Bioaugmented ensiling of sweet sorghum with Pichia anomala and cellulase and improved enzymatic hydrolysis of silage via ball milling.

Sweet sorghum, as a seasonal energy crop, is rich in cellulose and hemicellulose that can be converted into biofuels. This work aims at investigating the effects of synergistic regulation of Pichia anomala and cellulase on ensiling quality and microbial community of sweet sorghum silages as a storage and pretreatment method. Furthermore, the combined pretreatment effects of ensiling and ball milling on sweet sorghum were evaluated by microstructure change and enzymatic hydrolysis. Based on membership function analysis, the combination of P. anomala and cellulase (PA + CE) significantly improved the silage quality by preserving organic components and promoting fermentation characteristics. The bioaugmented ensiling with PA + CE restructured the bacterial community by facilitating Lactobacillus and inhibiting undesired microorganisms by killer activity of P. anomala. The combined bioaugmented ensiling pretreatment with ball milling significantly increased the enzymatic hydrolysis efficiency (EHE) to 71%, accompanied by the increased specific surface area and decreased pore size/crystallinity of sweet sorghum. Moreover, the EHE after combined pretreatment was increased by 1.37 times compared with raw material. Hence, the combined pretreatment was demonstrated as a novel strategy to effectively enhance enzymatic hydrolysis of sweet sorghum.

Identification of the major yeasts isolated from high moisture corn and corn silages in the United States using genetic and biochemical methods.

The objective of this study was to identify species of yeasts in samples of high moisture corn (HMC) and corn silage (CS) collected from farms throughout the United States. Samples were plated and colonies were isolated for identification using DNA analysis. Randomly selected colonies were also identified by fatty acid methyl esters (FAME) and by physiological substrate profiling (ID 32C). For CS, Candida ethanolica, Saccharomyces bulderi, Pichia anomala, Kazachstania unispora, and Saccharomyces cerevisiae were the predominant yeasts. Pichia anomala, Issatchenkia orientalis, S. cerevisiae, and Pichia fermentans were the prevalent species in HMC. The 3 identification methods were in agreement at the species level for 16.6% of the isolates and showed no agreement for 25.7%. Agreement in species identification between ID 32C and DNA analysis, FAME and ID 32C, and FAME and DNA analysis was 41.1, 14.4, and 2.2%, respectively. Pichia anomala and I. orientalis were able to grow on lactic acid, whereas S. cerevisiae metabolized sugars (galactose, sucrose, and glucose) but failed to use lactic acid. The yeast diversity in CS and HMC varied due to type of feed and location. Differences in species assignments were seen among methods, but identification using substrate profiling generally corresponded with that based on DNA analysis. These findings provide information about the species that may be expected in silages, and this knowledge may lead to interventions that control unwanted yeasts.

Bioprocess for efficient production of recombinant Pichia anomala phytase and its applicability in dephytinizing chick feed and whole wheat flat Indian breads.

The phytase of the yeast Pichia anomala (PPHY) is a suitable biocatalyst as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. The cell-bound nature and low phytase titres are the main bottlenecks for its utility in food and feed industries. In this investigation, we have overcome the problems by constitutive secretory expression of PPHY under glyceraldehyde phosphate dehydrogenase (GAP) promoter. A ~44-fold increase in rPPHY titre has been achieved after optimization of cultural variables by one-variable-at-a-time approach and two factorial statistical design. The use of GAP promoter makes the cultivation of the recombinant P. pastoris straight forward and eliminates the requirement of methanol for induction and hazards associated with its storage. Among metal-phytate complexes, Ca(2+) phytate is hydrolyzed more efficiently by rPPHY than Co(2+), Mn(2+), Mg(2+), Fe(3+) and Zn(2+) phytates. The enzyme is effective in dephytinizing whole wheat unleavened flat Indian breads (naan and tandoori) and different broiler feeds, thus mitigating anti-nutritional effects of phytates.

A transcriptome profile of Wickerhamomyces anomalus incubated with chitosan revealed dynamic changes in gene expression and metabolic pathways.

