CN117363671A - 一种发酵提高海参肠多糖提取率及生物活性的方法 - Google Patents
一种发酵提高海参肠多糖提取率及生物活性的方法 Download PDFInfo
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- CN117363671A CN117363671A CN202311371196.0A CN202311371196A CN117363671A CN 117363671 A CN117363671 A CN 117363671A CN 202311371196 A CN202311371196 A CN 202311371196A CN 117363671 A CN117363671 A CN 117363671A
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- Prior art keywords
- sea cucumber
- polysaccharide
- intestinal
- intestines
- enzymolysis liquid
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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Abstract
本发明属于生物技术领域,具体涉及一种发酵提高海参肠多糖提取率及生物活性的方法。本发明所公开的海氏肠球菌GS22(Enterococcus hirae GS22)能够提高从海参肠中提取多糖的得率,并改善海参肠多糖抗氧化和降糖的生物活性,在发酵海参肠方面有巨大应用潜力,为海参的高值化利用提供新途径。
Description
技术领域
本发明属于生物技术领域,具体涉及一株海氏肠球菌GS22(Enterococcus hiraeGS22)发酵海参肠酶解液后提取海参肠多糖,显著提高了从海参肠中提取多糖的得率及其抗氧化和体外降血糖功能。
背景技术
海参属于棘皮动物门,是一种珍贵的海洋食品,也是重要的药用食物来源,其含有多种生物活性物质,包括多糖、皂苷、多肽、蛋白质等,这些活性物质具有多种生理功能,对保持和提高人体健康水平十分重要。目前海参功能成分研究多以体壁为原料,对海参肠研究很少。海参肠是海参加工过程中的副产物,其加工利用度低,部分被大量丢弃造成资源浪费和环境污染。海参多糖是海参中重要的功能成分之一,海参不同身体部位,如体壁、触手/花、内脏等都含有多糖,研究已证实海参体壁多糖具有抗凝、降血脂、降血糖、抗氧化、抗肿瘤、抗癌的等多种生物活性,因此可以作为某些疾病治疗和预防的潜在药物,对于海参肠多糖目前还没有充分挖掘利用。
海参多糖常用的提取方法有化学水解法、酶解法和热水法。化学水解法和酶解法通过破坏多糖和蛋白质间的连结使二者分离。化学水解法操作简单,但会过度降解多糖分子并出现脱硫现象。酶解法在不改变糖链结构的基础上,使蛋白质与多糖实现分离,从而达到提取多糖的目的,但提取率较低。为了合理利用海参加工副产物资源,本发明提供了一种发酵法提取海参肠多糖。
发明内容
为了解决现有技术的不足,本发明目的是提供一种发酵提高海参肠多糖提取率及生物活性的方法。利用E.hiraeGS22发酵海参肠酶解液后提取海参肠多糖,目的在于提高多糖得率及其抗氧化和降血糖功能。
为了实现上述发明目的,本发明提供以下技术方案。
本发明提供了一种海参肠多糖SC-PF,其特征在于,所述海参肠多糖SC-PF分子量为32022 Da,单糖由甘露糖、核糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖及岩藻糖组成,其摩尔比为5.55:10.07:1.17:0.06:13.4:3.12:0.04:0.37:1.72。
进一步地,所述多糖从海参肠中提取获得。
进一步地,所述海参肠多糖SC-PF在制备抗氧化食品和/或药物中的应用。
进一步地,所述海参肠多糖SC-PF在制备降糖食品和/或药物中的应用。
本发明还提供了一种如上所述海参肠多糖SC-PF的提取方法,其特征在于,所述多糖通过E.hiraeGS22发酵海参肠酶解液后提取获得。
