CN111635915A - 一种冠突散囊菌黑色素发酵产物的制备方法与应用 - Google Patents
一种冠突散囊菌黑色素发酵产物的制备方法与应用 Download PDFInfo
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- CN111635915A CN111635915A CN202010602516.9A CN202010602516A CN111635915A CN 111635915 A CN111635915 A CN 111635915A CN 202010602516 A CN202010602516 A CN 202010602516A CN 111635915 A CN111635915 A CN 111635915A
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- melanin
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Abstract
一种冠突散囊菌黑色素发酵产物的制备方法与应用。本发明以冠突散囊菌为发酵菌种,通过特定的液体发酵培养基配方制备高黑色素含量的发酵产物,所述发酵产物包括冠突散囊菌黑色素发酵液及其浓缩物。该黑色素发酵产物具的pH适应范围广、热稳定性强和自然光下长期放置不褪色等特点。该黑色素发酵产物可用于长沙臭豆腐坯料的上色,还可广泛应用于pH>4.5的饮料、果冻、糖果等中低酸性食品及耗油和酱制品中。
Description
技术领域
本发明属于微生物发酵和食品领域,具体涉及一种冠突散囊菌黑色素发酵产物、其制备方法与应用。
背景技术
黑色素在食品加工中具有重要用途,饮料、果冻、饼干及冰激凌等多种食品及酱制品、蚝油等调味品在生产过程中都要使用黑色素来满足消费者的感官需求。另有一些特殊风味食品,如我国传统特色小吃长沙臭豆腐,则以呈墨黑色作为被消费者认可的主要特征。目前,食品中较多使用的黑色素以焦糖色为主,但焦糖色易沉淀、具有焦糊气味,限制了其使用范围;且焦糖色因含有有害物质4-甲基咪唑,其食用安全性在世界范围内备受争议;不法商贩在生产焦糖色时,大量使用铵盐催化来提高色率,使得4-甲基咪唑、氨氮等指标都超过了国家标准,长期食用将可能影响消费者健康。长沙臭豆腐现有上色工艺中,通过浸泡FeSO4溶液才能达到墨黑色的上色效果,FeSO4的使用不仅会严重腐蚀生产设备,而且不法商贩为了增强上色效果,在上色过程中过量添加FeSO4,也给臭豆腐的质量安全带来了隐患。因此,开发新型的、安全的和能满足臭豆腐等多种食品上色要求的黑色素,是当前的市场的技术需求。
冠突散囊菌(Eurotium cristatum)是茯砖黑茶生产过程中的优势发酵菌种,在茯砖茶“发花”过程中起主要作用。冠突散囊菌及其发酵产物不仅安全无毒,而且具有提高免疫力,调节肠道菌群、降血脂、抗氧化和抗菌等多种生物活性(苏凤. 冠突散囊菌接种发酵茯砖茶的初步研究及其安全性评价[D], 武汉:武汉工业学院硕士毕业论文, 2011; 覃金球, 罗美玲, 张旋,等. 冠突散囊菌医药价值研究进展[J]. 食品工业科技, 2018.)。冠突散囊菌在固体培养基上培养时通常会代谢产生色素类物质,初期所产色素以黄色素为主,随着培养时间的延长会有黑色素产生。黑色素是一类结构复杂的多酚或吲哚类生物大分子氧化聚合物,在医药、食品及化妆品等多个领域具有重要用途。
