CN113995068A - 苹果海盐发酵饮料及其制备方法 - Google Patents
苹果海盐发酵饮料及其制备方法 Download PDFInfo
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- CN113995068A CN113995068A CN202111119574.7A CN202111119574A CN113995068A CN 113995068 A CN113995068 A CN 113995068A CN 202111119574 A CN202111119574 A CN 202111119574A CN 113995068 A CN113995068 A CN 113995068A
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- apple
- culture medium
- fermented beverage
- sea salt
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Abstract
本发明公开了一种苹果海盐发酵饮料,所述饮料包括如下重量份数的组成成分:苹果230份、毕赤酵母20份、植物乳杆菌20份、罗伊氏乳杆菌10份、木糖醇20份、海盐10份、高活性微胶囊1份、磷酸二氢钾5份、α‑鼠李糖苷酶3份、木聚糖酶2份、柠檬酸钠5份、α‑淀粉酶4份。本发明针对发酵饮料营养价值低、口味单一及菌体活性保持差等问题进行改进,通过微胶囊包埋技术和新型加工手段,对发酵饮料感官品质及营养价值进行优化,力求发明一种风味佳、口感独特、利于消化吸收的发酵饮料。
Description
技术领域
本发明属于食品技术领域,尤其是一种苹果海盐发酵饮料及其制备方法。
背景技术
现有技术中饮料存在如下的问题:
1、苹果富含黄酮类化合物,具有抗氧化、降血脂等功效。黄酮类化合物主要由黄酮糖苷组成,由于黄酮糖苷的不易吸收性,导致苹果中黄酮类化合物生物利用率极低、不能被人体消化吸收,致使发酵饮料保健效果降低。
2、现有发酵饮料香味单一寡淡,制作过程中会损失大部分芳香物质,不利于发酵得到高香型发酵产品,不能满足人们的需求。
3、发酵果蔬汁发酵时间过长,且发酵乳酸菌的增殖速率、浓度低,得到的活性菌饮品,在储藏期内活性保持效果较差,发酵效率不高。
4、饮料中营养物质易发生氧化损失、酶褐变等不良变化影响品质。
通过检索,发现如下几篇与本发明专利申请相关的专利公开文献:
1、一种清爽型褐色脱脂发酵乳饮料及其生产方法(CN1046866),公开了一种发酵乳饮料及其生产方法,包括原料脱脂牛乳,葡萄糖,副干酪乳杆菌等,产品味道可口。
优点:味道可口、具有抗氧化保健功能、常饮用有利于健康。
问题:①功能因子组成少,保健效果差;②单一的乳杆菌菌株发酵环境抗逆性差;③香味单一寡淡。
对策:①采用复合菌种发酵,提高发酵效率,提高菌种活性。②采用二次发酵辅助复合增香技术,保持浓郁香气成分。
2、一种风味蓝莓汁的加工方法(CN107549557A),通过干酪乳杆菌和植物乳杆菌协同发酵,提高了饮料原有风味口感。经水浴杀菌,可抑制蓝莓果汁中的酶活性、杀死有害杂菌但不影响其风味。
优点:通过添加复合稳定剂,增强产品稳定性,结合均质、抽滤、冷藏等操作,增强口感,延长货架期。
问题:①发酵饮料在发酵过程中菌体增殖速率偏低且活性不高;②经水浴加热处理后,会减弱蓝莓饮料的香气,并影响其口感及营养价值;③以葡萄糖作为甜味剂虽利于吸收,但不利于糖尿病等特殊患者饮用。
对策:①通过对菌体进行微胶囊包埋,形成保护作用,可避免菌体在发酵后期受到损伤降低菌体活性;②采用巴氏杀菌可在低温条件下保持发酵饮料营养物质,且对其进行杀菌。③利用木糖醇代替甜味剂,在不改变饮料甜度的情况下,减少糖摄入量。
3、一种刺梨发酵饮料及其制作方法(CN108685002A),所述饮料主要由刺梨果汁、刺梨渣、苹果渣、铁皮石解渣、黄精渣、蒸馏水和山梨醇制成。
优点:制作的饮料营养成分均衡,且不易氧化褐变,稳定性好,口感好,无涩味。
问题:①发酵饮料在发酵过程中菌体增殖速率偏低且活性不高;③发酵时间长,发酵效率不高。
对策:①采用二次发酵辅助复合增香技术,保持浓郁香气成分;②通过对菌体进行微胶囊包埋,形成保护作用,可避免菌体在发酵后期受到损伤降低菌体活性。
通过对比,本发明专利申请与上述专利公开文献存在本质的不同。
发明内容
本发明目的在于克服现有技术中的不足之处,提供一种苹果海盐发酵饮料及其制备方法。
本发明解决其技术问题所采用的技术方案是:
一种苹果海盐发酵饮料,所述饮料包括如下重量份数的组成成分:
苹果230份、毕赤酵母20份、植物乳杆菌20份、罗伊氏乳杆菌10份、木糖醇20份、海盐10份、高活性微胶囊1份、磷酸二氢钾5份、α-鼠李糖苷酶3份、木聚糖酶2份、柠檬酸钠5份、α-淀粉酶4份。
