Microbial ecology studies of spontaneous fermentation: starter culture selection for prickly pear wine production

Abstract: A procedure for designing starter cultures for fermentation is illustrated for prickly pear wine production. The illustration includes kinetic studies on inoculated and spontaneous fermentation, microorganism identification studies based on molecular biology tools, and microbial ecology studies, which led to the selection of strains that are capable of synthesizing alcohol and desirable volatile compounds. Results show that a mixed starter inoculum containing Pichia fermentans and Saccharomyces cerevisiae leads to a fermented product that contains 8.37% alcohol (v/v). The gas chromatography and mass spectrometry (GC‐MS) analysis shows the presence of 9 major volatile compounds (Isobutanol, Isopentanol, Ethyl acetate, Isoamyl acetate, Ethyl octanoate, Ethyl decanoate, Ethyl 9‐decanoate, β‐Phenylethyl acetate, and Phenylethyl alcohol) that have ethereal, fruity, aromatic notes that are considered to be essential for a fine wine flavor. These compounds harmonically synergize with the alcohol to produce a fermented product with a unique flavor and taste. Several assays using the mixed culture show that the process is stable, predictable, controllable, and reproducible. Moreover, the results show that a mixed culture leads to a broader range of aromatic products than that produced by a single, pure culture. Therefore, we conclude that combinations of Saccharomyces strains and non‐Saccharomyces strains can be used to obtain high‐quality fermented beverages from prickly pear juice.     

Yeast and bacterial modulation of wine aroma and flavour

Wine is a highly complex mixture of compounds which largely define its appearance, aroma, flavour and mouth‐feel properties. The compounds responsible for those attributes have been derived in turn from three major sources, viz. grapes, microbes and, when used, wood (most commonly, oak). The grape‐derived compounds provide varietal distinction in addition to giving wine its basic structure. Thus, the floral monoterpenes largely define Muscat‐related wines and the fruity volatile thiols define Sauvignon‐related wines; the grape acids and tannins, together with alcohol, contribute the palate and mouth‐feel properties. Yeast fermentation of sugars not only produces ethanol and carbon dioxide but a range of minor but sensorially important volatile metabolites which gives wine its vinous character. These volatile metabolites, which comprise esters, higher alcohols, carbonyls, volatile fatty acids and sulfur compounds, are derived from sugar and amino acid metabolism. The malolactic fermentation, when needed, not only provides deacidification, but can enhance the flavour profile. The aroma and flavour profile of wine is the result of an almost infinite number of variations in production, whether in the vineyard or the winery. In addition to the obvious, such as the grapes selected, the winemaker employs a variety of techniques and tools to produce wines with specific flavour profiles. One of these tools is the choice of microorganism to conduct fermentation. During alcoholic fermentation, the wine yeast Saccharomyces cerevisiae brings forth the major changes between grape must and wine: modifying aroma, flavour, mouth‐feel, colour and chemical complexity. The wine bacterium Oenococcus oeni adds its contribution to wines that undergo malolactic fermentation. Thus flavour‐active yeasts and bacterial strains can produce desirable sensory results by helping to extract compounds from the solids in grape must, by modifying grape‐derived molecules and by producing flavour‐active metabolites. This article reviews some of the most important flavour compounds found in wine, and their microbiological origin.     

Volatile Compounds in Cider: Inoculation Time and Fermentation Temperature Effects

A cider fermentation at laboratory scale with controlled inoculation with selected yeasts and malolactic bacteria was performed. The composition of the major volatile compounds with low boiling points (<145°C) was determined by gas chromatography throughout the fermentation process, under the different operating conditions of inoculation time of the microorganisms (simultaneous or sequential) and fermentation temperature (15°C or 22°C). Temperature had a more important effect on the levels of certain volatile compounds when the simultaneous inoculation method was used. It was observed that when fermentation temperature increased to 22°C, using the simultaneous method, the final concentrations of ethyl acetate and some of the higher alcohols decreased, while others maintained similar levels. In the sequential inoculation model, after completion of the alcoholic fermentation at 15°C, an increase in the temperature of the malolactic fermentation (15°C to 22°C) showed no statistically significant differences in the profiles of the volatile compounds tested. Thus, malolactic fermentation could be conducted at 22°C, favouring malic acid degradation, without losses in the major volatile compounds, in relation to the levels measured at the lower temperature. Considering the traditionally recognized preference for low alcoholic fermentation temperatures in cider making, the results allow for the optimisation of the operating conditions.     