进一步地,所述E.hiraeGS22于2023年4月11日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC NO:26984。
进一步地,所述方法具体包括以下步骤:
步骤一海参肠酶解液的制备:
将冷冻的海参肠进行缓冻,后用蒸馏水将其清洗干净,并用剪刀将其剪碎;将剪碎后的海参肠与蒸馏水混合后添加木瓜蛋白酶酶解,酶解完成后离心取上清,将上清灭酶灭菌,得到海参肠酶解液;
步骤二海参肠酶解液发酵:
将E.hiraeGS22按照107CFU/mL添加于步骤一制备得到的海参肠酶解液中,置于37℃培养箱中发酵;
步骤三海参肠多糖的提取:
将步骤二所得发酵完成的海参肠酶解液进行灭菌后冷却至室温,添加等体积5%的三氯乙酸,置于4℃冰箱中静置过夜,离心取上清;向上清液中加入3倍体积预冷的无水乙醇,放于4℃冰箱中静置24~48h,去除上清取沉淀;将沉淀重溶于蒸馏水中,进行冷冻干燥得到多糖SC-PF。
本发明还提供了一种微生物发酵剂,其特征在于,所述微生物发酵剂包括E.hiraeGS22;所述E.hiraeGS22于2023年4月11日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC NO:26984。
进一步地,所述微生物发酵剂在制备海参肠多糖的产品中的应用。
更进一步地,所述海参肠多糖通过生物发酵剂发酵海参肠酶解液后提取获得。
与现有技术比,本发明的有益效果如下。
本发明首次提出菌株E.hiraeGS22能够提高从海参肠中提取多糖的得率,并改善海参肠多糖抗氧化和降糖的生物活性,在发酵海参肠方面有巨大应用潜力,为海参的高值化利用提供新途径。
相对于其他提取方法,本发明所公开的发酵法操作简单、成本低、得率高,为海参加工副产物资源利用提供理论支持。
本发明所提供的提取方法的原料来源于海参加工副产物,提取的海参多糖可用于制备抗氧化和降糖的食品和/或药物可提高海参产业附加值,并减少环境污染。
附图说明
图1为本发明未经菌株发酵和经菌株发酵后提取的海参肠多糖。
图2为本发明的海参肠多糖的傅里叶变换红外光谱分析。
图3为本发明的海参肠多糖的扫描电镜形态图。
图4为本发明的海参肠多糖的刚果红实验结果图。
图5为本发明的海参肠多糖对DPPH自由基清除活性图。
图6为本发明的海参肠多糖对超氧阴离子自由基清除活性图。
图7为本发明的海参肠多糖对α-淀粉酶抑制活性结果图。
图8为本发明的海参肠多糖对α-葡萄糖苷酶抑制活性结果图。
具体实施方式
以下结合具体实施例和说明书附图来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,本发明所用试剂和材料均为市购。
实施例1海参肠酶解液的制备。
将冷冻的海参肠置于4℃冰箱中进行缓冻,后用蒸馏水将其清洗干净,并用剪刀将其剪碎。将预处理完成的海参肠与蒸馏水混合(1:5),后添加木瓜蛋白酶(16000U/g),60℃酶解24h,酶解完成后离心(5000 rpm,15min)取上清,并将上清置于100℃水浴,30min灭酶灭菌,得到海参肠酶解液。
实施例2海参肠酶解液发酵。
将灭酶灭菌的海参肠酶解液分为两组,一组为未发酵组,一组为发酵组。发酵组将E.hiraeGS22按照107CFU/mL添加于海参肠酶解液中,后将两组海参肠酶解液均置于37℃培养箱中发酵48h。
实施例3海参肠多糖的提取。
将发酵完成的海参肠酶解液进行灭菌(100℃,30 min),后冷却至室温,添加等体积5%的三氯乙酸,置于4℃冰箱中静置过夜,离心(5000 rpm/min,15min)取上清,以除去蛋白。向除去蛋白的上清液中加入3倍体积4℃预冷的无水乙醇,放于4℃冰箱中静置24~48h,去除上清取沉淀。将沉淀重溶于蒸馏水中,进行冷冻干燥(样品温度-1.6℃,冷阱温度-53.4℃,真空度18.6Pa,冻干72h)得到粗多糖(图1)。
实施例4海参肠多糖的化学组成分析。
(1)苯酚硫酸法测定总糖含量。
(2)考马斯亮蓝法测定蛋白质含量。
(3)硫酸咔唑法测定糖醛酸含量。
(4)DNS法测定还原糖含量。
