Rapid screening of the novel bioactive peptides with notable α-glucosidase inhibitory activity by UF-LC-MS/MS combined with three-AI-tool from black beans.

This work aimed to propose a rapid method to screen the bioactive peptides with anti-α-glucosidase activity instead of traditional multiple laborious purification and identification procedures. 242 peptides binding to α-glycosidase were quickly screened and identified by bio-affinity ultrafiltration combined with LC-MS/MS from the double enzymatic hydrolysate of black beans. Top three peptides with notable anti-α-glucosidase activity, NNNPFKF, RADLPGVK and FLKEAFGV were further rapidly screened and ranked by the three artificial intelligence tools (three-AI-tool) BIOPEP database, PeptideRanker and molecular docking from the 242 peptides. Their IC50 values were in order as 4.20 ± 0.11 mg/mL, 2.83 ± 0.03 mg/mL, 1.32 ± 0.09 mg/mL, which was opposite to AI ranking, for the hydrophobicity index of the peptides was not included in the screening criteria. According to the kinetics, FT-IR, CD and ITC analyses, the binding of the three peptides to α-glucosidase is a spontaneous and irreversible endothermic reaction that results from hydrogen bonds and hydrophobic interactions, which mainly changes the α-helix structure of α-glucosidase. The peptide-activity can be evaluated vividly by AFM in vitro. In vivo, the screened FLKEAFGV and RADLPGVK can lower blood sugar levels as effectively as acarbose, they are expected to be an alternative to synthetic drugs for the treatment of Type 2 diabetes.

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus.

Diosgenin is an important raw material used in the synthesis of steroid drugs, and it is widely used in the pharmaceutical industry. The traditional method of producing diosgenin is through using raw materials provided via the plant Dioscorea zingiberensis C. H. Wright (DZW), which is subsequently industrially hydrolyzed using a high quantity of hydrochloric and sulfuric acids at temperatures ranging from 70 °C to 175 °C. This process results in a significant amount of unmanageable wastewater, creates issues of severe environmental pollution and consumes high quantities of energy. As an alternative, the enzymolysis of DZW to produce diosgenin is an environmentally and friendly method with wide-ranging prospects for its application. However, there are still only a few enzymes that are suitable for production on an industrial scale. In this study, three new key enzymes, E1, E2, and E3, with a high conversion stability of diosgenin, were isolated and identified using an enzyme-linked-substrate autography strategy. HPLC-MS/MS identification showed that E1, a 134.45 kDa protein with 1019 amino acids (AAs), is a zinc-dependent protein similar to the M16 family. E2, a 97.89 kDa protein with 910 AAs, is a type of endo-ß-1,3-glucanase. E3, a 51.6 kDa protein with 476 AAs, is a type of Xaa-Pro aminopeptidase. In addition, the method to immobilize these proteins was optimized, and stability was achieved. The results show that the optimal immobilization parameters are 3.5% sodium alginate, 3.45% calcium chloride concentration, 1.4 h fixed time, and pH 8.8; and the recovery rate of enzyme activity can reach 43.98%. A level of 70.3% relative enzyme activity can be obtained after employing six cycles of the optimized technology. Compared with free enzymes, immobilized enzymes have improved stability, acid and alkaline resistance and reusability, which are conducive to large-scale industrial production.

Water-Soluble Se-Containing Proteins from Chicken Alleviate DSS-Induced Ulcerative Colitis in Mice via Inhibiting TLR4/MyD88 Pathway and Protecting the Goblet Cell Pathway.

The influence of water-soluble selenium-containing proteins (WSSeP) in chicken on ulcerative colitis (UC) is not known. This work aims to investigate the effect of two WSSeP including h-Se with 1.78 µg Se/g and l-Se with 1.04 µg Se/g on mice UC induced by dextran sodium sulfate (DSS) versus 5-aminosalicylic acid (5-ASA). Seventy C57BL/6 mice were randomly divided into seven groups: groups 1 and 7 were given normal saline. Group 2 to group 4 were administrated orally 500, 1500, and 3000 mg/kg/day h-Se, respectively. Group 5 was given 1500 mg/kg/day l-Se as the control of group 3. From day 14 to day 21, groups 2 to 7 were fed with 3% DSS. Synchronously, group 6 was fed with 150 mg/kg/day 5-ASA. On day 21, the disease activity index, colon length, the histopathological changes, the expressions of claudin-1, occludin, ZO-1, TLR4, and MyD88 in colons, the levels of inflammatory cytokines (IFN-γ, IL-1ß, IL-6, TNF-α), and antioxidant markers (LPS, GSH-Px, SOD, MDA) in serum were determined. WSSeP can effectively improve the damages of DSS to the colon, thymus, and spleen, which present protein and Se dose-dependent. 1.50 g h-Se dose can significantly promote the expression levels of claudin-1, occludin, and ZO-1, to surround crypt gland and goblet and epithelial cells and inhibit the attack of DSS, suppress TLR4/MyD88 pathway, decrease the levels of IL-1ß, IL-6, TNF-α, IFN-γ, LPS, and MDA, and increase the activities of GSH-Px and SOD, which are better than those of 5-ASA. Therefore, WSSeP would be a natural and potential anti-inflammatory agent for UC.

