Selection of Yeasts as Starter Cultures for Table Olives

Ninety‐nine yeasts were isolated from Bella di Cerignola table olives; first, the strains were studied in relation to their ability to produce biogenic amines in a laboratory medium and 49 strains were positive to this assay and cut off from the research. The remaining 50 strains were characterized for their enzymatic traits (β‐glucosidase, catalase, pectolytic, xylanolytic, and lipolytic activities) and for their ability to grow at different temperatures, pHs, with salt or lactic/acetic acids added. Data were used for the evaluation of growth index and submitted to cluster and principal component analyses to choose the most promising 4 strains. In the final step of the research, the strains were inoculated as a cocktail in a model brine, containing different amounts of salt (4% to 12%) and glucose (0% to 3%), and adjusted to different pHs (4.0 to 9.0). Data analysis through a multiple regression procedure highlighted that salt, glucose, and pH acted in a different way within the storage and NaCl affected yeast growth only for few days, and then glucose and pH played a major role. Practical Application Olive fermentation relies upon a complex microflora, including lactic acid bacteria and yeasts; the selection of suitable strains of yeasts intended as starter cultures, as well as their inoculation in brines, could improve the fermentation.     

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.     

Controlled Fermentation of Spanish-type Green Olives

Pure culture fermentation of Spanish-type green olives was developed. The method used no heat treatment, included chlorination of both fermentor and olives, used sterile lye, water and brine, and acidification with lactic acid before inoculation. Lactobacillus pluntarum was used as test species. After 34 days fermentation, citric acid, mannitol and malic acid were completely degraded and ～ 90% of available glucose and fructose, but <30% sucrose, were utilized. Fermentation products were D- and L-lactic acid, ethanol, succinic, and acetic acid with a calculated carbon recovety of 107.5%. D-lactic predominaied over L-lactic acid. No differences were found between flavor of pure culture and naturally fermented olives, but there was a tendency towards preference of the latter.     

Evolution and perspectives of cultivar identification and traceability from tree to oil and table olives by means of DNA markers

In recent years, an increasing number of typicality marks has been awarded to high‐quality olive oils produced from local cultivars. In this case, quality control requires effective varietal checks of the starting materials. Moreover, accurate cultivar identification is essential in vegetative‐propagated plants distributed by nurseries and is a pre‐requisite to register new cultivars. Food genomics provides many tools for cultivar identification and traceability from tree to oil and table olives. The results of the application of different classes of DNA markers to olive with the purpose of checking cultivar identity and variability of plant material are extensively discussed in this review, with special regard to repeatability issues and polymorphism degree. The characterization of olive germplasm from all countries of the Mediterranean basin and from less studied geographical areas is described and innovative high‐throughput molecular tools to manage reference collections are reviewed. Then the transferability of DNA markers to processed products – virgin olive oils and table olives – is overviewed to point out strengths and weaknesses, with special regard to (i) the influence of processing steps and storage time on the quantity and quality of residual DNA, (ii) recent advances to overcome the bottleneck of DNA extraction from processed products, (iii) factors affecting whole comparability of DNA profiles between fresh plant materials and end‐products, (iv) drawbacks in the analysis of multi‐cultivar versus single‐cultivar end‐products and (v) the potential of quantitative polymerase chain reaction (PCR)‐based techniques. © 2016 Society of Chemical Industry     

DNA分子标记技术在酵母菌鉴定中的研究进展

介绍了RFLP、RAPD、AFLP以及微卫星DNA(Micro-satellite)4种DNA分子标记技术的方法和原理,及近年来在应用国内外酵母菌鉴定中的研究进展,指出了各种技术的优缺点.     

Analysis of Thermal Processing of Table Olives Using Computational Fluid Dynamics

In the present work, the thermal processing of table olives in brine in a stationary metal can was studied through computational fluid dynamics (CFD). The flow patterns of the brine and the temperature evolution in the olives and brine during the heating and the cooling cycles of the process were calculated using the CFD code. Experimental temperature measurements at 3 points (2 inside model olive particles and 1 at a point in the brine) in a can (with dimensions of 75 mm × 105 mm) filled with 48 olives in 4% (w/v) brine, initially held at 20 °C, heated in water at 100 °C for 10 min, and thereafter cooled in water at about 20 °C for 10 min, validated model predictions. The distribution of temperature and F‐values and the location of the slowest heating zone and the critical point within the product, as far as microbial destruction is concerned, were assessed for several cases. For the cases studied, the critical point was located at the interior of the olives at the 2nd, or between the 1st and the 2nd olive row from the bottom of the container, the exact location being affected by olive size, olive arrangement, and geometry of the container.     

