CN115572739A - 一种发酵菌体为原料制备水凝胶的方法 - Google Patents
一种发酵菌体为原料制备水凝胶的方法 Download PDFInfo
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- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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Abstract
本发明属发酵工程和化学工程领域,具体涉及发酵生产结束后发酵菌体资源化利用制备水凝胶。本发将发酵菌体在裂解酶或化学裂解剂的作用下适度裂解释放菌体大分子,裂解后的菌液中包含蛋白质,核酸,壳聚糖及肽聚糖等成分,通过菌体裂解液中的大分子与壳聚糖大分子进行交联制备水凝胶,实现废弃发酵菌体的产品化应用。同时,本发明制备的水凝胶产品可应用于种子包衣、农业吸水剂等领域,具有利于提高种子发芽率等特性。
Description
技术领域:
本发明属发酵工程和化学工程领域,具体涉及发酵生产结束后发酵菌体资源化利用制备水凝胶。
背景技术:
以谷氨酸棒状杆菌发酵生产谷氨酸、精制味精为例,按照每生产1吨味精可以从发酵液中提取干菌体200kg计算,则全国每年可产生的菌体蛋白约22×104吨(周大伟,肖冬光,郭学武,吕鸿雁.食品研究与开发,2012)。且中国每年由大肠杆菌生产的L-色氨酸超过10000吨,1吨L-色氨酸的生产伴随着1.3-1.5吨废细菌细胞(WBC)的生产(Qingyang Xu,Fang Bai,Ning Chen,and Gang Bai,Bioengineered,2019)。由米根霉发酵生产乳酸的同时,每获得1吨的乳酸会产生84.5kg的米根霉菌体(杨磊,李鑫,余世袁.林产化学与工业,2015)。
现有的发酵菌体处理工艺包括用废弃的菌体蛋白制备出营养成分全面的菌体蛋白粉,来替代酵母膏,降低发酵培养基的成本(中国专利申请CN106086093A;中国专利申请CN 110092528 A);制备出纯度较高的肽产品以及饲料产品,避免菌体原料的浪费(中国专利申请CN107011409A;中国专利申请CN107418897A);用氨基酸发酵废弃菌体制备液态有机肥(中国专利申请CN106116773A),以维生素C发酵废弃物生产菌体蛋白和多肽有机肥(中国专利申请CN111995467A)。
以革兰氏阴性菌为例,细胞壁由肽聚糖层和外壁层组成。外壁层主要由脂多糖和脂蛋白组成。磷脂双分子层是构成细胞膜的的基本支架。细胞膜的主要成分是蛋白质和脂质,含有少量糖类。水凝胶是亲水性聚合物交联网络后经水溶胀形成的一种高分子材料,通常由天然或合成高分子制备而成。可以通过万能材料试验机对其力学性能进行测试,采用红外光谱仪对其结构进行表征,借助差示扫描量热仪对其热学性能分析,利用扫描电镜观察其表面多孔结构及纵切面孔隙取向,并通过公式计算水凝胶溶胀率。
水凝胶通常由通过共价键、氢键和/或物理纠缠形成的三维亲水聚合物网络组成。它们具有良好的水溶性和生物相容性,因此在药物递送、组织工程和生物吸附剂中有应用。一些众所周知的例子是聚乙二醇(PEG)、聚丙烯酰胺(PAM)和聚乙烯醇(PVA),它们都是亲水的。然而,其固有毒性阻碍了健康利用。因此,壳聚糖、海藻酸盐和纤维素等生物聚合物已被视为替代品,以创建更具生物相容性、生物降解性和低毒性的消费者友好型凝胶。本发明制备的水凝胶产品可应用于种子包衣、农业吸水剂等方向。
发酵菌体现有处理方式通常存在处理成本高、环保压力大等问题,大部分发酵菌体尚未实现“变废为宝”。本发明通过充分利用菌体裂解后释放的蛋白质、多糖和核酸等大分子,并通过壳聚糖等可降解高分子物质与其进行交联制备可适用于种子包衣、绿植保温、以及肥料缓释等领域的水凝胶材料新产品。
发明内容:
本发明的目的是将发酵菌体在裂解酶或化学裂解剂的作用下释放菌体大分子,裂解后的菌液中包含蛋白质,核酸,壳聚糖及肽聚糖等成分,此外还包括各类一价、二价离子及细胞碎片,小分子肽及单糖、寡糖在裂解液中的占比低于10%,然后通过菌体裂解液中的大分子与壳聚糖大分子进行交联制备水凝胶,实现废弃发酵菌体的产品化应用。
