Elaboration of Tempranillo wines at two different pHs. Influence on biogenic amine contents

The aim of this work was to study the effect of pH on biogenic amine formation during the elaboration and conservation (7 months) of Tempranillo wines. Grapes at two pHs (3.4 and 3.7) were vinified to achieve this purpose. After alcoholic fermentation the wines were either inoculated with a commercial malolactic starter or not inoculated. Identification and clonal distribution of lactic acid bacteria and amino acid concentration were determined in an attempt to explain the biogenic amine generation. The results showed that the pH of the must influenced the clonal distribution of the Oenococcus oeni strains which conducted the malolactic fermentation and also the concentration of amino acids in the wines after alcoholic fermentation. These aspects could account for the higher biogenic amine formation in wines with the lowest pH during malolactic fermentation. In these wines, inoculation with a malolactic starter was favourable since it produced a lower biogenic amine concentration after malolactic fermentation. Furthermore, the pH also influenced the concentration of amino acids after malolactic fermentation and the lactic acid bacteria distribution during the conservation of the wines. These aspects allow explaining the greater formation of histamine, tyramine and putrescine in the wines with the lowest pH vinified via lactic acid bacteria inoculation after seven months of conservation.     

Potential of phenolic compounds for controlling lactic acid bacteria growth in wine

Lactic acid bacteria are important in enology since they undergo the malolactic fermentation, a process which main effect is the reduction of wine acidity and is almost indispensable in red wine-making. However, if this process is not well controlled during the elaboration of wine, alterations in wine quality due to bacteria metabolic activity can happen. Polyphenols are wine natural components in must and wine that can potentially affect the growth of lactic acid bacteria and the malolactic fermentation. In this paper, after describing the main features of the malolactic fermentation in wine, we review the use of different chemical substances to control growth of lactic acid bacteria in enology. Special attention is given to phenolic compounds, being revised the recent studies about the effect of polyphenols on the growth and metabolism of lactic acid bacteria in wine in order to establish the extent to which these compounds are involved in malolactic fermentation during wine-making. Finally, the potential use of phenolic extracts as new antimicrobial agents during wine-making, as a total or partial alternative to traditional treatments mainly using sulphur dioxide (SO₂) is discussed.     

Analysis of lactic acid bacteria populations during spontaneous malolactic fermentation of Tempranillo wines at five wineries during two consecutive vintages

The ecology and the population dynamics of lactic acid bacteria during malolactic fermentation of Tempranillo wine made at five wineries in Castilla-La Mancha were analysed for two consecutive vintages (years 2006 and 2007). This microbiota was typed using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) subsequently identified using both molecular and phenotypic methods. Oenococcus oeni was the predominant species. Genotypes varied considerably in both of the vintages from each winery, although a number of coincident genotypes were found in each one. Three genotypes were present in all the wineries in at least one of the vintages. These results confirm the existence of a coincident population of O. oeni genotypes that may be considered endemic to this wine region and that seem to play an important role in all the vinifications.     

Free amino acids and proteolysis involved in ‘salchichon’ processing

Changes in free amino acid and nitrogen fraction contents involved in salchichón manufactured by industrial processing at different times of curing process were analysed. An increase in the free amino acid concentration and non protein fraction was observed during the curing process, specially between the fermentation stage and the second week of drying while soluble protein nitrogen decreased. The predominant amino acids in the initial mix of salchichon were Glutamic acid, Cysteine, Carnosine, Alanine and Taurine summing up the 73.7% of the total. During the last two weeks of drying, the concentration of some of the free amino acids went on increasing (Aspartic acid, Glutamic acid, Serine, Asparragine, Glycine, Taurine, Threonine, Alanine, Carnosine, Tryptophan and Lysine) but some others decreased significantly (Proline, Tyrosine, Valine, Methionine, Cysteine, Isoleucine and Leucine). The curing stage of salchichon might be estimated approximately based on the concentration of Aspartic acid, Glutamic acid, Serine, Glycine, Asparragine, Threonine and the total free amino acid content amino acids. The majority of amino acids that showed the greatest concentration in the final product were recorded as having `bitter' flavour characteristics.     