进一步地,所述高活性微胶囊的制备步骤如下:
1)芯材:将植物乳杆活菌粉、乳清蛋白粉加入到的蒸馏水中,静置四十八小时,使其混合充分,得芯材液;植物乳杆活菌粉:乳清蛋白粉:蒸馏水的比例g:g:ml为2:1:5;
2)胶囊:将芯材液加入到浓度4.8g/100mL的可食性抗性糊精溶液中,再加入海藻酸钠,混合物中海藻酸钠的终浓度为16.5wt%,得混合物;以质量浓度1.5%的冰乙酸溶解并稀释成浓度为3.2g/100mL的氯化钙溶液,将氯化钙溶液加入可食性抗性糊精溶液中,氯化钙的终浓度为2.1g/100mL,调pH值至5.5,得可食性抗性糊精-氯化钙溶液;将混合物与壳聚糖的混合液用注射器逐滴滴入到可食性抗性糊精-氯化钙溶液中,进行成膜反应,过滤收集微胶囊,用灭菌的生理盐水洗涤微球,即得高活性微胶囊;
其中,可食性抗性糊精、海藻酸钠、壳聚糖的质量比为4:4:3。
进一步地,所述高活性微胶囊的直径为50微米。
如上所述的苹果海盐发酵饮料的制备方法,步骤如下:
(1)选择成熟苹果为原料,采用高温105-110℃高压饱和蒸汽对苹果恒温杀青,蒸汽成分包括醋酸、氯化钙、硫酸锌、抗坏血酸钠、柠檬酸、脱醛酶多物质组成的复合体,恒温循环20-25min,有效杀青;
其中,所述复合体中包含1.6mmol/L醋酸,1.6mmol/L氯化钙,0.8mmol/L硫酸锌,1.6mmol/L抗坏血酸钠,0.8mmol/L脱醛酶,0.8mmol/L柠檬酸;
(2)再采用低温0-5℃高压饱和雾化蒸汽对苹果恒温冷激护色,蒸汽成分包括碳酸钙、氯化钙、氯化铜多物质组成的复合体,恒定低温冷激循环5-10min;
其中,所述复合体中包含0.8mmol/L碳酸钙、1.6mmol/L氯化钙、1.6mmol/L氯化铜;
(3)多级酶解:
1)预处理:将步骤(2)得到的苹果进行低温破碎打浆;
2)多级复合酶解:添加高效浸提复合酶,提取温度40℃,提取时间6h,加酶量为打浆质量的0.4%;过滤后,浆液中加高色值复合酶,95℃,10min;在100HZ条件下,超声10min,即得苹果浆液,备用;
其中,高效浸提复合酶为果胶裂解酶2ml/L、果胶醋酶5ml/L、纤维素酶6ml/L;高色值复合酶为木瓜蛋白酶4ml/L、菠萝蛋白酶6ml/L和无花果蛋白酶7ml/L;
(4)发酵:
1)一次发酵:
毕赤酵母培养:
①将活化好的毕赤酵母以10%(V/V)的接种量接种到发酵培养基中,30℃发酵12h;
②活化好的种子液,以10%(V/V)的接种量接种到种子培养基中培养10h,使菌体处于对数生长的后期;
③取上述种子培养液12000r/min离心2min,收集菌体,然后用无菌生理盐水洗涤3次,最后将菌体重悬于无菌生理盐水中,使细胞的终浓度为1×107CFU/m L;
④将菌悬液稀释到105CFU/ml,接种到步骤(3)得到的苹果浆液中,添加α-鼠李糖苷酶、木聚糖酶、α-淀粉酶,发酵5h,然后添加高活性微胶囊,得发酵液;
2)二次发酵:
植物乳杆菌+罗伊氏乳杆菌:
①菌株富集培养:分别吸取制备好的植物乳杆菌、罗伊氏乳杆菌样品液,接种至MRS肉汤培养基,37℃恒温培养24h;
②菌株初筛:植物乳杆菌、罗伊氏乳杆菌分别采用溶钙圈法,将富集的菌液进行梯度稀释,取菌液涂布于MRS初筛培养基中,37℃培养24-36h,挑取具有透明溶钙圈的单菌落划线,获得形态单一的菌落;
③菌株复筛:将初筛获得的两种植物乳杆菌、罗伊氏乳杆菌菌株分别接种于MRS肉汤培养基,37℃培养24h,8000r/min离心10min,收集上清液,用0.22μm滤膜过滤除菌,制备无菌上清液;以短波单胞菌属(Brevundimonas sp.)CICC10994为指示菌,在营养琼脂培养基中添加短波单胞菌属CICC10994菌液,终浓度为106CFU/mL,制备指示菌平板,采用牛津杯琼脂扩散法,牛津杯中加样,4℃冰箱预扩散10h后,于37℃培养14h,测定抑菌圈大小,筛选抑菌圈最大的菌株;
④TPY培养:使用TPY培养基30℃培养24h;挑选透明圈/菌落直径最大的植物乳杆菌、罗伊氏乳杆菌于105CFU/ml接种到步骤(4)1)一次发酵得到的发酵液中;
(5)调酸调糖:向二次发酵液中添加木糖醇、海盐、柠檬酸钠、磷酸二氢钾,进行调配,再离心、杀菌、灌装后得到饮料成品。
进一步地,所述步骤(1)中苹果为9成熟苹果;
或者,所述步骤(2)中对苹果恒温冷激护色时采用喷射隧道式恒温冷激。
进一步地,所述步骤(4)2)①中的样品液为泡菜或酸菜样品汁液。
进一步地,所述步骤(4)1)①中发酵培养基的配方为:H3PO4质量浓度25g/L、K2HPO4质量浓度0.5g/L、NH4Cl质量浓度0.30g/L、Ca SO4·2H2O质量浓度0.25g/L、Na Cl质量浓度0.6g/L、K2SO4质量浓度3.0g/L、Mg SO4·7H2O质量浓度2.2g/L;
所述步骤(4)1)②中种子培养基的配方为:3.0g酵母提取物,3.0g麦芽提取物,10.0g葡萄糖,5.0g蛋白栋,20.0g琼脂,蒸馏水1000ml;
