Influence of nitrogen status on fermentation performances of non-Saccharomyces yeasts:a review

Nitrogen,one of the most crucial nutrients present in grapes and musts,plays a key role in yeast activities during alcoholic fermentation.Such influences are imposed on yeast growth and fermentation performances including the formation of secondary metabolites.Saccharomyces cerevisiae,the main yeast responsible for fermentation,has been studied extensively regarding nitrogen impacts.On the other hand,a similar study for non-Saccharomyces yeasts,whose contributions to winemaking have gradually been acknowledged,remains to be fully explored,with a few studies being reported.This review starts by discussing nitrogen impacts on non-Saccharomyces yeast growth and fermentation kinetics in different case scenarios,then proceeds to summarize the nitrogen preferences of individual yeast strains with regulation mechanisms elucidated by recent studies.Detailed discussions on the influences on the production of volatile compounds and proposed pathways therein are made,followed by future work suggested as the final section.In summarizing the nitrogen impacts on non-Saccharomyces yeasts throughout alcoholic fermentation,this review will be helpful in obtaining a more comprehensive view on these non-conventional wine yeasts in terms of nutrient requirements and corresponding volatile production.Research gaps will therefore be elucidated for future research.

Evaluation and selection of yeasts as potential aroma enhancers for the production of dry-cured ham

Yeasts play a critical role in the flavor formation of dry-cured ham.In this study,41 yeast isolates from the dry-cured ham at different processing stages were evaluated for their technological properties.Debaryomyces hansenii was the most dominant yeast and has been detected at each phase of dry-cured ham,followed by Candida zeylanoides which was mainly detected in salting phase.Yarrowia bubula and Yarrowia alimentaria were found at the first two-phase of dry-cured ham.All isolates of yeast showed enzymatic activities against milk protein and tributyrin,while only 4 strains displayed proteolytic activity on meat protein.Yeast strains were grown in a meat model medium and volatile compounds were identified.The result showed that inoculated yeast strains could promote the production of volatiles and there were significant differences among strains.D.hansenii S25 showed the highest production of volatile compounds,followed by the strain C.zeylanoides C4.D.hansenii S25 was the highest producer of alcohols showing the highest production of benzeneethanol and 3-(methylthio)-1-propanol.Based on OAV and PLS analysis,D.hansenii S25 was strongly correlated with overall flavor and key volatile compounds of dry-cured ham,which could be selected as potential starter cultures.

Marine Yeasts and Their Applications in Mariculture

The terrestrial yeasts have been receiving great attention in science and industry for over one hundred years because they can produce many kinds of bioactive substances. However, little is known about the bioactive substances of marine yeasts. In recent years, it has been found that marine yeasts have wide applications in mariculture and other fields. Therefore,marine yeasts, the bioactive substances from them and the applications of marine yeasts themselves and the bioactive substances they produced are reviewed in this paper.

Occurrence and Diversity of Marine Yeasts in Antarctica Environments

A total of 28 yeast strains were obtained from the sea sediment of Antarctica.According to the results of routine identi-fication and molecular characterization,the strains belonged to species of Yarrowia lipolytica,Debaryomyces hansenii,Rhodotorula slooffiae,Rhodotorula mucilaginosa,Sporidiobolus salmonicolor,Aureobasidium pullulans,Mrakia frigida and Guehomyces pullu-lans,respectively.The Antarctica yeasts have wide potential applications in biotechnology,for some of them can produce b-galactosidase and killer toxins.

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.

Alkaline Protease Production by a Strain of Marine Yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45 ℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min -1. Under the optimal conditions, 623.1 U mg -1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

Sensitivity of Four Yeasts to Fungicides and CO2 Concentrations and Their Antagonistic Ability in Combination with Fungicide to Pathogenic Fungi in vitro

The yeasts Cryptococcus laurentii, Trichosporon pullulans, Rhodotorula glutinis andTrichosporon sp.were used to investigate their sensitivity to four fungicides anddifferent concentrations of CO2, as well as their antagonistic ability to Penicilliumexpansumand Alternariaalternata in vitro when applied with fungicide. There were significantdifferences in sensitivity to the fungicides among the different yeasts(P=0.05). R.glutinis was more sensitive to Deccocil, Iprodione and Stroby as compared to otheryeasts. Combination antagonistic yeasts with fungicide could more significantly enhancebiocontrol ability of the yeasts against the pathogenic fungi in vitro(P = 0.05). C.laurentii was the most effective antagonist among the four yeasts and could completelycontrol spore germination of P.expansum and A.alternata when combined with Stroby at theconcentration of 100 L L-1. The yeasts, except R.glutinis, could grow well on nutrientyeast dextrose agar (NYDA) after 8 d incubation even at 20% CO2 concentration at 25℃.Particularly Trichosporon sp. showeda better adaptability to low temperature as comparedto other antagonists.

