Modeling the effects of initial nitrogen content and temperature on fermentation kinetics of hard cider

Hard apple cider manufactured as a value-added product will increase consumption of excess American apples and give impetus to local industry. For larger scale productions, a mechanistic model that could predict the fermentation kinetics would be a useful tool for understanding and designing processes. Hence, the objective of this study was to apply a simple mechanistic model to predict fermentation kinetics of hard cider from Michigan apples and study the combined effect of initial nitrogen and temperature on fermentation. Kinetic parameters of the primary mechanistic model based on Monod kinetics were estimated non-linearly via the Runge-Kutta method. Four coupled ordinary differential equations were used for each of the four dependent variables, yeast cell concentration, nitrogen, sugar and ethanol concentration. For the secondary model, it was proposed to model the effect of temperature and initial nitrogen content on the kinetic parameters of the primary model with an Arrhenius relationship. The current study is the first known application of a mathematical model to the hard cider fermentation process.     

Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22′ and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of β-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3′ and 19′) to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with β-glucosidase activity are reported for the first time in autochthonous cider yeasts.     

Saccharomyces cerevisiae associated with the spontaneous fermentation of tequila agave juice

Saccharomyces cerevisiae dominates the spontaneous fermentation of blue agave juice. Because of the batch heterogeneity, the aim of this work was to determine the strain diversity of S. cerevisiae among fermentations. During January and February 2015, agave juice was sampled in triplicate from four sampling points at a tequila distillery. The heterogeneity of yeast strains and the production of carbon dioxide were assessed during fermentation, whereas the amount of ethanol produced was measured at the end of the process. The fermentation cycle times varied widely (9 to 25 days), as did fermentation efficiency (2.5–45.5%). Yeast isolates were identified at the species level by ITS‐5.8S rRNA restriction fragment length polymorphism and differentiated at the strain level by random amplified polymorphic DNA. A total of 199 isolates were obtained and identified as S. cerevisiae, showing 69 different random amplified polymorphic DNA profiles. There was no clear dominance of any strain during fermentation. However, two strains (P1 and P2) were detected in all fermentation samples, suggesting their residency in the distillery, despite the deep‐cleaning applied to the tanks after each fermentation batch. According to the RAPD profiles, the number of strains isolated from fermentation samples increased from 17 in January to 25 in February. © 2018 The Institute of Brewing & Distilling     

茶树花苹果酒的发酵工艺研究

将茶树花加入苹果汁中进行发酵,研究茶树花苹果酒发酵工艺。在单因素实验的基础上,应用响应面分析法对茶树花苹果酒的发酵条件进行了优化,并确定最佳的发酵条件。结果表明,茶树花的添加量为苹果汁的1.0%(w/v)、酵母接种量12.5%(v/v)、初始糖度200g/L、果胶酶0.08g/L、温度27.0℃时,所制茶树花苹果酒色泽金黄,澄清透明无沉淀,具有浓郁的茶花香、酒香和果香,口感柔和,酒体醇和协调;各理化指标为:酒精度10.7%,可溶性固形物5.0%,还原糖2.32g/L,pH3.66,杂醇油397.89mg/L;是一种既风味独特又健康养生的低度果酒。      

Screening of cider yeasts for sparkling cider production (Champenoise method)

