CN111357953B - 益生菌发酵果蔬原浆产品 - Google Patents
益生菌发酵果蔬原浆产品 Download PDFInfo
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- CN111357953B CN111357953B CN202010127835.9A CN202010127835A CN111357953B CN 111357953 B CN111357953 B CN 111357953B CN 202010127835 A CN202010127835 A CN 202010127835A CN 111357953 B CN111357953 B CN 111357953B
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- probiotic
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Abstract
本发明属于饮用品技术领域,具体涉及一种益生菌发酵果蔬原浆产品。是将果蔬原浆,经干酪乳杆菌NCU215发酵所得。本发明提供的益生菌发酵果蔬原浆具备以下特点:(1)充分利用新鲜果蔬可食用部分进行生产,与现有果蔬原浆所采用的榨汁浓缩法相比,对原料更有效利用,生产过程中产出废料减少;(2)可产生天然柔和酸味,有效去除水果中的青涩味及蔬菜中的野蒿味,并对水果中较为不愉悦酸味进行柔和;(3)通过益生菌发酵可将果蔬中氨基酸含量提高20%以上,产生多种芳香物质,风味物质提高30%以上,有效提高产品风味和口感;(4)充分保留果蔬原料中的维生素、膳食纤维等营养成分;(5)延长产品保质期,防止腐败。
Description
技术领域:
本发明属于饮用品技术领域,具体涉及一种益生菌发酵果蔬原浆产品。
背景技术:
目前国内外市场上果蔬原浆主要产品为菠萝原浆,山楂原浆,芒果原浆,苹果原浆等,其生产工艺一般为清洗、打浆(榨汁)、浓缩、调配、灌装、灭菌。如CN 105995710 A公开了一种《植物益生菌发酵果蔬浆方法》,通过果蔬前处理、破碎、软化、果蔬制浆、调配、一次杀菌冷却、发酵、离心、脱气、均质、二次杀菌冷却、无菌灌装步骤完成。该发明通过不同的乳酸菌对酸性较弱的果蔬浆进行发酵,产生大量的乳酸等有机酸,从而使其pH降低,同时产生较好的发酵风味,降低了杀菌的条件,节约成本同时保留了果蔬本身的营养、增加发酵产物营养成分;同时通过卧螺离心机浆发酵后的果蔬原浆进行离心分离酵泥,有效提高发酵果蔬浆在饮料行业利用的稳定性。解决了低酸以及酸性果蔬原浆在保质期较短、产品风味不佳、加工成本高以及加工中营养损失严重的问题。CN 107136372A公开了《一种植物益生菌发酵雪莲果浆的方法》,通过前处理、破碎、软化、打浆、酶解、调配、一次杀菌冷却、发酵、离心、脱气、均质、二次杀菌冷却、无菌灌装步骤完成。通过不同的乳酸菌对酸性较弱的雪莲果浆进行发酵,产生大量的乳酸等有机酸,从而使其pH降低,同时产生较好的发酵风味,降低了杀菌的条件,节约成本同时保留了雪莲果本身的营养、增加发酵产物营养成分;同时通过卧螺离心机浆发酵后的果蔬原浆进行离心分离酵泥,有效提高发酵雪莲果浆的稳定性,拓宽了雪莲果在饮料行业的用途。
现有技术普遍存在产品中的风味主要是通过香精调配产生,在产品生产过程中果蔬原料中营养成分流失较多,产品香味不协调、生产工序复杂等问题。
本发明通过自主筛选的具有良好果蔬发酵性能的益生菌菌株对果蔬原浆进行发酵,有效保留了果蔬原料中营养成分,并通过发酵工艺使产品保持了主体香但增加了香气种类,果浆更醇香。产品中没有添加任何香精、色素、防腐剂,是一款新型绿色发酵果蔬原浆产品。
发明内容:
本发明的目的是提供一种以新鲜果蔬为原料,经过益生菌发酵,得到营养成分较丰富,具有一定功能的新型益生菌发酵果蔬原浆,该法制备的益生菌发酵果蔬原浆充分保留了新鲜果蔬中的果肉及果汁部分,实现对原料极大化利用,具有良好的发酵果蔬风味,色泽鲜亮,滋味浓郁,香气宜人,对于人体肠道功能有一定促进作用。
本发明所提供的益生菌发酵原浆,是由以下原料发酵而成的:果蔬原浆80~99.8份,糖浆或代用糖0~19.8份;
进一步地,原料还包括异维生素C钠或维生素C 0.01~0.5份;
进一步地,所述的糖浆或代用糖是白砂糖、葡萄糖、淀粉糖浆、麦芽糖浆、葡萄糖浆、麦芽糖醇、木糖醇、赤藓糖醇或低聚异麦芽糖;
采用上述原料发酵生产益生菌发酵果蔬原浆的方法如下:
(1)选择无腐烂、新鲜果蔬为原料,清洗后去除其不可食用部分,打浆或榨汁后得果蔬原浆,按以上组分和比例搅拌均匀,灭菌;
进一步地,新鲜果蔬经过预煮后再进行打浆或榨汁;
进一步地,灭菌温度为75~132℃,灭菌时间为2秒~50分钟;