所述步骤(4)2)②中MRS初筛培养基的配方为:蛋白胨10克,牛肉膏10克,酵母提取物5克,柠檬酸二铵2克,乙酸钠5克,葡萄糖20克,吐温80毫升,硫酸镁0.5克,硫酸锰0.25琼脂粉15克;
所述步骤(4)2)③中营养琼脂培养基的配方为:蛋白胨10g,牛肉膏粉10g,氯化钠5g,葡萄糖20g,吐温80ml,硫酸镁0.5g,硫酸锰0.25g,琼脂15g;
所述步骤(4)2)④中TPY培养基的配方为:胰蛋白胨8g,植物蛋白胨8g,酵母粉5g,氯化钠5g,葡萄糖5.0g,乙酸钠5.0g,柠檬酸二铵2.0g,吐温801.0g,K2HPO42.0g,MgSO4.7H2O 0.2g,MnSO4.H2O 0.05g,CaCO320.0g,葡萄糖20g,蒸馏水1.0L,pH6.8。
进一步地,所述步骤(5)中调配为将pH调至3.5-3.6,所述离心为常温离心3500r/min15min,所述杀菌为巴士杀菌20min,60℃。
进一步地,所述步骤(4)1)③得到的菌悬液还经过如下处理:
取上述菌悬液均匀涂布,晾干后将其置于多功能低强度感应电场联动表面等离子体系中,即电场和等离子的结合使用,1kv·cm-1电场场强辅助下,输入功率120W,照射间距3mm,氮气流量10L/min,处理时长90s;加入生理盐水,将菌种制成菌悬液;稀释到1×10-5梯度后,均匀涂到CaCO3培养基表面,30℃培养24h;挑选透明圈/菌落直径最大的毕赤酵母105CFU/ml接种到步骤(3)得到的苹果浆液中,发酵5h,然后添加高活性微胶囊;
其中,所述CaCO3培养基的组分及重量百分比为:0.05%的磷酸二氢钾,0.2%的碳酸钙,0.5%的蛋白胨,1%的葡萄糖,2%的酵母膏,溶剂为水;
进一步地,所述电场的电源能够提供从0kV到30kV连续变化的交流电压,频率为1kHz,采用1kv·cm-1场强;
或者,所述电场的上极板为针尖型电极,下极板为平板型电极,电场处理时两极间距离为0.8cm。
本发明取得的优点和积极效果为:
1、本发明针对发酵饮料营养价值低、口味单一及菌体活性保持差等问题进行改进,通过微胶囊包埋技术和新型加工手段,对发酵饮料感官品质及营养价值进行优化,力求发明一种风味佳、口感独特、利于消化吸收的发酵饮料。
2、本发明将苹果中富含的黄酮糖苷高效转化成相应的黄酮苷元,转化率达85%以上,使其更容易被人体吸收,大大提高苹果中黄酮类化合物的生物利用率,达到抗氧化的目的。另外β-葡萄糖苷酶在水解糖苷的同时还能释放出挥发性的香气成分,使发酵饮料富含酯香气。
3、本发明方法中苹果发酵后期也能起到增香的作用,将苹果中富含的少量脂肪物尽其用。
4、本发明延缓了发酵饮料中植物乳杆菌活性的损失,维持其高活性。
5、苹果切分后,果肉暴露于空气中后褐变迅速,致使后期发酵饮料颜色不鲜艳。发酵周期长易滋生有害细菌。本发明可增加苹果组织透性,提高组织内外物质交换的能力,钝化苹果所含酶的活性,抑制营养物质的氧化损失、酶褐变等不良变化。
6、本发明采用了恒温循环熏蒸协同循环冷激护色技术,采用高温(105-110℃)高压饱和蒸汽对苹果进行恒温循环熏蒸处理,并通过向熏蒸液中添加,醋酸、氯化钙、抗坏血酸钠、柠檬酸等多种护色保鲜物质,可提高苹果品质质量。随后,采用低温(0-5℃)高压饱和蒸汽对苹果进行恒温循环冷激,可有效护色,并可增加苹果组织透性,提高组织内外物质交换的能力,便于后续工序操作,钝化苹果所含酶的活性,抑制营养物质的氧化损失、酶褐变等不良变化。
7、本发明采用了二次发酵辅助复合增香技术,柠檬酸钠、酵母粉、磷酸二氢钾复配结合海盐作为发酵增殖关键因子,可以辅助植物乳杆菌和罗伊氏乳杆菌大量分泌脂肪酶,脂肪酶水解脂肪释放脂肪酸,同时产生系列挥发性呈味物质如游离脂肪酸、形成异戊醛、二乙酰、3-羟基丁酮等,在发酵后期起到增香的作用,将苹果中富含的少量脂肪物尽其用。
另外植物乳杆菌和罗伊氏乳杆菌生长最适pH值在5.5-6.2,发酵后期菌体生长环境的pH降低时,会对菌体存活造成不良影响,同时又会造成菌体活性降低。将发酵增殖关键因子添加到果汁中,可以提高苹果汁的pH和缓冲能力,有利于菌体的活性保持,对植物乳杆菌的增殖起到促进作用。
8、本发明采用了植物乳杆菌高活性保持技术,带有氨基、羧基和羟基的乳清蛋白可以与植物乳杆菌细胞膜表面的磷脂基团或蛋白质极性基团形成氢键,降低H+对菌体细胞的损伤。
采用乳清蛋白、抗性糊精及海藻酸钠对菌体具有保护作用的物质为基础材质对一部分植物乳杆菌进行包埋处理,所制备的微胶囊可在发酵后期减缓H+的侵入,对菌体起到保护兼缓冲作用,发酵后期及贮藏过程中延缓了植物乳杆菌活性的损失,维持其高活性。
9、本发明采用了多功能低强度感应电场联动表面等离子体系促进一次发酵,毕赤酵母菌的代谢作用可将糖类分解为乙醇等物质,进而可与苹果酱醅中的有机酸发生酯化反应,生成酯类等风味物质。
