Detailed Analysis of the Microbial Population in Malaysian Spontaneous Cocoa Pulp Fermentations Reveals a Core and Variable Microbiota

The fermentation of cocoa pulp is one of the few remaining large-scale spontaneous microbial processes in today''s food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality. Despite intensive research in the field, a detailed and comprehensive analysis of the microbiota is still lacking, especially for the expanding Asian production region. Here, we report a large-scale, comprehensive analysis of four spontaneous Malaysian cocoa pulp fermentations across two time points in the harvest season and two fermentation methods. Our results show that the cocoa microbiota consists of a “core” and a “variable” part. The bacterial populations show a remarkable consistency, with only two dominant species, Lactobacillus fermentum and Acetobacter pasteurianus. The fungal diversity is much larger, with four dominant species occurring in all fermentations (“core” yeasts), and a large number of yeasts that only occur in lower numbers and specific fermentations (“variable” yeasts). Despite this diversity, a clear pattern emerges, with early dominance of apiculate yeasts and late dominance of Saccharomyces cerevisiae. Our results provide new insights into the microbial diversity in Malaysian cocoa pulp fermentations and pave the way for the selection of starter cultures to increase efficiency and consistency.     

A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.     

Isolation and Characterization of Microorganisms Associated with the Traditional Sorghum Fermentation for Production of Sudanese Kisra

Sorghum flour obtained from Sudan was mixed with water in a 1:2 (wt/vol) ratio and fermented at 30°C for 24 h. The bacterial populations increased with fermentation time and reached a plateau at approximately 18 h. At the end of 24 h, sorghum batter pH had dropped from 5.95 to 3.95 and the batter had a lactic acid content of 0.80%. The microbial population during the 24 h of fermentation consisted of bacteria (Pediococcus pentosaceus, Lactobacillus confusus, Lactobacillus brevis, Lactobacillus sp., Erwinia ananas, Klebsiella pneumoniae, and Enterobacter cloacae), yeasts (Candida intermedia and Debaryomyces hansenii), and molds (Aspergillus sp., Penicillium sp., Fusarium sp., and Rhizopus sp.). P. pentosaceus was the dominant microorganism at the end of the 24-h fermentation. When three consecutive fermentations using an inoculum from the previous fermentation were carried out, the bacterial population increase plateaued at 9 h. The microbial populations in these fermentations were dominated by P. pentosaceus.     

Genetics of Ethanol-Producing Microorganisms

Abstract

Ethanol-Producing Microrganisms

A wide variety of microbial species are known to produce ethanol as a product of carbohydrate fermentation.¹ Organisms which have received attention in recent studies include a wide range of yeasts, some molds, and a number of specialized bacteria (Table 1). Traditionally, yeasts, particularly Saccharomyces cerevisiae, have been used for producing fermentation ethanol or alcoholic beverages in large-scale processes. In Table 1, Zymomonas mobilis, the predominant organism in fermentations producing Mexican “pulque” or palm wine,³⁴-⁴⁶ is the only bacterium of current economic significance. However, the development of interest in other species with the ability, for example, to convert xylose to ethanol or to ferment at high temperatures indicates that no existing strain of Saccharomyces or Zymomonas meets the specifications for all current and future uses. Certainly the use of alternative organisms, or even mixed cultures,⁴²⁴⁵ warrants investigation. However, this review will concentrate on proven ethanol producers (i.e., yeasts, particularly Saccharomyces spp., and Z. mobilis) and how these might be improved in a systematic way for ethanol production, using the wide range of genetic techniques which is now available.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

酱香型白酒发酵过程中核心酵母的鉴别及其功能

【背景】酵母是酱香型白酒发酵过程中最重要的微生物,但酵母群落中核心酵母的种类和功能尚不清晰。【目的】通过探索酱香型白酒发酵微生物群落中核心酵母的种类和功能,为揭示酱香型白酒酿造机制以及提升白酒品质提供理论支撑。【方法】联合使用未培养(内转录间隔区扩增子和宏转录组高通量测序技术)和可培养(菌种筛选和模拟发酵实验)技术对酱香型白酒发酵过程中核心酵母的结构和功能进行定性和定量分析。【结果】内转录间隔区扩增子和宏转录组测序结果显示,酱香型白酒发酵过程中涉及10个属的酵母微生物,其中在发酵过程中平均相对丰度大于0.2%的酵母有13种,有2种核心酵母分别为库德威兹氏毕赤酵母(Pichia kudriavzevii)和粟酒裂殖酵母(Schizosaccharomyces pombe)。在发酵过程中,P. kudriavzevii占总量的89%以上,Schi. pombe表达了占总量21%以上的功能基因。模拟发酵实验结果显示P. kudriavzevii在酵母群落中发挥耐受乳酸积累的作用,而Schi. pombe在酵母群落中发挥耐受乙醇积累的作用,这两种作用保证了酱香型白酒发酵过程中酵母群落的结构和功能的稳定性。【结论】P. kudriavzevii和Schi. pombe是酱香型白酒发酵过程中的优势酵母,这两种酵母在生产中发挥了各自不同的作用。本研究进一步加深了核心酵母对于整个生产过程贡献的认识,有助于研究者认识到白酒发酵过程中对核心微生物进行调控的重要性。     