(2)灭菌后将原料冷却至20~45℃后,将益生菌按照103~109cfu/mL的比例接种,25℃~45℃发酵6~96小时,pH值2.5~5.0为发酵终点;
进一步地,所述益生菌为一株干酪乳杆菌NCU215,所述干酪乳杆菌(Lactobacillus casei)NCU215,已于2019年10月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.18702;
干酪乳杆菌NCU215筛选自我国传统发酵泡菜,是一株具有优良果蔬发酵性能且耐消化道环境压力的益生菌株,该菌株具有以下生理特性:
①在pH 2.0的PBS中处理2h存活率为78.98%;
②在0.5%的胆盐环境中处理4h后的存活率为84.89%;
③在pH 3.0的模拟胃液中消化3h,随即转移至pH 8.0的模拟肠液中消化8h后,活力无显著下降;
④该菌株具有较好的表面性质及黏附肠上皮细胞的能力:24h的自凝集率为64.32%,表面疏水率为23.15%,对人结肠癌细胞Caco-2的黏附率为7.47%;
⑤该菌株具有较好的抗氧化活性:DPPH自由基清除率为11.91%,羟基自由基清除率为10.85%,总抗氧化能力相当于95.90μmol Trolox,总还原能力相当于0.28mM FeSO4;
⑥该菌株的24h发酵上清液对常见食源性致病菌具有优异的抑菌活性,尤其对单增李斯特菌及金黄色葡萄球菌的抑制活性最好,其抑菌圈直径分别为23.18mm和24.42mm;此外,该菌株的溶血活性检测显示不具溶血性;抗生素敏感性检测显示:该菌株对四环素、氨苄青霉素、阿莫西林、头孢噻吩、红霉素和青霉素敏感,对卡那霉素、环丙沙星、链霉素、庆大霉素耐受;
⑦菌落形态:橙黄色、圆形,扁平、表面光滑、边缘整齐、产酸强、1-3mm;革兰氏染色阳性、菌体呈短杆状(见图1);
结合生理生化特性及16SrRNA序列鉴定为干酪乳杆菌,系统发育树见图2;
进一步地,所述的pH值发酵终点,可以根据不同口味的要求确定;
(3)将发酵后的果蔬原浆进行标准化即为益生菌发酵果蔬原浆产品;
进一步地,所述标准化是按照行业、企业或者实际需求对产品的酸度、甜度进行标准化,通过标准化弥补每批发酵产品在酸度、糖度或糖酸比上的少许差异;
进一步地,益生菌发酵果蔬原浆成品可放入0℃~4℃的冰箱中冷藏,产品在0℃~4℃保质期为21天;也可以进行温度85℃~132℃,时间2秒~10分钟的超高温瞬时杀菌,无菌罐装,产品在常温下保质期为18个月;也可以罐装封口后进行温度75℃~132℃,时间20~40分钟杀菌,产品在常温下保质期为18个月。
在本发明中,所述果蔬为浆果类(包括草莓、蓝莓、桑葚、黑莓、覆盆子、蔓越莓等);瓜类(包括西瓜、哈密瓜、香瓜、甜瓜等);核果类(包括桃、黄桃、樱桃、李、杨梅、酸枣、橄榄、龙眼、荔枝等);仁果类(包括苹果、梨、柿子、枇杷等);柑橘类(包括橘子、柳丁、金桔、柠檬、葡萄柚、文旦、柚子等);根菜类(包括萝卜、胡萝卜、甘蓝、甜菜、生姜、葛根、山药、红薯、竹笋等);叶菜类(包括菠菜、茼蒿、芹菜等);果菜类(包括菱角、秋葵、番茄、辣椒、南瓜、苦瓜等)等果蔬中的任何一种或多种。
有益效果:
1、本发明所采用的菌株NCU215具有优良的发酵性能,在果蔬原料中产酸速度快,发酵时间相比其他干酪乳杆菌明显缩短;以胡萝卜浆为发酵原料接种NCU215,pH下降速度快,产酸能力强,菌株NCU215发酵8h后胡萝卜原浆pH值就降到3.8以下,24h后则降到了3.03,酸度达到4.34‰;
2、本发明所述生产出的益生菌发酵果蔬原浆有效保留了新鲜果蔬中营养成分,滋味浓郁,口感酸甜,功能性强,产品未添加任何香精、色素或防腐剂,满足大众对健康营养功能食品的需求。
3、本发明提供的益生菌发酵果蔬原浆具备以下特点:(1)充分利用新鲜果蔬可食用部分进行生产,与现有果蔬原浆所采用的榨汁浓缩法相比,对原料更有效利用,生产过程中产出废料减少;(2)可产生天然柔和酸味,有效去除水果中的青涩味及蔬菜中的野蒿味,并对水果中较为不愉悦酸味进行柔和;(3)通过益生菌发酵可将果蔬中氨基酸含量提高20%以上,产生多种芳香物质,风味物质提高30%以上,有效提高产品风味和口感;(4)充分保留果蔬原料中的维生素、膳食纤维等营养成分;(5)延长产品保质期,防止腐败。
附图说明:
图1 NCU215菌体形态;
图2系统发育树。
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仅用以解释本专利,并不用于限定本发明。
1、本发明所采用的菌株NCU215具有优良的发酵产能,在果蔬原料中产酸速度快,发酵时间相比其他干酪乳杆菌明显缩短。以胡萝卜浆为发酵原料接种NCU215,并以干酪乳杆菌ATCC393为发酵对照菌株。结果发现,菌株NCU215比ATCC393的pH下降速度快,产酸能力强,菌株NCU215发酵8h后胡萝卜原浆pH值就降到3.8以下,24h后则降到了3.03,酸度达到4.34‰,而对比菌株发酵8h后胡萝卜原浆pH值为4.23,24h后为3.56,酸度仅为3.15‰。
