双螺杆挤压对低值海参多肽提取率及抗氧化活性的影响

海地瓜(Acaudina molpadioides)属低值海参产品,为了提高其使用价值,本研究以海地瓜为原料,利用双螺杆挤压辅助不同浓度亚硫酸钠(Na2SO3)处理,并对挤出物多肽提取率、游离巯基含量和蛋白质溶解度进行测定;利用DA201-C型大孔吸附树脂对挤出物多肽进行分离纯化,并对多肽纯化组分的分子量分布和抗氧化能力进行测定。结果显示,双螺杆挤压辅助3.0% Na2SO3处理组多肽提取率、游离巯基含量和蛋白质溶解度分别为(57.12±0.62)%、(110.32±0.07)%和(28.72±0.13)%,较空白对照组(control check, CK)分别显著提升(7.66±0.35)%、(105.32±0.01)%和(4.91±0.15)% (P<0.05);粗肽经纯化得到纯化组分Ⅰ(1000~3000 u)和纯化组分Ⅱ(<1000 u)。CK组、双螺杆挤压辅助3.0% Na2SO3处理组及其经分离纯化后得到的的纯化组分Ⅰ、Ⅱ海地瓜多肽的DPPH自由基清除率的IC50值分别为25.51、12.72、6.58和9.02 mg/mL,对超氧阴离子自由基清除率的IC50值分别为25.56、13.51、11.87和8.44 mg/mL。纯化组分Ⅰ在浓度为80 μg/mL时,人正常肝细胞(human normal liver cell, LO2)细胞存活率较损伤组最大提高(20.33±0.41)%;纯化组分Ⅱ在浓度为20 μg/mL时,LO2细胞存活率较损伤组最大提高(17.07±1.18)%。综上所述,双螺杆挤压辅助亚硫酸钠处理海地瓜提高了多肽的提取率,并增强了海地瓜多肽抗氧化活性。     

海地瓜营养成分的分析及评价

对海地瓜进行营养成分测定,包括对海地瓜中主要营养组分、氨基酸组成、脂肪酸组成和矿物质元素的测定、分析和比较,并对其营养价值进行分析和评价。结果显示,海地瓜体壁中粗蛋白、粗脂肪、多糖和粗灰分的含量分别为81.22%、0.23%、3.62%和10.33%;羟脯氨酸含量达31.2 mg/g;18种氨基酸的总量为80.08%(干重),其中8种必需氨基酸占总氨基酸的23.95%,鲜味氨基酸占总氨基酸的54.48%,必需氨基酸与非必需氨基酸的比值为0.31,必需氨基酸指数为57.03,必需氨基酸的构成比例低于FAO/WHO的标准;不饱和脂肪酸(UFA)占脂肪酸总量的55.1%;矿物质元素中钙含量最高,为4 100 mg/kg。结果表明,海地瓜是一种低脂高蛋白的海洋生物资源。     

复合酶提取牡蛎抗氧化肽的工艺研究

以牡蛎为原料,首先从木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶中筛选出胰蛋白酶为内切酶,以水解度为指标,得到最佳的酶解工艺条件:时间4h,温度50℃,pH8.0,料水比1∶2,加酶量3％(7 500 U/g),水解度为49.50％;同时以水解度为指标,得到外切酶风味蛋白酶的最佳酶解工艺:时间5h,温度50℃,pH8.0,料水比1∶2,加酶量3％ (450 U/g),实际水解度50.95％.最后,以清除羟自由基能力和水解度为指标,探讨内切酶(胰蛋白酶)和外切酶(风味蛋白酶)不同复合酶解方式的抗氧化能力.最终确定,复合酶解制备牡蛎抗氧化肽效果最好,其酶解条件为胰蛋白酶为3％(7 500 U/g)、风味蛋白酶加酶量为3％ (450 U/g),pH8.0,时间5h,料水比1∶2,温度50℃时,水解度高达53.94％,体外清除·OH的EC50为0.56 mg/mL.     

