Effects of Inhibitory Environmental Factors on Growth of Oenococcus oeni CCSYU2068 for Malolactic Fermentation of Cider Production

The effects of several inhibitory factors (sulfur dioxide, pH and ethanol) on the growth of lactic acid bacteria and the subsequent malolactic fermentation (MLF) were studied by inoculation of different culture strains of Oenococcus oeni, the major lactic acid bacteria (LAB) in cider production. After comparing their organoleptic properties, three strains of Oenococcus oeni were selected from indigenous and commercial sources and their inhibitory effects on cell growth and MLF examined. The malolactic bacteria expressed variations in tolerance to the environmental conditions of pH, sulfur and ethanol concentration. Isolated from an indigenous cider production facility, O. oeni L4 had a better capacity with constant growth even when the concentration of SO₂ was 50 ppm, ethanol 10% (v/v) and pH 3.0. O. oeni L4 showed better properties for metabolizing the major acids: malic, lactic and acetic acid. The decomposition mean rate of malic acid was as high as 228.52 mg/L per day with a low acetic acid concentration of 101.78 mg/L under the stress conditions of cider production.     

苹果酒酿造中的苹果酸-乳酸发酵

在苹果酒酿造中进行苹果酸-乳酸发酵,乳酸菌通过分解苹果酸,产生乳酸,引起其他有机酸的变化,使苹果酒的口感、质量得以改善。pH、温度、SO_2、酒度通过影响乳酸菌的活动而影响苹果酸-乳酸发酵的进行。保证苹果酒苹果酸-乳酸发酵进行的条件为:温度16～18℃,总SO_2含量<70mg/L,pH<3.70,酒精体积分数低于13％。     

冷破碎红富士苹果浆发酵低度苹果酒工艺优化

以冷破碎红富士苹果浆为原料发酵制备低度苹果酒,通过分析不同酵母发酵周期的糖度、酒精度、pH和总酸筛选酵母菌株;在此基础上采用单因素与响应面试验以优化发酵工艺,确定冷破碎苹果浆发酵酒的最佳条件,通过分析酒品指标,与苹果清汁发酵酒的品质进行对比。结果表明,冷破碎苹果浆发酵酒的最佳工艺参数为:SY酵母发酵的初始糖度18%、酵母接种量0.1 g/L、发酵温度20℃;苹果浆发酵酒的残糖（7.5±0.46 g/L）、酒精度（7.8±0.1%）分别与苹果清汁发酵酒的残糖（7.3±0.31 g/L）、酒精度（8.0%±0.1%）差异不显著;苹果浆发酵酒的总酚（74.99±1.23 mg/100 mL）和有机酸（5.33±0.01 g/L）等成分含量高于苹果汁发酵酒（总酚47.5±2.2 mg/100 mL,有机酸5.01±0.03 g/L）。该结果可供苹果酒实际生产借鉴,也为苹果加工产业多元化发展提供思路。     

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.     

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.     

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.     

Effect of sulphur dioxide concentration added at different processing stages on volatile composition of ciders

This study evaluated the effect of sulphur dioxide (SO₂) added at different steps in the process on the volatile composition contributing to the fruity aroma of cider. Potassium metabisulphite was added (150 and 300 mg/L) at three processing steps (a) crushed apple, (b) apple must and (c) final product. The SO₂ added to the crushed apple either maintained or increased the fermentation rate, whereas the addition to the apple must slowed the fermentation rate. The addition of 300 mg/L of potassium metabisulphite to Golden Delicious apple must inhibited fermentation. The losses of volatile compounds varied from 23 to 46% and from 33 to 97% in all of the treatments with 150 and 300 mg/L of potassium metabisulphite, respectively. 3‐Methyl‐1‐butanol, acetaldehyde and ethyl ethanoate corresponded to >85% of the volatile compounds in the cider; 3‐methyl‐1‐butanol was not affected by the sulphur dioxide. Acetaldehyde showed the greatest reduction (>80%) with the addition of sulphur dioxide and ethyl ethanoate was reduced when the compound was added to the crushed apple and apple must. All of the interactions between the apple variety, the stage of SO₂ addition and the concentration of SO₂ showed significant negative impact on volatile compounds. © 2018 The Institute of Brewing & Distilling     

