泡菜中优良乳酸菌的筛选及高效混合菌株发酵剂研制
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摘要
泡菜作为一种传统的发酵食品,由于其口感独特,营养丰富而深受大众喜爱。因此,实现人工发酵对于提高泡菜品质,实现工业化具有重要意义。本文从泡菜中分离并筛选出产酸速率快、硝酸盐降解能力强的乳酸菌株,对其进行发酵性能和菌种优化搭配进行了研究;研究了泡菜发酵剂菌株适宜生长的廉价增殖培养基,优化筛选出适合工业化生产的冻干复合保护剂;研究了乳酸菌增殖培养、细胞浓缩分离、真空冷冻干燥的工艺技术;制备出高效浓缩型乳酸菌冻干发酵剂,并对冻干发酵剂在蔬菜盐水中的发酵活力进行了检测;对泡菜的产品质量进行了初步分析和评价。
本文从8种泡菜样品中分离出产酸菌株86株,经革兰氏染色镜检、接触酶反应及乳酸定性试验,并以产酸速度及亚硝酸盐降解能力进行了比较,筛选出五株产酸较快,亚硝酸盐降解能力较强的乳酸菌LS②、LN①、LN②、JL②和Z_(11)④,经形态特征、生理生化特性初步鉴定分别为短乳杆菌、肠膜明串珠菌葡聚糖亚种、戊糖乳杆菌、植物乳杆菌和植物乳杆菌。并对筛选出的优良菌株进行了发酵性能的研究,结果表明五株菌均适用于低温泡菜的发酵。
将筛选出的发酵性能较优的肠膜明串株菌LN①、植物乳杆菌JL②、短乳杆菌LS②三株菌按比例组合搭配,组成发酵剂,筛选出LN①:JL②=1:2、LN①:LS②=1:2、LN①:JL②:LS②:1:1:1作为优良发酵剂,并研究了三组发酵剂在不同温度条件下发酵泡菜试验,确定10℃为发酵泡菜的最适温度。
选择原料易得、价格低廉、易于分离细胞的番茄汁培养基、白菜汁培养基、胡萝卜汁培养基,通过细胞生长试验,确定了白菜汁培养基是泡菜优良发酵菌株的最适基础培养基。在此基础上,研究了在白菜汁中添加不同营养物质对细胞生长量的影响,优化筛选出白菜复合汁增殖培养基,其最佳配比为:在含有0.2%K_2HPO_4的白菜汁基础培养基中添加,1%蛋白胨、1%牛肉膏、0.7%玉米浆、0.5%葡萄糖;与白菜汁基础培养基相比LS②、LN①、JL②在此增殖培养基中的最大细胞生长量分别提高了39倍、42.5倍和54.5倍,与在液体MRS培养基的最高活菌数数量级相当。
通过研究离心力、离心时间对乳酸菌离心损失率、离心存活率及离心收得率的影响,确定了短乳杆菌LS②、肠膜明串株菌LN①和植物乳杆菌JL②细胞浓缩分离的最适离心条件为均为3672g(6000rpm),10min,活菌收得率分别为96.28%、97.52%和98.93%。
筛选出抗冻干、耐贮藏的复合冻干保护剂1种,冷冻干燥后,短乳杆菌LS②、肠膜明串株菌LN①和植物乳杆菌JL②冻干发酵剂活菌含量均在10~(11)cfu/g以上,冻干存活率均达95%以上。
肠膜明串株菌LN①、植物乳杆菌JL②、短乳杆菌LS②冻干发酵剂以万分之一的接种量接入蔬菜盐水中10℃发酵,6d左右即可达到成熟,pH为3.55,滋味浓郁,酸度适中,与传统液态混菌发酵剂相比无明显差别。
Pickling vegetable is a sort of traditionary ferment food, it has sepecial sense and abundance nutrition , it is populared for the people. In this paper, lactic acid bacteria were isolated and identified from many sorts of pickling vegetable for having excellent characteristics in acid production and the ability for degradation the nitrate. The fermentation characterisics of these srains were also studied. It has isolated strains and optimization collocation for fine lactic acid bacteria, studied the primary factors which affect manufacturing technology of pickling vegetable. The cheap enrichment medium suited for industrial processing and freeze-drying protectant were screened. The technology of enrichment culture for lactic acid bacteria, cell concentration, lyophilization were studied. The fermentative activity of the freeze-drying starter in cabbage brine were examined.