2、经干酪乳杆菌NCU215发酵后,能增加果蔬原浆中的鲜甜香味、降低苦味。以发酵芒果原浆和南瓜原浆为例,与对比菌株干酪乳杆菌ATCC393、CICC 6117、ATCC334相比,经干酪乳杆菌NCU215发酵后的芒果原浆和南瓜原浆中甜味、鲜味、芳香味氨基酸均显著提高,而苦味氨基酸显著降低,结果见表1、2。
芒果原浆发酵方法:选择新鲜、无腐烂的芒果为原料,洗净去皮去核后打浆,按照芒果浆85份、葡萄糖15份的组分及比例搅拌均匀,灭菌,灭菌温度为102℃,时间9分钟。灭菌后将料液冷却至37℃后,将干酪乳杆菌NCU215、ATCC393、CICC 6117、ATCC334的冻干菌粉分别按照106cfu/mL的比例接种到灭菌冷却后的芒果浆中,37℃发酵,pH值为3.8时为发酵终点。
南瓜原浆发酵方法:选择新鲜、无腐烂的南瓜为原料,预煮后打浆,按南瓜浆82份、白砂糖18份的组分及比例搅拌均匀,灭菌,灭菌温度为100℃、时间12分钟。灭菌后将料液冷却至38℃后,将干酪乳杆菌NCU215、ATCC393、CICC 6117、ATCC334的冻干菌粉分别按照106cfu/mL的比例接种到灭菌冷却后的南瓜浆中,37℃发酵,pH值为3.9时为发酵终点。
表1芒果原浆发酵前后氨基酸的变化
表2南瓜原浆发酵前后氨基酸的变化
3、经干酪乳杆菌NCU215发酵后,果蔬原浆中的抗氧化物质增加、抗氧化能力加强;对维生素影响较小。以前述“2”发酵芒果原浆为例,经干酪乳杆菌NCU215发酵后的芒果原浆中,多酚和黄酮等重要的抗氧化物质增加,且抗氧化能力增强。而经对比菌株ATCC393、CICC6117、ATCC334发酵后的芒果原浆中多酚和黄酮等重要的抗氧化物质和抗氧化能力都未发生明显增强。所有菌株发酵后芒果原浆中的维生素C、β-胡萝卜素和类胡萝卜总量均有所下降,但与对比菌株干酪乳杆菌ATCC393、CICC 6117、ATCC334相比,经干酪乳杆菌NCU215发酵后的芒果原浆中维生素C、β-胡萝卜素和类胡萝卜总量下降幅度最小,结果见表3、4。
表3芒果浆发酵前后的抗氧化物质含量
表4发酵前后芒果浆抗氧化能力的变化
4、干酪乳杆菌NCU215发酵后储存稳定性很好,以前述“2”芒果浆发酵相同的方法发酵完成后,132℃、3秒超高温瞬时灭菌,无菌灌装,测定相关物质含量。
表5发酵芒果浆贮藏期间品质的变化
由上表可知,益生菌NCU215发酵芒果浆产品贮藏期间糖类和有机酸含量变化不大,第6个月时,二者的保存率仍然很高,分别是96.8%和91.2%。蛋白质含量贮藏结束时保存率达到93%。维生素C含量整体呈下降趋势,第6个月时测得含量较贮藏初期减少了35%,维生素C极易氧化分解,相比于普通芒果饮料贮藏期间的VC变化趋势,发酵芒果浆的VC具有较高的保存率。
5、本发明提供的发酵果蔬原浆较普通果蔬原浆产品具备以下功能:(1)增强人体免疫力,预防肠炎和肠癌;(2)可调节血脂、降低胆固醇;(3)润肠通便;(4)含活菌的益生菌发酵果蔬原浆对人体肠道微生态平衡有重要调节作用。
6、菌株NCU215的16SrRNA序列如下(序列表SEQ ID NO.1):
CGGCAGTGCGGGTGCTATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGCCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGGTTTAATTCGAAGCAACGCGAAGAACCCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGTTTTCCCCTTTCGGGGCAAAATGACAGGTGGTGCATGATGTCGTCAGCCTCGTGTCGTGAGATGGTGGGGTAGGTCCCGCACGAGCGCACCTATGAACTAGTGCAGCATTAGTTGGTCACTCTAGTAGACTGCAGTGACGACCGGAGGCAACGTTGGAATGAACGGTTCAATTCATCAG。
7、测定方法
(1)风味物质的检测方法如下:
采用静态顶空-气相色谱-质谱法,利用Agilent三重串联四极杆气质联用仪进行风味物质的检测,其中气相色谱和质谱条件为:
色谱柱:HP-5石英毛细管柱(30m×0.25mm,1μm);
升温程序:初温50℃,保持3min,然后以2℃/min升温到120℃,最后以20℃/min升温到250℃,保持5min;载气(He),流速1.0mL/min,压力7.6522psi;进样量1μL;不分流。电子电离离子源;电子能量70eV;质量扫描范围m/z35~450,20scans/s;离子源温度230℃。
