Pichia Manshurica Research Articles

Effects of Pichia manshurica yeast supplementation on ruminal fermentation, nutrient degradability, and greenhouse gas emissions in aflatoxin B1 contaminated diets

Yeast feed additives present a natural approach for mitigating ruminal greenhouse gases (GHG) in an environmentally sustainable manner. This study aimed to isolate yeast strains from ruminal fluids capable of reducing GHG from Aflatoxin (AFB1) contaminated diets. Two isolates of Pichia manchuria (FFNLYFC1 and FFNLYFC2) were isolated and identified from the ruminal contents of dairy Zaraibi goats. An in vitro gas production assay was conducted to evaluate the impact of the yeast supplementations on a basal diet contaminated with AFB1 or not. The treatments were control (-AFB1; basal diet without supplements), control with AFB1 contamination (+ AFB1; basal diet containing 20 ppb AFB1), and yeast-supplemented diets (basal diet supplemented with Saccharomyces cerevisiae, and three treatments of P. manchuria [FFNLYFC1, FFNLYFC2, and their mixture at 1:1 ratio (Mix)]. High biological components were detected in abundance of both FFNLYFC1, FFNLYFC2 filtrates (e.g., diisooctyl phthalate). The Mix and FFNLYFC2 of P. manchuria reduced (P < 0.05) methane by 23.5 and 20.8%, respectively, while only Mix inhibited carbon dioxide by 44% compared to the + AFB1 diet. All yeast diets improved (P < 0.05) ammonia concentration, total protozoal and Entodinium spp. counts compared to + AFB1 diet. The Mix exhibited higher (P < 0.05) values of ruminal degraded cellulose, total short-chain fatty acids, acetate and propionate compared to the individual isolates diets. The results suggest synergistic interactions among P. manshurica isolates, leading to enhanced ruminal fermentation and reduced GHG emissions while alleviating the adverse effects of AFB1. Therefore, we recommended the Mix of P. Manchuria as a novel feed additive to ruminant diets.

Effect of carvacrol on the inhibitory of gas-producing spoilage microbes and volatile flavor in fermented pepper (Capsicum annuum L.)

In this study, two gas-producing spoilage microbes, namely CQ17 and CQ22, were isolated from fermented pepper. Combining the physiological and biochemical characteristics, molecular biological identification, and specific colony morphology, CQ17 was identified as Pichia manshurica while CQ22 was identified as Lentilactobacillus buchneri. Subsequently, our findings indicated that the CQ17 and CQ22 strains exhibited resistance to high temperature, low-pH, and high salinity environments. Meanwhile, our findings revealed that carvacrol, a commonly utilized preservative, exhibited remarkable inhibitory activities against Pichia manshurica and Lentilactobacillus buchneri with the EC50 values of 0.28 and 0.68 mg/L respectively, effectively suppressing the growth of gas-producing strains to prolong the shelf life of fermented pepper. Simultaneously, volatile components of the fermented pepper were analyzed using HS-GC-IMS technology resulting in identification of 55 volatile flavor compounds including acids, alcohols, esters, aldehydes, ketones, alkenes, and other compounds. Among them, (R/S)-linalool, 3-methyl-1-butanol acetate butanoic acid ethyl ester, 2-methyl butanoic acid ethyl ester, and ethyl 2-methylpropionate were identified as crucial components contributing to sensory attributes in fermented pepper as well as (E)-3-hexen-1-ol and l-Pentanol exhibited the highest VIP values and can be considered as the best reliable markers for differentiating fermented pepper samples with the addition of carvacrol as a preservative.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Diversity of Culturable Yeasts Associated with the Technification Level in the Process of Mezcal Production in the State of Durango