实验结果表明,未发酵组与发酵组提取多糖的得率分别为0.48±0.06%、0.63±0.06%,发酵过后多糖得率显著高于未发酵组(p<0.05)。其中SC-P与SC-PF总糖含量分别为44.10±0.10%、50.40±0.00%;蛋白质含量分别为3.30±0.00%、3.98±0.20%;还原糖含量分别为3.65±0.34%、5.10±0.63%、0.29±0.01%。发酵组总糖含量、糖醛酸含量均显著高于未发酵组(p<0.05),而蛋白质、还原糖含量两者并无显著性差异(p>0.05),可能与E.hiraeGS22代谢产物有关。E.hiraeGS22发酵改变了海参肠多糖得率,同时,发酵与未发酵组别化学组成相似但略有差异(表1)。
实施例5海参肠多糖结构特征。
(1)多糖分子量分布:为了测定多糖的分子量,使用高效凝胶渗透色谱法(HPGPC)平均分子量(Mw)进行检测。使用不同分子量的葡聚糖(6.7×105、4.1×105、2.7×105、5×104、2.5×104、1.2×104、5×103和1×103Da)作为标准品。使用HPGPC软件程序校准标准曲线。
(2)单糖组成:通过高效液相色谱(HPLC)分析多糖的单糖组成。将5mg多糖样品用2mol/L三氟乙酸(TFA)在100℃下水解6h。水解后,用甲醇除去过量的酸。将真空干燥的水解产物(100mg)溶解在100μL的0.3mol/L NaOH中,并加入到120μL的0.5mol/L 1-苯基-3-甲基-5-吡唑啉酮(PMP)在甲醇中的溶液中,并在70℃下反应1h。然后将混合物加入100μL0.3mol/L HCl中,剧烈振摇,并以2400×g离心5min。将混合物通过0.22μm膜过滤,并将10μL滤液注入C18柱中进行HPLC分析。移动的相为0.1mol/L磷酸二氢钾(pH10)-乙腈(83:17)。流速设定为1.0 mL/min,柱温保持在30℃。中性单糖用作外标,包括甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、葡糖醛酸(GlcA)、半乳糖醛酸(GalUA)、葡萄糖(Glu)、半乳糖(Gal)、木糖(Xyl)、阿拉伯糖(Ara)和岩藻糖(Fuc)。
(3)傅里叶变换红外光谱:将干燥的多糖与KBr粉末研磨,然后压制成薄片室温下记录其在4000-400cm-1的红外光谱。并通过数据软件对数据进行处理。
(4)扫描电镜观查海参肠多糖形态:将干燥后的海参肠多糖粘附于样品台上,后置于离子溅射仪中镀金,在5.0kV的加速电压下通过扫描电子显微镜进行观察,在不同放大倍数(500×,2000×,5000×和15000×)下观察海参肠多糖,获取多糖电镜照片。
(5)刚果红实验:配制浓度为2mg/mL的多糖溶液,同时配制浓度为0M、0.2M、0.4M、0.6M和0.8M的NaOH溶液。取1mL海参肠多糖样品溶液,3mL的NaOH溶液1.5mL 0.2mmol/L刚果红溶液混合,后续添加0.5mL蒸馏水立即混匀,静置1h后,在400~800nm波长下,通过紫外分光光度计进行扫描,记录NaOH溶液中混合液所拥有的最大吸收波长,后绘图。同时以未加多糖样品的刚果红溶液作为对照。
实验结果表明:SC-P与SC-PF分子量和单糖组分如表1所示。
表1不发酵和发酵提取海参肠的得率、化学组成、分子量和单糖组成
。
SC-P与SC-PF分子量分别为35964Da、32022Da,说明发酵降低了海参肠多糖的分子量。
单糖组成分析表明,SC-P由甘露糖、核糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖及岩藻糖组成,摩尔比为2.96:7.80:0.10:0.02:7.63:1.49:0.19:1.26,其中含量最高的为核糖其次为葡萄糖,含量最少的为半乳糖醛酸;SC-PF由甘露糖、核糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖及岩藻糖组成,其摩尔比为5.55:10.07:1.17:0.06:13.4:3.12:0.04:0.37:1.72,其中含量最高的为葡萄糖其次为核糖,含量最少的为木糖。结果表明,与SC-P相比发酵后单糖组成有一定改变,SC-PF单糖组成摩尔比都略高于SC-P,且木糖只存在SC-PF中,说明菌株发酵会影响海参肠多糖单糖组成和摩尔比。