风味物质表格中“-”均表示样品中未检测出。
(2)氨基酸检测方法是根据GB/T 5009.124-2003《食品中氨基酸的测定》,利用氨基酸分析仪测定样品的氨基酸种类及含量。
以下将结合具体实施例对本发明做进一步的解释说明。
实施例1:一种益生菌发酵梨原浆
一种益生菌发酵梨原浆,原料配比为:89.9份梨浆、10份葡萄糖、0.1份异维生素C钠。
选择新鲜、无腐烂的梨为原料,取果肉打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为105℃,时间2分钟。灭菌后将料液冷却至35℃后,将干酪乳杆菌NCU215的冻干菌粉按照104cfu/mL的比例接种到灭菌冷却后的原料中,35℃发酵72小时,pH值为3.0时为发酵终点。将发酵后的梨原浆进行甜、酸标准化即为益生菌发酵梨原浆成品。将标准化后的发酵梨原浆进行132℃、3秒超高温瞬时灭菌,无菌灌装,产品在常温下保质期为18个月。
实施例2:一种益生菌发酵哈密瓜原浆
一种益生菌发酵哈密瓜原浆,原料配比为:89.9份哈密瓜浆、10份麦芽糖浆、0.1份异维生素C钠。
选择新鲜、无腐烂的哈密瓜为原料,洗净去皮去籽后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为100℃,时间3分钟。灭菌后将料液冷却至40℃后,将干酪乳杆菌NCU215的冻干菌粉按照103cfu/mL的比例接种,37℃发酵90小时,pH值为2.8时为发酵终点。将发酵后的哈密瓜原浆进行甜、酸标准化即为益生菌发酵哈密瓜原浆成品。将标准化后的发酵哈密瓜原浆放入0~4℃冰箱中冷藏,产品在0~4℃保质期为21天。
实施例3:一种益生菌发酵桃原浆
一种益生菌发酵桃原浆,原料配比为:89.9份桃浆、10份赤藓糖醇、0.1份异维生素C钠。
选择新鲜、无腐烂的桃为原料,清洗去核后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为90℃,时间8分钟。灭菌后将料液冷却至37℃后,将干酪乳杆菌NCU215的冻干菌粉按照106cfu/mL的比例接种,37℃发酵72小时,pH值为3.1时为发酵终点。将发酵后的桃原浆进行甜、酸标准化即为益生菌发酵桃原浆成品。将标准化后的发酵桃原浆进行132℃、3秒超高温瞬时灭菌,无菌灌装,产品在常温下保质期为18个月。
实施例4:一种益生菌发酵苦瓜原浆
一种益生菌发酵苦瓜原浆,原料配比为:84.95份苦瓜浆、5份赤藓糖醇、0.05份异维生素C钠。
选择新鲜、无腐烂的苦瓜为原料,洗净去籽后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为132℃,时间2秒。灭菌后将料液冷却至30℃后,将干酪乳杆菌NCU215的冻干菌粉按照104cfu/mL的比例接种,32℃发酵82小时,pH值为3.0时为发酵终点。将发酵后的苦瓜原浆进行甜、酸标准化即为益生菌发酵苦瓜原浆成品。将标准化后的发酵苦瓜原浆放入0~4℃冰箱中冷藏,产品在0~4℃保质期为21天。
实施例5:一种益生菌发酵芹菜原浆
一种益生菌发酵芹菜原浆,原料配比为:80.95份芹菜浆、19份赤藓糖醇、0.05份异维生素C钠。
选择新鲜、无腐烂的芹菜为原料,洗净后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为112℃,时间1分钟。灭菌后将料液冷却至42℃后,将干酪乳杆菌NCU215的冻干菌粉按照105cfu/mL的比例接种,38℃发酵54小时,pH值为2.8时为发酵终点。将发酵后的芹菜原浆进行甜、酸标准化即为益生菌发酵芹菜原浆成品。将标准化后的发酵芹菜原浆罐装封口后进行115℃30分钟灭菌,产品在常温下保质期为18个月。
实施例6:一种益生菌发酵蓝莓原浆
一种益生菌发酵蓝莓原浆,原料配比为:82.1份蓝莓浆、17.8份葡萄糖、0.1份异维生素C钠。
选择新鲜、无腐烂的蓝莓为原料,洗净后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为85℃,时间10分钟。灭菌后将料液冷却至35℃后,将干酪乳杆菌NCU215的冻干菌粉按照109cfu/mL的比例接种,35℃发酵72小时,pH值为2.5时为发酵终点。将发酵后的蓝莓原浆进行甜、酸标准化即为益生菌发酵蓝莓原浆成品。将标准化后的发酵蓝莓原浆放入0~4℃冰箱中冷藏,产品在0~4℃保质期为21天。
实施例7:一种益生菌发酵番茄原浆
一种益生菌发酵番茄原浆,原料配比为:85.3份番茄浆、14.6份麦芽糖醇、0.1份异维生素C钠。
