Accepted Manuscript

Characterization of gelatin from bovine skin extracted using ultrasound subsequent to

bromelain pretreatment

Tanbir Ahmad, Amin Ismail, Siti Aqlima Ahmad, Khalilah Abdul Khalil, Elmutaz Atta
Awad, Teik Kee Leo, Jurhamid C. Imlan, Awis Qurni Sazili

PII: S0268-005X(17)31188-8
DOI: 10.1016/j.foodhyd.2018.01.036
Reference: FOOHYD 4250

To appear in: Food Hydrocolloids

Received Date: 12 July 2017

Revised Date: 27 December 2017
Accepted Date: 29 January 2018

Please cite this article as: Ahmad, T., Ismail, A., Ahmad, S.A., Khalil, K.A., Awad, E.A., Leo, T.K., Imlan,
J.C., Sazili, A.Q., Characterization of gelatin from bovine skin extracted using ultrasound subsequent to
bromelain pretreatment, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.01.036.

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 ACCEPTED MANUSCRIPT

Characterization of gelatin from bovine skin extracted using ultrasound

subsequent to bromelain pretreatment
Tanbir Ahmada, f, Amin Ismailb, g, Siti Aqlima Ahmadc, Khalilah Abdul Khalild, Elmutaz Atta
Awade, h, Teik Kee Leoa, , Jurhamid C Imlane, i, Awis Qurni Sazilia, e, g, *
a

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Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
b
Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia

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c
Faculty of Biotechnology and Molecular Sciences, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
d
Department of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah

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Alam, Selangor, Malaysia
e
Laboratory of Sustainable Animal Production and Biodiversity, Institute of Tropical Agriculture
and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
f

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ICAR-Central Institute of Post-Harvest Engineering and Technology, Ludhiana, Punjab-141004,
India
g
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Halal Products Research Institute, Putra Infoport, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
h
Department of Poultry Production, University of Khartoum, 13314, Khartoum North, Sudan
i
Department of Animal Science, College of Agriculture, University of Southern Mindanao,
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Philippines
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* Corresponding author: Department of Animal Science, Faculty of Agriculture, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Tel.: +60389474870; Fax: +60389381024
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E-mail address: awis@upm.edu.my (A. Q. Sazili)

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1 Characterization of gelatin from bovine skin extracted using ultrasound

2 subsequent to bromelain pretreatment
3

4 Abstract

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5 Bovine skin was pretreated with bromelain enzyme and ultrasound (53 kHz and 500 W) was

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6 used to extract gelatin for the time durations of 2, 4 and 6 h at 60 °C (samples were referred as

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7 UB2, UB4 and UB6, respectively). Control (UBC) gelatin was extracted using ultrasound for 6 h

8 at 60 °C without enzymatic pretreatment. Gelatin yield increased significantly (P<0.05) as the

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9 time duration of ultrasound treatment increased with UB6 giving the highest yield of 19.71%
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10 followed by UBC (18.67%). Gel strength and viscosity of UBC were 603.24 g and 16.33 mPa.s,

11 respectively. The corresponding values for UB6 were 595.51 g and 16.37 mPa.s, respectively.
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12 The amino acids content increased with longer duration of ultrasonic treatment and UBC

13 exhibited the highest content of the glycine (27.06%) and hydroxyproline (17.21%) compared to
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14 other samples. Protein pattern of the gelatin samples showed the progressive degradation of
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15 polypeptide chains as the time duration of ultrasound extraction increased. As demonstrated by

16 Fourier transform infrared (FTIR) spectroscopy, amide I band of gelatins extracted by ultrasound
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17 treatment exhibited higher wavenumbers than the commercial gelatin (CG) suggesting greater
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18 loss of molecular order in these samples. Longer duration of ultrasonic treatment resulted in
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19 denser, irregular, disorganized and more interconnected structure with increased porosity as

20 revealed by scanning electron microscopy (SEM) but structural integrity was retained in UBC

21 indicating degradation effect of bromelain enzyme in other samples. Finally, it was concluded

22 that the ultrasound assisted gelatin extraction using bromelain enzyme produced high yield with

23 good quality gelatin.

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24 1. Introduction

25 Gelatin is a high molecular weight biopolymer derived from collagen by thermal

26 denaturation. Insoluble collagen is required to be converted into soluble form by pretreatment

27 with either acid or alkali resulting in the loss of the ordered structure of native collagen which is

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28 swollen but still insoluble (Stainsby, 1987). Finally, conversion into gelatin takes place during

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29 extraction process due to the cleavage of hydrogen and covalent bonds by heat leading to helix-

30 to-coil transition (Djabourov, Lechaire, & Gaill, 1993). Cleavage of covalent and non-covalent

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31 bonds in sufficient numbers releases free α- chains and oligomers (Johnston-Banks, 1990). Apart

32 from this, hydrolysis of some amide bonds present in the collagen molecules take place (Bailey,

33
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1985). Therefore, the recovered gelatin has lower molecular weight components than native
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34 collagen and comprises of a mixture of polypeptide fragments having molecular weight in the

35 range of 16-150 kDa (Asghar & Henrickson, 1982).

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36 The molecular bonds present in the collagen are stable to thermal and acid treatment (Galea,
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37 Dalrymple, Kuypers, & Blakeley, 2000) resulting in a low gelatin yield (Nalinanon, Benjakul,
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38 Visessanguan, & Kishimura, 2008). Previously, to improve the gelatin extractability, some

39 proteases capable of breaking the collagen cross-links have been used (Nalinanon et al., 2008).
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40 Pepsin and proctase (isolated from Aspergillus niger) were used to extract the gelatin from
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41 bovine skin but the gelatin yield, its gel strengths and viscosities were low (Chomarat, Robert,
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42 Seris, & Kern, 1994). Papain was used to extract gelatin from rawhide splits but the obtained

43 gelatin showed low gel strength and viscosity (Damrongsakkul, Ratanathammapan, Komolpis, &

44 Tanthapanichakoon, 2008). Higher gelatin yield was obtained from raw hide by using crude

45 proteolytic enzyme from papaya latex and commercial papain enzyme but the gel strength was

46 relatively low and there was complete degradation of α- and β- chains in the recovered gelatin
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47 (Pitpreecha & Damrongsakkul, 2006). Although better gelatin yield was achieved with

48 proteolytic enzyme, the functional qualities of the obtained gelatin were compromised. Gelatin

49 with high molecular weight polymers (less degraded peptides) are reported to be better in

50 functional properties (Badii & Howell, 2006; Gómez-Guillén et al., 2002; Muyonga, Cole, &

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51 Duodu, 2004a; Zhang, Xu, & Wang, 2011). Therefore, novel enzymes capable of cleaving long

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52 chains of collagen only at few sites should be studied so that a long chain gelatin of high quality

53 can be produced (Ahmad et al., 2017). Bromelain enzyme at level of 20 units of enzyme per

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54 gram of skin had shown better gelatin yield and quality in our laboratory experiment

55 (unpublished results). Therefore, bromelain enzyme has been used in this study.

