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.     

The effect of UV‐C in combination with H2O2 treatments on microbial response and quality parameters of fresh cut pear discs

The response of Escherichia coli ATCC 11229, Listeria innocua ATCC 33090 and Zygosaccharomyces bailii NRRL 7256 in fresh‐cut pear to simultaneous and serial combined treatments involving H₂O₂ immersion (3% w/v; pH 3.0; 25 °C; 2.0 or 5.0 min) and UV‐C exposure (7.5 min; 3.7 kJ m^?2) was investigated. For selected treatments, native flora, sensory and colour changes were also evaluated. E. coli and L. innocua were more sensitive than Z. bailii. Serial H₂O₂/UV‐C treatments were more effective than the simultaneous arrangement. The single effect of UV‐C was enhanced by the combination with 5 min H₂O₂, hence avoiding the recovery of the surviving population. The combined treatment kept optimal microbial stability and exhibited more luminosity than the single UV‐C treatment. Texture profile analysis conducted using a trained panel showed that H₂O₂/UV‐C processed pear discs were perceived with significantly less hardness and fracturability but as juicy as untreated discs. Consumers found them pleasant.     

Osmotolerant yeast species differ in basic physiological parameters and in tolerance of non‐osmotic stresses

Osmotolerance is the ability to grow in an environment with a high osmotic pressure. In this study we compared the physiological parameters and tolerance to osmotic and non‐osmotic stresses of three osmotolerant yeast species, Debaryomyces hansenii, Pichia farinosa (sorbitophila) and Zygosaccharomyces rouxii, with those of wild‐type Saccharomyces cerevisiae. Although the osmotolerant species did not differ significantly in their basic parameters, such as cell size or growth capacity, they had different abilities to survive anhydrobiosis, potassium limitation or the presence of toxic cationic drugs. When their osmotolerance was compared, the results revealed that some of the species isolated as sugar/polyol‐tolerant (e.g. P. farinosa) are also highly tolerant to salts and, vice versa, some strains isolated from an environment with high concentration of salt (e.g. Z. rouxii ATCC 42981) tolerate high concentrations of sugars. None of the tested strains and species was osmophilic. Taken together, our results showed that P. farinosa (sorbitophila) is the most robust species when coping with various stresses, while Z. rouxii CBS 732, although osmotolerant in general, is not specifically salt‐tolerant and is quite sensitive to most of the tested stress conditions. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.     

A peculiar stimulatory effect of acetic and lactic acid on growth and fermentative metabolism of Zygosaccharomyces bailii

Stimulatory or protective effects of organic acids at low concentrations, e.g. acetic and lactic acid, on microorganisms have previously been reported. Especially in case of Zygosaccharomyces bailii, a peculiar growth stimulation by these two acids has recently been noticed. In order to elucidate this interesting phenomenon, growth and fermentative metabolism of Z. bailii was investigated in media with low pH (pH 4.0), high sugar (15% (w/v)) and different acetic and lactic acid concentrations. At both experimental temperatures (7 and 30 °C), a growth stimulation in the presence of 2.5% (v/v) lactic acid was observed. Furthermore at 7 °C, the yeast exhibited another unusual behaviour as it grew much faster in media containing 1.25% (v/v) acetic acid than in the control (without any acid). Production of fermentative metabolites was also increased together with the enhanced growth at both temperatures. These possible stimulatory effects of acetic and lactic acid should be taken into consideration when the acids are used at low doses for food preservative purpose. Presence of the acids may stimulate Z. bailii growth and fermentative metabolism, particularly at refrigeration temperature, consequently resulting in an earlier spoilage.     

Bacterial inactivation and quality changes of fresh‐cut avocados as affected by intense light pulses of specific spectra

Intense light pulses (ILP) treatments have good prospects for becoming an alternative to traditional thermal methods for decontamination of food surfaces. The aim of this work was to evaluate which ranges of the light spectrum are responsible for bacterial inactivation and their effect on the quality of fresh‐cut avocado. Results show that the effectiveness of ILP treatment decreases when the ultraviolet (UV) spectral region is blocked (particularly UV‐C). ILP treatments without UV‐C light (305–1100 nm) and an overall fluence of 10.68 J cm^?2 caused reductions of 2.47 and 1.35 log CFU g^?1 in the initial counts of inoculated Escherichia coli and Listeria innocua, respectively, in comparison with those treated using only VIS–NIR light (0.83 and 0.68 log CFU g^?1, respectively). Treatments applying light of a wavelength between 305 and 1100 nm had a more pronounced impact on colour, texture and headspace gas composition than treatments that did not contain UV light (400–1100 nm).     