SC-P和SC-PF的FT-IR光谱(图2)显示在3421、2935、1656、1415、1076和611cm-1附近有吸收峰。在3421cm-1附近的吸收是O-H的伸缩振动,2935cm-1附近的吸收则反映了岩藻糖甲基对C-H的伸缩振动。1656cm-1和1076cm-1处的条带分别是C=O和C-O-C的伸展振动,表明这两种多糖都含有糖醛酸。1415cm-1附近的吸收峰是由S=O的伸展振动引起的,894cm-1附近的吸收峰是由对称C-O-S的伸展引起的,611 cm-1附近的吸收峰是由S-O的伸展振动引起的,证实多糖发生硫酸酯取代。结果表明,SC-P和SC-PF的FT-IR光谱基本一致,表明SC-P在GS22发酵后主要结构特征并没有发生明显变化。上述结果表明,SC-P和SC-PF为含有岩藻糖、糖醛酸、硫酸基等取代基的多糖,发酵不改变多糖主要官能团结构。
扫描电镜分析:利用SEM对多糖样品进行成像,如图3所示,SC-P的SEM分析结果表明,在500×放大倍数下,SC-P样品呈现颗粒状和块状结构;在2000×放大倍数下,多糖样品表面有颗粒状聚集体,紧密堆积在一起,这可能是由于样品分子之间的强烈相互作用造成的;在5000×的放大倍数下,可以看到多糖样品的表面积聚了大量的球状体;在15000×放大倍数下多糖样品呈现疏松多孔的形态。SC-PF相较于SC-P结构相比比较疏松,空隙较多,说明发酵会改变多糖表面形态。
SC-P、SC-PF与刚果红络合后,在0~0.8mol/LNaOH终浓度下最大吸收波长如图4所示,刚果红SC-P与刚果红SC-PF络合溶液吸光度先增加后趋于平缓,而刚果红溶液在不同NaOH终浓度下呈现缓慢下降的趋势,SC-P与SC-PF均出现显著的红移现象,说明两者具有三股螺旋链构象。其功能特性也与其三螺旋结构紧密相关。
实施例6海参肠多糖功能特性分析。
1. 抗氧化活性研究。
(1)DPPH自由基清除活性:将多糖配置成不同浓度的溶液(0.3mg/mL、0.6mg/mL、1.2mg/mL、2.5mg/mL、5mg/mL),取2mL不同浓度的多糖溶液,加入2mL0.2mM的DPPH-无水乙醇溶液,充分混匀,室温下避光反应30min,在517nm处测定吸光度,计算DPPH自由基清除率(式1)。
。
(2)超氧阴离子清除活性:将1mL不同浓度的多糖溶液(0.3-5mg/mL)和0.3mL 3mM焦性没食子酸加入到4.5mL 50mM Tris-HCl缓冲液(pH8.2)中,将得到的混合物快速摇匀,然后在25℃下避光反应5min。然后加入10mM HCl终止反应,以Vc为阳性对照,在320nm处测定混合物的吸光度,计算超氧阴离子自由基清除活性(式2)。
。
2.α-淀粉酶抑制活性。
所有试剂均用PBS(pH6.9,0.1mol/L)配制。500μL的α-淀粉酶溶液(1U/mL)与每种浓度500μL的多糖在37℃下孵育10min。然后加入可溶性淀粉溶液500μL(1%,w/v),孵育10min。然后加入1mLDNS试剂,在沸水浴中加热5min使酶失活。最后,在多糖最终浓度分别为0.12、0.2、0.4、0.8和1.2mg/mL的混合物中加入10mL去离子水。在540nm处测量了混合物的吸光度(A)。用EQ法计算抑制率(式3)。
。
其中A3为混合物的吸光度,A1不含抑制剂,A2不含酶和抑制剂,A4不含酶。缺少的抑制剂与酶用等量的PBS代替。
3.α-葡萄糖苷酶抑制活性。
所有试剂均用PBS(pH6.9,0.1mol/L)。每种浓度40μL的多糖与40μL的α-葡萄糖苷酶混合,在37℃下孵育15min。然后加入20μL的4-硝基苯基-α-D-葡萄糖苷(PNPG,16mM),在37℃下孵育15min。最后加入150μLNa2CO3溶液(0.2mol/L)终止反应。多糖的最终浓度分别为0.8、1.6、3.2、4.8、6.4mg/mL。在405nm处测量了混合物的吸光度(A)。用EQ法计算抑制率(式4)。
。
其中A1是混合物的吸光度,A2是无α-葡萄糖苷酶的吸光度,A3是无多糖的吸光度,用等量的PBS替代多糖和酶。
实验结果表明,在多糖浓度为0.3mg/mL-2.5mg/mL范围内,SC-P与SC-PF的DPPH自由基清除活性均显著低于同等浓度下Vc清除活性。但在5.0mg/mL浓度下,SC-P、SC-PF和VC的DPPH自由基清除率分别为84.7%、96.3%和96.0%,其中,SC-PF清除活性最高(96.3%),其次是Vc(96%),最后是SC-P(84.7%),SC-PF与VC清除活性无显著性差异(p>0.05)。在试验范围内,SC-PF对DPPH的清除活性显著高于SC-P(p<0.05),这可能与SC-PF的羧基或乙酰基含量有关。SC-P与SC-PF对DPPH自由基清除活性均表现出浓度依赖性(图5)。