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56 There is no much published work on ultrasound assisted extraction (UAE) from animal tissue

57 (Vilkhu, Mawson, Simons, & Bates, 2008). UAE can enhance extraction efficiency and
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58 extraction rate particularly for aqueous extraction and lower processing temperatures can be

59 applied for enhanced extraction of heat sensitive bioactive and food components at lower
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60 processing temperatures (Vilkhu et al., 2008). Ultrasound has attracted the attention of food
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61 industry because of its promising in food science (Jia et al., 2010). High power ultrasound

62 (power >1W cm-2 and frequencies 20 to 500 kHz) can be applied for facilitating the extraction
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63 process of a variety of food components (e.g. herbal, oil, protein, polysaccharides) as well as
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64 bioactive ingredients (e.g. antioxidants) from plant and animal resources (Vilkhu et al., 2008).
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65 With ultrasonic treatment, high collagen yield was obtained from bovine tendon in significantly

66 less extraction time than the conventional pepsin isolation method (Li, Mu, Cai, & Lin, 2009).

67 Ultrasonic treatment of sea bass fish skin resulted in increased extraction yield of collagen (Kim,

68 Kim, Kim, Park, & Lee, 2012). Bighead carp scales treatment with ultrasound bath led to good
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69 quality gelatin with high yield (30.94-46.67%) and the presence of α- and β-chains was observed

70 in the resulting gelatin (Tu et al., 2015).

71 There is no published research work on the ultrasound assisted extraction of gelatin as well

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72 as on the ultrasound-enzyme assisted extraction of gelatin from bovine skin. Taking into

73 consideration all these facts, the objective of this study was to extract gelatin using ultrasound in

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74 conjugation with enzyme bromelain pretreatment and elucidate their effects on the quality

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75 parameters of the recovered gelatin.

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77 2. Materials and methods

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79 2.1 Chemicals

80 Acrylamide, sodium dodecyl sulphate (SDS), N,N,Nʹ,Nʹ-tetramethyl ethylene diamine

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81 (TEMED), coomassie brilliant blue R-250, 2-mercaptoethanol were purchased from Merck,
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82 Darmstadt, Germany. Other chemicals and reagents used were of analytical grade.
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83 Chromatographic column, mobile phase, reagents and amino acid standards were purchased from

84 Waters Corporation, MA, USA and hydroxyproline standard supplement was procured from
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85 Agilent Technologies, CA, USA. Bromelain enzyme, EC 3.4.22.32 (≥ 2.0 mAnsonU/mg)

86 extracted from pineapple (Ananas comosus) was obtained from Merck, Darmstadt, Germany.
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87 Commercial gelatin type B from bovine skin was purchased from Sigma, St. Louis, MO, USA.
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89 2.2 Preparation of skin

90 Three to four year old female Brahma cross skin was procured from a local commercial

91 ruminant abattoir located in Shah Alam, Selangor, Malaysia and transported in ice and stored at -
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92 20 °C. The subcutaneous fat was removed by scrapping. The skin was washed thoroughly and

93 stored at -20 °C until further used. It was thawed overnight at 4 °C before being used.

94

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95 2.3 Ultrasound assisted extraction of gelatin from bovine skin in conjugation with enzyme
96 bromelain

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98 2.3.1 Removal of non-collagenous proteins

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99 Bovine skin was soaked in the 0.1 M NaOH solution with stirring at the ratio of 1:5 (w/v) at

100 room temperature (25±1 °C) for 6 h to remove non-collagenous materials from the skin. The

101

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NaOH solution was changed every 2 h. Thereafter, the hairs on the skin were removed by
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102 scraping with scalpel and cut into 1 cm x 2 cm size. The skin was rinsed thoroughly with

103 distilled water until neutral pH wash water was obtained.

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104
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105 2.3.2 Ultrasound assisted gelatin extraction in conjugation with enzyme bromelain
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106 Skin was soaked in 1% HCl for 20 h with discontinuous stirring at the ratio of 1:10 (w/v) at

107 room temperature for swelling. The samples were washed thoroughly with distilled water until
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108 neutral wash water was obtained. The experiment in the laboratory had shown better yield and
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109 quality gelatin could be obtained from bromelain enzyme at the level of 20 units of bromelain
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110 enzyme/g of skin at its optimum temperature and pH of the enzyme (35.5 °C and 6.0,

111 respectively) (unpublished results). Therefore, the swollen skins were incubated with enzymes

112 bromelain for 48 h at the level of 20 units per g of wet skin at the optimum temperature and pH

113 of the enzyme (35.5 °C and 6.0, respectively) as indicated by the manufacturer.
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114 The swollen skin samples were kept in the optimum pH solution at skin to solution ratio of 1:

115 3 (w/v) and the enzyme was added. The mixture was stirred in the waterbath at 35.5 °C for 48 h.

116 Thereafter, the mixture was transferred to another waterbath at 90 °C for 15 min to terminate the

117 enzyme activity. Gelatin was extracted at 60 °C for the time duration of 2, 4 and 6 h in ultrasonic

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118 bath (SK8210HP, Kudos, China) using 53 kHz frequency and ultrasonic power of 500 W. The

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119 mixture was filtered using cheese cloth and then centrifuged (Beckman Coulter Avanti J-26 XPI)

120 at 12,800 x g for 20 min. The supernatant was dried using freeze drier (LABCONCO

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121 FreeZone18, KS, USA) and the obtained gelatin was stored at 4 °C for analysis. Control gelatin

122 was extracted using ultrasound treatment at 60 °C for 6 h without enzymatic treatment to the

123
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swollen skin as mentioned above. The extraction was performed in triplicate.
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125
126 2.4 Analyses of gelatin
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128 2.4.2 Yield

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130 The yield of the gelatin was calculated on the wet weight basis of the skin as reported by

131 Balti et al. (2011), Bougatef et al. (2012), Ktari et al. (2014) and Lassoued et al. (2014).
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133 Weight of the freeze dried gelatin (g)
134 Yield (%) = x 100
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135 Wet weight of the skin (g)

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138 2.4.3 Determination of colour
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140 Colour of the gelatin samples were measured by ColorFlex HunterLab (Hunter Associates

141 Laboratory Inc., Reston, VA, USA.). Three colour co-ordinates, namely L* (lightness), a*

142 (redness/greenness) and b* (yellowness/blueness) were used (Jamilah & Harvinder, 2002). The
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143 sample was filled in a 64 mm glass sample cup with three readings in the same place and

144 triplicate determinations were taken per sample.

