Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells

An integrated winemaking process – including sequential alcoholic and malolactic fermentations operated continuously – was developed. For the continuous alcoholic fermentation, yeast cells (Saccharomyces cerevisiae) were immobilized either on grape stems or on grape skins, while bacterial cells (Oenococcus oeni) used for conducting continuous malolactic fermentation were immobilized on grape skins only. The produced wines were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds). The final proposed integrated continuous process permitted the production of 960 mL/d of a dry white wine, with an alcoholic strength of about 13 vol%, by using two 1.5 L tower bed reactors packed with 260 g of grape skins. The produced wines revealed a good physicochemical quality. Moreover, 67% of the malic acid concentration could be reduced in the second reactor. Both fermentative processes proved to be much more efficient than those conducted traditionally with free cells or even with immobilized cells, but in the batch mode of operation.     

Evolution of malolactic bacteria and biogenic amines during spontaneous malolactic fermentations in a Greek winery

AIMS: To study the population dynamics of indigenous malolactic bacteria in a Greek winery and to examine their potential to produce detrimental levels of biogenic amines (BA) under winemaking conditions. METHODS AND RESULTS: Although the wines studied were of different vintage, grape variety and enological characteristics, molecular typing of malolactic bacteria revealed only a low number of strains within the single-species populations of Oenococcus oeni that developed during spontaneous fermentations. Strain MF1, originating primarily from the vineyards surrounding the winery invariably predominated in almost all samples. HPLC analysis showed a slight increase in the BA, putrescine, tyramine and phenylethylamine after malolactic conversion, while histamine, methylamine and ethylamine remained unaffected. No correlation could be established between the BA profiles and the bacterial compositions or the amino acid concentrations in wine samples studied. CONCLUSIONS: A certain regional bacterial flora is established in the winery that prevails in spontaneous malolactic fermentations (MLF) irrespective of the wine characteristics. In all cases, the BA content of the wines after malolactic conversion was within enologically acceptable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the malolactic bacteria occurring naturally in spontaneous MLF in Greek red wines and a preliminary assessment of their impact on wine safety in relation to BA.     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Use of an ion-exchange sponge to immobilise yeast in high gravity apple based (cider) alcoholic fermentations

Summary An ion-exchange sponge that can have a tailored surface charge has been used for yeast immobilisation in high original gravity (o.g. 1.106) cider fermentation. Continuous circulation of fermentation medium through columns containing weakly basic sponge encouraged yeast growth, decreased batch fermentation time and increased final ethanol concentration, possibly aided by sponge enhanced CO₂ removal from solution.     

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains.     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

Analysis of yeast diversity during spontaneous and induced alcoholic fermentations

The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae . In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.     

Biosynthesis of phenolic compounds and its regulation in apple

This paper summarises the information on the occurrence of phenolic compounds in apple Malus domestica leaves and fruits with special reference to their developmental changes and regulation of biosynthesis. Besides the ontogenetic variation in biosynthesis and accumulation, the stress-induced and pathogenesis-related changes are emphasised. Aspects of commercial importance are addressed, ranging from fruit colouration, through disease resistance, to the direct use of apple tissues, as raw material for the extraction of bioactive phenolic compounds.     

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts. Received 4 August 1998/ Accepted in revised form 9 December 1998     

Kinetic considerations about the study of alcoholic fermentations of starch hydrolysate

Alcoholic fermentations of starch hydrolysate by two different yeast strains, Saccharomyces cerevisiae(var. Vinal) and Saccharomyces oviformis(IMAP 383), have been studied in batch runs. In order to evaluate the different inhibition phenomena due to both substrate and product, a new kinetic equation is suggested.     

Two-step fermentation of acidic grape musts: Interactions between Schizosaccharomyces pombe and Saccharomyces spp.