86 strains of lactic acid bacteria were isolated and identified from eight pickling vegetable. Five strains were selected for having excellent characteristics in acid production and the ability for degradation the nitrate. For morphology appraisement and the test for physiology and biochemical event, there are two Lactobacillus plantarum, one, one Lactobacillus pentosus, one Leuconostoc mesenteroides subsp, dextrainicum. The fermentation characterisics of these five srains were also studied.
It make Leuconostoc mesenteroides subsp. LN①, Lactobacillus plantarum JL②and Lactobacillus brevis LS②pro rate combination arrange in pairs for starter. It sceen out LN①: JL②=1 : 2、LN①: LS②=1 : 2、LN①: JL②: LS②=1 :1 : 1 are choiceness starter, and make the three starter in different temperature ferment cabbage, make sure 10℃is the best temperature for the ferment cabbage .
Tomato juice medium, carrot juice medium and cabbage medium which are apt to gain and separate are obtained by cell groeth experimented. The result showed that cabbage juice was the best base medium for Lactobacillus brevis LS②. The effect of adding 12 kinds of different nutrient factors in cabbage juice medium. The result showed that the optimum enrichment medium obtained by orthogonality test L_9(3~4) consist of adding 1% peptone, 1% beeves, 0.7% corn liquor, 0.5% glucose and 0.2% K_2HPO_4 in cabbage juice medium. In the optimum enrichment medium, the living-cell number of LS②、LN①and JL②were increased by 39 times, 42.5 times and 54.5 times compared with cabbage juice base medium and the same with the living number in the MRS used in lab.
The effects of centrifuging force, time on the loss rate, survival rate and harvest rate of lactobacillus was studied. The results showed that the optimum conditions for centrifugation of Lactobacillus brevis LS②, Leuconostoc mesenteroides subsp. LN①and Lactobacillus plantarum JL②3672g(6000rpm)for 10min and the highest harvesting rate of living cells were 96.28%, 97.52%, 98.93%.
A complex protectant was screened, which could protect cell from damaging during freeze-dry. After freeze-drying, the living-cell number of starter, Lactobacillus brevis LS②, Leuconostoc mesenteroides subsp. LN①and Lactobacillus plantarum JL②were over 10~(11) cfu/g, freeze-dry livability were over 95%. The cabbage fermented by protectant in 10℃. The result were no differental significance compared with traditional culture which show the cabbage can be matured about 6 days and the pH was 3.55.
本文从8种泡菜样品中分离出产酸菌株86株,经革兰氏染色镜检、接触酶反应及乳酸定性试验,并以产酸速度及亚硝酸盐降解能力进行了比较,筛选出五株产酸较快,亚硝酸盐降解能力较强的乳酸菌LS②、LN①、LN②、JL②和Z_(11)④,经形态特征、生理生化特性初步鉴定分别为短乳杆菌、肠膜明串珠菌葡聚糖亚种、戊糖乳杆菌、植物乳杆菌和植物乳杆菌。并对筛选出的优良菌株进行了发酵性能的研究,结果表明五株菌均适用于低温泡菜的发酵。
将筛选出的发酵性能较优的肠膜明串株菌LN①、植物乳杆菌JL②、短乳杆菌LS②三株菌按比例组合搭配,组成发酵剂,筛选出LN①:JL②=1:2、LN①:LS②=1:2、LN①:JL②:LS②:1:1:1作为优良发酵剂,并研究了三组发酵剂在不同温度条件下发酵泡菜试验,确定10℃为发酵泡菜的最适温度。
选择原料易得、价格低廉、易于分离细胞的番茄汁培养基、白菜汁培养基、胡萝卜汁培养基,通过细胞生长试验,确定了白菜汁培养基是泡菜优良发酵菌株的最适基础培养基。在此基础上,研究了在白菜汁中添加不同营养物质对细胞生长量的影响,优化筛选出白菜复合汁增殖培养基,其最佳配比为:在含有0.2%K_2HPO_4的白菜汁基础培养基中添加,1%蛋白胨、1%牛肉膏、0.7%玉米浆、0.5%葡萄糖;与白菜汁基础培养基相比LS②、LN①、JL②在此增殖培养基中的最大细胞生长量分别提高了39倍、42.5倍和54.5倍,与在液体MRS培养基的最高活菌数数量级相当。
通过研究离心力、离心时间对乳酸菌离心损失率、离心存活率及离心收得率的影响,确定了短乳杆菌LS②、肠膜明串株菌LN①和植物乳杆菌JL②细胞浓缩分离的最适离心条件为均为3672g(6000rpm),10min,活菌收得率分别为96.28%、97.52%和98.93%。
筛选出抗冻干、耐贮藏的复合冻干保护剂1种,冷冻干燥后,短乳杆菌LS②、肠膜明串株菌LN①和植物乳杆菌JL②冻干发酵剂活菌含量均在10~(11)cfu/g以上,冻干存活率均达95%以上。
肠膜明串株菌LN①、植物乳杆菌JL②、短乳杆菌LS②冻干发酵剂以万分之一的接种量接入蔬菜盐水中10℃发酵,6d左右即可达到成熟,pH为3.55,滋味浓郁,酸度适中,与传统液态混菌发酵剂相比无明显差别。
