CN113045683B - A kind of preparation method of seaweed oligosaccharide and its application in fish feed - Google Patents

A kind of preparation method of seaweed oligosaccharide and its application in fish feed Download PDF

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CN113045683B
CN113045683B CN202110413226.4A CN202110413226A CN113045683B CN 113045683 B CN113045683 B CN 113045683B CN 202110413226 A CN202110413226 A CN 202110413226A CN 113045683 B CN113045683 B CN 113045683B
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王晶
王晓晴
张全斌
刘婧
吴宁
耿丽华
岳洋
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Qinhuangdao Yi Er Biological Technology Co ltd
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Abstract

The invention provides a preparation method of laver oligosaccharide and application of the laver oligosaccharide in fish feed. The laver oligosaccharide is mainly composed of 3-linked beta-D-galactose and 4-linked alpha-L-galactose-6-sulfate linkage, the polymerization degree is 2-20, the galactose content is 60-80%, and the sulfate content is 15-30%. The porphyra oligosaccharide is added into the fish feed, so that the meat quality of the cultured fish can be improved, the fat content of the fish body is reduced, and the protein content of the fish body is increased, so that the meat quality and flavor of the fish are improved. The porphyra oligosaccharide is a brand-new, green and safe fish feed additive, can effectively solve the problems of loose meat quality, low protein content and poor taste of cultured fish, can improve the immunity of the fish, and can broaden the market of the cultured fish.

Description

一种紫菜寡糖的制备方法及其在鱼饲料中的应用A kind of preparation method of seaweed oligosaccharide and its application in fish feed

技术领域technical field

本发明涉及水产养殖领域,具体涉及一种紫菜寡糖的制备方法及其在鱼饲料中的应用。The invention relates to the field of aquaculture, in particular to a preparation method of laver oligosaccharide and its application in fish feed.

背景技术Background technique

随着水产养殖业的发展,制约养殖生产的问题也日益突出。当前严重影响养殖鱼销量的问题之一在于养殖鱼风味较差,严重的腥味、松散的肉质、较高的脂肪含量都使消费者敬而远之。养殖鱼较低的品质,已经严重影响其商业价值和发展。养殖鱼肉质较差与养殖环境的恶劣和饲料品质低下直接相关。污染的水环境势必导致鱼病和腥味物质的积累,而普通的配方饲料能量偏高,导致鱼体脂肪偏高,瘦肉率低。因此,开发安全、无毒、绿色的水产养殖抗病害制剂已然形成一种趋势。目前养殖鱼类肉质的改善,一方面,可以通过调整饲料配方,另一方面也要注重养殖鱼免疫力的增强。有研究显示,微生物多糖、甲壳素、动植物提取物等对水产动物都有不同程度的免疫增强作用,但考虑制备成本和技术难度,目前这些免疫增强剂的应用还需进一步改良。With the development of aquaculture, the problems restricting aquaculture production have become increasingly prominent. One of the problems currently seriously affecting the sales of farmed fish is the poor flavor of farmed fish. The severe fishy smell, loose meat and high fat content make consumers stay away. The lower quality of farmed fish has seriously affected its commercial value and development. The poor meat quality of farmed fish is directly related to the harsh farming environment and low feed quality. The polluted water environment will inevitably lead to the accumulation of fish diseases and fishy substances, while the energy of ordinary formula feed is high, resulting in high body fat and low lean meat rate. Therefore, it has become a trend to develop safe, non-toxic and green aquaculture anti-disease preparations. At present, to improve the meat quality of farmed fish, on the one hand, we can adjust the feed formula, and on the other hand, we should also pay attention to the enhancement of the immunity of farmed fish. Studies have shown that microbial polysaccharides, chitin, animal and plant extracts, etc. have different degrees of immune-enhancing effects on aquatic animals. However, considering the preparation cost and technical difficulty, the application of these immune-enhancing agents needs to be further improved at present.

紫菜多糖是一种硫酸化的多糖,是紫菜属的植物所特有的多糖成分,它存在于紫菜的细胞壁中,占紫菜干重的20-40%。它的主要结构单元是→3)β-D-半乳糖-(1→4)-α-L-6-硫酸基-半乳糖-(1→。其中部分α-L-6-硫酸基-半乳糖被L-3,6-内醚半乳糖代替,形成结构单元:→3)-β-D-半乳糖-(1→4)-α-L-3,6内醚半乳糖-(1→。紫菜多糖具有抗凝血、降血糖、调血脂、抗血栓、抑制肿瘤及增强细胞免疫功能等多种生物活性,广泛地用于食品和医药等行业。紫菜寡糖是由紫菜直接制备或者紫菜多糖经过降解制备,聚合度为2-20。但是,目前紫菜寡糖的制备方法紫菜寡糖的制备方法主要有酸降解法、氧化降解法、微波、超声辅助降解法和酶降解法。化学降解法中过氧化氢降解前景最佳,此方法成本低,易实现工业化生产,然而氧化剂并没有完全参与到反应过程中,同时温度升高,产品也更加容易出现褐变;利用生物酶协助发生反应,可以有效地避免产生副作用,同时外部的反应条件也容易控制,然而使用酶反应导致成本上升;通过物理法降解反应不完全。分子结构决定其生物学功能,紫菜寡糖糖链在分子大小、序列排布、构型构象等微观结构的不均一性都可能造成其生物学功能的巨大差异。因此,建立紫菜寡糖的制备方法是紫菜寡糖应用的前提。Porphyra polysaccharide is a sulfated polysaccharide, which is a unique polysaccharide component of Porphyra. It exists in the cell wall of Porphyra and accounts for 20-40% of the dry weight of Porphyra. Its main structural unit is →3)β-D-galactose-(1→4)-α-L-6-sulfate-galactose-(1→. Part of α-L-6-sulfate-half Lactose is replaced by L-3,6-lactone galactose, forming the structural unit: →3)-β-D-galactose-(1→4)-α-L-3,6-lactone galactose-(1→ .Porphyra polysaccharide has various biological activities such as anti-coagulation, lowering blood sugar, regulating blood lipid, antithrombotic, inhibiting tumor and enhancing cellular immune function, and is widely used in food and medicine industries.Porphyra oligosaccharide is directly prepared from Porphyra or Porphyra Polysaccharides are prepared by degradation, and the degree of polymerization is 2-20. However, the current preparation methods of laver oligosaccharides mainly include acid degradation, oxidative degradation, microwave, ultrasonic-assisted degradation and enzymatic degradation. Chemical degradation In the method, hydrogen peroxide has the best degradation prospect. This method has low cost and is easy to realize industrial production. However, the oxidant does not fully participate in the reaction process, and at the same time, the temperature increases, and the product is more prone to browning; biological enzymes are used to assist the reaction. , can effectively avoid side effects, and the external reaction conditions are also easy to control, however, the use of enzymatic reactions leads to increased costs; the degradation reaction is incomplete through physical methods. The molecular structure determines its biological function, and the oligosaccharide chain of laver is in molecular size, The heterogeneity of microstructure such as sequence arrangement, configuration and conformation may cause huge differences in its biological functions. Therefore, the establishment of the preparation method of Porphyra oligosaccharides is the premise of the application of Porphyra oligosaccharides.

