Changes in sour rotten grape berry microbiota during ripening and wine fermentation

Available at ScienceDirectThis study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health statu

The microbial ecology of wine grapes

Available at ScienceDirectGrapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing. The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. Methods and Results: In sound grapes, the mean yeast counts ranged from 3Æ20 ± 1Æ04 log CFU g)1 to 5Æ87 ± 0Æ64 log CFU g)1. In honeydew grapes, the mean counts ranged from 3Æ88 ± 0Æ80 log CFU g)1 to 6Æ64 ± 0Æ77 log CFU g)1. In sour rot grapes counts varied between 6Æ34 ± 1Æ03 and 7Æ68 ± 0Æ38 log CFU g)1. Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera ⁄ Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). Conclusions: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. Significance and Impact of the Study: The impact of damaged grapes in the yeast ecology of grapes has been underestimated

Evaluation of the inhibitory effect of dimethyl dicarbonate (DMDC) against wine microorganisms

Several microbial species associated with wine were challenged against increasing concentrations of dimethyl dicarbonate (DMDC). The concentration inducing complete cell death upon addition to red wine was regarded as the minimum inhibitory concentration (MIC). In dry red wines with 12% (v/v) ethanol and pH 3.50, the inactivation depended on the initial cell concentration. For an initial inoculum of 500 CFU/ml, the MIC of the yeasts species Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae and Pichia guilliermondii was 100 mg/l. The most sensitive strains belong to Zygosaccharomyces bailii, Zygoascus hellenicus and Lachancea thermotolerans, with MIC of 25 mg/l DMDC. For inoculation rates of about 106 CFU/ml, the maximum dose of DMDC legally authorised (200 mg/l) was not effective against the most resistant species. The addition of 100 mg/l potassium metabisulphite (PMB), equivalent to 1 mg/l molecular sulphur dioxide, increased the inactivation effect of 100 mg/l DMDC over initial yeast populations of 106 CFU/ml but did not fully kill S. pombe and S. cerevisiae. Lactic acid and acetic acid bacteria were not killed by the addition of 300 mg/l of DMDC. Trials performed in wines before bottling showed that in most samples indigenous bacterial populations were not affected by 200 mg/l DMDC. Therefore, under winery practice, DMDC at the maximum dose legally permitted may be regarded as an efficient preservative to control low contamination rates of yeasts but ineffective against lactic acid and acetic acid bacteria

Microbial Characterization of Yellow Curing Process of Codfish

Research articleYellow cured codfish has a typical yellow colour, distinctive taste, and low salt content due to its special curing process of the raw salted codfish involving several soaks in water of the raw salted codfish, alternated with drying steps. The purpose of this study was to assess the main functional groups of bacteria involved in this process and relate them with physicochemical properties of the product. A total of 28 codfish from Iceland were supplied by two local companies. Seven stages of the curing process were analyzed. From each of these seven stages, four fish samples were collected to carry out the microbial and physicochemical analyses (moisture, salt content, pH, total volatile basic nitrogen (TVB-N), and trimethylamine nitrogen (TMA-N)). Bacteria counts were performed using the MPN method and adequate culture media for aerobic, proteolytic, sulphite-reducing, biogenic amine, and trimethylamine-producing and ammonifying bacteria. Strains isolated from the highest dilutions with microbial growth were used to characterize the predominant bacteria. The results showed that total aerobic counts increased from 3.9 log MPN/g in raw salted codfish to 5.9 log MPN/g in the final. Proteolytic, ammonifying, and trimethylamine bacteria producers also increased to 8, 7.5, and 6.5 log MPN/g, respectively. The salt content decreases (from 17% until 8%) and moisture increases (53% until 67%) during the salted-raw-codfish soaking, favoring sulphite-reducing and biogenic amine-producing species, confirming that desalting enhances potential spoilers. The subsequent drying step benefits proteolytic, ammonifying, and trimethylamine-producing bacteria, with a corresponding non-protein-nitrogen content (TVB-N and TMA-N) increase. The dominant bacteria during yellow curing belong to the genera Staphylococcus, Psychrobacter, Pseudomonas, and Alcaligenes with a clear positive correlation between the content of Staphylococcus and Psychrobacter and TVB-N and TMA-N concentration. Staphylococcus spp. are the dominant bacteria in the steps where the product has a higher salt concentration; thus, it could be particularly useful as an indicator to control the industrially yellow curing process and could have an important role in the development of the final characteristics of this productinfo:eu-repo/semantics/publishedVersio