A Novel Glucose-6-Phosphate Isomerase Exists in Chicken Breast Meat: A Selenium-Containing Enzyme that Should Be Re-recognized Through New Eyes.

Glucose-6-phosphate isomerase (GPI) is a highly conserved glycolytic enzyme in nature, and less information was available for GPI from hens. In this study a newly discovered selenocysteine (Sec)-containing GPI in common chicken breast meat was first isolated, purified and identified. Data about LC-MS/MS, FTIR and Se species analyses show that the molecular weight of the enzyme is 62,091 Da and only one Sec is inserted at the 403rd position in the highly conserved primary domain SIS_PGI with sugar conversion function. The enzyme shows excellent activity against hydroxyl radicals as vitamin C (Vc) in vitro. It is deduced that the Sec-containing GPI in the chicken meat may depend on Sec in its molecular structure to resist reactive oxygen species (ROS) stress produced by the accompanying biochemical reactions in cells, to protect its stability and maintain its efficient function that catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate in the critical glycolytic pathway.

Effect of the selenized yeast added in feed on selenium-containing proteins of albumins in egg yolk.

This work was aimed to study the effects of the selenized yeast added in feed on selenium-containing proteins of egg yolk. Two groups of the same little hens were given the ordinary grain feed either unsupplemented selenized yeast (Group O) or supplemented with 0.15% selenized yeast (Group Y), respectively. The water-soluble Se-containing proteins were isolated and purified from the two group eggs yolk using the same conditions. SeP1-1 and SeP1-I were purified from the yolk of Group Y and Group O, respectively. Sequences identified by HPLC-MS/MS showed that SeP1-1 was a highly homologous Se-containing protein with Se-free YGP-42 with 83% match, in which Se species include methylselenocysteine and selenocysteine. SeP1-I was a highly homologous Se-containing protein with Se-free ovalbumin with 78.2% match, in which Se species include selenomethionine and selenocysteine. It can be concluded that the selenized yeast can change the compositions and structures of Se-containing proteins in egg yolk.

The Effects of Catabolism Relationships of Leucine and Isoleucine with BAT2 Gene of Saccharomyces cerevisiae on High Alcohols and Esters.

This study sought to provide a theoretical basis for effectively controlling the content of higher alcohols and esters in fermented foods. In this work, isoleucine (Ile) or leucine (Leu) at high levels was used as the sole nitrogen source for a BAT2 mutant and its parental Saccharomyces. cerevisiae 38 to investigate the effects of the addition of amounts of Ile or Leu and BAT2 on the aroma components in the flavor profile using gas chromatography mass spectrometer (GC-MS). The results showed that 2-methyl-butyraldehyde, 2-methyl-1-butanol, and 2-methylbutyl-acetate were the products positively correlated with the Ile addition amount. In addition, 3-methyl-butyraldehyde, 3-methyl-1-butanol, and 3-methylbutyl-acetate were the products positively correlated with Leu addition amount. BAT2 deletion resulted in a significant decline in the yields of 2-methyl-butyraldehyde, 3-methyl-butyraldehyde,2-methyl-1-butanol, and 3-methyl-1-butanol, but also an increase in the yields of 2-methylbutyl-acetate and 3-methylbutyl-acetate. We speculated that BAT2 regulated the front and end of this metabolite chain in a feedback manner. Improved metabolic chain analyses, including the simulated energy metabolism of Ile or Leu, indicated that reducing the added amount of branched-chain amino acids, BAT mutation, and eliminating the role of energy cofactors such as NADH/NAD+ were three important ways to control the content of high alcohols and esters in fermented foods.

Identification and a phased pH control strategy of diosgenin bio-synthesized by an endogenous Bacillus licheniformis Syt1 derived from Dioscorea zingiberensis C. H. Wright.

