Characterization of gelatin from bovine skin extracted using ultrasound subsequent to bromelain pretreatment

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. 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 proof before it is published in its final 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.

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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, *

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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 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 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 ICAR-Central Institute of Post-Harvest Engineering and Technology, Ludhiana, Punjab-141004, India g 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, Philippines

* Corresponding author: Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

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Tel.: +60389474870; Fax: +60389381024

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E-mail address: [email protected] (A. Q. Sazili)

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Characterization of gelatin from bovine skin extracted using ultrasound subsequent to bromelain pretreatment

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Abstract

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

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

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

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time duration of ultrasound treatment increased with UB6 giving the highest yield of 19.71%

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followed by UBC (18.67%). Gel strength and viscosity of UBC were 603.24 g and 16.33 mPa.s,

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respectively. The corresponding values for UB6 were 595.51 g and 16.37 mPa.s, respectively.

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The amino acids content increased with longer duration of ultrasonic treatment and UBC

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exhibited the highest content of the glycine (27.06%) and hydroxyproline (17.21%) compared to

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other samples. Protein pattern of the gelatin samples showed the progressive degradation of

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polypeptide chains as the time duration of ultrasound extraction increased. As demonstrated by

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Fourier transform infrared (FTIR) spectroscopy, amide I band of gelatins extracted by ultrasound

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treatment exhibited higher wavenumbers than the commercial gelatin (CG) suggesting greater

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loss of molecular order in these samples. Longer duration of ultrasonic treatment resulted in

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denser, irregular, disorganized and more interconnected structure with increased porosity as

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

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

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

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good quality gelatin.

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1. Introduction Gelatin is a high molecular weight biopolymer derived from collagen by thermal

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

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

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

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

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

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

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

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1985). Therefore, the recovered gelatin has lower molecular weight components than native

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collagen and comprises of a mixture of polypeptide fragments having molecular weight in the

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

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The molecular bonds present in the collagen are stable to thermal and acid treatment (Galea,

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Dalrymple, Kuypers, & Blakeley, 2000) resulting in a low gelatin yield (Nalinanon, Benjakul,

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Visessanguan, & Kishimura, 2008). Previously, to improve the gelatin extractability, some

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proteases capable of breaking the collagen cross-links have been used (Nalinanon et al., 2008).

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Pepsin and proctase (isolated from Aspergillus niger) were used to extract the gelatin from

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bovine skin but the gelatin yield, its gel strengths and viscosities were low (Chomarat, Robert,

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Seris, & Kern, 1994). Papain was used to extract gelatin from rawhide splits but the obtained

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

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

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

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relatively low and there was complete degradation of α- and β- chains in the recovered gelatin

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(Pitpreecha & Damrongsakkul, 2006). Although better gelatin yield was achieved with

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

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

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

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

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

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

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

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

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

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(Vilkhu, Mawson, Simons, & Bates, 2008). UAE can enhance extraction efficiency and

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extraction rate particularly for aqueous extraction and lower processing temperatures can be

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applied for enhanced extraction of heat sensitive bioactive and food components at lower

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processing temperatures (Vilkhu et al., 2008). Ultrasound has attracted the attention of food

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industry because of its promising in food science (Jia et al., 2010). High power ultrasound

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(power >1W cm-2 and frequencies 20 to 500 kHz) can be applied for facilitating the extraction

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process of a variety of food components (e.g. herbal, oil, protein, polysaccharides) as well as

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bioactive ingredients (e.g. antioxidants) from plant and animal resources (Vilkhu et al., 2008).

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With ultrasonic treatment, high collagen yield was obtained from bovine tendon in significantly

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

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

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Kim, Kim, Park, & Lee, 2012). Bighead carp scales treatment with ultrasound bath led to good

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quality gelatin with high yield (30.94-46.67%) and the presence of α- and β-chains was observed

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in the resulting gelatin (Tu et al., 2015). There is no published research work on the ultrasound assisted extraction of gelatin as well

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

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

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

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parameters of the recovered gelatin.

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2. Materials and methods 2.1 Chemicals

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

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(TEMED), coomassie brilliant blue R-250, 2-mercaptoethanol were purchased from Merck,

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Darmstadt, Germany. Other chemicals and reagents used were of analytical grade.