Durango State has the denomination of origin for the production of mezcal, which is made from Agave durangensis, mainly in an artisanal way; therefore, differences in the fermentation process affect the quality of the final product. The main objective of the present study was to evaluate the diversity of culturable yeasts involved in the artisanal and semi-technified process of mezcal production in the State of Durango. Three distilleries with different production processes were monitored at different fermentation stages (beginning, mid-fermentation, and end of fermentation) in the spring and summer seasons. A greater diversity was found in the distillery of Nombre de Dios in both the spring and summer production seasons (H’ = 1.464 and 1.332, respectively), since it maintains an artisanal production process. In contrast, the distillery of Durango, where a Saccharomyces cerevisiae commercial inoculum is used to start fermentation, presented low diversity indexes (H’ = 0.7903 and 0.6442) and only S. cerevisiae, Kluyveromyces marxianus, and, sporadically, Pichia manshurica were found. Results suggest that the yeast microbiota involved in mezcal fermentation during the different seasons is affected by the type of inoculum; changes include the presence of some species that were only identified during a specific season in alcoholic fermentation, such as Torulaspora delbrueckii and Pichia kluyveri.

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.

Impact of single and mixed cultures of yeasts and lactic acid bacteria in coffee fermentation and cup quality

AbstractSaccharomyces cerevisiae LBTF 21.3, Pichia manshurica LBTF 21.1, and Leuconostoc lactis LBTF 21.1 were used as single and mixed cultures in coffee fermentations. Fermentations carried out in glass jars with P. manshurica LBTF 21.1/Leuc. lactis LBTF 21.1 showed higher fermentation rates than S. cerevisiae LBTF 21.3/P. manshurica LBTF 21.1 or S. cerevisiae LBTF 21.3/Leuc. lactis LBTF 21.1. The highest cup quality was reached with P. manshurica LBTF 21.1/Leuc. lactis LBTF 21.1 (83.0 points). Full factorial design (22) carried out with P. manshurica LBTF 21.1 revealed that the higher proportion of water added before the fermentation resulted in higher production of acetic acid. A contrary effect of this variable was observed on the production of lactic acid with Leuc. lactis LBTF 21.1. Fermentations conducted in bioreactor evidenced the production of acetaldehyde by P. manshurica LBTF 21.1/Leuc. lactis LBTF 21.1, P. manshurica LBTF 21.1, and Leuc. lactis LBTF 21.1. Additionally, the highest cup quality was reached in fermentations with P. manshurica LBTF 21.1 (82.5 points) followed by Leuc. lactis LBTF 21.1 (81.5 points) and P. manshurica LBTF 21.1/Leuc. lactis LBTF 21.1 (81.0 points). Fermentations of coffee with single and mixed cultures are interesting strategies for the improvement of the sensory quality.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Effects of film-forming Pichia and Candida yeasts on cider and wine as post-fermentation contaminants.

Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming yeasts in cider and wine and the effects on their chemical composition. Cider, partially and fully fermented wine were inoculated with strains of C. californica, P. fermentans, P. kluyveri, P. kudriavzevii, P. manshurica, and P. membranifaciens to simulate a post-fermentative contamination. The former three species grew only in cider. Pichia manshurica and P. kudriavzevii displayed high viability in wine up to 13.18% (v v-1) ethanol. Significant changes in odour-active molecules from different chemical groups were observed in cider and wine in the inoculated samples, compared to the non-inoculated ones. Cider is more susceptible to contamination by all of the species tested, due to its low alcohol content, while P. membranifaciens, P. manshurica, and P. kudriavzevii are additionally potential spoilage agents of wine. This study highlights the risk of cider and wine contamination by film-forming yeasts. Their impact on aroma profiles depends on their ability to grow and their metabolism. This study contributes to an understanding of the possible physiological and metabolic mechanisms responsible for film formation and chemical changes in alcoholic beverages.

The Potential of Cellulolytic Yeast Pichia manshurica UNJCC Y-123, Saccharomyces cerevisiae UNCC Y-84, and Saccharomyces cerevisiae UNJCC Y-83 to Produce Cellulase Enzyme by Using Substrate Skin Delignification of Cocoa (Theobroma cocoa)