Previous studies have shown that Wickerhamomyces anomalus can control postharvest diseases of fruits and incubation of the yeast with chitosan can improve its efficiency. In this study, transcriptome study was conducted to determine molecular mechanisms involved in the yeast-chitosan interaction. The bioinformatics analysis of the RNA-seq data confirmed that incubating W. anomalus with 1 % chitosan for 24 h significantly altered the expression of differential genes involved in yeast metabolic and cellular activities. Genes involved in ethyl acetate production, reactive oxygen species regulation, cell wall reinforcement, stress resistance, and signalling were all significantly up-regulated. Pathways which have significant role in the yeast growth and reproduction, energy production, cellular homeostasis, signal transduction, catalytic, and antioxidant activities were significantly enriched. In general, incubation of the yeast with chitosan genes metabolic pathways which are important for the yeast survival, adaptation, and reproduction. Molecular studies are important in providing fundamental theoretical foundation for the practical application of antagonistic yeasts for future uses. As a result, this research will be an input for use of the antagonistic yeast as microbial or biochemical pesticides instead of synthetic chemicals which have both health and environmental effects.

Fungemia by Wickerhamomyces anomalus-A Narrative Review.

Wickerhamomyces anomalus has been previously classified as Hansenula anomala, Pichia anomala, and Candida pelliculosa and was recently reclassified in the genus Wickerhamomyces after phylogenetic analysis of its genetic sequence. An increasing number of reports of human infections by W. anomalus have emerged, suggesting that this microorganism is an emerging pathogen. The present review aimed to provide data on the epidemiology, antifungal resistance, clinical characteristics, treatment, and outcomes of fungemia by W. anomalus by extracting all the available information from published original reports in the literature. PubMed/Medline, Cochrane Library, and Scopus databases were searched for eligible articles reporting data on patients with this disease. In total, 36 studies involving 170 patients were included. The age of patients with fungemia by W. anomalus ranged from 0 to 89 years; the mean age was 22.8 years, the median age was 2.2 years, with more than 37 patients being less than one month old, and 54% (88 out of 163 patients) were male. Regarding patients' history, 70.4% had a central venous catheter use (CVC), 28.7% were on total parenteral nutrition (TPN), 97% of neonates were hospitalized in the neonatal ICU (NICU), and 39.4% of the rest of the patients were hospitalized in the intensive care unit (ICU). Previous antimicrobial use was noted in 65.9% of patients. The most common identification method was the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in 34.1%, VITEK and VITEK 2 in 20.6%, and ID32 C in 15.3%. W. anomalus had minimal antifungal resistance to fluconazole, echinocandins, and amphotericin B, the most commonly used antifungals for treatment. Fever and sepsis were the most common clinical presentation noted in 95.8% and 86%, respectively. Overall mortality was 20% and was slightly higher in patients older than one year. Due to the rarity of this disease, future multicenter studies should be performed to adequately characterize patients' characteristics, treatment, and outcomes, which will increase our understanding and allow drawing safer conclusions regarding optimal management.

Fungemia With Wickerhamomyces anomalus: A Case Report and Literature Review.

We report the case of an 84-year-old man with a history of IgG4-related sclerosing cholangitis who was diagnosed with advanced esophageal cancer and underwent radiation and chemotherapy. An implantable central venous access port was placed for chemotherapy and total parenteral nutrition. The patient presented with a fever and received antimicrobial therapy for acute cholangitis but remained febrile, and subsequently, yeast was detected in the aerobic bottle of blood culture obtained from the central venous line. The yeast was identified as Wickerhamomyces anomalus. Liposomal amphotericin B was administered, and the central line access port was removed. After confirmation of negative blood cultures and 14 days post treatment, he underwent reinsertion of the central line access port. Due to persistent pain at the insertion site, fluconazole was added for an additional 14 days, and the patient was discharged and transferred to another hospital. Wickerhamomyces anomalus is a rare fungal infection with other synonyms including Pichia anomala, Hansenula anomala, and Candida pelliculosa. A literature review of 53 case reports of Wickerhamomyces anomalus, Pichia anomala, Hansenula anomala, and Candida pelliculosa was conducted, with a total of 211 cases reviewed. Fungemia was reported in 94% of cases, with central venous catheterization, parental feeding, low birth weight, and immunocompromised status identified as major risk factors. The majority of cases were pediatric, particularly neonatal, and there were reports of nosocomial infections causing outbreaks, with some cases involving the eye such as endophthalmitis or keratitis.