Antioxidant Effect of Natural Table Olives Phenolic Extract Against Oxidative Stress and Membrane Damage in Enterocyte‐Like Cells

The phenolic fraction of a naturally fermented cultivar of table olives, “Tonda di Cagliari,” was investigated for the ability to protect Caco‐2 cells against oxidative stress and membrane damage induced by tert‐butyl hydroperoxyde (TBH). TBH exposure resulted in an alteration of cellular redox status, with an increase in reactive oxygen species (ROS) and a decrease in reduced glutathione (GSH) level. A loss of the epithelial integrity, as indicated by the decrease of the transepithelial electrical resistance value, was also observed over time, together with an intense lipid peroxidation process. The olives phenolic extract significantly counteracted ROS generation and subsequent alteration of monolayer integrity and membrane oxidative damage. The protective action of the extract is likely due to the scavenging ability of its main components, as hydroxytyrosol, oleuropein, and verbascoside among the secoiridoids and derivatives. Since olives phenolic compounds concentrate in the intestinal lumen, they may be a useful tool in the prevention of intestinal disorders related to oxidative damage.     

食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。     

Molecular Identification of Yeasts Associated with Traditional Egyptian Dairy Products

ABSTRACT:  This study aimed to examine the diversity and ecology of yeasts associated with traditional Egyptian dairy products employing molecular techniques in yeast identification. A total of 120 samples of fresh and stored Domiati cheese, kariesh cheese, and "Matared" cream were collected from local markets and examined. Forty yeast isolates were cultured from these samples and identified using the restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region and sequencing of the domains D1 and D2 of the 26S rRNA gene. Yeasts were identified as  Issatchenkia orientalis  (13 isolates),  Candida albicans  (4 isolates),  Clavispora lusitaniae  ( Candida lusitaniae ) (9 isolates),  Kodamaea ohmeri  ( Pichia ohmeri ) (1 isolate),  Kluyveromyces marxianus  (6 isolates), and  Candida catenulata  (7 isolates). With the exception of  C. lusitaniae , the D1/D2 26S rRNA gene sequences were 100% identical for the yeast isolates within the same species. Phylogenetic reconstruction of  C. lusitaniae  isolates grouped them into 3 distinguished clusters. Kariesh cheese was found to be the most diverse in its yeast floras and contained the highest total yeast count compared with other examined dairy products. This was linked to the acidic pH and lower salt content of this cheese, which favor the growth and survival of yeasts in foodstuffs. Stored Domiati cheese also contained diverse yeast species involving isolates of the pathogenic yeast  C. albicans . This raises the possibility of dairy products being vehicles of transmission of pathogenic yeasts.     

自然发酵哈密瓜果汁中酵母分离与鉴定

从自然发酵的哈密瓜汁中分离得到25株形态较好的酵母菌株,经过初筛、复筛,得到1株产酒精能力好,产气较快,最适温度28℃,能耐受150mg／L的SO2,耐浓度为15％的酒精的酵母菌株HM02。经鉴定该酵母为酵母属的酿酒酵母（Saccharomyces cerevisive）。     

橙汁腐败酵母菌的分子鉴定及其形态特征分析

目的：结合多种酵母菌鉴定方法,分析腐败橙汁中酵母菌的种类,为快速检测和控制商品橙汁中酵母菌污染奠定基础,也为酵母菌种类的快速鉴定提供参考。方法：以分离自腐败橙汁的8株酵母菌株为材料,观察菌落和细胞形态。用引物ITS1/ITS4扩增菌株5.8S rDNA 及其ITS间隔区(5.8S-ITS),对扩增产物用HhaⅠ、Hae Ⅲ 和 HinfⅠ进行限制性酶切片段多态性(RFLP)分析和测序;用引物NL1/NL4扩增26S rDNA D1/D2区并测序。结果：菌落特征观察将菌株初分为6组,细胞学观察粗分为3组,分子方法都分成4组。用限制性内切酶HhaⅠ、Hae Ⅲ和HinfⅠ对5.8S-ITS区产物进行 RFLP分析,观察到4种不同的图谱类型。5.8S-ITS 区和26S rDNA D1/D2区的序列分析结果相似,8株分离菌与GenBank中的4种酵母菌参考菌株序列一致性达99%以上。分离株与参考菌株以两区序列构建的NJ系统树都分成4枝：Y1与克鲁维毕赤酵母(Pichia kluyveri),Y12-3、Y18-1与发酵毕赤酵母(Pichia fermentans),Y22、Y23、Y26和Y56与Meyerozyma guilliermondii,Y47与Wickerhamomyces anomalus分别聚为一枝。结论：结合形态分析和核酸分析,将8株腐败橙汁分离菌鉴定为P. kluyveri var. kluyveri、P. fermentans、M. guilliermondii和W. anomalus 4个种,其中M. guilliermondii在腐败橙汁中首见报道;ITS- RFLP分析、26S rDNA D1/D2区测序与菌株形态特征结合能有效鉴定酵母菌,核糖体DNA分析可鉴定酵母到生物学种,菌落形态特征可反映种以下的遗传差异,因此,采用分子方法鉴定时不能忽视形态特征分析的重要性。     