Bacterial dynamics during the spontaneous fermentation of cassava dough in gari production

The microbial dynamics and diversity during solid substrate fermentation of cassava, a case study of gari production in West Africa, was investigated. The 16S rDNA gene sequence analysis of the PCR-Denaturing Gradient Gel Electrophoresis (PCR–DGGE) analysis of microbial community DNA and Pulsed Field Gel Electrophoresis (PFGE) of selected isolates as well as culturing techniques using different selective media were used to monitor the bacterial dynamics during cassava fermentation. The V3 variable region of the 16S gene was analyzed and the closest relatives of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus casei were identified by sequencing of the DGGE band amplimers. The DGGE amplimers also revealed the succession and dynamics of LAB; there was a progressive increase in their population proportional to the fermentation period. The analysis of the PFGE band patterns showed that five diverse species of LAB were involved in the fermentation. The representative isolate of each of the PFGE clusters was phenotypicaly identified as L. plantarum, L. fermentum and Leuconostoc mesenteroides by the API 50 CHL sugar fermentation profile. These combinations of parameters identified heterofermentative LAB as bacteria that initiated the fermentation, reduced the pH below four and increased the acidity of the fermentation mash. Information such as this is relevant for the development of starter cultures and predictability of the process for traditional fermented foods and to aid their intermediate and large scale production.     

Survival of Listeria innocua in Minas Traditional Serro cheese during ripening

Cheese produced with raw milk can be a risk to consumer health. It is known that lactic acid bacteria present in raw milk and in natural starters can produce antimicrobial compounds against some foodborne bacteria. This work aimed to evaluate the survival of Listeria innocua in Minas Traditional Serro cheese during cheese ripening. The cheeses were inoculated with 10¹, 10² or 10³ CFU mL⁻¹ of the bacterium and were analyzed for 60 days of ripening at 30 °C. It was observed that the time and the dose of bacteria inoculated affected (p < 0.05) the survival of L. innocua. Even when the lowest dose was inoculated, at the end of the 60 days, approximately 10² CFU mL⁻¹ of L. innocua was detected in the cheese. The lactic acid bacteria present in the milk and in the natural starter were not sufficient to guarantee the absence of L. innocua in Minas Traditional Serro cheese even after 60 days of storage, as is required by Brazilian legislation.     

Effect of autochthonous starter cultures on microbiological and physico-chemical characteristics of Suan yu,a traditional Chinese low salt fermented fish

In this study, three groups of mixed starter cultures (S1: Lactobacillus plantarum 120, Staphylococcus xylosus 135 and Saccharomyces cerevisiae 31; S2: L. plantarum 145, S. xylosus 135 and S. cerevisiae 22; S3: Pediococcus pentosaceus 220; S. xylosus 135 and S. cerevisiae 22), isolated from Suan yu, were inoculated to produce the traditional fermented fish. After 42 days fermentation at 24 °C, Suan yu inoculated with different mixed starter cultures underwent rapid growth of lactic acid bacteria (LAB), declined pH, suppressed increase of thiobarbituric acid (TBARS) and total volatile base nitrogen (TVB-N) as well as growth of spoilage bacteria and pathogens. Besides, Suan yu had higher contents of non-protein nitrogen (NPN) and total free amino acids (FAA) compared to the control (P < 0.05). The muscle proteins were severely hydrolyzed during fermentation (SDS-PAGE). Moreover, the sensory evaluation indicates the fermented fish was more widely accepted than the control. The results suggest that the inoculation with S1, S2 and S3 reduced the lag time that fermentation began and improved the quality of Suan yu.     

Kinetics of traditional Turkish sausage quality aspects during fermentation

Changes in chemical (pH, moisture, salt, ash, fat, protein, free fatty acids (FFA), thiobarbituric acid reactive substances (TBARS) and residual nitrite contents), colour (Hunter L*, a* and b*, hue angle, chroma (saturation index), browning index (BI) and total colour difference (ΔE)) and microbiological (total mesophilic aerobic bacterial (TMAB), lactic acid bacteria (LAB), total Enterobacteriaceae (TE) and Staphylococcus and Micrococcus (SM)) quality characteristics of traditional fermented sausage “sucuk” during fermentation were investigated and kinetic modeling of these parameters were performed, in this study. The fermentation of the sucuk lasted 9 days. Analysis of the quality parameters was run on the beginning of the fermentation and on the 1st, 2nd, 3rd, 4th, 5th and 9th days of the fermentation process. Changes in the chemical and colour parameters were represented by zero, first and second order kinetic models. Microbial increments were represented by linear (first order kinetic) models and reductions were represented by both linear and Weibull distribution models.     

Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region

As a traditional alcoholic beverage, Chinese rice wine is quite popular for its unique flavor and high nutritional value. In this study, we investigated the changes of flavor compounds in varying stages of fermentation by HS-SPME-GC-MS and HPLC. In addition, 16s rDNA-PCR was utilized to analyze the changes of bacterial communities during fermentation of Chinese rice wine from the Shaoxing region. It was noted that the content of flavor compounds including esters, higher alcohols, amino acids and organic acids was different in varying stages of fermentation. In general, the flavor compounds of Chinese rice wine were mainly produced after pre-fermentation. The results also showed that the bacterial community structures and diversity of Chinese rice wine varied significantly during different fermentation stages, and more than 10 genera of bacteria was detected in Chinese rice wine fermentation broth. Among these specific bacteria identified in the study, Lactobacillus (the predominant genus) and Bacillus might take an active part in flavor development in Chinese rice wine. This implied that these bacteria might play significant contributions on the flavor of Chinese rice wine.     

Study of physicochemical parameters and spontaneous fermentation during traditional production of munkoyo, an indigenous beverage produced in Democratic Republic of Congo

The evolution of physicochemical parameters and spontaneous fermentation were investigated during traditional production of munkoyo. During munkoyo preparation, starch hydrolysis by amylolytic enzymes contained in munkoyo roots was performed at temperatures above 43 °C. After 90 h, pH decreased from 6.4-6.2 to 3.7-3.6; lactic acid concentration and acidity increased respectively from 4.4-15.0 mmol l⁻¹ to 42.0-44.0 mmol l⁻¹ and 0.05% to 0.45-0.6%; alcohol content did not exceed 394 mmol l⁻¹. When the temperature dropped from 43-42 °C to 29-25 °C, in the first 15 h of fermentation, the rate of pH decrease was significant, from 6.4-6.2 to 4.1-4.0. The acidification of munkoyo was due to lactic acid, largely in the form of the d (−) lactate isomer. At temperatures around 20 °C, secondary products also contributed to the acidification of the beverage. Lactic fermentation, observed from 43 °C in the wort, was promoted by thermophilic lactic acid bacteria. Lactobacillus delbrueckii lactis was identified as the representative lactic acid bacteria. Alcohol production in munkoyo, beginning only after 15 h of fermentation, is due mainly to Saccharomyces cerevisiae.     

A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage

By excluding the effects of organic acids and hydrogen peroxide, strain C2 producing a bacteriocin strongly inhibited Staphylococcus aureus ATCC 63589 and Escherichia coli ATCC 25922 was selected from 300 lactic acid bacteria isolated from traditional Chinese fermented cabbage. Strain C2 was identified as Lactobacillus sakei by phenotypical and physiological tests and 16S rDNA identification. This bacteriocin, which was designated sakacin C2, was purified by cold ethanol and Sephadex G50 column chromatography for further studies. The molecular weight of sakacin C2 was 5.5 kDa by Tris-Tricine SDS–PAGE, which was the largest among all known sakacins. Sakacin C2 exhibited a wide range of antimicrobial activity, strong heat stability (15 min at 121 °C) and pH stability (pH 3.0–8.0). Sakacin C2 was sensitive to protease but insensitive to lipase, α-amylase and β-amylase. These characters suggested that sakacin C2 was a novel Class II bacteriocin with a broad inhibitory spectrum and might broaden the application range of bacteriocins from LAB in the food industry.     

Fermented tofu: Enhancement of keeping quality and sensorial properties

In this study a new way to produce tofu by means of soymilk fermentation by specific lactic acid bacteria, Lactobacillus casei and Lactobacillus acidophilus alone or in combination, and subsequent precipitation has been developed, in order to prevent undesired microbial and chemical spoilage, as well as improve the stability and the quality of the product. In particular, the combination L. casei and L. acidophilus generated tofu having shelf life exceeding 20 days and able to prevent the growth of the spoilage strains inoculated. This fermented tofu was characterized by the production of antimicrobial molecules, such as acetic acid, limonene, 2-nonen-1-ol, 1-nonanol, 2(5H)-furanone, benzyl alcohol, phenylethyl alcohol and heptanoic acid. Depending on the Lactobacillus species used, the fermentation process generated different metabolites profiles and sensorial properties. Another promising properties conferred by the lactic acid bacteria fermentation was the inhibition of unsaturated fatty acids (UFAs) peroxidation or reduction of the aldehydes originated to their corresponding alcohols.     