和紫菜多糖相比紫菜寡糖具有更好的抗氧化、免疫刺激的活性和更好的吸收性,具有更广阔的应用前景。我们的研究结果显示将紫菜寡糖作为肉质改良剂作用于鱼体,不仅可以显著的改善养殖鱼的肉质,还可以提高养殖鱼的免疫力。Compared with seaweed polysaccharide, seaweed oligosaccharide has better antioxidant, immune stimulating activity and better absorption, and has broader application prospects. Our research results show that using laver oligosaccharide as a meat quality modifier on fish can not only significantly improve the meat quality of farmed fish, but also improve the immunity of farmed fish.

发明内容SUMMARY OF THE INVENTION

本发明针对现有技术的不足,一方面提供了一种紫菜寡糖的制备工艺,该制备工艺得率高,最大限度保留了寡糖中硫酸基含量,提高紫菜寡糖的活性。另一方面从改善鱼肉风味的角度,从鱼类肉质改善剂入手,提供了一种紫菜寡糖在鱼饲料中的应用。Aiming at the deficiencies of the prior art, the present invention provides a preparation process of laver oligosaccharide on the one hand, which has high yield, maximizes the retention of the sulfate group content in the oligosaccharide, and improves the activity of laver oligosaccharide. On the other hand, from the perspective of improving fish meat flavor, starting from a fish meat quality improving agent, an application of laver oligosaccharide in fish feed is provided.

为实现上述目的,本发明采用的技术方案如下:For achieving the above object, the technical scheme adopted in the present invention is as follows:

所述的紫菜寡糖的制备方法,具体制备步骤为:The preparation method of the laver oligosaccharide, the specific preparation steps are:

步骤1,将紫菜粉碎后加入20-40倍水,优选30倍;在溶液中加入固定化的纤维素酶,至终浓度为0.1-2%,优选0.8-1%;用稀HCl调整pH为4.5-6.5,优选5.0;在搅拌的情况下于30-60℃水解1-5h,优选50℃,水解3-4h;Step 1: After crushing the seaweed, add 20-40 times of water, preferably 30 times; add immobilized cellulase to the solution to a final concentration of 0.1-2%, preferably 0.8-1%; adjust the pH with dilute HCl to 4.5-6.5, preferably 5.0; hydrolyzed at 30-60°C for 1-5h under stirring, preferably 50°C, hydrolyzed for 3-4h;

步骤2,在步骤1酶解后的溶液中加入分子筛负载的Fenton催化剂,使催化剂的质量浓度为0.1-1%,优选0.5-0.7%,加入质量浓度为28-30%的过氧化氢至终浓度为10-50mM,优选30-40mM;离心除去沉淀,将得到的上清液纳滤脱盐,浓缩,干燥,得紫菜寡糖,寡糖得率为10-22%。In step 2, the molecular sieve-loaded Fenton catalyst is added to the solution after enzymatic hydrolysis in step 1, so that the mass concentration of the catalyst is 0.1-1%, preferably 0.5-0.7%, and hydrogen peroxide with a mass concentration of 28-30% is added to the end. The concentration is 10-50 mM, preferably 30-40 mM; the precipitate is removed by centrifugation, the obtained supernatant is desalted by nanofiltration, concentrated and dried to obtain laver oligosaccharide, and the yield of the oligosaccharide is 10-22%.

所述的固定化的纤维素酶的制备方法为:将干燥分子筛浸渍于质量浓度为0.5-5%的纤维素酶液中,优选2-3%;使分子筛的终浓度为0.5-5%,优选2-3%;室温搅拌6-24h,优选12-18h,离心分离并以蒸馏水洗涤,沉淀真空干燥后得到固定化酶催化剂;所述分子筛为M41S系列介孔分子筛MCM-41、MCM-48、MCM-50中的一种或几种。The preparation method of the immobilized cellulase is as follows: immersing the dry molecular sieve in a cellulase solution with a mass concentration of 0.5-5%, preferably 2-3%; making the final concentration of the molecular sieve 0.5-5%, Preferably 2-3%; stir at room temperature for 6-24h, preferably 12-18h, centrifuge and wash with distilled water, precipitate and vacuum dry to obtain immobilized enzyme catalyst; the molecular sieves are M41S series mesoporous molecular sieves MCM-41, MCM-48 , one or more of MCM-50.

所述的分子筛负载的Fenton催化剂的制备方法为:将分子筛浸渍到10-20倍体积的0.5-5M的硫酸铁水溶液中,优选12-15倍、2-3M;室温搅拌4-12h,优选8h,110℃干燥,马弗炉500-600℃焙烧2-5h后待用。The preparation method of the molecular sieve-loaded Fenton catalyst is as follows: immersing the molecular sieve in a 0.5-5M ferric sulfate aqueous solution of 10-20 times the volume, preferably 12-15 times, 2-3M; stirring at room temperature for 4-12h, preferably 8h , 110 ℃ drying, muffle furnace 500-600 ℃ roasting for 2-5h after use.

所述分子筛为M41S系列介孔分子筛MCM-41、MCM-48、MCM-50中的一种或几种。The molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50.

分子筛负载的Fenton催化剂的制备方法和固定化的纤维素酶的制备方法制备的分子筛可以一样也可以不一样。The molecular sieves prepared by the preparation method of the molecular sieve-supported Fenton catalyst and the immobilized cellulase can be the same or different.

所述的紫菜寡糖由3-连接的β-D-半乳糖-6-硫酸基和4-连接的α-L-半乳糖交替连接,寡糖聚合度为2-20,半乳糖含量为60-80%,硫酸基含量为15-30%。The laver oligosaccharide is alternately connected by 3-connected β-D-galactose-6-sulfate group and 4-connected α-L-galactose, the oligosaccharide polymerization degree is 2-20, and the galactose content is 60 -80%, sulfuric acid group content is 15-30%.

所述的紫菜寡糖作为饲料添加剂直接添加到鱼基础饲料中,添加剂量为0.05-5%,优选0.1-1%。The seaweed oligosaccharide is directly added to the basic fish feed as a feed additive, and the additive amount is 0.05-5%, preferably 0.1-1%.