Exploring the possibility of using agricultural waste as a substrate for the synthesis of cellulase enzymes for fuel and renewable energy is the main goal of this study, which is in line with the notions of sustainable development and environmental stewardship. The product of delignification of cocoa peel can be used as a substrate for cellulase enzymes producing by yeast isolated from Balinese palm wine. Cellulase enzymes made from yeast from Balinese palm wine can be produced using the delignified cocoa peel as a substrate. This study aims to analyze the cellulase enzyme activity of yeast from Bali's palm wine on delignified cocoa bark substrates. The tests carried out were delignification of cocoa bark (solvent variations: HCl, H2SO4 and NaOH; concentrations: 1, 1.5 and 2 %; biomass 1:15 and 1:20 (w/v)); screening of cellulolytic yeast from Balinese palm wine; molecular identification and morphological characteristics of yeast; and cellulase enzyme activity (variation of yeast isolates and fermentation time of 48, 72 and 96 h). The results of delignification of cocoa shells showed that the use of 1.5 % NaOH solvent with cocoa shell powder biomass of 1:15 (9.59 ± 0.11) significantly differed from the value of reducing sugar content. Based on the screening results of 6 yeast isolates, 3 yeast isolates with the highest cellulolytic index values were selected, namely UNJCC Y-83 (0.29 ± 0.01 mm), UNJCC Y-123 (0.24 ± 0.01 mm) and UNJCC Y-84 (0.23 ± 0.01 mm). For testing cellulase enzyme activity. S. cerevisiae UNJCC Y-83 (4.11 ± 0.41 U/mL); S. cerevisiae UNJCC Y-84 (4.11 ± 0.33 U/mL) and P. manshurica UNJCC Y-123 (4.06 ± 0.12 U/mL) at 96 h of fermentation had significantly different cellulase enzyme activity. The results of identification of yeast rDNA in the D1/D2 region with NL1/NL4 primers obtained the identity of Saccharomyces cerevisiae UNJCC Y-83; Saccharomyces cerevisiae UNJCC Y-84 (99.66 % homology); and Pichia manshurica (100 % homology). Theobroma cocoa rind can be used as a yeast substrate to produce cellulase enzymes for fuel and renewable energy. HIGHLIGHTS Screening of 6 yeast isolates, 3 yeast isolates with the highest cellulolytic index values were selected, namely UNJCC Y-83 (0.29 ± 0.01 mm), UNJCC Y-123 (0.24 ± 0.01 mm) and UNJCC Y-84 (0.23 ± 0.01 mm) for testing molecular identification and cellulase enzyme activity The result of rDNA sequence analysis in the D1/D2 region showed that the yeast isolates UNJCC Y-83 and UNJCC Y-84 had a homology level of 99.66 % as Saccharomyces cerevisiae with a bootstrap value of 92 %. UNJCC Y-123 yeast isolate has a homology level of 100 % as Pichia manshurica with a bootstrap value of 100 % cerevisiae UNJCC Y-83 (4.11 ± 0.41 U/mL); S. cerevisiae UNJCC Y-84 (4.11 ± 0.33 U/mL); and P. manshurica UNJCC Y-123 (4.06 ± 0.12 U/mL) at 96 h of fermentation had significantly different cellulase enzyme activity Theobroma cocoa rind can be used as a yeast substrate to produce cellulase enzymes for fuel and renewable energy GRAPHICAL ABSTRACT

Effective degradation of azo dyes and colorimetric detection of Hg2+ by biogenic silver nanoparticles of Pichia manshurica

A novel biogenic silver nanoparticle production has been done using a yeast Pichia manshurica, strain CD1 cell resuspension exudates. The characterization of AgNPs by DLS, Zeta Potential, FTIR, XRD, SEM, and TEM was done to obtain spherical particle morphology with 22 nM diameter. Biocatalysis of CD1-NPs was done to obtain the catalytic degradation of azo dyes, Congo red, and Malachite green. Degradation occurred within 30 min for Congo red and 40 min for Malachite green with degradation efficiency of 98.86 % and 97.54 % respectively. High reusability (approximately 94 %) was observed of CD1-NPs up to 5 recycles. CD1-NPs were further tested for selectivity and sensitivity towards Hg2+ by colorimetric detection with a limit of 21.04 nM in the linear range of 10–80 nM. The nanosensor has been tested for Hg2+ ions in mercury-treated tap water samples. The efficiency of the biocatalysts was accessed for practical applicability.

