Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.     

Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes

Twenty-six strains of yeasts, belonging to the genera Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces and Torulaspora previously isolated from wines, were screened for the production of extracellular pectinases, amylases, lipases, proteases and β-glucosidases. Some strains of Candida species and Hanseniaspora uvarum/Kloeckera apiculata produced extracellular proteolytic or lipolytic activities. Most yeasts exhibited β-glucosidase activity, but particularly high activity was observed in strains of Pichia anomala/Candida pelliculosa (formerly Hansenula anomala ) and Hanseniaspora uvarum/Kloeckera apiculata . The potential impact of these enzymes on wine quality is discussed.     

Yeast ecology in French cider and black olive natural fermentations

In this study, rDNA ITS restriction analysis was used to identify yeasts from two naturally fermented products: French ciders and black olives. In cider musts and bottled ciders, the PCR-RFLP method generated 15 different ITS/RFLP profiles for a total of 208 isolates. The predominant yeasts corresponded to Saccharomyces bayanus, Saccharomyces cerevisiae, Lachancea cidri and Dekkera anomala. Three identified species: Candida sake, Candida tropicalis and Kluyveromyces marxianus had never been described before in ciders. For the black olive fermentation, the method allowed for identification of 11 profiles for a total of 137 isolates. A sequential apparition of yeasts was observed with Pichia anomala, Candida boidinii and Debaryomyces etchellsii being the predominant species. Four isolates did not correspond to any known species based on the sequencing of the D1/D2 region of the 26S rRNA gene. By using the rDNA ITS method, valuable information on yeast population biodiversity and dynamics in the naturally fermented food products studied was obtained.     

Characterization of butter spoiling yeasts and their inhibition by some spices

This study was designed to identify the yeasts in packaged and unpackaged butters and screen antiyeast activity of spices, including marjoram (Origanum majorana L.), summer savory (Satureja hortensis L.), and black cumin (Nigella sativa L.) against the most dominant yeast species in the packaged and unpackaged butters. Mean total yeast populations were 5.40 log CFU/g in unpackaged butter samples and 2.22 log CFU/g in packaged butter samples, indicating better hygienic quality of packaged samples. Forty-nine yeast species were isolated and identified from butter samples with the most prevalent isolates belonging to genera Candida-C. kefyr, C. zeylanoides, and C. lambica-and with moderate number of isolates belonging to genera Cryptococcus, Rhodotorula, Saccharomyces, and Zygosaccharomyces. Black cumin exhibited the highest antiyeast activity against C. zeylanoides and C. lambica species, even inhibited these species, while summer savory inhibited C. kefyr. The results of this study revealed clear antimicrobial potential of black cumin against the yeast species isolated from butters. Marjoram, summer savory, and black cumin could be used as natural antimicrobial agents against spoilage yeasts in food preservation, especially in butter.     

Technological and spoiling characteristics of the yeast microflora isolated from Bella Di Cerignola table olives

This study focused to investigate the technological significance and the spoiling impact of diverse yeast strains, isolated from 'Bella di Cerignola' Italian table olives. Sixty-four isolates (belonging mainly to the species Candida famata and C. guilliermondii ) were studied to assess their growth at different temperatures (15, 25 and 37 °C), pHs (4.0, 5.0 and 9.5) and NaCl concentrations (0–10.0%) in lab medium. Their pectolytic, xylanolytic, lipolytic and catalase activities were also evaluated. Most of the yeasts showed a moderate pectolytic activity and were able to grow at pH 9.5 after 120 h of incubation. Salt and temperature were the most important environmental variables affecting yeast growth, which could exert a strong selective pressure on yeast population. Candida guilliermondii appeared more resistant to high salt concentrations, whereas C. famata was able to grow at 15 °C.     

Yeasts associated with Sardinian ewe's dairy products

In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.     

Role of yeasts in table olive production

Table olives are a traditional fermented vegetable of the Mediterranean countries, but their production and consumption are now spread all around the world. Yeasts can play a double role in this food. They are present throughout the fermentative process and it is generally accepted that they can produce compounds with important organoleptic attributes determining the quality and flavour of the final product. However, yeasts can also be spoilage microorganisms in olive fermentation/storage and packing causing gas pockets, swollen containers, cloudy brines and off-flavours and off-odours. Candida boidinii, Debaryomyces hansenii, Pichia anomala, P. membranifaciens, Rhodotorula glutinis and Saccharomyces cerevisiae are species isolated with a high frequency from olive processes. Scarce information is still available about the ecology, biochemistry and molecular biology of these important microorganisms in table olives. A wider knowledge about these aspects could facilitate the possible application of yeasts as a starter culture, due to their ability to produce aromatic compounds, antioxidants, enzymes, and improve the growth of lactic acid bacteria.     