本发明与现有技术比较拥有以下优点:Compared with the prior art, the present invention has the following advantages:

1、提高紫菜高值化利用水平。我国是紫菜养殖大国,后期紫菜口感较差,附加值低。本项目将紫菜寡糖应用于鱼饲料中可以极大的提高紫菜的附加值。1. Improve the level of high-value utilization of seaweed. my country is a big country of seaweed farming, and the taste of seaweed in the later stage is poor and the added value is low. The application of seaweed oligosaccharide in fish feed in this project can greatly improve the added value of seaweed.

2、紫菜寡糖制备工艺简单高效。首次提出了生化协同多相催化的紫菜寡糖高效制备新技术,该技术工艺简单,成本低,适合工业化生产,为紫菜寡糖的应用奠定了基础。2. The preparation process of seaweed oligosaccharide is simple and efficient. For the first time, a new technology for efficient preparation of laver oligosaccharides by biochemical synergistic heterogeneous catalysis is proposed. The technology is simple in process, low in cost, suitable for industrial production, and lays a foundation for the application of laver oligosaccharides.

3、固定化的纤维素酶和负载型催化剂技术比普通的纤维素酶和催化剂提高了酶和催化剂的使用效率,节约了成本。3. The immobilized cellulase and supported catalyst technology improves the use efficiency of enzymes and catalysts and saves costs compared with ordinary cellulase and catalysts.

4、拓宽了紫菜寡糖的应用领域。首次将紫菜寡糖添加到鱼类饲料中,可以改善肉质,适用于多种养殖鱼类,并且具有操作的简便性,不需改变原饲料配方。4. Broaden the application field of laver oligosaccharide. It is the first time that seaweed oligosaccharide is added to fish feed, which can improve meat quality, is suitable for a variety of farmed fish, and has the convenience of operation without changing the original feed formula.

5、紫菜寡糖天然、安全、无毒害作用。紫菜寡糖对使用对象和环境没有毒副作用,不会产生耐药性,不存在在鱼体中残留对人体健康造成伤害的风险。5. Seaweed oligosaccharide is natural, safe and non-toxic. Porphyra oligosaccharide has no toxic and side effects on the object of use and the environment, no drug resistance, and no risk of damage to human health caused by residues in fish.

6、紫菜寡糖生物活性强。紫菜寡糖不仅具有改善肉质和增强免疫力的作用,还具有抗氧化、抗衰老、抗肿瘤等药理活性。由于环境污染日益严重,鱼类的肿瘤病也越来越常见,使用紫菜寡糖有一定预防鱼类肿瘤的作用。6. Seaweed oligosaccharide has strong biological activity. Porphyra oligosaccharide not only has the effect of improving meat quality and enhancing immunity, but also has pharmacological activities such as antioxidant, anti-aging, and anti-tumor. Due to the increasingly serious environmental pollution, the tumor diseases of fish are becoming more and more common. The use of laver oligosaccharide has a certain effect on preventing fish tumors.

此外紫菜寡糖对养殖鱼类具有免疫调节作用,从而加快鱼体代谢,减少腥味物质的积累。该紫菜寡糖是一种全新、绿色和安全的鱼类饲料添加剂,能够有效解决养殖鱼类肉质疏松、蛋白含量偏低和口感较差的问题,同时能够提高鱼的免疫力,能够为养殖鱼拓宽市场。In addition, laver oligosaccharides have immunomodulatory effects on farmed fish, thereby accelerating the metabolism of fish and reducing the accumulation of fishy substances. The seaweed oligosaccharide is a brand-new, green and safe fish feed additive, which can effectively solve the problems of loose meat, low protein content and poor taste in farmed fish, at the same time, it can improve the immunity of fish, and can provide food for farmed fish. Broaden the market.

附图说明Description of drawings

图1紫菜寡糖饲料PS对石斑鱼鱼体粗蛋白和粗脂肪的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 1 Effects of seaweed oligosaccharide diet PS on crude protein and crude fat of grouper fish, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图2紫菜寡糖饲料PS对石斑鱼头肾呼吸爆发活性氧产生量的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 2 The effect of seaweed oligosaccharide diet PS on the production of reactive oxygen species in the head kidney of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图3紫菜寡糖饲料PS对石斑鱼增重率和存活率的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 3 The effect of seaweed oligosaccharide diet PS on the weight gain and survival rate of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图4紫菜寡糖饲料PS对鲈鱼鱼体粗蛋白和粗脂肪的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 4 Effects of seaweed oligosaccharide diet PS on crude protein and crude fat of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图5不同海藻寡糖饲料对鲈鱼头肾呼吸爆发活性氧产生量的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 5 Effects of different seaweed oligosaccharide diets on the production of reactive oxygen species in the head kidney of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图6不同海藻寡糖饲料对鲈鱼鱼体粗蛋白和粗脂肪的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 6 Effects of different seaweed oligosaccharide diets on crude protein and crude fat of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图7紫菜寡糖和紫菜多糖饲料对石斑鱼体粗蛋白和粗脂肪的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 7 Effects of laver oligosaccharide and laver polysaccharide diet on grouper body crude protein and crude fat, *P<0.05 compared with the control group, **P<0.01 compared with the control group

图8添加不同剂量紫菜寡糖OP的水产饲料对石斑鱼体粗蛋白和粗脂肪的影响,*和对照组相比P<0.05,**和对照组相比P<0.01Figure 8 The effect of adding different doses of laver oligosaccharide OP on the crude protein and crude fat of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

具体实施方式Detailed ways

下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本发明所限定的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the limited scope of the present invention.

实施例1,紫菜寡糖的制备Embodiment 1, the preparation of seaweed oligosaccharide

方法一、紫菜寡糖的生化协同生物催化高效制备技术(本发明专利使用的方法)Method 1. High-efficiency preparation technology of biochemical synergistic biocatalysis of laver oligosaccharide (the method used in the patent of the present invention)

①固定化酶催化剂(酶-MCM-48)的制备① Preparation of immobilized enzyme catalyst (enzyme-MCM-48)

称取3g纤维素酶加入到50mL醋酸-醋酸钠缓冲溶液(pH=4.5)中,搅拌1h,离心收集上清液,沉淀继续搅拌1h并离心,至全部溶解,溶液定容至100mL,制得纤维素酶酶液。称取10g干燥MCM-48分子筛浸渍于100mL 3%的纤维素酶液中室温搅拌12h,离心分离并以蒸馏水洗涤。沉淀真空干燥后得到固定化酶催化剂。Weigh 3 g of cellulase and add it to 50 mL of acetic acid-sodium acetate buffer solution (pH=4.5), stir for 1 hour, collect the supernatant by centrifugation, continue to stir the precipitate for 1 hour and centrifuge until it is completely dissolved, and make up the solution to 100 mL. Cellulase enzyme solution. 10 g of dry MCM-48 molecular sieves were weighed and immersed in 100 mL of 3% cellulase solution, stirred at room temperature for 12 h, centrifuged and washed with distilled water. The immobilized enzyme catalyst was obtained after the precipitate was vacuum-dried.