Identification and evaluation of thermotolerance of yeasts from milk in natura exposed to high temperature and slow and fast pasteurization.

Milk is considered one of the basic raw materials of animal origin; it must present hygienic quality and physical-chemical properties suitable for processing and human consumption. Thus, the ingestion of milk in natura when not properly treated can be characterized as an opportunistic route of transmission of possible microbial pathogens, which can offer risks to public health. The present study aimed the yeast identification, to analyze the thermo-resistance of yeasts isolated from fresh milk, and to trace the susceptibility profile of the isolates to antifungal agents. For this, 23 samples of fresh milk type B, collected by manual or mechanical milking, were stored in collective refrigeration tanks of farms located in the Metropolitan Region of Natal and nearby, State of Rio Grande do Norte (RN), Brazil. Twenty samples of fresh milk commercially traded in the city of Ceará-Mirim RN were also analyzed. The yeasts were quantified by count of colony-forming units (CFU). All isolated species were treated by slow pasteurization (62-64°C for 30min) and fast (72-75°C for 20s), as well as by boiling (100°C). Fifty yeast strains were obtained, and the species were identified as Candida tropicalis (28%), Candida parapsilosis (14%), Candida albicans (12%), Candida glabrata (10%), Candida krusei (10%), Kluyveromyces marxianus (10%), Candida guilliermondii (8%), Candida rugosa (2%), Candida orthopsilosis (2%), Pichia manshurica (2%), and Kodamaea ohmeri (2%). Five isolates showed resistance to the antifungal agents tested. Among all the isolates submitted to heat treatment, 80% were resistant to fast pasteurization and 60% to boiling, but none of them resisted the slow pasteurization. The milk collected through mechanical milking and stored in collective cooling tanks, presented higher rates of yeast contamination, compared to milk samples collected by manual milking and kept under the same storage conditions.

Antibiofilm mechanism of dielectric barrier discharge cold plasma against Pichia manshurica

Biofilm spoilage has become one of the most common concerns in fermented foods. The objective of the present study was to explore the biofilm-inhibitory effect and antibiofilm mechanism of dielectric barrier discharge (DBD) cold plasma on Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods). After plasma treatment (80 kV, 50 Hz) for 4.5 min and 7.5 min, viable counts decreased by 2.38 and 5.11 lg CFU/mL, respectively, and the biofilm-forming rate decreased to 73% and 48% of the control group, respectively, in microbiological media. In addition, DBD caused severe morphological damage, cell membrane permeabilization and metabolic changes. A number of amino acid metabolic pathways, the tricarboxylic acid cycle (TCA), and the synthesis of extracellular polymeric substance (EPS) were destroyed. The cell damage and changes in multiple metabolic activities led to significant inhibitory effects on Pichia manshurica and its biofilm-forming ability. Industrial relevanceThis study found that DBD can significantly inhibit the biofilm-forming ability of Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods) by causing severe cell damage and metabolic changes. The exploration of the biofilm inhibitory effect of DBD on Pichia manshurica and its mechanism can provide a theoretical basis for the application of DBD technology in the inhibition of biofilm spoilage in foods.

Three new yeast species from flowers of Camellia sinensis var. assamica collected in Northern Thailand and their tannin tolerance characterization.

Our recent research study focused on Miang fermentation revealed that tannin-tolerant yeasts and bacteria play vital roles in the Miang production process. A high proportion of yeast species are associated with plants, insects, or both, and nectar is one of the unexplored sources of yeast biodiversity. Therefore, this study aimed to isolate and identify yeasts of tea flowers of Camellia sinensis var. assamica and to investigate their tannin tolerance, which is a property essential to Miang production processes. A total of 82 yeasts were recovered from a total of 53 flower samples in Northern Thailand. It was found that two and eight yeast strains were distinct from all other known species within the genera Metschnikowia and Wickerhamiella, respectively. These yeast strains were described as three new species, namely, Metschnikowia lannaensis, Wickerhamiella camelliae, and W. thailandensis. The identification of these species was based on phenotypic (morphological, biochemical, and physiological characteristics) and phylogenetic analyses of a combination of the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) ribosomal RNA gene. The yeast diversity in tea flowers acquired from Chiang Mai, Lampang, and Nan provinces had a positive correlation with those acquired from Phayao, Chiang Rai, and Phrae, respectively. Wickerhamiella azyma, Candida leandrae, and W. thailandensis were the species uniquely found in tea flowers collected from Nan and Phrae, Chiang Mai, and Lampang provinces, respectively. Some of the tannin-tolerant and/or tannase-producing yeasts were associated with yeasts in the commercial Miang process and those found during Miang production, i.e., C. tropicalis, Hyphopichia burtonii, Meyerozyma caribbica, Pichia manshurica, C. orthopsilosis, Cyberlindnera fabianii, Hanseniaspora uvarum, and Wickerhamomyces anomalus. In conclusion, these studies suggest that floral nectar could support the formation of yeast communities that are beneficial for Miang production.

