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Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content.

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.

DNA, Fungal↗

Rapid detection and quantification of yeast species during spontaneous wine fermentation by PCR-RFLP analysis of the rDNA ITS region.

PCR-RFLP analysis of the rDNA-ITS (internal transcribed spacer) region was applied to 174 yeast strains belonging to 30 species of oenological significance and including 27 type strains in order to define a rapid identification protocol for yeast colonies. DraI-or HaeIII-PCR-RFLP patterns were species-specific with the exception of teleomorphic and anamorphic forms. An improved protocol taking about 30 h was used for the detection and quantification of yeast species occurring in the course of a spontaneous wine fermentation at industrial level. Wine samples were taken and plated daily on an agar medium and the developed colonies were analysed by PCR-RFLP after 24 h of incubation. A representative sample of these colonies was also identified by traditional methods. Both procedures gave identical results. However, PCR-RFLP analysis allowed a more precise enumeration of the yeast populations, proving to be a reliable and simple method for monitoring the development of the yeast community throughout wine fermentation.

Journal Article↗

Phenotypic and technological diversity of lactic acid bacteria and staphylococci isolated from traditionally fermented sausages in southern Greece.

The physicochemical and microbiological characteristics of spontaneously fermented sausages made by two medium-sized enterprises (MSE) located in southern Greece have been studied. A total of 300 lactic acid bacteria and 300 staphylococcal strains have been isolated and identified by their physiological characteristics. Lactobacillus plantarum strains were found to dominate the lactic acid bacteria microbiota in most of the cases with L. sakei strains prevailing in some of them and L. rhamnosus strains occasionally accompanying the dominant lactic acid bacteria microbiota. On the other hand, S. saprophyticus strains were found to dominate the staphylococcal microbiota in all spontaneously fermented sausages with of S. simulans, S. xylosus, S. gallinarum and S. cohnii cohnii strains being sporadically present. Following the identification, an evaluation of their technological properties, namely proteolytic and lipolytic capacities as well as production of biogenic amines and antimicrobial compounds, took place. None of the lactic acid bacteria and staphylococci was found to possess lipolytic activity whereas a total of 6 lactic acid bacteria and 51 staphylococci strains were found to be able to hydrolyse either the sarcoplasic, myofibrillar or both protein fractions. Furthermore, only one L. sakei strain and 185 staphylococci strains were found to possess decarboxylase activity against lysine, tyrosine, ornithine or histidine. Finally none of the staphylococcal microbiota and 3 lactic acid bacteria strains were found to be able to produce antimicrobial compounds of proteinaceous nature against Listeria monocytogenes.

Animals↗

Microbiological and biochemical characterization of cassava retting, a traditional lactic Acid fermentation for foo-foo (cassava flour) production.

The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C(inf2) to C(inf4)) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products.

Journal Article↗

Effectiveness of a Lactobacillus sakei starter culture in the reduction of biogenic amine accumulation as a function of the raw material quality.

The effectiveness of an amine-negative starter culture (Lactobacillus sakei CTC494) in the reduction of biogenic amine production during the ripening of fermented sausages was examined. Four batches were manufactured in parallel: spontaneously fermented and starter-mediated sausages were manufactured from two lots of raw materials of different hygienic quality. Besides the biogenic amine contents, changes in the microbial counts, nitrogenous fractions, pH, and water content were measured at several sampling points during the ripening process. In sausages manufactured from good quality meat, the starter strain of L. sakei reduced and even inhibited biogenic amine accumulation during sausage fermentation, the end products showing extremely low biogenic amine contents (tyramine levels less than 15 mg/kg of dry matter and putrescine and cadaverine levels less than 5 mg/kg of dry matter). Nevertheless, starter-mediated sausages made from poorer-quality raw materials showed much higher amine contents (308, 223, and 36 mg/kg of dry matter of cadaverine, tyramine, and putrescine, respectively), which were only slightly lower than those of the spontaneously fermented sausages made from the same raw materials. The relatively high bacterial numbers of raw materials of poorer-hygienic quality diminished the beneficial effect of the starter strain. Therefore, the effectiveness of the starter was strongly dependent on the hygienic quality of the raw materials used.

Animals↗

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece.

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.

DNA, Mitochondrial↗

Evaluation of danger from fermentation-induced spontaneous ignition of wood chips.

