浓缩苹果汁全产业链高渗酵母分离鉴定及电子鼻快速检测方法研究
发布时间：2018-08-28 20:40
【摘要】：高渗酵母是浓缩苹果汁产业中关键的质量安全因子之一,对果汁的生产加工及出口贸易构成了严重威胁。了解高渗酵母在浓缩苹果汁生产链条中的分布,并研究建立高效、快速的检测方法,及时识别苹果汁中高渗酵母污染,切实保障苹果汁产品品质安全,对苹果加工产业稳定、健康、可持续发展具有重要意义。本文以陕西洛川和白水县两大苹果主产区作为样品采集区,同时也在以上述主产区苹果为主要加工原料的果汁加工厂进行采样。样品富集后对高渗酵母进行分离纯化,通过ITS1-5.8S-ITS2或26S rDNA D1/D2测序进行鉴定。分别采用生理方法和随机引物扩增多态性分析方法(RAPD-PCR)对分离菌株的生物多样性进行考察,并通过研究分离菌株在高浓度糖环境中的生长状况和产气性对其污染能力进行揭示。以具有较大潜在危害(能够在70%w/v葡萄糖环境中生长)的11株分离菌株为对象,通过研究温度、pH、糖含量和抑菌物质等环境因素对其生长的影响进一步考察其污染能力。然后研究了糖含量及pH对污染能力最强的鲁氏接合酵母(Zygosaccharomyces rouxii)在模拟培养基和浓缩苹果汁中生长的联合抑制效应。最后,以Z.rouxii分离株及购买于ATCC的模式菌株为对象,研究了电子鼻技术定性及定量检测其在高糖培养基(PYGF60培养液)和苹果汁中污染的可行性。论文的主要研究结果如下:(1)自陕西苹果主产区果园及浓缩苹果汁加工企业环境共采集196个样品,40个样品检出高渗酵母(阳性),检出率(阳性率)为20.4%,其中果园环境检出率为5%,果汁加工厂环境检出率为44.7%。从上述阳性样品中共分离出66株高渗酵母,其中13株来自果园环境,其余53株来自果汁加工厂环境。果园环境中高渗酵母主要来自果实(5株)与土壤(4株),果汁加工厂环境中高渗酵母主要来自车间空气(20株)与储存浓缩汁(7株)。66株高渗酵母分布于14属23种,包含1个类酵母种Aureobasidium pullulans。分离菌株中常见的种有Kluyveromyces marxianus(8株)、Candida tropicalis(6株)、Z.rouxii(6株)、Hanseniaspora uvarum(5株)、Saccharomyces cerevisiae(5株)和Pichia kudriavzevii(5株)。仅有Z.rouxii为嗜高渗酵母,其余分离菌株均为耐高渗酵母。(2)C.tropicalis、H.opuntiae、K.marxianus、P.kudriavzevii、S.cerevisiae和Z.rouxii种内菌株基于生理方法分为2-3个类群,各类群之间的遗传相似性高于95%,与RAPD-PCR分析结果基本吻合。A.pullulans、C.tropicalis(来源于浓缩苹果汁)、C.glabrata、C.orthopsilosis、C.zemplinina、Debaryomyces hansenii、Meyerozyma caribbica、M.guilliermondii、Torulaspora delbrueckii、Wickerhamomyces anomalus和Z.rouxii能够在70%w/v葡萄糖环境中生长,并且大多能够在高糖环境中代谢产气,对果汁产品具有较大潜在危害。(3)11株测试菌株在4 oC环境中无法生长;测试菌株的最大耐受温度介于46-55oC之间;食品中的糖类物质能够减弱高温对测试菌株的损害,使其最大耐受温度提高至52-67 oC。pH降低至2.0时,测试菌株的生长被大幅抑制;测试菌株的最大耐受pH介于1.7-2.4之间。仅有鲁氏接合酵母能够在70 oBrix苹果汁中生长,表现出极强的耐糖性;将鲁氏接合酵母的高糖培养液作为接种体时,其最高耐糖性从73提高至75 oBrix,表现出高糖环境适应性。在苹果汁(pH=4.0)中添加4.0 mM山梨酸钾、6.0 mM苯甲酸钠、1.5 mM肉桂酸、20 mM香草醛、10 mM阿魏酸或者12 mM对香豆酸能够有效降低测试菌株的污染。随着糖含量增加或者pH值降低,鲁氏接合酵母菌株在模拟培养基和浓缩苹果汁中的μmax值逐渐减小,λ和TFS值逐渐增加。变温环境(海外船运环境)比恒温环境(25 oC)更有助于鲁氏接合酵母在浓缩苹果汁中生长。响应面模型表明pH对鲁氏接合酵母菌株μmax、λ和TFS值的影响最大,是影响其生长的主要限制因素。(4)鲁氏接合酵母污染PYGF60培养液48 h后,对应的细胞浓度分别为3.68(B-WHX-12-54)、3.16(ATCC 2623)和2.98(ATCC 8383)lg CFU/mL,污染苹果汁12 h后,对应的细胞浓度小于2.3 lg CFU/mL,基于线性判别分析(LDA)电子鼻能够正确识别污染,而感官评定人员在上述时间点无法正确区分空白与污染样品。2号以及6-9号传感器对电子鼻识别污染具有重要作用。2号以及6-9号传感器对电子鼻识别污染具有重要作用。电子鼻响应信号分别与污染时间(h)、细胞浓度(lg CFU/mL)具有良好的线性关系。对于PYGF60样品而言,模型R2介于0.92-0.99,SEC分别介于0.11-0.14 lg CFU/mL以及3.44-4.96 h,SEP分别介于0.19-0.34 lg CFU/mL以及8.94-12.06 h。对于苹果汁样品而言,模型R2介于0.94-0.99,SEC分别介于0.16-0.28 lg CFU/mL以及1.91-2.98 h,SEP分别介于0.20-0.26 lg CFU/mL以及1.49-2.69 h。鲁氏接合酵母污染PYGF60培养液48 h后代谢生成的乙醛、丙酮、3-甲基丁醛、乙醇和乙酸乙酯以及污染苹果汁12 h后引起果汁固有挥发性物质含量的降低对于电子鼻早期识别污染具有重要作用。
[Abstract]:Hypertonic yeast is one of the key quality and safety factors in apple juice concentrate industry, which poses a serious threat to the production, processing and export of apple juice. The quality and safety of juice products are of great significance to the stability, health and sustainable development of apple processing industry. In this paper, two major apple producing areas, Luochuan and Baishui, Shaanxi Province, were selected as sample collecting areas, and apple juice processing factories were sampled as the main processing raw materials. The isolates were identified by ITS1-5.8S-ITS2 or 26S rDNA D1/D2 sequencing. The biodiversity of the isolates was investigated by physiological methods and random primer amplified polymorphism analysis (RAPD-PCR), and their contamination ability was revealed by studying the growth status and gas production of the isolates in high glucose environment. Eleven isolates with potential hazards (able to grow in 70% w/v glucose) were selected to study the effects of