Mutation of Acetobacter pasteurianus by UV irradiation under acidic stress for high‐acidity vinegar fermentation

To obtain a mutant with higher vinegar production, a wild‐type industrial strain Acetobacter pasteurianus CICIM B7003 was pretreated in 50 g L^?1 acetic acid for 1 h and cultured for 12 h without adding any acetic acid; then, the enriched cells were mutated by UV under acidic stress (60 g L^?1 acetic acid). Using this method, A. pasteurianus CICIM B7003‐02, a mutant exhibiting best performance, was obtained. Notably, after repeated experiments, it was confirmed that B7003‐02 accumulated 103.81 ± 1.17 g L^?1 acetic acid within 160‐h batch cultivation in Erlenmeyer flasks, which was 49.2% higher than that of the wild type. Repeated batch fermentations with A. pasteurianus B7003‐02 were carried out for several runs in a Frings 8‐L acetator, and high‐acidity vinegars (90 ± 0.39 g L^?1) were produced. This work reveals the potential value for improvement in industrial vinegar production.     

Characterization of plasma membrane proton-ATPase from salt-tolerant yeast Zygosaccharomyces rouxii cells

Plasma membrane was isolated from the salt-tolerant yeast Zygosaccharomyces rouxii and from Saccharomyces cerevisiae. The ATPase in the plasma membrane of Z. rouxii cells was a typical proton-ATPase as judged by testing with various ATPase inhibitors. There were slight differences in the pH optima of activities and in the sensitivity to sodium chloride (NaCl) and potassium chloride (KCl) of the ATPase from Z. rouxii and S. cerevisiae. The specific ATPase activity from Z. rouxii was higher in cells grown in a medium containing 2 _M-NaCl than in those not containing NaCl. No in vivo activation by incubation with glucose was observed in Z. rouxii cells and the specific ATPase activity was independent of the growth phase, unlike S. cerevisiae cells.     

From wheat sourdough to gluten‐free sourdough: a non‐conventional process for producing gluten‐free bread

Gluten‐free (GF) sourdough was prepared from wheat sourdough and analysed both in fresh (GFS) and dried forms (DGFS). The gluten content in each GF sourdough sample was <20 mg kg^?1. The dough leavening capacity and the properties of the bread samples were investigated and compared to those of bread prepared using bakery yeast (Saccharomyces cerevisiae). Two commercial rice‐based mixtures (different for the presence/absence of buckwheat flour) were used to prepare bread samples. In GFS, lactic acid bacteria (LAB) and yeasts were found in amounts corresponding to 10⁸ and 10⁷ CFU g^?1, respectively, whereas both LAB and yeasts were detected in lower amounts (about 10⁶ CFU g^?1) in DGFS. When used in bread‐making, both GFS types produced significant dough acidification and exhibited good dough development during proofing, resulting in loaves with specific volume values between 3.00 and 4.12 mL g^?1, values similar to those obtained for reference bread (3.05÷4.15 mL g^?1). The use of GFS was effective in lowering the bread staling rate during storage for up to 7 days.     

Co‐inoculation of yeast and lactic acid bacteria to improve cherry wines sensory quality

This study examined the effect of co‐inoculation of yeast and lactic acid bacteria (LAB) on the chemical and sensory characteristics of cherry wines, in comparison with a traditional sequential culture. Three LABs were investigated, including two O. oeni (SG26 and Viniflora) and one L. plantarum (PL18). All co‐inoculations significantly shortened the fermentation time (average 8 days earlier) to reach a stable level of residual sugar (<2 g L^?1) and L‐malic acid (<0.5 g L^?1), and no inhibitory effect on the yeast proliferation was observed. For volatiles determined, co‐culture with SG26 produced the greatest amount of volatile components (138.5 mg L^?1), whereas sequential inoculation with PL18 had the lowest level (119.6 mg L^?1). PCA result revealed that different LABs had diverse influences on the volatile profile of cherry wines, and sensory analysis confirmed that these samples presented distinct sensory profiles, and particularly, a stronger note of fruity was perceived when co‐culture was used.     