A total of 350 colonies isolated from a cider cellar in Asturias (Spain) were identified by rDNA ITS-RFLP restriction analysis. Saccharomyces spp. strains were characterized by mitochondrial DNA (mtDNA) restriction analysis. Fifty-four different Saccharomyces spp. strains were identified and tested to ascertain their capacity to carry out secondary fermentation of sparkling ciders. The screening of yeasts to determine their principal enological characteristics (tolerance to ethanol, production of volatile acidity and hydrogen sulphide) was accomplished by means of rapid, non-expensive assays (plate agar). As a result, 13 (24%) of the 54 initial Saccharomyces spp. yeast strains were eliminated. The technological properties assessed were flocculation capacity, ethanol and sulphite tolerance, and production of major volatiles. Ten Saccharomyces cerevisiae strains were characterized as true flocculants; all of these strains were able to grow in ethanolic medium and in the presence of 200mg/l of sulphite. Applying cluster analysis to the production of amyl alcohols, isobutanol, propanol and 2-phenylethanol, the strains were classified in two natural groups. Two flocculent yeast strains referred to as 3' and 50', representative of the each statistical group, were selected together with two reference strains (Saccharomyces bayanus C6 and S. cerevisiae Levuline CHP) to elaborate four sparkling ciders by the Champenoise method. The analysis of variance (p<0.01) among ciders revealed that glycerol, acetaldehyde, ethyl acetate, methanol, propanol, i-butanol and 2-phenylethanol were significantly influenced by the secondary yeast strain. The results of sensory analysis indicated that all the sparkling ciders were scored as good. No significant differences among sparkling ciders were found for odour attributes and taste intensity.     

苹果酒发酵工艺对比研究

利用德国生产的5L全自动发酵罐和0.5L三角瓶,以5个栽培苹果品种的果实(‘鲁加1号’、‘鲁加4号’、‘鲁加5号’、‘国光’、‘新红星’)为原料,发酵温度设定为20℃,酵母菌接种量为5%,进行苹果酒发酵工艺初步的放大试验。测定5个品种在陈酿过程中的酒精度、单宁、总酚、可滴定酸、透光率、可溶性固形物变化情况。结果表明,当发酵体积扩大10倍后,发酵罐中苹果酒发酵效果与三角瓶中小体积状态下的发酵效果无显著差异,总的变化趋势趋于一致。     

Determination of organic acids evolution during apple cider fermentation using an improved HPLC analysis method

An efficient method for analyzing ten organic acids in food, namely citric, pyruvic, malic, lactic, succinic, formic, acetic, adipic, propionic and butyric acids, using HPLC was developed. Boric acid was added into the mobile phase to separate lactic and succinic acids, and a post-column buffer solution [5 mmol/L p-toluensulfonic acid (p-TSA) + 20 mmol/L bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (bis–tris) + 100 μmol/L sodium ethylenediaminetetraacetic (EDTA-2Na)] was used to improve the sensitivity of detection. The average spiked recoveries for the ten organic acids ranged from 82.9 to 127.9% with relative standard deviations of 1.44–4.71%. The linear ranges of determination were from 15 to 1,000 mg/L with correlation coefficients of 0.9995–0.9999. The metabolism of organic acids in cider, and the effect of nutrients including diammonium phosphate (DAP), thiamine, biotin, niacinamide and pantothenic acid on their metabolism, were studied using this method of analysis. We found that before cider brewing, additions of 200 mg/L DAP and 0.3 mg/L thiamine to apple juice concentrate results in a high quality cider. F. Zhou and B. Ji contributed equally to this work.     

霞多丽葡萄自然发酵过程中酵母种群的分布与变化规律

采用WL形态学归类分析和5.8S-ITS rDNA PCR-RFLP分子生物学鉴定方法,对我国2个主要产区所产白葡萄品种霞多丽自然发酵过程中的酵母种群进行了系统研究。结果表明,从霞多丽葡萄发酵液中分离到7个酵母菌种,不同产区霞多丽发酵液中分布酵母菌的种类和数量有一定差别,但在发酵过程中酵母种群的变化趋势是一致的。     