Protein haze formation in wines revisited. The stabilising effect of organic acids

The effect on the wine protein haze potential of five organic acids commonly encountered in wines (L(+)- tartaric, L( )-malic, citric, succinic and gluconic acids) was assessed. All five acids, tested at 20 mM, reduced dramatically the haze potential of proteins, either in wine or dissolved in water, throughout the range of pH values typical of wines (i.e., from 2.8 through 3.8). Subtle differences among the acid effects did not correlate with the number of their carboxyl groups, but were attributed to electrostatic interactions that depend upon the acid pKa values, the protein pI values and the medium pH. These results invalidate or question the validity of all experiments on wine proteins involving wine model solutions containing organic acids. Overall, the results obtained in the present work clearly indicate that organic acids with a common occurrence in wines exhibit a stabilising effect upon the haze potential of the wine proteins

Zymological indicators: a new concept applied to the detection of potential spoilage yeast species associated with fruit pulps and concentrates

DOI:10.1006/fmic.2000.0360; available online at http://www.idealibrary.comIn a survey of the microbial quality of raw materials used in fruit juice processing, yeast counts in fruit concentrates and pulps were found to range from51to 2?96103 cfu g71. Ascomycetous yeasts were representedby 76%of the isolateswhile 24%were basidiomycetes.The identi¢cation of strains isolated by the simpli¢ed identi¢cation system(SIM) revealed19 yeast species representing12 genera.Themost frequently isolated yeasts belonged to the genera Saccharomyces, Pichia, Cryptococcus, Kluyvero- myces and Candida. Fatty acid yeast composition allowed the separation of contaminating yeasts into one of threemajor groups. Group I included yeasts without linoleic (C 18:2) and linolenic (C 18:3) fatty acids such as Saccharomyces cerevisiae. Group II comprised yeasts without C 18:3 fatty acid like Zygosaccharo- myces rouxii and Torulaspora delbrueckii, and group III included yeasts with C18:2 and C18:3 acids that belong, among others, to one of the following yeast genera: Pichia, Candida, Kluyveromyces or Cryptococcus. Species-speci¢c PCR primers were used for the rapid detection and identi¢cation of the most dangerous species a¡ecting fruit concentrate stability. The simpli¢ed protocol used consisted of PCR-ampli¢cation of conserved tracts in the ITS region of the rDNA unit, thus enabling the detection ofpotentially dangerous £ora such as Zygosaccharomyces species andT. delbrueckii in contaminated fruit concentrates. Results from PCR-typing were in full agreement with the fatty acid compositions of these species. The grouping of contaminant yeasts into threemain groups showed that fatty acid compositionmay be used to di¡erentiate yeasts according to their technological signi¢cance.Yeasts isolated in thiswork as being most dangerous to product stability belong to either group II ( Z. rouxii and T. delbrueckii) or group I (Saccharomyces spp.). Group III was comprised of several species regarded as indicators of de¢ciencies in `good manufacturing practices'.Thus, each of the groups delineated may be considered to be a zymological indicator of technological signi¢cance.The conjugation of fatty acid pro¢les with PCR-typing methods may be used as a rapid detection system for contaminant yeasts. The fatty acid pro¢les provide a preliminary identi¢cation of yeasts potentially dangerous to product stability present within 48 h. of isolation. Whereas the PCR-typing method is mainly used to confirm isolate identity, when required, after the initial diagnosis has been performed, over a period of 4 h

Growth and 4-ethylphenol production by the yeast Pichia guilliermondii in grape juices