选择新鲜、无腐烂的番茄为原料,洗净后打浆,滤掉皮后按照以上组分及比例搅拌均匀后灭菌,灭菌温度为105℃,时间2分钟。灭菌后将料液冷却至37℃后,将干酪乳杆菌NCU215的冻干菌粉按照105cfu/mL的比例接种,38℃发酵72小时,pH值为2.8时为发酵终点。将发酵后的番茄原浆进行甜、酸标准化即为益生菌发酵番茄原浆成品。将标准化后的发酵番茄原浆进行132℃、3秒超高温瞬时灭菌,无菌灌装,产品在常温下保质期为18个月。
实施例8:一种益生菌发酵生姜原浆
一种益生菌发酵生姜原浆,原料配比为:84.07份生姜浆、15.9份淀粉糖浆、0.03份异维生素C钠。
选择新鲜、无腐烂的生姜为原料,洗净去皮后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为85℃,时间10分钟。灭菌后将料液冷却至38℃后,将干酪乳杆菌NCU215的冻干菌粉按照107cfu/mL的比例接种,32℃发酵96小时,pH值为2.7时为发酵终点。将发酵后的生姜原浆进行甜、酸标准化即为益生菌发酵生姜原浆成品。将标准化后的发酵生姜原浆进行132℃、3秒超高温瞬时灭菌,无菌灌装,产品在常温下保质期为18个月。
实施例9:一种益生菌发酵红薯原浆
一种益生菌发酵红薯原浆,原料配比为:85.09份红薯浆、14.9份麦芽糖浆、0.01份异维生素C钠。
选择新鲜、无腐烂的红薯为原料,预煮去皮后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为101℃,时间3分钟。灭菌后将料液冷却至40℃后,将干酪乳杆菌NCU215的冻干菌粉按照104cfu/mL的比例接种,37℃发酵50小时,pH值为2.6时为发酵终点。将发酵后的红薯原浆进行甜、酸标准化即为益生菌发酵红薯原浆成品。将标准化后的发酵红薯原浆罐装封口后进行115℃,30分钟灭菌,产品在常温下保质期为18个月。
实施例10:一种益生菌发酵香蕉原浆
一种益生菌发酵香蕉原浆,原料配比为:87.99份香蕉浆、12份淀粉糖浆、0.01份异维生素C钠。
选择新鲜、无腐烂的香蕉为原料,去皮后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为85℃,时间10分钟。灭菌后将料液冷却至40℃后,将干酪乳杆菌NCU215的冻干菌粉按照107cfu/mL的比例接种,34℃发酵60小时,pH值为3.0时为发酵终点。将发酵后的香蕉原浆进行甜、酸标准化即为益生菌发酵香蕉原浆成品。将标准化后的发酵香蕉原浆罐装封口后进行115℃,30分钟灭菌,产品在常温下保质期为18个月。
实施例11:一种益生菌发酵菠萝原浆
一种益生菌发酵菠萝原浆,原料配比为:84.95份菠萝浆、5份低聚异麦芽糖、0.05份异维生素C钠。
选择新鲜、无腐烂的菠萝为原料,去皮后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为132℃,时间2秒。灭菌后将料液冷却至32℃后,将干酪乳杆菌NCU215的冻干菌粉按照105cfu/mL的比例接种,32℃发酵48小时,pH值为2.8时为发酵终点。将发酵后的菠萝原浆进行甜、酸标准化即为益生菌发酵菠萝原浆成品。将标准化后的发酵菠萝原浆放入0~4℃冰箱中冷藏,产品在0~4℃保质期为21天。
实施例12:一种益生菌发酵枸杞原浆
一种益生菌发酵枸杞原浆,原料配比为:81.97份枸杞浆、18份赤藓糖醇、0.03份异维生素C钠。
选择新鲜、无腐烂的枸杞为原料,洗净后打浆,按照以上组分及比例搅拌均匀后灭菌,灭菌温度为112℃,时间1分钟。灭菌后将料液冷却至37℃后,将干酪乳杆菌NCU215的冻干菌粉按照106cfu/mL的比例接种,37℃发酵46小时,pH值为2.7时为发酵终点。将发酵后的枸杞原浆进行甜、酸标准化即为益生菌发酵枸杞原浆成品。将标准化后的发酵枸杞原浆罐装封口后进行115℃,30分钟灭菌,产品在常温下保质期为18个月。
实施例13保质期评价标准
本发明的保质期通过以下指标进行确定:
未灭菌发酵果蔬原浆0~4℃储存过程中感官及微生物数量变化
灭菌发酵原浆常温储存过程中感官及微生物数量变化
实施例15 NCU215生理生化特性测定
①耐酸性测试——在pH 2.0的PBS中处理2h存活率为78.98%;
将挑选好的NCU215用MRS液体培养基活化两次,然后按2%(v/v)的接种量37℃培养24h,10000g离心5min以收集菌体。将菌体用pH 2.0的无菌PBS重悬并调节活菌浓度至108CFU/mL,于37℃下孵育2h,采用稀释涂布法测定孵育前后的活菌数以测定其存活率。存活率按下式计算
其中N1为孵育后存活菌数,N0为初始菌数。
②耐胆盐测试——在0.5%的胆盐环境中处理4h后的存活率为84.89%;
将培养24h后的NCU215在4℃、10000g下离心5min以收集菌体,获得的菌体用含0.5%胆盐的无菌PBS重悬并调节活菌浓度至108CFU/mL,在37℃孵育4h,用稀释涂布平板法测定活菌数以评估NCU215的耐胆盐能力。根据下式计算NCU215存活率