Pickling vegetable is a sort of traditionary ferment food, it has sepecial sense and abundance nutrition , it is populared for the people. In this paper, lactic acid bacteria were isolated and identified from many sorts of pickling vegetable for having excellent characteristics in acid production and the ability for degradation the nitrate. The fermentation characterisics of these srains were also studied. It has isolated strains and optimization collocation for fine lactic acid bacteria, studied the primary factors which affect manufacturing technology of pickling vegetable. The cheap enrichment medium suited for industrial processing and freeze-drying protectant were screened. The technology of enrichment culture for lactic acid bacteria, cell concentration, lyophilization were studied. The fermentative activity of the freeze-drying starter in cabbage brine were examined.
86 strains of lactic acid bacteria were isolated and identified from eight pickling vegetable. Five strains were selected for having excellent characteristics in acid production and the ability for degradation the nitrate. For morphology appraisement and the test for physiology and biochemical event, there are two Lactobacillus plantarum, one, one Lactobacillus pentosus, one Leuconostoc mesenteroides subsp, dextrainicum. The fermentation characterisics of these five srains were also studied.
It make Leuconostoc mesenteroides subsp. LN①, Lactobacillus plantarum JL②and Lactobacillus brevis LS②pro rate combination arrange in pairs for starter. It sceen out LN①: JL②=1 : 2、LN①: LS②=1 : 2、LN①: JL②: LS②=1 :1 : 1 are choiceness starter, and make the three starter in different temperature ferment cabbage, make sure 10℃is the best temperature for the ferment cabbage .
Tomato juice medium, carrot juice medium and cabbage medium which are apt to gain and separate are obtained by cell groeth experimented. The result showed that cabbage juice was the best base medium for Lactobacillus brevis LS②. The effect of adding 12 kinds of different nutrient factors in cabbage juice medium. The result showed that the optimum enrichment medium obtained by orthogonality test L_9(3~4) consist of adding 1% peptone, 1% beeves, 0.7% corn liquor, 0.5% glucose and 0.2% K_2HPO_4 in cabbage juice medium. In the optimum enrichment medium, the living-cell number of LS②、LN①and JL②were increased by 39 times, 42.5 times and 54.5 times compared with cabbage juice base medium and the same with the living number in the MRS used in lab.
The effects of centrifuging force, time on the loss rate, survival rate and harvest rate of lactobacillus was studied. The results showed that the optimum conditions for centrifugation of Lactobacillus brevis LS②, Leuconostoc mesenteroides subsp. LN①and Lactobacillus plantarum JL②3672g(6000rpm)for 10min and the highest harvesting rate of living cells were 96.28%, 97.52%, 98.93%.
A complex protectant was screened, which could protect cell from damaging during freeze-dry. After freeze-drying, the living-cell number of starter, Lactobacillus brevis LS②, Leuconostoc mesenteroides subsp. LN①and Lactobacillus plantarum JL②were over 10~(11) cfu/g, freeze-dry livability were over 95%. The cabbage fermented by protectant in 10℃. The result were no differental significance compared with traditional culture which show the cabbage can be matured about 6 days and the pH was 3.55.
引文
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