The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

Biocontrol ability and action mechanisms of Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 against Penicillium digitatum DSM2750 and Penicillium expansum DSM62841 causing postharvest diseases

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 10⁶ and 10⁸ cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 10⁸ cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.     

Comparison of Industrial Scale Ethanol Production from a Palmyrah‐Based Carbon Source by Commercial Yeast and a Mixed Culture from Palmyrah Toddy

Palmyrah (Borassus flabellifer) based products were used as an alternative carbon source for industrial scale ethanol production. The fermentation medium was enriched with spent wash obtained from a distillation column. The performance of a commercially available baker's yeast in the media was compared with a ‘palmyrah toddy mixed culture’ where the organisms were obtained from the sedimentation of palmyrah toddy. In a laboratory scale study, the ethanol produced from a palmyrah fruit pulp extract, diluted with distilled water, was 16.5 gL^?1 (36 h) and 13.0 gL^?1 (48 h) with ‘palmyrah toddy mixed culture’ and baker's yeast respectively. The ‘palmyrah toddy mixed culture’ performed better than the baker's yeast with palmyrah fruit pulp extract, diluted either with distilled water or spent wash. Among the different palmyrah based carbon sources, both cultures preferred molasses diluted with spent wash and both performed best in the medium containing the spent wash supplemented with sucrose. In a 5,000 L industrial scale fermentation of 20° Brix molasses supplemented with 10 gL^?1 ammonium sulphate, 72 gL^?1 and 65 gL^?1 ethanol was produced by the ‘palmyrah toddy mixed culture’ (72 h) and the baker's yeast (90 h) respectively. As the performance of the ‘palmyrah toddy mixed culture’ was better than that of the baker's yeast, the former was selected for the industrial scale studies of molasses fermentation media diluted with spent wash. In these studies the temperature reached 42°C by 36 h and resultant cell death was observed. However ethanol production was higher and more rapid in the molasses diluted with spent wash, rather than in the molasses diluted with tap water and supplemented with (NH₄)₂SO₄. Cell recycle operation obviated the interruption in fermentation caused by temperature induced cell death and increased rates and efficiency of ethanol production were observed.     

Quantification of Viable Plesiomonas shigelloides in a Mixture of Viable and Dead Cells Using Ethidium Bromide Monoazide and Conventional PCR

A rapid and efficient method for quantitative detection of viable Plesiomonas shigelloides in pure culture containing a mixture of viable and heat-killed cells was developed using ethidium bromide monoazide (EMA) in combination with the polymerase chain reaction (PCR). The addition of EMA (1 μg/ml) to mixtures of viable and heat-killed cells of P. shigelloides inhibited the PCR amplification of DNA derived from the dead cells, but did not inhibit the PCR amplification of DNA derived from the viable cells. EMA at 5 μg/ml or less had little or no inhibition on the PCR amplification of DNA derived from viable cells of P. shigelloides. After EMA treatment, the DNA from viable P. shigelloides cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was DNA from 24 genomic targets per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 2.4 × 10¹ to 2.4 × 10⁴ DNA targets from viable cells per PCR.     

Bacteria Associated with Spoilage of Braunschweiger

Refrigerated braunschweiger was spoiled mainly by Gram positive, catalase negative coçci identified as Streptococcus faecalis and Pediococcus pentosaceus. P. pentosaceus was isolated from sausage without nitrite held at refrigeration for 12 wk and from sausages made with 156 ppm nitrite stored at 22°C for 21 days. It produced souring and a low pH in the sausage. S. faecalis was isolated from refrigerated sausages and produced a perfumy or scented odor under anaerobic conditions. S. faecalis survived 65°C for 5 min and 60°C for 60 min. P. pentosaceus at levels of 10⁸ cells/ml of broth survived 65°C for 5 min but did not survive when the number of cells was 10³/ml.     

Impedimetric detection of yeast and mold

An automated impedance test was developed for the rapid detection of yeast and mold. Various mycological media were evaluated using the capacitance (C) and conductance (G) components of the impedance signal. C and G were compared as to their effectiveness in monitoring microbial growth-induced changes in the media. Similarly, agar vs. broth media were compared. 0·1 ml aliquots of yeast and mold food isolates were loaded into duplicate Bactomatic module wells pre-filled with media (0·5 ml agar; 1·0 ml broth) to give 10²–10³ cells well⁻¹. Each organism-medium combination was monitored by C and G at 28°C. C measurement was more effective than G measurement was. C monitoring was characterized by a sharp acceleration (i.e., detection) in the resulting curves as the cell concentration reached 10⁴–10⁵ cells ml⁻¹. Conversely, weak, irregular responses to G obscured detections. The agars, particularly Bactomatic Yeast Medium and Wort Agar, gave the best results. Broth curves exhibited ambiguous detections and less percent change/h. An impedimetric test detects mold in 1–2 days compared to a 5 day standard plate count. Selection of appropriate media and signal (C) optimizes impedimetric results for yeast and mold. An agar surface supports mold and yeast growth better than does a broth.     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