Characterization of selected species of Pichia and Candida for their growth capacity in apple and grape must and their biofilm parameters.

Pichia and Candida species include biofilm-forming yeasts able to spoil foods and beverages. Strains belonging to 10 Pichia and Candida species isolated from apples, grape musts, and wines were analysed. They were subjected to molecular typing and characterized for their ability to grow and ferment must for cider and wine production, and for their biofilm properties. All strains grew similarly in apple and grape must. Glucose-fermenting strains displayed differentiated fermentation performances. Great variation in SO2 and ethanol sensitivity was observed among the strains. Pichia manshurica strains showed high tolerance to both molecules. Eleven and five surface-spreading biofilm (MAT) phenotypes were identified in solid and liquid media, respectively. Strains produced biofilms with variable thicknesses and widths in culture tubes. Cell adherence and aqueous-hydrocarbon biphasic hydrophobicity assays were carried out. Some Pichia manshurica and P. membranifaciens strains exhibited a high capacity to form a thick biofilm and had high cell adherence and hydrophobicity values. These strains could be more likely to colonize the internal surfaces of tanks. This study evidenced that some Pichia and Candida strains can proliferate during apple and grape must fermentation and may be detrimental the beverage quality, due to their specific biofilm properties.

Laser-assisted bioprinting of microorganisms with hydrogel microdroplets: peculiarities of Ascomycota and Basidiomycota yeast transfer.

Laser-assisted bioprinting of microbial cells by hydrogel microdroplets is a rapidly developing and promising field that can contribute to solving a number of issues in microbiology and biotechnology. To date, most research on the use of laser bioprinting for microorganism manipulation and sorting has focused on prokaryotes; the bioprinting of eukaryotic microorganisms is much less understood. The use of hydrogel allows solving two fundamental problems: creating comfortable environments for living microorganisms and imparting the necessary rheological properties of the gel for the stable transfer of microdroplets of a preset size. Two main problems were solved in this article. First, the parameters of the hydrogel based on hyaluronic acid and laser fluence to ensure stable transfer of single drops are selected. Second, possible differences in the bioprinting by hyaluronic acid hydrogel microdroplets with yeasts of various taxonomy (Ascomycota vs Basidiomycota), which form and do not form polysaccharide capsules and evaluated. We have performed laser induced forward transfer of 8 yeast species (Goffeauzyma gilvescens, Lipomyces lipofer, Lipomyces starkey, Pichia manshurica, Saitozyma podzolica, Schwanniomyces occidentalis var. occidentalis, Sterigmatosporidium polymorphum, Vanrija humicola) and assessed its viability based on colony formation on the nutrient medium. It is shown that after laser-induced transfer in hydrogel microdroplets the mean viability rate was 77% with some strains showing relatively high viability rates exceeding 90%. Effect of capsules presence on colony formation after laser bioprinting was not revealed. Differences in laser transfer of the yeast of various phyla were found-basidiomycetes formed a greater number of colonies than ascomycetes. The causes and mechanisms of these effects require detailed studies. The data obtained contributes to the knowledge about the bioprinting of eukaryotic microorganisms and can be useful in the studies of single microbial cells and inter-organism interactions.

Prevention of microbes-induced spoilage in sodium chloride-free cucumber fermentations employing preservatives.

