Entire Fermentation Process Research Articles

Comparative evaluation of several Dynamics of accumulation of lactic and acetic acids in the process of directed fermentation of white cabbage of the Parus variety

Relevance and methodology. Today, white cabbage is the most popular type of vegetable raw materials used for fermentation not only in Russia, but also in other countries. To obtain a variety of flavors and shades of flavor, various vegetables (carrots, sweet peppers), fruit (apples, cranberries) and spicy-aromatic additives (cumin, dill seed, etc.) are added to the cabbage during fermentation. During fermentation, the product contains not only the initial components of the raw material (vitamin C, macro-and micronutrients), but also significantly increases the number of lactic acid microorganisms – the main "participants" in the process.One of the main products of the metabolism of lactic acid microorganisms is lactic acid. At a concentration of 0.5%, it begins to inhibit the development of many microorganisms. When reaching a concentration of 1-2% microbiological enzymatic hydrolysis, as a rule, ceases. The accumulation of acetic acid allows us to judge the intensity of the heterofermentative stage of fermentation using strains of lactic acid microorganisms.Results. The L. plantarum + L. casei consortium has shown a synergistic interaction in the accumulation of lactic acid, since the amount of acid produced by the consortium significantly exceeds the amount of acid produced by each strain separately. This point is also noted for the accumulation of acetic acid. In the L. brevis + L. casei consortium, the opposite situation is observed: when using cultures separately, the accumulation of lactic acid occurs to a greater extent than when using two cultures simultaneously. There are no obvious differences between monocultures and the consortium in terms of acetic acid accumulation. In the case of the L. plantarum + L. brevis consortium, we observe a synergistic interaction, with the dominant role in this consortium played by L. plantarum, and the contribution of L. brevis to the accumulation of lactic acid is insignificant. At the same time, the total resulting accumulation of acetic acid is significantly less than the result of each strain separately, which may indicate a negative mutual effect of the participants in the process. Acid degradation of the polysaccharide matrix of cell walls does not occur during the entire fermentation process.

Robustness of fermented carrot juice against Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7

Artisanal vegetable fermentations are regaining popularity in industrialized countries, but they could be prone to contamination with foodborne pathogens. By simulating home or small-scale restaurant fermentations, we evaluated the microbiological safety of spontaneous carrot juice fermentations. Raw carrot juice was spiked with Listeria monocytogenes, Salmonella enterica subsp. enterica Typhimurium and Escherichia coli O157:H7, and the microbial dynamics were followed throughout the entire fermentation process by cultivation and amplicon sequencing. In addition, the behavior of these pathogens was also monitored after addition of raw cucumber juice and storage under refrigerated conditions to mimic post-contamination issues. Although the numbers of the pathogens increased during the first phase of the fermentation, the pathogens were not able to persist throughout the fermentation. Their numbers fell below the detection limit after 8 days of fermentation at 20 °C. Further investigation using amplicon sequencing also showed that there was no major impact on the general microbial dynamics of the spontaneous carrot juice fermentation. This indicates that the artisanal carrot juice fermentation is a robust process which resists the persistence of pathogens. More caution is needed however when mixing the final fermented product with a raw juice. When simulating pathogen post-contamination, both Salmonella enterica and Escherichia coli were able to survive in the refrigerated fermented juice up to 10 days after the fermentation. Listeria monocytogenes was detected up to 8 days in the refrigerated juice. Pasteurization of the raw juice before adding it to the fermented product is thus recommended.

Composition and Metabolic Functions of the Microbiome in Fermented Grain during Light-Flavor Baijiu Fermentation.