Starter culture fermentation of Chinese sauerkraut: Growth,acidification and metabolic analyses

Chinese sauerkraut is a kind of traditional and typical fermented food. Four lactic acid bacteria (LAB) strains, Leuconostoc mesenteroides NCU1426, Lactococcus lactis NCU1315, Lactobacillus plantarum NCU1121 and Lactobacillus casei NCU1222 isolated from Chinese sauerkraut, were used in single starter cultures. Microbiological changes and pH values were monitored during fermentation. Metabolic substrates and products during the fermentation were examined using high performance liquid chromatography (HPLC) technology. Results have shown that Leu. mesenteroides and Lc. lactis grew faster, produced lactic acid earlier and were poorly acid-resistant, whereas Lb. plantarum and Lb. casei produced much more lactic acid throughout fermentation and showed better acid-tolerance. Two Lactococcus had outstanding performance in sucrose utilization while the other two Lactobacillus were likely to use glucose and fructose during fermentation. Unexpectedly, Leu. mesenteroides and Lc. lactis showed weak citric acid metabolism in fermentation. All the four LAB strains were able to utilize malic acid in early fermentation. In conclusion, these LAB strains have shown notable differences in growth and fermentative properties during starter culture fermentation of Chinese sauerkraut, probably resulting from LAB fermentative function and a mixture of complex substrates.     

Microbial composition of turbid rice wine (Makgeolli) at different stages of production in a real processing line

The aim of this study was to examine for changes in microbial populations during the production of turbid rice wine (also known as Makgeolli) at different production plants and to identify critical points for the control of microbial quality. Samples from raw ingredients (water, rice and wheat flour), materials from different production stages (steamed ingredients, the base of fermentation, primary and secondary fermentation stages at different time points), and the final rice wine products (non–sterilized and sterilized) were analyzed. The microbiological content of samples was assessed by quantitative (aerobic plate counts, lactic acid bacteria, fungi, acetic acid bacteria, coliforms and Bacillus cereus) and qualitative (Escherichia coli and eight foodborne pathogens) analyses. Aerobic plate counts for rice and wheat flour were relatively low (3.1 and 1.9 log CFU/g, respectively), as were those for lactic acid bacteria (1.6 and 2.1 log CFU/g), and fungi counts (2.2 and 0.7 log CFU/g). All counts increased to 7.8–7.9 log CFU/ml in the base of fermentation after the addition of Nuruk and Koji, and these counts were maintained at 8–9 log CFU/ml during fermentation. Acetic acid bacteria were not detected in the ingredients, but were isolated from the base of fermentation (1.2–2.8 log CFU/ml). Heat sterilization reduced aerobic pate counts significantly from 8.4 to 2.1 log CFF/ml, and lactic acid bacteria, fungi and acetic acid bacteria were reduced to non-detectable or negligible levels. Isolated microorganisms were considered as autochthonous to the environment or were artificially introduced with the starter culture. B. cereus was widely distributed throughout the manufacturing process, and may have been introduced from the raw material (present in 100% rice and 41.7% wheat flour samples). B. cereus counts were not significantly affected by sterilization, suggesting that it may exist at low levels as spores in the final products. No coliforms and other pathogens were detected in any samples. The raw materials, the base of fermentation, and sterilization stage were identified as important points for the control of microbial quality during the fermentation process.     

UV-C-inactivation of microorganisms in naturally cloudy apple juice using novel inactivation equipment based on Dean vortex technology

A novel UV-C irradiation device in laboratory scale was tested for its potential to inactivate bacteria in naturally cloudy apple juice. In this device, liquid flows through a helically wound tubing wrapped around a quartz glass tube containing a 9 W UV lamp with an irradiation intensity of 60 W/m² at 254 nm. The equipment was capable of reducing numbers of inoculated Escherichia coli and Lactobacillus brevis from an initial concentration of approximately 10⁶ CFU/ml or 10⁴ CFU/ml to below detectable limits in commercial naturally cloudy apple juice at a flow rate of 2 l/h, and to well below 1 × 10² also at higher flow rates of 4 and 8 l/h. Numbers of Saccharomyces cerevisiae could be reduced from an initial level of ca. 1 × 10⁴–1 × 10² CFU/ml or less at flow rates of 2 and 4 l/h. Although E. coli could be effectively inactivated also in self-extracted, as well as industrially processed apple juice, contaminating yeast and lactic acid bacteria were not completely eliminated.     

Molecular characterisation and antimicrobial activity of bacteria associated with submerged lactic acid cassava fermentation