Wickerhamomyces Anomalus Postoperative Endophthalmitis.

INTRODUCTION: We describe the case of a postoperative fungal endophthalmitis caused by Wickerhamomyces anomalus, an opportunistic yeast common in nature but rare as a causative agent in ocular infections. This would be the second reported case of W. anomalus endophthalmitis. CASE REPORT: A patient came to our hospital with a dense hemovitreous caused by an inadvertent ocular perforation and retinal detachment during a cataract surgery. In the days following the first vitreoretinal surgery a chronic postoperative endophthalmitis was evidenced. Only after several surgeries as well as intravitreal antibiotics and sample takings, Wickerhamomyces was detected. The treatment with oral voriconazole and an intraocular lens extraction controlled the infection. DISCUSSION: Wickerhamomyces anomalus, despite not being particularly aggressive in our case, was resistant to various consequent vitreoretinal surgeries. The unresponsiveness to treatment led us to contemplate the fungal etiology, and fortunately the cultures were positive for this yeast. In conjunction to antifungal therapy, it is possible that the IOL explantation played an important role in the treatment. CONCLUSION: A high index of suspicion must be held in cases of fungal endophthalmitis. Both IOL explantation and oral antifungal therapy are useful treatment options in cases of W. anomalus endophthalmitis.

Effects on Cell Membrane Integrity of Pichia anomala by the Accumulating Excessive Reactive Oxygen Species under Ethanol Stress.

Ethanol stress to yeast is well recognized and exists widely during the brewing process of alcohol products. Pichia anomala is an important ester-producing yeast in the brewing process of Chinese Baijiu and other alcohol products. Therefore, it is of great significance for the alcohol products brewing industry to explore the effects of ethanol stress on the growth metabolism of P. anomala. In this study, the effects of ethanol stress on the growth, esters production ability, cell membrane integrity and reactive oxygen species (ROS) metabolism of P. anomala NCU003 were studied. Our results showed that ethanol stress could inhibit the growth, reduce the ability of non-ethyl ester compounds production and destroy the cell morphology of P. anomala NCU003. The results also showed that 9% ethanol stress produced excessive ROS and then increased the activities of antioxidant enzymes (superoxide dismutase, catalase, aseorbateperoxidase and glutathione reductase) compared to the control group. However, these increased antioxidant enzyme activities could not prevent the damage caused by ROS to P. anomala NCU003. Of note, correlation results indicated that high content of ROS could promote the accumulation of malondialdehyde content, resulting in destruction of the integrity of the cell membrane and leading to the leakage of intracellular nutrients (soluble sugar and protein) and electrolytes. These results indicated that the growth and the non-ethyl ester compounds production ability of P. anomala could be inhibited under ethanol stress by accumulating excessive ROS and the destruction of cell membrane integrity in P. anomala.

Isolation and characterization of a Pichia anomala strain: a promising candidate for bioethanol production.

A yeast strain designated as Y-1 was isolated and characterized from wine yeast ("Jiuqu"). Based on the morphological and biochemical results, along with the rDNA internal transcribed spacer region (ITS), Y-1 was identified to be a Pichia anomala strain. Y-1 is an ethanol-tolerant strain, enduring ethanol concentrations of up to 14 %. Y-1 growth medium conditions were optimized, results showing good growth in medium with pH ranges from 3.5-6.5, temperature ranges from 25-30 °C, and inoculums range of 8 %-12 %, while optimum growth conditions were reached at a temperature of 30 °C, pH 5.0, and inoculums of 10 %. Furthermore, when the alkaline hydrolyzed Shatian pummelo peel solutions were inoculated with 10 % Y-1 and fermented at 30 °C for 6 d, 4.7 % pure ethanol (w/w) was produced, as evidenced by gas chromatography analysis. Our present study shows potential for the Y-1 strain to be a promising candidate for bioethanol production.