鱼蛋白粉酶水解产物对DPPH自由基清除能力的研究

以鱼蛋白粉为原料,碱性蛋白酶(Alcalase 2.4 L)为水解用酶,研究了底物浓度、加酶量、水解p H、温度和时间对鱼蛋白粉水解产物的DPPH自由基清除率的影响。结果显示,5个因素对DPPH自由基清除率均有不同程度的影响。在单因素试验的基础上,采用响应面法对鱼蛋白粉的酶水解条件进行优化,得到的最佳水解工艺条件为:底物浓度(w/v)18.05%,加酶量(w/w)2.40%,水解p H 8.04、温度49.0℃、时间4.0 h。在此条件下水解度为9.81%,鱼蛋白粉水解液的DPPH自由基清除率为76.10%,与浓度为120μg/m L的还原型谷胱甘肽的DPPH自由基清除率相当。结果表明,鱼蛋白粉酶水解产物具有较好的清除DPPH自由基的能力。     

鳜传染性脾肾坏死病毒重组主衣壳蛋白免疫效果的初步验证

将大肠杆菌重组表达的鳜(Siniperca chuatsi)传染性脾肾坏死病毒(ISKNV)主衣壳蛋白(MCP),以不同剂量(20μg,尾、50μg/尾、100μg/尾)腹腔注射免疫幼鳜(2月龄),测定各免疫组的血清抗体效价变化规律、头肾淋巴细胞增殖刺激指数(SI)、肝脏Mx蛋白表达及相对免疫保护率(RPS).结果表明,各免疫组抗体效价在第14天时达到峰值,其中50 ug/尾免疫组的抗体效价最高,随后各组的效价均下降,至第35天时与对照组无差异;头肾淋巴细胞经LPS、ConA刺激后,50 μg/尾免疫组及100 μg/尾免疫组的刺激指数均升高,其中50 μg/尾免疫组的刺激指数最高,20μg/尾免疫组与对照组无差异;在免疫后48 h各剂量免疫组Mx蛋白均有低量表达,对照组无表达,各免疫组表达量无显著差异;免疫后第36天攻毒,50μg/尾免疫组的相对保护率最高,为64.3%.研究结果表明,重组MCP蛋白有较好的免疫原性,可以激发鳜特异性免疫及非特异性免疫应答,当免疫剂量为50μg/尾时,免疫保护效果最好.     

提取方法对淡水鱼油提取率及品质的影响

以白鲢(Hypophthalmichthys molitrix)内脏为原料,采用复合蛋白酶(Protemax)水解法提取鱼油,研究酶解条件和提取方法对鱼油提取率、品质及脂肪酸组成的影响,以获取高品质的鱼油制品.结果表明:提取方法和条件对鱼油的提取率、酸价及过氧化值(POV)均有明显影响;采用复合蛋白酶水解法提取鱼油时,其适宜的条件为酶解起始pH 7.5,酶解温度50 ℃,酶用量2 000 U/g粗蛋白,酶解时间3 h,鱼油的提取率达到85.67%,鱼油品质较好.复合蛋白酶水解法和碱性蛋白酶(Alcalase)水解法的提取率高于稀碱水解法,且复合蛋白酶水解法得到的n-3系列多不饱和脂肪酸含量更高.     

鳕鱼下颌肉水解蛋白制备工艺优化

通过单因素实验(蛋白酶种类、加酶量、料液比、水解时间、温度和pH值),对鳕鱼下颌肉进行酶解分析,以水解度(DH)为指标,确定最适蛋白酶为复合风味蛋白酶,其单因子最佳水解条件为:加酶量为1 400 U/g(相对于鱼糜质量)、料液比为1:3、水解时间为5 h、温度为43℃、pH 7.6。经正交试验验证,复合风味蛋白酶的最佳酶解工艺条件为:pH7.0,料液比1∶2,加酶量为1 400 U/g,水解8 h,DH可达42.62%。     

