Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

Isolation and Characterization of Pepsin-Soluble Collagen from the Skin of Peru Squid (Dosidicus gigas)

Pepsin-soluble collagen (PSC) was isolated from Peru squid (Dosidicus gigas) skin and physicochemical properties of the PSC were determined. The PSC exhibited a maximum absorbance at 220 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested the collagen containing α1 and α2 chain was classified as type I collagen. Amino acid composition indicated that the collagen had lower amino acid content than that of mammalian collagen. Denaturation temperature (T_d) of the PSC was 26.8°C. The PSC had relatively high solubility in alkaline condition or NaCl concentrations below 2%. Fourier transform infrared spectroscopy investigations showed the existence of helical arrangements of collagen. The lyophilized collagen had a uniform and regular network structure. These results suggested that Peru squid skin was a potential source of collagen for further research and application.     

Extraction and Characterization of Pepsin Soluble Collagen from the Body Wall of Sea Cucumber Acaudina leucoprocta

Sea cucumber Acaudina leucoprocta is a potential alternative collagen source. However, the high levels of heavy metals contained in the body wall restricts its utilization. In this work, an efficient method was established to remove the heavy metals accumulated in the body wall of A.leucoprocta by demineralizing with 0.2 M ethylenediaminetetraacetic acid (EDTA). The resulting body wall of A.leucoprocta was then used for extracting pepsin-soluble collagen (PSC) with pepsin proteolysis. The PSC from the body wall of A.leucoprocta was obtained with a yield of 43.99 ± 0.65% (dry weight) and high purity. Maximum and minimum solubility for the isolated PSC in 0.5 M acetic acid was observed at pH 2.66 and 4.43, respectively. The solubility was remarkably decreased in the presence of NaCl. The denaturation temperature of PSC rehydrated in 0.5 M acetic acid was measured as 25.4°C. The PSC was characterized as type I collagen, which consists of three α₁ chains without α₂ chain. Interestingly, α₁ chain in PSC showed two isoforms with the pI values of 4.02 and 4.29. The heavy metals existing in PSC were all below the contaminant limit of edible gelatin. The PSC isolated from the body wall could be an alternative to mammalian collagens.     

Thermal and Chemical Properties of Gelatin from Tilapia (Oreochromis niloticus) Scale

ABSTRACT

Gelatin from the scale of Nile tilapia was extracted and characterized chemically, thermally, and structurally. Scales of Nile tilapia were subjected to acid pretreatment and extraction with water at 60°C. The process yielded 12.1% (m/m) on a dry basis. The 6.67% gelatin solution had a gel strength of 233.5 ± 14g. The amino acid composition revealed that 20.02% was comprised of imino acids and glycine (34.49%) as the most abundant amino acids. The scale gelatin had α1, α2, and β subunits and was thermally stable up to 200ºC. The Fourier transform infrared spectrum showed absorption in the regions of amides A, I, II, and III. The characterization of tilapia scale gelatin showed that the obtained product was similar to commercial gelatin, thereby demonstrating its potential application.     

Extraction and Physicochemical Characterization of Pepsin Soluble Collagens from Golden Pompano (Trachinotus blochii) Skin and Bone

Pepsin-soluble collagen (PSC) was extracted and purified from wasted skin and bone of the golden pompano by acetic acid-pepsin method. The result showed that the PSCs extraction yields of skin and bone were 21.81% and 1.25% (wet weight), 62.21% and 1.78% (on the basis of lyophilized dry weight), respectively. Golden pompano skin and bone PSCs contained the typical chain of α and β dimers, and they were preliminarily judged to belong to type I collagen. The skin PSC had similar amino acid composition to bone PSC, which is rich in glycine, alanine, proline, and hydroxyproline. After addressing the pepsin, three helical structure of PSCs were intact, and their natural structures largely remained. The denaturation temperatures of skin and bone PSCs were 37.04°C and 38.23°C, respectively. Solubility results showed that the skin and bone PSCs solubility was the largest at pH = 3. The solubility of skin and bone PSCs was stable at NaCl concentrations lower than 3%. In addition, two PSCs in acid and low salt conditions had good solubility. This study demonstrated that golden pompano skin and bone could be used as good materials to extract PSC, representing an economic benefit and added value.