苹果酸-乳酸发酵对军果酒风味的影响

介绍了苹果酸 -乳酸发酵 (简称MLF)的机理、苹果酒发酵过程中苹果酸 -乳酸发酵的控制及苹果酸-乳酸发酵对苹果酒风味的影响     

冰葡萄在自然发酵过程中酵母的动态变化

为了探讨云南德钦产区冰葡萄自然发酵过程中酵母的动态变化,采用WL培养基对不同阶段发酵液中的511株酵母进行形态划分。通过分析5.8S rDNA-ITS、18S rDNA以及26S rDNA的D1/D2区基因序列对酵母进行分子鉴定。结果表明:发酵过程中共存在5种酵母,葡萄破碎后立即取样Cryptococcus flavescens,Cryptococcusamylolentus,Hyphopichiapseudoburtonii,分别占50%,16.7%和33.3%。48h后,Cryptococcus amylolentus迅速减少甚至消失,Hyphopichiapseudoburtonii成为优势菌种,同时分离到Hanseniasporauvarum。待发酵启动后,发酵液中只存在Hanseniasporauvarum与Saccharomyces cerevisiae。随着发酵的进行,Hanseniasporauvarum所占的比例呈动态减少趋势。当还原糖下降至172 g/L,酒精含量上升为11.6%时,发酵液中只分离到Saccharomyces cerevisiae,直至发酵结束。     

Yeast community associated with the solid state fermentation of traditional Chinese Maotai-flavor liquor

Yeasts are the most important group of microorganisms contributing to liquor quality in the solid-state fermentation process of Chinese Maotai-flavor liquor. There occurred a complex yeast community structure during this process, including stages of Daqu (the starter) making, stacking fermentation on the ground and liquor fermentation in the pits. In the Daqu making stage, few yeast strains accumulated. However, the stacking fermentation stage accumulated nine yeast species with different physio-biochemical characteristics. But only four species kept dominant until liquor fermentation, which were Zygosaccharomyces bailii, Saccharomyces cerevisiae, Pichia membranifaciens, and Schizosaccharomyces pombe, implying their important functions in liquor making. The four species tended to inhabit in different locations of the stack and pits during stacking and liquor fermentation, due to the condition heterogeneity of the solid-state fermentation, including the different fermentation temperature profiles and oxygen density in different locations. Moreover, yeast population was much larger in the upper layer than that in the middle and bottom layers in liquor fermentation, which was in accordance with the profile of reducing sugar consumption and ethanol production. This was a systematical investigation of yeast community structure dynamics in the Maotai-flavor liquor fermentation process. It would be of help to understand the fermentative mechanism in solid-state fermentation for Maotai-flavor liquor.     

茶多酚苹果酒的发酵新工艺中试研究

以浓缩苹果汁为原料,采用固定化酵母发酵技术,加入茶多酚作为抗氧化剂,进行苹果酒中试发酵试验,通过测定发酵过程中的酒精度、酸度、透光率、残糖等理化指标的变化情况,考察中试后发酵速度和发酵效果的变化。结果表明:当发酵体积扩大50倍后,苹果酒发酵效果与小试样小体积状态下的发酵效果无显著差异,总的变化趋势趋于一致。     

Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.     

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。     

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.     

Yeast flora associated with fermentation of mahua flowers

Nine morphologically different strains of yeasts were isolated from fresh, fermenting and fermented juice of mahua flowers and identified by both morphological and biochemical characteristics. The yeasts belonged to six genera namely, Kloeckera (Kl. apiculata), Candida (C. krusei and C. tropicalis), Torulopsis (T. apicola and Torulopsis sp.), Pichia (P. terricola), Saccharomyces (S. chevalieri and S. cerevisiae) and an unidentified genus.     

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l’Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.     

制备苹果酒酵母固定化载体ACA的研究

以苹果酒酵母固定化载体ACA的机械强度和通透性为指标,通过单因素试验观察了海藻酸钠浓度、氯化钙浓度、壳聚糖浓度、针头型号、固化时间和液化时间对制备固定化载体ACA的影响,并通过正交试验优化了载体ACA的制备工艺。试验结果表明,4个因素中海藻酸钠浓度(A)对载体ACA的机械强度和通透性有极显著影响,氯化钙浓度(B)对载体ACA的机械强度影响显著,壳聚糖浓度(C)对载体ACA的通透性影响显著,而交互作用(A×B)、液化时间(D)对载体ACA的机械强度和通透性影响均不显著。制备苹果酒酵母固定化载体ACA的最佳条件为:2.0%海藻酸钠溶液,2.0%CaCl2溶液,0.2%壳聚糖溶液,液化时间10min。