The metabolism and accumulation of flavor compounds in Chinese Baijiu are driven by microbiota succession and their inter-related metabolic processes. Changes in the microbiome composition during Baijiu production have been examined previously; however, the respective metabolic functions remain unclear. Using shotgun metagenomic sequencing and metabolomics, we examined the microbial and metabolic characteristics during light-flavor Baijiu fermentation to assess the correlations between microorganisms and their potential functions. During fermentation, the bacterial abundance increased from 58.2% to 97.65%, and fermentation resulted in the accumulation of various metabolites, among which alcohols and esters were the most abundant. Correlation analyses revealed that the levels of major metabolites were positively correlated with bacterial abundance but negatively with that of fungi. Gene annotation showed that the Lactobacillus species contained key enzyme genes for carbohydrate metabolism and contributed to the entire fermentation process. Lichtheimia ramosa, Saccharomycopsis fibuligera, Bacillus licheniformis, Saccharomyces cerevisiae, and Pichia kudriavzevii play major roles in starch degradation and ethanol production. A link was established between the composition and metabolic functions of the microbiota involved in Baijiu fermentation, which helps elucidate microbial and metabolic patterns of fermentation and provides insights into the potential optimization of Baijiu production.

Study on the dynamic changes and formation pathways of metabolites during the fermentation of black waxy rice wine.

Black waxy rice wine fermentation metabolites are closely related to the product's final quality. However, little is known about dynamic metabolite changes during fermentation. Here, we used gas chromatography time‐of‐flight mass spectrometry (GC‐TOF‐MS) metabolomics and multivariate statistical analysis to explore the relationship between metabolites and fermentation time. A total of 159 metabolites were identified during the entire fermentation process. The PCA analysis revealed a clear separation between the samples after 4 days and 2 days, and the samples after 4–24 days clustered together. This indicated that BGRW fermentation progresses rapidly in the first 48 hr of fermentation. A total of 40 metabolites were identified as differential during fermentation (VIP > 1 and p < .05), including 12 organic acids, four amino acids, one fatty acid, 17 sugars and sugar alcohols, one alcohol, and five other metabolites. Pathway analysis showed that the differential metabolites were involved in 28 metabolic pathways, and the most commonly influenced pathways (impact value > 0.1 and p < .05) were galactose metabolism, pyruvate metabolism; starch and sucrose metabolism; alanine, aspartic acid, and glutamate metabolism; the tricarboxylic acid cycle, glyoxylic acid, and dicarboxylic acid metabolism; and amino sugar and nucleotide sugar metabolism. Moreover, the integrated metabolic pathway was generated to understand the transformation and accumulation of differential metabolites. Overall, these results provide a comprehensive overview of metabolite changes during black waxy rice wine fermentation.

Combined Application of High-Throughput Sequencing and Metabolomics Reveals Metabolically Active Microorganisms During Panxian Ham Processing.

Panxian ham, a traditional Chinese dry-cured ham, is protected by national geographical indication. Similar to other fermented foods, the microbial population of dry-cured ham is pivotal to taste and flavor formation. This study aimed to establish the relationship between microorganisms and metabolites during the spontaneous fermentation of Panxian ham. Multivariate analysis based on metabolomics data revealed that continuous metabolic changes occurred during the entire fermentation process, with the most significant changes occurring in the initial stage of ripening. Thirty-one significantly different metabolites (SDMs) were identified as discriminant factor, and pathway analysis suggested that these metabolites were involved in 30 pathways, including alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; and arginine and proline metabolism. Microbial community analysis using the Illumina MiSeq platform indicated that the bacterial community was more complex than the fungal community, and their succession regulation differed during processing. At the genus level, 11 bacteria and five fungi were identified as core microbes, of which Staphylococcus was the dominant bacteria and Debaryomyces and Aspergillus were the dominant fungi. Further, statistical redundancy analysis (RDA) indicated that Staphylococcus, Debaryomyces, and Chromohalobacter promoted the production of amino and fatty acids; Cobetia and Aspergillus were associated with sugar metabolism, and Kushneria, Penicillium, and Yamadazyma were closely related with organic acids. These findings provide fundamental knowledge regarding the metabolically active microorganisms in Panxian ham, helping industrial processors to develop effective strategies for standardizing quality parameters.

