CN110226666A - 利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法 - Google Patents
利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法 Download PDFInfo
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Abstract
本发明公开了一种利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法,该方法以马克思克鲁维酵母为发酵菌,以超声预处理的乳清蛋白为底物进行液体发酵,发酵完成后,得到低致敏性乳清蛋白。本发明方法所用微生物安全性较高、水解蛋白能力较强,获得的低致敏性乳清蛋白不仅致敏性低、而且安全无毒。
Description
技术领域
本发明涉及生物工程和微生物发酵技术领域,尤其涉及一种利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法。
背景技术
食物过敏是一个主要的健康问题,影响全球1~3%的成年人和4~6%的儿童,并且在过去的二十年中发病率大大增加。牛乳是联合国粮农组织(FAO)认定的八大类主要过敏食物之一,其引起的过敏严重威胁婴幼儿健康,严重时甚至导致死亡。牛乳蛋白过敏(cow’s milk protein allergy,CMPA),是临床上一个或多个牛乳蛋白引起的免疫反应,主要是由IgE介导。据调查显示,学龄前儿童对牛乳过敏占0.6%~2.5%,5~16岁人群占0.3%,成年人小于0.5%。牛乳中主要致敏蛋白包括酪蛋白(casein)、β-乳球蛋白(β-lactoglobulin,β-LG)、α-乳白蛋白(lactalbumin,α-LA),乳清蛋白中主要含有β-LG和α-LA,可引起过敏性鼻炎、哮喘、湿疹、腹泻、胃肠出血等症状。
目前,降低牛奶致敏性的方法包括热加工、高压、超声、辐照、发酵、糖基化、基因法和酶改性等。利用低频率高振幅超声波(频率20-100KHz,功率10-1000W/cm2)是一种具有很大潜力的新兴食品加工技术,目前已经证实超声预处理能提高蛋白的酶解敏感性;目前国内外利用乳酸菌、酵母发酵牛乳蛋白来降低其致敏性的报道很多,可将β-LG和α-LA抗原性降低70%-90%,但是其与IgE的结合能力并未显著减弱。菌株、发酵基质、发酵条件、发酵产物的处理方法对降低菌株的致敏性都有较大影响,还有很大的研究空间。
发明内容
本发明提供了一种利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法,所用微生物安全性较高、水解蛋白能力较强,获得的低致敏性乳清蛋白不仅致敏性低、而且安全无毒。
一种利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法,包括:
(1)将乳清蛋白溶液进行超声处理,处理完成后,加入乳糖溶液,混匀,灭菌,得到发酵培养基;
(2)将活化的马克思克鲁维酵母(Kluyveromyces marxianus)接入步骤(1)制得的发酵培养基中进行发酵培养,培养完成后,通过后处理从发酵液中分离纯化得到低致敏性乳清蛋白。
与其他微生物相比,食源性菌株马克思克鲁维酵母在生物转化过程中的安全性更高、水解蛋白能力更强,以马克思克鲁维酵母作为发酵菌株、以浓缩乳清蛋白为底物进行发酵获得低致敏性乳清蛋白,易于产品的推广。
步骤(1)中,所述乳清蛋白可以为浓缩乳清蛋白WPC80。
作为优选,所述超声处理条件为:超声间歇比2s:1s~2s:3s,超声功率100~300W,超声时间5~15min。
更优选地,所述超声处理条件为:超声间歇比2s:1s、超声功率200W、处理时间10min。
作为优选,所述发酵培养基中乳清蛋白的浓度为3~8g/100mL,乳糖的浓度为2~6g/100mL,酵母利用率较好,其中乳糖作为碳源促进菌株生长。
步骤(2)中,所述马克思克鲁维酵母(Kluyveromyces marxianus)可以为马克思克鲁维酵母(Kluyveromyces marxianus)CGMCC 2.68,购于中国普通微生物菌种保藏管理中心。