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Chromatographic column, mobile phase, reagents and amino acid standards were purchased from

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Waters Corporation, MA, USA and hydroxyproline standard supplement was procured from

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Agilent Technologies, CA, USA. Bromelain enzyme, EC 3.4.22.32 (≥ 2.0 mAnsonU/mg)

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extracted from pineapple (Ananas comosus) was obtained from Merck, Darmstadt, Germany.

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Commercial gelatin type B from bovine skin was purchased from Sigma, St. Louis, MO, USA.

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2.2 Preparation of skin

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

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ruminant abattoir located in Shah Alam, Selangor, Malaysia and transported in ice and stored at -

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20 °C. The subcutaneous fat was removed by scrapping. The skin was washed thoroughly and

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

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2.3

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

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2.3.1

Removal of non-collagenous proteins

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

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

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NaOH solution was changed every 2 h. Thereafter, the hairs on the skin were removed by

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scraping with scalpel and cut into 1 cm x 2 cm size. The skin was rinsed thoroughly with

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distilled water until neutral pH wash water was obtained.

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2.3.2

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Ultrasound assisted gelatin extraction in conjugation with enzyme bromelain

Skin was soaked in 1% HCl for 20 h with discontinuous stirring at the ratio of 1:10 (w/v) at

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room temperature for swelling. The samples were washed thoroughly with distilled water until

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neutral wash water was obtained. The experiment in the laboratory had shown better yield and

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quality gelatin could be obtained from bromelain enzyme at the level of 20 units of bromelain

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enzyme/g of skin at its optimum temperature and pH of the enzyme (35.5 °C and 6.0,

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

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

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of the enzyme (35.5 °C and 6.0, respectively) as indicated by the manufacturer.

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The swollen skin samples were kept in the optimum pH solution at skin to solution ratio of 1:

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

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

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

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

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

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

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

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

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swollen skin as mentioned above. The extraction was performed in triplicate.

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2.4.2 Yield

The yield of the gelatin was calculated on the wet weight basis of the skin as reported by Balti et al. (2011), Bougatef et al. (2012), Ktari et al. (2014) and Lassoued et al. (2014).

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2.4 Analyses of gelatin

Weight of the freeze dried gelatin (g)

Yield (%) =

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x 100

Wet weight of the skin (g)

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

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

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(redness/greenness) and b* (yellowness/blueness) were used (Jamilah & Harvinder, 2002). The

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sample was filled in a 64 mm glass sample cup with three readings in the same place and

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triplicate determinations were taken per sample.

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2.4.4 Determination of pH

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

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

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

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

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2.4.5 Determination of amino acid composition

To determine the amino acid (AA) content of the gelatin samples using high performance liquid chromatography (HPLC), slightly modified method of Awad, Zulkifli, Farjam, & Loh

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(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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110 °C for 24 h. Upon cooling, 4 ml of internal standard (α-aminobutyric acid; AABA) was

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added to the hydrolysate and aliquot was paper and syringe filtered. Ten microlitre of the filtered

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sample was mixed with 70 µl of borate buffer and 20 µl of ACCQ reagent (Waters Corporation,

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Milford, MA, USA). Internal standard was spiked with hydroxyproline and a mixture of amino

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acid standard H (Waters Corporation, MA, USA) was used to determine the concentration of all

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AA except methionine, cysteine and tryptophan. An AA column (AccQ Tag 3.9 150 mm; Waters

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

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

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

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2.4.6 Electrophoretic analysis The molecular weight distributions of the extracted gelatins were determined by sodium

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

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

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

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

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

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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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stained with 0.1% (w/v) coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid

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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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blue background were clear. Prestained protein ladder (BLUeye, GeneDireX, Taoyuan, Taiwan)

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was used to estimate the molecular weight distributions of the gelatins. The gel was scanned with

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a GS-800 Calibrated Densitometer (Model: PowerLook 2100XL- UB, Bio-Rad Laboratories,

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CA, USA) gel imaging system.

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2.4.7 Determination of turbidity

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

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

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Japan) was to measure the absorbance at 660 nm.

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

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2.4.8 Determination of gel strength

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

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

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

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

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

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

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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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The speed of the plunger was 0.5 mm/s. All determinations are means of three measurements.

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

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2.4.9 Determination of viscosity

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

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water and heated to 60 °C. RheolabQC (Anton Paar, Graz, Austria) viscometer was used to

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measure the viscosity of the samples. The measurement was performed in triplicate.