②分子筛负载的Fenton催化剂(Fe-MCM-48)的制备② Preparation of molecular sieve supported Fenton catalyst (Fe-MCM-48)

配置2M硫酸铁水溶液100mL,称取10g干燥MCM-48分子筛浸渍其中,室温搅拌8h,110℃干燥,马弗炉500℃焙烧3h小时后待用。Prepare 100 mL of 2M ferric sulfate aqueous solution, weigh 10 g of dry MCM-48 molecular sieve and immerse it in it, stir at room temperature for 8 hours, dry at 110 °C, and roast in a muffle furnace at 500 °C for 3 hours before use.

③酶-MCM-48和Fe-MCM-48协同降解紫菜寡糖的工艺研究③Study on the process of synergistic degradation of Porphyra oligosaccharides by enzyme-MCM-48 and Fe-MCM-48

首先采用酶-MCM-48对紫菜进行酶解。将粉碎后的紫菜加入30倍水,加入酶-MCM-48至终浓度为1%,调pH为5.0,50℃搅拌水解3h,反应结束后滤除催化剂。在酶解后的溶液中加入Fe-MCM-48至终浓度为0.5%,加入30%的过氧化氢至终浓度为30mM。在搅拌的情况下于60℃水解3h,离心除去沉淀,将得到的上清液纳滤脱盐,浓缩,干燥,得紫菜寡糖OP,得率为15.4%。经测定,OP中半乳糖含量为66.2%,硫酸基含量为24.3%,聚合度为2-20。Firstly, the enzyme-MCM-48 was used for enzymatic hydrolysis of seaweed. Add 30 times water to the pulverized seaweed, add enzyme-MCM-48 to a final concentration of 1%, adjust the pH to 5.0, stir and hydrolyze at 50°C for 3 hours, and filter out the catalyst after the reaction. Fe-MCM-48 was added to the solution after enzymolysis to a final concentration of 0.5%, and 30% hydrogen peroxide was added to a final concentration of 30 mM. Under stirring, hydrolyzed at 60° C. for 3 hours, centrifuged to remove the precipitate, the obtained supernatant was desalted by nanofiltration, concentrated and dried to obtain laver oligosaccharide OP with a yield of 15.4%. It was determined that the content of galactose in OP was 66.2%, the content of sulfate group was 24.3%, and the degree of polymerization was 2-20.

方法二、紫菜寡糖的硫酸水解制备技术(文献中紫菜寡糖的制备方法)Method 2. Preparation technology of sulfuric acid hydrolysis of laver oligosaccharide (preparation method of laver oligosaccharide in literature)

①紫菜多糖的制备①Preparation of seaweed polysaccharide

将粉碎后紫菜加入20倍水,120度,高压锅提取3h,4000r/min离心15min,上清液直接透析,透析结束后,收集透析液,浓缩,干燥,得紫菜多糖(P),得率为18%。Add 20 times water to the pulverized laver, 120 degrees, extract in a pressure cooker for 3 hours, centrifuge at 4000 r/min for 15 minutes, and dialyze the supernatant directly. After the dialysis, collect the dialysate, concentrate, and dry to obtain laver polysaccharide (P). The yield is 18%.

②紫菜寡糖的制备②Preparation of seaweed oligosaccharides

5g紫菜多糖加150mL水,室温过夜搅拌溶解。取5.435mL浓硫酸稀释至50mL,加入紫菜多糖溶液,边加边搅拌混匀,80度搅拌反应3小时(加回流)。反应结束称取31.546g Ba(OH)2·8H2O,配成饱和溶液中和(持续搅拌)。4000r/min,8min离心,取上清过滤,浓缩,截留分子量为1kDa的透析袋透析,冻干,得紫菜寡糖OM,得率为55%,按紫菜换算得率为9.9%。经测定,OM中半乳糖含量为55.6%,硫酸基含量为14.3%,聚合度为2-20。Add 5 g of seaweed polysaccharide to 150 mL of water, stir and dissolve at room temperature overnight. Take 5.435 mL of concentrated sulfuric acid and dilute it to 50 mL, add laver polysaccharide solution, stir and mix while adding, and stir and react at 80 degrees for 3 hours (add reflux). At the end of the reaction, 31.546 g of Ba(OH) 2 ·8H 2 O was weighed and made into a saturated solution for neutralization (continuous stirring). Centrifuge at 4000 r/min for 8 min, take the supernatant for filtration, concentrate, dialyze the dialysis bag with a molecular weight cut-off of 1 kDa, and freeze-dry to obtain OM with a yield of 55%, which is 9.9% according to the conversion of laver. It was determined that the content of galactose in OM was 55.6%, the content of sulfate groups was 14.3%, and the degree of polymerization was 2-20.

比较方法一和方法二,方法一制备获得的紫菜寡糖OP得率、半乳糖和硫酸基含量都比方法二获得的紫菜寡糖OM高,而且方法一步骤简单,更适合工业生产。同时,我们还尝试只用纤维素酶、琼胶酶等酶解的方法和只用H2O2氧化降解的方法制备紫菜寡糖,这两种方法获得的样品分子量均大于5000Da,未能获得聚合度低的寡糖样品。综合比较,本发明专利建立的紫菜寡糖的制备方法简单高效,获得的寡糖样品质量好,适合工业放大生产,有很好的应用潜力。Comparing method 1 and method 2, the yield of laver oligosaccharide OP, galactose and sulfate group content prepared by method 1 are higher than those of laver oligosaccharide OM obtained by method 2, and method 1 has simple steps and is more suitable for industrial production. At the same time, we also tried to prepare laver oligosaccharides only by enzymatic hydrolysis methods such as cellulase and agarase, and by only using H 2 O 2 oxidative degradation methods. Oligosaccharide samples with low degree of polymerization. Comprehensive comparison, the preparation method of laver oligosaccharide established by the patent of the present invention is simple and efficient, the obtained oligosaccharide sample is of good quality, suitable for industrial scale-up production, and has good application potential.