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146 2.4.4 Determination of pH

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148 The BS 757 of British Standard Institute method was followed (Eastoe & Leach, 1977). One

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149 percent (w/v) of gelatin solution (0.2 g in 20 ml distilled water) was prepared and it was cooled

150 to room temperature of about 25 ºC. The pH meter (Mettler Toledo, AG 8603, Switzerland) was

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151 standardized with pH 4.0 and 7.0 buffers and pH determination was carried out in triplicates.

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153
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155 2.4.5 Determination of amino acid composition
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157 To determine the amino acid (AA) content of the gelatin samples using high performance
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158 liquid chromatography (HPLC), slightly modified method of Awad, Zulkifli, Farjam, & Loh
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159 (2014) was followed. Shortly, 5 ml of 6 N HCl was used to hydrolyze 0.1 to 0.2 g of sample at
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160 110 °C for 24 h. Upon cooling, 4 ml of internal standard (α-aminobutyric acid; AABA) was

161 added to the hydrolysate and aliquot was paper and syringe filtered. Ten microlitre of the filtered
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162 sample was mixed with 70 µl of borate buffer and 20 µl of ACCQ reagent (Waters Corporation,

163 Milford, MA, USA). Internal standard was spiked with hydroxyproline and a mixture of amino
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164 acid standard H (Waters Corporation, MA, USA) was used to determine the concentration of all
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165 AA except methionine, cysteine and tryptophan. An AA column (AccQ Tag 3.9 150 mm; Waters

166 Corporation, MA, USA) was used to separate peaks. Peaks were detected by a fluorescent

167 detector (2475; Waters Corporation, MA, USA). Triplicate determinations were performed and

168 data corresponds to mean values. Standard deviations in all cases were lower than 2%.

169
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170 2.4.6 Electrophoretic analysis

171 The molecular weight distributions of the extracted gelatins were determined by sodium

172 dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by (Laemmli,

173 1970). Dry gelatin (10 mg) was dissolved in distilled water (1 ml) at 60 °C. The sample solution

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174 was mixed in a 1:2 (v/v) ratio with loading buffer (0.5 M Tris-HCl, pH 6.8, glycerol, 10% (w/v)

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175 SDS, 0.5% (w/v) bromophenol blue and 5% 2-mercaptoethanol). The mixed solution was heated

176 in waterbath (95 °C) for 5 min before loading into 4% stacking gel and 7.5% resolving gel. The

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177 gelatin samples were separated using Mini-PROTEAN Tetra System (Bio-Rad Laboratories, CA,

178 USA) at a constant current of 15 mA/gel for 15 min, followed by 25 mA/gel until the

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bromophenol blue dye reached the bottom of the gel. Following electrophoresis, the gel was
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180 stained with 0.1% (w/v) coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid

181 for 2 h and destained with 30% (v/v) methanol and 10% (v/v) acetic acid until the zones on the
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182 blue background were clear. Prestained protein ladder (BLUeye, GeneDireX, Taoyuan, Taiwan)
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183 was used to estimate the molecular weight distributions of the gelatins. The gel was scanned with
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184 a GS-800 Calibrated Densitometer (Model: PowerLook 2100XL- UB, Bio-Rad Laboratories,

185 CA, USA) gel imaging system.

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186
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187 2.4.7 Determination of turbidity

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189 Turbidity of the gelatin samples was determined by slightly modified method of Cho et al.

190 (2004). Gelatin sample (0.025 g) was dissolved in distilled water (5 ml) at 60 °C to make 0.5%

191 (w/w) solution. Spectrophotometer (Shimadzu UV Spectrophotometer, Model UV-1800, Kyoto,

192 Japan) was to measure the absorbance at 660 nm.

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194 2.4.8 Determination of gel strength

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196 The method of Fernàndez-Diaz, Montero, & Gòmez-Guillèn (2001) was slightly modified to

197 determine the gel strength of the extracted gelatin. Gelatin (2.0 g) was dissolved in 30 ml of

distilled water at 60 °C using 50 ml-beaker (SCHOTT DURAN, Mainz, Germany) to get the

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199 final concentration of 6.67% (w/v). The solution was stirred until gelatin was solubilized

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200 completely, and kept at 7 °C for 16–18 h for gel maturation. Texture Analyzer (Stable Micro

201 Systems, Model: TA-XT2i, Surrey, UK) was used to measure the bloom strength using a load

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202 cell of 5 kN equipped with a 1.27 cm diameter flat-faced cylindrical Teflon plunger (P/0.5R).

The dimensions of the sample were 3.8 cm in diameter and 2.7 cm in height. The maximum

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force (in grams) was recorded when the probe penetrated a distance of 4 mm inside the sample.
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205 The speed of the plunger was 0.5 mm/s. All determinations are means of three measurements.
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207
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208 2.4.9 Determination of viscosity

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209 Gelatin solution of 6.67% was prepared by dissolving 1.34 g of gelatin in 20 ml of distilled

210 water and heated to 60 °C. RheolabQC (Anton Paar, Graz, Austria) viscometer was used to
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211 measure the viscosity of the samples. The measurement was performed in triplicate.

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213 2.4.9 Fourier transform infrared (FTIR) spectroscopy

214 FTIR spectra were obtained using spectrometer (Perkin Elmer Ltd., Model: Spectrum 100,

215 AZ, USA) equipped with a deuterated triglycine sulphate (DTGS) detector. The attenuated total

216 reflectance (ATR) accessory was mounted into the sample compartment. Diamond internal

217 reflection crystal had a 45° angle of incidence to the IR beam. Resolution of 4 cm-1 was used to
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218 acquire the spectra and 4,000-500 cm-1 (mid-IR region) was chosen as measurement range at

219 room temperature. Automatic signals were collected in 16 scans at a resolution of 4 cm-1 and

220 were normalized against a background spectrum recorded from the clean, empty cell at 25 °C.