The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains （HN3.11, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 was found to have the highest antioxidant activity （61.4%） and the highest angiotensin I converting enzyme （ACE）-inhibitory activities （68.4%）. The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

Biocontrol Efficacy of Two Antagonistic Yeasts Against Postharvest Diseases in Peach Fruits During Storage Periods

Two antagonistic yeasts, Thichosporon pullulans and Cryptococcus laurentii, were investigated for their biocontrol potential to blue mold rot and rhizopus rot on harvested peach fruits(Prunus persica L. Batsch, cv. Okubao), alone or in combination with a low dose of iprodione(50 μg ml-1). The results indicated that T. pullulans and C. laurentii were effective at reducing disease incidence and severity of blue mold rot and rhizopus rot in peach fruits. Biocontrol efficacy of C. laurentii and T. pullulans were significantly enhanced by combination with a low dose of iprodione(50 μg ml-1)against blue mold and rhizopus rot in peach fruits. T. pullulans and C. laurentii combined with a low dose of iprodione(50 μg ml-1)resulted in better disease control than either iprodione or the yeasts used alone. Dipping fruits in suspensions of antagonist cells showed the similar control effect as the treatment with iprodione(500μg ml-1).

Screening Yeasts from Ruminal Fluid of Dairy Heifer Fed a Different Ratio Roughage to Concentrate Diets

The objective of these studies were to identify ruminal yeast in varying ratios of roughage to concentrate in TMR diets in order to explore yeast diversity by using molecular technique with similarity of rDNA sequence. The experiment was assigned to four 98.6% of cross bred Holstein Friesian heifers with 2 levels and two replicates of roughage to concentrate ratios as： 10：90 （T1） and 50：50 （T2）. The experimental period was 14 days. Rumen fluid sample was collected by stomach tube for total DNA extraction by using silica gel method, and analysis of quantity and quality of DNA by Nanovue and agarose gel electrophoresis. The divergent DI/D2 domain of 26S rDNA was amplified by primers NL-1（5＇-GCA TAT CAA TAA GCG GAG GAA AAG-Y） and NL4 （5＇-GGT CCG TGT TTCAAG ACG G-3＇） by polymerase chain reaction （PCR）. The nucleotide sequences of D l/D2 domain of 26S rDNA were determined using PCR products. Generated sequences were aligned with related species by using the CLUSTAL W. The result showed that an average dry matter intake of TI was 7.00 kg/d and T2 was 6.99 kg/d. DNA concentrate from TIRI, TIR2, T2RI and T2R2 were 106, 131.5, 84 and 182.5 ng//aL, respectively. The purity of DNA was 1.57, 1.76, 1.78 and 1.86, respectively. The divergent D1/D2 domain of 26S rDNA of treatment could be amplified for T1R1 and T2R1 but could not for T1R2 and T2R2. The sequences of D1/D2 domain of 26S rDNA were compared with nucleotide database by BLAST programs （http：//www.ncbi.nlrn.nih.gov/BLAST）, the T2RI yeast-strain was closest to Yarrowia lipolytica. However, yeast strain in T1R1 could not be specifically identified because D 1/132 domain of 26S rDNA seem to represent variable region with number of nucleotide sequence showing 2-3 substitution from known species. The phylogenetic tree based on the sequences of the DI/D2 domain of 26S rDNA showed that TIR! was related to Pichia and Candida （96%） and T2R1 was related to Yarrowia lypolytica （100%）. This study indicated that ruminal yeast strains could be found varying in different ratio of roughage to concentrate.