CN111088300A公开了一种冠突散囊菌黑色素发酵制备方法;具体公开了“从茯砖茶中分离的高产黑色素的冠突散囊菌株发酵液除去菌丝体后,加入3 mol/L盐酸使pH=3,低温下放置4h,黑色素分子自然沉淀出来,离心后收集存留在沉淀中的黑色素,用0.01 mol/L的NaOH溶液溶解黑色素,再次加入3 mol/L盐酸使pH=3,沉淀黑色素分子,通过3-5次的沉淀和复溶解过程,黑色素分子得到纯化,最后一次的沉淀用超纯水洗涤3~5次后,冷冻干燥使得黑色素分子彻底干燥。”
杨妮等人(杨妮,刘素纯,王继刚等. 冠突散囊菌产胞外黑色素发酵条件优化及稳定性研究[J].食品与发酵工业, 2020.4.27网络首发;杨妮,刘素纯,王继刚等. 冠突散囊菌胞外黑色素碱法提取工艺的研究[J], 中国酿造,2020,39(3):63-67)对以马铃薯葡萄糖液体培养基培养条件下冠突散囊菌产胞外黑色素的发酵条件进行了优化,并且对酸沉碱溶法提取冠突散囊菌黑素的工艺进行了研究。
二者均采用了碱溶酸沉法对冠突散囊菌黑色素进行提取和纯化,得到冠突散囊菌黑色素。但经发明人研究发现,此种方法获得的冠突散囊菌黑色素溶解性较差,难溶于中性或酸性溶液,也不溶于有机溶剂,只有在强的碱性溶液中才能较好溶解。且其热稳定性及光稳定性也较差。不仅如此,现有技术中发酵法生产冠突散囊菌黑色素的周期较长,所得发酵产物中色素浓度较低。这些都不利于冠突散囊菌黑色素在实际生产中的应用,限制了该黑色素的商业化进程。
发明内容
本发明所要解决的技术问题是,克服以上不足,提供一种以冠突散囊菌为发酵菌种,通过特定的有利于产黑色素的培养基配方来制备黑色素发酵产物的方法。该方法制备的黑色素发酵产物可广泛应用于pH>4.5的液体或固体食品中,并且热稳定性强,自然光下长期放置不褪色。
本发明解决其技术问题所采用的技术方案如下:
一种冠突散囊菌黑色素发酵产物的制备方法,包括以下步骤:
(1)将经活化的冠突散囊菌菌种接种至马铃薯葡萄糖(Potato dextrose, PD)液体培养基中,进行扩大培养,得冠突散囊菌扩培菌种;
(2)将步骤(1)所得冠突散囊菌扩培菌种接种至液体发酵培养基中,26-32℃条件下培养5d以上,得黑色素发酵中间产物;按重量计,所述液体发酵培养基的配方为:新鲜去皮马铃薯5-20份(煮后取汁)、碳源0.5-3份、蛋白胨0.5-2份、酪氨酸 0.1-0.5份和水100份;
(3)对步骤(2)所得黑色素发酵中间产物进行压滤或离心处理,然后经微孔滤膜过滤除菌,得黑色素发酵原液;然后对黑色素发酵原液进行浓缩,获得不同浓度的冠突散囊菌黑色素发酵产物。
优选的,步骤(3)中,所述浓缩为真空浓缩,浓缩的温度为50~75℃。
优选的,步骤(3)中,所述微孔滤膜的孔径为0.22~0.45 μm。
优选的,步骤(2)中,所述接种的接种量为3-8mL冠突散囊菌扩培菌种/100 mL液体发酵培养基。
优选的,步骤(2)中,所述碳源为葡萄糖、蔗糖、麦芽糖或糖蜜,优选葡萄糖。
优选的,步骤(1)中,所述扩大培养为在马铃薯葡萄糖(Potato dextrose, PD)液体培养基中, 28-30℃,180 r/min条件下培养2-4 d。
优选的,步骤(1)中,所述活化为:将冷藏的冠突散囊菌菌种接种至新鲜的马铃薯葡萄糖琼脂(Potato dextose agar, 简称PDA)斜面上,28-30℃培养5-7d,得经活化的冠突散囊菌菌种。
本发明所述突散囊菌菌种购自中国工业微生物菌种保藏管理中心,菌种保藏编号为:CICC41041。所述冠突散囊菌菌种也可从市售的茯砖黑茶中自行分离,分离方法如下:在无菌操作条件下,将小块的茯砖黑茶用镊子或茶刀撬开,在新撬开面选取金花比较旺盛的部位,用接种钩在此部位挑取金花接种在事先制备好的PDA平板上,28-30℃恒温培养5-7d,经镜检及纯化后转接至新鲜的PDA斜面上,28-30℃恒温培养7d后,冷藏保存。