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2.4.9 Fourier transform infrared (FTIR) spectroscopy

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

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

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

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reflection crystal had a 45° angle of incidence to the IR beam. Resolution of 4 cm-1 was used to

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acquire the spectra and 4,000-500 cm-1 (mid-IR region) was chosen as measurement range at

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

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

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2.4.10 Microstructure analysis of gelatin

The microstructure of extracted gelatins was elucidated using scanning electron microscope

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

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

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SCD 005 sputter coater, Schalksmühle, Germany). An acceleration voltage of 10 kV was used to

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observe the specimen at 30x.

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All statistical analyses were carried out using GLM procedure of Statistical Analysis System

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package (SAS) Version 9.4 software (Statistical Analysis System, SAS Institute Inc., Cary, NC,

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USA) and statistical significance was set at p<0.05. Significant differences between means were

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evaluated by Duncan’s Multiple Range Test.

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3. Results and discussion

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3.1 Effect of ultrasound assisted gelatin extraction in conjugation with bromelain

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

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

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15.65 and 19.71%, respectively. The higher yield of gelatin with increasing time was due to

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cavitation and mechanical effect of ultrasound (Tu et al., 2015). Increased extraction yield

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

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

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

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

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

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

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

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

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

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consistent with the finding of Arnesen & Gildberg (2007) and Tu et al. (2015) who reported that

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longer extraction time from Atlantic salmon skin and bighead carp scales, respectively resulted

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in higher yield of gelatin. More energy was provided by increasing time to destroy the stabilizing

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bonds present in the collagen structures and peptide bonds of α-chains resulting in helix-to-coil

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transformation (Nagarajan, Benjakul, Prodpran, Songtipya, & Kishimura, 2012).

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3.2 Colour

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

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

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

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

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(a* and b* values, respectively) was observed for UB6 and UBC. Non-enzymatic browning

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reaction due to longer extraction time might be held responsible for the higher yellowness (b*

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

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3.3 pH

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

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

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

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

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(Park et al., 2013).

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3.4 Amino acid composition of gelatin

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Principally, amino acid composition and molecular weight distribution determines the

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properties of gelatin (Gomez-Guillen et al., 2009). The most abundant amino acid in gelatin is

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glycine (Arnesen & Gildberg, 2002). Triple peptides which consist of repeating chains of Gly-X-

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Y, where X is generally proline and Y is mainly hydroxyproline, make up to 50-60% of α-chains

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(Asghar & Henrickson, 1982). Gelatins extracted from warm-blooded animal tissues have been

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found to be rich in proline and hydroxyproline (imino acid) amino acids content, particularly

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hydroxyproline (Norland, 1990).

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

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

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and hydroxyproline (Hyp) content for UBC, UB2, UB4 and UB6 was 27.06, 12.44 and 17.21%,

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20.84, 11.91 and 15.99, 21.30, 12.08 and 16.74% and 25.88, 12.75 and 17.05%, respectively

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

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

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

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

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

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

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

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

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to increase in the amino acids content (Chandrapala, Zisu, Kentish, & Ashokkumar, 2012; Li et

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al., 2016).

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

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and 9.6% of glycine, proline and hydroxyproline content out of the total amino acids,

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respectively in food grade halal bovine gelatin. Our amino acid results are expressed in terms of

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percentage of sample (mg/100 mg of sample) which may be the reason for the difference.

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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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glycine, proline and hydroxyproline, respectively (Ward & Courts, 1977). Differences in

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manufacturing processes of gelatin might give rise to variations in the amino acid contents (Zhou

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& Regenstein, 2006).

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

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

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

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stability to the triple-stranded collagen helix by its ability to form hydrogen bond through its

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hydroxyl group (Burjandze, 1979; Ledward, 1986; Mizuno, Hayashi, & Bächinger, 2003). The

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

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

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

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

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

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

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strength and viscosity of the recovered gelatin.

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3.5

SDS-PAGE analysis of gelatin

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Functional properties of gelatin are affected by the amino acid composition, the molecular

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weights distribution, structure and compositions of its subunits (Balti el at., 2011). SDS-PAGE

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analysis was used to elaborate the molecular weight pattern of pretreated skin samples (PS),

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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-

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

326

treatment time duration. α1- and low intensity α2-chains were observed in UB4 sample whereas

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only faint presence of α1- and α2-chains along with lower weight polypeptides were observed in

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

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

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protein fraction (Gülseren, Güzey, Bruce, & Weiss, 2007; Hu et al., 2013; Krise, 2011; Karki et

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al., 2010; O'Sullivan, Arellano, Pichot, & Norton, 2014; Yanjun et al., 2014;), the ultrasound

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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

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

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

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

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

338

(O'Sullivan et al., 2014).