The interactions between Schizosaccharomyces pombe and Saccharomyces spp. (S. cerevisiae, S. cerevisiae sake, S. bayanus, S. uvarum) were investigated by growing the yeasts in sterile, partially fermented glucose asparagine medium in flasks, and also in the Ecologen containing either synthetic medium or grape must be separating the adjacent chambers with membranes which allow free movement of medium but not of cells. The growth of Sch. pombe was inhibited by Saccharomyces spp. to a varied extent, but the reverse was not observed. Saccharomyces uvarum, and S. cerevisiae more strongly inhibited Sch. pombe than the other species tested. All three strains of Sch. pombe (ICV-M, BG, ATCC-16979) were inhibited by S. cerevisiae although ICV-M and ATCC strains were more sensitive than BG. The higher growth rate of S. cerevisiae resulted in the exhaustion of nutrients, and its metabolic products could possibly be responsible for the growth inhibition of Sch. pombe. In the light of the present experimental results, the suitability of a two-step fermentation process for making better quality wines from acidic grape musts is discussed.     

Monitoring of volatiles in alcoholic fermentations on molasses via a gas membrane sensor

Summary Gas membrane sensors, connected to a gas chromatography, have been tested in fermentation on complex media of industrial interest. Selectivity, sensitivity and reliability have been assessed: the calibration on fermentation medium is recommended. The use of such a sensor is demonstrated in the study of the kinetics of batch and fed-batch alcoholic fermentations on beet molasses. The kinetics of acetaldehyde are especially pointed out in relation to ethanol, fusel alcohols and carbon dioxide production.Offprint requests to: M.-N. Pons     

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.     

Chemistry and vegetation of highly acidic streams

An account is given of the water chemistry and photosynthetic flora of waters in England with a pH value of 3.0 or less. Of the fifteen sites found, thirteen were associated with coal-mining, one with a barytes mine and one was an industrial effluent. Flowing waters predominated, but in some cases pools in direct contact with the streams were also present. All waters carried high levels of heavy metals and silicate, and most carried moderately high levels of phosphate and combined inorganic nitrogen. The total flora consisted of twenty-four algae, two mosses and two flowering plants. Of these, three algae and one flowering plant were found in pools only. Euglena mutabilis was both the most widespread species, and often also the most abundant. The number of species present in a reach showed a positive correlation with pH during late summer, and a negative correlation with total acidity in winter. Only four of the algal species found in England are certainly included in the U.S.A. literature. Some, but not all, the apparent differences between the two floras are very probably due simply to taxonomic problems.     

Arginase activity is a useful marker of nitrogen limitation during alcoholic fermentations

Nitrogen deficiency in musts is one of the causes of sluggish or stuck fermentations. In this work we propose that arginase activity determination can be useful for detecting nitrogen starvation early in vinification. CAR1 and YGP1 genes are not specifically induced under conditions of nitrogen starvation. However, a significant increase in the enzymatic activity of arginase, the product of the CAR1 gene, is detected in vinifications carried out with musts containing limiting amounts of nitrogen. Moreover, on adding ammonia to a nitrogen-deficient vinification, even at late stages, this enzymatic activity is repressed, and growth rate is restored simultaneously. We also investigate the role of ethanol toxicity in nitrogen starvation. The results suggest that ethanol produced during vinification or exogenously added up to 8% (v/v) concentration does not cause nitrogen starvation under the conditions tested because arginase activity is not increased.     

Design of a laboratory automatic system for studying alcoholic fermentations in anisothermal enological conditions

Summary Equipment in which the temperature changes during industrial-scale wine fermentations can be simulated on a laboratory scale is described.     

Ecophysiology of algae living in highly acidic environments

Highly acidic environments are inhabited by acidophilic as well as acidotolerant algae. Acidophilic algae are adapted to pH values as low as 0.05 and unable to grow at neutral pH. A prerequisite for thriving at low pH is the reduction of proton influx and an increase in proton pump efficiency. In addition, algae have to cope with a limited supply of carbon dioxide for photosynthesis because of the absence of a bicarbonate pool. Therefore, some algae grow mainly in near terrestrial situations to increase the CO₂-availability or actively move within the water body into areas with high CO₂. Beside these direct effects of acidity, high concentrations of heavy metals and precipitation of nutrients cause indirect effects on the algae in many acidic environments.     