environmental factors such as temperature, pH, sugar content and antimicrobial substances on their growth. Then, the contamination ability of Zygosaccharomyce was studied. The combined inhibitory effect of s rouxii on growth in simulated medium and apple juice concentrate was studied. Finally, the feasibility of qualitative and quantitative detection of contamination in high glucose medium (PYGF60) and apple juice by electronic nose was studied with Z. rouxii isolates and ATCC-purchased model strains. A total of 196 samples were collected from orchards and concentrated apple juice processing enterprises in Shaanxi apple production area, 40 samples were found to be hypertonic yeast (positive), the detection rate (positive rate) was 20.4%, of which the detection rate was 5% in orchard environment and 44.7% in juice processing plant environment. In the orchard environment, the remaining 53 strains came from the environment of the juice processing plant. In the orchard environment, the osmotic yeast mainly came from the fruit (5 strains) and the soil (4 strains). In the juice processing plant environment, the osmotic yeast mainly came from the workshop air (20 strains) and the storage concentrate (7 strains). 66 strains of the osmotic yeast distributed in 14 genera and 23 species, including 1 kind of yeast Aureobasidium pullulans. Kluyveromyces marxianus (8 strains), Candida tropicalis (6 strains), Z. rouxii (6 strains), Hansenia aspora uvarum (5 strains), Saccharomyces cerevisiae (5 strains) and P ichia kudriavzevii (5 strains). Only Z. rouxii was hypertonic yeast, and the other isolates were hypertonic yeast. Driavzevii, S. cerevisiae and Z. rouxii strains were divided into 2-3 groups based on physiological methods. The genetic similarity between these groups was more than 95%, which was consistent with RAPD-PCR analysis. A. pullulans, C. tropicalis (from apple juice concentrate), C. glabrata, C. orthopsilosis, C. zemplinina, Debaryomyces hansenii, Meyer caribbica, M. G. Uilliermondii, Torulaspora delbrueckii, Wickerhamomyces anomalus and Z. rouxii can grow in 70% w / V glucose environment, and most of them can metabolize gas in high glucose environment, which is potentially harmful to fruit juice products. When the maximum tolerance temperature increased to 52-67 oC. pH decreased to 2.0, the growth of the tested strain was significantly inhibited; the maximum tolerance pH of the tested strain was between 1.7 and 2.4. Only the conjugated yeast could grow in 70 oBrix apple juice, showing strong resistance. Sugar tolerance; the highest sugar tolerance of conjugated yeast was increased from 73 to 75 oBrix, showing high sugar environmental adaptability. Adding 4.0 mM potassium sorbate, 6.0 mM sodium benzoate, 1.5 mM cinnamic acid, 20 mM vanillin, 10 mM ferulic acid or 12 mM coumaric acid to apple juice (pH = 4.0) could effectively reduce the test results. Contamination of strains. With the increase of sugar content or the decrease of pH value, the micron ax value of conjugated yeast strains in simulated culture medium and apple juice concentrate gradually decreased, while the values of lambda and TFS gradually increased. Temperature changing environment (overseas shipping environment) was more conducive to the growth of conjugated yeast strains in apple juice concentrate than constant temperature environment (25oC). PH had the greatest effect on the micron ax, lambda and TFS values of the conjugated yeast strain, and was the main limiting factor for its growth. (4) After 