Diosgenin is widely used as one precursor of steroidal drugs in pharmaceutical industry. Currently, there is no choice but to traditionally extract diosgenin from Dioscorea zingiberensis C. H. Wright (DZW) or other plants. In this work, an environmentally friendly approach, in which diosgenin can be bio-synthesized by the endophytic bacterium Bacillus licheniformis Syt1 isolated from DZW, is proposed. Diosgenin produced by the strain was identified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). The thermal gravimetric analysis (TGA) showed that the melting point of the diosgenin product was 204 °C. The optical rotation measurement exhibited that the optical rotation was α20589 = - 126.1° ± 1.5° (chloroform, c = 1%): negative sign means that the product is left-handed, which is very important to further produce steroid hormone drugs. Cholesterol may be the intermediate product in the diosgenin biosynthesis pathway. In the batch fermentation process to produce diosgenin using the strain, pH values played an important role. A phased pH control strategy from 5.5 to 7.5 was proved to be more effective to improve production yield than any single pH control, which could get the highest diosgenin yield of 85 ± 8.6 mg L-1. The proposed method may replace phyto-chemistry extraction to produce diosgenin in the industry in the future.Key points• An endophytic Bacillus licheniformis Syt1 derived from host can produce diosgenin.• A dynamic pH industrial control strategy is better than any single pH control.• Proposed diosgenin-produced method hopefully replaces phyto-chemistry extraction.

Inactivation Effect of Thymoquinone on Alicyclobacillus acidoterrestris Vegetative Cells, Spores, and Biofilms.

Alicyclobacillus acidoterrestris (A. acidoterrestris), a spore-forming bacterium, has become a main challenge and concern for the juices and acid beverage industry across the world due to its thermo-acidophilic characteristic. Thymoquinone (TQ) is one of the active components derived from Nigella sativa seeds. The objective of this study was to investigate antibacterial activity and associated molecular mechanism of TQ against A. acidoterrestris vegetative cells, and to evaluate effects of TQ on A. acidoterrestris spores and biofilms formed on polystyrene and stainless steel surfaces. Minimum inhibitory concentrations of TQ against five tested A. acidoterrestris strains ranged from 32 to 64 µg/mL. TQ could destroy bacterial cell morphology and membrane integrity in a concentration-dependent manner. Field-emission scanning electron microscopy observation showed that TQ caused abnormal morphology of spores and thus exerted a killing effect on spores. Moreover, TQ was effective in inactivating and removing A. acidoterrestris mature biofilms. These findings indicated that TQ is promising as a new alternative to control A. acidoterrestris and thereby reduce associated contamination and deterioration in the juice and acid beverage industry.

Antimicrobial and anti-biofilm activity of thymoquinone against Shigella flexneri.

Shigella flexneri (Sh. flexneri), a common foodborne pathogen, has become one of the main threats to food safety and human health due to its high pathogenicity and persistent infection. The objective of this study was to explore the antimicrobial and anti-biofilm activities and the possible mechanism of thymoquinone (TQ) against Sh. flexneri. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of TQ against Sh. flexneri were 0.4 and 0.5 mg/mL, respectively. TQ showed bactericidal activity against Sh. flexneri in culture medium and milk system. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) observations demonstrated that TQ could induce abnormal cell morphology and destroy cell membrane. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested that TQ could inhibit protein synthesis in Sh. flexneri. Also, at sub-inhibitory concentrations (SICs), TQ exhibited an inhibitory effect on Sh. flexneri biofilm formation, which was confirmed by crystal violet quantitative analysis and SEM observation. Real-time quantitative PCR (RT-qPCR) analyses revealed that TQ downregulated the expression of genes involved in Sh. flexneri biofilm formation. Thus, TQ has potential as a natural antimicrobial and anti-biofilm agent to address the contamination and infection caused by Sh. flexneri. KEY POINTS: • Antimicrobial and anti-biofilm activity of TQ on Shigella flexneri were investigated. • TQ inhibited biofilm formation by Shigella flexneri. • TQ provided a new strategy for Shigella flexneri control.

Mechanochemical activation on selective leaching of arsenic from copper smelting flue dusts.

A novel application, including mechanochemical pre-treating and alkali leaching, for arsenic selective leaching from copper smelter flue dusts (CSFUs) was developed to overcome the disadvantages of hydrometallurgical methods. Compared with raw CSFU powders, the mechanical-activated ones showed higher maximum arsenic leaching efficiency (increased by ~20%), and lower apparent activation energy (decreased by ~7 kJ·mol-1). Furthermore, this novel process only consumed half of alkali and sulfides and needed one-third of the leaching time to compare with the ones used in the traditional alkali leaching process. The promoting effect of mechanical force on arsenic leaching firstly relied on the physical property changes of CSFU powders, including a decrease of particle sizes and an increase of the specific surface. Secondly, mechanochemical force converted As5+ species into reduced phases (e.g. As2O3, NaAsO2), and thio-arsenates (e.g. AsO2S23-, AsO3S3-), which could spur its leaching due to their stronger mobilities in the alkali solution within sulfides. Finally, mechanochemical activation could be facilitated to separate discrete soluble arsenic species or incorporated ones from sulfate minerals in the CSFUs. This work may have important implications for the development of new eco-friendly technologies for purifying arsenic-bearing materials.