Variation in phenolic compounds and antioxidant activity in apple seeds of seven cultivars

Polyphenols are the predominant ingredients in apple seeds. However, few data are available on the phenolic profile or antioxidant activity in apple seeds in previous researches. In this study, low-molecular-weight phenolic compounds and antioxidant activity in seeds, peels, and flesh of seven apple cultivars grown in northwest China were measured and analyzed using HPLC and FRAP, DPPH, ABTS assays, respectively. HPLC analysis revealed phloridzin as the dominant phenolic compound in the seeds with its contents being 240.45–864.42 mg/100 gDW. Total phenolic content (TPC) measured by the Folin–Ciocalteu assay in apple seed extracts of seven cultivars ranged from 5.74 (Golden Delicious) to 17.44 (Honeycrisp) mgGAE/gDW. Apple seeds showed higher antioxidant activity than peels or flesh; antioxidant activity in seeds varied from 57.59 to 397.70 μM Trolox equivalents (TE)/g FW for FRAP, from 37.56 to 64.31 μM TE/g FW for DPPH, and from 220.52 to 708.02 μM TE/g FW for ABTS. TPC in apple seeds was significantly correlated with all three assays. Principal component analysis (PCA) indicated that Honeycrisp was characterized with high contents of total polyphenols and phloridzin. Our findings suggest that phenolic extracts from apple seeds have good commercial potential as a promising antioxidant for use in food or cosmetics.     

Influence of phenolic acids on phosphorus mobilisation in acidic and calcareous soils

Phenolic acids and phenols are abundant in soils. However, little information is available on the role of these compounds in mobilisation of soil phosphorus (P). The present study examined the effects of three phenolics on P mobilisation in comparison with citric acid in three soils differing in chemical properties. The soils were incubated with organic compounds at concentrations of 0 to 100 mol g^–1 soil for 30 min. While the addition of phenolic acid anions and phenol decreased soil pH, citrate either increased or did not affect soil pH depending on the soil type. All the organic compounds increased the amounts of easily-mobilised P fractions in the order of catechol citric acid proto-catechuic acid caffeic acid for the acid soils and citric acid > catechol = proto-catechuic acid > caffeic acid for the calcareous soil. Phosphorus mobilisation did not correspond to the amounts of Ca, Fe or Al ions released from the soils. These results suggest that organic acids and phenols altered the P fractions from stable, sparingly-soluble forms to easily dissolvable forms, and that chelation or precipitation of cations with organic ligands occur before biodegradation of the ligands.     

Development of a modified Respiration Activity Monitoring System for accurate and highly resolved measurement of respiration activity in shake flask fermentations

ABSTRACT: BACKGROUND: The Respiration Activity Monitoring System (RAMOS) is an established device to measure on-line the oxygen transfer rate (OTR), thereby, yielding relevant information about metabolic activities of microorganisms and cells during shake flask fermentations. For very fast-growing microbes, however, the RAMOS technique provides too few data points for the OTR. Thus, this current study presents a new model based evaluation method for generating much more data points to enhance the information content and the precision of OTR measurements. RESULTS: In cultivations with E.coli BL21 pRSET eYFP-IL6, short diauxic and even triauxic metabolic activities were detected with much more detail compared to the conventional evaluation method. The decline of the OTR during the stop phases during oxygen limitations, which occur when the inlet and outlet valves of the RAMOS flask were closed for calibrating the oxygen sensor, were also detected. These declines reflected a reduced oxygen transfer due to the stop phases. In contrast to the conventional calculation method the new method was almost independent from the number of stop phases chosen in the experiments. CONCLUSIONS: This new model based evaluation method unveils new peaks of metabolic activity which otherwise would not have been resolved by the conventional RAMOS evaluation method. The new method yields substantially more OTR data points, thereby, enhancing the information content and the precision of the OTR measurements. Furthermore, oxygen limitations can be detected by a decrease of the OTR during the stop phases.