2.35 (0.06) b 1.79 (0.05) 2.02 (0.05)a 3.08 (0.08)ab 3.04 (0.08)ab 1.70 (0.07) 1.70 (0.07)
18.81 (0.21)
19.55 (0.23)
19.37 (0.27)
19.17 (0.27)
0.1832
33.95 (0.16) 7.80 (0.19)b 25.18 (0.35) 38.40 (0.24)
33.47 (0.17) 8.70 (0.21)a 23.82 (0.40) 38.69 (0.27)
34.05 (0.20) 8.39 (0.25)ab 24.05 (0.46) 38.65 (0.31)
33.97 (0.20) 8.64 (0.25)ab 23.90 (0.46) 38.45 (0.31)
0.1639 0.0402 0.0879 0.8460
Means within a row with different letters are significantly different at the mentioned P value
ND = not detected Standard error of mean in parentheses
25
P value
0.0779 0.0844 0.0001 0.5671 0.0066 0.3749 0.1776 0.0008 0.0649 0.1527 0.1994 0.0737 0.3336 0.6756 0.0403 0.0110 0.6858