本发明提供的技术方案之一,是一种采用发酵废弃菌体制备水凝胶材料的方法,包括以下步骤:
(1)微生物发酵液提取目的产物之前或之后,收集菌体;
进一步地,在发酵液中添加絮凝剂收集菌体;
进一步地,所述絮凝剂为壳聚糖,添加量为发酵液的0.01%~1%(w/v);
进一步地,絮凝条件为:搅拌速度100-300r/min、絮凝温度为45-85℃,搅拌5-20min后调整pH至6~10;
更进一步地,壳聚糖添加量为0.06%~0.3%,氢氧化钙调节pH至7~8.5;
进一步地,采用板框过滤、膜过滤或离心收集菌体;
进一步地,所述发酵液为以大肠杆菌为发酵菌种的乳酸发酵液;
(2)将收集的菌体,通过裂解酶,和/或化学裂解剂处理后制备菌体裂解液;
进一步地,所述菌体裂解液中细胞破碎率为84%~99.5%,同时裂解液中蛋白含量范围为62~239mg/g,核酸含量为6~75mg/g,肽聚糖含量为1.2~14.95mg/g;
进一步地,所述裂解酶为溶菌酶、蜗牛酶、纤维素酶或溶壁酶中的至少一种;
进一步地,所述化学裂解剂包括但不限于NaOH、KOH、SDS、Triton X-100等;
进一步地,裂解条件为:40℃~100℃,裂解10~30min;
更进一步地,在50℃~100℃,pH 7~14条件下裂解15-30min;
进一步地,将收集的菌体重悬后加入终浓度为0.5~3M的氢氧化钠和0.25~1.0%(w/v)的SDS作为裂解剂;
更进一步地,收集的菌体重悬后加入终浓度为0.5~1.5M的氢氧化钠和0.25~1%(w/v)的SDS作为裂解剂;
(3)菌体裂解液静置制备水凝胶;
进一步地,菌体裂解液静置4~12h后形成水凝胶。
本发明还提供由上述方法制备的水凝胶材料。
本发明还提供上述水凝胶材料的应用,特别是在种子包衣、绿植保温、以及肥料缓释等领域的应用。
本发明取得的有益效果:
本发明能够将发酵料液中的菌体高效收集、适度裂解并用于制备水凝胶,实现发酵产物与发酵菌体来源水凝胶的同步生产,彻底解决现有工业生产体系中发酵菌体废弃物的再利用问题。由此也可以显著提升发酵生产的经济效率和环境效益并显著降低发酵产物生产综合成本。
本发明还可应用于其它有机酸如乳酸、柠檬酸、苹果酸、丁二酸等,或氨基酸如赖氨酸、谷氨酸、苏氨酸、丙氨酸等生产过程的菌体回收利用。
本发明在乳酸发酵结束进行菌体收集时,以壳聚糖为生物絮凝剂并维持一定温度和pH,絮凝剂会全部随菌体一起被收集不影响乳酸后续分离精制,且收集后的菌体经过简单裂解,即可生产流变性能和溶胀性能较好的水凝胶。工艺的关键点是发酵菌体裂解程度的控制,即体系中细胞破碎程度,蛋白、核酸、肽聚糖浓度的控制。
本发明制备的水凝胶产品可应用于种子包衣、农业吸水剂等方向。
本发明通过充分发挥菌体裂解后释放的蛋白质、多糖和核酸等大分子,并通过壳聚糖等可降解高分子物质与其进行交联制备可适用于种子包衣、绿植保温、以及肥料缓释等领域的水凝胶或共混膜材料新产品。本发明制备的水凝胶与单独使用壳聚糖制备的水凝胶相比,蛋白会增加水凝胶体系的韧性,且作为种子包衣或农业吸水剂过程中,可以释放蛋白等营养物质供种子摄取,具有利于提高种子发芽率等特性。
具体实施方式:
为了使本专利的目的、技术方案及优点更加清楚明白,以下结合具体实施例,对本专利进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本专利,并不用于限定本发明。
本发明以钙盐法生产乳酸的发酵液为例,在乳酸发酵结束后,将发酵液升温至45℃~85℃,加入生物絮凝剂壳聚糖并搅拌维持反应5-20min,然后通过过滤进行固液分离,收集固体部分即为菌体,收集液体部分即为含乳酸单体的游离乳酸液,获得的菌体可用于后续的水凝胶的制备,获得的游离乳酸液经过过滤、浓缩等,即为乳酸粗成品,可用于后续如纳滤、脱色、离交等精制,获得高纯度乳酸单体。
本发明采用的主要实验方法如下:
(1)发酵料液制备-菌体收集(絮凝)。钙盐法乳酸发酵液的制备按照发明专利(王正祥等,ZL201580000781.7)方法进行,发酵菌种为CGMCC 11059或CGMCC11060,其中菌种CGMCC 11059用于D-乳酸的发酵生产,菌种CGMCC11060用于L-乳酸的发酵生产(王正祥等,ZL201580000781.7)。在发酵起始阶段,向发酵基本培养基中加入葡萄糖至终浓度10~50g/L,培养在30℃~37℃、pH 5.5~7.5、通风0.1~2.0vvm、100~1000r/min搅拌下进行;培养时间5~15h,菌体量达到10~50OD;关闭通风,降低搅拌速度到0~300r/min,提高发酵温度到37℃~50℃,补加终浓度16%~25%的葡萄糖溶液,流加速度控制在3g/(L h)~25g/(Lh),同步流加5%~35%氢氧化钙,控制发酵pH在5.0~8.0之间。絮凝是向发酵液中加入生物絮凝剂壳聚糖0.1-10g/L、搅拌时间为5-20min、搅拌速度100-300r/min、絮凝温度为45-85℃,用氢氧化钙将发酵液pH调到7.0-8.5,过滤收集菌体。
(2)菌体裂解。使用NaOH溶液和SDS溶液对大肠杆菌进行破壁处理,加入NaOH终浓度为0.5~3M、SDS终浓度为0.25~1.5%(w/v),温度为40℃-100℃,pH7-14,10-30min。
(3)水凝胶制备。将上述裂解后的菌体裂解液于室温下静置4~12h即获得水凝胶。
以下通过具体实施例对本发明作进一步地解释说明。
实施例1一种D-乳酸发酵方法
将D-乳酸生产菌种CGMCC NO.11059的甘油管冻藏物,接种于50mL的LB液体培养基中,37℃、200r/min摇床培养12h,作为一级种子液。将一级种子液接种于5L以葡萄糖为碳源的M9液体培养基中,起始糖浓度为0.5%,37℃、200r/min摇床培养10h,作为二级种子液。将二级种子液按照起始OD值0.3的接种量接种于装有M9液体培养基的发酵罐中,接种后5m3发酵罐初始体积为2.5m3,初始转化糖浆添加量为3%,开始乳酸单体的发酵生产。发酵起始温度控制为37℃,用氨水维持pH 6.5,菌体生长过程中调节通气量为1.5vvm,搅拌转速为600r/min,当菌体浓度达到OD600 30后,关闭通风,发酵温度控制在40℃,搅拌转速调为200r/min,流加25%氢氧化钙悬浊液维持pH在7.0,补加总量6.0kg葡萄糖,残余糖浓度低于0.5g/L后即发酵结束。
M9培养基组成:NaCl 0.5g/L,NH4Cl 1g/L,KH2PO4 3g/L,Na2HPO4·12H2O 15.1g/L,其余为水。
以获得的乳酸发酵液为原料,在反应釜中将其加热至60℃并维持此温度,100r/min搅拌下加入壳聚糖溶液2g/L,全部壳聚糖溶液加完后,维持反应15min后,添加20%氢氧化钙将发酵液pH调到7.5;反应结束后,反应液的固液分离采用过滤孔径6μm板框过滤装置进行,收集固体部分即为菌体。
实施例2-1:不同浓度裂解剂下的裂解效果
将实施例1获得的菌体和水以1:4(w/v)的比例在重悬三次后,50℃温度下分别加入终浓度为0.25~1.5M NaOH和0.5%的SDS溶液,以及0.25~1%的SDS溶液和1M的NaOH进行破壁处理20min,裂解后裂解液中各裂解物成分如下表1、2。并将菌体裂解液放于离心管中均在室温下静置6h,倒立离心管观察水凝胶成型情况。
表1不同NaOH浓度对应裂解数据(0.5%的SDS溶液)
表2不同SDS浓度对应裂解数据(1M的NaOH)
实施例2-2:不同温度下的裂解效果
将实施例1获得的菌体和水以1:4(w/v)的比例在重悬三次后,加入终浓度为1MNaOH和0.5%的SDS溶液在不同温度(20-90℃)条件下进行破壁处理20min,裂解后裂解液中各裂解物成分如下表。并将菌体裂解液放于离心管中均在室温下静置6h,倒立离心管观察水凝胶成型情况。
表3不同裂解温度对应裂解数据
实施例2-3:不同裂解时间下的裂解效果
将实施例1获得的菌体和水以1:4(w/v)的比例在重悬三次后,加入终浓度为1MNaOH和0.5%的SDS溶液在70℃条件下进行破壁处理不同时间(5-35min),裂解后裂解液中各裂解物成分如下表。并将菌体裂解液放于离心管中均在室温下静置6h,倒立离心管观察水凝胶成型情况。