在低强度感应电场辅助下,毕赤酵母菌体细胞膜中的磷脂分子链重新定向排列,镶嵌在双分子层上的蛋白质亚基离子通道发生形变,另外通过添加海盐、氯化钙,不仅增加了发酵饮料的风味,同时增强Ca2+,Na+等离子进出细胞的活动,强化了生长分裂繁殖进程,最终提升了毕赤酵母的存活率。
α-鼠李糖苷酶,木聚糖酶,α-淀粉酶组成的复合酶法预处理,结合表面等离子体系促进毕赤酵母发酵高产的β-葡萄糖苷酶,可以将黄酮糖苷高效转化成相应的黄酮苷元,转化率达85%以上,使其更容易被人体吸收,大大提高苹果中黄酮类化合物的生物利用率,达到抗氧化的目的。另外β-葡萄糖苷酶在水解糖苷的同时还能释放出挥发性的香气成分,使发酵饮料富含酯香气。
附图说明
图1为本发明中电场的一种结构连接示意图;
图2为本发明中所使用的等离子体的一种设备照片示意图。
具体实施方式
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。
一种苹果海盐发酵饮料,所述饮料包括如下重量份数的组成成分:
苹果230份、毕赤酵母20份、植物乳杆菌20份、罗伊氏乳杆菌10份、木糖醇20份、海盐10份、高活性微胶囊1份、磷酸二氢钾5份、α-鼠李糖苷酶3份、木聚糖酶2份、柠檬酸钠5份、α-淀粉酶4份。
较优地,所述高活性微胶囊的制备步骤如下:
1)芯材:将植物乳杆活菌粉、乳清蛋白粉加入到的蒸馏水中,静置四十八小时,使其混合充分,得芯材液;植物乳杆活菌粉:乳清蛋白粉:蒸馏水的比例g:g:ml为2:1:5;
2)胶囊:将芯材液加入到浓度4.8g/100mL的可食性抗性糊精溶液中,再加入海藻酸钠,混合物中海藻酸钠的终浓度为16.5wt%,得混合物;以质量浓度1.5%的冰乙酸溶解并稀释成浓度为3.2g/100mL的氯化钙溶液,将氯化钙溶液加入可食性抗性糊精溶液中,氯化钙的终浓度为2.1g/100mL,调pH值至5.5,得可食性抗性糊精-氯化钙溶液;将混合物与壳聚糖的混合液用注射器逐滴滴入到可食性抗性糊精-氯化钙溶液中,进行成膜反应,过滤收集微胶囊,用灭菌的生理盐水洗涤微球,即得高活性微胶囊;
其中,可食性抗性糊精、海藻酸钠、壳聚糖的质量比为4:4:3。
较优地,所述高活性微胶囊的直径为50微米。
如上所述的苹果海盐发酵饮料的制备方法,步骤如下:
(1)选择成熟苹果为原料,采用高温105-110℃高压饱和蒸汽对苹果恒温杀青,蒸汽成分包括醋酸、氯化钙、硫酸锌、抗坏血酸钠、柠檬酸、脱醛酶多物质组成的复合体,恒温循环20-25min,有效杀青,提高了杀青质量;
其中,所述复合体中包含1.6mmol/L醋酸,1.6mmol/L氯化钙,0.8mmol/L硫酸锌,1.6mmol/L抗坏血酸钠,0.8mmol/L脱醛酶,0.8mmol/L柠檬酸;
(2)再采用低温0-5℃高压饱和雾化蒸汽对苹果恒温冷激护色,蒸汽成分包括碳酸钙、氯化钙、氯化铜多物质组成的复合体,恒定低温冷激循环5-10min,有效护色;
其中,所述复合体中包含0.8mmol/L碳酸钙、1.6mmol/L氯化钙、1.6mmol/L氯化铜;
(3)多级酶解:(高效浸提复合酶解及高色值复合酶解)
1)预处理:将步骤(2)得到的苹果进行低温破碎打浆;
2)多级复合酶解:添加高效浸提复合酶,提取温度40℃,提取时间6h,加酶量为打浆质量的0.4%;过滤后,浆液中加高色值复合酶,95℃,10min;在100HZ条件下,超声10min,即得苹果浆液,备用;
其中,高效浸提复合酶为果胶裂解酶2ml/L、果胶醋酶5ml/L、纤维素酶6ml/L;高色值复合酶为木瓜蛋白酶4ml/L、菠萝蛋白酶6ml/L和无花果蛋白酶7ml/L;
(4)发酵:
1)一次发酵:
毕赤酵母培养:
①将活化好的毕赤酵母以10%(V/V)的接种量接种到发酵培养基中,30℃发酵12h;
②活化好的种子液,以10%(V/V)的接种量接种到种子培养基中培养10h,使菌体处于对数生长的后期;
③取上述种子培养液12000r/min离心2min,收集菌体,然后用无菌生理盐水洗涤3次,最后将菌体重悬于无菌生理盐水中,使细胞的终浓度为1×107CFU/m L;
④将菌悬液稀释到105CFU/ml,接种到步骤(3)得到的苹果浆液中,添加α-鼠李糖苷酶、木聚糖酶、α-淀粉酶,发酵5h,然后添加高活性微胶囊,得发酵液;
2)二次发酵:
植物乳杆菌+罗伊氏乳杆菌:
①菌株富集培养:分别吸取制备好的植物乳杆菌、罗伊氏乳杆菌样品液,接种至MRS肉汤培养基,37℃恒温培养24h;
②菌株初筛:植物乳杆菌、罗伊氏乳杆菌分别采用溶钙圈法,将富集的菌液进行梯度稀释,取菌液涂布于MRS初筛培养基中,37℃培养24-36h,挑取具有透明溶钙圈的单菌落划线,获得形态单一的菌落;
③菌株复筛:将初筛获得的两种植物乳杆菌、罗伊氏乳杆菌菌株分别接种于MRS肉汤培养基,37℃培养24h,8000r/min离心10min,收集上清液,用0.22μm滤膜过滤除菌,制备无菌上清液;以短波单胞菌属(Brevundimonas sp.)CICC10994为指示菌,在营养琼脂培养基中添加短波单胞菌属CICC10994菌液,终浓度为106CFU/mL,制备指示菌平板,采用牛津杯琼脂扩散法,牛津杯中加样,4℃冰箱预扩散10h后,于37℃培养14h,测定抑菌圈大小,筛选抑菌圈最大的菌株;