生姜猕猴桃自然酒精发酵醪微生物多样性研究

为了探讨和评估生姜猕猴桃自然酒精发酵醪酵母菌系多样性及其产品的安全性,采用微生物可培养方法对生姜猕猴桃自然酒精发酵中的酵母菌进行分离,并利用5.8S rDNA ITS序列信息分析不同时期生姜猕猴桃自然酒精发酵醪的酵母菌株α多样性和β多样性。结果表明,生姜猕猴桃自然酒精发酵中酵母菌有8属11种,包括假丝酵母、孢汉逊酵母、德巴利酵母、毕赤酵母、伊萨酵母、柯达酵母、梅奇酵母、Meyerozyma。毕赤酵母是生姜猕猴桃自然酒精发酵醪初期和后期的优势酵母菌群,Myerozyma是生姜猕猴桃自然酒精发酵醪初期和中期的优势酵母菌群。α多样性、β多样性分析结果显示,生姜猕猴桃自然酒精发酵中期物种多样性最高,发酵后期酵母菌数与独有酵母菌数最多,物种丰富度最高。     

新疆传统馕发酵面团中酵母菌的多样性分析

采用平板分离与纯化、26S rDNA D1/D2区域序列测定和系统发育分析方法,对新疆乌鲁木齐、阿勒泰、伊犁、喀什、阿克苏及和田6 个地区民间传统馕面团发酵酵母菌多样性进行了研究。从中分离得到227 株酵母菌,它们隶属于10 个属14 个种：Saccharomyces cerevisiae、Saccharomyces servazzii、Pichia fermentans、Pichia membranifacies、Pichia kudriavzevii、Pichia anomala、Candida humilis、Hanseniaspora uvarum、Torulasporadelbrueckii、Wickerhamomyces anomalus、Cladosporium ramotenellum、Cryptococcus albidus、Kodamaea ohmeri、Issatchenkia orientalis;其中Saccharomyces cerevisiae为优势种,占总分离株的55.51%（126 株）,在系统发育树上形成2 个大分枝4 个小分枝,A1-1分枝由115 株菌株构成,是馕面团发酵的优势品系菌群。研究结果阐明了新疆民间传统馕面团中酵母菌的多样性,为开发利用传统馕面团的酵母菌资源提供了理论依据。     

接种不同嗜杀特性的酿酒酵母对赤霞珠发酵中酵母多样性的影响

以赤霞珠葡萄为原料,分别接种不同嗜杀特性的酿酒酵母(Saccharomyces cerevisiae)菌株NXU17-26(中性)、UCD522(敏感菌株)和UCD2610(嗜杀菌株),并以自然发酵为对照,研究各菌株对赤霞珠葡萄酒的发酵特征及发酵中酵母菌多样性的影响。结果表明,接种发酵在启酵和发酵速度上显著快于自然发酵。WLN培养基将分离到的480株酵母菌鉴定为7种类型,26S rDNA D1/D2序列分析进一步将其鉴定为4属5种:葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、克鲁维毕赤酵母(Pichia kluyveri)、伯顿丝孢毕赤酵母(Hyphopichia burtonii)、S.cerevisiae、库徳毕赤酵母(Pichiakudriavzevii)。这4属5种的酵母均存在于自然发酵中,而接种发酵中仅有H.uvarum和S.cerevisiae两种酵母,接种发酵中酵母菌多样性较低。Interdelta指纹图谱分析表明,所接种的酿酒酵母菌株是相应发酵中的优势菌株:接种中性酵母NXU17-26的发酵中,NXU17-26的基因型占比为63.46%;接种敏感菌株...     

Influence of Yeasts on the Oil Quality Indexes of Table Olives

After moisture, fat is the major constituent of table olives. However, scarce studies have been carried out to determine the influence of microorganisms and type of processing on the modification of their quality indexes. The present survey studies the influence of lipolytic (Candida boidinii TOMC Y5 and Wickerhamomyces anomalus TOMC Y10) and nonlipolytic (Debaryomyces etchellsii TOMC Y9 and Pichia galeiformis TOMC Y27) yeasts on the oil quality indexes of Manzanilla and Hojiblanca green fruits processed as directly brined and lye‐treated table olives. Overall, the inocula scarcely used available sugars, except the lipolytic C. boidinii strain in lye‐treated olives. Acetic acid production was limited in all conditions, except for the D. etchellsii strain in directly brined Manzanilla fruits. Ethanol formation was also reduced, although the W. anomalus (in both types of elaboration) and the C. boidinii (in lye‐treated olives) strains produced significantly higher proportions. Apparently, changes in the oil quality indexes of processed olives were not related to the presence of yeasts, and hence, could have been caused by the endogenous activity of the fruits. A principal component analysis using the microbiological, physicochemical, and oil quality data supported this hypothesis, grouping treatments according to olive variety and type of elaboration, while segregation due to yeast inocula was not observed.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