表4不同裂解时间对应数据
蛋白质含量测定参考考马斯亮蓝法,核酸含量测定采用紫外吸收法,肽聚糖含量测定采用葡萄糖胺标曲法(曾敏,谢为天,潘丽媚,徐春厚.饲料工业.2010)。
细胞破碎率=[1-(裂解后菌体OD600/裂解前菌体OD600)]×100%。
其中,1OD=0.462×109细胞/mL。(KangmingTian,DandanNiu,ZhengxiangWang.Biotechnologyand Bioengineering.2015)。
经过上述实验,确定影响成胶的主要因素为细胞破碎率,体系中的蛋白浓度、核酸浓度以及肽聚糖浓度。在上述实验的基础上,发明人又经过大量实验验证,最终确定菌体裂解液能够形成水凝胶的较佳条件为:细胞破碎率为84%~99.5%,同时裂解液中蛋白含量范围为62~239mg/g,核酸含量为6~75mg/g,肽聚糖含量为1.2~14.95mg/g。
且形成水凝胶适宜的裂解条件为:NaOH终浓度0.5~3M,SDS浓度0.25~1%,温度40~100℃,裂解时间在10min~30min之间。
实施例3:不同裂解条件下形成的水凝胶
将实施例1获得的菌体和水以1:4(w/v)的比例在重悬三次后,加入一定终浓度的NaOH和SDS溶液在一定温度下进行破壁处理一定时间,裂解后裂解液中各裂解物成分如下表。并将菌体裂解液放于离心管中均在室温下静置6h,倒立离心管观察水凝胶成型情况。
表5不同裂解条件对应数据
实施例4:水凝胶流变学表征
水凝胶系统中大分子的结构形成可以通过动态测量确定粘弹性来解释,对水凝胶的粘弹特性,进行了流变学测量。使用旋转流变仪对菌体裂解液水凝胶进行流变测试,测试温度为25℃,振幅扫描测试条件ω为10rad/s,振幅范围0.01~100%;频率扫描测试条件为0.1~100rad/s,振幅1%。
其中弹性由储能模量G'表示,而粘性由损耗模量G"解释。在实施例2-1、2-2和2-3形成的水凝胶中,G'总是高于G",储能模量G'值是损耗模量G"值的3~5倍左右,G'≈104,G"≈103,表明所有水凝胶具有稳定的结构和良好的粘弹性。
随着应变的增加,储能模量和损耗模量出现交点,网络结构被破坏。
实施例5:本发明水凝胶产品的性能参数
本发明实施例2和3获得的部分水凝胶,对应的溶胀率及力学性能数据如下表:
相关参数测定参考文献(许梦媛,刘让同,李亮,刘淑萍,李淑静.印染,2022)
壳聚糖水凝胶制备:称取2.5g的1%壳聚糖溶液于25mL容器中,加入去离子水至10mL并通过磁力搅拌器在90℃下搅拌使壳聚糖溶解呈透明澄清溶液,静置6h。本发明实施例1中于发酵液中加入0.2%的壳聚糖溶液,絮凝后壳聚糖占菌体含量的1%,菌体裂解后壳聚糖含量占菌体裂解液的0.25%,因此本实施例壳聚糖水凝胶设置的壳聚糖浓度为0.25%。
牛血清蛋白水凝胶制备:称取1.55g牛血清蛋白于25mL容器中,加入8.45g去离子水(牛血清白蛋白终浓度155mg/g)并通过磁力搅拌器在90℃下搅拌使牛血清蛋白溶解呈透明澄清溶液,静置6h。
壳聚糖+牛血清蛋白水凝胶制备:称取2.5g的1%壳聚糖溶液和1.55g牛血清白蛋白于25mL容器中,加入去离子水补足至10g并通过磁力搅拌器在90℃下搅拌使溶解呈透明澄清溶液,静置6h。
由上述实验可知,在壳聚糖或蛋白浓度相当的情况下,由本发明菌体裂解液制备的水凝胶具有更好的溶胀率、拉伸强度和断裂伸长率,性能更加优越。水凝胶能迅速吸水溶胀而不在水中溶解,与水结合时,能表现出高度溶胀,拥有良好的保水性能,其高保水性能可以为种子提供良好的环境,防止水分丢失,延长货架期;较好的拉伸强度和断裂伸长率可以使水凝胶在种子包衣过程中不易断裂,与种子更好的接触并形成包衣。
实施例6:本发明水凝胶作为种子包衣的应用
将本发明裂解条件为1.5M NaOH、0.5% SDS、40℃、20min(实施例3);1M NaOH、0.5% SDS、70℃、25min(实施例2-3);1.5M NaOH、0.75% SDS、90℃、15min;2.5M NaOH(实施例3)、0.75% SDS、100℃、25min(实施例3)制备的水凝胶应用于种子包衣,具体如下:
将黄豆种子均匀浸入水凝胶中,充分搅拌,静置30min后捞出,摊开晾干,待种子表面形成一层薄膜后即可,约1~2h。
同时将同样的种子裹上以壳聚糖制备的水凝胶、以牛血清蛋白制备的水凝胶以及壳聚糖+牛血清白蛋白制备的水凝胶,以未经任何处理的种子作为对照,并将所有种子催芽(用清水浸泡14h,捞出控除多余水分,用两层湿滤纸覆盖种子表面,每天洒水保持滤纸微湿润,三四天)播种,计算种子发芽率,获得的数据如下:
壳聚糖水凝胶1、2、3制备:称取2.5g的1%壳聚糖溶液于25mL容器中,加入去离子水至10mL并通过磁力搅拌器在70℃、90℃、100℃下搅拌使壳聚糖溶解呈透明澄清溶液,静置6h。
牛血清蛋白水凝胶1、2、3制备:称取1.10g、1.55g、2.30g牛血清蛋白于25mL容器中,加入去离子水补足10g(牛血清白蛋白终浓度110mg/g、155mg/g、230mg/g)并通过磁力搅拌器在分别在70℃、90℃、100℃下搅拌使牛血清蛋白溶解呈透明澄清溶液,静置6h。
壳聚糖+牛血清蛋白水凝胶1、2、3制备:分别称取2.5g的1%壳聚糖溶液和1.10g、1.55g、2.30g于25mL容器中,分别加入去离子水补足10g并通过磁力搅拌器分别在70℃、90℃、100℃下搅拌使壳聚糖、蛋白溶解呈透明澄清溶液,静置6h。
由上述实验可知,在同等壳聚糖或蛋白浓度下,本发明由菌体裂解液制备的水凝胶作为种子包衣更有利于种子发芽。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (10)
1.一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,包括以下步骤:
(1)微生物发酵液提取目的产物之前或之后,在发酵液中添加壳聚糖作为絮凝剂收集菌体;
(2)将收集的菌体,通过裂解酶,和/或化学裂解剂处理后制备菌体裂解液;
所述菌体裂解液中细胞破碎率为84%~99.5%,同时裂解液中蛋白含量为62~239mg/g,核酸含量为6~75mg/g,肽聚糖含量为1.2~14.95mg/g;
(3)菌体裂解液静置制备水凝胶。
2.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,絮凝条件为:搅拌速度100-300r/min、絮凝温度为45-85℃,搅拌5-20min后调节pH6~10。
3.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,所述絮凝剂为壳聚糖,添加量为发酵液的0.01%~1%。
4.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,壳聚糖添加量为0.06%~0.3%,反应5-20min后氢氧化钙调节pH至7~8.5。
5.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,所述裂解酶为溶菌酶、蜗牛酶、纤维素酶或溶壁酶中的至少一种;所述化学裂解剂包括NaOH、KOH、SDS或Triton X-100。
6.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,裂解条件为:40℃~100℃,裂解10~30min。
7.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,将收集的菌体重悬后加入终浓度为0.5~3M的氢氧化钠和0.25~1.0%的SDS作为裂解剂。
8.如权利要求1所述的一种采用发酵废弃菌体制备水凝胶材料的方法,其特征在于,菌体裂解液静置4~12h后形成水凝胶。
9.权利要求1-8任意一项所述方法制备的水凝胶材料。
10.权利要求9所述水凝胶材料的应用,其特征在于,应用于种子包衣、绿植保温、或肥料缓释技术领域。
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