Huxuan Wang

Characterization of Osmotolerant Yeasts and Yeast-Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast-like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S-ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD-PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast-like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.

Early detection of Zygosaccharomyces rouxii--spawned spoilage in apple juice by electronic nose combined with chemometrics.

Spoilage spawned by Zygosaccharomyces rouxii can cause sensory defect in apple juice, which could hardly be perceived in the early stage and therefore would lead to the serious economic loss. Thus, it is essential to detect the contamination in early stage to avoid costly waste of products or recalls. In this work the performance of an electronic nose (e-nose) coupled with chemometric analysis was evaluated for diagnosis of the contamination in apple juice, using test panel evaluation as reference. The feasibility of using e-nose responses to predict the spoilage level of apple juice was also evaluated. Coupled with linear discriminant analysis (LDA), detection of the contamination was achieved after 12h, corresponding to the cell concentration of less than 2.0 log 10 CFU/mL, the level at which the test panelists could not yet identify the contamination, indicating that the signals of e-nose could be utilized as early indicators for the onset of contamination. Loading analysis indicated that sensors 2, 6, 7 and 8 were the most important in the detection of Z. rouxii-contaminated apple juice. Moreover, Z. rouxii counts in unknown samples could be well predicted by the established models using partial least squares (PLS) algorithm with high correlation coefficient (R) of 0.98 (Z. rouxii strain ATCC 2623 and ATCC 8383) and 0.97 (Z. rouxii strain B-WHX-12-53). Based on these results, e-nose appears to be promising for rapid analysis of the odor in apple juice during processing or on the shelf to realize the early detection of potential contamination caused by Z. rouxii strains.

A novel method to quantify the activity of alcohol acetyltransferase Using a SnO2-based sensor of electronic nose

Alcohol acetyltransferase (AATFase) extensively catalyzes the reactions of alcohols to acetic esters in microorganisms and plants. In this work, a novel method has been proposed to quantify the activity of AATFase using a SnO2-based sensor of electronic nose, which was determined on the basis of its higher sensitivity to the reducing alcohol than the oxidizing ester. The maximum value of the first-derivative of the signals from the SnO2-based sensor was therein found to be an eigenvalue of isoamyl alcohol concentration. Quadratic polynomial regression perfectly fitted the correlation between the eigenvalue and the isoamyl alcohol concentration. The method was used to determine the AATFase activity in this type of reaction by calculating the conversion rate of isoamyl alcohol. The proposed method has been successfully applied to determine the AATFase activity of a cider yeast strain. Compared with GC-MS, the method shows promises with ideal recovery and low cost.

Detection of Zygosaccharomyces rouxii and Candida tropicalis in a High-Sugar Medium by a Metal Oxide Sensor–Based Electronic Nose and Comparison with Test Panel Evaluation

Osmotolerant yeasts are primarily responsible for spoilage of sugar-rich foods. In this work, an electronic nose (e-nose) was used to diagnose contamination caused by two osmotolerant yeast strains (Zygosaccharomyces rouxii and Candida tropicalis) in a high-sugar medium using test panel evaluation as the reference method. Solid-phase microextraction gas chromatography with mass spectrometry (GC-MS) was used to determine the evolution of the volatile organic compound fingerprint in the contaminated samples during yeast growth. Principal component analysis and linear discriminant analysis revealed that the e-nose could identify contamination after 48 h, corresponding to the total yeast levels of 3.68 (Z. rouxii) and 3.09 (C. tropicalis) log CFU/ml. At these levels, the test panel could not yet diagnose the spoilage, indicating that the e-nose approach was more sensitive than the test panel evaluation. Loading analysis indicated that sensors 8 and 6 were the most important for detection of these two yeasts. Based on the result obtained with the e-nose, the incubation time and total yeast levels could be accurately predicted by established multiple regression models with a correlation of greater than 0.97. In the sensory evaluation, spoilage was diagnosed after 72 h in samples contaminated with C. tropicalis and after 48 to 72 h for samples contaminated with Z. rouxii. GC-MS revealed that compounds such as acetaldehyde, acetone, ethyl acetate, alcohol, and 3-methyl-1-butanol contributed to spoilage detection by the e-nose after 48 h. In the high-sugar medium, the e-nose was more sensitive than the test panel evaluation for detecting contamination with these test yeast strains. This information could be useful for developing instruments and techniques for rapid scanning of sugar-rich foods for contamination with osmotolerant yeasts before such spoilage could be detected by the consumer.

Accessing spoilage features of osmotolerant yeasts identified from kiwifruit plantation and processing environment in Shaanxi, China.

Osmotolerant yeasts originating from kiwifruit industrial chain can result in spoilage incidences, while little information is available about their species and spoilage features. This work identified possible spoilage osmotolerant yeasts from orchards and a manufacturer (quick-freeze kiwifruit manufacturer) in main producing areas in Shaanxi, China and further characterized their spoilage features. A total of 86 osmotolerant isolates dispersing over 29 species were identified through 26S rDNA sequencing at the D1/D2 domain, among which Hanseniaspora uvarum occurred most frequently and have intimate relationships with kiwifruit. RAPD analysis indicated a high variability of this species from sampling regions. The correlation of genotypes with origins was established except for isolates from Zhouzhi orchards, and the mobility of H. uvarum from orchard to the manufacturer can be speculated and contributed to spoilage sourcing. The manufacturing environment favored the inhabitance of osmotolerant yeasts more than the orchard by giving high positive sample ratio or osmotolerant yeast ratio. The growth curves under various glucose levels were fitted by Grofit R package and the obtained growth parameters indicated phenotypic diversity in the H. uvarum and the rest species. Wickerhamomyces anomalus (OM14) and Candida glabrata (OZ17) were the most glucose tolerant species and availability of high glucose would assist them to produce more gas. The test osmotolerant species were odor altering in kiwifruit concentrate juice. 3-Methyl-1-butanol, phenylethyl alcohol, phenylethyl acetate, 5-hydroxymethylfurfural (5-HMF) and ethyl acetate were the most altered compound identified by GC/MS in the juice. Particularly, W. anomalus produced 4-vinylguaiacol and M. guilliermondii produced 4-ethylguaiacol that would imperil product acceptance. The study determines the target spoilers as well as offering a detailed spoilage features, which will be instructive in implementing preventative measures to increase production safety of kiwifruit.

Protein abundance changes of Zygosaccharomyces rouxii in different sugar concentrations

Zygosaccharomyces rouxii is a yeast which can cause spoilage in the concentrated juice industries. It exhibits resistance to high sugar concentrations but genome- and proteome-wide studies on Z. rouxii in response to high sugar concentrations have been poorly investigated. Herein, by using a 2-D electrophoresis based workflow, the proteome of a wild strain of Z. rouxii under different sugar concentrations has been analyzed. Proteins were extracted, quantified, and subjected to 2-DE analysis in the pH range 4-7. Differences in growth (lag phase), protein content (13.97-19.23mg/g cell dry weight) and number of resolved spots (196-296) were found between sugar concentrations. ANOVA test showed that 168 spots were different, and 47 spots, corresponding to 40 unique gene products have been identified. These protein species are involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, protein transport and vesicle organization, cell morphogenesis regulation, transcription and translation, nucleotide metabolism, amino-sugar nucleotide-sugar pathways, oxidoreductases balancing, and ribosome biogenesis. The present study provides important information about how Z. rouxii acts to cope with high sugar concentration at molecular levels, which might enhance our global understanding of Z. rouxiis high sugar-tolerance trait.