Recently we conducted investigations in biological wastes because large pile-up storage of waste wood chips and others caused many fires in Japan. This paper shows the experimental results on wood chips with thermal analysis, by using a Thermogravimetry-differential thermal analysis (TG-DTA) and micro calorimeters, and as well with spontaneous ignition measurements, such as a UN wire mesh cube tester and a spontaneous ignition tester (SIT). Exothermic reaction of wood chips was observed during 45-60 degrees C only by the high sensitive microcalorimeters, TAM and MS 80. This reaction is far apart from the second major reaction by oxidation and is not easy to be recognized by the conventional detectors, like the TG-DTA and the wire mesh cube tester, because their sensitivity cannot meet the strict requirement. Correspondingly, experimental results under the adiabatic condition in the SIT confirmed this theory, in which the onset temperature of spontaneous ignition of wood chips was measured as 50-80 degrees C. This implies that the weak initial reaction at ambient temperatures mainly results from microbial fermentation in the presence of its inherent moisture and possibly gives rise to the further intense combustion sustained by a chemical reaction if the heat cannot be removed from the large scale storage of wood chips.

Fermentation↗

Mixed starter cultures to control biogenic amine production in dry fermented sausages.

Several combinations of an amine-negative Lactobacillus sakei strain, along with proteolytic Staphylococcus carnosus or Staphylococcus xylosus strains, were used to study the influence of mixed starter cultures on biogenic amine production during the manufacture of dry fermented sausages. Changes in pH, water content, proteolysis, microbial counts, and biogenic amine contents were simultaneously examined in a spontaneously fermented batch and in three mixed starter-mediated batches. A double-controlled microbial charge initially inoculated as mixed starter culture of L. sakei and Staphylococcus spp. (all amine-negative strains) drastically reduced tyramine, cadaverine, and putrescine accumulation. No production of other aromatic amines such as histamine, phenylethylamine, or tryptamine was observed in any batch. The polyamines, spermine and spermidine, were found in raw materials and their levels decreased slightly in the spontaneously fermented batch. No correlation between proteolysis and biogenic amine production was observed. The use of proper technological conditions favoring starter development and the use of the raw materials with good hygienic quality make it possible to produce fermented sausages nearly free of biogenic amines.

Animals↗

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal.

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.

Agriculture↗

Multiple strains of Saccharomyces cerevisiae on a single grape vine.

On the basis of the levels of secondary product formation four different phenotypes were represented among the 28 strains of Saccharomyces cerevisiae isolated during the spontaneous fermentation of grape juice. The genetic analysis indicated that four different strains, representing each phenotypic class, were derived, one from the other, by mutation. The spontaneous fermentation of a Malvasia must was dominated by different strains of Saccharomyces cerevisiae at different stages of fermentation.

Acetaldehyde↗

The microflora of fermented nixtamalized corn.

Nixtamalization is a traditional process that improves the nutritional quality of corn. To provide a means of utilizing the nutritional benefits of nixtamalized corn and improve product acceptability, lactic acid fermentation was applied. The objective of the study was to study the microbial profile and establish the important lactobacilli of fermenting nixtamalized corn dough. Two batches of cleaned whole corn were subjected to the process of nixtamalization, using two concentrations of lime (0.5 or 1.0%), milled, made into a dough (50% moisture) and fermented spontaneously for 72 h. A control sample was prepared without alkaline treatment. pH and titratable acidity of the dough were measured. Aerobic mesophiles, lactic acid bacteria, yeasts and molds were enumerated on Plate Count Agar (PCA), deMan, Rogossa and Sharpe (MRS) Agar and Malt Extract Agar (MEA), respectively. The identity of lactobacilli present was established at the species level using API 50 CHL. The pH of all the fermenting systems decreased with fermentation time with concomitant increase in titratable acidity. Lactic acid bacteria in numbers of 1.6 x 10(9), 2.3 x 10(9) and 1.8 x 10(9) cfu/g, respectively yeasts and molds, and numbers of 8.0 x 10(7), 5.0 x 10(5) and 1.7 x 10(5) cfu/g, respectively were observed in the control and the two nixtamalized (0.5% and 1.0% lime) samples after 48 h of fermentation. Lactobacilli identified in the fermenting nixtamalized corn dough were Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus cellobiosus as well as Pediococcus spp. The study demonstrates that nixtamalized corn though alkaline in nature can be subjected to spontaneous fermentation to produce a sour product.

Colony Count, Microbial↗

Characterization of the bacterial flora of Sudanese sorghum flour and sorghum sourdough.