Influence of pH and ethanol on malolactic fermentation and volatile aroma compound composition in white wines

The present study investigated the influences of pH and ethanol on malolactic fermentation (MLF) and the volatile aroma profile of the subsequent white wines from Riesling and Chardonnay inoculated with two different Oenococcus oeni strains. In all cases MLF was induced after completion of alcoholic fermentation (AF). Partial MLF occurred under low pH 3.2 and high alcohol (118.3 g/L) conditions. In the cases with complete MLF, the time required for each strain varied from 13 to 61 days and was dependent on bacterial culture, cultivar and wine parameter. Chemical properties of each wine were determined after AF, complete and partial MLF. The wines showed significant differences in total higher alcohols, esters and acids that are important for the sensory profile and quality of wine. This work demonstrated that the wine matrix as well as the pH and alcohol concentration affects MLF and the final volatile aroma profile. Results indicate that changes in volatile aroma composition are not necessarily related to complete MLF and that partial MLF already has distinct influences on the wine aroma profile of white wines.     

雪梨枇杷果酒主发酵工艺优化及挥发性成分分析

采用雪梨和枇杷作为原材料,探讨雪梨枇杷复合果酒主发酵的最佳工艺条件。以酒精度、pH、总酸、透光率、还原糖和感官评价为指标,通过单因素实验和正交试验确定雪梨与枇杷体积比、酵母接种量、SO₂添加量,对成品进行微生物指标的测定,并且利用顶空固相微萃取结合GC-MS分析雪梨枇杷果酒中挥发性成分种类以及相对含量。结果表明,以雪梨和枇杷为原料进行发酵,雪梨枇杷体积比为6:4,发酵原浆初始pH为3.5,SO₂添加量为80 mg/L,酵母接种量为0.4 g/L,5 d发酵结束后,得到果酒酒精度为9.51%vol,透光率达到91.5%,总酸为7.60 g/L,pH为3.56,还原糖为8.32 g/L,在此工艺条件下发酵果酒感官评价得分为91分,各因素对果酒酒精度影响次序为:初始pH > 酵母接种量 > 雪梨枇杷体积比 > SO₂添加量。并且测得该雪梨枇杷复合果酒的菌群总数为11 CFU/mL以及大肠菌群数小于10^-9MPN/mL,均符合标准。通过分析其挥发性成分,雪梨枇杷果酒共检测出30种挥发性化合物,相对含量较高的为醇类和酯类,占总挥发性成分的87.82%,对果酒的香味起着积极作用。     

贺兰山东麓不同子产区赤霞珠葡萄自然发酵对葡萄酒香气的影响

为探究宁夏贺兰山东麓不同子产区自然发酵对葡萄酒香气的影响,利用顶空固相微萃取-气相色谱-质谱联用技术对青铜峡、红寺堡及银川3个子产区自然和接种发酵的赤霞珠葡萄酒中挥发性成分进行了定性定量测定,并采用显著性分析、主成分分析及聚类分析的统计学方法进行了分析。结果表明,3个子产区的挥发性化合物以酯类和醇类为主。银川产区挥发性化合物的总量在3个子产区中最高,自然酒精发酵和接种酒精发酵后挥发性化合物的含量分别为339.05和453.90 mg/L,自然苹乳发酵和接种苹乳发酵后挥发性化合物的含量分别为307.01和379.08 mg/L。青铜峡产区自然酒精发酵的葡萄酒中挥发性化合物的总量(312.8 mg/L)显著高于接种发酵(296.1 mg/L),自然苹乳发酵的葡萄酒中的挥发性化合物的总量(215.78 mg/L)显著高于接种发酵(202.10 mg/L)。对3个子产区葡萄酒中挥发性成分的聚类分析发现,对葡萄酒中挥发性化合物影响程度从高到低依次为产区、发酵阶段和发酵方式。     

