| Osmotolerant yeast is one of the most important quality and safety factors of concentrated apple juice and has become a major issue in the fruit juice processing industry. Acquisition of the knowledge about the distribution of osmotolerant yeast in the production chain of concentrated apple juice and the development of rapid and accurate methods for the detection and identifying of osmotolerant yeast in apple juice products is of great importance to the stable and sustainable development of the apple processing industry. In this study, two big apple producing regions located in luochuan and baishui county of Shaanxi province were chosen as the sample collection region, and samples were also collected from the juice processing plants which mainly use the apples from the both counties as the raw materials. After enrichment, osmotolerant yeasts were isolated and purified. Identification was performed through their ITS1-5.8S-ITS2 or 26 S r DNA D1/D2 amplification and sequencing. The biodiversity of isolates was investigated by the physiological method and RAPD-PCR amplification. Their growth conditions and gas production capabilities in high concentrations of glucose were studied to reveal their contamination potential. In order to get a further investigation of the contamination capacity of the 11 species which could grow in 70% w/v glucose, the effects of environmental factors including temperature, p H, sugar content and antimicrobial substances on their growth were studied. The combined inhibitory effects of sugar content and p H on the growth of Z. rouxii strain in high glucose medium and concentrated apple juice were studied. Finally, the feasibility of rapid detection of the spoilage in high glucose medium and apple juice caused by three Z. rouxii strains(one isolate and two ATCC strains) using electronic nose(e-nose) was evaluated.The main research results are as follows:(1) A total of 196 samples were collected from the apple orchards and apple juice processing plants located in apple producing areas of Shaanxi province, and 40 samples were detected with osmotolerant isolates(positive samples), with a positive ratio of 20.4%. The ratio of positive samples from apple orchards was 5%, and the ratio of positive samples from apple juice processing plants was 44.7%. In total, 66 osmotolerant isolates were recovered from the positive samples, 13 of which were from the apple orchards, and the remaining 53 were from the juice processing plants. For the apple orchard environments, osmotolerant yeast isolates were mainly isolated from the apple surface(5 strains) and the soil(4 strains). Regarding the plant environments, osmotolerant yeast isolates were mainly obtained from the workshop air(20 strains) and the stored concentrated apple juice(7 strains). The 66 osmotolerant yeast isolates were distributed in 14 genera and 23 species, including one yeast-like mold Aureobasidium pullulans. Kluyveromyces marxianus(8 strains), Candida tropicalis(6 strians), Z. rouxii(6 strians), Hanseniaspora uvarum(5 strains), Saccharomyces cerevisiae(5 strains), and Pichia kudriavzevii(5 strains) were the species that were frequently isolated. Only strains of Z. rouxii belonged to osmophilic yeast, while the other isolates were osmotolerant yeast.(2) Strains of C. tropicalis, H.opuntiae, K. marxianus, P. kudriavzevii, S. cerevisiae, and Z. rouxii were divided into two to three cluster in each species with a similarity of higher than 95%, and the similar strain composition in each cluster was observed based on the traditional physiological-biochemical method and RAPD-PCR technique. Strains of A. pullulans, C. tropicalis(from stored concentrated apple juice), C. glabrata, C. orthopsilosis, C. zemplinina, D. hansenii, M. caribbica, M. guilliermondii, Tp. delbrueckii,W. anomalus, and Z. rouxii were capable of growing in 70%(w/v) glucose, and most of them could produce gas in high concentration of glucose, posing great potential hazard to high-sugar foods.(3) The 11 osmotolerant isolates tested could not grow at 4 oC. The minimum temperature required to completely kill all the initial yeast inoculum ranged from 46 to 55 oC depending on the isolates. However, the environment of high concentration glucose could decrease the damage of high temperature to test strains, and enabled test strains to survival in what would have been a lethal treatment(52-67 oC). A p H value of 2 had a significant inhibitory effect on their growth. The maximum p H required to completely kill all the initial yeast inoculum ranged from 1.7 to 2.4 depending on the isolates. The most tolerant species towards sugar content was Z. rouxii for which the growth in apple juice at 70 oBrix was observed, whereas the growth of other species was inhibited by 45-60 oBrix. The sugar tolerance of Z. rouxii increased to 75 oBrix when the yeast culture adapted by growth in apple juice at 73 oBrix was used as inoculums, exhibiting the ability to adapt to high sugar content. Addition of antimicrobial components(4.0 m M of sorbic acid, 6.0 m M of benzoic acid, 1.5 m M of cinnamic acid, 20 m M of vanillin, 10 m M of ferulic acid or 12 m M of p-coumaric acid) was an applicable strategy to decrease the spoilage spawned by test strains. As the p H decreased or sugar content increased, the μmax values reduced and λ and TFS values added, suggesting an inhibitory effect of both factors on the growth of the test strain. The TFS values from the combinations under non-isothermal condition were smaller than those obtained under isothermal condition(p < 0.05). The results of standardized effect(standardized regression coefficients) of the studied environmental factors show that the main limiting factor that affects the growth of test strain was the p H.(4) Linear discriminant analysis(LDA) showed that e-nose could identify the contamination in PYGF60 broth after 48 h, corresponding to the total yeast count of 3.68(Z. rouxii B-WHX-12-54), 3.16(Z. rouxii ATCC 2623), and 2.98(Z. rouxii ATCC 8383) lg CFU/m L, the level at which the test panel could not yet diagnose the spoilage. As for apple juice samples, e-nose could identify the contamination after 12 h, corresponding to the total yeast count of less than 2.3 lg CFU/m L, the level at which the test panel could not yet diagnose the spoilage. Loadings analysis indicated that sensors 2 and 6-9 were the most important in the detection of the three Z. rouxii isolates. Based on the response of e-nose, the contamination time(h) or total yeast counts(lg CFU/m L) in PYGF60 broth could be well predicted by established partial least squares(PLS) models with high R2 of more than 0.98 and low standard error of calibration(SEC) of 3.44-4.96 h and 0.11-0.14 lg CFU/m L. As for apple juice samples, high R2 of more than 0.94 and low SEC of 1.91-2.98 h and 0.16-0.28 lg CFU/m L were obtained. The established models were also applied to predict the external test samples, and the results show that the correlation between the values estimated by the output of the e-nose and the values determined by reference method was more than 0.92, with low standard error of perdition(SEP) values of 0.19-0.34 lg CFU/m L and 8.94-12.06 h for PYGF60 broth samples. As for apple juice samples, high R2 of more than 0.95 and low SEP values of 0.20-0.26 lg CFU/m L and 1.49-2.69 h were obtained. Gas Chromatography-Mass Spectrometer(GC-MS) analysis showed that compounds including acetaldehyde, acetone, ethyl acetate, alcohol and 3-methyl-1-butanol contributed to the discrimination of the spoilage in PYGF60 broth by e-nose after 48 h, while the proliferation of the three Z. rouxii strains decreased the relative amount of components in the apple juice at early contamination stage, which contributed to the discrimination of the contamination in apple juice by e-nose after 12 h. |
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