This study evaluated preservatives to stabilize sodium chloride (NaCl)-free-cucumber fermentations. The brining of air-purged laboratory cucumber fermentations with 100.0 mM calcium chloride (CaCl2 ) and 25.0 mM acetic acid resulted in immediate rises in pH, the chemical reduction of the medium, and malodors. Supplementation with 3.0 mM sodium benzoate or 3.0 mM potassium sorbate enabled a decline in pH, a continuous oxidative state of the medium, and delayed rising pH spoilage. However, lactic and acetic acids eventually disappeared in fermentations supplemented with preservatives. The amount of preservatives needed to suppress growth of brined-cucumber-spoilage microbes was determined in Fermented Cucumber Juice Medium (FCJM). Supplementation of FCJM with 10.0 mM sodium benzoate was inhibitory for the spoilage yeasts, Issatchenkia occidentalis and Pichia manshurica, and the lactobacilli, Lentilactobacillus buchneri and Lentilactobacillus parafarraginis, but not of Zygosaccharomyces globiformis. Potassium sorbate inhibited the spoilage yeasts at 15.0 mM in FCJM but not the lactobacilli. Supplementation of FCJM with 20.0 mM fumaric acid had a bactericidal effect on the spoilage-associated lactobacilli. As expected, NaCl-free-commercial cucumber fermentations brined with 100 mM CaCl2 , no acetic acid, and 6 mM potassium sorbate resulted in complete fermentations, but supported rising pH, microbially induced spoilage during long-term storage. Post-fermentation supplementation with 12 mM sodium benzoate, 10 mM fumaric acid, a combination of the two, or 10 mM fumaric acid and 2 mM AITC prevented microbial activity during long-term bulk storage. PRACTICAL APPLICATION: Several preservative-based strategies for stabilizing NaCl-free cucumber fermentation in a commercial production setting were developed, enabling the implementation of a processing technology that reduces wastewater volumes and environmental impact.

Non-Saccharomyces Are Also Forming the Veil of Flor in Sherry Wines

Biological ageing is an essential process for obtaining some distinctive Sherry wines, such as Fino and Manzanilla. It occurs after the fermentation of the grape must due to the appearance of a biofilm on the surface of the wine called “veil of flor”. Yeasts belonging to the Saccharomyces cerevisiae species mainly comprise such biofilm. Although other species have also been found, these have been traditionally considered spoilage. Indeed, it has even been hypothesised that they may not be able to form biofilm on their own under such conditions. In the present work, four different non-Saccharomyces yeasts isolated from barrels in the Jerez area under biological ageing have been characterised through their physiological abilities, including extracellular enzymatic and biofilm-forming capabilities. Results showed not only a surprising ethanol tolerance, above 15.5% in all cases, but also a significant degree of extracellular enzyme production, highlighting the urease and proteolytic activities found in Pichia manshurica, as well as lipolytic activity in Pichia kudriavzevii, Pichia membranifaciens and Wicherhamomyces anomalus. As a conclusion, these non-Saccharomyces could be very interesting in the oenological field, beyond improving the organoleptic characteristics as well as technological features in these wines.

Saccharomyces cerevisiae and Pichia manshurica from Amazonian biome affect the parameters of quality and aromatic profile of fermented and dried cocoa beans.

The use of yeasts as starter cultures is a promising alternative to produce fermented cacao with particular characteristics regarding the quality of aromas and physical and chemical characteristics that are accepted by the chocolate market. This study aimed to evaluate the physical and chemical transformations of cocoa beans during fermentation after inoculation with starter cultures of yeast species Pichia manshurica (PF) and Saccharomyces cerevisiae (SF), both previously isolated in cocoa bean fermentations in the Brazilian Amazon, in comparison with a fermentation without the inoculum addition (CF). During the fermentation time, which was carried out on a cocoa farm in Igarapé-Miri (Amazon biome, Pará, Brazil), the contents of phenolic compounds (catechin and epicatechin), sugars (glucose, fructose, and sucrose), acetic acid, and ethanol were monitored by HPLC, and the volatile compounds profiles were assessed by GC-MS. The starter culture of P. manshurica was able to produce fermented cocoa beans with highly desirable characteristics for the production of good quality chocolate: low acidity, a broad variety of aromatic compounds with floral, fruity, and sweet characteristics, in addition to showing high contents of catechin and epicatechin, which are known by their antioxidant properties. Therefore, the use of starter cultures with species of yeasts isolated in the Amazon region, during cocoa fermentation, is an alternative to obtain fermented seeds with high quality favoring the commercial agreements in the chocolate market by cocoa producers. PRACTICAL APPLICATION: The addition of starter cultures was able to produce cocoa beans with good quality. Yeasts species isolated and identified in Amazonian cocoa fermentation can improve the profiles of aromatic compounds. Catechin and epicatechin contents are higher in inoculated cocoa beans fermentations.