High-energy ball milling treatment of soybean for Bacillus thuringiensis culture media

Soybean meal has been intensively used as a substrate in culture media for several microorganisms. However, the fermentable sugar containing the soybean needs to be released from the solid matrix through different processes. Against this backdrop, the present study explores the use of high-energy ball milling as a one-step treatment method for expedited production of fermentable sugars of textured soybean. The best result is observed after only 5min of milling, obtaining 34.1 times more fermentable sugars than untreated textured soybean, and 2.5 times more than commercially used soybean meal. Notably, the textured soybean ball-milled has been used as a substrate for Bacillus thuringiensis var. kurstaki HD-73 fermentation. The cell and spore production is also compared with a standard Rowe media. The maximum cell concentration obtained in the entire fermentation process using ball-milled textured soybean media is found to be higher than the concentration obtained using the standard Rowe media. In addition, it is observed that there is a direct correlation between maximum cell production and reducing sugar concentration generated by the high-energy ball milling treatment. No fermentation inhibitors or by-products are generated during the physical treatment.

Lactobacillus paracasei HD1.7 used as a starter modulates the bacterial community and metabolome profile during fermentation of Chinese cabbage.

Pickled Chinese cabbage is a very traditional and popular food in Asia and where Asians migrate. High-throughput sequencing and GC-MS have been used to investigate the effects of Lactobacillus paracasei HD1.7 as a starter on the bacterial community and metabolome during pickled Chinese cabbage fermentation. Lactobacillus paracasei HD1.7 accelerated acidification and resulted in server acidity. Lactobacillus dominated the entire fermentation process. Lactobacillus paracasei HD1.7-inoculated fermented cabbage had several advantages over naturally fermented cabbage, including a shorter fermentation process, fewer pathogens and more plentiful flavours. This research suggested that L. paracasei HD1.7 would be a desirable starter in the fermentation of other vegetables.

A Novel Dual-cre Motif Enables Two-Way Autoregulation of CcpA in Clostridium acetobutylicum.

The master regulator CcpA (catabolite control protein A) manages a large and complex regulatory network that is essential for cellular physiology and metabolism in Gram-positive bacteria. Although CcpA can affect the expression of target genes by binding to a cis-acting catabolite-responsive element (cre), whether and how the expression of CcpA is regulated remain poorly explored. Here, we report a novel dual-cre motif that is employed by the CcpA in Clostridium acetobutylicum, a typical solventogenic Clostridium species, for autoregulation. Two cre sites are involved in CcpA autoregulation, and they reside in the promoter and coding regions of CcpA. In this dual-cre motif, creP, in the promoter region, positively regulates ccpA transcription, whereas creORF, in the coding region, negatively regulates this transcription, thus enabling two-way autoregulation of CcpA. Although CcpA bound creP more strongly than creORFin vitro, the in vivo assay showed that creORF-based repression dominates CcpA autoregulation during the entire fermentation. Finally, a synonymous mutation of creORF was made within the coding region, achieving an increased intracellular CcpA expression and improved cellular performance. This study provides new insights into the regulatory role of CcpA in C. acetobutylicum and, moreover, contributes a new engineering strategy for this industrial strain.IMPORTANCE CcpA is known to be a key transcription factor in Gram-positive bacteria. However, it is still unclear whether and how the intracellular CcpA level is regulated, which may be essential for maintaining normal cell physiology and metabolism. We discovered here that CcpA employs a dual-cre motif to autoregulate, enabling dynamic control of its own expression level during the entire fermentation process. This finding answers the questions above and fills a void in our understanding of the regulatory network of CcpA. Interference in CcpA autoregulation leads to improved cellular performance, providing a new useful strategy in genetic engineering of C. acetobutylicum Since CcpA is widespread in Gram-positive bacteria, including pathogens, this dual-cre-based CcpA autoregulation would be valuable for increasing our understanding of CcpA-based global regulation in bacteria.