缢蛏蛋白组分Y3的提取及抑癌活性研究

以新鲜缢蛏Sinonovacula constrzcta为材料,经25 mmol/L Tris-HCl(pH 7.5,含0.14mol/L NaCl)缓冲液提取缢蛏粗蛋白、透析、DEAE离子交换层析、Sepharose CL-6B凝胶层析,得到1种具有抑癌生物活性的缢蛏蛋白组分Y3.SDS-PAGE电泳测得该蛋白组分的分子量约为64 kD.采用MTT比色法检测Y3对4种肿瘤细胞增殖的影响.结果显示,Y3对Hela和BGC803细胞有明显的抑制作用,而对SKOV3和HepG2抑制作用不明显.此外,探讨了Y3对羟基自由基和超氧阴离子自由基的清除能力.结果显示,该蛋白组分清除自由基的能力很强,在浓度为100 μg/ml时,Y3对羟基自由基清除率达到92.71％.在3μg/ml下,对超氧阴离子自由基的清除率达到59.73％.     

外源激素诱导唇(鱼骨)排卵作用的研究

于2005年4月在浙江湖州地区进行了外源激素单一剂种和2种、3种剂型配伍对唇(鱼骨)的诱导排卵作用试验.结果表明,单一的DOM诱导,剂量为10 mg/kg时,其排卵率为80%,LRH-A剂量为20 μg/kg,排卵率为20%,HCG剂量为2 000 IU/kg时,排卵率为0.当各组的剂量减至1/4时,其排卵率均为0;2种以上激素配伍诱导时,凡有DOM加入的试验组及对照组均有较好的排卵效果,DOM剂量为5 mg/kg时,排卵率为70%～100%.     

低值淡水鱼的酶法水解

本文研究了胰蛋白酶对低值淡水鱼的水解效果,结果表明:当酶用量为600u/100g 鱼肉、水解温度为45℃、水解时间为1.5～2.0小时,鱼肉蛋白质的水解度达30～40%;水解产物营养成分测定表明:水解鱼肉蛋白粉蛋白质含量达85.1%,必需氨基酸含量占总氨基酸的40%,同时富含Ca、P、Fe、Zn 等矿质元素,是理想的食物蛋白添加剂。     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Production of Interesting Peptide Fractions by Enzymatic Hydrolysis of Tuna Dark Muscle By-Product Using Alcalase

ABSTRACT

A protein hydrolysate was prepared from proteins of tuna dark muscle by-product. The hydrolysis conditions (time, temperature, pH, and enzyme concentration) using Alcalase was optimized by response surface methodology (RSM). The regression coefficient close to 1.0, observed during experimental and validation runs, indicated the validity of the model. The hydrolysate produced under the optimum conditions determined by RSM has a low rate of peptide fraction of molecular weight of 4–1 kDa. Meanwhile, the results obtained by hydrolysis under optimal conditions determined by a complementary study (temperature 55°C, time 60 min, 1% enzyme concentration, and pH 8.5) show that the hydrolysate produced has a height rate of the peptide fraction of molecular weight of 4–1 kDa. The amino acid composition of the protein hydrolysate prepared proved to have the potential for application as an ingredient in balanced fish diets and as a source of nitrogen in microbial growth media.     

Antioxidant Properties of Protein Hydrolysate Obtained from Oyster Saccostrea cucullata (Born, 1778)

The antioxidant activities of enzymatically hydrolyzed (protease from Bacillus cereus SU12) oyster (Saccostrea cucullata) protein were studied. The hydrolysate exhibited a strong antioxidant potential in 1, 1-diphenyl-2-picrylhydrazyl (DPPH, 85.7 ± 0.37%), followed by hydrogen peroxide radical scavenging activity (81.6 ± 0.3%), hydroxyl radical scavenging activity (79.32 ± 0.6%), and reducing power assay (2.63 ± 0.2 OD at 700 nm) at a concentration of 1 mg/mL. Due to the high antioxidant potential, the hydrolysate was purified in Sephadex G-25 gel filtration chromatography. The active peptide fraction was identified by DPPH and reducing power assay. The amino acid content of the purified active peptide fraction was analyzed by high performance liquid chromatography. The active fraction contained a good quantity of both essential and nonessential amino acids. The present study revealed that oyster (S. cucullata) protein hydrolysate is a potential source for natural antioxidants.     

Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

SUMMARY: A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg²⁺-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg²⁺-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg²⁺-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.     

Recovery of Bioactive Peptides and Omega-3 Fatty Acids-Containing Phospholipids from Squid Processing By-Product Hydrolysate

ABSTRACT

The present study examined whether bioactive peptides and omega-3 fatty acids-containing phopholipids could be recovered from squid processing by-product (SPB) hydrolysate. The hydrolysate was produced at 55°C for varying times with endogenous proteases and centrifuged to yield peptides-containing supernatant and phospholipids-containing precipitate. The supernatant showed angiotensin I-converting enzyme (ACE) inhibitory activity with half maximal inhibitory concentration (IC₅₀) values decreasing with hydrolysis time from 2.11 mg (0 h) to 1.71 mg (1 h), 1.38 mg (1.5 h), and 1.34 mg (2 h). Two-hour squid hydrolysate was further fractionated to isolate the most active fraction whose molecular weight was found to be below 10 kDa with IC₅₀ of 0.32 mg. The phospholipids-containing precipitate (45.6 g/100 g oil) was analyzed for a fatty acid profile, with the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being 16.86 and 29.24 g/100 g oil, respectively. The ACE inhibition by hydrolysate peptides and omega-3 fatty acid recovery support the nutraceutical potential of the SPB hydrolysate.     

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.     

Assessing the Antioxidant Activity of the Ultrafiltration Fractions From Silver Carp Protein Hydrolysate by Different Antioxidant Methods

Silver carp protein hydrolysate (SH) was fractionated by 5- and 1-kDa cutoff ultrafiltration (UF) membrane, and three ultrafiltrates were obtained: SHI (>5 kDa), SHII (1–5 kDa), and SHIII (<1 kDa) which were screened for antioxidant activity by five in vitro assays. The SHIII showed higher 2, 2-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) values, inhibiting effects for liposome oxidation induced by Fe³⁺/V_C. The SHII showed higher ferric reducing power (FRAP) and inhibiting effects for liposome oxidation induced by 2, 2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH). However, the SHI showed the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity. Therefore, the antioxidant activity of silver carp protein hydrolysates was mainly associated with their molecular weight distribution, and the antioxidant assays used to test different samples gave comparable results.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.     

Microencapsulation of Fish Oil Using Fish Protein Hydrolysate,Maltodextrin, and Gum Arabic: Effect on Structural and Oxidative Stability

ABSTRACT

Microencapsulated fish oil was prepared by spray drying using fish protein hydrolysate, sodium caseinate, maltodextrin, and gum Arabic as wall material. Fish protein hydrolysate was prepared from pink perch meat, and its physical and functional properties were studied. Microencapsulates prepared with a combination of sodium caseinate, maltodextrin, and gum Arabic were kept as control. The encapsulation efficiency and oil release behavior of microencapsulates was evaluated. Surface morphology and thermal properties of microencapsulates were determined by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) analysis, respectively. Fourier-transformed infrared spectroscopy analysis indicated that the spectral pattern of microencapsulates showed a similar structural pattern with a minor band shift for both control and fish protein hydrolysate containing microencapsulates. Oxidative stability of fish oil microencapsulates indicated that the sample stored under was 4°C was more stable than microencapsulates stored under 60°C and 28 ± 2°C temperature conditions. Moreover, microencapsulates containing fish protein hydrolysate had a lower thiobarbituric acid value. Results suggest that the incorporation of fish protein hydrolysate along with other wall material could improve the oxidative stability of microcapsules during storage.