Microbial Characterization of Yellow Curing Process of Codfish.

Yellow 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 product.

Historical Wines of Portugal: The Classification, Consumer Associations and Marketing Implications.

Geographical origin, use of traditional varieties and ancestral viticulture/oenology practices characterize wines classified as Historical Wines of Portugal (HWP). This study identifies the authenticity attributes consumers associate with this classification and assesses the relative strength of associations. A review of brand authenticity research and interviews with Portuguese wine producers (n = 3) and consumers (n = 12) were conducted to identify HWP classification attributes. Strength of attribute association was subsequently evaluated in an online questionnaire with a convenience sample of Portuguese wine consumers (n = 641), which included a measure of general wine knowledge and questions about the adequacy of different contexts for HWP purchase and consumption. Wine knowledge markedly affected the nature and strength of consumer associations. Compared to Aspirational Explorers, wine connoisseurs emerged as Heritage Gatekeepers, associating origin, cultural heritage, quality, production and at-home consumption more strongly with HWP, and tradition, wine age and out-of-home consumption less strongly. Market recognition of HWP as a novel and distinctive table wine classification, with well-defined and unique attributes, is thus likely to depend on consumers' general wine knowledge. Related promotional activities targeting wine novices should first focus on educating them on HWP classification, whereas those directed at savvier consumers should emphasize wine authenticity cues instead.

Sulfite concentration and the occurrence of headache in young adults: a prospective study.

BACKGROUND/OBJECTIVES: Sulfites are additives commonly used in food and wine industries that are associated to adverse clinical effects such as headaches. The objective of this study is to investigate the possible association between sulfite concentration in wine and the occurrence of headaches in young adults. SUBJECTS/METHODS: Eighty volunteers, aged between 18 and 25 years, were evaluated. Sub-groups (with or without previous headaches related with wine) were created and volunteers were submitted to two wine tests (minimum and maximum sulfite concentration accordingly to weight). A questionnaire was handed out after the test regarding the presence or not of headaches, their main characteristics, as well as other symptoms associated. RESULTS: Subjects that refer a previous headache history upon wine ingestion presented a risk 2266 greater of developing headaches after wine ingestion with a greater sulfite concentration. Those that refer constant headaches related to wine ingestion previous to the test present a risk of 6232 times more of developing headaches compared to those who refer sporadic headaches related to wine consumption. CONCLUSIONS: In our group of subjects, sulfite concentration in wine is related to the risk of developing headaches in individuals who are susceptible to wine induced headaches.

Peculiar H⁺ homeostasis of Saccharomyces cerevisiae during the late stages of wine fermentation.

Intracellular pH (pH(in)) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H(+) homeostasis is accomplished by an industrial wine strain of Saccharomyces cerevisiae while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH(in) 5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H(+) via H(+)-ATPase and of keeping H(+) out by means of an impermeable membrane were evaluated as potential mechanisms of H(+) homeostasis. At 30°C, H(+) efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H(+). Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H(+) remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H(+) permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H(+) homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.

New insights into the ecological interaction between grape berry microorganisms and Drosophila flies during the development of sour rot.

In this work, we studied the ecological interactions between grape berry microorganisms and Drosophila sp. flies involved in sour rot disease during grape ripening. After veráison the total microbial counts of grape berries affected by sour rot increased from about 2 log CFU/g of berries to more than 7 log CFU/g. Berry damage provoked a clear shift in yeast diversity from basidiomycetes to ascomycetous fermentative species. The latter were mostly Pichia terricola, Hanseniaspora uvarum, Candida zemplinina, and Zygoascus hellenicus. However, these species were not able to produce the metabolites characteristic of sour rot (gluconic and acetic acids) in inoculated berries. On the contrary, the acetic acid bacteria Gluconacetobacter saccharivorans produced high levels of these acids, mainly when berries were incubated in the presence of the insect Drosophila sp. Sour rot was not observed when grape bunches were physically separated from insects, even when berries were artificially injured. The wounds made in berry skin healed in the absence of insects, thus preventing the development of sour rot. Therefore, in the vineyard, the induction of sour rot depends on the contamination of wounded berries by a microbial consortium--yeasts and acetic acid bacteria--transported by drosophilid insects which disseminate sour rot among damaged berries. In the absence of these insects, plant defense mechanisms are effective and lead to skin healing, preventing disease spread. Thus, we showed that Drosophila sp. act as a vector for microorganisms associated with grape sour rot disease.