Isolation and Identification of Indigenous Yeasts from “<i>Rabil&eacute;</i>”, a Starter Culture Used for Production of Traditional Beer “<i>dolo</i>”, a Condiment in Burkina Faso

Rabilé is a ferment of dolo, a traditional sorghum beer of Burkina Faso and it is harvested at the end of dolo fermentation process. It is a significant source of proteins and it is used like a condiment and food seasoning by some communities. The present study characterized and identified the yeasts isolated from Rabilé. A total of 70 samples were collected from 7 localities of Burkina Faso with dolo sellers according to the ethnic groups. The aerobic mesophilic flora count from Rabilé varied from 8.34 ± 0.72 to 10.07 ± 0.51 log10 CFU·g-1 and yeast varied from 7.24 to 8.28 log10 UFC·g-1. Based on morphological, cultural, sexual and biochemical (carbon and nitrogen assimilation) profiles, 50 yeast strains were identified and found to belong to 4 genera and 7 species. Saccharomyces cerevisiae was found as most predominant yeasts species of yeast in of Rabilé with 64 %, Rhodotorula muciloginosa, (8%), Candida pseudorhangii (6%), Candida heliconiae (12%), Candida utilis (4%), Shizosaccharomyces pombe (4%) and Sporobolomyces odoratus (2%). This activity has been carried out in the Laboratory of Biotechnology for Food and Nutritional Sciences, University Ouaga I Pr Josph KY-ZERBO, Burkina Faso, and it was done from June to October 2018.

Predominant yeasts in Chinese Dong fermented pork(Nanx Wudl)and their aroma-producing properties in fermented sausage condition

Yeasts are one of the predominant microbial groups in fermented meats.In this study,yeast communities of Chinese Dong fermented pork(Nanx Wudl)were investigated and the technological properties of 73 yeast isolates were evaluated.Through culture-dependent and high-throughput sequencing methods,the main yeast species identified included Pichia membranifaciens,Kazachstania bulderi,Millerozyma farinosa,Candida zeylanoides,Kazachstania exigua,Candida parapsilosis and Saccharomyces cerevisiae.Among these yeasts,P.membranifaciens,M.farinosa,K.exigua and K.bulderi were detected in fermented meats for the first time.A total of 73 yeast isolates was investigated for their lipolytic and proteolytic activities.All yeast species showed lipolytic activity,while proteolytic activity against meat protein was only detected in S.cerevisiae.Assay of aroma-producing potential was performed in a model simulating fermented sausage condition.Inoculation of yeast strains increased volatiles production,especially esters and alcohols.The highest ester production was observed in S.cerevisiae Y70 strain,followed by K.exigua Y12 and K.bulderi Y19.C.zeylanoides Y10 and S.cerevisiae Y70 were the highest producers of benzeneethanol and 3-methyl-1-butanol.S.cerevisiae Y70 with its highest production of branched alcohols and esters could be a promising candidate as aroma enhancer in the manufacture of fermented sausages.

Probiotic yeasts: Anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice

AIM: To evaluate the in vitro immunomodulation capacity of various non-pathogenic yeast strains and to investigate the ability of some of these food grade yeasts to prevent experimental colitis in mice.METHODS: In vitro immunomodulation was assessed by measuring cytokines [interleukin (IL)-12p70,IL-10,tumor necrosis factor and interferon γ] released by human peripheral blood mononuclear cells after 24 h stimulation with 6 live yeast strains (Saccharomyces ssp.) and with bacterial reference strains.A murine model of acute 2-4-6-trinitrobenzene sulfonic acid (TNBS)-colitis was next used to evaluate the distinct prophylactic protective capacities of three yeast strains compared with the performance of prednisolone treatment.RESULTS: The six yeast strains all showed similar non-discriminating anti-inflammatory potential when tested on immunocompetent cells in vitro .However,although they exhibited similar colonization patterns in vivo ,some yeast strains showed significant anti-inflammatory activities in the TNBS-induced colitis model,whereas others had weaker or no preventive effect at all,as evidenced by colitis markers (body-weight loss,macroscopic and histological scores,myeloperoxidase activities and blood inflammatory markers).CONCLUSION: A careful selection of strains is required among the biodiversity of yeasts for specific clinical studies,including applications in inflammatory bowel disease and other therapeutic uses.