Abbreviations: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; FTIR: Fourier transform infrared; DSC: Differential scanning calorimetry; XRD: X-ray diffraction     

Characteristics of Collagen from Rohu (Labeo rohita) Skin

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196–202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (α1)₂α2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T_max value of ASC and PSC was 36.40 and 35.48°C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.     

Characterization of Acid-Soluble Collagen From Bone of Pacific Cod (Gadus macrocephalus)

Acid-soluble collagen (ASC) was isolated from Pacific cod (Gadus macrocephalus) bone. The ASC was rich in glycine. The amount of imino acid was lower than that of calf skin collagen, as was the transition temperature (48.6°C). Electrophoresis revealed two different α chains (α₁ and α₂), β-component, and γ-component. Fourier transform infrared spectroscopy measurement showed that ASC was in triple-helix structure. ASC had a solubility greater than 90% in a very acidic pH range (pH 1–4), and the solubility decreased with increasing NaCl concentrations up to 3%. Lyophilized ASC had a network ultrastructure with lace-like fibers, similar to calf skin collagen sponge.     

Characteristics and Physicochemical Properties of Gelatin Extracted from Scales of Seabass (Lates calcarifer) and Grey Mullet (Mugil cephalus) in Vietnam

In this study, gelatin was obtained from the scales of seabass (Lates calcarifer) and grey mullet (Mugil cephalus) collected in Vietnam by using Alcalase® 2.4 L FG for pretreatment and electrochemically activated water with pH 2.0–2.5 for extraction. The characteristics and physicochemical properties of gelatin were investigated. The results indicated that the gelatin had a high protein content (88.6–90.0%) with ash (1.43–1.55%) and no fat. The gelatin was identified as type A due to its pH value. The yields of gelatin were 14.1–15.2%. The gel strength and melting point were 270.3 g and 26.7°C, respectively, for gelatin from seabass scales and 249.1 g and 25.9°C, respectively, for samples from grey mullet scales. In contrast, the viscosities of gelatin were found to be 6.97 cP for seabass and 8.73 cP for grey mullet. Both gelatins contained α-chain and β-chain as the major components. Circular dichroism spectra indicated that the triple helical structure in collagen was almost transformed to the random coil structure in gelatin. The proportion of imino acids was high (197–205 residues/1,000 residues). So, gelatin from seabass and grey mullet scales could be used as a potential replacement for mammalian gelatin.     

Effects of Dietary β-Hydroxy-β-Methylbutyrate on Growth and Survival of Nile Tilapia,Oreochromis niloticus,Vaccinated Against Streptococcus iniae

Abstract

β-hydroxy-b-methylbutyrate (HMB), a leucine catabolite, has been shown to cause increased disease resistance and growth in animal production. A vaccine produced from formalin killed bacteria and concentrated extracellular products of the ARS-98-60 Streptococcus iniae isolate has been used for the prevention of streptococcal disease in Nile tilapia, Oreochromis niloticus. In the present study, the effects of feeding HMB were determined in tilapia vaccinated by intraperitoneal (IP) injection of the S. iniaevaccine or unvaccinated (controls). Nile tilapia were fed diets containing either 0, 12.5, 25, or 50 mg HMB/kg diet for 14 days. The mean daily growth rate and feed efficiency showed no significant (P> 0.05) differences between the treatment groups. Dietary HMB supplementation did not enhance antibody production in unvacci-nated Nile tilapia following challenge. Dietary HMB supplementation did not enhance the survival of vaccinated Nile tilapia following challenge injection with 1 X10⁸ CFU of S. iniae.     