实施例2,紫菜寡糖饲料(PS、PO)、紫菜多糖饲料(PP)和紫菜粉饲料(PM)的制备Example 2, preparation of laver oligosaccharide feed (PS, PO), laver polysaccharide feed (PP) and laver powder feed (PM)

将实施例1方法一得到的紫菜寡糖提取物(OP)按饲料总重0.05-5%添加到饲料中,在这个范围内的任何一浓度都可以显著改善养殖鱼的肉质和风味,增进机体细胞免疫力,提高其抗病害能力,实施例中仅以几种不同浓度作为代表样品。制备方法为:先将紫菜寡糖提取物制成8-12%的溶液,优选10%;然后在搅拌下加入无水乙醇使乙醇的总浓度为10%-20%,优选15%;将制得的溶液均匀的喷雾与饲料上,制得紫菜寡糖占饲料总重量的0.1%的饲料,烘干或者晾干后保存备用,记为PS。按同样方法制备含实施例1方法二得到的紫菜寡糖OM 0.1%的饲料PO,实施例1方法二得到的含紫菜多糖3%的饲料PP和含紫菜粉20%的饲料PM。The seaweed oligosaccharide extract (OP) obtained by the first method in Example 1 is added to the feed at 0.05-5% of the total weight of the feed. Any concentration within this range can significantly improve the meat quality and flavor of the farmed fish, and enhance the body. Cell immunity, improve its ability to resist disease, only a few different concentrations are used as representative samples in the examples. The preparation method is as follows: firstly, the laver oligosaccharide extract is made into a solution of 8-12%, preferably 10%; then anhydrous ethanol is added under stirring so that the total concentration of the ethanol is 10%-20%, preferably 15%; The obtained solution was evenly sprayed on the feed to obtain a feed with laver oligosaccharides accounting for 0.1% of the total weight of the feed, which was dried or dried and stored for later use, which was recorded as PS. The same method was used to prepare feed PO containing 0.1% laver oligosaccharide OM obtained in method 2 of Example 1, feed PP containing 3% laver polysaccharide obtained in method 2 of Example 1, and feed PM containing 20% laver powder.

实施例3,饲喂含有紫菜寡糖的水产饲料对石斑鱼肉质改善和免疫调节的作用Example 3, the effect of feeding the aquatic feed containing seaweed oligosaccharide on the improvement of grouper meat quality and immune regulation

本试验使用含有紫菜寡糖的水产饲料喂养石斑鱼,通过石斑鱼体成分变化、肌肉质构、生理功能相关指标变化,来说明其对石斑鱼肉质改善和免疫调节的作用。In this experiment, aquafeed containing seaweed oligosaccharide was used to feed grouper, and the changes in body composition, muscle texture, and physiological function-related indicators of grouper were used to illustrate its effects on the improvement of grouper meat quality and immune regulation.

试验方法:选择健康均匀的石斑鱼随机分成3组,分别使用不同的饵料,按正常的饲养管理程序进行。第1组为市售水产饲料F对照组,第2组为PM对照组,第3组为PS实验组,试验期为42天,每隔14天取9尾鱼采血,并无菌取头肾用于测定头肾巨噬细胞呼吸爆发。在超净工作台中取出鱼的头肾,放入含有1ml PBS的1.5ml离心管中,于冰上研碎,使用100目的细胞筛,除去组织团块,500g离心去上清,用PBS洗2次,重悬制备单细胞悬液,细胞计数不少于106个细胞。将单细胞悬液中加入DCFH-DA荧光探针孵育。后续操作依照南京建成活性氧(ROS)测试盒说明书操作。试验结束后对增重率、存活率进行测定,并取背肌(2cm×2cm×1.5cm)进行肌肉质构(TPA)特性分析,未取组织鱼体用于粗蛋白、粗脂肪测定。血清中抗氧化酶(SOD、CAT)总补体CH50、碱性磷酸酶AKP的活性采用南京建成试剂盒测定。Test method: Select healthy and uniform groupers and randomly divide them into 3 groups, use different bait respectively, and carry out according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the PM control group, and the third group was the PS experimental group. The test period was 42 days. Blood was collected from 9 fish every 14 days, and the head and kidney were aseptically removed. Used to measure head kidney macrophage respiratory burst. Take out the head kidney of the fish from the ultra-clean workbench, put it into a 1.5ml centrifuge tube containing 1ml PBS, grind it on ice, use a 100-mesh cell sieve to remove tissue clumps, centrifuge at 500g to remove the supernatant, and wash with PBS for 2 Next, resuspend to prepare a single cell suspension, and the cell count is not less than 10 6 cells. The single cell suspension was incubated with DCFH-DA fluorescent probe. Follow-up operations were performed in accordance with the Nanjing Jiancheng reactive oxygen species (ROS) test kit instructions. After the experiment, the weight gain rate and survival rate were determined, and the back muscles (2cm×2cm×1.5cm) were taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat. The activities of total complement CH50 of antioxidant enzymes (SOD, CAT) and alkaline phosphatase AKP in serum were determined by Nanjing Jiancheng kit.

结果:result:

试验期间,石斑鱼的增重率和存活率如图1所示,第3组增重率较基础饲料组显著提高,并且其增重率也高于20%紫菜粉添加组,证明紫菜寡糖对石斑鱼的增重有明显促进作用且其增重效果优于直接添加高剂量紫菜粉。试验期间第3组石斑鱼的存活率也比基础饲料组和20%紫菜粉添加组更高。During the test, the weight gain rate and survival rate of grouper were shown in Figure 1. The weight gain rate of group 3 was significantly higher than that of the basal diet group, and its weight gain rate was also higher than that of the 20% seaweed powder addition group, which proved that the oligarch of seaweed Sugar can obviously promote the weight gain of grouper, and its weight gain effect is better than adding high-dose seaweed powder directly. The survival rate of grouper in group 3 was also higher than that in the basal diet group and the 20% laver powder-supplemented group during the test period.

由表1可以看出,饲喂添加有紫菜寡糖的第3组比对照组超氧化物歧化酶SOD活性均有显著提高,在前28天饲喂过程中,第3组超氧化物歧化酶SOD活性始终呈上升趋势,但28天后有所下降,这也与对照组趋势一致。对于总补体CH50值,第3组较基础饲料组也有明显增加,但总体趋势与对照组相似,随饲喂实验的延长而减少。对于过氧化氢酶CAT,添加紫菜寡糖的实验组较对照组更高,随时间推移先减少后升高。对于碱性磷酸酶AKP,三组均随时间的延长而降低,但第三组较对照组和紫菜粉添加组活性更高。值得一提的是紫菜寡糖添加组的所有指标均优于紫菜粉添加组。SOD、CH50、CAT、AKP与石斑鱼的免疫能力密切相关,这些指标的升高证明了添加了紫菜提取物的饲料具有积极的免疫调节作用。It can be seen from Table 1 that the superoxide dismutase SOD activity of the third group fed with laver oligosaccharide was significantly improved compared with the control group. During the first 28 days of feeding, the third group of superoxide dismutase The SOD activity always showed an upward trend, but decreased after 28 days, which was also consistent with the trend of the control group. For the total complement CH 50 value, the third group also increased significantly compared with the basal diet group, but the overall trend was similar to the control group, and decreased with the prolongation of the feeding experiment. For catalase CAT, the experimental group with added laver oligosaccharide was higher than the control group, which decreased at first and then increased over time. For alkaline phosphatase AKP, the three groups all decreased with time, but the third group had higher activity than the control group and the laver powder-added group. It is worth mentioning that all the indexes of the seaweed oligosaccharide-added group were better than those of the seaweed powder-added group. SOD, CH 50 , CAT and AKP are closely related to the immune ability of grouper, and the increase of these indicators proves that the feed supplemented with seaweed extract has a positive immune regulation effect.