降解苹果酸酵母菌的筛选及鉴定

使用YPD培养基从猕猴桃果园的土壤、猕猴桃叶片、猕猴桃果浆和猕猴桃酒酒渣中筛选出酵母菌,通过传代培养及镜检获得纯培养物。用苹果酸降解指示培养基厌氧培养,通过培养液蓝色的深浅筛选出能降解苹果酸的酵母菌,检测酵母菌的降酸能力,进行分子生物学鉴定和同源性分析,再测试酵母菌对酒精、SO2、酸、糖的耐受性,最终得到具有较强苹果酸降解能力的酵母菌株ZJ22、PJ34、ZG13,对苹果酸的降解比例分别为28.36%、25.92%和25.68%。经鉴定,菌株ZJ22和菌株PJ34是酿酒酵母,菌株ZG13是毕赤酵母。耐受性试验表明:酿酒酵母-ZJ22对酒精、SO2、酸和糖的耐受性较好,具有潜在使用价值。     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

Genetic polymorphism of kappa‐casein,growth hormone and prolactin genes in Turkish native cattle breeds

A total of 259 cattle of four Turkish native cattle breeds, East Anatolian Red (EAR), South Anatolian Red (SAR), Turkish Grey (TG) and Anatolian Black (AB), Holstein and Brown Swiss (BS) breeds were genotyped for kappa‐casein (CSN3), bovine growth hormone (GH1) and prolactin (PRL) polymorphism by the polymerase chain reaction and restriction length polymorphism (PCR‐RFLP). The degree of genetic differentiation between populations F_ST was calculated as 0.053 and was found to be significant (P < 0.001). According to the genetic distance values (Nei), the highest genetic difference was found between SAR and EAR in four Turkish native cattle breeds and this difference was significant.     

低产尿素与高级醇黄酒酵母菌株的筛选、鉴定与发酵

通过发酵实验比较分离得到的5 株酵母菌株产尿素与高级醇的能力,筛选出低产尿素和高级醇的菌株AW001和J-16-2,并利用实时定量聚合酶链式反应技术验证,5 株酵母菌的尿素产量与精氨酸吸收有关的CAR2基因拷贝数呈正相关。通过生理生化以及rDNA ITS序列的分析,鉴定其均为酿酒酵母。结合实验室模拟黄酒发酵实验的理化指标检测结果表明,菌株AW001、J-16-2发酵所得样品中总糖、总酸等含量均满足国标中黄酒发酵指标。因此,该2 株菌为特性优良的酿酒酵母,有进一步开发利用的价值。     

浓香型白酒发酵旺盛期糟醅中酵母菌菌群研究

充分解析并挖掘浓香型白酒糟醅中的酵母菌株资源,对五粮液发酵旺盛期中的糟醅进行高通量测序以及纯培养。其中通过高通量测序技术共检测出11个种属的酵母菌群,包括:Kazachstania exigua、Saccharomyces cerevisiae、Kazachstania humilis、Pichia kudriavzevii、Zygosaccharomyces bailii、Saccharomycopsis fibuligera、Trichosporon ovoides、Debaryomyces hansenii、Naumovozyma castellii、Pichia manshurica、Geotrichum silvicola。利用7种培养基筛选五粮液发酵旺盛期糟醅中的酵母菌株,通过合并共获得57株纯培养酵母菌株,通过26S rDNA序列鉴定,发现其分属于Pichia kudriavzevii、Saccharomyces cerevisiae、Candida glabrata和Candida rugosa。结果表明:浓香型白酒发酵旺盛期糟醅中的酵母群体多样性丰富,但与高通量测序相比,纯培养法仅能分离到糟醅酵母群体中的少数种属,因此要充分挖掘浓香型白酒糟醅中的酵母菌株资源,还需进一步优化分离、培养方法。     

