Apple Aminoacid Profile and Yeast Strains in the Formation of Fusel Alcohols and Esters in Cider Production

The amino acid profile in dessert apple must and its effect on the synthesis of fusel alcohols and esters in cider were established by instrumental analysis. The amino acid profile was performed in nine apple musts. Two apple musts with high (>150 mg/L) and low (<75 mg/L) nitrogen content, and four enological yeast strains, were used in cider fermentation. The aspartic acid, asparagine and glutamic acid amino acids were the majority in all the apple juices, representing 57.10% to 81.95%. These three amino acids provided a high consumption (>90%) during fermentation in all the ciders. Principal component analysis (PCA) explained 81.42% of data variability and the separation of three groups for the analyzed samples was verified. The ciders manufactured with low nitrogen content showed sluggish fermentation and around 50% less content of volatile compounds (independent of the yeast strain used), which were mainly 3‐methyl‐1‐butanol (isoamyl alcohol) and esters. However, in the presence of amino acids (asparagine, aspartic acid, glutamic acid and alanine) there was a greater differentiation between the yeasts in the production of fusel alcohols and ethyl esters. High contents of these aminoacids in dessert apple musts are essential for the production of fusel alcohols and most of esters by aromatic yeasts during cider fermentation.     

Killer strains of Saccharomyces: application for apple wine production

The aim of this study was to evaluate possible application of killer strains Saccharomyces paradoxus CBS 3702 (K1), S. cerevisiae CBS 6505 (K2) and S. capensis CBS 7903 (K3) for apple wine production. Apple musts were obtained from Jonagold apples. The optimal pH value of killer toxins activity, the temperature of their formation and the spectrum of activity against selected wine spoilage fungal strains were analysed. The influence of yeast strains on the chemical composition, volatile profile and sensory properties of apple wines was determined using high‐performance liquid chromatography and gas chromatography methods. The pH values of 4.2 (K1, K3) and 4.6 (K2), and a temperature of 28 °C, were optimal for the action of toxins. The analysed killer strains inhibited growth of seven of the 11 tested wine spoilage fungal strains. The samples obtained using the S. cerevisiae killer strain were characterized by the highest fermentation rate, highest ethanol concentration and a balanced level of volatiles, but their taste was described as bitter. S. capensis gained the highest scores in the sensory evaluation. It could be used for semi‐sweet or semi‐dry cider production, because of the lower amounts of ethanol formation but higher synthesis of glycerol, volatile esters and higher alcohols. Copyright © 2016 The Institute of Brewing & Distilling     

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.     

产香酵母的筛选及其苹果酒发酵特性

从分离自赤霞珠葡萄酿酒不同时期发酵液中的酵母菌中筛选产香菌株,并对其苹果酒发酵特性进行研究。结果表明,经感官评定初筛,获得7株产香较优的酵母菌,并对其进行耐酒精、耐SO2和耐盐性能分析,其中菌株B2-13具有较好的发酵性能,经分子生物学鉴定菌株B2-13为葡萄酒有孢汉逊酵母（Hanseniaspora uvarum）。菌株B2-13发酵苹果酒酒精度为4.2%vol～5.5%vol,糖度为2.6～44 g/L,共鉴定出32种香气成分,主要有酯类（6种）、醛酮类（4种）、醇类（6种）、酸类（8种）等,其中异戊醇、苯乙醇含量最高,分别为0.36 mg/mL和0.16 mg/mL,另外还有赤霞珠干红葡萄酒的特有香气物质3-羟基-2-丁酮（0.16 mg/mL）。菌株B2-13是一株具有较好的苹果酒发酵和产香性能的酵母菌株,有良好的工业化生产应用前景。     

模糊综合评判结合响应面法优化苹果酒发酵工艺

采用模糊综合评判结合响应面法优化苹果酒发酵工艺参数,并用顶空固相微萃取气质联用（HS-SPME/GC-MS）技术分析其香气成分。以红富士苹果为原料,结合单因素试验结果,选取了酵母接种量、初始糖度、初始pH值三个主要工艺参数,采用中心组合试验设计,以苹果酒品质的模糊评判结果为目标,构建主因素突出型综合评判模型,进行响应面法分析。得到苹果酒最佳发酵工艺条件为：初始糖度19 g/100 mL,初始pH值3.5,接种量10%,发酵温度22 ℃。此条件下的苹果酒颜色金黄,酒体澄清有光泽,口感丰满协调、风格良好。苹果酒中共检测到33种香气成分,结合气味活性值（OAV）法鉴定出10种关键香气组分,这些香气物质构成了苹果酒的独特的风味。     