④TPY培养:使用TPY培养基30℃培养24h;挑选透明圈/菌落直径最大的植物乳杆菌、罗伊氏乳杆菌于105CFU/ml接种到步骤(4)1)一次发酵得到的发酵液中;
(5)调酸调糖:向二次发酵液中添加木糖醇、海盐、柠檬酸钠、磷酸二氢钾,进行调配,再离心、杀菌、灌装后得到饮料成品。
较优地,所述步骤(1)中苹果为9成熟苹果;
或者,所述步骤(2)中对苹果恒温冷激护色时采用喷射隧道式恒温冷激。
较优地,所述步骤(4)2)①中的样品液为泡菜或酸菜样品汁液。
较优地,所述步骤(4)1)①中发酵培养基的配方为:H3PO4质量浓度25g/L、K2HPO4质量浓度0.5g/L、NH4Cl质量浓度0.30g/L、Ca SO4·2H2O质量浓度0.25g/L、Na Cl质量浓度0.6g/L、K2SO4质量浓度3.0g/L、Mg SO4·7H2O质量浓度2.2g/L;
所述步骤(4)1)②中种子培养基的配方为:3.0g酵母提取物,3.0g麦芽提取物,10.0g葡萄糖,5.0g蛋白栋,20.0g琼脂,蒸馏水1000ml;
所述步骤(4)2)②中MRS初筛培养基的配方为:蛋白胨10克,牛肉膏10克,酵母提取物5克,柠檬酸二铵2克,乙酸钠5克,葡萄糖20克,吐温80毫升,硫酸镁0.5克,硫酸锰0.25琼脂粉15克;
所述步骤(4)2)③中营养琼脂培养基的配方为:蛋白胨10g,牛肉膏粉10g,氯化钠5g,葡萄糖20g,吐温80ml,硫酸镁0.5g,硫酸锰0.25g,琼脂15g;
所述步骤(4)2)④中TPY培养基的配方为:胰蛋白胨8g,植物蛋白胨8g,酵母粉5g,氯化钠5g,葡萄糖5.0g,乙酸钠5.0g,柠檬酸二铵2.0g,吐温801.0g,K2HPO42.0g,MgSO4.7H2O 0.2g,MnSO4.H2O 0.05g,CaCO320.0g,葡萄糖20g,蒸馏水1.0L,pH6.8。
较优地,所述步骤(5)中调配为将pH调至3.5-3.6,所述离心为常温离心3500r/min15min,所述杀菌为巴士杀菌20min,60℃。
较优地,所述步骤(4)1)③得到的菌悬液还经过如下处理:
取上述菌悬液均匀涂布(平板表面皿),晾干后将其置于多功能低强度感应电场联动表面等离子体系中,即电场和等离子的结合使用,1kv·cm-1电场场强辅助下,输入功率120W,照射间距3mm,氮气流量10L/min,处理时长90s;等离子体中含有氮正离子N+、原子态氧O、OH-自由基等活性成分;加入生理盐水,将菌种制成菌悬液;稀释到1×10-5梯度后,均匀涂到CaCO3培养基表面,30℃培养24h;挑选透明圈/菌落直径最大的毕赤酵母105CFU/ml接种到步骤(3)得到的苹果浆液中,发酵5h,然后添加高活性微胶囊;
其中,所述CaCO3培养基的组分及重量百分比为:0.05%的磷酸二氢钾,0.2%的碳酸钙,0.5%的蛋白胨,1%的葡萄糖,2%的酵母膏,溶剂为水;
较优地,所述电场的电源能够提供从0kV到30kV连续变化的交流电压,频率为1kHz,采用1kv·cm-1场强;
或者,所述电场的上极板为针尖型电极,下极板为平板型电极,电场处理时两极间距离为0.8cm。其结构可以如图1所示。所使用的等离子体可以为MPMS多功能等离子体诱变系统,具体设备可以如图2所示。
具体地,相关制备及检测实施例如下:
一种苹果海盐发酵饮料,所述饮料包括如下重量份数的组成成分:
苹果230份、毕赤酵母菌20份、植物乳杆菌20份、罗伊氏乳杆菌10份、木糖醇20份、海盐10份、氯化钙8份、柠檬酸钠5份、α-鼠李糖苷酶3份,木聚糖酶2份,α-淀粉酶4份、酵母粉3份、磷酸二氢钾5份、乳清蛋白5份、抗性糊精4份、海藻酸钠4份、醋酸2份、抗坏血酸钠2份、柠檬酸7份、高活性微胶囊1份。
其中,上述高活性微胶囊的制备如下:
①芯材:将20g植物乳杆活菌粉、10g乳清蛋白粉加入到50ml的蒸馏水中,静置四十八小时,使其混合充分。
②胶囊:将处理好的混合液加入到浓度4.8g/100mL的可食性抗性糊精溶液中,再加入5g海藻酸钠,混合物的终浓度为16.5wt%,以1.5%冰乙酸溶解并稀释成浓度3.2g/100mL的氯化钙溶液,加入可食性抗性糊精溶液,其浓度为2.1g/100mL,调p H值至5.5,将混合物与壳聚糖的混合液用注射器逐滴滴入到可食性抗性糊精-氯化钙溶液中进行成膜反应,过滤收集微胶囊,用灭菌的生理盐水洗涤微球,即得高活性微胶囊(胶囊直径50微米)。
上述苹果海盐发酵饮料的制备工艺流程可以如下:
苹果原料→高温(105-110℃)高压饱和蒸汽→低温(0-5℃)高压饱和蒸汽→低温破碎打浆→酶解→过滤→巴氏杀菌→毕赤酵母结合植物乳杆菌和罗伊氏乳杆菌进行两次发酵→糖酸调配→离心→巴氏杀菌→灌装.