其中N1为存活菌数,N0为初始菌数。
③模拟胃肠液耐受性
将培养24h后得NCU215在4℃、10000g下离心5min以收集菌体,并用无菌PBS清洗两次;随即将其重悬至pH 3.0的模拟胃液中37℃孵育3h,并于第0,1,2,3h测定活菌数;然后将1mL培养物加入9mL模拟肠液中37℃孵育8h,并于第0,2,4,8h时测定培养物的活菌数。根据下式计算NCU215存活率
其中N1为存活菌数,N0为初始菌数。
结果显示,在pH 3.0的模拟胃液中消化3h,随即转移至pH 8.0的模拟肠液中消化8h后,活力无显著下降,在模拟胃液中处理3h存活率为101.52±1.67%,在模拟肠液中处理8h存活率为100.27±2.05%。
④-1自凝集能力测试
将过夜培养的乳酸菌在10000g下离心5min收集菌体,并用无菌PBS缓冲液洗涤两次。随后,将获得的菌体细胞用无菌PBS重悬并调整A600=0.6±0.05(A0),涡旋振荡10s后在37℃孵育24h。在第0、2、4、6、12和24h时取上层菌悬液,于600nm下测定吸光度(At),每次测定三个平行,重复三次。按照下式计算细菌的自凝集能力。
其中,A0表示菌体初始吸光值,At代表t时刻上层菌悬液的吸光值。
结果显示,该菌株24h的自凝集率为64.32%。
④-2表面疏水性测试
利用微生物黏着碳氢化合物法(Bacteria Adhesion To Hydrocarbons,BATH)进行乳酸菌表面疏水性的测定。首先将过夜培养的乳酸菌在10000g下离心5min收集菌体细胞,并用无菌PBS缓冲液洗涤两次;随后,将获得的菌体细胞用0.1mol/L KNO3重悬并调整A600=0.6±0.05(A0)。然后取1mL二甲苯加入至3mL调整好浓度的菌体细胞悬液中,混合后在室温下预孵育10min,涡旋振荡2min,然后在室温下孵育30min分层,小心吸取水相,以无菌PBS为空白测定OD600值(A)。按下式计算细菌的疏水性
其中A0表示菌体初始吸光值;A为二甲苯处理后下层水相吸光值。结果显示该菌株表面疏水率为23.15%。
④-3黏附性测试
将培养好的Caco-2细胞用胰酶-EDTA消化液消化,再用DMEM完全培养液调整细胞浓度为1.0×105个/mL,装于6孔组织培养板中,每孔2mL,于CO2培养箱(5%CO2、95%空气)中37℃孵育至细胞长至分化的单层,每两天换液一次。弃去组织培养板中各孔的DMEM培养液,以无菌PBS缓冲液清洗培养板2遍,加入1mL用DMEM不完全培养液重悬的乳酸菌菌悬液(108CFU/mL;OD=1)(Caco-2细胞:细菌数量≧1:100),于37℃孵育2h。孵育完成后弃去组织培养板中各孔的混合液,用无菌PBS缓冲液洗涤5次,以除去未黏附的菌体细胞。加入0.5mL0.25%的胰酶在37℃孵育5min以消化细胞,然后将细胞消化液进行梯度稀释,涂布于MRS固体平板上计算黏附的细菌数。黏附能力用下式计算
黏附率(%)=Nt/N0×100
其中,Nt表示黏附于Caco-2细胞上的细菌数,N0表示加入的NCU215总细菌数。结果显示该菌株对人结肠癌细胞Caco-2的黏附率为7.47%。
⑤抗氧化活性测试(NCU215样品液菌体浓度是109CFU/mL)
DPPH自由基清除能力
将1.0mL NCU215样品液加入到1mL的乙醇DPPH自由基溶液(0.1mM)中,混匀后在室温下黑暗环境中温育30min。8000g离心10min后,以PBS和DPPH溶液作为对照,乙醇溶液和NCU215样品作为空白,在517nm下测量所得溶液的吸光度。清除能力用下式表示:
其中As为样品在517nm下的吸光度,Ab为空白在517nm下的吸光度,Ac为对照在517nm下的吸光度。
羟基自由基清除能力
将1mL 2.5mM的邻菲罗啉,1mLPBS(pH 7.4),1mL 2.5mM FeSO4和1mL的NCU215样品简单混合。通过加入1mL 20mM H2O2并在37℃下孵育90min开始反应。在536nm下测量混合物的吸光度,并按下式计算羟基自由基清除活性。
其中Asample是在样品和H2O2都存在下的吸光度,Acontro1是无样品和H2O2存在下的吸光度,Ablank是在无样品和有H2O2存在下对照的吸光度。
ABTS自由基清除能力
根据ABTS自由基检测试剂盒(碧云天)说明书配制ABTS工作母液,室温避光放置16h,然用PBS调整吸光值为A734=0.7±0.01(ABTS工作液)。96孔板的每个检测孔中加入200μL ABTS工作液。空白对照孔中加入10μL PBS;标准曲线检测孔内分别加入10μL的0.15、0.3、0.6、0.9、1.2和1.5mM的Trolox标准溶液;样品检测孔内加入10μL NCU215样品,轻轻混匀。室温避光孵育6min后测定A734。根据标准曲线计算出样品的总抗氧化能力。