48 hours of contamination with PYGF60 culture medium by conjugated yeast, the corresponding cell concentrations were 3.68 (B-WHX-12-54), 3.16 (ATCC 2623) and 2.98 (ATCC 8383) LG CFU/mL, respectively. After 12 hours of contamination with apple juice, the corresponding cell concentrations were less than 2.3 LG CFU/mL. CFU / mL, based on linear discriminant analysis (LDA) electronic nose can correctly identify pollution, and sensory assessors can not correctly distinguish between blank and contaminated samples at the above time points. 2 and 6-9 sensors play an important role in the identification of pollution. 2 and 6-9 sensors play an important role in the identification of pollution. For PYGF60 samples, model R2 is between 0.92-0.99, SEC is between 0.11-0.14 LG CFU/mL and 3.44-4.96 h, SEP is between 0.19-0.34 LG CFU/mL and 8.94-12.06 h, respectively. The contents of acetaldehyde, acetone, 3-methylbutyraldehyde, ethanol and ethyl acetate in the juice were decreased after 48 h of PYGF60 contaminated by conjugated yeast and 12 h of apple juice, respectively. Early identification of pollution in the nose plays an important role.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS255.44
，
本文编号：2210555
[Abstract]:Hypertonic yeast is one of the key quality and safety factors in apple juice concentrate industry, which poses a serious threat to the production, processing and export of apple juice. The quality and safety of juice products are of great significance to the stability, health and sustainable development of apple processing industry. In this paper, two major apple producing areas, Luochuan and Baishui, Shaanxi Province, were selected as sample collecting areas, and apple juice processing factories were sampled as the main processing raw materials. The isolates were identified by ITS1-5.8S-ITS2 or 26S rDNA D1/D2 sequencing. The biodiversity of the isolates was investigated by physiological methods and random primer amplified polymorphism analysis (RAPD-PCR), and their contamination ability was revealed by studying the growth status and gas production of the isolates in high glucose environment. Eleven isolates with potential hazards (able to grow in 70% w/v glucose) were selected to study the effects of environmental factors such as temperature, pH, sugar content and antimicrobial substances on their growth. Then, the contamination ability of Zygosaccharomyce was studied. The combined inhibitory effect of s rouxii on growth in simulated medium and apple juice concentrate was studied. Finally, the feasibility of qualitative and quantitative detection of contamination in high glucose medium (PYGF60) and apple juice by electronic nose was studied with Z. rouxii isolates and ATCC-purchased model strains. A total of 196 samples were collected from orchards and concentrated apple juice processing enterprises in Shaanxi apple production area, 40 samples were found to be hypertonic yeast (positive), the detection rate (positive rate) was 20.4%, of which the detection rate was 5% in orchard environment and 44.7% in juice processing plant environment. In the orchard environment, the remaining 53 strains came from the environment of the juice processing plant. In the orchard environment, the osmotic yeast mainly came from the fruit (5 strains) and the soil (4 strains). In the juice processing plant environment, the osmotic yeast mainly came from the workshop air (20 strains) and the storage concentrate (7 strains). 