上述苹果海盐发酵饮料的制备方法,包括如下步骤:
1、采用高温(105-110℃)高压饱和蒸汽:采用采用高温(105-110℃)高压饱和蒸汽对苹果恒温杀青,蒸汽成分包括醋酸、氯化钙、硫酸锌、抗坏血酸钠、柠檬酸、脱醛酶等多物质组成的复合体,恒温循环,有效杀青,提高了杀青质量。
2、低温(0-5℃)高压饱和雾化蒸汽:采用低温(0-5℃)高压饱和雾化蒸汽喷射隧道式恒温冷激,蒸汽成分包括碳酸钙、氯化钙、氯化铜多物质组成的复合体,恒定低温循环,有效护色。
3、多级酶解:(高效浸提复合酶解及高色值复合酶解)
(1)预处理:选择9成熟苹果为原料,将其低温破碎打浆。
(2)多级复合酶解:添加高效浸提复合酶,提取温度40℃,提取时间6h,加酶量为打浆量的0.4%。过滤后,浆液中加高色值复合酶,95℃,10min。在100HZ条件下,超声10min,即得苹果浆液,备用。高效浸提复合酶为果胶裂解酶2ml/L、果胶醋酶5ml/L、纤维素酶6ml/L。高色值复合酶为木瓜蛋白酶4ml/L、菠萝蛋白酶6ml/L和无花果蛋白酶7ml/L。
4、发酵:
(1)一次发酵:
毕赤酵母培养:
①将活化好的毕赤酵母以10%(V/V)的接种量接种到含有50m L发酵培养基的500m L三角瓶中,30℃发酵12h。
②活化好的种子液,以10%(V/V)的接种量接种到种子培养基中培养10h,使菌体处于对数生长的后期。
③取1m L上述种子培养液12000r/min离心2min,收集菌体,然后用无菌生理盐水洗涤3次,最后将菌体重悬于无菌生理盐水中,使细胞的终浓度为1×107CFU/m L。
④取上述菌悬液10μL均匀的涂布晾干后将其置于多功能低强度感应电场联动表面等离子体系中,1kv·cm-1场强辅助下,输入功率120W,照射间距3mm,氮气流量10L/min,处理时长90s;等离子体中含有氮正离子N+、原子态氧O、OH-自由基等活性成分。加入100μL生理盐水,将菌种制成菌悬液。稀释到1×10-5梯度后,取100μL均匀涂到CaCO3培养基表面,30℃培养24h。挑选透明圈/菌落直径比较大的短乳杆菌105CFU/ml接种到发酵液中,然后添加高活性微胶囊,得发酵液。
(2)二次发酵:
植物乳杆菌+罗伊氏乳杆菌:
①菌株富集培养:分别吸取制备好的样品液(泡菜或酸菜类样品汁液),接种至MRS肉汤培养基,37℃恒温培养24h。
②菌株初筛:采用溶钙圈法,将富集的菌液进行梯度稀释,取100μL适当稀释度的菌液涂布于MRS初筛培养基中,37℃培养24-36h,挑取具有透明溶钙圈的单菌落划线,获得形态单一的菌落。
③菌株复筛:将初筛获得的菌株接种于MRS肉汤培养基,37℃培养24h,8000r/min离心10min,收集上清液,用0.22μm滤膜过滤除菌,制备无菌上清液。以S.uureu.s ATCC14458为指示菌,在营养琼脂培养基中添加S.uureu.s ATCC 14458菌液,终浓度为106CFU/mL,制备指示菌平板,采用牛津杯琼脂扩散法,牛津杯中加样50μL,4℃冰箱预扩散10h后,于37℃培养14h,测定抑菌圈大小,筛选抑菌活力高的菌株。
④TPY培养基:胰蛋白胨8g,植物蛋白胨8g,酵母粉5g,氯化钠5g,葡萄糖5.0g,乙酸钠5.0g,柠檬酸二铵2.0g,吐温801.0g,K2HPO42.0g,MgSO4.7H2O 0.2g,MnSO4.H2O0.05g,CaCO320.0g,葡萄糖20g,蒸馏水1.0L,pH6.8。
5、调酸调糖:对二次发酵液进行调配、离心、杀菌、灌装后得到饮料成品。
本发明的相关检测:
1、用漂烫冷激护色技术制备的发酵饮料(A)与未用漂烫冷激护色技术制备的发酵饮料(B)的颜色比较,说明该技术对苹果发酵饮料有护色作用(L*值越大,表示亮度越高),如下表所示。
2、GC-MS气象色谱质普联用仪分析:
采用二次发酵辅助复合增香技术制备的发酵饮料(A)与未采用二次发酵辅助复合增香技术制备的发酵饮料(B)的谱图进行比较得出,采用二次发酵辅助复合增香技术使异戊醛、二乙酰、3-羟基丁酮化合物相对含量增加,使发酵饮料富含酯香气,如下表所示。
3、黄酮苷元含量表
