Detection and characterization of the Pichia manshurica biofilm on the traditionally produced homemade apple vinegar.

Pichia yeasts are capable of forming biofilms during vinegar production and causing spoilage in various beverages. In addition, there exists a significant likelihood of encountering yeast contamination which can prevent vinegar production. The present study investigates the detection and characterization of the Pichia manshurica (P. manshurica) biofilm on traditionally produced homemade apple vinegar. The unique characteristics of vinegar were analyzed with a focus on the constituent, known as the "mother of vinegar", whose composition is comprised of cellulosic biofilm and acetic acid bacteria, including Gluconobacter oxydans (G. oxydans) Briefly, P. manshurica was isolated from apple vinegar and characterized in terms of the effect of biofilm formation on the surface of the cellulosic film on vinegar production. Microbial identification of vinegar with/without contamination by P. manshurica was analyzed through MALDI-TOF mass spectrometry (MS), and biofilm was characterized by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and crystal violet staining. Accordingly, MS spectrum of isolates was identified as G. oxydans and P. manshurica with a ratio of 2.01 and 1.94, respectively. The FTIR analysis indicated that the peaks within the range of 1150-900 cm-1 revealed a high content of polysaccharide in P. manchuria-contaminated biofilm, which is attributed to the stretching vibration of C-C and C-O bonds. The spectral region from 2921.51 to 2853.71 cm-1 exhibited the characteristic of lipids in bacterial cell walls and membranes. SEM images of bacterial biofilms revealed a three-dimensional network composed of ultrafine fibers with a ribbon-like shape; however, the condensed reticulated structure was observed in contaminated biofilms. The presence of two microbial populations was detected regarding the morphological analysis. Crystal violet staining of contaminated-cellulosic biofilms visualized bacterial and yeast colonization. Concisely, this study emphasizes that the proliferation of Pichia during apple fermentation has the potential to adversely affect the quality of the homemade vinegar, due to its distinct biofilm characteristics.

Fate of Spoilage and Pathogenic Microorganisms in Acidified Cold-Filled Hot Pepper Sauces.

Consumption of spicy foods and hot sauces is currently a popular trend worldwide. Shelf-stable acidified sauces are commonly hot-filled to ensure commercial sterility, but cold-fill-hold processes might also be suitable if microbial safety and stability are ensured. For this study, model acidified hot pepper sauces were developed and characterized. The effects of sauce pH and of two different organic acids on the survival of Pichia manshurica and Lactobacillus curvatus isolated from contaminated commercial hot sauces and on pathogenic Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes were assessed. Full factorial designs with three levels for pH (3.2, 3.5, and 3.9) and two for organic acid (citric and acetic) were used to determine the effects of these factors and their interactions on the survival of the microorganisms. Commercially sterile sauces were independently inoculated and kept at ambient temperature. Microbial counts were determined at different sampling times, depending on the treatment evaluated. Sauces acidified to pH 3.2 with citric or acetic acid were inoculated with cocktails of five strains or serotypes of the three pertinent pathogens, and inactivation curves were determined. Trials were performed in triplicate. A greater than 5-log reduction of P. manshurica and L. curvatus was achieved in less than 6 h in sauces adjusted to pH 3.2 with acetic acid. Greater than 5-log reductions of pathogenic bacteria were achieved 0.5 h after inoculation in sauces acidified to pH 3.2 with acetic acid. In contrast, at least 48 h was required to guarantee the same inactivation for the most tolerant pathogen when citric acid was used. Thus, a cold-fill-hold process may be a suitable alternative for acidified hot pepper sauces. Based on survival of the microorganisms evaluated in this study, microbial safety and stability can be achieved by adjusting the pH to 3.2 or less by the addition of acetic acid.

Culture-based analysis of fungi in leaves after the primary and secondary fermentation processes during Ishizuchi-kurocha production and lactate assimilation of P. kudriavzevii.

Ishizuchi-kurocha is a Japanese traditional fermented tea that is produced by primary aerobic and secondary fermentation steps. The secondary fermentation step of Ishizuchi-kurocha is mainly mediated through lactic acid bacteria. Here, we performed quantitative analyses of the culturable fungal communities at each step and identified several morphologically representative fungal isolates. While filamentous fungi (median, 3.2 × 107 CFU/g sample) and yeasts (median, 3.7 × 107 CFU/g) were both detected after the primary fermentation step, only yeasts (median, 1.6 × 107 CFU/g) were detected in the end of the secondary fermentation step, suggesting that the fungal community in tea leaves are dramatically changed between the two steps. Pichia kudriavzevii and Pichia manshurica, the prevalent fungal species at the end of the secondary fermentation step, grew well in exudate from the secondary fermentation step. P. kudriavzevii also grew well in media containing d- or l-lactate as the sole carbon source. The growth of the disruptant of cyb2A encoding a cytochrome b2 lactate dehydrogenase in P. kudriavzevii was severely impaired on medium supplemented with l-lactate, but not d-lactate, suggesting that Cyb2Ap plays a crucial role in the use of l-lactate, and P. kudriavzevii efficiently uses both l- and d-lactate as carbon sources. Thus, lactate assimilation seems to be a key phenotype to become a prevalent species in the secondary fermentation step, and Cyb2Ap has a pivotal role in l-lactate metabolism in P. kudriavzevii. Further understanding and engineering of P. kudriavzevii and P. manshurica will contribute to the control of lactic acid bacteria fermentation during the fermented tea production and also to other industrial uses.

Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon

The purpose of this study was to identify the yeasts involved in spontaneous fermentation of cocoa from the Brazilian Amazon region. The fermentation process was carried out experimentally with cocoa seeds from two sites (Medicilândia and Tucumã), State of Pará, northern Brazil, during a six-day period. Totals of 44 yeasts were isolated from Medicilândia and 29 from Tucumã. Molecular identification was carried out by sequencing the D1/D2 region fragment of the rRNA 26S gene, expanded with universal primers for the NL1GC and LS2 eukaryotes. Pichia manshurica and Saccharomyces cerevisiae were identified in Medicilândia and five yeast species (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae and Zygosaccharomyces bailii) were identified in Tucumã. The results showed that P. manshurica and S. cerevisiae may have potential for use as starter cultures in future studies to improve the quality of cocoa seeds fermented in the Brazilian Amazon region. (AU)

A proposta deste estudo foi identificar as leveduras envolvidas na fermentação espontânea de cacau da Amazônia brasileira. A fermentação foi realizada em Medicilândia e Tucumã, Pará, Brasil, durante 6 dias. Em total foram obtidos 44 isolados de leveduras de Medicilândia e 29 de Tucumã. A identificação molecular foi realizada por sequenciamento do fragmento da região D1/D2 do gene rRNA 26S, amplificado com primers universais para eucariotos NL1GC e LS2. Em Medicilândia, foram identificadas Pichia manshurica e Saccharomyces cerevisiae. Em Tucumã foram identificadas cinco espécies (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae e Zygosaccharomyces bailii). Os resultados sugerem que P. manshurica e S. cerevisiae podem ter potencial para uso como culturas starter em estudos futuros, para melhorar a qualidade das sementes de cacau fermentadas na Amazônia brasileira.(AU)