221

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222 2.4.10 Microstructure analysis of gelatin

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223 The microstructure of extracted gelatins was elucidated using scanning electron microscope

224 (SEM) (JEOL JSM-IT100 InTouchScope, Tokyo, Japan). Dried gelatin samples having a

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225 thickness of 2–3 mm were mounted on a bronze stub and sputter-coated with gold (BAL-TEC

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226 SCD 005 sputter coater, Schalksmühle, Germany). An acceleration voltage of 10 kV was used to
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227 observe the specimen at 30x.

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229 2.5 Statistical analysis

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231 All statistical analyses were carried out using GLM procedure of Statistical Analysis System
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232 package (SAS) Version 9.4 software (Statistical Analysis System, SAS Institute Inc., Cary, NC,
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233 USA) and statistical significance was set at p<0.05. Significant differences between means were
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234 evaluated by Duncan’s Multiple Range Test.

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237 3. Results and discussion
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238

239 3.1 Effect of ultrasound assisted gelatin extraction in conjugation with bromelain

240 The effects of ultrasound assisted extraction in conjugation with enzyme bromelain on

241 gelatin yield are shown in the Table 1. UB6, UB2, UB4 and UB6 gelatin yield was 18.67, 7.09,

242 15.65 and 19.71%, respectively. The higher yield of gelatin with increasing time was due to
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243 cavitation and mechanical effect of ultrasound (Tu et al., 2015). Increased extraction yield

244 obtained from UAE is basically due to acoustic cavitations (Wang, Sun, Cao, Tian, & Li, 2008)

245 as it releases more energy to wash out the gelatin from the skin sample. Collagen extraction

246 improved with ultrasound treatment due to cavitation which opened up the collagen fibrils and

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247 increased the dispersal of enzyme aggregates and thus facilitating the transport of pepsin

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248 molecules to collagen substrate surface and subsequent hydrolysis (Li et al., 2009). Apart from

249 this, mechanical effect of ultrasound increases the contact surface area between sample matrix

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250 and solvent and thus enabling greater penetration of liquid medium into the solid phase for

251 extraction (Rostagno, Palma, & Barroso, 2003).

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252 Gelatin yield increased significantly (P<0.05) with increase in time duration. This is in

253 consistent with the finding of Arnesen & Gildberg (2007) and Tu et al. (2015) who reported that
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254 longer extraction time from Atlantic salmon skin and bighead carp scales, respectively resulted

255 in higher yield of gelatin. More energy was provided by increasing time to destroy the stabilizing
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256 bonds present in the collagen structures and peptide bonds of α-chains resulting in helix-to-coil
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257 transformation (Nagarajan, Benjakul, Prodpran, Songtipya, & Kishimura, 2012).

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259 3.2 Colour

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260 UB2 sample exhibited significantly (P<0.05) higher L* value (lightness) and significantly

261 (P<0.05) lower a* and b* colour coordinates than the other samples indicating less redness and

262 browning in UB2 (Table 2). Sinthusamran, Benjakul, & Kishimura (2014) reported highest L*

263 for gelatin extracted for short time (3 h). Significantly (P<0.05) higher redness and yellowness

264 (a* and b* values, respectively) was observed for UB6 and UBC. Non-enzymatic browning
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265 reaction due to longer extraction time might be held responsible for the higher yellowness (b*

266 value) recorded for UB6 and UBC samples (Sinthusamran et al., 2014).

267

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268 3.3 pH

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269 The highest pH of 2.51 was recorded for UB4 and lowest for UB2 (2.40) (Table 1). Mohtar,

270 Perera, & Quek (2010) reported that the pH of bovine gelatin was 5.48. The low pH obtained for

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271 these samples could be due to HCl solution used for swelling the skin. The relationship between

272 the processing method used to extract gelatin and the pH of gelatin has yet not been established

273 (Park et al., 2013).

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275 3.4 Amino acid composition of gelatin

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276 Principally, amino acid composition and molecular weight distribution determines the
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277 properties of gelatin (Gomez-Guillen et al., 2009). The most abundant amino acid in gelatin is

278 glycine (Arnesen & Gildberg, 2002). Triple peptides which consist of repeating chains of Gly-X-
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279 Y, where X is generally proline and Y is mainly hydroxyproline, make up to 50-60% of α-chains

280 (Asghar & Henrickson, 1982). Gelatins extracted from warm-blooded animal tissues have been
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281 found to be rich in proline and hydroxyproline (imino acid) amino acids content, particularly
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282 hydroxyproline (Norland, 1990).

283 There is very few published research work on the effects of duration of high power

284 ultrasound treatment on the amino acid content. In present study, glycine (Gly), proline (Pro)

285 and hydroxyproline (Hyp) content for UBC, UB2, UB4 and UB6 was 27.06, 12.44 and 17.21%,
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286 20.84, 11.91 and 15.99, 21.30, 12.08 and 16.74% and 25.88, 12.75 and 17.05%, respectively

287 (Table 3). Generally, the amino acids content increased with the increase in time duration of

288 ultrasound treatment and UBC exhibited the highest content of glycine and hydroxyproline. With

289 respect to glycine, proline and hydroxyproline content, UB6 and UBC amino acid composition

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290 was almost similar. UBC had slightly higher glycine and hydroxyproline and lower proline

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291 content than UB6 (P>0.05). Hydrophobic amino acids content of rice dreg protein (RDP)

292 extracted from rice dreg flour (RDF) increased with ultrasound treatment (Li, Ma, Li, Zhang, &

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293 Dai, 2016). Cavitation effect caused the microfractures, molecule unfolding and protein structure

294 changes by producing high-intensity shock waves, microjets, shear forces and turbulence leading

295
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296 al., 2016).
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297 Lassoued et al. (2014) and Balti et al. (2011) reported 34.48, 13.39 and 9.54% and 34.1, 12.3

298 and 9.6% of glycine, proline and hydroxyproline content out of the total amino acids,
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299 respectively in food grade halal bovine gelatin. Our amino acid results are expressed in terms of
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300 percentage of sample (mg/100 mg of sample) which may be the reason for the difference.