利用紫外-可见光谱仪对本发明所制得的黑色素发酵液在200-1000nm波长范围进行光谱扫描的结果显示,该黑色素在300nm附近有最大吸收峰。测定不同稀释倍数的黑色素发酵液在300nm处的光密度值(OD300nm),并进行稀释倍数和OD300nm的相关性分析,结果表明产品的稀释倍数与OD300nm呈良好正相关(相关系数为0.9947),因此该发酵液中黑色素的浓度可以用其OD300nm值来表示。
上述方法制备的制备的冠突散囊菌黑色素发酵产物也属于本发明的保护范围。
本发明还提供一种食品着色剂,包括上述制备方法制备的冠突散囊菌黑色素发酵液及其浓缩物,所述食品着色剂用于对pH>4.5的固态或液态食品的着色。
优选的,所述食品着色剂用于生产外表呈墨黑色的臭豆腐。
与现有技术相比,本发明有益效果在于:
(1)现有技术中,采用碱溶酸沉法对发酵产物进行进一步提纯,得到冠突散囊菌黑色素,经研究发现该方法得到的黑色素仅能溶解于强碱性溶液中,因此不适用于pH<7的中性或酸性的介质;本发明得到的黑色素发酵原液或其浓缩物,可广泛应用pH>4.5的多种介质中;
(2)本发明方法制备的黑色素发酵产物具有良好的热稳定性和光稳定性,在室内自然光照射条件下放置6个月,肉眼观察色素颜色无明显变化。通过测定样品在放置6个月前后的OD300nm值,计算可得室内自然光下放置6个月后,色素损失率仅为5%左右;在中性至碱性水溶性介质中,经高温121℃杀菌20min,色素损失率仅1.5%左右,颜色几乎无变化;
(3)本发明方法制备的黑色素发酵产物安全性高:所用菌种冠突散囊菌本身安全无毒,发酵培养基中所有成分均可采用食品级原料,发酵完成后仅需过滤、离心或浓缩操作,无需添加或使用其他任何化学试剂,是一款绿色健康的 “零添加”型产品;
(4)本发明方法制备的黑色素发酵产物用于食品着色,着色后,稳定性高,不易褪色;加入该色素的面团经100℃左右高温蒸制30min无明显褪色,经该色素发酵液浸泡上色后的豆腐块经200℃高温油炸3min后无明显褪色;
(5)采用本发明方法制备的黑色素发酵产物作为着色剂,不仅能够对食品进行上色,缓解食品工业中传统使用的焦糖色含有4-甲基咪唑及长沙臭豆腐传统上色工艺中使用的FeSO4等物质可能给消费者健康造成的安全隐患问题;同时还可以有效延长着色产品的货架期,能够减少有害微生物污染引起的腐败变质及涨袋等质量问题;
(6)本发明通过培养基配方的优化研究,获得步骤(2)中所示液体发酵培养基配方,可以在较短发酵时间内获得高浓度的黑色素发酵产物。采用该培养基在接种量为5mL扩培菌种/100mL培养基条件下, 28℃静止培养6d,所得发酵原液稀释至原体积20倍后,其OD300nm仍在0.7以上。
具体实施方式
以下结合实施例对本发明进行进一步的说明。
实施例 1
本实施例所用冠突散囊菌购自中国工业微生物菌种保藏管理中心,菌种保藏编号为:CICC 41041。
本实施例包括以下步骤:
(1)冠突散囊菌菌种的活化和扩培:将冠突散囊菌冷藏菌种接种至新鲜的PDA斜面上,28℃培养7d,得经活化的冠突散囊菌菌种;将经活化的冠突散囊菌菌种接种至马铃薯葡萄糖液体培养基中,28℃,180 r/min条件下培养3d,得冠突散囊菌扩培菌种;