Isolation and identification of three water-soluble selenoproteins in Se-enriched Agaricus blazei Murrill.

Selenoproteins in selenium (Se)-enriched vegetables play an important role in human health. In this study, three water-soluble selenoproteins PR-Se-1, PR-Se-2 and PR-Se-3 in Agaricus blazei Murrill (ABM) were isolated by anion exchange chromatography, gel filtration chromatography and SDS-PAGE. Sequence analyses performed by HPLC-MS/MS showed that PR-Se-1, a 114024 Da selenoprotein with 1019 amino acids (AAs), is an isoenzyme of isocitrate dehydrogenase. PR-Se-2, a 53983 Da selenoprotein with 508 AAs, is a kind of dihydrolipoyl dehydrogenase. PR-Se-3, a 47179 Da selenoprotein with 415 AAs, is a kind d-proline reductase. Se content is high at 26.1 µg/g, and selenocystine is the predominant Se unit in the three selenoproteins. Se content of ABM is 9.15 µg/g, and the organic form of Se accounts for ~81% of total Se content. ABM could be a promising source of Se in Se-poor regions.

Benefit-risk assessment of dietary selenium and its associated metals intake in China (2017-2019): Is current selenium-rich agro-food safe enough?

Dietary consumption of selenium-rich agro-food is an effective way to avoid selenium deficiency diseases, however, over consumption of selenium-rich agro-food will result in potential risk of selenosis and problems with associated metals. In this study, we measured the concentrations of selenium and its associated metals in 2756 common and 4894 selenium-rich agro-food samples in 10 regions of China. We found that selenium-rich rice, flour, edible fungi and algae, meat, and tea contain higher levels of associated metals than other selenium-rich agro-food samples. Increasing the consumption of selenium-rich agro-food could make the actual intakes (AIs) of selenium for all population to meet respective recommended daily intakes (RDIs). Benefit-risk assessment results indicated that increasing the consumption of selenium-rich agro-food make AIs of selenium for all populations meet RDIs, chromium intakes for people under 18 years old exceed provisional tolerated daily intake (PTDIs), while arsenic and cadmium intakes are close to PTDIs. The main dietary contributors of selenium, chromium, arsenic and cadmium were meat, edible fungi and algae, rice, and rice, respectively. The study supported the consumption of selenium-rich agro-food for effective selenium supplement, but also emphasized potential risk from associated metals in selenium-rich agro-food, especially chromium.

Determination of Selenium in Common and Selenium-Rich Rice from Different Areas in China and Assessment of Their Dietary Intake.

In this study, 41 common rice varieties and 211 selenium-rich rice varieties from ten representative areas in China were collected in 2017-2019. The selenium contents of rice were analyzed with optimized inductively coupled plasma mass spectrometry (ICP-MS). Selenium concentrations of common rice and selenium-rich rice ranges were 0.81-7.26 and 0.76-180.73 µg/100 g, respectively. The selenium contents in selenium-rich rice from different areas were significantly different (p < 0.001) while those in common rice from different areas were not. The selenium-rich rice in Harbin and Keshan showed the lowest selenium level and those from selenium-rich areas (Enshi and Ankang) were highest. Based on the estimation of the risk assessment software @risk7.0 (Palisade Corporation, New York, NY, USA), the consumption of selenium-rich rice can effectively increase dietary selenium intake for the population. However, the risk index of P95 (Percentile 95) selenium exposure at the tolerable upper intake level for children at 2-14 years old exceeded 100%, with potential risk currently. Therefore, the consumption of selenium-rich rice should be properly monitored for young children and adolescents.

Characterization of selenium-containing polysaccharides isolated from selenium-enriched tea and its bioactivities.

Two novel selenium polysaccharide fractions (SeTPS-1 and SeTPS-2) were isolated purified, characterized from Se-enriched tea. The results showed that the molecular weights and Se content of SeTPS-1 and SeTPS-2 were 1.7 × 104 Da, 1.3 × 104 Da, and 23.50 µg/g and 13.47 µg/g, respectively. SeTPS-1 and SeTPS-2 had absorption spectra typical of selenium esters. SeTPS-1 was composed of glucose and galactose at a molar ratio of 80.1:2.3, respectively, while SeTPS-2 was composed of arabinose, glucose, galactose and galacturonic acid with a molar ratio of 2.04: 48.83: 3.21: 1.30, respectively. Both SeTPS-1 and SeTPS-2 adopted a random coil conformation. Importantly, in vitro assessment of the antioxidant capacity revealed that SeTPS-1 is a more potent antioxidant compared to SeTPS-2. Both compounds were effective at reducing DNA damage induced by H2O2. The promising data suggesting that these compounds confer natural protection against DNA-damaging agents, thereby contributing to the functional food qualities of tea.

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.

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.

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.

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.

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.