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3.6 Turbidity

Except for UBC, turbidity decreased as the ultrasound treatment time increased (Table 1).

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The significantly (P<0.05) higher turbidity of UB2 showed its lower quality than other samples

343

(Montero, Fernandez-Diaz, & Gomez-Guillen, 2002). UB4 and UB6 had significantly (P<0.05)

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

346

of high intensity ultrasound treatment. This might be due to size reduction of the suspended

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insoluble aggregates by ultrasound (Jambrak, Mason, Lelas, Paniwnyk, & Herceg, 2014).

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Another possible explanation for the decrease in turbidity after ultrasonication was attributed to

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disruption in the protein-protein interactions resulting in small aggregates formation leading to

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

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3.7 Gel strength

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

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

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

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

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

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

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

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

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

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especially hydroxyproline through its ability to form hydrogen bonding by –OH group (Montero,

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Fernández-Dı́az, & Gómez-Guillén, 2002).

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The gel strength values of all the gelatins extracted using ultrasound samples were high

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(Table 1). UBC, UB2, UB4 and UB6 demonstrated the gel strength value of 603.24, 475.26,

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631.90 and 595.51 g, respectively. There was non significant (P>0.05) difference between UBC

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and UB6. The UB2 sample revealed the presence of α1- and α2- chains along with lower

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molecular weight polypeptides. The protein chains experienced progressive degradation with

369

increase in time duration of ultrasound treatment leading to faint presence of α1- and α2- chains

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in UB6 and UBC samples.

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

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

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

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

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al., 2011). Further, the gel strength is also dependent on the polypeptide chains configuration and

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the inter-junction zones formed during the maturation process (Ahmad & Benjakul, 2011).

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

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between gelatin gel strength and molecular weight distribution in high strength gelatin. Apart from all these factors, amino acid composition and the type of extraction treatments

380

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

381

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

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

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

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

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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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to other samples. The high gel strength of UB4 than other samples could be explained in term of

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

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of imino acid. Apart from the similar imino acid and hydroxyproline content, UBC and UB6

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demonstrated similar molecular distribution pattern which could be the deciding factors for

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exhibiting the almost same gel strength for these two gelatin samples.

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3.8 Viscosity

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The second most important commercial physical property of gelatin is viscosity (Wards &

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Courts, 1977). Destruction of hydrogen and electrostatic bonds present in collagen causes

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

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

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water (Badii & Howell, 2006).

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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

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

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

403

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

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

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

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

407

viscosities obtained for these samples might be due to particle size denatured collagen recovered

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during ultrasonic extraction attributed to cavitation effect which caused impingement by micro-

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

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3.9 FTIR spectra

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Generally, FTIR spectroscopy is used to study the functional groups and secondary structure

413

of gelatin samples and the most important for infrared spectroscopic analysis of the secondary

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structure of proteins is the amide I band (Kittiphattanabawon, Benjakul, Visessanguan, &

415

Shahidi, 2010). C=O stretching vibration hydrogen bonding coupled to contributions from the

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

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

425

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

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

428

wavenumber along with higher amplitude indicated greater loss of molecular order due to

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

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

433

crosslink resulting in more loss of molecular order in UB4 gelatins compared to other ultrasound

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

436

pretreatment than the UB4 caused the protein-protein linkages being formed in UB6 due to

437

unfolding of functional groups (Jiang et al., 2014). The shorter but uniform length of protein

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

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

447

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

448

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

449

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

450

implied higher involvement of NH group in hydrogen bond formation with neighboring

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

453

amplitude than CG suggesting greater participation of NH group in H-bonding.

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C-N stretching and N-H deformation in plane bending occurring due to amide linkages and

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-

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,

459

1,234.44 and 1,235.93 cm−1, respectively. Amide III band around 1,233-1,234 cm−1 indicated

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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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

469

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

470

1995).

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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

473

wavenumbers, generally near the 3,300 cm−1, when the N-H group of a peptide is involved in a

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,

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

478

with high amplitude of amide A suggested gelatin degradation (Nagarajan et al., 2012). UB2 and

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.