Indigenous Bacteria and Fungi Drive Traditional Kimoto Sake Fermentations

Sake (Japanese rice wine) production is a complex, multistage process in which fermentation is performed by a succession of mixed fungi and bacteria. This study employed high-throughput rRNA marker gene sequencing, quantitative PCR, and terminal restriction fragment length polymorphism to characterize the bacterial and fungal communities of spontaneous sake production from koji to product as well as brewery equipment surfaces. Results demonstrate a dynamic microbial succession, with koji and early moto fermentations dominated by Bacillus, Staphylococcus, and Aspergillus flavus var. oryzae, succeeded by Lactobacillus spp. and Saccharomyces cerevisiae later in the fermentations. The microbiota driving these fermentations were also prevalent in the production environment, illustrating the reservoirs and routes for microbial contact in this traditional food fermentation. Interrogating the microbial consortia of production environments in parallel with food products is a valuable approach for understanding the complete ecology of food production systems and can be applied to any food system, leading to enlightened perspectives for process control and food safety.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

Influence of aeration during propagation of pitching yeast on fermentation and beer flavor

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.     

Wine yeasts for the future

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

The cocoa bean fermentation process: from ecosystem analysis to starter culture development

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro‐organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well‐fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp‐bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa‐derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.     

The effects of a pesticide program on microbial populations from apple leaf litter

The leaf litter microbial community was quantitatively and qualitatively changed when a standard pesticide schedule that comprised an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees in the summer. Effects were observed for two winters by four indirect assays and three direct methods. Populations were altered qualitatively both years, but the most striking difference was the quantitative impact from year to year. Bacteria, filamentous fungi, and yeasts from treated leaves were reduced 10- to 10 000-fold between November 1976 and April 1977 and did not recover until snow cover had melted in March. Reductions in 1977-1978 were negligible. The marked seasonal difference is attributed to meteorological influences. Fluorescent pseudomonads were among the bacteria depressed by chemicals. Of the 49 genera of fungi and yeasts isolated, Coniothyrium sp., Penicillium spp., Arthrobotrys spp., and Nodulisporium sp. were appreciably reduced, whereas Typhula spp., Pleurophomella sp., Sporobolomyces spp., and Rhodotorula spp. were substantially enhanced by the spray program.     

Study of the micro-organisms associated with the fermented bread (khamir) produced from sorghum in Gizan region, Saudi Arabia

Traditional bread (khamir) was made from sorghum flour of two local varieties, Bayadh and Hamra. The bread was prepared by mixing the sorghum flour with water and spices (onion, garlic, lemon juice and fenugreek) in a 1:0.8 (w/w) ratio and fermented for 24 h at 30 degrees C. Two other fermentations were carried out using an inoculum from the previous fermentation. The micro-organisms were isolated from different plates and identified using different characterization systems. Both total bacterial populations and lactic acid bacteria increased with fermentation time and reached the highest number at 16 h (first fermentation) and at 8 h (second and third fermentation). The content of lactic acid was increased with time to reach 1.2%, but the increase was higher for the second and third fermentations (1.6% each). The pH dropped with time from 6.77 to 4.35 in the first fermentation and from 6.65 to 4.18, and 6.57-3.93, in the second and third fermentations, respectively. The microorganisms, which were isolated and characterized during the 24 h fermentation, included: bacteria (Pediococcus pentosaceus, Lactobacillus brevis, Lact. lactis subsp. lactis, Lact. cellobiosus, Klebsiella oxytoca, Kl. pneumoniae, Enterobacter aerogenes, Ent. sakazakii, Serratia marcescens and Ser. odourifera), moulds (Penicillium sp., Rhizopus sp., Aspergillus niger, Alternaria sp., Fusarium sp. and Mucor sp.) and yeasts (Candida parapsilosis, C. orvegnsis and Rhodotorula glutinis).     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Microbial community structure and dynamics of dark fire-cured tobacco fermentation

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.     

Occurrence and Growth of Killer Yeasts during Wine Fermentation

Sixteen wine fermentations were examined for the presence of killer yeasts. Killer property and sensitivity to killer action were found in isolates of Saccharomyces cerevisiae but not in isolates of Kloeckera, Candida, Hansenula, and Torulaspora spp. Several killer and killer-sensitive strains of S. cerevisiae were differentiated by colony morphology, and this property was used to monitor their growth kinetics in mixed cultures in grape juice. Killer-sensitive strains died off within 24 to 48 h during mixed-strain fermentation. Killer action was demonstrated at pH 3.0 and pH 3.5 and over the range of 15 to 25°C but depended on the proportion of killer to killer-sensitive cells at the commencement of fermentation. The dominance of killer strains in mixed-strain fermentations was reflected in the production of ethanol, acetic acid, and glycerol.     

Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.     

Yeast diversity and persistence in botrytis-affected wine fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by SACCHAROMYCES: In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of CANDIDA: Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate.

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.