Biological effect of magnetic field on the fermentation of wine

During the transformation process of matter is produced energy, which afterwards interacts with matter itself, and other forms of energy. Energy induced electromagnetic appliances may affect the processes occurring in biological systems. In our study we have evaluated the wine fermentation process of the magnetic field with different amplitudes of electromagnetic induction, the constant exposure of 30 minutes a day for 10 days. The device for inducing magnetism was constructed at the Department of Fruit Growing, Viticulture and Enology at Slovak University of Agriculture in Nitra for research purposes. Essence of the device lies in the way of the management of direct current, which flows through the coil. Volume of direct current is regulated by network auto-transformer. Output of network autotransformer is rectified by two-way bridge rectifier. The coil is powered by a direct current voltage pulse. This device has a maximum value of the magnetic induction at 150 mT. At full power it must be supplied from three-phase socket with a rated current of 32 A. For our experiment, we chose wine grape variety of Hibernal, from Nitra wine region. The magnetic field induced by the electromagnetic device has an impact on the process of fermentation and sensory characteristics of a young wine. As part of the sensory profile, we noticed higher levels of residual sugar and speed up of the fermentation process and the process of purifying of the young wine. The influence of magnetic field on grape juice during the entire fermentation process and production of wine is a convenient way to improve the quality of wine without side effects or any chemical additives.

Microbial ecology involved in the ripening of naturally fermented llama meat sausages. A focus on lactobacilli diversity

Llama represents for the Andean regions a valid alternative to bovine and pork meat and thanks to the high proteins and low fat content; it can constitute a good product for the novel food market. In this study, culture-dependent and independent methods were applied to investigate the microbial ecology of naturally fermented llama sausages produced in Northwest Argentina. Two different production technologies of llama sausage were investigated: a pilot-plant scale (P) and an artisanal one (A). Results obtained by High-Throughput Sequencing (HTS) of 16S rRNA amplicons showed that the production technologies influenced the development of microbial communities with a different composition throughout the entire fermentation process. Both sequencing and microbiological counts demonstrated that Lactic Acid Bacteria (LAB) contributed largely to the dominant microbiota. When a total of 230 isolates were approached by RAPD-PCR, presumptive LAB strains from P production exhibited an initial variability in RAPD fingerprints switching to a single profile at the final of ripening, while A production revealed a more heterogeneous RAPD pattern during the whole fermentation process. The constant presence of Lactobacillus sakei along the fermentation in both productions was revealed by HTS and confirmed by species-specific PCR from isolated strains. The technological characterization of Lb. sakei isolates evidenced their ability to grow at 15°C, pH4.5 and 5% NaCl (95%). Most strains hydrolyzed myofibrillar and sarcoplasmic proteins. Bacteriocins encoding genes and antimicrobial resistance were found in 35% and 42.5% of the strains, respectively. An appropriate choice of a combination of autochthonous strains in a starter formulation is fundamental to improve and standardize llama sausages safety and quality.

Selection and use of pectinolytic yeasts for improving clarification and phenolic extraction in winemaking

Pectinase enzymes have shown a considerable influence in both, sensitive and technological properties of wines. They can help to improve clarification process, releasing more color and flavor compounds entrapped in grape skin, facilitating the liberation of phenolic compounds. This work aims to find yeasts that, because of their native pectinases, can be applied on combined fermentations with Saccharomyces cerevisiae obtaining significant benefits over single-inoculated traditional fermentations. 462 yeast strains isolated from wineries were identified and tested for several enzymatic activities of recognized interest for enology industry. Considering the 7 identified species, only Aureobasidium pullulans, Metschnikowia pulcherrima and Metschnikowia fructicola showed polygalacturonase activity. Because of its interest in winemaking, due to its reported incidence in wine flavor, the impact of M. pulcherrima as a source of pectinolytic enzymes was analyzed by measuring its influence in filterability, turbidity and the increase on color, anthocyanin and polyphenol content of wines fermented in combination with S. cerevisiae. Among the strains screened, M. pulcherrima NS-EM-34 was selected, due to its polygalacturonase activity, for further characterization in both, laboratory and semi-industrial scale assays. The kinetics concerning several metabolites of enological concern were followed during the entire fermentation process at microvinification scale. Improved results were obtained in the expected parameters when M. pulcherrima NS-EM-34 was used, in comparison to wines fermented with S. cerevisiae alone and combined with other pectinolytic and non-pectinolytic yeasts (A. pullulans and Lachancea thermotolerans, respectively), even working better than commercial enzymes preparations in most parameters. Additionally, M. pulcherrima NS-EM-34 was used at a semi-industrial scale combined with three different S. cerevisiae strains, confirming its potential application for red wine improvement on the mentioned sensorial and technological properties.

Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite

Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions.In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite.Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must.

Characterization of lactic acid bacteria isolated from Bosnian artisanal dry fermented sausage (sudzuk) during fermentation

Bosnian sudzuk is a dry fermented sausage produced in a rural household near the town of Visoko in central Bosnia and Herzegovina. This kind of sausage was manufactured only from beef and spices in a traditional way without the addition of a starter cultures. To identify lactic acid bacteria (LAB), a total number of 160 Lstrains were isolated from five samples of Bosnian sudzuk collected over 28 days of fermentation. Preliminary identification by phenotypic tests and 16S rDNA sequencing were performed for all 160 of the Lisolates. Identification of Lstrains from traditionally produced Bosnian sausage at the species level revealed the presence of six genera: Lactococcus sp., Enterococcus sp., Leuconostoc sp., Lactobacillus sp., Pediococcus sp. and Weissella sp.. Among the 15 distinct species identified, the species Lactobacillus plantarum, Leuconostoc mesenteroides, Lactococcus lactis, Enterococcus faecalis and Enterococcus durans were present throughout the entire process of fermentation. Leuconostoc mesenteroides, Lactobacillus plantarum and Lactococcus lactis prevailed, with 21.8%, 19.3% and 13.1%, respectively, of total Lstrains during the entire fermentation process. Significant negative correlations (r = 0.892 and r = 0.829, respectively) between the presence of Weissella sp. and Lactobacillus sp., and between the presence of Weissella sp. and Lactococcus sp. were recorded. Lactobacillus plantarum, Enterococcus durans and Leuconostoc mesenteroides were the best producers of aromogenic compounds while 32.3% of Lactobacillus plantarum and 28.6% of Leuconostoc mesenteroides were produced exopolysaccharides.

Change of pH during excess sludge fermentation under alkaline, acidic and neutral conditions

The change in pH during excess sludge (ES) fermentation of varying sludge concentrations was investigated in a series of reactors at alkaline, acidic, and neutral pHs. The results showed that the changes were significantly affected by fermentative conditions. Under different conditions, pH exhibited changing profiles. When ES was fermented under alkaline conditions, pH decreased in a range of (10±1). At the beginning of alkaline fermentation, pH dropped significantly, at intervals of 4h, 4h, and 5h with sludge concentrations of 8665.6mg/L, 6498.8mg/L, and 4332.5mg/L, then it would become moderate. However, under acidic conditions, pH increased from 4 to 5. Finally, under neutral conditions pH exhibited a decrease then an increase throughout entire fermentation process. Further study showed short-chain fatty acids (SCFAs), ammonia nitrogen and cations contributed to pH change under various fermentation conditions. This study presents a novel strategy based on pH change to predict whether SCFAs reach their stable stage.