3种品系尼罗罗非鱼生长及高密度胁迫后生理响应变化的比较

以‘吉富’、‘新吉富’、‘埃及尼罗’3种不同品系尼罗罗非鱼(Oreochromis niloticus)为研究对象, 通过不同品系之间生长与高密度胁迫反应的比较, 探讨3种尼罗罗非鱼的生长特点与应激后生理响应的变化规律。将初始规格基本一致的3种品系尼罗罗非鱼饲养100 d后, 吉富罗非鱼特定生长率最高, 新吉富其次, 两者间无显著差异(P>0.05), 埃及尼罗罗非鱼特定生长率显著低于吉富罗非鱼(P<0.05)。吉富与新吉富罗非鱼的内脏比显著高于埃及尼罗(P<0.05); 3种尼罗罗非鱼的肥满度之间无显著差异(P>0.05)。养殖实验结束后, 进行48 h的急性高密度(100 g/L)应激实验。应激48 h内, 吉富与新吉富罗非鱼血清总蛋白、葡萄糖、谷草转氨酶、胆固醇与溶菌酶活力/水平以及肝HSP70mRNA水平呈先上升后下降的变化。应激48 h后, 埃及尼罗罗非鱼血清皮质醇水平与应激前相比无显著差异(P>0.05), 吉富与新吉富罗非鱼的皮质醇水平显著高于应激前(P<0.05); 埃及尼罗罗非鱼血清溶菌酶活力与肝HSP70 mRNA水平在应激后48 h内始终高于应激前(P<0.05)。研究结果表明, 短期高密度胁迫可提高3种尼罗罗非鱼血清葡萄糖与甘油三酯的利用, 并诱发肝损伤。埃及尼罗罗非鱼的抗高密度应激能力高于吉富和新吉富罗非鱼。

     

尼罗罗非鱼肌肉蛋白质双向电泳体系的建立

为建立1种尼罗罗非鱼肌肉组织蛋白质的双向电泳体系,实验将尼罗罗非鱼肌肉组织蛋白质提取后,用双向电泳(2-DE)分离蛋白质,分别对蛋白质样品的制备方法、IPG胶条长度及p H范围、SDS-PAGE凝胶浓度及染色方法4个关键因素进行了探索和优化。结果显示,采用液氮研磨-丙酮沉淀法制备样品蛋白质,使用24 cm p H 4~7的IPG胶条进行第一向等电聚焦电泳,第二向SDS-PAGE电泳采用浓度为12.5%的凝胶进行,双向电泳后的凝胶采用硝酸银染色法进行染色处理,该条件下扫描所得尼罗罗非鱼肌肉蛋白质二维电泳图谱具有蛋白质分离程度好、斑点清晰、分辨率高及横纹少等优点。研究表明,技术体系适用于尼罗罗非鱼肌肉蛋白质的分离,可用于后续罗非鱼肌肉蛋白质组学研究。     

Physicochemical Properties and the Radical Scavenging Capacities of Pepsin-Solubilized Collagen from the Body Wall of Starfish (Asterina pectinifera)

Pepsin-soluble collagen was successfully prepared from the body wall of starfish (Asterina pectinifera) (PSCBWS). Amino acid composition suggested that the collagen might be classified as type I collagen. Ultraviolet-visible and Fourier transform infrared spectroscopic analyses showed that the PSCBWS was a high-purity collagen that maintained the intact triple-stranded helices. Physical and chemical characterizations of the PSCBWS showed an isoelectric point (pI) of 5.0 ± 0.2, superior moisture-absorption and retention capacities compared with glycerol, a minimum solubility at pH 5.0 in 0.5 M acetic acid, and a sharp decrease in solubility in the presence of low concentration of NaCl. The PSCBWS was evaluated for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals. Results indicated that PSCBWS possessed DPPH and hydroxyl radicals scavenging capacity in a dose-dependent manner. These results indicated that PSCBWS might be used as a new collagen resource in the food and cosmetic industries.     

Characteristics and Select Functional Properties of Collagen from Golden Pompano (Trachinotus ovatus) Skins

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) from golden pompano skins were extracted and characterized. The molecular weight of ASC was about 130 kDa for α₁ and 115 kDa for α₂, which were slightly higher than those of PSC. Similar amino acid composition and Fourier transform infrared (FTIR) spectra were observed in both collagens, but slight differences were found in the peptide maps of collagen digested by V8 protease and trypsin. The denaturation temperatures (Tds) of ASC and PSC calculated from the reduced viscosity were 31.8 and 30.0°C, while the transition temperature (T_m) of ASC and PSC analyzed by DSC were 33.0 and 32.0°C, respectively. ASC has a lag phase, a growth phase, and a plateau phase in the turbidity–time curve, while PSC does not have similar phenomenon. It was found that the fibril gel of ASC could be formed at 25°C, leading to improved thermal stability.     