(2)冠突散囊菌黑色素的发酵培养:将步骤(1)得到的冠突散囊菌扩培菌种接种至液体发酵培养基中, 30℃条件下静置培养8d,发酵液的颜色变为墨黑色,终止发酵,得发酵中间产物; 接种量为5mL冠突散囊菌扩培菌种/100 mL液体发酵培养基;液体发酵培养基配方为:新鲜去皮马铃薯10.0g(煮后取汁)、葡萄糖2.0g、 蛋白胨1.5g、 酪氨酸 0.3g、水100mL;
(3)制备黑色素发酵产物:步骤(2)中所得发酵中间产物,经压滤去除冠突散囊菌菌丝体,再经0.45 μm滤膜过滤除菌,即得黑色素发酵液;所得黑色素发酵液在75℃进行不同真空浓缩或完全浓缩,得冠突散囊菌黑色素浓缩液或冠突散囊菌黑色素固体。
本实施例所制得黑色素发酵原液的pH为5.5,该发酵原液经纯水稀释至原体积20倍的稀释液的OD300nm为0.786+0.004。在室温条件下可稳定放置6个月以上。
实施例 2
该实施例中冠突散囊菌菌种分离自市售茯砖黑茶。本实施例包括以下步骤:
(1)冠突散囊菌菌种的活化和扩培:将冷藏的冠突散囊菌菌种活化至新鲜的PDA斜面上,30℃培养5d,得活化的冠突散囊菌菌种;将经活化的冠突散囊菌菌种接种至马铃薯葡萄糖液体培养基中,30℃,180 r/min条件下培养2d,得冠突散囊菌扩培菌种;
(2)冠突散囊菌的发酵培养:将步骤(1)得到的冠突散囊菌扩培菌种接种至液体发酵培养基中,28℃,180r/min条件下培养6d,发酵液的颜色变为墨黑色,终止发酵,得发酵中间产物;接种量为8mL冠突散囊菌扩培菌种/100 mL液体发酵培养基;液体发酵培养基配方为:新鲜去皮马铃薯20.0g(煮后取汁)、麦芽糖1.0g、 蛋白胨0.5g、 酪氨酸 0.5g、水100mL;
(3)制备黑色素发酵产物:步骤(2)中所得发酵中间产物,经5000r/min离心15min去除冠突散囊菌菌丝体,再经0.22 μm滤膜过滤除菌,即得黑色素发酵原液;该发酵原液稀释至20倍体积后的OD300nm值为0.735±0.005。
实施例2所得黑色素发酵液在真空度0.09Mpa左右和55℃条件下进行真空浓缩或真空干燥,得冠突散囊菌黑色素浓缩液或冠突散囊菌黑色素固体。
对比例 1
本对比例中所用菌种同实施例2。本对比例包括以下步骤:
(1)冠突散囊菌菌种的活化和扩培:同实施例2 中步骤(1);
(2)冠突散囊菌的发酵培养:同实施例2中步骤(2);
(3)冠突散囊菌黑色素的制备:步骤(2)中所得发酵中间产物,经5000r/min离心15min去除冠突散囊菌菌丝体,得发酵液,参照CN 111088300A中的酸沉碱溶法制得黑色素固体。
对比例 2
本对比例中所用菌种同实施例2。本对比例包括以下步骤:
(1)冠突散囊菌菌种的活化和扩培:同实施例2 中步骤(1);
(2)冠突散囊菌的发酵培养:将步骤(1)得到的冠突散囊菌扩培菌种分别接种至液体发酵培养基A和B中,液体发酵培养基A配方为:新鲜去皮马铃薯20.0g(煮后取汁)、葡萄糖2.0g、水100mL;液体发酵培养基B配方为:硝酸钠3g、蔗糖30g、磷酸氢二钾1g、硫酸镁0 .5g、氯化钾0.5 g、硫酸亚铁0 .01g、蒸馏水1 L、pH 5.5。接种量为8mL冠突散囊菌扩培菌种/100mL液体发酵培养基。培养条件同实施例2,即:28℃,180r/min条件下培养6d。发酵培养结束后,观察发现液体发酵培养基配方A中发酵液的颜色为橙黄色,配方B中发酵液的颜色为黄褐色。采用这两个配方替代本发明的液体培养基配方,其他条件与实施例2相同的情况下,所得发酵液均未见明显产生黑色素。