Microbiota dynamics related to environmental conditions during the fermentative production of Fen-Daqu, a Chinese industrial fermentation starter

Chinese Daqu is used as a starter for liquor and vinegar fermentations. It is produced by solid state fermentation of cereal–pulse mixtures. A succession of fungi, lactic acid bacteria and Bacillus spp. was observed during the production of Daqu. Mesophilic bacteria followed by fungi, dominated the first phase of fermentation. Next, lactic acid bacteria increased in relative abundance, resulting in an increase of the acidity of Daqu. At the final stages of fermentation, Bacillus spp. and thermophilic fungi became the dominant groups, possibly due to their tolerance to low water activity and high temperature. Both culture-dependent and culture-independent analyses confirmed that Bacillus spp. were ubiquitous throughout the process. Yeast species such as Wickerhamomyces anomalus, Saccharomycopsis fibuligera and Pichia kudriavzevii were present throughout almost the entire fermentation process, but the zygomycetous fungus Lichtheimia corymbifera proliferated only during the final stages of fermentation. Canonical correspondence analysis (CCA) revealed the significance of acidity, moisture content and temperature in correlation with the composition of the microbial communities at different stages.

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q(p) for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l(-1) in 48 h with the yield of 0.051 g g(-1) by applying this strategy, which were 20.5 and 21.4% over the best results controlled by constant agitation speeds.

Characterization and dynamic behavior of wild yeast during spontaneous wine fermentation in steel tanks and amphorae.

We studied the dynamic behavior of wild yeasts during spontaneous wine fermentation at a winery in the Valais region of Switzerland. Wild yeasts in the winery environment were characterized using a PCR-RFLP method. Up to 11 different yeast species were isolated from the vineyard air, whereas only seven were recovered from the grapes surface. We initially investigated a cultureindependent method in pilot-scale steel fermentation tanks and found a greater diversity of yeasts in the musts from two red grape varieties compared to three white grape varieties. We found that the yeasts Metschnikowia pulcherrima, Rhodotorula mucilaginosa, Pichia kluyveri, P. membranifaciens and Saccharomyces cerevisiae remained active at the end of the fermentation. We also studied the dynamic behavior of yeasts in Qvevris for the first time using a novel, highlysensitive quantitative real-time PCR method. We found that non-Saccharomyces yeasts were present during the entire fermentation process, with R. mucilaginosa and P. anomala the most prominent species. We studied the relationship between the predominance of different species and the output of the fermentation process. We identified so-called spoilage yeasts in all the fermentations, but high levels of acetic acid accumulated only in those fermentations with an extended lag phase.

Gas–liquid transfer of aroma compounds during winemaking fermentations

We precisely monitored the production kinetics of 16 volatile carbon compounds corresponding to the predominant higher alcohols and esters produced during the alcoholic fermentation of wine using an on-line GC system. We studied the gas–liquid partitioning of isobutanol, isoamyl acetate and ethyl hexanoate and showed that CO2 stripping had no impact on the partition coefficient (ki). We formulated a predictive model for ki changes during the fermentation and calculated the gas–liquid balances of these volatile compounds throughout the entire fermentation process. Losses in the off-gas were highly compound-dependent. They were negligible for higher alcohols but very high for esters, with losses of up to 70% for ethyl hexanoate at 30 °C. The rates of volatile compound production, accumulation and loss could also be calculated with high precision. Loss rates were maximal at the end of fermentation, indicating that high final temperatures, although helpful in avoiding sluggish fermentations, can be very detrimental to aroma losses.

English

The number of laccase isoenzymes and laccase activities of Pleurotus ostreatus in buffered and non-buffered media were determined. In both culture media, the initial pH was adjusted to 3.5. Similar laccases activity (100-500 U/L) was observed in the range of 100-400 h (approximately) of fermentation in both media. Buffered culture medium yielded higher laccases activities (3200 U/L) at 500 h of fermentation than those observed in non-buffered culture medium (450 U/L). One laccase isoenzyme was produced during the entire fermentation process in both media. In the non-buffered medium, an additional isoenzyme of lower molecular weight than that produced in the entire fermentation process was produced at the beginning of the exponential phase of growth when the pH reached a value of 6.5. These results suggest that the fungus produces metabolites that modify the pH of the medium and that some laccases isoenzymes are regulated by pH signals. This is the first report on zymogram patterns from laccases activity of P. ostreatus at low pH. Key words: Laccases, fermentation, Pleurotus ostreatus, zymogram.

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0–6 h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12–54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.