Comparative time-course study on pituitary and branchial response to salinity challenge in Mozambique tilapia (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) and Nile tilapia (<Emphasis Type="Italic">O</Emphasis>. <Emphasis Type="Italic">niloticus</Emphasis>)

The physiological response of Mozambique and Nile tilapia transferred from fresh to brackish (15 ppt) water was compared during a one-week time course. Response in the pituitary was measured by the gene expression pattern of prolactin (PRL I), growth hormone (GH), and calcium-sensing receptor (CaSR), while the response in the gills was measured by the gene expression pattern of the prolactin receptor (PRL-R), Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) and Na⁺/Cl⁻ cotransporter (NCC), and by activity and expression of Na⁺/K⁺-ATPase (NKA). The time-course curves of plasma osmolality levels indicate a rapid elevation 24 h after transfer, which later decreased and maintained at stable level. PRL I expression decreased in both species, but with stronger response in the Nile tilapia, while no differences were found in the slightly elevated levels of GH mRNA. The branchial response demonstrated a faster up-regulation of NKA and NKCC in the Mozambique tilapia, but similar levels after a week, while Nile tilapia had stronger and constant down-regulation of NCC. The time-course response of the measured osmoregulatory parameters indicate that 24 h after transfer is a critical time point for brackish-water adaptation. The differences in responses to saltwater challenge between Mozambique and Nile tilapia shown in this study may be associated with the differences in saltwater tolerance between these two tilapiine species.     

Comparison of Stunning Methods on the Physicochemical Properties of Frozen Nile Tilapia (Oreochromis niloticus) Fillets

This study compares how CO₂ narcosis and thermal shock affect the physicochemical aspects of Nile tilapia fillets held frozen (?18°C) for up to 6 months. The characteristics of tilapia fillets subjected to CO₂ narcosis were those of higher lightness and whiteness and lower redness. The hardness values of fillets of tilapia subjected to thermal shock were lower than those subjected to CO₂ narcosis. During frozen storage to both treatments, the drip loss, denaturation of myosin, and lipid oxidation increased, while fillet water holding capacity and hardness decreased. The physicochemical properties of frozen fillets of tilapia stunned by CO₂ narcosis were considered more desirable; therefore, the method can be a potential replacement to thermal shock.     

Ultraviolet-induced androgenesis in Nile tilapia, Oreochromis niloticus (L.), and hybrid Nile × blue tilapia, O. aureus (Steindachner)

Fertilization of ultraviolet (UV) irradiated oocytes of Nile tilapia, Oreochromis niloticus (L.), with sperm from O, niloticus or blue tilapia, O, aureus (Steindachner), and subsequent suppression of the first cleavage of fertilized eggs successfully induced androgenesis in Nile and blue tilapia. The optimal doses of UV irradiation to denucleate a female genome of Nile tilapia prior to androgenesis ranged from 5940 to 6930 erg mm^?2 for 54-63 s at a fixed intensity of 110 erg mm^?2 s^?1. Putative androgenetic fish were created from eggs which were irradiated at various times and several durations of heat-shock. Eggs which were treated for 5 min at 41.6 ^oC at 2 7.5 min after fertilization were the most successful at suppressing the first cleavage and producing viable androgenetic diploids in Nile or hybrid Nile X blue tilapia. The maximal survivals of putative androgenetic diploids in relation to the control were 1.60% and 0.90% in Nile and hybrid Nile X blue tilapia, respectively. The androgenetic offspring established exhibited active feeding and normal growth.     

Effect of Alpha-Linolenic Acid Sources in Diets for Nile Tilapia on Fatty Acid Composition of Fish Fillet Using Principal Component Analysis

This study evaluated the effect of feed supplementation with chia and canola oils as a substitute for soybean oil on the composition of Nile tilapia muscle tissue using chemometrics. Diets were supplemented with 2.1% of each oil and were provided to fish for 15 and 30 days. Compared to soybean oil, supplementation with canola and chia oils significantly increased (P < 0.05) the contents of eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3) in Nile tilapia fillet. At 30 days, DHA content increased 97% in Nile tilapia fed chia oil and 91% in treatment with canola oil. The highest EPA content correlated to treatment with chia oil (7.33 mg 100 g^?1). The long-chain polyunsaturated fatty acids (LC-PUFAs) precursors, linoleic acid and α-linolenic acid, were observed to increase according to treatment type and feed supplementation duration. The principal component analysis resulted in a two-principal-component model that described 92.07% of the total data variance. Also, it highlighted that the replacement of soybean oil with canola and chia oils in Nile tilapia diets contributed to increasing the n-3 LC-PUFA concentration in Nile tilapia fillets, improving its nutritional value.     