表1对石斑鱼血清中特异性指标影响Table 1 Effects of specific indicators in grouper serum

Figure BDA0003024772910000061
Figure BDA0003024772910000061

Figure BDA0003024772910000071
Figure BDA0003024772910000071

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

由图2可以看出,紫菜寡糖添加组头肾巨噬细胞呼吸爆发产生的活性氧ROS水平显著高于对照组和紫菜粉添加组,表明其非特异性免疫能力显著提升。It can be seen from Figure 2 that the level of reactive oxygen species ROS generated by the respiratory burst of macrophages in the head kidney in the laver oligosaccharide-added group was significantly higher than that in the control group and the laver powder-added group, indicating that its non-specific immunity was significantly improved.

由图3,可以看出相较于对照组,紫菜寡糖添加组有效的提高了鱼体粗蛋白含量,降低了鱼体粗脂肪含量。From Figure 3, it can be seen that compared with the control group, the seaweed oligosaccharide addition group effectively increased the crude protein content of the fish and reduced the crude fat content of the fish.

由表2可以看出PS组在肌肉硬度、弹性、胶着性、咀嚼性、回复性均优于对照组和紫菜粉添加组。It can be seen from Table 2 that the PS group is superior to the control group and the laver powder-added group in muscle hardness, elasticity, adhesiveness, chewiness, and recovery.

表2石斑鱼肌肉质构(TPA)特征测定结果Table 2. Results of determination of grouper muscle texture (TPA) characteristics

Figure BDA0003024772910000072
Figure BDA0003024772910000072

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

实施例4,饲喂含有紫菜寡糖的水产饲料对鲈鱼肉质改善和免疫调节的作用Example 4, the effect of feeding the aquatic feed containing seaweed oligosaccharide on the improvement of sea bass meat quality and immune regulation

本试验使用含有紫菜寡糖的水产饲料喂养鲈鱼,通过鲈鱼体成分变化、肌肉质构、生理功能相关指标变化,来说明其对鲈鱼肉质改善和免疫调节的作用。In this experiment, the aquatic feed containing seaweed oligosaccharide was fed to sea bass, and the changes in body composition, muscle texture, and physiological function-related indicators of sea bass were used to illustrate its effects on the improvement of sea bass meat quality and immune regulation.

试验方法:experiment method:

选择健康均匀的鲈鱼随机分成3组,分别使用不同的饵料,按正常的饲养管理程序进行。第1组为市售水产饲料F对照组,第2组为20%紫菜粉添加饲料(PM)对照组,第3组为PS实验组,试验期为28天,分别在0天、14天和28天随机取各组试验鱼10尾,尾静脉取血,每尾0.5ml,全血室温静置3h后,于4℃静置5h,离心收集血清,-20℃贮存备用样本,用于血清非特异性免疫指标测定(SOD、AKP、LZM)。饲喂实验结束后取背肌(2cm×2cm×1.5cm)进行肌肉质构(TPA)特性分析,未取组织鱼体用于粗蛋白、粗脂肪测定。Select healthy and uniform sea bass and randomly divide them into 3 groups, using different bait respectively, according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the 20% seaweed powder added feed (PM) control group, and the third group was the PS experimental group. The test period was 28 days. 10 experimental fish from each group were randomly selected for 28 days, and blood was collected from the tail vein, 0.5 ml per tail. After the whole blood was allowed to stand at room temperature for 3 hours, it was allowed to stand at 4 °C for 5 hours, and the serum was collected by centrifugation. The spare samples were stored at -20 °C for serum. Determination of non-specific immune indicators (SOD, AKP, LZM). After the feeding experiment, the dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat.

结果:result:

由图4可以看出紫菜寡糖添加组相较于对照组鲈鱼鱼体粗蛋白含量显著增加,粗脂肪含量显著降低。It can be seen from Figure 4 that the crude protein content of the sea bass in the seaweed oligosaccharide addition group was significantly increased compared with the control group, and the crude fat content was significantly reduced.

由表3可以看出,与对照组相比,PS组的非特异性免疫指标均有所增加且比PM组增加更为明显,说明添加紫菜寡糖能够有效的提升水产动物非特异性免疫能力As can be seen from Table 3, compared with the control group, the non-specific immune indexes of the PS group increased and the increase was more obvious than that of the PM group, indicating that the addition of seaweed oligosaccharides can effectively improve the non-specific immunity of aquatic animals.

表3对鲈鱼非特异性免疫指标的影响Table 3 Effects on non-specific immune indexes of sea bass

Figure BDA0003024772910000081
Figure BDA0003024772910000081

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

由表4可以看出紫菜粉添加组和紫菜寡糖添加组在硬度、黏着性、胶着度、咀嚼度四个方面均优于对照组,其中紫菜寡糖添加组肌肉性质改善更为显著,在鲈鱼肌肉黏着度方面,三个组未表现出显著性差异。It can be seen from Table 4 that the laver powder-added group and the laver oligosaccharide-added group are better than the control group in four aspects: hardness, stickiness, adhesiveness, and chewiness, and the muscle properties of the laver oligosaccharide-added group are improved more significantly. There was no significant difference among the three groups in terms of muscle adhesion of sea bass.