Molecular identification of microorganisms associated with submerged cassava fermentation was carried out and isolates of lactic acid bacteria (LAB) were examined for antimicrobial activity, including ability to produce antimicrobial peptides as a first step to define starter cultures for controlled cassava fermentations. A total of 75 isolates, including 41 LAB, 31 aerobic bacteria (AB) and three anaerobic bacteria were isolated from unfermented and fermenting cassava roots, cassava leaves and fermented cassava dough and identified by a combination of phenotypic tests and sequencing of 16S rRNA, rpoA, rpoB and pheS genes. Microbial diversity at interspecies and intraspecies level was screened by, respectively, PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence based PCR (rep-PCR). Antimicrobial activity of LAB cultures and supernatants against indicator bacteria; Escherichia coli, Salmonella enterica serotype Typhimurium (S. Typhimurium), Bacillus cereus and Staphylococcus aureus was studied using agar diffusion tests. Furthermore, inactivation of indicator bacteria was investigated in both liquid medium and during controlled cassava fermentation. Results revealed a diversity of bacterial genera, species and subspecies associated with submerged cassava fermentation. DNA sequencing enabled identification of LAB isolates as Lactobacillus plantarum, Weissella confusa, Weissella paramesenteroides, Lactobacillus rhamnosus, Lactobacillus hilgardii, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecium, Enterococcus casseliflavus, and Pediococcus acidilactici. Lactobacillus spp. were the predominant LAB and were present in all cassava samples studied. Aerobic bacteria were predominantly Bacillus spp., including Bacillus subtilis, Bacillus amyloliquefaciens and B. cereus. Other species identified included Staphylococcus pasteuri and Clostridium beijerinkii. Cells, supernatants and cell free supernatants (CFS) of selected LAB isolates were able to inhibit both Gram positive and Gram negative pathogenic bacteria. LAB isolates inactivated all indicator organisms during controlled cassava fermentations, with a 4–6 log reduction after 48 h fermentation. The antimicrobial effect of the LAB was attributed to acid production.     

Survival of Escherichia coli O157:H7 and Listeria monocytogenes during kimchi fermentation supplemented with raw pork meat

Kimchi, a traditional Korean food, is fermented by lactic acid producing bacteria. Among the many types of kimchi, soongchimchae is a typical type of kimchi that combines fermented vegetables and meat. We aimed to investigate the survival of Escherichia coli O157:H7 and Listeria monocytogenes during the fermentation of kimchi supplemented with pork meat. Regardless of whether it was assessed in cabbage kimchi or radish kimchi, in which pH levels differ, the population of E. coli O157:H7 gradually decreased during the fermentation at 4 °C and was no longer detected in cabbage samples after 14 post-fermentation days (PFDs). The pH of cabbage kimchi and radish kimchi were 5.8 and 6.0, respectively, at 0 PFDs, changing to 3.9 and 4.1 at 15 PFDs. Although the population of L. monocytogenes gradually decreased, L. monocytogenes did not survive in cabbage kimchi or radish kimchi after 15 PFDs. Compared with radish kimchi, the pH of cabbage kimchi dropped rapidly during the early fermentation period and reached a pH of 3.9 at 15 PFDs. We observed that pH changes during fermentation were associated with a reduction in foodborne bacterial pathogens. Antimicrobial factors and inhibitory mechanisms against foodborne bacteria need to be investigated in the different types of kimchi.     

Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties

The ability of lactic acid bacteria (LAB) to inhibit Aspergillus, Fusarium, and Penicillium, the main contaminants in bread, was evaluated. Only four strains (Lactobacillus plantarum CRL 778, Lactobacillus reuteri CRL 1100, and Lactobacillus brevis CRL 772 and CRL 796) from 95 strains tested displayed antifungal activity. The major antifungal compounds were acetic and phenyllactic acids. The fermentation quotient (FQ = 2.0) and the leaven volume (80 cm³) of doughs with LB and yeasts were higher than doughs without LB. The inclusion of antifungal LAB strains in the starter culture allowed a reduction in the concentration of calcium propionate by 50% while still attaining a shelf life similar to that of traditional bread containing 0.4% CP.     

Ripening of Ossau-Iraty cheese: determination of free amino acids by RP-HPLC and of total free amino acids by the TNBS method

The free amino acids and sulphosalicylic acid-soluble N fraction in the French ewes’-milk cheese, Ossau-Iraty, were determined to evaluate the degree of proteolysis during ripening of the cheese. RP-HPLC was used to assess the free amino acids, the trinitrobenzenesulphonic acid method to assess the sulphosalicylic acid-soluble N fraction. The total free amino acids content as determined by RP-HPLC ranged from 200 mg/100 g dry matter on the first day of ripening to 2200 mg/100 g dry matter after 120 d of ripening. The major amino acids were glutamic acid, asparagine, glutamine, valine, leucine, phenylalanine and lysine, which accounted for between 50% (on day 1) and 70% (on day 120) of the total free amino acids. Correlations between all the main free amino acids and ripening time were good. Regression of the sulphosalicylic acid-soluble N fraction as determined by the trinitrobenzenesulphonic acid method on the total free amino acids as determined by RP-HPLC yielded a correlation coefficient value of 0.985.