Characterization of the ß-Glucosidase activity in indigenous yeast isolated from wine regions in China.

ß-glucosidase is a pivotal enzyme that hydrolyzes bound volatile aromatic compounds. However, the activity of ß-glucosidase in winemaking and the mechanism by which it affects the flavor and taste of wines have not been fully investigated. In this study, we profiled the characteristics of ß-glucosidase derived from wine-related yeasts isolated from different wine-making regions in China, and analyzed the enzyme activity from different parts of the cells under aerobic and anaerobic conditions. A total of 56 strains of wine-related yeasts producing ß-glucosidases were screened using the YNB-C medium (YNB 6.7 g L-1 , cellobiose 5 g L-1 , pH 5.0). We found that strain Clavispora lusitaniae C117 produced the highest enzyme activity (152.39 µmol pNP ml-1 h-1 ). In most strains, ß-glucosidase were located in whole cells (periplasmic space) and permeabilized cells (intracellular). The non-Saccharomyces species had the highest enzymatic activity in a strain-dependent manner. Under aerobic conditions, C. lusitaniae C117, Hanseniaspora guilliermondii A27-3-4, Metschnikowia pulcherrima F-1-6, and Pichia anomala C84 had the highest ß-glucosidase activity. We further investigated the ß-glucosidase activity during the wine fermentation and the effects of sugar, pH, temperature, and ethanol on the enzyme activities of P. anomala C84 and commercial Saccharomyces yeast strains RC212 and VL1. The presence of fructose, glucose, and sucrose strongly inhibited enzyme activity. Similarly, low pH and low temperature inhibited the activity of ß-glucosidase, whereas ethanol promoted enzyme activity. Our findings provide a theoretical basis on understanding the different yeast characteristics of ß-glucosidase and their potential application for further improving wine aroma complexity.

Isolation, Identification and Function of Pichia anomala AR2016 and Its Effects on the Growth and Health of Weaned Pigs.

This study isolates and identifies Pichia anomala (P. anomala) AR2016, and studies its effect on the growth and health of weaned pigs. A P. anomala strain from solid wine koji is isolated and identified using 26S rDNA analysis, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, artificial gastric, and intestinal juice tolerance are evaluated. In our methodology, thirty 28 d Large White × Landrace × Rongchang weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days. A P. anomala strain was identified and named P. anomala AR2016. P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our results show that compared with the CK group, orally administered P. anomala AR2016 increases average daily gain, the ileal villus height, the ileal mucosal concentrations of occludin and zonula occluens-1, the serum glucose and total protein concentration, total superoxide dismutase, glutathione peroxidase, and total antioxidative capacity activity, the trypsin and lipase activity in jejunal and ileal contents, the jejunal and ileal mucosa mRNA levels of ALP, TNF-α, and TLR-2, and the relative abundance of Bacteroidetes, Actinobacteria, Succinivibrionaceae, Lachnospiraceae, and Prevotellaceae (p < 0.05). Compared with the CK group, oral administration of P. anomala AR2016 decreased the incidence of diarrhea, aspartate aminotransferase activity, alanine amino-transferase-activity, malondialdehyde, D-lactic acid and endotoxin content in serum, the mRNA level of aminopeptidase N of ileum mucosa, and the relative abundance of Proteobacteria, Clostridiaceae, Campylobacteraceae, Vibrionaceae, Bacillus, and Pseudon (p < 0.05). Collectively, the study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. Oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.

Pichia anomala Induced With Chitosan Triggers Defense Response of Table Grapes Against Post-harvest Blue Mold Disease.