UV INACTIVATION OF E. COLI O157:H7 IN APPLE CIDER: QUALITY, SENSORY AND SHELF-LIFE ANALYSIS

Increasing concern about food safety following contamination of unpasteurized apple cider with Escherichia coli O157:H7 reinforces the need for using the best technologies in apple cider production. Pasteurization of apple cider with ultraviolet irradiation (UV) is a low‐cost alternative to heat pasteurization for small processing operations. UV treatment efficacy applied to raw unpasteurized apple cider was examined through evaluation of physical parameters, exposure time and treatment dosage. A UV light processing system was used to treat apple cider. The apple cider received a calculated average dosage of 8777 µW‐s/cm ² per pass through the system. UV light (at 254.7 nm) was effective in reducing bacteria‐inoculated apple cider by an average of 2.20 logs per pass. In multiple passes, the 5‐log reduction mandated by the Food and Drug Administration was achieved. Sensory analysis yielded no significant differences between the UV‐treated and control apple ciders. Experiments with UV‐treated apple cider indicated a significant extension of product shelf life through inhibition of yeast and mold growth. For low throughput apple cider processing operations, this technology is a viable cost‐effective alternative.     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

优良野生葡萄酒酵母的筛选及性能评价

以河北昌黎葡萄酒产区4个不同品种的葡萄为野生酵母分离源,分离到了58株酵母。以此作为出发菌株,通过酒精耐受性试验、SO2耐受性试验、模拟发酵试验,以发酵力、发酵速率、糖利用率等为标准初步筛得4株具有应用价值的野生酵母。用这4株酵母在酒厂进行了小型的葡萄酒发酵试验,将所得酒样进行理化分析及感官品评,并采用气-质联用仪(GC-MS)分析其香气成分,最终获得了对这4株酵母发酵性能的综合评价,认为其中2株具有应用于生产的潜能。     

香格里拉本土酿酒酵母冰酒发酵产香特征分析

选取云南香格里拉葡萄酒产区4株耐受性好的本土酿酒酵母（Saccharomyces cerevisiae）（编号为CZ10、CZ11、BZL10、BZL12）及目前被广泛用于冰葡萄酒发酵的商业酵母DV10进行赤霞珠冰酒发酵,利用顶空固相微萃取气质联用（HS-SPME-GC-MS）技术检测4株酵母与商业酵母DV10发酵赤霞珠冰酒产品的挥发性香气物质,结合感官评定比较其品质优劣。结果表明,5个试验样品中共检测到67种挥发性化合物,主要包括醇类、脂肪酸乙酯类、乙基酯类、酸类、酮类、萜烯类。其中酵母BZL12生成的香气物质种类最多（62种）,含量最高为490.02 mg/L,其中酯类和醇类总量均分别高于商业酵母DV10 1.61%和4.52%。酵母BZL12发酵葡萄酒的感官评分最高（95分）,理化指标满足相关国标要求,具有生产不同风味特征葡萄酒的应用潜力。     

Genome comparison and evolutionary analysis of different industrial lager yeasts (Saccharomyces pastorianus)

Fermented beverages, especially beer, have accompanied human civilizations throughout our history. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. For hundreds of years, lager yeast (Saccharomyces pastorianus) has been subjected to multiple rounds of domestication owing to artificial selection during beer production. As a result, this species comprises a genetically diverse collection of strains that are used in different breweries. However, the scope of genetic diversity captured during the domesticated evolution of this species remains to be determined. To begin to address this, we collected the genome information of the only four lager strains that had been whole‐genome sequenced. For the first time, genome comparison was conducted between lager yeasts and clear signatures were found that defined each industrial yeast strain. The genetic variation comprises both single nucleotide polymorphisms and insertions and deletions. In addition, the core–pan genome was introduced for the first time to the genomic analysis of lager yeasts, detecting numerous strain‐specific and species‐shared genes. Furthermore, phylogenetic tree and synteny analysis results obtained in this study revealed information regarding the evolutionary relationship and group differentiation of studied strains. Genome comparison of the lager strains will, therefore, enable the characterization of the overall genetic diversity of this species, assist in the identification of genomic loci that play important roles in regulating key industrial phenotypes, and highlight the understanding of the hybrid nature and evolutionary details. Copyright © 2016 The Institute of Brewing & Distilling     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Sulphite binding in ciders