Identification and characterization of yeast isolated from the elaboration of seasoned green table olives

The purpose of this study was to investigate the yeast population during the processing of green table olives. In the fresh olives, yeast were found at concentrations of around 3.0 log cfu/g, with Cryptococcus spp. being predominant. In the brine, the yeast concentrations were greater than 4.9 log cfu/ml, with Pichia anomala, Kluyveromyces marxianus, and Saccharomyces cerevisiae being the predominant species. Unlike the yeast isolated from the fresh olives, the strains obtained from the olive brine mostly showed low pectolytic but high catalase activities. Some of these strains also exhibited other biochemical desirable properties for the fermentation of green table olives, including their lipolytic activities and their assimilation or production of organic acids in the brine. Seven strains in particular of P. anomala, K. marxianus, S. cerevisiae, and Candida maris showed the best properties for use in trials as starter culture in pilot fermenters.     

Screening of non-Saccharomyces wine yeasts for the production of beta-D-xylosidase activity

Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D-xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl-beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D-xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes.     

Detection and identification of wild yeast in Koumiss

Koumiss is a slightly alcoholic fermented mare's milk beverage, originally obtained by using a natural mixed starter of lactic acid bacteria and yeasts. Yeast is an important component of Koumiss processing which can affect the aroma, texture, as well as the nutrients beneficial to human health, but few reports have examined the yeast ecology of local ecosystems. The purpose of this study was to isolate and identify the yeast present in Koumiss from three representative regions of China using a polyphasic method. A total of 655 yeast isolates were obtained from 96 Koumiss samples collected from three regions in China. Koumiss harbored yeast populations at 5-7 log CFU/ml. Twelve different yeast species belonging to nine genera were detected in the Koumiss samples tested, including Candida pararugosa, Dekkera anomala, Geotrichum sp., Issatchenkia orientalis, Kazachstania unispora, Kluyveromyces marxianus, Pichia deserticola, Pichia fermentans, Pichia manshurica, Pichia membranaefaciens, Saccharomyces cerevisiae and Torulaspora delbrueckii. Kluyveromyces marxianus, Kazachstania unispora and Saccharomyces cerevisiae were the dominant species present in this traditional fermented dairy product. This study is the first to identify the yeast communities associated with Koumiss in China. The results enrich our knowledge of yeast in Koumiss, give us a more complete picture of the microbial diversity in Koumiss and can be used to promote the development of the local dairy industry.     

Effects of pectinolytic yeast on the microbial composition and spoilage of olive fermentations

This study resulted in the identification of pectinolytic yeasts in directly brined Sicilian-style green olive fermentations and examination of the influence of those yeasts on the microbial composition and quality of fermented olives. Firstly, defective olives processed in Northern California from 2007 to 2008 and characterized by high levels of mesocarp tissue degradation were found to contain distinct yeast and bacterial populations according to DNA sequence-based analyses. Strains of (pectinolytic) Saccharomyces cerevisiae, Pichia manshurica, Pichia kudriavzevii, and Candida boidinii isolated from directly brined olives were then inoculated into laboratory-scale olive fermentations to quantify the effects of individual yeast strains on the olives. The pH, titratable acidity, and numbers of lactic acid bacteria (LAB) and yeasts varied between the fermentations and fermentations inoculated with P. kudriavzevii and C. boidinii promoted the development of LAB populations. Olive tissue structural integrity declined significantly within 30, 74, and 192 days after the inoculation of pectinolytic S. cerevisiae, P. manshurica and C. boidinii, respectively. In comparison, tissue integrity of olives in control fermentations remained intact although pectinolytic yeasts were present. Notably, pectinolytic yeasts were not found in fermentations inoculated with (non-pectinolytic) P. kudriavzevii and olives exposed to a 1:1 ratio of P. kudriavzevii and P. manshurica exhibited no significant tissue defects. This study showed that pectinolytic yeast are important components of directly brined green olive fermentations and damage caused by pectinolytic yeasts might be prevented by other microbial colonists of the olives.     

Indigenous wine killer yeasts and their application as a starter culture in wine fermentation

Wine yeast strains were isolated from seven fermentations of the red wines ‘Refošk’ and ‘Teran’, produced in the southwestern part of Slovenia. Among 613 isolated yeast strains, 22 expressed killer activity against the supersensitive strain Saccharomyces cerevisiae. Killer strains were isolated at different stages of wine fermentation but did not dominate in any of them. Species identification was based on the combination of RFLP analysis of an amplified rDNA region and biochemical–physiological tests. Killer isolates were identified as S. cerevisiae, Pichia anomala, Pichia kluyveri, Pichia pijperi, Hanseniaspora uvarum and Candida rugosa. Electrophoretic karyotyping was used to differentiate strains of the same species. Fermentation properties of four S. cerevisiae strains that possessed stable killer activity were characterized in fermentations of Malvasia must by studying their population dynamics and chemical composition and by sensory analysis of the wines produced. In order to compare the results, spontaneous fermentations and fermentations induced by commercial yeast starters were performed concomitantly. The local killer strain S_S12/10 showed the best fermentation properties and produced wine with favourable characteristics.     

