摘要
由于鲢鱼具有生长快、养殖成本低和抗病能力强等特点，近年来越来越受到人们的关注。该鱼种主要生长在中国、其他亚洲国家和美国。为了控制其过度繁殖并减少对以浮游生物为食的本土物种的威胁，有关鲢鱼市场开发相关的研究已经开始，将鲢鱼加工为具有附加值的人类消费品不失为一个很好的选择。
豆豉是一种发酵大豆产品，具有很高的营养和药用价值，是中国、日本和其他东南亚国家传统的食品调味品，并且美国的许多中餐中也常见豆豉的身影。豆豉在许多发酵食品（如日本豆面酱）中被作为发酵剂使用，从而生产出适用于鱼肉发酵的如蛋白酶、淀粉酶、脂酶等多种酶类。
为了扩展豆豉的应用范围，本文设计了一系列实验以验证能否使用豆豉培养基为发酵剂提高发酵鲢鱼肉的质量以及加速发酵过程。在实验1中，将活性预发酵豆豉培养基加入到鱼肉酱中后发酵30天，使其利于风味物质的生成（CulF）。在实验2中，将发酵30天后的豆豉添加到鱼肉酱然后再一同发酵30天（PSF）。在实验3中，未添加豆豉的鱼酱也发酵30天（ConF）。在CulF、PSF和ConF中，游离氨基酸总量分别增加了68.0%、68.6%和78.8%（P <0.05），其中谷氨酸、天冬氨酸、丙氨酸、赖氨酸和亮氨酸含量较高（>100mg/mL）。甲醛活性氮和氨基氮在发酵期间按照ConF
香气和滋味。这种豆豉接种发酵可以作为新技术来扩展鲢鱼的利用、消费和经济价值。
此外，进一步研究了接种豆豉培养基（CulF）和空白（ConF）的发酵鱼酱中的蛋白降解和风味物质的生成情况。CulF中的蛋白酶活性比ConF样品中的高(P <0.05)，并且在发酵15天后有3倍的差距。提取出的组分中主要含有低分子量的肽（<1.3kDa）。SDS–PAGE显示CulF和ConF样品提取物中的肌球蛋白重链分别在发酵1天和5天后完全降解。CulF的游离氨基酸和TCA可溶肽的含量高于ConF（P <0.05）。酶抑制剂测定显示出该发酵体系中主要含有酸性和丝氨酸蛋白酶。甲醇、硅甲烷二醇、吡嗪、酚和甲酸是CulF主要的挥发性化合物，而丁醇、丁酸和醋酸在ConF中占多数。鉴于豆豉接种发酵可以生成明显的滋味和香味物质，它应该优于鱼酱生产中的天然发酵。
由于自由基清除活性是控制脂类过氧化的重要机制，本实验中接种豆豉培养基（CulF）和空白（ConF）鲢鱼酱样品发酵后，测定液体提取物中清除2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)自由基（ABTS）和1,1-二苯基-2-三硝基苯肼自由基（DPPH）的能力和金属螯合力，以及使用亚麻酸体系测定提取液的对脂质氧化的抑制力。结果显示出CulF在蛋白浓度为0.2mg/mL时可以清除高于50%的ABTS和DPPH自由基。CulF样品的亚铁离子螯合力高于ConF (P <0.05)。自由基清除能力跟发酵时间有关，除了DPPH都随着时间的增加而增加。CulF样品发酵15天后显示出很强的DPPH清除能力。尽管CulF展现出良好的抗氧化能力，但是显著(P <0.05)低于豆豉的抗氧化能力。CulF和ConF都可以稳定自由基并减少了80%的脂类被破坏。游离脂肪酸测定显示发酵30天后，CulF中EPA和DHA的含量明显高于ConF(P <0.05)，可以推测这应得益于使用了豆豉发酵剂。
由于提取液中的低分子量肽有部分抗氧化活性，从而对接种豆豉培养基的提取液（CulF）做进一步的研究。组分分析中有八种组分可以淬灭ABTS自由基，其淬灭能力随着分子量的增大而减弱。其中，7号组分呈现出很强的抗氧化活性。这些短肽也作为反应物质对风味形成起重要作用。
微生物分析发现鱼酱中含有大量的乳酸菌，后者正是使得鱼酱有较低的pH并形成风味。这种格兰仕阳性菌（micrococci）在发酵鱼酱中大量增殖。另外，该研究发现CulF具有较高的过氧化值（16.8meq/kg鱼肉）和挥发性碱（TVB-N，111.9mg/100g）
因此，本研究结果显示出豆豉作为新型发酵剂可以用来生产发酵鲢鱼酱，并具有极佳的风味和消费者接受度。发酵时间降至30天。该结果可以应用于其他鱼类品种以生产相似的发酵产品。
Silver carp (Hypophthalmichthys molitrix) has attracted much attention in recent years due to itsfast growth rate, a low feed demand, and resistance to diseases. Silver carp is intensively grown inChina, other Asian countries and U.S. To control over-population thereby reducing its threat tonative plankivorous species, research incentives and initiatives have been proposed toward marketdevelopment of silver carp. Processing silver carp to produce value-added products for humanconsumption is considered to be an attractive alternative.