赤霞珠葡萄酒苹果酸-乳酸发酵过程中香气成分动态变化

为了研究苹果酸-乳酸发酵过程对葡萄酒香气的影响,采用顶空固相微萃取（HS-SPME）结合气相色谱-质谱联用（GC-MS）对赤霞珠葡萄酒苹乳发酵过程中香气物质动态的变化进行了监测。结果表明：在葡萄酒苹乳发酵过程中共检出55种不同的香气成分,包括22种酯类、10种醇类、8种酸类、7种醛类、4种酮类、3种萜烯类和1种酚类,其中酯类、醇类和酸类化合物为苹乳发酵过程中主要香气成分。相比于发酵起始阶段（0 d）,发酵末期（18 d）葡萄酒中的酯类和酸类化合物的质量分数由20.7%和1.08%上升至25.61%和2.33%,主要包括乙酸乙酯、己酸乙酯、月桂酸等,这类香气物质能够赋予葡萄酒浓郁的果香,而由于酯化反应醇类化合物的质量分数由42.89%下降至29.01%,主要变化为异戊醇等高级醇质量分数的降低。由此看出,葡萄酒经过苹乳发酵不仅增强了酒体的水果类香气还提高了葡萄酒的安全性。     

显齿蛇葡萄提取物抗菌作用的研究

为推进梨酒减硫增质进程,促进天然抗氧化剂在梨酒中的应用。本文通过单独使用二氢杨梅素（Dihydromyricetin,DMY）或与SO₂联合使用酿造库尔勒香梨酒,分别测定各组梨酒自由基清除率、总酚含量、总黄酮含量、酒体色度及风味物质,研究了DMY对梨酒抗氧化活性以及风味物质的影响。结果表明,各组梨酒基础理化指标无显著性差异（P>0.05）,发酵均能正常完成;H4组（SO₂ 30 mg/L、DMY 100 mg/L）抗氧化活性最佳,DPPH自由基清除率为70.08%,ABTS⁺自由基清除率达到95.89%;单独使用DMY（浓度为150或200 mg/L）或与SO₂联合使用时,均能促进酒体总酚及总黄酮的生成（P<0.05）。此外,DMY对梨酒挥发性风味物质影响较大,DMY浓度越高,越有益于主体香气物质的生成;DMY和SO₂联合使用亦能提高挥发性物质的种类和总量,其中H3组（SO₂ 30 mg/L、DMY 75 mg/L）风味物质种类达到24种,总含量高达11846.14 μg/L,乙酸乙酯和辛酸乙酯含量相比CK组分别增加了107.61、420.27 μg/L。综合来看,H3组总酚含量更高、颜色更加稳定,酒体香气更加浓郁、层次感愈加分明,感官品评高达85.7分 。 DMY与SO₂的混合使用在抑制褐变、促进总酚方面综合效果最佳,适量DMY可增加改善香梨酒香气成分,提高其品质。     