481

Low amide A wavenumbers of bromelain pretreated samples compared to UBC could be

482

attributed to high hydrogen bonding involvement of a N-H group in α chain.

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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.

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3.10 Microstructure of gelatin

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

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

other samples. Gelatin structure changed to denser, smaller particle sized, irregular, disorganized

497

and more interconnected structure with increased porosity as the duration of ultrasound treatment

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

500

functional groups resulting in protein aggregation and network formation (Jiang et al., 2014).

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

503

microstructure.

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4. Conclusions

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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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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

514

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

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.

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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Acknowledgements

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Indian Council of Agricultural Research, New Delhi, India and Department of Agricultural

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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-

526

EQR/Edn (pt.III) dated October 28, 2014 and granting study leave to him (letter number F. No.

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

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

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January 5, 2016).

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Figure captions:

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Fig. 1. SDS-PAGE pattern of pretreated skin (PS) sample along with gelatin extracted using ultrasound for the time duration of 2, 4 and 6 h (UB2, UB4 and UB6, respectively) from bovine skin with bromelain enzyme pretreatment. UBC: control gelatin extracted using ultrasound without enzyme pretreatment. CG: commercial gelatin. M denotes the marker.

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Fig. 2. FTIR spectra 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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760 761 762

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Fig. 3. SEM images 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.

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Table 3

Hydroxyproline (Hyp)

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

Aspartic acid (Asp) Serine (Ser) Glutamic acid (Glu)

4.44a 3.34a 8.22a

Glycine (Gly) Histidine (His)

27.06a 20.84b 21.30b 25.88a 0.89a 0.90a 0.89a 0.86a

4.34a 3.36a 8.16a

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4.46a 3.32a 7.92a

10.28a 8.42b 9.01ab 9.11ab 1.90a 1.89a 2.01a 1.98a 8.00a 8.70a 8.75a 8.50a 12.44a 11.91a 12.08a 12.75a 0.73a 0.65a 0.74a 0.72a 2.30a 2.39a 2.42a 2.37a 3.91a 3.61a 3.66a 3.59a 1.47a 1.46a 1.51a 1.51a 3.14a 3.06a 3.17a 3.13a

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Arginine (Arg) Threonine (Thr) Alanine (Ala) Proline (Pro) Tyrosine (Tyr) Valine (Val) Lysine (Lys) Isoleucine (Ile) Leucine (Leu) Phenylalanine (Phe)

4.86a 3.27a 8.53a

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Amino Acids

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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.

2.28a

2.14a

2.27a

2.22a

EP

Imino acids (Pro+Hyp) 29.65a 27.90a 28.82a 29.80a

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Table 4

UBC 1635.64 1535.34 1230.58 3309.85 2943.37

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Amide I Amide II Amide III Amide A Amide B

Peak wavenumber (cm-1) UB2 UB4 UB6 1631.78 1635.64 1631.78 1531.48 1535.34 1531.48 1230.58 1242.16 1234.44 3294.42 3302.13 3294.42 2931.80 2947.23 2924.09

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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.

GC 1629.83 1532.36 1235.93 3278.74 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.

UBC

18.67±0.11

UB2

7.09±0.20

UB4

15.65±0.20

pH

b

b

2.46±0.01

d

b

a

Viscosity (mPa.s)

b

b

a

c

c

30.35±0.28 475.26±3.26 15.90±0.06

c

2.51±0.18

a

Gel strength (g)

22.45±0.10 603.24±3.01 16.33±0.09

2.40±0.01 c

UB6

Turbidity (ppm)

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Yield (%) of gelatin

c

a

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Sample

a

21.07±0.18 631.90±1.85 16.77±0.09

b

d

b

b

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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. 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.

Table 2

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Colour of gelatin extracted using ultrasound from bovine skin pretreated with enzyme bromelain. Values are presented as mean±SE from triplicate determination. Treatment

L*

a*

b*

c

a

65.33±0.63 1.22±0.08

UB2

73.40±0.77 0.29±0.17

UB4

68.71±0.39 0.42±0.03

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UBC

UB6

a b

a

13.31±0.15

b

c

9.02±0.36 b

d

61.01±0.34 1.43±0.04

b

10.65±0.07 a

a

12.99±0.11

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

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Longer duration of UT increased amino acids content of the extracted gelatin Degradation of gelatin protein chains as duration of extraction increased

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UT resulted in greater loss of molecular order in gelatins