301 Nonetheless, ox hide and calf skin contained 27.6, 16.5 and 13.4% and 26.9, 14.0 and 14.6%
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302 glycine, proline and hydroxyproline, respectively (Ward & Courts, 1977). Differences in
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303 manufacturing processes of gelatin might give rise to variations in the amino acid contents (Zhou
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304 & Regenstein, 2006).

305 The amount of imino acids (Pro + Hyp) greatly determines the stability of the triple helix

306 structure of the renatured gelatins as imino acids rich regions are likely to be involved in the

307 formation of nucleation zones (Ledward, 1986). In addition to that, Hyp is thought to provide

308 stability to the triple-stranded collagen helix by its ability to form hydrogen bond through its
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309 hydroxyl group (Burjandze, 1979; Ledward, 1986; Mizuno, Hayashi, & Bächinger, 2003). The

310 imino acid content in bovine gelatin was 21.90% (Lassoued et al., 2014; Balti et al., 2011) or

311 23.3% (Kasankala, Xue, Weilong, Hong, & He, 2007). UBC, UB2, UB4 and UB6 samples

312 showed imino acid (Pro+Hyp) content as 29.65, 27.90, 28.82 and 29.80%, respectively and

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313 corresponding Hyp content was 17.21, 15.99, 16.74 and 17.05%, respectively. This was higher

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314 than the Hyp content of halal bovine gelatin as reported by Lassoued et al. (2014) and Balti et al.

315 (2011) (9.6 and 9.54%, respectively). The high imino acid content was reflected in the high gel

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316 strength and viscosity of the recovered gelatin.

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318 3.5 SDS-PAGE analysis of gelatin
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319 Functional properties of gelatin are affected by the amino acid composition, the molecular

320 weights distribution, structure and compositions of its subunits (Balti el at., 2011). SDS-PAGE
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321 analysis was used to elaborate the molecular weight pattern of pretreated skin samples (PS),
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322 UBC, UB2, UB4, and UB6 samples (Fig. 1). Molecular distribution pattern of PS showed the

323 presence of γ- (covalently linked α-chains trimers), β- (covalently linked α-chains dimers), α1-
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324 and α2-chains whereas only α1- and α2-chains along with lower molecular weight peptides were

325 found in UB2. There was progressive degradation of polypeptide chains with ultrasonic
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326 treatment time duration. α1- and low intensity α2-chains were observed in UB4 sample whereas
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327 only faint presence of α1- and α2-chains along with lower weight polypeptides were observed in

328 UB6 and UBC samples indicating that bromelain pretreatment did not affect the protein pattern.

329 Although ultrasonic treatment of various food products did not cause much difference in the

330 protein fraction (Gülseren, Güzey, Bruce, & Weiss, 2007; Hu et al., 2013; Krise, 2011; Karki et
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331 al., 2010; O'Sullivan, Arellano, Pichot, & Norton, 2014; Yanjun et al., 2014;), the ultrasound

332 treatment was only for few minutes in these cases. Ultrasound treatment of 20 and 40 kHz for 30

333 min degraded the protein molecules present in whey protein concentrate (WPC) and whey

334 protein isolate (WPI) (Jambrak et al., 2014) and in α-lactalbumin (Jambrak, Mason, Lelas, &

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335 Kresic, 2010). Ultrasound assisted extraction of gelatin from bighead carp scales for long

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336 duration resulted in α chains degradation (Tu et al., 2015). Degradation of protein molecular

337 structure might be due to higher shear stress and turbulence effects of ultrasound treatment

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338 (O'Sullivan et al., 2014).

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339
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340 3.6 Turbidity
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341 Except for UBC, turbidity decreased as the ultrasound treatment time increased (Table 1).

342 The significantly (P<0.05) higher turbidity of UB2 showed its lower quality than other samples
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343 (Montero, Fernandez-Diaz, & Gomez-Guillen, 2002). UB4 and UB6 had significantly (P<0.05)
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344 lower turbidity than the UBC. The result is consistent with the finding of Malik, Sharma, & Saini

345 (2017). They reported decrease in turbidity of 10% sunflower isolate solution with time duration
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346 of high intensity ultrasound treatment. This might be due to size reduction of the suspended

347 insoluble aggregates by ultrasound (Jambrak, Mason, Lelas, Paniwnyk, & Herceg, 2014).
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348 Another possible explanation for the decrease in turbidity after ultrasonication was attributed to
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349 disruption in the protein-protein interactions resulting in small aggregates formation leading to

350 turbidity reduction (Martini, Potter, & Walsh, 2010).

351

352 3.7 Gel strength

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353 Gel strength is the most important functional property of gelatin. According to Gómez-

354 Guillén et al. (2002), gel strength is function of complex interaction determined by molecular

355 weight distribution. It is a function of complex interactions between amino acid composition and

356 α- chain ratio and quantity of β- components (Balti et al., 2011). Differences in molecular weight

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357 distribution and amino acid composition give rise to differences in gel strength (Nagarajan et al.,

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358 2012) and generally, high gel strength is exhibited by the gelatin having high molecular weight

359 polypeptides (Badii & Howell, 2006) because low molecular weight peptides might not be able

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360 to form inter-junction zones effectively. Gel strength is also controlled by the imino acid (proline

361 and hydroxyproline) content (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2006),

362
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especially hydroxyproline through its ability to form hydrogen bonding by –OH group (Montero,
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363 Fernández-Dı́az, & Gómez-Guillén, 2002).
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364 The gel strength values of all the gelatins extracted using ultrasound samples were high

365 (Table 1). UBC, UB2, UB4 and UB6 demonstrated the gel strength value of 603.24, 475.26,
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366 631.90 and 595.51 g, respectively. There was non significant (P>0.05) difference between UBC
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367 and UB6. The UB2 sample revealed the presence of α1- and α2- chains along with lower

368 molecular weight polypeptides. The protein chains experienced progressive degradation with
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369 increase in time duration of ultrasound treatment leading to faint presence of α1- and α2- chains
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370 in UB6 and UBC samples.

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371 There are several factors influencing the gel strength of gelatin. The presence of crosslinked

372 two α-chains and the β-component facilitate the chains to form triple helices upon cooling and

373 thereby to helix growth during gel maturation (Balti et al., 2011). In addition to molecular weight

374 distribution, protein aggregation between gelatin molecules also affects the gel strength (Balti et

375 al., 2011). Further, the gel strength is also dependent on the polypeptide chains configuration and
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376 the inter-junction zones formed during the maturation process (Ahmad & Benjakul, 2011).

