摘要
浙江温州沿海滩涂养殖面积达6.5万公顷,辽阔的养殖面积造就了水产加工企业的兴起,与此同时,也导致了大量鱼类废弃物的产生。这些鱼类废弃物的丢弃不仅造成了一定的经济损失,也造成了环境的污染。近年来,随着人们对蛋白质资源需求越来越大,充分利用这些下脚料可提高鱼类加工附加值,获得良好的经济和社会效益。小黄鱼作为一种典型的低值鱼类,研究小黄鱼下脚料水产调味品,探讨小黄鱼下脚料酶解产物的生理活性具有一定的现实意义。
本研究以小黄鱼下脚料为原料,一方面通过生物酶解技术结合热反应技术制备水产调味料,以小黄鱼下脚料风味物质为主要评价指标,追踪调味料加工工艺流程中氨基酸含量、风味物质种类与含量的变化；另一方面,通过生物酶解及超滤技术,对小黄鱼下脚料酶解液的生理活性(抗氧化和抗凝血能力)进行了初步研究。本工作的主要研究内容与结果如下：
1)采用同时蒸馏萃取法(Simultaneous Distillation Extraction,简称SDE)与固相微萃取法(Solid-Phase Microextraction,简称SPME)两种方法提取挥发性风味物质,并运用气质联用仪鉴定小黄鱼下脚料的挥发性风味物质,通过内标法对SDE提取的风味物质进行定量分析。实验分析共鉴定出57种挥发性风味物质,其中醇酮类物质含量较高,醛类与烷烯类物质含量次之；选取其中10种对小黄鱼下脚料风味具有主要贡献的化合物作为调味料加工的特征性风味成分。
2)采用生物酶解技术,提取小黄鱼下脚料中的滋味成分与风味前体物质。经过单因素实验与响应曲面分析,确定了碱性蛋白酶(Alcalase)和风味复合蛋白酶(Flavourzyme)同步酶解工艺,同步酶解的最优条件为料液比1：7,酶解温度55℃,酶解时间6.5 h,初始pH 8.0,碱性蛋白酶用量2.50%,风味蛋白酶用量3.00%。在此条件下,水解度达到40.11%。经分析发现,小黄鱼下脚料酶解液中氨基酸含量达40.03g/100g,其中必需氨基酸15.20g/100g,鲜味和甘味氨基酸44.44g/100g。与酶解前相比,氨基酸含量明显增加,氨基酸总量增加39.77%,其中必需氨基酸增加51.85%,呈味氨基酸增加49.75%,产品具有浓郁的小黄鱼鲜香味。结果表明,酶解产物是以鲜味为主,包括咸、苦、甜等多种味觉的复杂呈味体系。
3)为进一步探讨热反应技术在制备水产调味料工艺流程中发挥的作用,通过运用DPS v7.05软件进行热反应均匀实验设计,以反应温度、时间、pH值、氨基酸、糖为因素,采用模糊矩阵法进行感官评分,得到热反应工艺最优条件为酶解液40 g,温度110℃,时间25min,初始pH 9.0,葡萄糖6.00 g,Glu3.00g,Gly0.80g,Met 0.70 g, Pro 0.70 g,Ala 3.00 g,Arg 0.20 g,Cys 1.78 g。在此基础上,进一步优化热反应液的口味,考察VC、蒜粉、柠檬酸对热反应液风味的改善作用。结果表明,Vc的最适添加量为0.10%,蒜粉的最适添加量为0.10%,柠檬酸的最适添加量为1.50%。同时,氨基酸分析表明,氨基酸总量为40.29g/100g,必需氨基酸含量为10.44g/100g。胱氨酸含量降低20.00%,呈鲜味氨基酸谷氨酸、甘氨酸和丙氨酸分别升高16.43%、33.91%和33.10%。经热反应处理后,产品风味醇厚,具有浓郁的鱼肉香味。
4)对加工流程中的风味成分变化进行追踪分析,比较小黄鱼下脚料原料、酶解产物和热反应产品中风味物质种类和含量的变化。结果表明,热反应产品的风味强度与滋味强度都有所提高,总体可接受性较强。风味方面,烷烯类、羧酸类和酯类对调味料产品的风味贡献不大,醛类、杂环类化合物这类阈值较低的风味物质则明显增强了调味料产品的风味强度,具有特征性鱼腥臭味物质明显减少。
5)除了研究酶解液风味及加工适用性,同时也初步探讨了酶解液的生理活性。通过对不同酶解时间酶解液清除DPPH能力,清除超氧阴离子自由基能力,清除羟基自由基的能力和抗凝血.能力这几个指标的测定,得到初步结果,经Master Flex超滤系统分离后不同组分对DPPH·,O2-和OH-都具有不同程度的抑制能力,对凝血时间也有一定延长作用。在所测浓度范围内,各组分清除DPPH能力均达到半抑制浓度,各组分清除O2-·能力部分达到半抑制浓度,各组分样品对OH-基本无抑制作用,并有一定程度的抗凝血能力。虽然酶解液的抗氧化和抗凝血活性不高,但是作为废弃资源再利用,我们在调味料的风味基础上又赋予调味料产品以部分功能性,进一步提高了产品的营养价值。
The cultivation area in coastal beach of Wenzhou in Zhejiang exceeds 65,000 hectares. The vast cultivation area has made the aquatic products processing enterprises springing up, which produces lots of fish scraps at the same time. These waste fish scraps not only pollutes environment but also makes direct economic loss. With the larger demands of fish protein sources, the full use of these scraps can rise the added value of fish, and achieve excellent economic effect and social effect. The small yellow croaker is one of the typical low value fish. Therefore, there is a practical significance to study the preparation of aquatic flavoring and investigation of the physiolagical activity from small yellow croaker scraps.