Growth performance,digestibility, and blood biochemical parameters of Nile tilapia (Oreochromis niloticus) reared in floating cages and fed poultry by-product meal

ABSTRACT

A 120-day feeding trial was conducted in floating cages to evaluate growth performance and digestibility of Nile tilapia (Oreochromis niloticus) juveniles fed diets containing different levels of poultry by-product meal (PBM) as a replacement for fishmeal. Nile Tilapia with an average body weight of 56.6 ± 0.28 g were used in this experiment. Five diets were formulated to contain graded levels of PBM at 0%, 5%, 10%, 20%, and 30% poultry by-product meal (PBM) and are referred as PBM0, PBM5, PBM10, PBM20, and PBM30, respectively. Groups of 25 Nile tilapia were fed close to the apparent satiation, three times a day. The results showed that growth performance, survival and whole-body composition for fish fed the experimental diets were not significantly different (P > .05) than fish fed the control diet. Apparent digestibility of dry matter, protein, lipid and amino acids of fish fed the PBM20 and PBM30 were lower compared to the other treatments (P < .05). The PBM dietary levels significantly lowered total protein, high-density-lipoprotein (HDL) and cholesterol concentration. The results of this study indicated that good quality PBM can be used up to 30% in commercial diets for Nile tilapia without any adverse effect.     

Purification and immunochemical detection of a quantitatively major collagen in the dermis of sea cucumber Apostichopus japonicus

Pepsin-solubilized collagen (PSC) was prepared from the dermis of sea cucumber Apostichopus japonicus (green type) by performing pepsin digestion to collagen fiber pretreated with disaggregating solution (0.1 M Tris–HCl, pH 8.0, containing 0.5 M NaCl, 0.05 M ethylenediaminetetraacetic acid (EDTA), and 0.2 M 2-mercaptoethanol) and 0.1 M NaOH. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the PSC clearly showed two alpha bands under phosphate buffer system in the presence of 3.5 M urea. An antiserum was raised against chromatographically purified major molecular species in the PSC, and immunoblot analyses were performed for the soluble fractions at 4 M guanidine hydrochloride treatment and disaggregation as well as the collagen fiber before and after treatment. These fractions and collagen fibers showed quite similar band patterns to that of the PSC, showing mainly two alpha bands. These combined results suggest that the major molecular species of collagen contains at least two distinct alpha components and that the effect of pepsin digestion is relatively small on the structure of this collagen type.

     

Gustatory sensitivity of the external taste buds of Oreochromis niloticus L. to amino acids

The gustatory sensitivity of the Nile tilapia, Oreochromis niloticus L., to different amino acids was studied using an electrophysiological approach. The electrical responses were recorded from a branch of the facial nerve innervating the external taste buds of the upper lip. The relative stimulatory effectiveness (RSE) of nine amino acids and betaine were determined at a concentration of 1 mm and all of them elicited neural responses. This species responded well to the neutral, basic and acidic amino acids. The most stimulatory amino acids were L‐histidine, L‐arginine, L‐serine, L‐methionine and L‐glutamine; L‐proline and betaine were the least stimulatory. The results of this study suggest that the Nile tilapia has high external gustatory sensitivity to some amino acids as a physiological adaptation to search effectively for their sources. The effect of the pH, ranging from 4.0 to 9.0, on the RSE of three neutral amino acids and artificial pond water (APW) was also studied. The RSE increased below pH 6.0 and was relatively unaffected from 7.0 to 9.0, indicating that acidified stimulants are highly stimulatory in this species. Nile tilapia did not discriminate the pH of APW as effectively as some of the species studied earlier.