所述活化的马克思克鲁维酵母可以采用活化马克思克鲁维酵母的菌悬液,在发酵培养基中的接种量可以为2~14%,优选为6~10%,更优选为10%。
作为优选,所述发酵培养的条件为温度20~45℃,初始pH3.0~9.0,转速0~250rpm,时间1~3天。此条件范围有利于菌株生长和代谢。
更优选地,所述发酵培养的条件为温度25~35℃,初始pH6.7~7.3,转速150rpm。
最优选地,所述发酵培养的条件为温度35℃,初始pH7.25。
本发明还提供了一种采用上述方法制备得到的低致敏性乳清蛋白。该产物致敏性较低,易分离,口感温和,可直接用于配方奶粉。
本发明还提供了上述低致敏性乳清蛋白在低致敏性婴幼儿配方奶粉中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明以马克思克鲁维酵母作为发酵菌株、以超声波预处理的浓缩乳清蛋白为底物进行发酵,获得的低致敏性乳清蛋白不仅致敏性低、而且安全无毒。与其他微生物相比,马克思克鲁维酵母在生物转化过程中的安全性更高、水解蛋白能力更强,易于产品的推广。
附图说明
图1为本发明利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法流程图。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例中所使用的菌种为马克思克鲁维酵母(Kluyveromyces marxianus)CGMCC 2.68,购于中国普通微生物菌种保藏管理中心。本发明不涉及菌种的保藏。
实施例1-18
(1)菌种活化:将配置好的活化培养基进行高温高压灭菌,冷却并倒好平板,将冷冻保存的马克思克鲁维酵母(Kluyveromyces marxianus)CGMCC 2.68菌株接种至灭菌后的活化培养基平板上,在25℃培养箱中,培养7d,至平板布满菌丝为止,平板备用。
其中,活化培养基的成分为:YPD50g/L,琼脂20g/L,水。
(2)菌悬液制备:取活化后的菌种接种于液体培养基,30℃,150rpm摇床培养36h,培养液于6000×g,4℃离心15min,弃去上清液,用预先灭菌的0.9%(w/v)生理盐水清洗菌体,于6000×g,4℃离心15min收集菌体,重复3次。最后悬浮于同体积的无菌0.9%(w/v)生理盐水中,制成菌悬液。
其中,液体培养基由以下组分构成:YPD50g/L,水。
(3)超声预处理:将浓缩乳清蛋白WPC80配成5g/100mL溶液,以2s:1s、200W、10min作为超声条件进行处理,得到预处理产物,向其中加入4g/100mL乳糖,经108℃,15min灭菌处理后得到发酵培养基。
(4)发酵培养:将准备好的菌悬液以10%接种量接种于装有100mL发酵培养基的250mL三角瓶中,150r/min摇床培养48h后,产物存于4℃冰箱24h,存于-20℃冰箱。
其中,发酵培养基的初始pH、发酵温度及接种量下表1。其中实施例1~17为响应面优化实验,实施例18为验证实验。
(5)蛋白水解能力的测定:配置OPA试剂:A液:19.05g硼砂,500mg十二烷基磺酸钠(SDS)完全溶于375mL蒸馏水。B液:160mg邻苯二甲醛(98%)(OPA)溶于10mL无水乙醇。C液:440mg二硫苏糖醇(DTT)溶于50mL蒸馏水。将配制好的B液全部加入A液,用去离子水冲洗,充分混匀,然后将C液加入巳混合好的A液B液混合物中,用去离子水冲洗,蒸馏水定容至500mL。标准曲线测定:分别配制浓度为0.04、0.08、0.12、0.16、0.20mg/mL丝氨酸标准溶液。400μL丝氨酸标准溶液加入到3mLOPA试剂,混合均匀,室温静置2min后,在340nm下测定吸光度值。以蒸馏水作空白对照,每个样品重复测定5次,取平均值绘制标准曲线。样品测定:取200μL发酵乳于2mL离心管中,加入400μL蒸馏水混匀,再加入1mL0.75mol/L三氯乙酸(TCA)溶液,混匀后静置l0min,3000xg离心5min,取上清液。取该上清液100μL加入到0.75mLOPA试剂中,混匀后室温反应2min,340nm测定吸光度值,以未接种的脱脂牛奶培养基作为对照。OPA指数(OPA index)定义为:用OPA法测量340nm吸光度的差值所对应于标准曲线丝氨酸的量,用于表示发酵乳蛋白的水解程度。