不同酿造阶段添加富含谷胱甘肽酵母衍生物对梨酒品质的影响

为探究在不同酿造时间添加不同质量浓度富含谷胱甘肽酵母衍生物(glutathione-enriched inactive dry yeast,g-IDY)对梨酒品质的影响,以库尔勒香梨为原料,分别在乙醇发酵前、苹果酸-乳酸发酵前及陈酿前添加10、20、30 mg/L g-IDY进行梨酒酿造,待各组梨酒陈酿2个月后,对梨酒总酚及单体酚含量、抗氧化活性、有机酸含量、香气成分及感官评价进行分析。结果表明：在乙醇发酵前或陈酿前添加g-IDY能显著提高梨酒总酚含量、抗氧化能力及降低颜色指数(P<0.05);添加g-IDY能提高梨酒中苹果酸、乳酸及柠檬酸等主要风味有机酸含量及提高梨酒总风味物质含量,在乙醇发酵前或陈酿前添加g-IDY有利于芳香族等香气化合物的形成并有助于减少部分不良气体的产生,但会导致更多H₂S生成。总体表明：在乙醇发酵前添加20 mg/L g-IDY所酿梨酒品质最好,总酚质量浓度为282 mg/L,总风味物质质量浓度为13 453.22 mg/L,感官评价达到88.7分。     

Impact of malolactic fermentation on the volatile composition of Turkish Kalecik karası red wines

The effects of spontaneous malolactic fermentation (MLF) and a malolactic starter culture (Oenococcus oeni) on the volatile compounds of Kalecik karas? red wines were investigated. The volatile compounds were extracted using liquid–liquid extraction and quantified by GC–MS–flame ionisation detection. Starter cultures of O. oeni had a positive effect in shortening MLF and producing lower volatile acidity. After MLF, the total volatile compounds increased in both the inoculated wines and the spontaneously fermented wine. Ethyl lactate, diethyl succinate and γ‐butyrolactone increased upon inoculation with cultures of O. oeni strains PN4 and VP41. MLF increased the diacetyl concentration in all Kalecik karas? wines. Strain dependent differences were found in the level of diacetyl, with O. oeni PN4 producing more diacetyl than O. oeni VP41. After MLF, the concentrations of acetoin, 4‐ethylguaicol and 4‐ethylphenol increased in spontaneously fermented wine compared with wines inoculated with O. oeni. Accordingly, the use O. oeni PN4 is recommended for a controlled MLF to produce better quality Kalecik karas? wines. © 2018 The Institute of Brewing & Distilling     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

Improvement of volatile composition of wines by controlled addition of malolactic bacteria

The effect of malolactic fermentation (MLF) on the volatile composition of red wines was studied by inoculation with selected lactic acid bacteria. Four wines were inoculated with different Oenococcus oeni (syn. Leuconostoc oenos) strains, the major malolactic species found in wines, and one was inoculated with a Lactobacillus sp. strain. A non inoculated wine was also analyzed to act as a control. Malolactic fermentation and evolution of non volatile compounds were followed by HPLC and after the depletion of the malic acid present in wine the volatile compounds were extracted and analyzed by gas chromatography with flame ionization and mass spectrometry. Wines which had undergone the MLF showed a significant increment in total higher alcohols, esters and acids that are important in the sensory properties and quality of wine.     

酵母菌混合发酵制备米酒及其工艺优化

为了降低米酒的高级醇含量,提高米酒品质,以异常威克汉姆酵母（Wickerhamomyces anomalus）、扣囊复膜酵母（Saccharomyces fibuligera）和酿酒酵母（Saccharomyces cerevisiae）为菌株混合发酵制备米酒,对其挥发性风味物质及感官品质进行分析,并采用单因素试验及响应面试验对其发酵条件进行优化。结果表明,W. anomalus、S. fibuligera、S. cerevisiae按1∶1∶1比例制备米酒品质较好,米酒高级醇含量为164.95 mg/L,感官评分为87.3分。米酒最优发酵工艺条件为：接种量3×106 CFU/mL、后发酵温度21 ℃、后发酵时间2.5 d。在此优化条件下,米酒感官评分为90.1分,高级醇含量为153.1 mg/L,与优化前相比,其高级醇含量下降了7.2%,感官品质有所提高。     