To study the mechanism by which Pichia anomala induced with chitosan (1% w/v) controls blue mold disease in table grapes caused by Penicillium expansum, this study evaluated alterations in three yeast enzymatic activities. The changes in the five primary disease defense-related enzymes and two non-enzyme activities of table grapes were assayed. The results of the study showed that chitosan (1% w/v) significantly increased the yeast ß-1,3-glucanase, catalase (CAT), and malondialdehyde (MDA) activities. Furthermore, P. anomala alone or induced with chitosan (1% w/v) significantly increased the table grapes enzymatic activities of Polyphenol oxidase (PPO), phenylalanine (PAL), peroxidase (POD), and catalase (CAT) compared to the control. The RT-qPCR results also confirmed that the genes of these major disease defense enzymes were up-regulated when the table grapes were treated with P. anomala. The highest results were recorded when the fruit was treated by yeast induced with chitosan (1% w/v). The phenolic compounds, in addition to their nutritional value, can also increase the antimicrobial properties of table grapes. The current experiment determined that the total phenol and flavonoid contents of table grapes showed the highest results for fruits treated by P. anomala induced with chitosan compared with the control. Generally, the increment of these fruit enzymatic and non-enzymatic activities shows improved table grape defense against the pathogenic fungus. The induction of the yeast with chitosan also increases its bio-control efficacy against the pathogen. This study will enable future detailed investigation in the yeast pathogen control mechanisms and the use of yeasts as bio-pesticides.

Transcriptome Characterization and Expression Profiles of Disease Defense-Related Genes of Table Grapes in Response to Pichia anomala Induced with Chitosan.

Transcriptome analysis (TA) was conducted to characterize the transcriptome changes in postharvest disease-related genes of table grapes following treatment with Pichia anomala induced with chitosan (1% w/v). In the current study, the difference in the gene expression of table grapes after treatment with P. anomala induced with chitosan and that of a control group was compared 72 h post-inoculation. The study revealed that postharvest treatment of table grapes with P. anomala induced with chitosan could up-regulate genes that have a pivotal role in the fruit's disease defense. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results also confirmed that GO terms and the KEGG pathways, which have pivotal roles in plant disease resistance, were significantly enriched. The up-regulated genes of the treatment group have a unique function in the fruit's disease resistance compared to the control group. Generally, most genes in the plant-pathogen interaction pathway; the plant Mitogen-activated protein kinase (MAPK) signaling pathway; the plant hormone signal transduction pathway; the pathway of glutathione metabolism; the pathway of phenylalanine, tyrosine, and tryptophan biosynthesis; and the pathway of flavonoid biosynthesis were all up-regulated. These up-regulations help the fruit to synthesize disease-resistant substances, regulate the reactive oxygen species (ROS), enhance the fruit cell wall, and enrich hormone signal transduction during the pathogen's attack. This study is useful to overcome the lags in applying transcriptomics technology in postharvest pathology, and will provide insight towards developing other alternative methods to using bio-pesticides to control postharvest diseases of perishables.

Validation of a predictive model for the growth of chalk yeasts on bread.

The present study focused on the effects of temperature, T, and water activity, aw, on the growth of Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera on Sabouraud Agar Medium. Cardinal values were estimated by means of cardinal models with inflection. All the yeasts were xerophilic, and they exhibited growth at 0.85 aw. The combined effects of T, aw, and pH on the growth of these species were described by the gamma-concept and validated on bread in the range of 15-25 °C, 0.91-0.97 aw, and pH 4.6-6.8. The optimum growth rates on bread were 2.88, 0.259, and 1.06 mm/day for H. burtonii, P. anomala, and S. fibuligera, respectively. The optimal growth rate of S. fibuligera on bread was about 2 fold that obtained on Sabouraud. Due to reproduction by budding, P. anomala exhibited low growth on Sabouraud and bread. However, this species is of major concern in the baker's industry because of the production of ethyl acetate in bread.

Pichia anomala, a new species of yeast-like endosymbionts and its variation in small brown planthopper (Laodelphax striatellus).