将对比例1中制得的黑色素按照CN 111088300A的方法,用0.01mol/L的NaOH溶解制成黑色素溶液,用纯水稀释该黑色素溶液使其对应的OD300nm值与实施例2制得的黑色素发酵液接近。对比该黑色素溶液和实施例2制得的黑色素发酵液对臭豆腐生产所用白豆腐坯料的上色效果(上色过程与实施例3相同)。结果发现经对比例1制成的黑色素溶液浸泡30min后,豆腐坯料出现部分溶解现象,原有的块形和质感被破坏,说明该溶液不能用于臭豆腐坯料的上色。而本发明所制得的黑色素发酵液对臭豆腐生产用坯料具有良好的上色效果(见实施例3),且不会破坏坯料原有的块形和质感。
另外,还对实施例2和对比例1制得的黑色素的进行热稳定性和光稳定性测定。测定方法为:将对比例1制得的黑色素用 0.01mol/L的NaOH溶解制成黑色素溶液,用纯水稀释该黑色素溶液使其OD300nm值与实施例2制得的黑色素发酵液接近,并将该样品标记为样品1,将实施例2制得的黑色素发酵液标记为样品2。热稳定性的测定:在100℃条件下将样品1和样品2分别加热30、60和90min,通过测定样品加热处理前后的OD300nm计算色素的损失率。光稳定性测定:将样品1和样品2分别放置在室内自然光照射条件下放置3、6、9d,通过测定样品在光照处理前后的OD300nm计算色素的损失率。
色素损失率计算公式为:色素损失率 = (样品处理前的OD300nm值-样品经光或热处理后的OD300nm值)/ 样品处理前的OD300nm值
热稳定实验的数据见表1.
表1 100℃加热不同时间对两种样品黑色素稳定性的影响
注:为避免OD值过大超出与浓度呈线性相关的范围,测定前将各样品稀释至原体积的20倍。
由表1中OD300nm值计算可得,样品1加热30min色素损失率为5.7%,加热90min后色素损失率为25.6%。方差分析(P<0.05)结果表明,本实验条件下样品1的光密度值显著降低,而样品2的光密度值变化不显著,可以认为样品中黑色素无损失。
光稳定性实验数据见表2。由表2中OD300nm值计算可得,样品1在自然光下照射3d时的色素损失率为14.5%,照射9d时的色素损失率已达到30.4%。方差分析(P<0.05)结果表明,样品1的OD300nm值显著降低,而样品2的OD300nm值变化不显著,可以认为自然光照射9d时样品2中黑色素无损失。
表2 自然光照射对两种样品黑色素稳定性的影响
注:为避免OD值过大超出与浓度呈线性相关的范围,测定前将各样品稀释至原体积的20倍。
实施例 3
采用实施例2制备的冠突散囊菌发酵原液作为着色剂对长沙臭豆腐进行着色(所用发酵原液稀释20倍后的OD300nm为0.735±0.005)。过程如下:
(1)pH调节:用质量百分浓度为5%的食品级NaOH溶液将冠突散囊菌黑色素发酵原液的pH值调至8.5-11.0(本实施例选择9.0);
(2)上色:将生产臭豆腐用的新鲜白豆腐坯块沥干水分,浸泡在步骤(1)所制得的pH9.0,温度为40-70℃(本实施例选择45℃)的冠突散囊菌黑色素发酵液中,浸泡30-120min(本实施例选择70min),捞出后沥干;
(3)卤水浸泡及油炸:按照臭豆腐生产的常规工艺进行卤水浸泡和油炸,即得臭豆腐成品。
经黑色素发酵液浸泡后,白色豆腐块成功上色,形成具有均匀的墨黑色泽臭豆腐坯,与传统工艺上色后豆腐块的色差值在0.5-2.0之间,用肉眼看不出两者颜色上的明显差别;上色后经卤水浸泡2h,200℃下油炸3min后,豆腐块呈墨黑色,进一步证明本发明制备的黑色素具有优异的热稳定性。
另外,发明人意外发现,本实施例的制备的臭豆腐坯,在未经任何杀菌处理的情况下,在4℃条件下放置7d以上其质感和气味均无任何不良变化,而相同条件下未经上色处理的臭豆腐坯料有明显腐败并有酸臭味。由此可见,冠突散囊菌黑色素发酵液同时还具有抗菌和抗氧化的功能,对于防止臭豆腐变酸腐败具有良好效果。