在5.0mg/mL浓度下,SC-P、SC-PF和Vc的超氧阴离子清除率分别达到19.7%、36.5%和98.9%,其中Vc(98.9%)清除活性最高,其次为SC-PF(36.5%),最后是SC-P(19.7%)(图6)。在试验范围内,多糖对超氧阴离子的清除效果与其浓度呈正相关,且SC-PF的清除活性显著高于SC-P(p<0.05)。其清除超氧自由基的机制可能与O-H键的离解能有关。
两种多糖对α-淀粉酶抑制活性如图7所示。结果表明,在0.5mg/mL-4.0mg/mL浓度下SC-PF组α-淀粉酶抑制活性略高于SC-P组但差异不显著(p>0.05);在6mg/mL浓度下,SC-P与SC-PF对α-淀粉酶的抑制作用分别为64.40%、49.00%,其中SC-PF抑制活性显著高于SC-P(p<0.05)。SC-P与SC-PF对α-淀粉酶的抑制活性均具有浓度依赖性。
两种多糖对α-葡萄糖苷酶抑制活性如图8所示。结果表明,在多糖浓度为10mg/mL时,SC-P与SC-PF对α-葡萄糖苷酶的抑制率分别为33.40%、42.83%,其中SC-PF对α-葡萄糖苷酶抑制活性显著高于SC-P(p<0.05)。同时,在多糖浓度为40mg/mL时,SC-P组与SC-PF组α-葡萄糖苷酶抑制活性分别为61.03%、66.63%,SC-PF抑制活性显著高于SC-P。两组多糖对α-葡萄糖苷酶的抑制活性均具有浓度依赖性。
Claims (10)
1.一种海参肠多糖SC-PF,其特征在于,所述海参肠多糖SC-PF分子量为32022 Da,单糖组成为甘露糖、核糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖及岩藻糖,其摩尔比为5.55:10.07:1.17:0.06:13.4:3.12:0.04:0.37:1.72。
2.根据权利要求1所述的海参肠多糖SC-PF,其特征在于,所述多糖从海参肠中提取获得。
3.根据权利要求1所述的海参肠多糖SC-PF在制备抗氧化食品和/或药物中的应用。
4.根据权利要求1所述的海参肠多糖SC-PF在制备降糖食品和/或药物中的应用。
5.一种权利要求1所述海参肠多糖SC-PF的提取方法,其特征在于,所述多糖通过海氏肠球菌GS22(Enterococcus hirae GS22)发酵海参肠酶解液后提取获得。
6.根据权利要求5所述的提取方法,其特征在于,所述E.hiraeGS22于2023年4月11日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC NO:26984。
7.根据权利要求5所述的提取方法,其特征在于,所述方法具体包括以下步骤:
步骤一海参肠酶解液的制备:
将冷冻的海参肠进行缓冻,后用蒸馏水将其清洗干净,并用剪刀将其剪碎;将剪碎后的海参肠与蒸馏水混合后添加木瓜蛋白酶酶解,酶解完成后离心取上清,将上清灭酶灭菌,得到海参肠酶解液;
步骤二海参肠酶解液发酵:
将E.hiraeGS22按照107 CFU/mL添加于步骤一制备得到的海参肠酶解液中,置于37℃培养箱中发酵;
步骤三海参肠多糖的提取:
将步骤二所得发酵完成的海参肠酶解液进行灭菌后冷却至室温,添加等体积5%的三氯乙酸,置于4℃冰箱中静置过夜,离心取上清;向上清液中加入3倍体积预冷的无水乙醇,放于4℃冰箱中静置24~48h,去除上清取沉淀;将沉淀重溶于蒸馏水中,进行冷冻干燥得到多糖SC-PF。
8.一种微生物发酵剂,其特征在于,所述微生物发酵剂包括E.hiraeGS22;所述E.hiraeGS22于2023年4月11日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCCNO:26984。
9.根据权利要求8所述的微生物发酵剂,其特征在于,所述微生物发酵剂在制备海参肠多糖产品中的应用。
10.根据权利要求9所述的微生物发酵剂,其特征在于,所述海参肠多糖通过生物发酵剂发酵海参肠酶解液后提取获得。
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