Yeast-like symbionts (YLS) are endosymbionts that promote the growth of delphacid planthoppers (Hemiptera: Delphacidae), some of which are pests on cultivated rice. Identification and characterization of YLS growth can be helpful for pest control, because it has been demonstrated that there is a variety of YLS in rice planthopper and they affected the planthopper's growth and virulence to plant hosts. So, elucidation of the species of YLS in planthopper is crucial for exploiting a new way to control planthopper. In this study, a new isolated of YLS was obtained from the small brown planthopper, Laodelphax striatellus, which was cultured in vitro, simultaneously identified as Pichia anomala based on its phylogenetic analysis. In order to confirm the existence of P. anomala in the L. striatellus body, we used the denaturing gradient gel electrophoresis (DGGE) to identify the YLS and obtain the specific bands for P. anomala. The quantification and localization of P. anomala in L. striatellus samples were determined by fluorescent in situ hybridization (FISH) using genus-specific 18S rDNA targeted probe. The result confirmed that a certain number of P. anomala exist in L. striatellus's abdomen. Subsequently, the variation and copy number of P. anomala in different L. striatellus instars was measured by using absolute quantitative real-time PCR (qPCR), the results indicated that the new isolated strain was closely related to the developmental process of L. striatellus.

A model for the effect of pH on the growth of chalk yeasts.

Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera were isolated from spoiled packaged sliced bread. These chalk yeasts were characterized by a wide range of pH for which growth was almost optimum. Thus, the curve growth vs pH exhibited plateau and sharp profiles close to the minimum and the maximum pH. This study described a chalk yeast model (CYM) for the effect of pH derived from a new germination model for fungi (Dantigny, P., Nanguy, S., P.-M., Judet-Correia, D., and Bensoussan, M. 2011, International Journal of Food Microbiology, 146, 176-181). The CYM is asymmetric, versatile, based on parameters with biological significance, and compatible with the gamma concept. The CYM was compared to the cardinal pH model (CPM) which is widely used to describe the effect of pH on microbial growth. The CYM exhibited RMSE values two fold less than those obtained with the CPM for H. burtonii, and S. fibuligera for which plateaus were clearly observed. For P. anomala, the plateau was less obvious, but the RMSE value obtained with the CYM was similar to that found with the CPM. The CYM could extend its use to represent the effect of pH on mold growth.

Microbiological Characteristics of Wild Yeast Strain Pichia anomala Y197-13 for Brewing Makgeolli.

Makgeolli is a traditional cloudy-white Korean rice wine with an alcohol content of 6~7%. The present study investigated the morphological characteristics, carbon-utilizing ability, fatty acid composition, alcohol resistance, glucose tolerance, and flocculence of Saccharomyces cerevisiae Y98-5 and Pichia anomala Y197-13, non-S. cerevisiae isolated from Nuruk, which is used in brewing Makgeolli. Similar morphological characteristics were observed for both isolated wild yeast strains; and the carbon source assimilation of Y197-13 differed from that of other P. anomala strains. Strain Y197-13 was negative for D-trehalose, mannitol, arbutin, I-erythritol, and succinic acid. The major cellular fatty acids of strain Y197-13 included C18:2n6c (33.94%), C18:1n9c (26.97%) and C16:0 (20.57%). Strain Y197-13 was Crabtree-negative, with 60% cell viability at 12% (v/v) ethanol. The flocculation level of strain Y197-13 was 8.38%, resulting in its classification as a non-flocculent yeast.

Identification and Partial Characterization of Antilisterial Compounds Produced by Dairy Yeasts.

Food-grade yeasts make significant contributions to flavor development in fermented foods. Some yeast species also inhibit undesirable bacteria, yeasts and molds, apparently by producing antimicrobial compounds called mycocins. The aim of this study was to evaluate the ability of wild yeasts, isolated from raw milk and cheese in the Quebec province area, to produce antilisterial compounds. Based on an agar-membrane screening test, 22 of 95 isolates, namely one Candida catenulata, one Candida parapsilosis, five Candida tropicalis, four Debaryomyces hansenii, one Geotrichum candidum, nine Pichia fermentans and one Pichia anomala, exhibited a significant inhibitory effect against Listeria ivanovii HPB28. Four in particular, namely C. tropicalis LMA-693, D. hansenii LMA-916, P. fermentans LMA-256 and P. anomala LMA-827, produced substances extractable from culture supernatant and capable of decreasing 18-h growth of L. ivanovii by, respectively, 97, 92, 84 and 78 %. Heating the extracted material (100 °C for 10 min) decreased these values to 72, 62, 58 and 31 %, respectively, while treatment with trypsin or pronase E decreased them to as little as 27 %. The extracts reduced the numbers of viable Listeria monocytogenes by as much as four log cycles within an hour. Transmission electron microscopy revealed a high proportion of lysis among the cells, apparently due to pore formation. This study clearly shows the potential of these four yeast isolates for use as bio-preservatives in a variety of dairy products.