实施例 4
实施例1制备的冠突散囊菌发酵液浓缩后得到的黑色素固体作为食品着色剂在饼干生产中的应用:
饼干生产的工艺流程通常包括原辅料选择、配粉和面、成型、烘烤、冷却灭菌、包装和检验等工艺步骤。在配粉和面步骤中,可根据对颜色的要求添加本专利之冠突散囊菌黑色素发酵产物,制成特有的黑色饼干。本实施例以以下步骤说明:
(1)将本专利实施例1制得的黑色素固体按重量比1:(10-50)加水溶解;
(2)按照饼干生产的原有要求加入各种配料,用步骤(1)配制的黑色素溶液和面,反复糅合使形成色泽均匀的面团;
(3)按照饼干生产原有工艺进行成型、烘烤、冷却灭菌、包装和检验。得到成品饼干。
本实施例中所生产的黑色饼干,色泽均匀,在经过200℃左右焙烤温度后不褪色。且由于冠突散囊菌黑色素发酵产物本身具有抗氧化、降脂等功能,有利于减缓饼干中脂类的氧化变质和增强饼干的营养功能。
Claims (10)
1.一种冠突散囊菌黑色素发酵产物的制备方法,其特征在于,包括以下步骤:
(1)将经活化的冠突散囊菌菌种接种至马铃薯葡萄糖液体培养基中,进行扩大培养,得冠突散囊菌扩培菌种;
(2)将步骤(1)所得冠突散囊菌扩培菌种接种至液体发酵培养基中,26-32℃条件下培养5d以上,得黑色素发酵中间产物;按重量计,所述液体发酵培养基的配方为:新鲜去皮马铃薯5-20份(煮后取汁)、碳源0.5-3份、蛋白胨0.5-2份、酪氨酸 0.1-0.5份和水100份;
(3)对步骤(2)所得黑色素发酵中间产物进行压滤或离心处理,然后经微孔滤膜除菌,得黑色素发酵原液,可根据需求再对黑色素发酵原液进行浓缩,获得不同浓度的冠突散囊菌黑色素发酵产物。
2.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(3)中,所述浓缩为真空浓缩,浓缩的温度为50~75℃。
3.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(3)中,所述微孔滤膜的孔径为0.22~0.45 μm。
4.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(2)中,所述接种的接种量为3~8mL冠突散囊菌扩培菌种/100 mL液体发酵培养基。
5.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(2)中,所述碳源为葡萄糖、蔗糖、麦芽糖或糖蜜,优选葡萄糖。
6.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(1)中,所述扩大培养为在马铃薯葡萄糖液体培养基中, 28-30℃,180r/min条件下培养2-4d。
7.根据权利要求1所述冠突散囊菌黑色素发酵产物的制备方法,其特征在于,步骤(1)中,所述活化为:将冷藏的冠突散囊菌菌种接种至新鲜的马铃薯葡萄糖琼脂斜面上,28-30℃培养5-7d,得经活化的冠突散囊菌菌种。
8.一种冠突散囊菌黑色素发酵产物,其特征在于,由权利要求1~7任一项所述制备方法制备。
9.一种食品着色剂,其特征在于,包括权利要求1~7任一项所述制备方法制备的冠突散囊菌黑色素发酵产物,所述食品着色剂用于对pH>4.5的固态或液态食品着色。
10.权利要求9食品着色剂,其特征在于,所述食品着色剂用于生产外表呈墨黑色的臭豆腐。
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