(6)牛α-LA、β-Lg酶联免疫测定:蛋白质的提取:取发酵样品0.2mL,加入0.8mL蒸馏水,10000RPM离心5min,上清液-20℃保存。标准品的稀释与加样:在酶标包被板上设标准品孔10孔,在第一、第二孔中分别加标准品100μL,然后再第一、第二孔中加标准品稀释液50μL,混匀;然后从第一、第二孔中各取100μL分别加到第三孔和第四孔,再在第三、第四孔分别加标准品稀释液50μL,混匀;然后在第三孔和第四孔中先各取50μL弃掉,再各取50μL分别加到第五、第六孔中,再在第五、第六孔中分别加标准品稀释液50μL,混匀,以此类推,直至十个孔逐步稀释,并且每个孔余下50μL。加样:分别设空白孔(不加样品及酶标试剂,其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中县加样品稀释液40μL,然后再加待测样品10μL(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动摇匀。加酶:每孔加入酶标试剂50μL,空白孔除外。温育:用封板膜封板后置37℃温育30min。配液:将30(48T的20倍)倍浓缩洗涤液用蒸馏水30(48T的20倍)倍稀释后备用。洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,拍干。显色:每孔先加入显色剂A50μL,再加入显色剂B50μL,轻轻震荡混匀,37℃避光显色10min。终止:每孔加终止液50μL,终止反反应(此时蓝色立转为黄色)。测定:以空白孔调零,450nm波长依序测定各孔的吸光度(OD值)。测定应在加终止液15min以内进行。
表1实施例1-18发酵条件及致敏性降低率
(7)发酵产物分离纯化:采用超滤分离设备对发酵液进行超滤分离。
其中,超滤条件为:pH6.5,滤膜截留分子量为10kDa和1kDa。
综上所述,通过将乳清蛋白溶液超声处理后加入乳糖溶液,混匀灭菌得到发酵培养基,接入马克思克鲁维酵母(Kluyveromyces marxianus)进行发酵培养,发酵完成后即可从发酵液中分离纯化得到低致敏性乳清蛋白。产物安全无毒,并且主要致敏蛋白α-LA致敏性降低29.07±1.53%,β-LG致敏性降低53.07±3.04%。
Claims (10)
1.一种利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法,包括:
(1)将乳清蛋白溶液进行超声处理,处理完成后,加入乳糖溶液,混匀,灭菌,得到发酵培养基;
(2)将活化的马克思克鲁维酵母(Kluyveromyces marxianus)接入步骤(1)制得的发酵培养基中进行发酵培养,培养完成后,通过后处理从发酵液中分离纯化得到低致敏性乳清蛋白。
2.如权利要求1所述的方法,其特征在于,步骤(1)中,所述超声处理条件为:超声间歇比2s:1s~2s:3s,超声功率100~300W,超声时间5~15min。
3.如权利要求2所述的方法,其特征在于,所述超声处理条件为:超声间歇比2s:1s、超声功率200W、处理时间10min。
4.如权利要求1所述的方法,其特征在于,步骤(1)中,所述发酵培养基中乳清蛋白的浓度为3~8g/100mL,乳糖的浓度为2~6g/100mL。
5.如权利要求1所述的方法,其特征在于,步骤(2)中,所述马克思克鲁维酵母(Kluyveromyces marxianus)为马克思克鲁维酵母(Kluyveromyces marxianus)CGMCC2.68。
6.如权利要求1所述的方法,其特征在于,步骤(2)中,所述活化的马克思克鲁维酵母采用活化马克思克鲁维酵母的菌悬液,在发酵培养基中的接种量为2~14%。
7.如权利要求1所述的方法,其特征在于,步骤(2)中,所述发酵培养的条件为温度20~45℃,初始pH3.0~9.0,转速0~250rpm,时间1~3天。
8.如权利要求7所述的方法,其特征在于,所述发酵培养的条件为温度25~35℃,初始pH6.7~7.3,转速150rpm。
9.一种采用如权利要求1所述的方法制备得到的低致敏性乳清蛋白。
10.如权利要求9所述的低致敏性乳清蛋白在低致敏性婴幼儿配方奶粉中应用。
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