Aislamiento e identificación de levaduras degradadoras de hidrocarburos aromáticos, presentes en tanques de gasolina de vehículos urbanos / Isolation and identification of aromatic hydrocarbon degrading yeasts present in gasoline tanks of urbans vehicles

RESUMEN Se obtuvieron aislamientos de levaduras a partir de muéstreos en tanques de combustible de vehículos urbanos, con el objeto de evaluar su potencial actividad de degradación de hidrocarburos aromáticos derivados del petróleo. Se realizaron ensayos de crecimiento en medio mínimo mineral sólido utilizando distintos hidrocarburos (benceno, tolueno, naftaleno, fenantreno, y pireno). Los aislamientos que presentaron crecimiento notorio en alguno de los hidrocarburos aromáticos policíclicos fueron identificados mediante secuenciación Sanger de los marcadores moleculares ITS1 e ITS2 del ARNr. Se obtuvieron 16 aislados de levaduras, de las cuales tres presentaron crecimiento conspicuo con hidrocarburos aromáticos como única fuente de carbono. Las cepas identificadas pertenecen al género Rhodotorula y corresponden a las especies Rhodotorula calyptogenae (99,8% de identidad) y Rhodotorula dairenensis (99,8% de identidad). Dichas cepas presentaron crecimiento en benceno, tolueno, naftaleno, fenantreno. En este estudio se reporta por primera vez la presencia de levaduras del género Rhodotorula que habitan los ductos y tanques de gasolina de vehículos urbanos, así como su capacidad para utilizar distintos hidrocarburos aromáticos que son contaminantes para el medio ambiente. Estos resultados sugieren que dichas levaduras constituyen potenciales candidatos para la degradación de éstos compuestos, como parte de estrategias de biorremediación.

ABSTRACT Yeast isolates were obtained from fuel tanks of vehicles in order to assess their potential use in the degradation of aromatic hydrocarbons. Growth assays were performed in minimum mineral medium using different aromatic hydrocarbons (benzene, toluene, naphthalene, phenanthrene, and pyrene) as the sole carbon source. Isolates that showed growth in any of the tested polycyclic aromatic hydrocarbons were identified by Sanger sequencing of the ITS1 and ITS2 rDNA molecular markers. A total of 16 yeasts strains were isolated, and three showed remarkable growth in media with aromatic hydrocarbons as the sole carbon source. These strains belong to the genus Rhodotorula, and correspond to the species Rhodotorula calyp-togenae (99,8% identity) and Rhodotorula dairenensis (99,8% identity). These strains grew in benzene, toluene, naphthalene, phenanthrene and pyrene. This study demonstrates for the first time that yeasts of the genus Rhodotorula inhabit pipelines and fuel tanks of vehicles and that remove aromatic hydrocarbons that are environmental pollutants. Our results suggest that these yeasts are potential candidates for aromatic hydrocarbon degradation as part of bioremediation strategies.

Physiological Functionality and Enzyme Activity of Biomass from Pichia anomala Grown on Ginseng-Steaming Effluent.

A novel biomass was prepared from Pichia anomala KCCM 11473, which grew well in ginseng-steaming effluent (GSE), and its physiological functionalities and enzyme activities were determined. When the strain was cultured in the GSE (pH 6.0) at 30℃ for 48 h, 1.6 mg of biomass per ml-cultures was produced. The cell-free extract of the biomass showed high antihypertensive angiotensin I-converting enzyme inhibitory activity of 72.0% and anticholesteromia HMG-CoA reductase inhibitory activity of 46.5%. The cell-free extract also showed 13.0 U per ml and 8.5 U per ml of neutral protease activity and alkaline protease, respectively.