表4鲈鱼肌肉质构(TPA)特征测定结果Table 4. Results of determination of texture of perch muscle texture (TPA) characteristics

组别group 硬度(N)Hardness (N) 弹性(mm)Elasticity(mm) 黏着性(g)Adhesion (g) 胶着度(N)Adhesion (N) 咀嚼度(N)Chewability (N) 对照组control group 47.87±0.6247.87±0.62 0.33±0.020.33±0.02 0.21±0.010.21±0.01 6.38±0.076.38±0.07 1.72±0.231.72±0.23 PM组PM group 46.23±1.0346.23±1.03 0.35±0.040.35±0.04 0.23±0.020.23±0.02 7.72±0.12<sup>*</sup>7.72±0.12<sup>*</sup> 2.14±0.03<sup>*</sup>2.14±0.03<sup>*</sup> PS组PS group 50.21±0.33<sup>*</sup>50.21±0.33<sup>*</sup> 0.38±0.01<sup>*</sup>0.38±0.01<sup>*</sup> 0.22±0.030.22±0.03 8.92±0.08<sup>**</sup>8.92±0.08<sup>**</sup> 3.32±0.16<sup>**</sup>3.32±0.16<sup>**</sup>

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

实施例5,几种添加不同海洋寡糖水产饲料对鲈鱼肉质改善和免疫调节的作用的对比Example 5, the comparison of the effects of several additions of different marine oligosaccharide aquatic feeds on the improvement of sea bass meat quality and immune regulation

本试验分别使用含有0.1%壳寡糖,岩藻寡糖、褐藻胶寡糖、紫菜寡糖的水产饲料喂养鲈鱼,通过鲈鱼体成分变化、肌肉质构、生理功能相关指标变化,来说明不同寡糖对鲈鱼肉质改善和免疫调节的作用。In this experiment, the aquatic feed containing 0.1% chitosan oligosaccharide, fucoid oligosaccharide, alginate oligosaccharide and seaweed oligosaccharide was used to feed perch, and the changes in body composition, muscle texture and physiological function of perch were used to explain the different oligosaccharides. Effects of sugar on meat quality improvement and immune regulation in sea bass.

选择健康均匀的鲈鱼随机分成5组,分别使用不同的饵料,按正常的饲养管理程序进行。第1组为市售水产饲料F对照组,第2组为PS实验组,第3组为添加0.1%壳寡糖实验组,第4组为添加0.1%岩藻寡糖实验组,第5组为添加0.1%褐藻胶寡糖实验组。试验期为28天。取头肾测定呼吸爆发,未取组织鱼体用于粗蛋白、粗脂肪测定。Select healthy and uniform sea bass and randomly divide them into 5 groups, using different bait respectively, according to the normal feeding and management procedures. The first group is the commercial aquatic feed F control group, the second group is the PS experimental group, the third group is the 0.1% chitosan oligosaccharide addition experimental group, the fourth group is the 0.1% fucoid oligosaccharide addition experimental group, and the fifth group For the experimental group added with 0.1% alginate oligosaccharide. The trial period was 28 days. The head kidney was taken to measure the respiratory burst, and the fish body was not taken for the determination of crude protein and crude fat.

结果:result:

由图5可以看出,寡糖添加组相较基础饲料对照组均对鲈鱼头肾巨噬细胞呼吸爆发产生的活性氧ROS水平有促进作用,其中紫菜寡糖添加组ROS水平最高,壳寡糖次之,该实验表明紫菜寡糖能够有效提升鲈鱼非特异性免疫能力,并且比壳寡糖、岩藻寡糖、褐藻胶寡糖有更好的促进作用。It can be seen from Figure 5 that the oligosaccharide supplementation group has a promoting effect on the reactive oxygen species ROS level produced by the macrophage respiratory burst in the perch head kidney compared with the basic diet control group. Secondly, this experiment shows that seaweed oligosaccharide can effectively improve the non-specific immunity of sea bass, and has a better promoting effect than chitosan oligosaccharide, fucoid oligosaccharide, and algin oligosaccharide.

由图6可以看出紫菜寡糖添加组鱼体粗蛋白含量与对照组相比有显著增加,鱼体粗脂肪含量与对照组相比显著降低,其他寡糖体成分改变并不显著,褐藻胶寡糖添加组鱼体粗蛋白与对照组相比略有降低。It can be seen from Figure 6 that the crude protein content of the fish body in the seaweed oligosaccharide addition group was significantly increased compared with the control group, and the crude fat content of the fish body was significantly reduced compared with the control group. The composition of other oligosaccharides did not change significantly. Compared with the control group, the crude protein of fish in the oligosaccharide-added group decreased slightly.

实施例6,添加紫菜寡糖和紫菜多糖的水产饲料对石斑鱼肉质改善作用的对比Example 6, the comparison of the effect of adding seaweed oligosaccharide and seaweed polysaccharide on the improvement of grouper meat quality

本试验分别使用含有实施例1制备的紫菜寡糖的水产饲料(PS和PO)和未降解的紫菜多糖的水产饲料(PP)喂养石斑鱼,通过石斑鱼体成分变化、肌肉质构变化,来说明其紫菜寡糖和紫菜多糖对石斑鱼肉质改善作用的差异。In this experiment, the aquatic feeds (PS and PO) containing laver oligosaccharides prepared in Example 1 and the aquafeeds (PP) containing undegraded laver polysaccharides were used to feed groupers, and the changes in body composition and muscle texture of groupers were observed. , to illustrate the difference in the improvement of seaweed oligosaccharides and seaweed polysaccharides on grouper meat quality.

选择健康均匀的石斑鱼随机分成4组,分别使用不同的饵料,按正常的饲养管理程序进行。第1组为市售水产饲料F对照组,第2组为PS实验组,第3组为PO实验组,第4组为PP实验组。试验期为28天。取背肌(2cm×2cm×1.5cm)进行肌肉质构(TPA)特性分析,未取组织鱼体用于粗蛋白、粗脂肪测定。Select healthy and uniform groupers and randomly divide them into 4 groups, use different bait respectively, and carry out the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the PS experimental group, the third group was the PO experimental group, and the fourth group was the PP experimental group. The trial period was 28 days. The dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat.

由图7可知,PS组相较于基础饲料组鱼体粗蛋白含量更高,粗脂肪含量降低,PO组和紫菜多糖添加组相较基础饲料组鱼体成分并未表现出显著性差异。说明不同工艺制备的紫菜寡糖活性是不同,本发明采用的工艺能够最大程度保留紫菜寡糖的生物活性。这可能是因为本工艺对硫酸基的破坏较小,硫酸基是紫菜寡糖发挥活性的重要基团。It can be seen from Figure 7 that compared with the basal feed group, the PS group had higher crude protein content and lower crude fat content, and the PO group and the laver polysaccharide added group showed no significant difference in fish body composition compared with the basal feed group. It shows that the activities of laver oligosaccharides prepared by different processes are different, and the process adopted in the present invention can retain the biological activity of laver oligosaccharides to the greatest extent. This may be because this process has less damage to the sulfate group, which is an important group for the activity of the laver oligosaccharide.

由表5可知,PO和PP组在肌肉质构特征指标方面与对照组相比并未表现出显著性差异,这可能是由于紫菜多糖分子量较大,不容易被水产动物吸收,而寡糖PO组因为含有的硫酸基较低,活性也不显著。紫菜寡糖PS组则在弹性、胶着度、咀嚼度、回复性方面均表现出了显著提升。It can be seen from Table 5 that the PO and PP groups did not show significant differences in muscle texture characteristics compared with the control group, which may be due to the large molecular weight of laver polysaccharides, which are not easily absorbed by aquatic animals, and the oligosaccharide PO. The activity of the group is not significant because of the lower sulfate group content. The laver oligosaccharide PS group showed significant improvement in elasticity, adhesiveness, chewiness, and recovery.