Douchi, a fermented soybean product, is a traditional food flavoring ingredient commonly usedin China, Japan, and other southeastern Asian countries, it is also used in many Chinese cuisines inthe U.S due its rich nutritional and medicinal compounds. Douchi find its practical applications inmany of fermented food products such as miso where it serves as starter culture, producing varietiesof enzymes including proteases, amylases and lipases which are suitable in fish fermentation.
To expand the applications of douchi, numbers of experiments were conducted to test thehypothesis that douchi cultures could serve as a potential starter for enhancing the quality attributesof fermented silver carp meat and accelerate the fermentation processes. In experiment1, an active,pre-fermented douchi culture was incorporated into a fish paste to aid in the fish fermentation (30days) and facilitates biochemical production of extractive flavor components (CulF). In experiment2, a fully fermented (30days) douchi was added to a fish paste and the mixture was fermented for30days (PSF). In experiment3, a fish paste without the douchi culture was fermented for30d(ConF). Total extracted free amino acids increased by68.0,68.6, and78.8%(P <0.05) from theirinitial levels to2930,2422, and1573mg/mL after30days of fermentation for CulF, PSF, andConF fish pastes, respectively, of which, glutamic acid, aspartic acid, alanine, lysine, and leucinewere the major amino acids (>100mg/mL). The concentrations of both formaldehyde-reactivenitrogen and ammonia nitrogen extractives increased significantly (P <0.05) during fermentation,following the order of CulF> PSF> ConF. Low amounts of biogenic amines (<25ppm) wereproduced in all samples. Sensory panel evaluation showed that CulF fish pastes had desirable aromaand taste. The douchi-inoculated fermentation could be a novel technique for expanding theutilization, consumption, and the economic values of silver carp meats.
Furthermore, silver carp fish pastes inoculated with a douchi starter culture (CulF) and withoutstarter culture (ConF) were fermented, and the consequent protein degradation and flavorcompound production were investigated. The protease activity in CulF, higher (P <0.05) than inConF, increased3-fold after15days of fermentation. The extractive fraction was largely comprisedof low-molecular-weight (<1.3kDa) peptides. SDS–PAGE analysis revealed complete degradationof myosin heavy chain in the extract after1and5days for CulF and ConF, respectively. CulF had ahigher (P <0.05) content of free amino acids and TCA–soluble peptides than ConF. Enzymeinhibitor assays showed that the fermentation system was dominated by acidic and serine proteases.Ethanol, silanediol, pyrazine, phenol, and formic acid were prevalent volatile compounds in CulF,while butanol, butanoic acid and acetic acid were abundant in ConF. For its strong capability to synthesize distinct taste and aroma compounds, douchi–inoculated fermentation was considered tobe superior to natural fermentation in the production of savory fish pastes.
Free radical scavenging activity is an important mechanism for controlling peroxidation of lipids.Silver carp fish pastes inoculated with douchi starter culture (CulF) or without douchi starter culture(ConF) were further fermented, and the liquid extracts tested towards scavenging of2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),1,1-diphenyl-2-picrylhydrazyl (DPPH), andmetal chelating ability. The extract was also tested for inhibition of lipid oxidation in linolenicmodel system. The results indicated that CulF was superior to scavenging over50%of ABTS,DPPH at0.2mg/mL protein. The ferrous ions (Fe2+) chelating ability, showed that CulF wasstronger (P <0.05) than ConF. Radical scavenging capacity was fermentation dependent and itincreased with time except for DPPH. A strong DPPH scavenging ability was registered on day15for CulF. Although CulF exhibited good antioxidative capacity, it was significantly (P <0.05) lesspotent than douchi. The inhibition of lipid oxidation showed that both CulF and ConF couldstabilize radicals to minimize over80%lipid damage. The free fatty acid assay showed that CulFpossessed significantly higher (P <0.05) amounts of EPA and DHA than ConF after30dfermentation, inferring the advantage of douchi inclusion as starter culture.
Since low molecular weight (MW) peptides of liquid extracts are known to play parts inantioxidantive activity of protein-derived antioxidants, further investigation was carried up to theliquid extracts (LE) of fish paste inoculated with douchi starter culture (CulF). The fractionationassay based on molecular weight revealed eight fractions which were able to scavenge the ABTSradicals. The scavenge capacity decrease with increases of molecular weights. Fraction7exhibiteda strong antioxidant activity among others. These low MW peptides also were considered to playsignificant role as reactants for flavor development.
The microbial analysis found that fish pastes were largely dominated by lactic acid bacteriawhich played a supporting role in lowering pH and flavor developments. The gram positive bacteria(micrococci) populated the fermented fish pastes. Furthermore, the study found that CulF containedsubstantial amount of peroxide value (16.8meq/kg of fish), and total volatile base (TVB-N) of111.9mg/100g, however paint-like smell was not detected into the product.
Therefore, the findings from the present study indicated that douchi as a novel starter can be usedto produce fermented silver carp fish pastes with excellent flavor and consumer acceptability.Fermentation time was reduced to30days. The results may be applicable to other fish species toproduce similar fermented products.
引文
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