苹果酸-乳酸发酵对威代尔冰酒香气的影响

以酒精发酵与苹果酸-乳酸发酵同时进行的威代尔冰酒为实验组,以单一酒精发酵的冰酒为对照,采用液相色谱（LC）、顶空固相微萃取（HS-SPME）和气相色谱-质谱联用（GC-MS）技术,对有机酸含量和挥发性香气成分进行定性定量检测,同时进行感官评定,探究苹果酸-乳酸发酵对威代尔冰酒香气的影响。结果表明,苹果酸-乳酸发酵能使冰酒中苹果酸含量降低26%,乳酸含量增加50%,显著提高冰酒中脂肪酸乙酯类（34.71%）和萜类香气物质含量（15.2%）（P＜0.05）,显著降低戊醇等高级醇类含量（12.19%）（P＜0.05）,提高冰酒的花香、果香、焦糖香,显著降低化学味。感官分析结果表明,苹果酸-乳酸发酵能增加冰酒中的果香、花香,降低冰酒酸度,提高糖酸平衡感,从而提高其整体接受度。     

Fermentation temperature and the phenolic and aroma profile of persimmon wine

Persimmon wine may offer nutritional and medicinal value as it contains compounds that may be beneficial to health. Product quality is impacted by fermentation and this study investigated the influence of different fermentation temperatures on the phenolic content, aroma profile and antioxidant activity of persimmon wine. Low ethanol concentration and high residual sugar content were found in wine fermented at low temperature (15°C) leading to sluggish fermentation. The majority of phenolic compounds determined by spectrophotometric and HPLC methods increased as temperature increased from 15 to 25°C, and then decreased at 30°C. However, total tannins, total flavonids and antioxidant activity, including reducing power and radical scavenging with DPPH, hydroxyl and ABTS, were enhanced by elevated temperature. For wine aroma, GC–MS demonstrated increased isoamyl alcohol, phenylethyl alcohol, acetic acid 2‐phenylethyl ester, decanoic acid ethylester and 1‐butanol‐3‐methyl‐acetate. These results provided some basic insights for the quality control and commercial development of persimmon wine. © 2018 The Institute of Brewing & Distilling     

几株乳酸菌对干红葡萄酒挥发性成分的影响

以完成酒精发酵的干红葡萄酒为原料,选用实验筛选的4株乳酸菌进行苹果酸-乳酸发酵（Malolactic fermentation,MLF）,以未进行MLF的试样为对照,采用顶空固相微萃取、气相色谱-质谱（HS-SPME-GC/MS）联用技术分析试样挥发性组分。结果显示,4个试样中共检出70种挥发性组分,其中酒样C5检出42种,N8检出46种,N9检出46种,N13检出48种。CK检出35种,MLF试样挥发性物质种类及挥发量均较对照多,表明MLF过程中有新的挥发性组分形成,对葡萄酒香气品质有修饰作用。4种乳酸菌中,筛自泡菜的C5菌株（Lactobacillus plantarum）用于MLF后酒样酯类组分最高,相对含量达56.48%,酸类最低,占1.23%,酒样挥发性组分构成优于其它菌株。      

The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine

Free and glycosidically bound volatile compounds of red wine were measured after malolactic fermentation (MLF) with four different commercial starter cultures of Oenococcus oeni. MLF resulted in a significant decrease in the concentration of total glycosides, expressed as phenol‐free glycosyl glucose. Gas chromatographic analyses of wine enzyme hydrolysates showed that the extent of hydrolysis of glycosides during MLF was dependent on both bacterial strain and chemical structure of the substrate. The highest decrease was observed for glycosidic precursors of primary terpene alcohols. Glycoside‐related aroma compounds such as linalool, farnesol, and β‐damascenone were increased after MLF with all the bacterial strains tested. Two of the strains were also able to release significant amounts of vinylphenols during MLF. Copyright © 2006 Society of Chemical Industry     

Influence of yeast on the yield of fermentation and volatile profile of ‘Węgierka Zwykła’ plum distillates