Using a polynomial model for fungi from table olives

Table olives are one of the most important fermented vegetables in the food industry because of their worldwide economic importance. Enterobacteriaceae, lactic acid bacteria and yeasts are among the most relevant microorganisms involved in olive fermentation determining safety, quality and flavour of the final product. Fungi can play a double role during table olive processing acting as spoilage or desirable microorganisms. This article addresses the effects of some factors (pH, NaCl, temperature, phenols) on the growth/survival of yeasts and moulds, both some wild isolates from olive brine and some yeasts from a public collection (Pichia guilliermondii, P. holstii, Wickerhamomyces anomalus). The research was divided into two steps: a 1st step was aimed at assessing fungal growth by evaluating separately each factor; in a 2nd step, NaCl, pH and temperature were combined through a simple DoE (design of experiment). For this last step, W. anomalus and P. guilliermondii were used as test fungi. Generally, fungi were not inhibited either by temperature, salt or acidic/alkaline pHs. Moreover, the approach of DoE pinpointed that the effect of some factors could experience a shift and change over the time.     

Biodiversity of Yeast Mycobiota in “Sucuk,” a Traditional Turkish Fermented Dry Sausage: Phenotypic and Genotypic Identification,Functional and Technological Properties

In this study, yeasts from Turkish fermented sucuks were identified and their functional and technological properties were evaluated. Two hundred fifty‐five yeast isolates were obtained from 35 different sucuk samples from different regions of Turkey. The yeast isolates were determined as genotypic using 2 different polymerase chain reaction (PCR) methods (rep‐PCR and RAPD‐PCR). Functional and technological properties of including proteolytic, lipolytic, and catalase activities, tolerance to NaCl and bile, as well as growing rates at different temperature and pH conditions selected yeast strains were also evaluated. Candida zeylanoides and Debaryomyces hansenii were dominant strains in sucuk samples. All C. zeylanoides and D. hansenii tested could grow at the condition of 15% NaCl and 0.3% bile salt. However, none of the strains were able to grow at 37 °C, even though catalase activity, weak proteolytic and lipolytic activities was still observed. D. hansenii were able to grow only at pH 3, while some of C. zeylanoides could grow at lower pH levels (pH 2). Three and 4 strains of C. zeylanoides showed β‐hemolysis activity and nitrate reduction ability to nitrite, respectively. D. hansenii did not have properties, which are β‐hemolysis, nitrate reduction, or hydrogen sulfide production. Overall, diverse yeast mycobiota present in Turkish fermented sucuk and their functional and technological properties were revealed with this study.     

The effect of pH,sodium chloride,sucrose, sorbate and benzoate on the growth of food spoilage yeasts

The effects of pH, concentration of NaCl, concentration of sucrose and concentrations of sorbic and benzoic acids on growth were examined for 30 strains of food spoilage yeasts, representingDebaryomyces hansenii, Yarrowia lipolytica, Kloeckera apiculata, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia anomalaandSaccharomyces cerevisiae.Zygosaccharomyces bailiidid not grow at pH 7.0 andZ. rouxii, Kl. apiculataandP. membranaefaciensdid not grow at pH 8.0. OnlyKl. apiculatagrew at pH 1.5–2.0. The remaining species grew at pH 2.5–8.0. None of the species grew at 20% NaCl but strains ofD. hansenii, Z. rouxiiandP. anomalagrew at 15% NaCl.S. cerevisiaeandK. marxianuswere the least salt tolerant, showing no growth at 7.5% NaCl and 10% NaCl, respectively. Medium pH influenced growth in the presence of NaCl. ForY. lipolytica, D. hanseniiandS. cerevisiae, greatest tolerance of NaCl occurred at pH 5.0–7.0, but forKl.apiculata, D. membranaefaciensandZ. bailiibest tolerance occurred at pH 3.0.K. apiculatagrew in the presence of 12.5% NaCl at pH 2.0. All yeasts grew at 50% sucrose, withZ. rouxii, Z. bailiiandP. anomalaandD. hanseniigrowing at 60–70% sucrose. Medium pH in the range 2.0–7.0 had little effect on ability to grow in the presence of high sucrose concentrations.Z. bailiiandY. lipolyticawere the species most tolerant of sorbate and benzoate preservatives (750–1200 mg l⁻¹) at pH 5.0.