66 strains of the osmotic yeast distributed in 14 genera and 23 species, including 1 kind of yeast Aureobasidium pullulans. Kluyveromyces marxianus (8 strains), Candida tropicalis (6 strains), Z. rouxii (6 strains), Hansenia aspora uvarum (5 strains), Saccharomyces cerevisiae (5 strains) and P ichia kudriavzevii (5 strains). Only Z. rouxii was hypertonic yeast, and the other isolates were hypertonic yeast. Driavzevii, S. cerevisiae and Z. rouxii strains were divided into 2-3 groups based on physiological methods. The genetic similarity between these groups was more than 95%, which was consistent with RAPD-PCR analysis. A. pullulans, C. tropicalis (from apple juice concentrate), C. glabrata, C. orthopsilosis, C. zemplinina, Debaryomyces hansenii, Meyer caribbica, M. G. Uilliermondii, Torulaspora delbrueckii, Wickerhamomyces anomalus and Z. rouxii can grow in 70% w / V glucose environment, and most of them can metabolize gas in high glucose environment, which is potentially harmful to fruit juice products. When the maximum tolerance temperature increased to 52-67 oC. pH decreased to 2.0, the growth of the tested strain was significantly inhibited; the maximum tolerance pH of the tested strain was between 1.7 and 2.4. Only the conjugated yeast could grow in 70 oBrix apple juice, showing strong resistance. Sugar tolerance; the highest sugar tolerance of conjugated yeast was increased from 73 to 75 oBrix, showing high sugar environmental adaptability. Adding 4.0 mM potassium sorbate, 6.0 mM sodium benzoate, 1.5 mM cinnamic acid, 20 mM vanillin, 10 mM ferulic acid or 12 mM coumaric acid to apple juice (pH = 4.0) could effectively reduce the test results. Contamination of strains. With the increase of sugar content or the decrease of pH value, the micron ax value of conjugated yeast strains in simulated culture medium and apple juice concentrate gradually decreased, while the values of lambda and TFS gradually increased. Temperature changing environment (overseas shipping environment) was more conducive to the growth of conjugated yeast strains in apple juice concentrate than constant temperature environment (25oC). PH had the greatest effect on the micron ax, lambda and TFS values of the conjugated yeast strain, and was the main limiting factor for its growth. (4) After 48 hours of contamination with PYGF60 culture medium by conjugated yeast, the corresponding cell concentrations were 3.68 (B-WHX-12-54), 3.16 (ATCC 2623) and 2.98 (ATCC 8383) LG CFU/mL, respectively. After 12 hours of contamination with apple juice, the corresponding cell concentrations were less than 2.3 LG CFU/mL. CFU / mL, based on linear discriminant analysis (LDA) electronic nose can correctly identify pollution, and sensory assessors can not correctly distinguish between blank and contaminated samples at the above time points. 2 and 6-9 sensors play an important role in the identification of pollution. 2 and 6-9 sensors play an important role in the identification of pollution. For PYGF60 samples, model R2 is between 0.92-0.99, SEC is between 0.11-0.14 LG CFU/mL and 3.44-4.96 h, SEP is between 0.19-0.34 LG CFU/mL and 8.94-12.06 h, respectively. The contents of acetaldehyde, acetone, 3-methylbutyraldehyde, ethanol and ethyl acetate in the juice were decreased after 48 h of PYGF60 contaminated by conjugated yeast and 12 h of apple juice, respectively. Early identification of pollution in the nose plays an important role.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS255.44
，
本文编号：2210555
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