Gluten‐free bread: multiple time–intensity analysis,physical characterisation and acceptance test

The formulation of gluten‐free breads is challenging, once the gluten network is responsible for the physical and sensory properties of bread. This study presents a novel concept for making gluten‐free breads using sugar substitutes. The quality of gluten‐free breads was analysed by means of physical (specific volume and colour), textural (firmness, elasticity and chewiness) and sensory properties (time‐intensity analysis and acceptance test). Time‐intensity analysis showed that the sample developed with raw sugar had a higher intensity of sweetness, and the sample with stevia had a higher intensity of yeast flavour. The sample with frutooligosaccharides (FOS) presented a higher acceptance by consumers. Sample with stevia presented the higher intensity of yeast flavour (8.10) and the lesser mean of specific volume (2.83 cm³.g^?1); whereas sample with sugar who presented higher intensity of sweetness stimulus (5.20) also presented the higher mean of specific volume (3.80 cm³.g^?1). The addition of prebiotic and sweetener opens up new opportunities to develop gluten‐free breads that may present similar properties to those of wheat‐based breads.     

Characteristics of yeast flora in Chinese strong‐flavoured liquor fermentation in the Yibin region of China

The yeast community in the Chinese strong‐flavoured liquor region of Yibin was investigated and the ethanol producing abilities and extracellular enzymes activities of the isolates were tested. A total of 110 yeast were isolated on Wallerstein Laboratory medium and through 26S rRNA D1/D2 region sequence analysis identified as 13 yeast species. These were Wickerhamomyces anomalus, Debaryomyces hansenii, Issatchenkia orientalis, Lodderomyces elongisporus, Clavispora lusitaniae, Saccharomyces cerevisiae, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Torulaspora delbrueckii, Trichosporon insectorum, Trichosporonoides megachiliensis, Zygosaccharomyces bailii, and one uncertain species. These yeast species, composed of various strains, formed the special yeast community in the Yibin region. Approximately 73.6% of the strains belong to the four dominant species: W. anomalus, D. hansenii, I. orientalis and L. elongisporus. The 110 yeast strains produced 0.6–9.0% (v/v) alcohol (average of 5.4%, v/v) in a grain medium, and 0.2–7.2% (v/v) alcohol (average value of 2.9%, v/v) in a yeast extract–peptone–dextrose medium. Furthermore, the 49 strains that produced pectinase, lipase, cellulase, amylase or protease generally showed better ethanol‐producing ability than those strains that do not produce extracellular enzymes. This work profiles the ethanol‐producing ability and the organic matter utilization of the yeast community in Chinese strong‐flavoured liquor produced in the Yibin region and provides a better understanding of Chinese strong‐flavoured liquor fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

Microbial Safety and Shelf Life of UV‐C Treated Freshly Squeezed White Grape Juice

The effects of UV‐C irradiation on the inactivation of Escherichia coli K‐12 (ATCC 25253), a surrogate of E. coli O157:H7, and on the shelf life of freshly squeezed turbid white grape juice (FSWGJ) were investigated. FSWGJ samples were processed at 0.90 mL/s for 32 min by circulating 8 times in an annular flow UV system. The UV exposure time was 244 s per cycle. The population of E. coli K‐12 was reduced by 5.34 log cycles after exposure to a total UV dosage of 9.92 J/cm² (1.24 J/cm² per cycle) at 0.90 mL/s flow rate. The microbial shelf life of UV‐C treated FSWGJ was extended up to 14 d at 4 °C. UV exposure was not found to alter pH, total soluble solid, and titratable acidity of juice. There was a significant effect (P < 0.05) on turbidity, absorbance coefficient, color, and ascorbic acid content. Furthermore, all physicochemical properties were altered during refrigerated storage. The microbial shelf life of FSWGJ was doubled after UV‐C treatment, whereas the quality of juice was adversely affected similarly observed in the control samples.     