In this study, small yellow croaker scraps were chosen as the raw material to prepare aquatic flavoring by enzymolysis and thermal reaction. The characteristic flavor compounds of small yellow croaker scraps were used as the main evaluation indicators to track the change of amino acids as well as characteristic flavor types and contents in the flavor processing scheme. On the other hand, physiological activity (including the antioxidant and the anticoagulation activity) of protein hydrolysate from small yellow croaker scraps by enzymolysis and ultrafiltration were preliminary studied. Our detailed works are mainly concentrated on the following aspects:
1) Volatile aroma compounds of small yellow croaker scraps were extracted by the simultaneous distillation extraction method and solid phase micro-extraction method. The extracts were isolated and identified by gas chromatograph mass spectrometry, and the contents of aroma compounds were determined via internal standard method by gas chromatography. Fifty seven kinds of small yellow croaker scraps flavor compounds were isolated and identified successfully. The result showed that alcohol and ketone compounds were the major flavor compounds, aldehyde and alkene compounds came second. Among these flavor compounds, ten kinds of flavor compounds which mainly contributed to the characteristic flavor of small yellow croaker scraps were regarded as evaluating indictors.
2) The taste compounds and flavor precursors were extracted after enzymolysis of small yellow croaker scraps. The optimum hydrolysis conditions of alcalase and falvorzyme were determined respectively by single factor experiments and response surface analysis. The results showed that, the optimum enzymatic hydrolysis condition was material-water ratio 1/7, Alcalase enzyme dosage 2.5%, Flavourzyme enzyme dosage 3.0%, temperature 55℃, initial pH 8.0, and hydrolysis time 6.5 h. Under this condition, the degree of hydrolysis (DH) was 40.11 %, the concentration of free amino acids in the hydrolysate was 40.03 g/100g, the concentration of the essential amino acids was 15.20 g/100g, and the concentration of flavor acids was 44.44%. The amino acid content in the products increased significantly as compared with that before enzymolysis. The concentration of the total free acid increased by 39.77%, the essential amino acids increased by 51.85%, and the flavor acids increased by 49.75%. The products had a unique flavor of small yellow croaker. And the umami taste was significantly strengenthed by enzymatic hydrolysis and the products had an enjoyable complex, mostly umami, including salty, sweet and bitter taste.
3) To study thermal reaction of enzymatic hydrolysate to prepare aquatic flavoring, the effect of reaction temperature, reaction time, pH value, exogenous amino acids, and exogenous sugar were investigated. The uniform designs and fuzzy matrix were used to compare the products' taste. The optimum thermal reaction condition was as follows: enzymatic hydrolyzate 40 g, the initial pH 9.0, the reaction time 25 min, the reaction temperature 110℃, glucose 6.00 g, Glu 3.00 g, Gly 0.80 g, Met 0.70 g, Pro 0.70 g, Ala 3.00 g, Arg 0.20 g, and Cys 1.78 g. To further optimize the thermal reaction product flavor, the effect of Vc, garlic powder and citric acid were investigated. The results showed that the suitable additional quantity of Vc, garlic powder, and citric acid was 0.1%,0.1% and 1.5% respectively. The result of amion acids analysis indicated that the concentration of free amino acids in the aquatic flavoring was 40.29 g/100g, and that of the essential amino acids was 10.44 g/100g. After thermal reaction, the content of Cys was reduced by 20.00%, and that of the flavor acids of Glu, Gly and Ala were increased by 16.43%,33.91%,33.10%, respectively.
4) In order to analysis the change of flavor compounds in the processing of small yellow croaker scraps, the flavor compounds of raw mateials, enzymolysis product and the thermal reaction product were investigated. The results showed that compared with the raw materials, the thermal reaction product was better in the aroma intensity and tastiness intensity, and easily accepted. The contribution to flavor of alkenes, acids compounds and ester compounds were little, the contribution of aldehyde compounds and sulfur-containing compounds with low aroma threshold were obvious. Besides, the stink odour compounds were significantly reduced.
5) In addition to analysis of the processing applicability and the flavor of enzymolysate, the physiological activity of enzymolysate was also discussed. The effect of DPPH·scavenging activity, O2-·scavenging activity, HO·scavenging activity and anticoagulation activity of the enzymolysates with different hydrolysis time were investigated. The results showed that, the different components separated from the Master Flex ultrafiltration system had a different degree of suppression on DPPH·, O2-·, OH·, and had some effect on the clotting time prolongation. Among the measured concentration range, the DPPH·scavenging capacity of each component was up to half inhibitory concentration, the O2-·scavenging capacity of each component partly reached half-inhibitory concentration, but each components almost had no inhibitory effect on the OH·. What's more, they had anti-clotting ability in a certain extent. Although the antioxidant activity and the anticoagulant activity was not high, but we enhanced the product's nutritional value by giving the functionality of products as a re-use waste of resources.
引文
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