Bacterial Histamine Production as a Function of Temperature and Time of Incubation

Optimal temperature, lower temperature limit, extent, and rate of histamine production in a tuna fish infusion broth (TFIB) varied for the strains of Proteus morganii, Klebsiella pneumoniae, Hafnia alvei, Citrobacter freundii, and Escherichia coli studied. P. morganii and K. pneumoniae produced large quantities of histamine in a relatively short incubation period (<24 hr) at 15°C, 30°C, and 37°C; production was fastest at 37°C. H. alvei, C. freundii, and E. coli produced toxicologically significant levels of histamine (>2500 nmoles/ml) only at 30°C and 37°C on prolonged incubation (≥48 hr). At 72 hr of incubation, optimal temperature for histamine production was 37°C for E. coli and C freundii; 30°C for P. morganii strain 110SC2, K. pneumoniae, and H. alvei; and 15°C for P. morganii strain JM. The lower temperature limits for production of toxicologically significant levels of histamine in TFIB were 7°C for K. pneumoniae; 15°C for both P. morganii strains; and 30°C for H. alvei, C. freundii, and E. coli.     

SUPPRESSION OF α-ACETOLACTATE FORMATION IN BREWING WITH IMMOBILIZED YEAST

Immobilized yeast cells extensively produced the diacetyl precursor, α-acetolactate, during alcohol fermentation. The activity of acetohydroxy acid synthetase, which is responsible for the formation of α-acetolactate from pyruvic acid, was high in cell-free extracts of immobilized yeast cells compared with that of free yeast cells. It was suggested that the expression of AHA synthase of immobilized yeast cells was increased during growth in the carrier as compared with free yeast cells. When the initial immobilizing yeast cell concentration was changed from 1.0 × 10⁶ cells/ml to 1.0 × 10⁹ cells/ml, production of α-acetolactate was reduced from 0.94 mg/l to 0.30 mg/l. Furthermore, during continuous fermentation for 10 d, the concentration of α-acetolactate in beer was 0.30 mg/l.     

Effect of milk supplementation and culture composition on acidification, textural properties and microbiological stability of fermented milks containing probiotic bacteria

In the present work, the combined effect of milk supplementation and culture composition on acidification, textural properties, and microbiological stability of fermented milks containing probiotic bacteria, was studied. Three powders (whey, casein hydrolysate, and milk proteins) were tested as supplementation. Two strains of probiotic bacteria, Lactobacillus acidophilus (LA5) and Lactobacillus rhamnosus (LC35), were used in pure culture, and in mixed culture with Streptococcus thermophilus (ST7). Acidifying activity was enhanced with mixed cultures, compared to pure cultures resulting in a shorter time to reach pH 4.5. Acidifying activity was greatly improved with casein hydrolysate, with a reduction of the fermentation time by about 55% by comparison with the other supplementations. The stability of probiotic bacteria was weakly affected by milk supplementation and culture composition. However, pure cultures were more stable than mixed cultures. The texture of the fermented products was not dependent on culture composition, but strongly dependent on milk supplementation. Sweet whey supplementation gave products with lower firmness and viscoelasticity than products supplemented with casein hydrolysate or milk proteins (decrease by 70%). It was observed that all products containing probiotic counts over 2.2×10⁷ CFU mL⁻¹ are suitable for the development of a lactic beverage containing probiotics.     

Hygienic status and biogenic amine content of mung bean sprouts

 Microbiological analyses of commercial mung bean sprouts showed the total, viable microbiological population to exceed 10⁸ cfu/g. Enterobacter cloacae, Klebsiella pneumoniae and Enterobacter agglomerans were found to be the dominant and most frequently isolated microbial species. Putrescine, cadaverine, spermidine and spermine were detected in all samples investigated. Formation of biogenic amines by pure culture isolates was studied in a modified decarboxylase medium at different temperatures, pH values and atmospheres. Highest activities were found under aerobic conditions at 20  °C. K. pneumoniae 861 produced 1.2 mg cadaverine/ml after an incubation period of 24 h and E. cloacae 862 produced 2 mg putrescine/ml after 48 h of incubation. For E. agglomerans 863, no biogenic amines were detected under these conditions. Production of cadaverine by E. cloacae 862 and K. pneumoniae 861 under aerobic conditions is presumably related to lysine decarboxylase activities. Although highest decarboxylase activities have usually been found at acidic pH values, amine production reached a maximum at pH 7. Under anaerobic conditions, E. cloacae 862 produced only about half the amount of putrescine as under aerobic conditions, whilst K. pneumoniae 861 produced significantly less cadaverine but was able to produce putrescine. Received: 10 October 1997 / Revised version: 21 January 1998     

