Collagen and Gelatin from Golden Carp Skin and Squalene fromFish Liver Extracted with the Aid of Ultrasound:Properties and Applications

Abstract

Acid solubilized collagen (ASC) and pepsin solubilized collagen (PSC) were isolated from the skin and scales of golden carp (Probarbus jullieni). Yield of ASC and PSC from skin was 21.16 and 39.05%, respectively, while the scales had a yield of 0.42 and 1.16%, respectively (based on the dry weight basis). Both ASC and PSC from skin and scales were composed of a and a chains and were characterized as type I collagen. Imino acid contents of ASC and PSC from skin were 197 and 199 residues/1000 residues, respectively, scales had 197 and 202 residues per 1000 residues, respectively. Fourier transform infrared (FTIR) and Circular dichroism (CD) spectra of both the samples were almost similar between ASC and PSC. Specifically, ASC and PSC from skin showed Tmax of 36.28 °C and 37.87 °C, respectively, which were higher than those of most temperate and cold-water fish collagens. The maximum solubility for both collagens was found at acidic pH (1-3). When ultrasonication was implemented as the extraction aid, yield of ASC and PSC increased as ultrasonication (20 kHz) time and amplitude increased (p<0.05). ASC and PSC with ultrasound aided process, named as UASC and UPSC, had the yields of 81.53 and 94.88%, while ASC and PSC prepared with typical method showed the yields of 51.90 and 79.27%, respectively. Amino acid compositions of all the collagens were similar. Overall, ultrasound treatment under appropriate condition effectively improved the extraction efficiency of ASC and PSC without affecting their physiochemical properties. Gelatins from golden carp skin pretreated using different acids (acetic acid and sulfuric acid + acetic acid), with and without prior-ultrasonication, extracted at 55 °C for 3 and 6 h, were characterized. Prior-ultrasonication (20 kHz) with amplitude of 80% and time 30 min increased the yield of gelatins by 110.9% and 174.8%, respectively compared with the corresponding controls (without prior-ultrasonication), when extracted for 6 h. Nevertheless, gelatin extracted from skin with prior- ultrasonication showed slightly higher content of imino acids, regardless of acid pretreatments with coincidentally higher gelling and melting temperature along with finer gel network than those produced by conventional method. Thus, appropriate acid pretreatment in combination with prior-ultrasonication effectively improved the extraction efficiency and gelling properties of gelatin from golden carp skin. Squalene from livers of four fish species including, L. calcarifer, K. pelamis, C. sorrah and C. griseum were extracted using ultrasound-assisted direct in- situ saponification (U-DS) process, in comparison with the conventional process. Based on Box-Behnken experimental design (BBD), optimal condition involved mass/methanol ratio (1:6, w/v), 50% KOH volume (6.5 ml) and sonication time (15 min). Higher yield was achieved with U-DS from 0.13±0.03 to 6.86±0.05 g (100 g)1 depending on the fish species. After extraction, squalene was further concentrated up to > 60% of purity from all the fish species via fractional crystallization (yield of squalene concentrate ranged from 48.35-74.49%) and purified using a silica gel column with a maximum recovery up to 98%. Entire extraction processes yielded squalene with a purity of ≥ 94%. Fourier transform infrared (FTIR) analysis confirmed the native structure of squalene with six nonconjugated bonds, suggesting no degradation of squalene taken place during U-DS process. Golden carp skin gelatin films incorporated with squalene rich fraction from shark liver (SRF) at various levels (5-25%, w/w) were characterized in comparison with palm oil (PO) incorporated film and control film (CON) (without oil addition). SRF films added with 25% SRF showed higher EAB as well as TS, compared to both CON and PO films (p < 0.05). SRF films showed significantly lower water vapor permeability (WVP) and lower lightness (L*) value than CON and PO films (p< 0.05). SEM images showed that CON and SRF films exhibited relatively smoother surface, compared to PO film. Both SRF and PO films demonstrated lower degradation (Ta), glass transition (Tg) and melting transition (Tmax) temperatures than CON film. SRF could render stronger films and enhanced flexibility with improved moisture barrier property of gelatin films. When gelatin films prepared by using glycerol (GLY) and squalene (SQ) at different ratios (10:0, 7:3, 5:5, 3:7 and 0:10; w/w) as plasticizer were characterized, incorporation of SQ reduced the moisture content of the gelatin film (p < 0.05). Films plasticized with GLY/SQ at a ratio of 5:5 (w/w) had highest tensile strength (TS), which was 61.7% higher than that using GLY as plasticizer (GLY). Nevertheless, continuous decrease in EAB were attained with increasing SQ ratios (p < 0.05). SQ films had a decrease in WVP and oxygen permeability (OP), compared to GLY film (p < 0.05). Based on FTIR and DSC spectra, SQ decreased gelatin-gelatin interactions associated with disordered structure. In addition, SQ film demonstrated lower Tmax, and enthalpy (AH), but a slightly increased Tg. Thermal degradation behavior of films showed that GLY film possessed a greater number of gelatin-gelatin interaction than that incorporated with SQ. Thus, SQ could replace GLY up to 50% to render stronger films with improved WVP as well as OP of gelatin films. The influence of bags from fish skin gelatin film prepared by substituting glycerol (GLY) with squalene (SQ) at 50% (GLY/SQ) was evaluated on the quality characteristics of fried fish crackers (FFC) stored for 30 days at 28 +0.5 °C and 70 ± 5% RH, in comparison with bags from control (CON) gelatin film and nylon/LLDPE film. After 30 days, GLY/SQ and nylon/LLDPE stored FFC had lower PV and TBARS than others. In addition, GLY/SQ and nylon/LLDPE stored FFC had less volatile compounds, especially those formed from autoxidation and non-enzymatic browning reaction. GLY/SQ bag lowered the oxidation and loss of polyunsaturated fatty acids, mainly n-3 and n-6 when compared to other bags. Nevertheless, FFC stored in nylon/LLDPE and GLY/SQ bags exhibited negligible changes in color and texture. GLY/SQ bag had an excellent oxygen barrier property and could lower water vapor permeability (WVP), thus lowering migration of reactants and oxidizing radicals. The prepared GLY/SQ bag could therefore improve oxidative stability of FFC. Therefore, value-added products from fish processing by-products could be potentially recovered with the aid of ultrasound and further uses of those products can be maximized via appropriate optimization.