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

This 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 status.

Analytical and sensorial characterization of the aroma of wines produced with sour rotten grapes using GC-O and GC-MS: identification of key aroma compounds.

In the present work, the aroma profiles of wines elaborated from sound and sour rot-infected grapes as raw material have been studied by sensory analysis, gas chromatography-olfactometry (GC-O), and gas chromatography-mass spectrometry (GC-MS), with the aim of determining the odor volatiles most likely associated with this disease. The effect of sour rot was tested in monovarietal wines produced with the Portuguese red grape variety Trincadeira and in blends of Cabernet Sauvignon and sour rotten Trincadeira grapes. Wines produced from damaged berries exhibited clear honey-like notes not evoked by healthy samples. Ethyl phenylacetate (EPhA) and phenylacetic acid (PAA), both exhibiting sweet honey-like aromas, emerged as key aroma compounds of sour rotten wines. Their levels were 1 order of magnitude above those found in controls and reached 304 and 1668 µg L(-1) of EPhA and PAA, respectively, well above the corresponding odor thresholds. Levels of γ-nonalactone also increased by a factor 3 in sour rot samples. Results also suggest that sour rot exerts a great effect on the secondary metabolism of yeast, decreasing the levels of volatiles related to fatty acids and amino acid synthesis. The highest levels of γ-decalactone of up to 405 µg L(-1) were also found in all of the samples, suggesting that this could be a relevant aroma compound in Trincadeira wine aroma.

The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine.

The wine spoilage yeast Dekkera bruxellensis was evaluated for the production of 4-ethylphenol under low concentrations (0.02-20 g L(-1)) of glucose and fructose in synthetic media. Measurable amounts of 4-ethylphenol were produced over 0.2 g L(-1) of each sugar. The yeast growth rate and amount of biomass formed increased from 0.2 to 20 g L(-1) of glucose or fructose, being accompanied by increasing production of 4-ethylphenol. In red wines, the production of 4-ethylphenol was only observed in the presence of growing populations of indigenous or inoculated strains of D. bruxellensis. The production rate of 4-ethylphenol varied between 22 and 93 mug day(-1) either with inoculated strains or wild populations in bottled wines. The production rate of 4-ethylphenol as a function of the increase in the number of cells varied from 349 to 1882 mug L(-1) per one log CFU mL(-1). The effect of temperature on cellular viability and 4-ethylphenol production was tested in red wines with indigenous or inoculated strains of D. bruxellensis. Incubation temperatures of 15, 20 and 25 degrees C allowed cellular growth and volatile phenol production. Increasing incubation temperatures to 36 degrees C induced full viability loss of 10 strains of D. bruxellensis within <12 h.

Sour rot-damaged grapes are sources of wine spoilage yeasts.

Yeast species of sound and sour rot-damaged grapes were analysed during fermentation and grape ripening in the vineyard, using general and selective culture media. During 2003 and 2004 vintages, microvinifications were carried out with sound grapes to which different amounts of grapes with sour rot were added. The wine spoilage species Zygosaccharomyces bailii was only recovered during fermentations with sour rot, reaching 5.00 log CFU mL(-1) (2003) and 2.48 log CFU mL(-1) (2004) at the end of fermentation. The study of yeast populations during the sour rot ripening process (2005 vintage) showed that the veraison-damaged grapes always exhibited higher total yeast counts and a much greater diversity of species. From a total of 22 ascomycetous species, 17 were present only in damaged grapes. The most frequent species were Issatchenkia occidentalis and Zygoascus hellenicus. The spoilage species Z. bailii and Zygosaccharomyces bisporus were consistently isolated exclusively from damaged grapes. This work demonstrates that one of the most dangerous wine spoilage species, Z. bailii, is strongly associated with sour rot grapes and survives during fermentation with Saccharomyces cerevisiae. The use of selective media provides a more accurate characterization of grape contamination species.

The diversity of pathogenesis-related proteins decreases during grape maturation.