Hygienic status and biogenic amine content of mung bean sprouts

 Microbiological analyses of commercial mung bean sprouts showed the total, viable microbiological population to exceed 10⁸ cfu/g. Enterobacter cloacae, Klebsiella pneumoniae and Enterobacter agglomerans were found to be the dominant and most frequently isolated microbial species. Putrescine, cadaverine, spermidine and spermine were detected in all samples investigated. Formation of biogenic amines by pure culture isolates was studied in a modified decarboxylase medium at different temperatures, pH values and atmospheres. Highest activities were found under aerobic conditions at 20  °C. K. pneumoniae 861 produced 1.2 mg cadaverine/ml after an incubation period of 24 h and E. cloacae 862 produced 2 mg putrescine/ml after 48 h of incubation. For E. agglomerans 863, no biogenic amines were detected under these conditions. Production of cadaverine by E. cloacae 862 and K. pneumoniae 861 under aerobic conditions is presumably related to lysine decarboxylase activities. Although highest decarboxylase activities have usually been found at acidic pH values, amine production reached a maximum at pH 7. Under anaerobic conditions, E. cloacae 862 produced only about half the amount of putrescine as under aerobic conditions, whilst K. pneumoniae 861 produced significantly less cadaverine but was able to produce putrescine. Received: 10 October 1997 / Revised version: 21 January 1998     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Increasing the levels of 2-phenylethyl acetate in wine through the use of a mixed culture of Hanseniaspora osmophila and Saccharomyces cerevisiae

The impact of mixed cultures of Hanseniaspora osmophila and Saccharomyces cerevisiae with different initial yeast ratios on wine composition has been examined. The mixed culture significantly affected sugar consumption, the main enological parameters and ester concentrations, with the exception of glycerol, isoamyl acetate and diethyl succinate levels. Remarkably, in wines obtained with mixed cultures the concentration of 2-phenylethyl acetate was approximately 3- to 9-fold greater than that produced by S. cerevisiae pure culture. Moreover sensory evaluation revealed a stronger fruity character in wines fermented with mixed cultures than in control wines. Independently of the mixed culture used, all wines showed concentrations of acetic acid and ethyl acetate within the ranges described for wines. Our data suggest that a mixed culture of H. osmophila and S. cerevisiae can be used as a tool to increase 2-phenylethyl acetate in wine and that its concentration can be controlled by modulating the initial yeast ratio in the culture.     

SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (10⁷–10⁸ cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4–6 days, while Sc remained culturable (about 10⁸ cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.     

Quantification of Plesiomonas shigelloides in Pure Culture and Clams by Competitive PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and clams based on the competitive polymerase chain reaction was developed. This is the first report for quantitative detection of P. shigelloides by competitive PCR. Forward (PS-F), reverse (PS23RV3), and hybrid primers were designed, and the specificity of the forward and reverse primer for P. shigelloides was proven. An internal standard DNA sequence was synthesized by PCR, with the hybrid primer as the forward primers and PS23RV3 as the reverse primer. A single concentration (0.588 pg/PCR) of internal standard (IS) was used for competitive PCR. The lowest level of detection of P. shigelloides was 80 CFU per PCR in pure culture, 240 CFU/g of clam tissue without enrichment, and 40 CFU/g of clam tissue after 7 hrs. nonselective enrichment at 37°C. There was a linear relationship between the log of the ratio of the relative fluorescent intensities of amplified target DNA bands to the internal standard DNA bands (IS) and the log of the CFU within a certain range in pure cultures and in clam tissue either with or without enrichment. The linear range with cells from a pure culture was the DNA derived from 8.0 × 10¹ to 8.0 × 10⁴ CFU per PCR while with clam tissue, the linear range was the DNA derived from 2.4 × 10² to 2.4 × 10⁵ CFU/g of tissue (1.2 × 10¹ to 1.2 × 10⁴ CFU per PCR) without enrichment, and 4.0 × 10¹ to 1.2 × 10⁴ CFU/g of clam tissue with enrichment, respectively.     

Bacteria and food poisoning organisms in milk

Individual milk samples of 50 goats, 50 ewes and 50 cows were examined for the total viable count, coliform bacteria, staphylococci and salmonellae. Growth of enterotoxin A producing Staphylococcus aureus NCTC 10 652 in the milk of the three animal species was also studied. The average total count was 1.9 × 10⁷ cells/ml for cow's, 7.7 × 10⁶ for goat's and 2.7 × 10⁶ for ewe's milk with micrococci staphylococci, rods and streptococci being the predominant organisms in the three milks, respectively. Goat's milk contained the lowest numbers of coliforms and ewe's milk the highest numbers. Staphylococcus aureus could not be detected in goat's milk, whilst 16 and 26% of the ewe's and cow's milk samples contained 100 and 80 cells/ml, respectively. Out of 39 coagulase positive staphylococci, 27 were thermonuclease positive, 18 produced lecithinase and 15 fermented mannitol. Red blood cells of sheep origin were much more resistant to lysis by ewe's strains compared to bovine strains. Growth curves of Staphylococcus aureus were nearly linear at 17°C but exponential at 31°C without lag phase. Hazardous numbers of about 10⁶ cells/ml were readily reached at 31°C after 6 h and at 17°C after 18 h. Salmonellae could not be detected in any of the samples examined. Out of 19 enterobacteria suspected to be salmonellae 11 proved to be Proteus and 8 Citrobacter.