苹果酸-乳酸发酵细菌乙醇胁迫应答机制研究进展

由乳酸菌引起的苹果酸-乳酸发酵是生产优质葡萄酒的一个重要工艺过程。在这个过程中,乙醇是乳酸菌活性的主要抑制因子,特别是随着全球气候变暖带来的葡萄含糖量的增加,乙醇对乳酸菌的抑制作用越来越成为人们的研究热点。乙醇胁迫使得乳酸菌细胞结构发生变化,影响苹果酸-乳酸发酵的顺利进行。因此,乳酸菌在乙醇胁迫下的生长、代谢对葡萄酒生产是非常重要的。该文对苹果酸-乳酸发酵过程中常见乳酸细菌在乙醇胁迫下的应答机制进行了阐述,为乳酸菌乙醇胁迫耐受性机制的研究和优良菌株的选育提供参考。     

Identification of <Emphasis Type="Italic">Zygosaccharomyces mellis</Emphasis> strains in stored honey and their stress tolerance

To screen yeast with high sugar tolerance and evaluate their stress tolerance, six yeast strains were selected from 17 stored honey samples. The species were identified through 26S rRNA sequencing. Their stress tolerance was determined via the Durham fermentation method and ethanol production ability was determined via flask fermentation. The results demonstrated that all the six strains were Zygosaccharomyces mellis. Their sugar, ethanol, and acid tolerance ranges were 500–700 g/L, 10–12% (v/v), and pH 2.5–4.5, respectively. The SO₂ tolerance was 250 mg/L. Among the six strains, 6-7431 had the best stress tolerance with sugar tolerance of 700 g/L, ethanol tolerance of 12% (v/v), and acid tolerance of pH 2.5. Furthermore, the strain of 6-7431 had the highest percentage of ethanol production at the same initial sugar content as the other strains. Therefore, the selected six yeast strains would be promising fermentation yeasts for wine-making, ethanol production, or other fermentation purposes.     

肠膜明串珠菌在苹果酒苹果酸乳酸发酵中的应用探讨

通过在苹果酒中添加肠膜明串珠菌启动苹果酸乳酸发酵,研究了温度、pH和酒精度等因子对苹果酸-乳酸发酵的影响表明,肠膜明串珠菌能够启动苹果酸-乳酸发酵,其中温度、pH和酒精度均对苹果酒的柔和指数和感官评价有显著影响,不同发酵条件下酿造的苹果酒柔和指数在1.33～7.94之间,感官评分在73.7～91.3之间.添加肠膜明串珠菌后,苹果酸的浓度变化在322.45mg/L～692.44mg/L之间,pH值提高了约0.06～0.11,苹果酒的口感变得柔和,风味得到明显改善.     

Preliminary study of the production of apple pomace and quince jelly

Apple pomace, a cheap by-product of apple juice and cider production, is rich in pectins and flavour compounds. Thus, a possible way of valorization of apple pomace was using it for jelly production. Fresh apple pomace from Braeburn, Gala, Golden Delicious and Granny Smith was used for the preparation of jelly to study the amount of sugar and quince content and the cooking time effect. The response surface methodology was used to study the preparation of apple pomace jelly and responses studied were textural characteristics and overall acceptability. The hardness of jelly was not affected by the factors studied whereas cohesiveness and overall acceptability were affected by the concentration of quince and sugar. The most appreciated jellies were those prepared with the highest quince concentration and the lowest sugar concentration.     

苹果酿造酒工艺流程及酒精生成规律的研究(Ⅰ)

通过对苹果原汁制备、发酵和醪液澄清各工艺方法或参数的筛选 ,制定了苹果酿造酒生产的适宜工艺流程。经分析测定发现 ,果汁接种酵母后 ,7d内酒精含量由 0升为 6 3% ,TSS、还原糖分别由 19.6 %、4 7%降为 5 6 %、0 53%。     

发酵过程果酒成分对苹果酸乳酸发酵的影响

苹果酸-乳酸发酵（MLF）是果酒（葡萄酒、苹果酒）酿造中非常重要的二次发酵过程。本研究采用人工模拟果酒，研究果酒成分对MLF的影响。结果表明，葡萄糖抑制乳酸菌的MLF，果糖却有促进作用，而且果糖可解除葡萄糖对酿酒酒球菌MLF的抑制作用，果酒中葡萄糖和果糖的浓度及其比例是预测和控制MLF的重要参数；高浓度乙醇、低pH抑制乳酸菌的MLF，其抑制作用的大小与使用的菌种有关；酒中酚类物质（没食子酸和阿魏酸）对酿酒酒球菌的MLF几乎无任何显著影响；酒精发酵完成后果酒中营养的缺乏会抑制乳酸菌的MLF。     