377 Contrary to these reports, Muyonga, Cole, & Duodu (2004a) found that there is no relationship

378 between gelatin gel strength and molecular weight distribution in high strength gelatin.

379 Apart from all these factors, amino acid composition and the type of extraction treatments

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380 also influence the gel strength of gelatin (Balti et al., 2011). Difference in gel strength could also

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381 be attributed to differences in the imino acid composition (Gudmundsson, 2002). Triple helices

382 are partially recovered during maturation of gel and the stability to triple helices is provided by

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383 the regions rich in Gly-Pro-Hyp (Ahmad, Benjakul, Ovissipour, & Prodpran, 2011). The imino

384 acid content of UBC, UB2, UB4 and UB6 were 29.65, 27.90, 28.82 and 29.80%, respectively.

385
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Significantly (P<0.05) low gel strength of UB2 could be due to low imino acid content compared
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386 to other samples. The high gel strength of UB4 than other samples could be explained in term of

presence of α1-chains and faint presence of α2-chains as well as comparatively moderate amount
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387

388 of imino acid. Apart from the similar imino acid and hydroxyproline content, UBC and UB6
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389 demonstrated similar molecular distribution pattern which could be the deciding factors for
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390 exhibiting the almost same gel strength for these two gelatin samples.

391
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392 3.8 Viscosity

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393 The second most important commercial physical property of gelatin is viscosity (Wards &
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394 Courts, 1977). Destruction of hydrogen and electrostatic bonds present in collagen causes

395 denaturation destroying the triple helical structure of collagen and formation of random chains

396 consisting of one, two or three chains gelatin molecules resulting in high viscosity solution in

397 water (Badii & Howell, 2006).

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398 Viscosity was 16.33, 15.90, 16.77 and 16.37 mPa.s for UBC, UB2, UB4 and UB6,

399 respectively (Table 1). There was non-significant (P<0.05) difference between UBC and UB6.

400 Viscosity increased with time and then decreased at 6 h of ultrasonic treatment. Bromelain

401 enzyme pretreatment seemed to have insignificant effect on viscosity. Viscosity of the

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402 commercial bovine gelatin was 9.80 cP (Mohtar et al., 2010). Generally, it is controlled by

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403 molecular weight and polydispersity meaning high molecular weight polypeptides leads to high

404 viscosity gelatin (Gudmundsson & Hafsteinsson, 1997). Low viscosity gelatin produces a short

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405 and brittle texture gel whereas tough and extensible gel is formed by the high viscosity gelatin

406 with more commercial value (Zhou, Mulvaney, & Regenstein, 2006). Comparatively high

407
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viscosities obtained for these samples might be due to particle size denatured collagen recovered
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408 during ultrasonic extraction attributed to cavitation effect which caused impingement by micro-

jets that resulted in surface peeling, erosion and particle breakdown (Vilkhu et al., 2008).
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409

410
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411 3.9 FTIR spectra

412 Generally, FTIR spectroscopy is used to study the functional groups and secondary structure
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413 of gelatin samples and the most important for infrared spectroscopic analysis of the secondary

414 structure of proteins is the amide I band (Kittiphattanabawon, Benjakul, Visessanguan, &
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415 Shahidi, 2010). C=O stretching vibration hydrogen bonding coupled to contributions from the
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416 CN stretch, CCN deformation and in-plane NH bending modes give rise to Amide-I band

417 (Bandekar, 1992). Its exact location is determined by hydrogen bonding and the conformation of

418 protein structure (Uriarte-Montoya et al., 2011). The amide I band was found between 1,600 and

419 1,700 cm-1 (Muyonga et al., 2004b). Absorption peak at 1,633 cm−1 is the characteristic of the
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420 coiled structure of gelatin (Yakimets et al., 2005) and this is in the agreement with our

421 observation of the amide-I peak obtained in the range of 1,631-1,635 cm-1.

422 FTIR spectra exhibited that the major peaks of UBC, UB2, UB4 and UB6 were detected in

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423 the amide regions (Fig. 2) and peak position of different bands has been presented in Table 4.

424 These spectra were in accordance with those reported by Muyonga et al. (2004b). For

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425 comparative reason, FTIR spectra of commercial gelatin (CG) have also been included. Amide I

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426 bands for UBC, UB2, UB4, UB6 and CG were detected at the wave numbers of 1,635.64,

427 1,631.78, 1,635.64, 1,631.78 and 1,629.83 cm−1, respectively. Shift of the band to higher

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428 wavenumber along with higher amplitude indicated greater loss of molecular order due to
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429 thermal uncoupling of inter-molecular crosslink (Ahmad & Benjakul, 2011). Although amide I

430 for UBC was found at higher wavenumber but its amplitude was relatively lower than the other
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431 ultrasound treated samples. The higher wavenumber with high amplitude of UB4 indicated that

432 longer duration of ultrasound treatment caused increased thermal uncoupling of inter-molecular
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433 crosslink resulting in more loss of molecular order in UB4 gelatins compared to other ultrasound
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434 treated samples. UB6 gelatin displayed the amide I band at lower wavenumber along with lower

435 amplitude. This might be due to higher duration of ultrasound treatment along with bromelain
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436 pretreatment than the UB4 caused the protein-protein linkages being formed in UB6 due to
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437 unfolding of functional groups (Jiang et al., 2014). The shorter but uniform length of protein
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438 chains produced as a result of long duration ultrasound treatment possibly aided in the better

439 linkages formation in UB6 (Damrongsakkul et al., 2008). The higher wavenumber and amplitude

440 of amide I band for the treatment samples compared to CG suggested greater loss of molecular

441 order in these samples. The higher wavenumber for UBC but similar amplitude to that of CG
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442 suggested the absence of intervening role of bromelain pretreatment. Amide I band of gelatin

443 samples extracted by ultrasound treatment exhibited higher wavenumber (Tu et al., 2015).

444 The characteristic amide II absorption bands of UBC, UB2, UB4, UB6 and CG gelatins were

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445 observed at 1,535.34, 1,531.48, 1,535.34, 1,531.48 and 1,532.36 cm−1, respectively. Amide II

446 band indicates an out-of phase combination of CN stretch and in-plane NH deformation modes

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447 of the peptide group (Lavialle, Adams, & Levin, 1982; Ahmad & Benjakul 2011). Dry collagen

had the amide II band in the infrared spectrum range of 1,530–1,540 cm−1, and often had minor

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448

449 bands at lower frequencies (Tu et al., 2015). The lower wavenumber with lower amplitude

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450 implied higher involvement of NH group in hydrogen bond formation with neighboring
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451 molecules (Ahmad et al., 2011). Although UB2, UB6 and CG showed lower amide II

452 wavenumbers but only UB2 and UB6 exhibited relatively lower wavenumber as well as lower
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453 amplitude than CG suggesting greater participation of NH group in H-bonding.