Exploration, screening and identification of indigenous yeast from some palm juices for bioethanol production

Abstract. Widyaningrum T, Febrianti N, Prastowo I, Saifuddin MF, Permadi A. 2022. Exploration, screening and identification of indigenous yeast from some palm juices for bioethanol production. Biodiversitas 23: 3984-3990. The major energy sources usually employed are originated mainly from fossil which can run out. Therefore, the creation of ecologically beneficial and long-lasting forms of alternative energy sources, such as bioethanol, is an absolutely necessary. This study aimed to explore, screen and to identify indigenous yeasts from some palm juices for bioethanol production. The isolates were screened in coconut water media for the degree of bioethanol production, the content of reducing sugar based on a DNS method, and the cell number based on optical density (600 nm). Furthermore, identification was performed using ITS and candidates of the highest bioethanol-producing ability including A3A, A11E from Arenga pinnata, K1C1, K1A, K2C from Cocos nucifera L., N3E, N3D, N1A from Nypa fruticans, S1A, and S2D from B. flabellifer L were obtained. The respective bioethanol contents were 13.4%, 12.8%, 13%, 16%, 14.2%, 13.2%, 12%, 12.6%, 14.4%, and 13.35%. The results showed that the isolates of A3A and N3E were similar to those of Pichia deserticola CBS 7119T. Meanwhile, the isolates of K1A and S1A were similar to that of Pichia manshurica CBS 240T and Candida tropicalis ZA 021T.

Exploring microbial communities of Spanish-style green table olives of Conservolea and Halkidiki cultivars during modified atmosphere packaging in multi-layered pouches through culture-dependent techniques and metataxonomic analysis

Τhe aim of this study was to investigate the microbiological and physicochemical changes of pitted green olives (cvs. Conservolea and Halkidiki) elaborated in the Spanish style method and packaged in two types of multi-layered pouches under nitrogen atmosphere (100% N2) for a period of 12 months. Moreover, the evolution of microbial consortia at the beginning (0 days), middle (180 days) and final (360 days) time points of storage was elucidated by plating, genotyped and identified through RAPD-PCR and ITS region amplicon sequencing, respectively, and subsequently metataxonomic analysis (for fungal communities only). Results showed that no enterobacteria could be detected on olive drupes, whereas the dominant microbiota from the onset of storage in both pouches consisted of LAB in populations ranging between ca. 4.2–6.6 log CFU/g. Although yeasts were initially enumerated at ca. 5.5 log CFU/g, they declined rapidly and could not be detected by plate counting after 30 days. The pH values increased from 4.11 to 4.24 and 4.03 to 4.12 at the beginning and end of storage for cvs. Halkidiki and Conservolea green olives, respectively. The total color difference index (ΔΕ*) presented a perceivable change in visual color (ΔΕ* > 2.0) only in cv. Conservolea olives during storage. Finally, molecular fingerprinting RAPD-PCR and 16S amplicon based identification revealed the dominance of five LAB species, namely Pediococcus ethanolidurans, Lactiplantibacillus pentosus, Lentilactobacillus rapi, Lentilactobacillus parafarraginis and Lentilactobacillus buchneri. Regarding the identification of yeasts, the metagenetic amplicon sequencing approach revealed fungal complexity in the olive samples. Pichia manshurica and Pichia membranifaciens prevailed during the first and middle stages of storage, whereas at the final stage higher complexity was noticeable.