原汁苹果醋酸发酵饮料的研究

试验以浓缩苹果汁为原料,分别采用酵母1450、醋酸菌株AM2进行酒精发酵及醋酸发酵,对醋酸发酵进行不同酒精度及不同糖度影响实验。结果认为,较高的酒精度和糖度对醋酸发酵有抑制作用,在酒精度3%,糖度7.5%的条件下进行醋酸发酵,可制得风味理想的原汁苹果醋酸发酵饮料。     

Influence of a malolactic starter on the quality of the cider produced on an industrial scale

The application of starter culture technologies to induce the malolactic fermentation in cidermaking are not yet established, as is already the case in winemaking. In this work, a selected indigenous cider malolactic strain was tested as a starter culture to assess its implementation and its influence on cider production on an industrial scale (10,000 l). The results showed that the culture, under the fermentation conditions tested, allowed successful achievement of the malolactic fermentation. Analysis of L-malic acid in the fermenting apple must medium revealed a highly significant difference in the rate of consumption of this acid from the moment of bacterial inoculation when compared with that of the control. The concentration of the malolactic starter culture maintained the inoculation level (10⁶ cfu/ml) during the process, the level necessary to ensure the malolactic fermentation. Thus, the cider malolactic starter culture showed good adaptation to the harsh environmental conditions found in the fermenting apple must, such as low temperature and the presence of SO₂ and ethanol. Several parameters were monitored during the process (specific gravity, temperature, pH, free SO₂ content, microbiological count, malic acid degradation rate, lactic acid formation, other major organic acids and volatile compounds) under inoculated and not inoculated conditions.     

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。     

Influence of Enzymatic Clarification with a Pectin Methylesterase on Cider Fermentation

In this study, the effect of two apple juice treatments (clarification with a pectin methylesterase in the presence and absence of sulphiting) on the cidermaking process was investigated. Pre‐fermentative clarification with the pectin methylesterase (Rapidase CPE) slowed the alcoholic fermentation in respect to traditional fermentation, the greatest effect being found for the combination of the enzymatic treatment with sulphur dioxide addition. With this treatment the start of the fermentation was delayed seven days. Enzymatic clarification delayed malolactic fermentation (MLF) and the interaction of both treatments produced a cider in which malolactic fermentation had not occurred. Both treatments ensured that the volatile acid levels during the maturation phase were significantly lower than in the traditional fermentation. Enzymatic clarification led to lower levels of yeasts during the active phase of alcoholic fermentation with respect to the control. When this treatment was combined with sulphiting, a decrease in apiculate yeast numbers during alcoholic fermentation was observed and a better survival of these species was noted after 59 days of fermentation.     

金红苹果醋饮料的加工工艺研究

以金红苹果为原料,经过酒精发酵、醋酸发酵等工艺酿制出金红苹果醋。通过调配、杀菌、灌装得到金红苹果醋饮料。试验结果表明,醋酸发酵的最佳条件为：醋酸菌的接种量为10％、发酵温度32℃、发酵液酒精含量6％。金红苹果醋饮料的最佳配方为：果酸原汁5％、醋酸0.15%、白砂糖8％。     

苹果酒发酵过程中绿原酸胁迫对酿酒酵母生理特性的影响

在苹果酒发酵过程中,酿酒酵母会对各种胁迫和环境变化做出代谢应答.绿原酸是苹果和苹果酒中含量相对较高的一种苹果多酚类物质.本文以绿原酸为代表性苹果多酚,考察不同质量浓度的绿原酸胁迫对酿酒酵母生理特性的影响.结果表明:酿酒酵母CICC 31084在绿原酸胁迫下,主要通过改变胞内超氧化物歧化酶(SOD)活性、过氧化氢酶(CA...     

以苹果渣生产苹果醋的工艺研究

以苹果湿渣为主要原料,经酒精发酵和醋酸发酵制得苹果醋.采用单因素和正交试验,研究了发酵过程中糖度、酒精度和酸度的变化,确定了最佳工艺参数.酒精发酵最佳条件为:温度28℃,糖浓度12％,接种量10％,发酵时间5天;醋酸发酵最佳条件为:温度32℃,初始酒精度6°,接种量8％,发酵时间6天.