总还原能力
根据FRAP总抗氧化能力检测试剂盒(碧云天)说明书配制FRAP工作液。新鲜配置0.15、0.3、0.6、0.9、1.2和1.5mM的。96孔板的每个检测孔中加入180μLFRAP工作液;空白对照孔中加入5μL PBS溶液;标准曲线检测孔内加入5μL新鲜配置的0.15、0.3、0.6、0.9、1.2和1.5mM FeSO4标准溶液;样品检测孔内加入5μL NCU215样品,轻轻混匀。37℃孵育5min后测定A593。根据标准曲线计算出样品的总抗氧化能力。
结果显示,该菌株具有较好的抗氧化活性:DPPH自由基清除率为11.91%,羟基自由基清除率为10.85%,总抗氧化能力相当于95.90μmol Trolox,总还原能力相当于0.28mMFeSO4。
⑥-1抑菌活性测试
NCU215在MRS培养基中37℃下培养24h,并于4℃下10000g离心10min。发酵上清液用0.22μm滤膜过滤除菌后得到无细胞上清液(CFS)并保存在-80℃下备用。利用打孔法测定NCU215 CFS对致病菌的抑菌活性,即取200μL NCU215 CFS加入涂有大肠杆菌、沙门氏菌、单增李斯特菌、铜绿假单胞菌、阪崎肠杆菌、蜡样芽孢杆菌和金黄色葡萄球菌的LB平板孔中,37℃下孵育12h测定抑菌圈直径。
⑥-2溶血活性测试
挑取测试菌株单菌落划线至哥伦比亚血琼脂平板上并在37℃下培养48h后确定溶血活性。发酵乳杆菌CECT5716为阳性对照,金黄色葡萄球菌为阴性对照,实验重复三次。
⑥-3抗生素敏感性测试
采用K-B(药敏纸片琼脂扩散法)进行药敏试验来评价菌株的抗生素耐药性。将过夜培养的乳杆菌发酵液调节菌浓至108CFU/mL,吸取100μL涂布于MRS平板上。分别将链霉素(10mg/mL),氨苄青霉素(10mg/mL),红霉素(15mg/mL),四环素(30mg/mL),庆大霉素(10mg/mL),卡那霉素(30mg/mL),青霉素(10mg/mL),头孢噻吩(15mg/mL),环丙沙星(5mg/mL)和阿莫西林(30mg/mL)的药敏纸片轻轻放置于涂有乳杆菌菌液的MRS平板上,37℃培养24h,游标卡尺测定抑菌圈直径,确定测试乳杆菌对上述抗生素的敏感性。
结果显示,该菌株的24h发酵上清液对常见食源性致病菌具有优异的抑菌活性,尤其对单增李斯特菌及金黄色葡萄球菌的抑制活性最好,其抑菌圈直径分别为23.18mm和24.42mm;此外,该菌株的溶血活性检测显示不具溶血性;抗生素敏感性检测显示:该菌株对四环素、氨苄青霉素、阿莫西林、头孢噻吩、红霉素和青霉素敏感,对卡那霉素、环丙沙星、链霉素、庆大霉素耐受。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以权利要求为准。
序列表
<110> 南昌大学
<120> 益生菌发酵果蔬原浆产品
<130> 1
<141> 2020-02-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1194
<212> DNA
<213> 干酪乳杆菌(Lactobacillus casei)
<400> 1
cggcagtgcg ggtgctatac atgcaagtcg aacgagttct cgttgatgat cggtgcttgc 60
accgagattc aacatggaac gagtggcgga cgggtgagta acacgtgggt aacctgccct 120
taagtggggg ataacatttg gaaacagatg ctaataccgc atagatccaa gaaccgcatg 180
gttcttggct gaaagatggc gtaagctatc gcttttggat ggacccgcgg cgtattagct 240
agttggtgag gtaatggctc accaaggcga tgatacgtag ccgaactgag aggttgatcg 300
gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360
cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaaggct ttcgggtcgt 420
aaaactctgt tgttggagaa gaatggtcgg cagagtaact gttgccggcg tgacggtatc 480
caaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540
gttatccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600