Isolation and Identification of Yeasts from Luzhou Flavor Daqu

A yeast strain had been isolated by dilution-plate from the Daqu samples in our study. The strain was identified as a strain of Rhodotorula aurantiaca through observation of its morphological features, micromorphological observation and biolog identification system.

Bioethanol Production from Molasses by Yeasts with Stress-Tolerance Isolated from Aquatic Environments in Japan

Bioethanol is a safe and renewable source of energy that continues to be a research focus, since fossil fuels have been linked to global warming and nuclear energy sources are affected by the increased safety concerns following the 2011 nuclear power plant accident in Japan. In general, bioethanol is converted from a biomass by yeast fermentation. The production efficiency of this bioethanol is not sufficiently high, and its practical use as a substitute for fossil fuels and nuclear energy is thus limited. For the industrial production of bioethanol, the yeast fermentation of biomass cultures containing high concentration sugar, NaCl, and ethanol is necessary, but this might induce phenomena in which the stresses arising in the yeasts weaken their cells during fermentation. As described herein, we isolated 1028 strains of yeasts from natural aquatic environments: Japan’s Tama River and Lake Kasumigaura. Among them, 412 strains were fermentative yeasts and 31 strains showed high fermentation ability under a 30% sorbitol + 10% ethanol condition. These strains were identified as Torulaspola delbrueckii, Wickerhamomyces anomalus, Candida glabrata, Pichia kudriavzevii, Saccharomyces cf. cerevisiae/paradoxus, and Lachancea kluyveri. The strains T. delbrueckii, W. anomalus, and C. glabrata also showed tolerance against 15% NaCl. Most importantly, S. cf. cerevisiae/paradoxus H28 and L. kluyveri F2-67 produced 57.4 g/L and 53.9 g/L ethanol from molasses (sucrose 104.0 g/L, fructose 33.4 g/L, and glucose 24.8 g/L) within 48 hrs at 25°C, respectively.

Molecular Phylogeny and Phenotypic Characterization of Yeasts with a Broad Range of pH Tolerance Isolated from Natural Aquatic Environments

In this study, yeasts with broad range of pH tolerance were isolated and characterized from natural aquatic environments in Japan. Only a few basic and application studies of alkali-tolerant yeasts have been reported, despite the unmet industrial needs. First, we surveyed alkali-tolerant yeasts from natural aquatic environments at pH 7.6 - 9.4. We isolated 35 yeast strains that grew in pH 9.0 medium, from seven genera and nine species: 25 strains (N1, N2, through N6, N9, K1, K3 through K19) were Rhodotorula mucilaginosa;one (N7) was Rhodosporidium fluvial;one (N8) was Scheffersomyces spartinae;two (N10 and N13) were Wicherhamonyces anomalus;one (N11) was Cyberlindnera saturnus;one (S1) was Candida sp.;two (S2 and S4) were Candida intermedia;one (S3) was Candida quercuum;and one (K2) was Cryptococcus liquefacience. We examined the effects of pH on the growth of representative yeast strains. Strains K12 and S4 showed high growth at pH 3 - 10. Strains N7, N8, N10, N11, and S3 showed high growth at pH 3 - 9. Strains K2 and S1 showed high growth at pH 4 - 8. All nine of these strains had neutralizing activities from acidic media at pH 3 - 5 to pH 6 - 8. We previously isolated acid-tolerant yeasts (Cryptococcus sp. T1 [1] and Candida intermedia CeA16 [2]) from extremely acidified environments;they showed high growth at pH 3 - 9 and neutralizing activities of acidic media by releasing ammonium ions. Thus, alkali-tolerant yeasts and acid-tolerant yeasts were found to be similar species and have both high growth at a broad range of pH and neutralizing activities of acid media. Previously, we also isolated acid-tolerant, acid-neutralizing yeasts from neutral natural environments (26 strains, 12 species) [3]. Next, we constructed the phylogenetic trees of both acid-tolerant yeasts and alkali-tolerant yeasts. All were situated in the same classification position. Similar yeast species with a broad range of pH tolerance were living in natural aquatic environments at pH values from alkali to acid.