The character of plum brandies depends on a unique aroma profile of the plum and the microbiota present on the surface of the fruits, as well as yeast used for fermentation. In this study, an evaluation of the effect of microorganisms applied for the fermentation of W?gierka Zwyk?a var. plum mashes and processing temperature (18 °C, 30 °C) on its efficiency and volatile profile, as well as taste and flavour of distillates obtained was performed. An estimation of the odour activity values (OAVs) of the volatile compounds was also conducted. Regardless of whether the fermentation was carried out using Saccharomyces bayanus wine yeast or by native microflora present on plums as well as raisins, the efficiency of this process was high and ranged between 91.7 and 96.7% of the theoretical efficiency. Especially rich in esters (among others ethyl acetate and isoamyl acetate) was the distillate derived after fermentation with the microflora of plums and raisins, at 18 °C. An evaluation of the individual aromatic effect of chemical compounds present in tested distillates, in terms of their OAVs, revealed that the highest OAVs were reached with isovaleraldehyde. Other compounds that showed aroma values >1 and possibly had an effect on the overall aroma of tested plum distillates were the following: hexanal, benzaldehyde, ethyl acetate, isoamyl acetate, ethyl benzoate, ethyl hexanoate, 1‐propanol, 2‐methyl‐1‐butanol, 3‐methyl‐1‐butanol and 1‐hexanol. The performed sensory ranking showed that the best rated distillate was the one obtained after fermentation with the indigenous microflora of plums and raisins, at 18 °C. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of macerate enzymes on the yield,quality, volatile compounds and rheological property of prickly pear juice

Pectinase and cellulase enzymes were used to investigate efficacy for improving juice yield, stability and quality from prickly pear fruit. Pectinase improved the yield, stable color, color‐assayed as release of anthocyanins or carotinoids and clarity of the juice. A significant increase in the effectiveness of pectinase was observed as the concentration was increased from 0.05 to 0.50% v/w. However, at concentration >0.25% v/w they tended to impart a bitter flavor in the juice. Among three concentrations of pectinase and cellulase, pectinase at 0.50% v/w produced higher yield, a sediment‐free clear juice and high‐quality juice. The results indicated that depectinated clarified prickly pear juice behaves as a Newtonian fluid. It was found that the activation energy (Ea) for viscous flow was in the range of 5.02×10³–20.06×10³ kJ/mol depending on the concentrations of pectinase and cellulase enzyme treatment of prickly pear juice, in contrast to 22.15×10³ kJ/mol in untreated juice. Volatile compound concentrations of twelve compounds were not affected by pectinase and cellulase treatment. Overall the quality of prickly pear juice was better in pectinase‐treated juice compared with untreated and cellulase‐treated juice.     

Malolactic Fermentation and Secondary Metabolite Production by Oenoccocus oeni Strains in Low pH Wines

Abstract: Different wine varieties, including some with low pH, were studied to determine the ability to grow and produce secondary metabolites of a previously selected autochthonous Oenococcus oeni strain (C22L9), compared with a commercial strain. Monitoring of malolactic fermentation (MLF) was carried out by microbiological and chemical analysis of wines. The concentration of some major volatile compounds and biogenic amines in wines before and after malolactic fermentation was also determined. The results showed major differences in MLF duration both between wines and strains, although the differences between strains were slight for most of the analyzed compounds. Statistically significant differences in citric acid degradation were found in all wine varieties and it was confirmed that O. oeni C22L9 is a poor degrader of citric acid; this means that MLF can be prolonged without the risk of producing high concentrations of diacetyl and acetoin. Sensory analysis of wines after MLF showed similar characteristics in wines from both strains. This study thus shows that O. oeni C22L9 possesses even better sensory and fermentation properties than the commercial strain and can be used in wines with different characteristics, which makes it highly valuable for industrial use. Practical Application: The increasingly use of grape varieties of low pH in winemaking and the higher alcohol content of wines, as a consequence of the climatic change, make interesting the study of the behavior during MLF of O. oeni strains in order to determine their ability to grow, when growth conditions are not optimal, and to produce secondary metabolites. A comparative study was conducted using an autochthonous O. oeni strain (C22L9) and a commercial O. oeni strain and 4 wine varieties.