gccctcggct taaccgagga agcgcatcgg aaactgggaa acttgagtgc agaagaggac 660
agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720
ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag catgggtagc gaacaggatt 780
agataccctg gtagtccatg ccgtaaacga tgaatgctag gtgttggagg gtttccgccc 840
ttcagtgccg cagctaacgc attaagcatt ccgcctgggg agtacgaccg caaaggttga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtgggtt taattcgaag 960
caacgcgaag aacccttacc aggtcttgac atcttttgat cacctgagag atcagttttc 1020
ccctttcggg gcaaaatgac aggtggtgca tgatgtcgtc agcctcgtgt cgtgagatgg 1080
tggggtaggt cccgcacgag cgcacctatg aactagtgca gcattagttg gtcactctag 1140
tagactgcag tgacgaccgg aggcaacgtt ggaatgaacg gttcaattca tcag 1194
Claims (9)
1.一种益生菌发酵果蔬原浆产品,其特征在于,是由以下原料发酵而成的:果蔬原浆80~99.8份,糖浆或代用糖0~19.8份;
所述的益生菌为干酪乳杆菌(Lactobacillus casei)NCU215,保藏编号为CGMCCNo.18702。
2.如权利要求1所述的一种益生菌发酵果蔬原浆产品,其特征在于,原料还包括异维生素C钠或维生素C 0.01~0.5份。
3.如权利要求1所述的一种益生菌发酵果蔬原浆产品,其特征在于,所述的糖浆或代用糖是白砂糖、葡萄糖、淀粉糖浆、麦芽糖浆、葡萄糖浆、麦芽糖醇、木糖醇、赤藓糖醇或低聚异麦芽糖。
4.如权利要求1所述的一种益生菌发酵果蔬原浆产品,其特征在于,制备方法如下:
(1)选择无腐烂、新鲜果蔬为原料,清洗后去除其不可食用部分,打浆或榨汁后按组分和比例搅拌均匀,灭菌;
(2)灭菌后将原料冷却至20~45℃后,将益生菌按照103~109cfu/mL的比例接种,25℃~45℃发酵,pH值2.5~5.0为发酵终点。
5.如权利要求4所述的一种益生菌发酵果蔬原浆产品,其特征在于,灭菌温度为75~132℃,灭菌时间为2秒~50分钟。
6.如权利要求4所述的一种益生菌发酵果蔬原浆产品,其特征在于,将发酵后的果蔬原浆进行标准化即为益生菌发酵果蔬原浆产品。
7.如权利要求6所述的一种益生菌发酵果蔬原浆产品,其特征在于,所述标准化是按照行业、企业或者实际需求对产品的酸度、甜度进行标准化。
8.如权利要求4所述的一种益生菌发酵果蔬原浆产品,其特征在于,发酵完成后,益生菌发酵果蔬原浆成品放入0℃~4℃的冰箱中冷藏;或进行85℃~132℃,2秒~10分钟的超高温瞬时杀菌,无菌罐装;或罐装封口后进行75℃~132℃,20~40分钟杀菌。
9.如权利要求1-8任意所述的一种益生菌发酵果蔬原浆产品,其特征在于,所述果蔬为浆果类、瓜类、核果类、仁果类、柑橘类、根菜类、叶菜类、果菜类中的任何一种或多种。
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US16/833,642 US20210007380A1 (en) | 2020-02-28 | 2020-03-29 | Probiotic fermented fruit and vegetable pulp product |
US17/035,811 US11717008B2 (en) | 2020-02-18 | 2020-09-29 | Probiotic fermented fruit and vegetable pulp composition containing active bacteria and preparation method thereof |
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CN104605277A (zh) * | 2013-11-05 | 2015-05-13 | 熊涛 | 一种功能性益生菌发酵果蔬粉的制备方法 |
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