表5石斑鱼肌肉质构(TPA)特征测定结果Table 5. Results of determination of grouper muscle texture (TPA) characteristics

Figure BDA0003024772910000101
Figure BDA0003024772910000101

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

实施例7,添加不同剂量紫菜寡糖OP的水产饲料对石斑鱼肉质改善作用的对比Example 7, the comparison of the effect of adding different doses of seaweed oligosaccharide OP on the improvement of grouper meat quality

本试验按实施例2的方法制备含有0.05%、0.1%、1%、5%、10%和20%紫菜寡糖的水产饲料PS1、PS2、PS3、PS4、PS5和PS6喂养石斑鱼,通过石斑鱼体成分变化、肌肉质构变化,来说明其紫菜寡糖和紫菜多糖对石斑鱼肉质改善作用的差异。In this experiment, aquafeeds PS1, PS2, PS3, PS4, PS5 and PS6 containing 0.05%, 0.1%, 1%, 5%, 10% and 20% of seaweed oligosaccharides were prepared according to the method of Example 2 and fed to groupers. The changes of body composition and muscle texture of grouper were used to illustrate the difference of the improvement effects of seaweed oligosaccharides and seaweed polysaccharides on grouper meat quality.

选择健康均匀的石斑鱼随机分成7组,分别使用不同的饵料,按正常的饲养管理程序进行。第1组为市售水产饲料F对照组,第2-7组分别为PS1-PS6实验组,试验期为28天。取背肌(2cm×2cm×1.5cm)进行肌肉质构(TPA)特性分析,取鱼体组织用于粗蛋白、粗脂肪测定。Select healthy and uniform groupers and randomly divide them into 7 groups, use different bait respectively, and carry out according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, and the second to seventh groups were the PS1-PS6 experimental groups respectively, and the experimental period was 28 days. The dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body tissue was taken for the determination of crude protein and crude fat.

由图8可知,含有不同剂量寡糖的PS组相较于基础饲料组鱼体粗蛋白含量更高,粗脂肪含量降低,当紫菜寡糖添加量大于5%时,各组对鱼体粗蛋白和粗脂肪含量影响不大。当紫菜寡糖添加量小于5%时,随着紫菜寡糖添加量的增加,各组可以逐渐增加鱼体粗蛋白含量,逐渐减少鱼体粗脂肪含量。这说明当紫菜寡糖添加量超过一定剂量时,随着添加剂量的增加对鱼体粗蛋白和粗脂肪成分也不会造成影响,添加量为0.05%-5%较为合适。It can be seen from Figure 8 that the PS group containing different doses of oligosaccharides has higher crude protein content and lower crude fat content than the basic feed group. and crude fat content have little effect. When the added amount of seaweed oligosaccharide was less than 5%, with the increase of the added amount of seaweed oligosaccharide, each group could gradually increase the content of crude protein in fish body and reduce the content of crude fat in fish body gradually. This shows that when the added amount of seaweed oligosaccharide exceeds a certain dose, the crude protein and crude fat components of fish will not be affected with the increase of the added dose, and the addition amount of 0.05%-5% is more suitable.

由表6可知,和对照组相比,含有不同剂量寡糖的PS组在肌肉质构特征指标方面表现出显著性差异。在弹性、胶着度、咀嚼度、回复性方面均表现出了显著提升。各指标随着当紫菜寡糖添加量大于5%时,各指标变化相对趋于平稳,这说明紫菜寡糖添加0.05-5%比较合适。As can be seen from Table 6, compared with the control group, PS groups containing different doses of oligosaccharides showed significant differences in muscle texture characteristics. The elasticity, stickiness, chewiness and recovery have all been significantly improved. When the amount of laver oligosaccharide added was more than 5%, the changes of each index tended to be relatively stable, which indicated that the addition of 0.05-5% of laver oligosaccharide was more appropriate.

表6石斑鱼肌肉质构(TPA)特征测定结果Table 6. Results of determination of grouper muscle texture (TPA) characteristics

Figure BDA0003024772910000111
Figure BDA0003024772910000111

*和对照组相比P<0.05,**和对照组相比P<0.01 * P<0.05 compared to the control group, ** P<0.01 compared to the control group

以上为对本发明实施例的描述,通过对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. A preparation method of laver oligosaccharide is characterized in that:
the preparation method comprises the following specific steps:
step 1, pulverizing laver and adding 20-40 times of water; adding immobilized cellulase into the solution to a final concentration of 0.1-2%; adjusting pH to 4.5-6.5 with HCl with concentration of 0.1-1M; hydrolyzing at 30-60 deg.C for 1-5h under stirring;
step 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.1-1%, and adding hydrogen peroxide with the mass concentration of 28-30% to the final concentration of 10-50 mM; centrifuging to remove precipitate, nano-filtering the obtained supernatant for desalting, concentrating, and drying to obtain laver oligosaccharide; the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 0.5-5%; the final concentration of the molecular sieve is 0.5-5%; stirring for 6-24h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain an immobilized enzyme catalyst; wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50;
the preparation method of the molecular sieve supported Fenton catalyst comprises the following steps: soaking the molecular sieve into 10-20 times volume of 0.5-5M ferric sulfate aqueous solution; stirring at room temperature for 4-12h, drying at 110 deg.C, and roasting in muffle furnace at 500-600 deg.C for 2-5 h.
2. The method of claim 1,
in the step 1, adding immobilized cellulase into the solution until the final concentration is 0.8-1%; hydrolyzing at 50 deg.C for 3-4h under stirring; and 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.5-0.7%, and adding hydrogen peroxide with the mass concentration of 28-30% to ensure that the final concentration is 30-40 mM.
3. The method of claim 1,
the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 2-3%; the final concentration of the molecular sieve is 2-3%; stirring for 12-18h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain the immobilized enzyme catalyst.
4. The method according to claim 1, wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48, MCM-50.
5. Use of the porphyra oligosaccharide prepared by the preparation method of any one of claims 1 to 4 in fish feed.
6. The use of the porphyra oligosaccharide in fish feed according to claim 5, wherein the porphyra oligosaccharide is directly added to the fish basal feed as a feed additive in an amount of 0.05-5%.
7. The use of the porphyra oligosaccharide in fish feed according to claim 6, wherein the porphyra oligosaccharide is directly added into the basic feed of fish as a feed additive, and the addition amount is 0.1-1%.
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