It was recently shown that wines contain typically a huge diversity of structurally similar polypeptides that exhibit a high degree of homology to pathogenesis-related (PR) proteins. This observation suggested the existence of one or a few precursors in mature grapes, common to most or all the wine PR proteins. Limited proteolysis and chemical modification of the precursor(s) during fruit ripening and winemaking could then generate the large number of distinct wine polypeptides. However, the patterns of PR proteins extracted from grape berries regularly harvested from the onset of development until maturity did not confirm the previous hypothesis. Two different methodologies, involving 2-D immunoblotting and a combination of FPLC cation/anion exchange chromatographies with 1-D immunoblotting, indicate that the total concentration of PR proteins is increased but its diversity is reduced from the early stages of berry development until maturity. These results indicate that PR proteins are synthesized in a wide variety of forms from the early stages of grape development, eliminating the hypothesis previously formulated on the existence of one or few precursors common to the wine proteins.

Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources.

A total of 63 strains of Dekkera bruxellensis and 32 strains of Pichia guilliermondii isolated from wine related environments were identified by restriction analysis of the 5.8S-ITS region of the rDNA. These strains were subjected to intraspecific discrimination using mtDNA restriction and RAPD-PCR analysis. The isolates identified as D. bruxellensis yielded 3 different molecular patterns of mtDNA restriction using the endonuclease HinfI. The pattern A was the most frequent (58 strains) among strains from different sources, regions and countries. Pattern B (4 strains) and C (one strain) were determined in isolates from Portuguese wines. The discrimination among the pattern A strains was achieved by a RAPD-PCR assay with 3 primers (OPA-2, OPA-3 and OPA-9). A total of 12 haplotypes were obtained with the combination of the patterns provided by the 3 OPAs. The pattern 2 was the most frequent and extensively distributed being found in strains from different countries and from different sources like wine, barrique wood and insects. The strains of P. guilliermondii were characterized with restriction of mtDNA using the endonuclease HinfI yielding 7 different restriction patterns. These patterns were associated with different efficiencies of 4-ethylphenol production. Patterns A to D corresponded to 19 strains producing low levels of 4-ethylphenol (<1 mg/l) while patterns F and G grouped 13 strains producing high levels of 4-ethylphenol (>50 mg/l), when grown in synthetic media supplemented with 100 mg/l of p-coumaric acid. The high degree of polymorphism observed shows that intraspecific typing is essential for accurate yeast dissemination studies in wine related environments.

Environmental conditions during vegetative growth determine the major proteins that accumulate in mature grapes.

Despite recent reports in the literature that chitinases comprise approximately 50% of the soluble proteins present in mature Vitis vinifera L. (cv. Moscatel) grapes, with the other major proteins being thaumatin-like proteins, a careful inspection of the published data reveals inconsistencies as to which proteins actually accumulate in mature grapes. Mature Moscatel grapes were harvested in the same vineyard in two consecutive years, 1999 and 2000. The grapes exhibited widely distinct polypeptide patterns when analyzed by either FPLC cation exchange chromatography or two-dimensional electrophoresis: whrereas the 2000 grapes possessed a much higher protein content (1.96 versus 1.11 mg g(-)(1) of fresh weight), the 1999 grapes contained a greater heterogeneity of polypeptides. In addition, highly specific antibodies that recognize the pathogenesis-related proteins present in the grapes demonstrated that the 2000 harvest grapes had a wider variety of pathogenesis-related polypeptides. N-Terminal sequencing of the major polypeptides revealed differences in the relative abundance and amino acid sequence of thaumatin-like and osmotin-like polypeptides present in the 1999 and 2000 harvest Moscatel grapes and did not detect the presence of chitinase. As a whole, the data indicate that the expression and accumulation of the major proteins in grapes is essentially determined by the environmental and/or pathological conditions prevailing during grape development and maturation. The potential physiological and technological implications are discussed. The results of the present work suggest that it is not possible to base varietal differentiation of grapes on the profile of the pathogenesis-related polypeptides present in the mature berries.

Influence of selected factors on browning of Camembert cheese.

Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+ thus leading to 16 different variants of cheese. Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body). Mn2+ promoted a significant increase of browning at both locations, whereas Yar. lipolytica had the opposite effect. Outside browning was significantly more intense when using the Pen. candidum strain with higher proteolytic activity. A significant interaction was found between Yar. lipolytica and Pen. candidum. The yeast had no effect in combination with a low proteolytic strain of Pen. candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen. candidum. We further confirmed that both strains of Pen. candidum were able to produce brown pigments from tyrosine and thus both are presumably responsible for the browning activity in this type of cheese.