Summary The extent of sulphite binding was measured in commercial ciders, and experimentally observed binding curves were compared with theoretically derived curves based on assessment of the levels of individual sulphite binding compounds determined in the ciders. Subsequently, experimental ciders were fermented under various controlled conditions using nine different strains of cider yeasts. The results indicated considerable differences both in the levels of sulphite binding compounds produced, and in the ability of different yeast strains to produce SO₂ in the cider. Juice concentration and the presence of cloud in juice had little or no effect on sulphite binding. Other factors which affected sulphite binding included the type and condition of juice (especially the effect of pectinase treatment) and, in some instances, the use of added yeast nutrients. Significant sulphite binding was also attributable to unaccountable components, probably derived from poor quality fruit, which were present in the apple juice prior to fermentation.     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.     

甘蔗酒酿造酵母的筛选

选择常用商业果酒酵母菌株KD、DV10、Q23、EC1118、安琪和H7Y7作为供试酵母菌株,以川蔗17为原料发酵生产甘蔗酒,通过分析发酵液中糖含量变化情况及酒精度、澄清度,比较不同菌株发酵特性,结果发现EC1118菌株发酵彻底,产酒能力最强,发酵后甘蔗酒的高级醇含量最低;H7Y7菌株发酵的甘蔗酒中酯类物质最高,为其他菌株的3～6倍;安琪酵母发酵液最易澄清,但其发酵后的甘蔗酒中甲醇含量为其他菌株的5倍以上。感官评分结果显示,不同酵母菌株发酵的甘蔗酒感官评分依次为H7Y7EC1118DV10安琪酵母Q23KD。研究结果表明,供试酵母菌株中EC1118和H7Y7更适宜用于甘蔗酒酿造。     

Microbiological and Fermentative Properties of Baker's Yeast Starter Used in Breadmaking

This study assessed the levels of microbial contaminants in liquid, compressed and dry commercial baker's yeasts used as starters in breadmaking. Eumycetes, Enterobacteriaceae, total and fecal coliforms, Bacillus spp., and lactic acid bacteria (LAB), in particular enterococci, were quantified. Results obtained in this study highlighted that baker's yeast could represent a potential vehicle of spoilage and undesirable microorganisms into the baking environment, even if these do not influence the leavening activity in the dough, as ascertained by rheofermentometer analysis. Different microbial groups, such as spore‐forming bacteria and moulds, were found in baker's yeast starters. Moreover, different species of LAB, which are considered the main contaminants in large‐scale yeast fermentations, were isolated and identified by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA sequencing. The most recurrent species were Lactobacillus plantarum, Enterococcus faecalis, and Enterococcus durans, isolated from both compressed and dry starters, whereas strains belonging to Leuconostoc and Pediococcus genera were found only in dry ones. Nested‐Polymerase Chain Reaction (Nested‐PCR) and Randomly Amplified Polymorphic DNA–PCR (RAPD‐PCR) were also used to highlight the biodiversity of the different commercial yeast strains, and to ascertain the culture purity.     

Analysis of key aroma components in cider from Shaanxi (China) Fuji apple

Aroma components play an important role in the quality of cider. To understand the contribution of every component to cider aroma quality, major aroma components present in ten ciders processed with different technological methods with different yeasts and apple orchards were determined by Headspace-solid phase microextraction–gas chromatography–mass spectrometry and internal standard method. Experimental results by principal component analysis indicated that ethyl acetate, acetic acid isobutylester, isopentylacohol acetate, ethyl caprylate, ethyl 4-hydroxybutanoate, isopentylalcohol, 3,4,5-trimethyl-4-heptanol, nonyl alcohol, 3-methylthio-1-propanol serve as the key aroma components in cider from Shaanxi (China) fuji apple. A model was established and verified for evaluating aroma quality based on factor analysis by comparing with the sensory evaluation method.