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454 C-N stretching and N-H deformation in plane bending occurring due to amide linkages and
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455 absorptions arising from the wagging vibrations of CH2 groups from the glycine back bone and

456 proline side chains is represented by amide III which is generally seen in the region of 1,200-
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457 1,400 cm−1 (Jackson, Choo, Watson, Halliday, & Mantsch, 1995). Amide III spectra for UBC,

458 UB2, UB4, UB6 and CG gelatins were detected at wavenumber of 1,230.58, 1,230.58, 1,242.16,
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459 1,234.44 and 1,235.93 cm−1, respectively. Amide III band around 1,233-1,234 cm−1 indicated
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460 loss of triple helical structure due to gelatin molecules disorder (Sinthusamran et al., 2014).

461 Lower amplitude amide III band signifies the disruption in the native state of α helical structure

462 to random coiled structure associated with the loss of triple helix structure due to denaturation of

463 collagen into gelatin (Muyonga et at., 2004b). Simultaneous lower amplitude of UB6 and its

464 occurrence near 1,234 cm−1 pointed towards transformation of α helical structure of UB6 to
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465 random coiled structure possibly due to concurrent effect of long duration ultrasound along with

466 bromelain pretreatment. Amide III of CG also had highest amplitude implying least loss of triple

467 helix configuration. Lowest amplitude of amide III for UBC might be due to absence of

468 bromelain pretreatment. Some additional peaks were observed for all the samples at lower than

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469 amide III regions indicating C ̶ O stretching vibrations of the short peptide chains (Jackson et al.,

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470 1995).

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471 Amide A band arising from NH-stretching coupled with hydrogen bonding are observed in

472 the range of wavenumber of 3,400–3,440 cm−1 (Muyonga et at., 2004b) and it is shifted to lower

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473 wavenumbers, generally near the 3,300 cm−1, when the N-H group of a peptide is involved in a
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474 hydrogen bond (Doyle, Bendit, & Blout, 1975; Nagarajan et al., 2012; Tu et al., 2015:). Amide A

475 appeared at 3,309.85, 3,294.42, 3,302.13, 3,294.42 and 3,278.74 cm−1, respectively for UBC,
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476 UB2, UB4, UB6 and CG. The lower wavenumber of UB2, UB6 and CG suggested higher

477 hydrogen bonding involvement of a N-H group in α-chain. The low wavenumber concurrently
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478 with high amplitude of amide A suggested gelatin degradation (Nagarajan et al., 2012). UB2 and
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479 UB6 showed comparatively low amide A wavenumber with low amplitude whereas CG

480 exhibited lower amide A wavenumber with high amplitude suggesting degradation of gelatin.
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481 Low amide A wavenumbers of bromelain pretreated samples compared to UBC could be
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482 attributed to high hydrogen bonding involvement of a N-H group in α chain.

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483 Asymmetric stretching vibration of =C-H as well as NH3+ is represented by amide B bands

484 (Ahmad & Benjakul, 2011). The amide B for UBC, UB2, UB4, UB6 and CG were detected at

485 2,943.37, 2,931.80, 2,947.23, 2,924.09 and 2,947.12 cm−1, respectively. Relatively lower

486 wavenumber of UB2 and UB6 suggested higher interaction of -NH3 group between peptide

487 chains in UB2 and UB6 (Ahmad & Benjakul, 2011; Nagarajan et al., 2012). Thus, it was
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488 concluded that the secondary structures and functional groups were affected by the ultrasound

489 duration and enzyme pretreatment.

490

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491 3.10 Microstructure of gelatin

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492 Microstructure of gelatin is related to the physical properties of the gelatin. Longer duration

493 of ultrasonic treatment with enzymatic pretreatment decreased the structural integrity of the

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494 gelatin samples but structural integrity is retained in UBC indicating degradation effect of

495 bromelain enzyme in other gelatins (Fig. 3). UB2 showed relatively less dense structure than the

496
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other samples. Gelatin structure changed to denser, smaller particle sized, irregular, disorganized
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497 and more interconnected structure with increased porosity as the duration of ultrasound treatment
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498 increased. High-power ultrasonication resulted in partial unfolding of protein whereby functional

499 groups (such as hydrophobic groups) were exposed and this led to immediate interaction among
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500 functional groups resulting in protein aggregation and network formation (Jiang et al., 2014).
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501 UBC microstructure was smooth having bigger size particles. The result indicated that

502 ultrasound extraction of gelatin from enzyme pretreated collagen resulted in change in the gelatin
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503 microstructure.
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504
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505
506 4. Conclusions

507 Bromelain pretreatment of bovine skin followed by ultrasound assisted extraction at 60 °C

508 for 6 h resulted in significant increase in gelatin yield. The recovered gelatin showed good gel

509 strength and viscosity. There was complete absence of β- component in all the samples. α1- and
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510 α2- chains along with lower molecular weight peptides were noticed in UB2. Degradation of

511 protein chains were observed with increase in time duration of ultrasonic treatment. Although

512 decreased structural integrity of the gelatin samples were observed with increase in ultrasound

513 extraction time, UBC did not lose its structural integrity. This might be due to proteolytic activity

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514 of bromelain on collagen chains cross links. The gel strength of gelatin is influenced by the

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515 higher extend of molecular order of triple-helix structure, presence of high molecular weight

516 protein components as well as the imino acid content especially hydroxyproline content.

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517 Financial implication of ultrasound-enzyme treatment to extract could be lowered down at viable

518 level if applied at the industrial scale.