通过采用多功能低强度感应电场联动表面等离子体系促进一次发酵制备的发酵饮料(A)与未采用多功能低强度感应电场联动表面等离子体系促进一次发酵制备的发酵饮料(B)的黄酮苷元总含量进行比较得出,采用多功能低强度感应电场联动表面等离子体系促进一次发酵制备的发酵饮料中黄酮苷元大量积累,具有保健功能,适合人体吸收。苹果发酵物甲醇提取物酸水解后用高效液相色谱法HPLC测定,以甲醇-水(60:40)为流动相,检测波长为368nm,流速为1.0mL·min-1,得到每100g苹果发酵物中含有黄酮苷元的含量表如下所示。
4、采用本发明技术制备的发酵饮料(A)与未采用本发明技术制备的发酵饮料(B)的各项指标参数比较,如下表所示,说明本发明技术对苹果发酵饮料营养品质的促进作用。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (10)
1.一种苹果海盐发酵饮料,其特征在于:所述饮料包括如下重量份数的组成成分:
苹果230份、毕赤酵母20份、植物乳杆菌20份、罗伊氏乳杆菌10份、木糖醇20份、海盐10份、高活性微胶囊1份、磷酸二氢钾5份、α-鼠李糖苷酶3份、木聚糖酶2份、柠檬酸钠5份、α-淀粉酶4份。
2.根据权利要求1所述的苹果海盐发酵饮料,其特征在于:所述高活性微胶囊的制备步骤如下:
1)芯材:将植物乳杆活菌粉、乳清蛋白粉加入到的蒸馏水中,静置四十八小时,使其混合充分,得芯材液;植物乳杆活菌粉:乳清蛋白粉:蒸馏水的比例g:g:ml为2:1:5;
2)胶囊:将芯材液加入到浓度4.8g/100mL的可食性抗性糊精溶液中,再加入海藻酸钠,混合物中海藻酸钠的终浓度为16.5wt%,得混合物;以质量浓度1.5%的冰乙酸溶解并稀释成浓度为3.2g/100mL的氯化钙溶液,将氯化钙溶液加入可食性抗性糊精溶液中,氯化钙的终浓度为2.1g/100mL,调pH值至5.5,得可食性抗性糊精-氯化钙溶液;将混合物与壳聚糖的混合液用注射器逐滴滴入到可食性抗性糊精-氯化钙溶液中,进行成膜反应,过滤收集微胶囊,用灭菌的生理盐水洗涤微球,即得高活性微胶囊;
其中,可食性抗性糊精、海藻酸钠、壳聚糖的质量比为4:4:3。
3.根据权利要求2所述的苹果海盐发酵饮料,其特征在于:所述高活性微胶囊的直径为50微米。
4.如权利要求1至3任一项所述的苹果海盐发酵饮料的制备方法,其特征在于:步骤如下:
(1)选择成熟苹果为原料,采用高温105-110℃高压饱和蒸汽对苹果恒温杀青,蒸汽成分包括醋酸、氯化钙、硫酸锌、抗坏血酸钠、柠檬酸、脱醛酶多物质组成的复合体,恒温循环20-25min,有效杀青;
其中,所述复合体中包含1.6mmol/L醋酸,1.6mmol/L氯化钙,0.8mmol/L硫酸锌,1.6mmol/L抗坏血酸钠,0.8mmol/L脱醛酶,0.8mmol/L柠檬酸;
(2)再采用低温0-5℃高压饱和雾化蒸汽对苹果恒温冷激护色,蒸汽成分包括碳酸钙、氯化钙、氯化铜多物质组成的复合体,恒定低温冷激循环5-10min;
其中,所述复合体中包含0.8mmol/L碳酸钙、1.6mmol/L氯化钙、1.6mmol/L氯化铜;
(3)多级酶解:
1)预处理:将步骤(2)得到的苹果进行低温破碎打浆;
2)多级复合酶解:添加高效浸提复合酶,提取温度40℃,提取时间6h,加酶量为打浆质量的0.4%;过滤后,浆液中加高色值复合酶,95℃,10min;在100HZ条件下,超声10min,即得苹果浆液,备用;
其中,高效浸提复合酶为果胶裂解酶2ml/L、果胶醋酶5ml/L、纤维素酶6ml/L;高色值复合酶为木瓜蛋白酶4ml/L、菠萝蛋白酶6ml/L和无花果蛋白酶7ml/L;
(4)发酵:
1)一次发酵:
毕赤酵母培养:
①将活化好的毕赤酵母以10%(V/V)的接种量接种到发酵培养基中,30℃发酵12h;
②活化好的种子液,以10%(V/V)的接种量接种到种子培养基中培养10h,使菌体处于对数生长的后期;
③取上述种子培养液12000r/min离心2min,收集菌体,然后用无菌生理盐水洗涤3次,最后将菌体重悬于无菌生理盐水中,使细胞的终浓度为1×107CFU/mL;
④将菌悬液稀释到105CFU/ml,接种到步骤(3)得到的苹果浆液中,添加α-鼠李糖苷酶、木聚糖酶、α-淀粉酶,发酵5h,然后添加高活性微胶囊,得发酵液;
2)二次发酵:
植物乳杆菌+罗伊氏乳杆菌:
①菌株富集培养:分别吸取制备好的植物乳杆菌、罗伊氏乳杆菌样品液,接种至MRS肉汤培养基,37℃恒温培养24h;