The microflora of a Sudanese sorghum flour, a spontaneously fermented sourdough and a long-term sourdough produced in a Sudanese household by consecutive re-inoculations, was studied. The dominant contaminants of sorghum flour were Gram-negative, catalase-positive, rod-shaped bacteria with counts of about 10(5) cfu g-1. The spontaneously fermented sorghum sourdough showed a bacterial succession from Gram-negative, catalase-positive contaminants to Enterococcus faecalis, Lactococcus lactis, Lactobacillus fermentum and Lact. reuteri. The total bacterial count reached about 10(10) cfu g-1 and the pH dropped from 6.4 to 3.35 in about 42 h. In this phase, only the Latter two species remained dominant in a ratio of 1:1. From the Sudanese long-term dough, seven strains of Lactobacillus were isolated, representing the dominant flora. Sequence comparison of partial 16S rRNA gene sequences were used to clarify their phylogenetic positions. Five strains were classified as Lact. vaginalis and could be regarded as heterogeneous biovars of this species. The other two strains could be assigned to Lact. helveticus. RAPD-PCR and sugar fermentation patterns were useful in differentiation of these strains.

Bacteria↗

Degradation of cyanogenic glycosides by Lactobacillus plantarum strains from spontaneous cassava fermentation and other microorganisms.

Strains of Lactobacillus plantarum, Leuconostoc mesenteroides, Candida tropicalis and Penicillium sclerotiorum were screened for 19 enzymatic activities using the commercial kit API zym (Bio Mérieux). This activity was compared to the ability of degrading the toxic cyanogenic glycosides amygdalin, linamarin, and linseed cyanogens (a mixture of linustatin and neolinustatin). Good correlation between the beta-glucosidase activity found in the API zym screening and the ability to degrade the cyanogenic glycosides was found for the first three species mentioned. P. sclerotiorum strains exhibited very high activity in the API zym test (substrate: 6-Br-2-naphthyl-beta-D-glucopyranoside), but proved unable to degrade any of the cyanogenic substrates. Among the seven strains of L. plantarum tested, a great variation was seen in the beta-glucosidase activity as well as in the ability to degrade the cyanogens. This was also the case for the strains of C. tropicalis. However, all the glucosidase positive strains of these species were also able to degrade all of the cyanogens tested and at approximately the same rate. A co-culture of the most active strain of L. plantarum and C. tropicalis seemed to degrade linamarin faster than the mono cultures. L. plantarum LPI (originally isolated from fermented cassava) was investigated in further detail. The hydrolytic activity of this strain was intracellular or cell bound, and beta-bis-glycosides such as amygdalin were hydrolysed by a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Finally, inoculation of extracted linseed meal (containing linustatin and neolinustatin) with L. plantarum LPI resulted in hydrolysis of the glycosides.

Amygdalin↗

Analysis and dynamics of the chromosomal complements of wild sparkling-wine yeast strains.

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations.

Blotting, Southern↗

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard.

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.

Agriculture↗

Biochemical characterization of lactobacilli from dry fermented sausages.

The characterization of 254 strains of lactobacilli isolated from dry spontaneously fermented sausages from 15 different producers at two different stages of ripening time is reported. The species identified were Lactobacillus sake 55%, L. curvatus 26%, L. bavaricus 11% and L. plantarum 8%. The main criteria for the identification of isolates to species level were: production of lactic acid isomers, presence of mDpm acid in cell walls, deamination of arginine and fermentation of mannitol and melibiose. The composition of the populations of lactobacilli were the same for the two stages of ripening. The deamination of arginine was tested in aerobic and anaerobic cultures and in different media by checking the production of ammonia and detecting the production of citrulline. In 94% of strains tested both methods gave identical results. In two L. sake strains arginine catabolism was dependent on culture media; for two other L. sake strains the deamination of arginine only occurred when oxygen was scarce.

Animals↗

Metabolic characterization of Kloeckera apiculata strains from star fruit fermentation.

A total of 37 strains of Kloeckera apiculata was isolated during the spontaneous fermentation of star fruit must. Each strain was differentiated from the others on the basis of its capacity to produce acetaldehyde, ethyl acetate, higher alcohols, acetoin and acetic acid. All the strains were characterized by the low production of higher alcohols and the high production of ethyl acetate, whereas consistent differences in the production of acetaldehyde, acetoin and acetic acid served to differentiate star fruit apiculate strains into six different phenotypes, present at different stages of the fermentation process. The metabolic strain diversity found can be interpreted as a natural consequence of environmental conditions, which influenced the frequency and selection of specific apiculate strains. From the biotechnological point of view the different metabolic biotypes represent an important source of strains for potential use as starter cultures for star fruit fermentation.

Acetaldehyde↗