羊羔美酒大曲中酵母菌多样性及分子鉴定

目的：分离和鉴定羊羔美酒大曲中的酵母菌,探寻酵母菌群多样性组成,为深入研究羊羔美酒的风味特征奠定基础。方法：对羊羔美酒大曲实施多点采样、混合研磨、无菌水梯度稀释、平板画线分离、挑取酵母菌单菌落。酵母菌的形态鉴定采取菌落特征和显微细胞特征结合的方法,分子鉴定采用5.8S rDNA-ITS区域限制性内切酶片段长度多态性（restriction fragment length polymorphism,RFLP）分析及序列分析法。结果：从羊羔美酒大曲中共分离出474 株酵母菌,传统形态学鉴定为14 种形态类型,经5.8S rDNA-ITS区域RFLP分析法区分为6 种分子类型。经基因序列分析,将其鉴定为分属于6 个属的6 种酵母菌,分别为：异常毕赤酵母（Pichia anomala）、酿酒酵母（Saccharomyce cerevisia）、阿氏丝孢酵母（Trichosporon asahii）、黏质红酵母（Rhodotorula mucilaginosa）、浅白隐球酵母（Cryptococcus albidus）、东方伊萨酵母（Issatchenkia orientalis）。结论：羊羔美酒大曲中酵母菌多样性丰富,除酿酒酵母外,还含有多种酵母菌辅助代谢产生各种风味物质,其中酿酒酵母为主要优势菌群。     

新疆石榴酒专用酵母的选育及应用研究

以石榴树叶、石榴皮及石榴园土壤为分离源,共分离到71 株酵母菌,经过三级筛选,获得两株适宜酿造石榴果酒的酵母菌,编号分别为SL18 和SL20 。发酵性能测试结果表明,SL20 菌株在25℃时的发酵周期比对照菌株和SL18 少1d,酒精度达到9.7%(V/V),并且所酿造的石榴酒香味浓郁纯正,而SL18 在25℃时虽然发酵周期与对照菌株相同但所酿造的石榴酒香味比对照菌株要浓郁纯正。30℃时,3 株菌起酵速度都比较快,SL20 和SL18 的发酵周期比对照菌株少1d,SL20 菌株的酒精度高于SL18 和对照菌株,为10.1%(V/V),但3 株菌发酵的石榴酒香味没有20℃和25℃浓郁,说明自选菌株SL20 和SL18 适于25℃发酵并且所酿制的石榴酒明显优于常用的酿酒活性干酵母,并且两株菌能耐受12%(V/V)的酒精度,经鉴定均为酵母属(Saccharomyces) 的酿酒酵母(Saccharomyces cerevisive) 。     

Determination of allele frequencies of growth hormone AluI polymorphism in Brown Swiss,Holstein, native East Anatolian Red and Turkish Grey breeds

A total of 177 cattle of four breeds were genotyped for the bovine growth hormone (BGH)‐AluI polymorphism by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). The genotype and gene frequencies for each breed were determined and tested to be in Hardy–Weinberg equilibrium. According to breeds, frequencies of allele L gene were 0.905 for Brown Swiss, 0.898 for Holstein, 0.976 for East Anatolian Red and 0.893 for Turkish Grey Breeds. The allele L was predominant and variant VV was not detected in the breeds studied. BGH‐AluI genotypes were found to be in equilibrium within and among breeds.     

产2-苯乙醇酵母的鉴定及其在酱油发酵中的应用

研究酱油中特征香气成分2-苯乙醇的合成途径并探索提高酱油发酵醪中2-苯乙醇含量,从酱油发酵醪分离得到1 株产2-苯乙醇酵母菌株922-1。通过形态观察、生理生化测试、转录间隔区序列测序和系统进化树分析鉴定922-1为Candida oceani,C. oceani 922-1可以在高含盐（18% NaCl）培养基中生长;采用唯一氮源实验证明了C. oceani 922-1主要通过艾氏途径,利用苯丙氨酸作为底物合成苯丙氨酸。C. oceani 922-1可以在酱油发酵醪中大量合成2-苯乙醇（44.70 mg/L）,添加1%苯丙氨酸可以将酱油发酵醪中2-苯乙醇质量浓度进一步提高到对照组的17.3 倍,并能一定程度改善发酵生酱油醇香和厚味,显示出C. oceani 922-1在高盐发酵调味品风味改善方面的应用前景。