The behavior of Pichia guilliermondii strains producing high levels of 4-ethylphenol in synthetic media was studied in wines and grape juices. These strains lost their viability and did not produce 4-ethylphenol after 24 hr of inoculation in red wines with ethanol adjusted to 10 or 12 % (v/v) and pH 3.5, in the absence of free sulphite. Under the same conditions, at 12 % (v/v) ethanol, growth of Dekkera bruxellensis was observed. When grown in single culture in grape juices, selected strains of P. guilliermondii produced high levels of 4-ethylphenol. In mixed grape juice fermentations with Saccharomyces cerevisiae, P. guilliermondii began to die after starter inoculation at 107 cfu/mL and did not produce 4-ethylphenol. Low starter inoculation rates (102 cfu/mL) added 72 hr after P. guilliermondii inoculation resulted in high production of 4-ethylphenol. In conditions mimicking cold pre-fermentative maceration processes, at 10ºC for 72 hr, P. guilliermondii did not grow, while at 25ºC growth attained a 104 fold increase. At this temperature, addition of 200 mg/L potassium metabisulfite after grape crushing did not eliminate P. guilliermondii inoculated at 104 cfu/mL in grape juice of pH 3.57. The possibility that high levels of 4-ethylphenol in wines are due to the activity of P. guilliermondii should be mostly related with uncontrolled growth in contaminated grape juices before starter inoculation. In wines, its ability to produce 4-ethylphenol seems to be much lower than that of D. bruxellensis

Characterisation of yeast flora isolated from an artisanal Portuguese ewe's cheese

www.elsevier.nl/locate/ijfoodmicroThe evolution of the yeast flora was studied for an artisanal semi-hard ewes’ cheese made from raw milk. Mean log10 22 yeast counts per gram of cheese body ranged from 2.7 to 6.4, with the higher counts observed after a ripening period of 30 23 days. The yeast population decreased thereafter and, at the end of curing process, reached values similar to those of the 24 beginning. A total of 344 yeasts strains were randomly isolated from the curd and cheese body during the 60 days long 25 ripening period. Esterase activity was common to almost all isolates (98%) while proteolysis was observed in 12% of the 26 total yeast population. The proportion of strains with positive glucose fermentation increased from 21% in the curd to 75% at 27 the end of the ripening period. A total of 150 isolates representative of the physiological characteristics tested were examined 28 with the API ID 32C system showing different degrees of quality of identification. Only 15% of the strains (23 isolates) were 29 excellently identified being assigned to the species Candida zeylanoides. The most frequent species appeared to be 30 Debaryomyces hansenii (anamorph Candida famata) and Candida intermedia. These two species amounted to 9% of the 31 yeasts in the curd increasing to 86% at the end of the ripening period

A response surface methodology study on the role of factors affecting growth and volatile phenol production by Brettanomyces bruxellensis ISA 2211 in wine

The present study was aimed at determining the effect of glucose, ethanol and sulphur dioxide on the growth and volatile phenol production by Brettanomyces bruxellensis in red wines using a response surface methodology approach. Sulphur dioxide proved to have a significant (p < 0.05) negative linear and quadratic effect on growth and 4-ethylphenol production. Concentrations of sulphur dioxide higher than 20 mg L 1, at pH 3.50, induced immediate loss of cell culturability under growth permissive levels of ethanol. Under high ethanol concentrations (14% v/v), the lag phase increased from 3 to 10 days, growth being fully arrested at 15% (v/v). Glucose up to 10 g L 1 was found to be a significant factor (quadratic level) in biomass increase under low ethanol (<12.5% v/v) and low sulphite concentrations. However, when cells were inactivated by sulphur dioxide and ethanol, glucose (up to 10 g L 1) did not prevent cell death. Production of more than 50 mg L 1 day 1 of 4-ethylphenol was only observed in the presence of high numbers (106 CFU mL 1) of culturable cells, being stimulated by increasing glucose concentrations.info:eu-repo/semantics/publishedVersio