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519

520
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521
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522 Acknowledgements
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523 Indian Council of Agricultural Research, New Delhi, India and Department of Agricultural
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524 Research & Education, Ministry of Agriculture, Government of India are duly acknowledged by

525 first author for providing ICAR-International Fellowship vide letter number F. No. 29-1/2009-
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526 EQR/Edn (pt.III) dated October 28, 2014 and granting study leave to him (letter number F. No.
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527 7-46/2014-IC II dated January 23, 2015), respectively. A special thanks to Director, ICAR-

528 Central Institute of Post-Harvest Engineering and Technology, Ludhiana, Punjab, India for

529 relieving the first author to pursue Ph. D. study. The research work was funded by Universiti

530 Putra Malaysia, Malaysia (Putra Grant vide letter no. UPM/700-2/1/GP-IPS/2015/9467000 dated

531 January 5, 2016).

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532

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749 Figure captions:

750 Fig. 1. SDS-PAGE pattern of pretreated skin (PS) sample along with gelatin extracted using
751 ultrasound for the time duration of 2, 4 and 6 h (UB2, UB4 and UB6, respectively) from bovine
752 skin with bromelain enzyme pretreatment. UBC: control gelatin extracted using ultrasound

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753 without enzyme pretreatment. CG: commercial gelatin. M denotes the marker.

754

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755 Fig. 2. FTIR spectra of gelatin extracted using ultrasound for the time duration of 2, 4 and 6 h
756 (UB2, UB4 and UB6, respectively) with bromelain enzyme pretreatment. UBC: control gelatin
extracted using ultrasound without enzyme pretreatment. For comparative reason, the spectra for

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758 commercial gelatin (CG) are also included.

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Fig. 3. SEM images of gelatin extracted using ultrasound for the time duration of 2, 4 and 6 h
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761 (UB2, UB4 and UB6, respectively) with bromelain enzyme pretreatment. UBC: control gelatin
762 extracted using ultrasound without enzyme pretreatment.
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Table 3

Amino acid composition (% of gelatin sample) of gelatin samples. UB2, UB4 & UB6 refers to
gelatin extracted using ultrasound for the time duration of 2, 4 and 6 h, respectively from bovine
skin pretreated with enzyme bromelain. UBC: control gelatin extracted using ultrasound without
enzymatic pretreatment.

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Amino Acids Gelatin samples
UBC UB2 UB4 UB6
17.21a 15.99a 16.74a 17.05a

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Hydroxyproline (Hyp)
Aspartic acid (Asp) 4.44a 4.86a 4.46a 4.34a
Serine (Ser) 3.34a 3.27a 3.32a 3.36a

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Glutamic acid (Glu) 8.22a 8.53a 7.92a 8.16a
Glycine (Gly) 27.06a 20.84b 21.30b 25.88a
Histidine (His) 0.89a 0.90a 0.89a 0.86a
Arginine (Arg)
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10.28a 8.42b 9.01ab 9.11ab
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Threonine (Thr) 1.90a 1.89a 2.01a 1.98a
Alanine (Ala) 8.00a 8.70a 8.75a 8.50a
Proline (Pro) 12.44a 11.91a 12.08a 12.75a
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Tyrosine (Tyr) 0.73a 0.65a 0.74a 0.72a

Valine (Val) 2.30a 2.39a 2.42a 2.37a
3.91a 3.61a 3.66a 3.59a
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Lysine (Lys)
Isoleucine (Ile) 1.47a 1.46a 1.51a 1.51a
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Leucine (Leu) 3.14a 3.06a 3.17a 3.13a

Phenylalanine (Phe) 2.28a 2.14a 2.27a 2.22a
Imino acids (Pro+Hyp) 29.65a 27.90a 28.82a 29.80a
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All the data expressed in the unit of mg/100 g of gelatin. Means with different superscripts in the
same row indicate significant difference at p<0.05. Standard deviations were in all cases lower
than 2%.
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Table 4

FTIR spectra peak position of gelatin extracted using ultrasound for the time duration of 2, 4 and
6 h (UB2, UB4 and UB6, respectively) with bromelain enzyme pretreatment. UBC: control
gelatin extracted using ultrasound without enzyme pretreatment. For comparative reason, the
spectra for commercial gelatin (CG) are also included.

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Peak wavenumber (cm-1)
Band

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UBC UB2 UB4 UB6 GC
Amide I 1635.64 1631.78 1635.64 1631.78 1629.83
Amide II 1535.34 1531.48 1535.34 1531.48 1532.36

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Amide III 1230.58 1230.58 1242.16 1234.44 1235.93
Amide A 3309.85 3294.42 3302.13 3294.42 3278.74
Amide B 2943.37 2931.80 2947.23 2924.09 2947.12

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Table 1

Yields, pH, turbidity, gel strength and viscosity of gelatin extracted using ultrasound from bovine
skin pretreated with enzyme bromelain. Values are presented as mean±SE from triplicate
determination.

Sample Yield (%) pH Turbidity Gel strength Viscosity

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of gelatin (ppm) (g) (mPa.s)
b b b b b
UBC

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18.67±0.11 2.46±0.01 22.45±0.10 603.24±3.01 16.33±0.09
d a a c c
UB2 7.09±0.20 2.40±0.01 30.35±0.28 475.26±3.26 15.90±0.06
c c c a a

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UB4 15.65±0.20 2.51±0.18 21.07±0.18 631.90±1.85 16.77±0.09
a b d b b
UB6 19.71±0.16 2.46±0.20 10.53±0.20 595.51±3.81 16.37±0.07
Means with different superscripts in the same column indicate significant difference at p<0.05.

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UB2, UB4 & UB6 refers to ultrasound assisted gelatin extracted for the time duration of 2, 4 and
6 h, respectively using bromelain enzyme pretreatment. UBC: control gelatin extracted using
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ultrasound without enzymatic pretreatment.
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Table 2

Colour of gelatin extracted using ultrasound from bovine skin pretreated with enzyme bromelain.
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Treatment L* a* b*
c a a
UBC 65.33±0.63 1.22±0.08 13.31±0.15
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a b c
UB2 73.40±0.77 0.29±0.17 9.02±0.36
b b b
UB4 68.71±0.39 0.42±0.03 10.65±0.07
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d a a
UB6 61.01±0.34 1.43±0.04 12.99±0.11
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Means with different superscripts in the same column indicate significant difference at p<0.05.
UB2, UB4 & UB6 refers to ultrasound assisted gelatin extracted for the time duration of 2, 4 and
6 h, respectively using bromelain enzyme pretreatment. UBC: control gelatin extracted using
ultrasound without enzymatic pretreatment.
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Highlights

Ultrasound assisted gelatin extracted from bovine skin using bromelain pretreatment
Significant increase in yield as duration of ultrasound treatment (UT) increased
Longer duration of UT increased amino acids content of the extracted gelatin

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Degradation of gelatin protein chains as duration of extraction increased
UT resulted in greater loss of molecular order in gelatins

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