②菌株初筛:植物乳杆菌、罗伊氏乳杆菌分别采用溶钙圈法,将富集的菌液进行梯度稀释,取菌液涂布于MRS初筛培养基中,37℃培养24-36h,挑取具有透明溶钙圈的单菌落划线,获得形态单一的菌落;
③菌株复筛:将初筛获得的两种植物乳杆菌、罗伊氏乳杆菌菌株分别接种于MRS肉汤培养基,37℃培养24h,8000r/min离心10min,收集上清液,用0.22μm滤膜过滤除菌,制备无菌上清液;以短波单胞菌属(Brevundimonas sp.)CICC10994为指示菌,在营养琼脂培养基中添加短波单胞菌属CICC10994菌液,终浓度为106CFU/mL,制备指示菌平板,采用牛津杯琼脂扩散法,牛津杯中加样,4℃冰箱预扩散10h后,于37℃培养14h,测定抑菌圈大小,筛选抑菌圈最大的菌株;
④TPY培养:使用TPY培养基30℃培养24h;挑选透明圈/菌落直径最大的植物乳杆菌、罗伊氏乳杆菌于105CFU/ml接种到步骤(4)1)一次发酵得到的发酵液中;
(5)调酸调糖:向二次发酵液中添加木糖醇、海盐、柠檬酸钠、磷酸二氢钾,进行调配,再离心、杀菌、灌装后得到饮料成品。
5.根据权利要求4所述的苹果海盐发酵饮料,其特征在于:所述步骤(1)中苹果为9成熟苹果;
或者,所述步骤(2)中对苹果恒温冷激护色时采用喷射隧道式恒温冷激。
6.根据权利要求4所述的苹果海盐发酵饮料,其特征在于:所述步骤(4)2)①中的样品液为泡菜或酸菜样品汁液。
7.根据权利要求4所述的苹果海盐发酵饮料,其特征在于:所述步骤(4)1)①中发酵培养基的配方为:H3PO4质量浓度25g/L、K2HPO4质量浓度0.5g/L、NH4Cl质量浓度0.30g/L、CaSO4·2H2O质量浓度0.25g/L、NaCl质量浓度0.6g/L、K2SO4质量浓度3.0g/L、MgSO4·7H2O质量浓度2.2g/L;
所述步骤(4)1)②中种子培养基的配方为:3.0g酵母提取物,3.0g麦芽提取物,10.0g葡萄糖,5.0g蛋白栋,20.0g琼脂,蒸馏水1000ml;
所述步骤(4)2)②中MRS初筛培养基的配方为:蛋白胨10克,牛肉膏10克,酵母提取物5克,柠檬酸二铵2克,乙酸钠5克,葡萄糖20克,吐温80毫升,硫酸镁0.5克,硫酸锰0.25琼脂粉15克;
所述步骤(4)2)③中营养琼脂培养基的配方为:蛋白胨10g,牛肉膏粉10g,氯化钠5g,葡萄糖20g,吐温80ml,硫酸镁0.5g,硫酸锰0.25g,琼脂15g;
所述步骤(4)2)④中TPY培养基的配方为:胰蛋白胨8g,植物蛋白胨8g,酵母粉5g,氯化钠5g,葡萄糖5.0g,乙酸钠5.0g,柠檬酸二铵2.0g,吐温80 1.0g,K2HPO4 2.0g,MgSO4.7H2O0.2g,MnSO4.H2O 0.05g,CaCO3 20.0g,葡萄糖20g,蒸馏水1.0L,pH6.8。
8.根据权利要求4所述的苹果海盐发酵饮料,其特征在于:所述步骤(5)中调配为将pH调至3.5-3.6,所述离心为常温离心3500r/min 15min,所述杀菌为巴士杀菌20min,60℃。
9.根据权利要求4至8任一项所述的苹果海盐发酵饮料,其特征在于:所述步骤(4)1)③得到的菌悬液还经过如下处理:
取上述菌悬液均匀涂布,晾干后将其置于多功能低强度感应电场联动表面等离子体系中,即电场和等离子的结合使用,1kv·cm-1电场场强辅助下,输入功率120W,照射间距3mm,氮气流量10L/min,处理时长90s;加入生理盐水,将菌种制成菌悬液;稀释到1×10-5梯度后,均匀涂到CaCO3培养基表面,30℃培养24h;挑选透明圈/菌落直径最大的毕赤酵母105CFU/ml接种到步骤(3)得到的苹果浆液中,发酵5h,然后添加高活性微胶囊;
其中,所述CaCO3培养基的组分及重量百分比为:0.05%的磷酸二氢钾,0.2%的碳酸钙,0.5%的蛋白胨,1%的葡萄糖,2%的酵母膏,溶剂为水。
10.根据权利要求9所述的苹果海盐发酵饮料,其特征在于:所述电场的电源能够提供从0kV到30kV连续变化的交流电压,频率为1kHz,采用1kv·cm-1场强;
或者,所述电场的上极板为针尖型电极,下极板为平板型电极,电场处理时两极间距离为0.8cm。
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