Influence of pH on Inhibitory Activity of Acetic Acid on Osmophilic Yeasts Used in Brine Fermentation of Soy Sauce

The inhibitory activity of acetic acid on osmophilic yeasts (Saccharomyces rouxii and Torulopsis versatilis) used for the brine fermentation of soy sauce increased remarkably as the pH of the medium was lowered. This increased toxicity could not be attributed to a hydrogen ion effect alone.     

Lactic acid bacteria population dynamics during spontaneous fermentation of radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) roots in brine

The aim of the present study was to assess the microecosystem development and the dynamics of the lactic acid bacteria population during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine at 20 and 30?°C. In both temperatures, lactic acid bacteria prevailed the fermentation; as a result, the pH value was reduced to ca. 3.6 and total titrable acidity increased to ca. 0.4% lactic acid. Enterococci population increased and formed a secondary microbiota while pseudomonads, Enterobacteriaceae and yeasts/molds populations were below enumeration limit already before the middle of fermentation. Pediococcus pentosaceus dominated during the first days, followed by Lactobacillus plantarum that prevailed the fermentation until the end. Lactobacillus brevis was also detected during the final days of fermentation. A succession at sub-species level was revealed by the combination of RAPD-PCR and rep-PCR analyses. Glucose and fructose were the main carbohydrates detected in brine and were metabolized into lactic acid, acetic acid and ethanol.     

Antimicrobial Properties of Oleuropein and Products of Its Hydrolysis from Green Olives

Oleuropein, the bitter glucoside in green olives, and products of its hydrolysis were tested for antibacterial action against certain species of lactic acid bacteria involved in the brine fermentation of olives. Oleuropein was not inhibitory, but two of its hydrolysis products, the aglycone and elenolic acid, inhibited growth of the four species of lactic acid bacteria tested. Another hydrolysis product, beta-3,4-dihydroxyphenylethyl alcohol, was not inhibitory. The aglycone of oleuropein and elenolic acid were much more inhibitory when the broth medium contained 5% NaCl; 150 mug of either compound per ml prevented growth of Lactobacillus plantarum. A crude extract of oleuropein, tested by paper disk bioassay, was inhibitory to 3 of 17 species of bacteria screened, none of which were lactic acid bacteria. The acid hydrolysate of the extract was inhibitory to 11 of the bacteria, which included four species of lactic acid bacteria and other gram-positive and gram-negative species. Neither crude preparation was inhibitory to growth of the seven species of yeasts tested. A possible explanation is given for the previously reported observation that heating (3 min, 74 C) olives prior to brining renders them more fermentable by lactic acid bacteria. Results of a brining experiment indicated that oleuropein is degraded to antibacterial compounds when unheated olives are brined.     

Fermentation of the Yugoslavian Pickled Cabbage

In Yugoslavia, cabbage is preserved by fermentation using a variety of methods. Bacteriological study of these fermentations has shown that the relative influence of the various lactic acid producing bacteria varies somewhat with the method of preservation. The heterofermenters, Leuconostoc mesenteroides and Lactobacillus brevis, exert a greater effect when cabbage is shredded than when the whole heads are packed in salt brine. Whole heads of cabbage preserved by fermentation in salt brine are a good source of ascorbic acid and provide a flavorful food.     

Evolution of Yeasts and Lactic Acid Bacteria During Fermentation and Storage of Bordeaux Wines

The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.     

Microbiological Analyses of Traditional Alcoholic Beverage (Chhang) and its Starter (Balma) Prepared by Bhotiya Tribe of Uttarakhand,India

Present article depicts microbiology of starter (Balma) used in traditional solid-state fermentation of alcoholic beverage (Chhang) by Bhotiya tribe of Uttarakhand. It also highlights the importance of herbs in Balma preparation and kinetics of lactic acid and ethanol fermentation under Chhang preparation using Balma. Balma contains 214 × 10⁶ cfu/g yeasts, 2.54 × 10⁶ cfu/g lactic acid bacteria (LAB) and 1.4 × 10⁶ cfu/g other mesophilic bacteria. ITS sequence analysis revealed a rich diversity of yeast comprising of Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Saccharomycopsis malanga in Balma. 16S rDNA sequence analysis revealed Lactobacillus pentosus and Pediococcus pentosaceus among LAB, while amylolytic Bacillus subtilis and B. aerophilus among other bacteria in Balma. Based on the results, it is speculated that herbs such as Inula cuspidata, Micromeria biflora, Origanum vulgare, Rubus sp. and Thymus linearis used earlier by Bhotiya in Balma preparation contribute as a source of yeasts, LAB and amylolytic bacilli. Study also demonstrates that Bhotiya tribe is rational in preparation of starter as they have circumvented the need of plants by using previous year Balma as inoculum and possibility of deficient quality of Balma due to weak colonization of phyllosphere and rhizosphere microbiota. Results suggest that simultaneous saccharification and lactic acid–ethanol fermentation take place in traditional cereal based Chhang fermentation system of Bhotiya.     

Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

Aims: To assess the yeast community structure and dynamics during Greek‐style processing of natural black Conservolea olives in different brine solutions. Methods and Results: Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0·5% (w/v) glucose, (ii) 0·2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5·8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro‐organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. Conclusions: By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Significance and Impact of the Study: Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well‐controlled process with minimization of product’s losses.     

Novel Method of Lactic Acid Production by Electrodialysis Fermentation

In lactic acid fermentation by Lactobacillus delbrueckii, the produced lactic acid affected the lactic acid productivity. Therefore, for the purpose of alleviating this inhibitory effect, an electrodialysis fermentation method which can continuously remove produced lactic acid from the fermentation broth was applied to this fermentation process. As a result, the continuation of fermentation activity was obtained, and the productivity was three times higher than in non-pH-controlled fermentation. In electrodialysis fermentation, the amount of produced lactic acid was 82.2 g/liter, which was about 5.5 times greater than that produced in non-pH-controlled fermentation. It was concluded that these good results were obtained on account of alleviating the lactic acid inhibitory effect by electrodialysis fermentation. However, the fouling of anion-exchange membranes by cells was observed in electrodialysis fermentation.     

Effects of Fermentation and Its Control on the Sensory Characteristics of Conservolea Variety Green Olives

The various factors assumed to retard or even prevent the lactic acid fermentation of Conservolea variety green olives have been investigated. Of these, the sugar supplement along with the appropriate lactic starter culture supported a complete lactic acid fermentation. Acidification, particularly of starting brine, was shown to have little or no effect, whereas heat shock treatment improved the olive fermentability slightly without modifying the course of fermentation. These findings indicate that Conservolea olives do not contain phenolic compounds in concentrations inhibitory to lactic acid bacteria.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

Bloater Formation by Gas-forming Lactic Acid Bacteria in Cucumber Fermentations

The formation of “bloaters” (hollow stock) in cucumbers brined for salt-stock purposes at 5 to 10% salt has been associated with gaseous fermentation caused chiefly by yeasts. Recently, serious early bloater damage, not attributable to yeasts, has been observed in commercial-scale experiments on control of bloaters in overnight dill pickles brined in 50-gal barrels at 3.0 to 4.5% salt. Growth of fermentative species of yeasts was effectively controlled by the addition of 0.025, 0.05, and 0.1% sorbic acid or its sodium salt. In contrast to this, the fermenting brines showed extremely high populations of acid-forming bacteria, identified as Lactobacillus plantarum, L. brevis, and Pediococcus cerevisiae. The gas-forming species (i.e., L. brevis) constituted a high proportion of the total populations. Representative isolates from 36 barrels of overnight dill pickles were tested for their ability to produce bloaters in 1-quart jars of pasteurized cucumbers equilibrated at 4 to 5% salt, 0.25% lactic acid, and pH 4.0. Bloaters, identical with those made by yeast cultures, were produced in all jars inoculated with L. brevis. No bloaters were produced by L. plantarum and P. cerevisiae. These results suggest that the control of bloater damage in cucumber fermentations, particularly at low salt concentrations, may necessitate inhibition of gas-forming lactic acid bacteria.     

Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.     

Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation

After the appearance of “Etudes sur le vin” by Pasteur, in enology lactic acid bacteria have been considered as deteriorating agents for more than 50 years. About 1920, Ferré in Burgundy and Ribéreau-Gayon in Bordeaux demonstrated the enological importance of the transformation of malic to lactic acid. This notion is now generally accepted in most vinicultural areas. Malolactic fermentation is encouraged, especially for red wines, for two reasons: a) it eliminates the taste of malic acid and lowers the acidity of the wine, b) it assures the biological stability of wines conserved with a minimum of sulphurous anhydride. In traditional vinification, malolactic fermentation is the result of bacterial growth. It is spontaneous, that means induced by the endogenous lactic acid bacteria of grapes and winery equipment. In the must, yeasts and bacteria develop simultaneously; in the antagonism between yeasts and bacteria the bacterial population is more often becoming dominant than being suppressed. The grapes are sulphited so that bacterial growth occurs only after complete exhaustion of sugars by the yeasts. Consequently, alteration of the wine, as a result of sugar fermentation by the bacteria, is prevented. In a well-controlled vinification lactic acid bacteria can complete their growth cycle in the wine. Wine, however, is a poor culture medium and the bacteria multiply under restricted nutritional, physical and chemical conditions. As a consequence, malolactic fermentation is difficult to control in practice, in spite of all the research done for more than 30 years. For a long time, one has tried to stimulate malolactic fermentation by inoculating wine with bacteria. Until now, the problem has been to determine the biomass of bacteria, sufficient for fermentation to take place as well as the quality required. The desired physiological state of the bacteria in the inoculum is also not known.     

Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine

AIMS: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation. METHODS AND RESULTS: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp. in the brine decreased whilst lactic acid bacteria and yeast populations increased. Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation. Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh. Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation. CONCLUSIONS: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine. SIGNIFICANCE AND IMPACT OF THE STUDY: These are the first observations of the micro-organisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

Antifungal activity of Leuconostoc citreum and Weissella confusa in rice cakes

The antifungal activity of organic acids greatly improves the shelf life of bread and bakery products. However, little is known about the effect of lactic acid fermentation on fungal contamination in rice cakes. Here, we show that lactic acid fermentation in rice dough can greatly retard the growth of three fungal species when present in rice cakes, namely Cladosporium sp. YS1, Neurospora sp. YS3, and Penicillium crustosum YS2. The antifungal activity of the lactic acid bacteria against these fungi was much better than that of 0.3% calcium propionate. We found that organic acids including lactic and acetic acid, which are byproducts of lactic fermentation or can be artificially added, were the main antifungal substances. We also found that some Leuconostoc citreum and Weissella confusa strains could be good starter species for rice dough fermentation. These results imply that these lactic acid bacteria can be applicable to improve the preservation of rice cakes.     

Microbial interactions associated with secondary cucumber fermentation

Aims

To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation.

Methods and Results

Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4·6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3·2), but only E. cloacae was able to produce a secondary product.

Conclusions

The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids.

Significance and Impact of the Study

Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.     

Microbiological and Biochemical Study of Coffee Fermentation

The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous. The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation, 60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic microorganisms. Received: 21 August 2000 / Accepted: 25 September 2000     

酱香型白酒发酵中两株主要乳酸菌对酿造微生物群体的影响

【目的】研究从酱香型白酒发酵酒醅中分离得到的2株主要乳酸菌Lactobacillus homohiochii XJ-L1和Lactobacillus buchneri XJ-L2对酱香型白酒发酵中酿造微生物群体的作用,并探索该种相互作用对酱香型白酒品质的影响。【方法】结合抑菌实验和组合发酵实验研究L. homohiochii XJ-L1和L. buchneri XJ-L2对酿造微生物群体生长的影响,通过对纯培养和共培养体系中代谢物的比较,研究2株优势乳酸菌对主要酿造酵母风味相关代谢产物的影响。【结果】L. buchneri XJ-L2能够抑制3株芽孢杆菌(Bacillus amyloliquefaciens XJ-B1,Bacillus subtilis XJ-B2,Bacillus licheniformis XJ-B3)、5株霉菌(Aspergillus oryzae XJ-M1,Aspergillus niger XJ-M2,Aspergillus flavus XJ-M3,Aspergillus albicans XJ-M4,Rhizopus oryzae XJ-M5)、2株酵母(Schizosaccharomyces pombe XJ-Y4,Geotrichum candidum XJ-Y5)的生长;L. homohiochii XJ-L1和L. buchneri XJ-L2能够促进3株主要酵母(Saccharomyces cerevisiae XJ-Y1,Zygosaccharomyces bailii XJ-Y2,Pichia galeiformis XJ-Y3)的生长,同时促进其酸类、醇类、酯类等风味物质的代谢。【结论】L. homohiochii XJ-L1和L. buchneri XJ-L2可促进3株主要酵母的生长代谢,同时L. buchneri XJ-L2明显抑制细菌、霉菌和少数酵母的生长,以此促进和维持主要酵母在酱香型白酒发酵过程中的生态地位,从而影响酒中酸类、醇类、酯类等风味物质的形成,保证酱香型白酒的品质。因此,适当比例的乳酸菌对维持酿造微生物区系平衡,生产典型酱香品质白酒具有重要意义。     

Identification and characterization of yeasts in sugarcane silages

Aims: To enumerate the micro‐organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. Method: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. Conclusions: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. Significance and Impact of the Study: Knowledge of the population of micro‐organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.