High Ethanol Production by Marine-Derived Yeasts-<i>Saccharomyces cerevisiae</i>under Stress Pressures

For practical applications of bioethanol, the uses of both highly concentrated biomass materials and their effective fermentation by yeasts are indispensable in order to produce ethanol at low costs. However, as the saccharified products of those biomass generally contain abundant sugars, the yeasts are affected by the compounds and are inclined to decrease their physiological activities. In the process of fermentation, ethanol is gradually produced by the yeasts in the culture;the concentrated metabolic product also damages itself, and inhibition of the fermentation frequently occurs. The application of yeasts with high fermentative activities under stress pressures such as sugars and ethanol is thus desired for bioethanol production. In this study, various types of high-fermentative yeasts under stress pressures were isolated mainly from coastal waters in Japan and characterized. All yeast strains with high fermentative activities under 20% v/v ethanol were found to be Saccharomyces cerevisiae. The HK21 strain isolated from Tokyo Bay and identified as S. cerevisiae had the highest fermentation activity under 30% w/v sorbitol and under 20% v/v ethanol, and it produced approx. 70 g/l (9% v/v) ethanol from the 15% w/v glucose solution at 25 oC within 5 days.

Isolation of aromatic yeasts(non-<i>Saccharomyces</i><i>cerevisiae</i>) from Korean traditional <i>nuruks</i>and identification of fermentation characteristics

Ethyl caproate and isoamyl alcohol are important for the quality of Yakju, one of the Korean traditional alcoholic beverages. From Korean traditional fermentation agent, Nuruk, we have isolated 8 yeasts which produced rich aromatic compounds using YEPD agar plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) containing 50 uL cerulenin at 30℃. The isolated aromatic yeasts are identified as Pichia anomala (4 strains), Pichia fabianii (2 strains), Pichia farinose (1 strains), Geotrichum candidum (1 strains). We conducted alcohol fermentation with each of the aromatic yeasts and the compounds (ethyl caproate and isoamyl alcohol) producing range were 59.5 - 193.2 ppm and 10.8 - 91.6 ppm respectively. As a control, Fermivin&reg;, famous aromatic wine yeast, was 89.4 ppm and 16.2 ppm respectively. We also find that the isolated Pichia anomala could produce higher level ethanol (4.2% - 5.0%, v/v) than other species (0.4% - 2.2%, v/v). Using the aromatic yeasts in fermentation industries, we expect to improve the quality of traditional alcoholic beverages.

THE PHYLOGENY OF YEASTS--A CLADISTIC ANALYSIS

Cladistic analysis was used to clarify the phylogeny of 16 genera of yeasts whose great morphological differences and inclusion in different classification systems resulted in controversies over the taxonomy of seven genera such as Crypeococcus, etc. Some scholars suggest that they belong to Ascomycetes. but others think they belong to fungi imperfecti. After comprehensive cladistic analysis of many genetic characters, the authors consider that the above-mentioned seven genera of yeasts developed in parallel with Ascomycetes so that they should belong to one and the same developmental system.

Characterization of oral yeasts isolated from healthy individuals attended in different Colombian dental clinics

The aim of this study was to identify the most frequent yeasts in the oral cavity of adult individuals without immune disorders and to associate the presence of these oral yeasts with different characteristics of each individual. Oral rinse samples were obtained from 96 healthy adults and cultured in Sabouraud dextrose agar media and CHROMagar. Yeasts were identified by sequencing the D1/D2 region of the 28 S rRNA gene. Probable association among the socio-demographic characteristics, body mass index, family and personal medical history,oral hygiene, tobacco and/or alcohol consumption habits and presence of oral fungi was analyzed. Contingency tables and logistic regression were employed to evaluate possible relationships between the presence of oral fungi and mixed colonization with these variables. 57.3% of the healthy individuals had oral yeasts and 21.8% had mixed colonization. The most prevalent yeasts were Candida albicans(52%), C. parapsilosis(17.9%), and C.dubliniensis(7.57%). Yeasts with most frequently mixed colonization were C. albicans and C. parapsilosis. No relationships were found among the variables analyzed. However, the presence of mixed colonization was related to the presence of dental prostheses(P<0.006), dental apparatuses(P=0.016) and O’Leary index(P=0.012). This is the first study that characterized oral yeasts in Colombian healthy individuals, determined the most prevalent oral yeasts C. albicans, C. parapsilosis and C. dublinensis and an association of mixed colonization with the use of dental prostheses and aparatology and poor hygiene.