Antagonism between Saccharomyces cerevisiae and Williopsis mrakii and significance for mixed yeast alcoholic beverage fermentations

This research was carried out to investigate the antagonism between Saccharomyces cerevisiae and Williopsis saturnus var. mrakii. When glucose level was ≥12% (w/v) (potential ethanol ≥7.2% v/v), W. mrakii that was co‐inoculated with S. cerevisiae died early, while W. mrakii alone could not survive in broth with ≥8% (v/v) ethanol. Moreover, when glucose was ≥15% (w/v) (potential ethanol ≥9.0% v/v), higher inoculums of S. cerevisiae induced faster cell decline of W. mrakii; when glucose was ≤2% (w/v), there was no early cell decline of W. mrakii at any inoculum ratio, but there was inoculum ratio‐dependent growth retardation. These results indicate that ethanol was at least one of the factors causing early cell decline of W. mrakii in co‐culture with S. cerevisiae. Further, S. cerevisiae was inhibited when it was inoculated sequentially into a W. mrakii monoculture; the former yeast was also suppressed when it was inoculated into the supernatants of a W. mrakii monoculture. This suggests that S. cerevisiae was sensitive to the mycosin (killer toxin) produced by W. mrakii. These findings have implications for controlling mixed yeast alcoholic beverage fermentations.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

Optimisation of the combined treatments of aqueous chlorine dioxide,fumaric acid and ultraviolet‐C for improving the microbial quality and maintaining sensory quality of common buckwheat sprout

The effects of the combined treatments with aqueous chlorine dioxide (ClO₂), fumaric acid and ultraviolet‐C (UV‐C) on the microbial quality of common buckwheat sprouts were examined using a response surface methodology. The populations of total aerobic bacteria, yeast and mould, and coliform decreased with increasing aqueous ClO₂ and fumaric acid concentrations and increasing UV‐C irradiation dose. However, the increase in the UV‐C irradiation dose had a negative effect on the sensory quality. Therefore, the optimal combined treatment condition of 100 ppm aqueous ClO₂, 0.31% fumaric acid and 1.9 kJ m^?2 UV‐C was selected for the buckwheat sprouts by providing reductions of 3.9, 1.8 and 2.4 log CFU g^?1 on the populations of total aerobic bacteria, yeast and mould, and coliform, respectively. The combined treatment also maintained an acceptable sensory quality. These results suggest that the optimised combined treatment can be used as a microbial inactivation method for buckwheat sprouts.     

Effect of ultraviolet‐B irradiation on antioxidative properties of aqueous extracts from shiitake (Lentinus edodes) mushrooms

Antioxidant properties of aqueous extracts from Lentinus edodes treated with UV‐B irradiation were examined in vitro systems including DPPH, ABTS and oxygen radical absorbance capacity (ORAC) assays and in riboflavin‐photosensitised oil‐in‐water (O/W) emulsions. Changes in total phenolics, total flavonoids and vitamin C were also analysed. Lentinus edodes receiving 25 kJ m^?2 UV‐B treatment showed high radical scavenging ability based on DPPH and ABTS assays compared with samples with 0, 50 and 75 kJ m^?2, while those with 50 kJ m^?2 had higher antioxidant capacities than other samples from ORAC assays. Samples with 25 kJ m^?2 UV treatment had significantly 7.1% higher total phenolic content, 12.0% higher total flavonoid content and 8.0% higher vitamin C content than UV‐B‐untreated sample (P < 0.05), respectively. In O/W emulsions under riboflavin photosensitisation, 25 and 50 kJ m^?2 UV treatment significantly increased the oxidative stability compared with other samples based on headspace oxygen content and lipid hydroperoxide analyses (P < 0.05). Aqueous extracts of UV‐B‐treated mushrooms possessed enhanced antioxidant properties compared with untreated mushrooms.     

Antimicrobial activities of high molecular weight water‐soluble chitosans against selected gram‐negative and gram‐positive foodborne pathogens

Antimicrobial activities of high molecular weight water‐soluble chitosans (HMWWS) against selected Gram‐negative and Gram‐positive foodborne pathogens (initial inoculation of ca. 6.5 Log CFU mL^?1) were evaluated. Chitosans with 789 kDa and/or 1017 kDa were dissolved in aspartic acid (AS) to obtain 1–4% w/v solutions. Among HMWWS, only 4% 789 kDa AS chitosan reduced E. coli counts by 2 Log CFU mL^?1 from 7.33 at 0 h to 5.16 Log CFU mL^?1 at 96 h, and they were not effective against S. Typhimurium. Depending on the concentrations, HMWWS completely inhibited V. cholerae, V. vulnificus and V. parahaemolyticus as well as B. cereus and L. monocytogenes after 48 h or 96 h of incubation. Compared with the control (no HMWWS), 2% or 3% 1017 kDa AS chitosans showed about 3 Log CFU mL^?1 lower (4.72–4.86 vs. 7.71) for S. aureus at 96 h of incubation.