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.     

Pure culture fermentation of green olives

The method previously developed by us for the pure-culture fermentation of brined cucumbers and other vegetables has been applied successfully to Manzanillo variety olives. Field-run grade fruit was processed first by conventional procedures to remove most of the bitterness. Then the relative abilities of Lactobacillus plantarum, L. brevis, Pediococcus cerevisiae, and Leuconostoc mesenteroides to become established and produce acid in both heat-shocked (74 C for 3 min) and unheated olives, brined at 4.7 to 5.9% NaCl (w/v basis), were evaluated. The heat-shock treatment not only proved effective in ridding the fruit of naturally occurring, interfering, and competitive microbial groups prior to brining and inoculation, but also made the olives highly fermentable with respect to growth and acid production by the introduced culture, particularly L. plantarum. Of the four species used as inocula, L. plantarum was by far the most vigorous in fermentation ability. It consistently produced the highest levels of brine acidity (1.0 to 1.2% calculated as lactic acid) and the lowest pH values (3.8 to 3.9) during the fermentation of heat-shocked olives. Also, L. plantarum completely dominated fermentations when used in two-species (with P. cerevisiae) and three-species (with P. cerevisiae and L. brevis) combinations as inocula. In contrast, when L. plantarum was inoculated into the brines of unheated olives it failed to become properly established; the same was true for the other species tested, but even to a more pronounced degree. L. brevis was the only species used that failed to develop in brines of both heat-shocked and unheated olives. Modification of the curing brine by the addition of lactic acid at the outset, either with or without dextrose, led to a much earlier onset of fermentation with accompanying acid development, as compared to treatments with dextrose alone or nonadditive controls. Reasons for the marked improvement of the fermentability of Manzanillo olives receiving the prebrining heat-shock treatment are discussed.     

Biological ensiling of sugarcane tops

The following lactic acid bacteria were isolated from sugarcane tops silage:Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus delbrueckii, Pediococcus cerevisiae, Leuconostoc mesenteroides andStreptococcus lactis. The isolates were grown in a synthetic medium and the final pH, sugar uptake and the effect of adding CaCO₃ and L1, L2 and L3 factors was observed. The importance of the buffering capacity of the ensiled material for the silage process with a view to the occurrence of lactic acid bacteria is discussed.     

Molds in Brined Cucumbers: Cause of Softening During Air-Purging of Fermentations

Softening of cucumbers in fermentations purged at high air-flow rates was caused by molds growing in the brined cucumbers, not in the brine. This conclusion is based on the following results: (i) no microorganisms were isolated in significant numbers from brines that caused softening of pasteurized brined cucumbers, (ii) no pectinolytic enzyme activities were produced in cucumber brines in the absence of cucumbers, (iii) the pickles in some air-purged fermentations became very soft without the appearance of any pectinolytic enzyme activity in the brine, (iv) mold hyphae were consistently observed in tissues of soft pickles, (v) molds consistently developed in cultures of slices of surface sterilized cucumbers taken from fermentations in which soft pickles were subsequently found, and (vi) molds belonging to the genera Alternaria, Fusarium, and Mucor isolated from slices all softened pasteurized brined cucumbers.     

Production of probiotic cabbage juice by lactic acid bacteria

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.     

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.     

Entrance and Growth of Lactic Acid Bacteria in Gas-Exchanged, Brined Cucumbers

Entrance of lactic acid bacteria into the interior of brined cucumbers was found to be greatly influenced by gas composition of the cucumbers before brining. Exchange of the internal gas of fresh cucumbers with O₂ resulted in absorption of bacteria into the subsequently brined fruit within a few hours. Bacteria were absorbed into nonexchanged cucumbers to a lesser extent. Little bacterial absorption occurred in N₂-exchanged cucumbers. Stomata of the cucumber skin appeared to be a likely port for bacterial entry. When Pediococcus cerevisiae or Lactobacillus plantarum cells were added to the brine of O₂-exchanged cucumbers, the respective cell types colonized in large numbers within intercellular spaces and vascular elements of mesocarp tissue during fermentation of the cucumbers. Implications of these observations, particularly with regard to bloater formation in brined cucumbers, are discussed.     

Synthesis of gamma-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses

The concentrations of gamma-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg(-1). Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum, and two strains of Pediococcus cerevisiae. Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.     

Metabolic flux analysis of carbon balance in Lactobacillus strains

Metabolic flux analyses were performed based on the carbon balance of six different Lactobacillus strains used in this study. Results confirmed that L. delbrueckii, L. plantarum ATCC 21028, L. plantarum NCIMB 8826 ΔldhL1, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB, and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB metabolized glucose via EMP: whereas, L. brevis metabolized glucose via PK pathway. Xylose was metabolized through the PK pathway in L. brevis, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB. Operation of both EMP and PK pathways was found in L. brevis, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB, and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB when glucose plus xylose were used as carbon source. The information of detailed carbon flow may help the strain and biomass selection in a designed process of lactic acid biosynthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1397–1403, 2016     

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.     

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA

AIMS: The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). METHODS AND RESULTS: Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. CONCLUSIONS: Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.     

Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.     

Microbial dynamics of commercial makgeolli depending on the storage temperature

Market fresh makgeolli was stored at different temperatures of 4°C and 25°C to assess the change of the microbial diversity according to the storage temperature and period. Yeast counts increased until day 3 of storage and decreased thereafter. General and lactic acid bacterial counts continuously increased during storage. The data indicated that the control of growth of microorganisms, particularly general bacteria and lactic acid bacteria (LAB), is essential. Total acid levels started to decrease in the makgeolli stored at 4°C, and increased from day 6 of storage in the makgeolli stored at 25°C. The increase of total acid in the non-refrigerated condition greatly affected the quality of makgeolli. In both the fresh makgeolli samples stored at 4°C and 25°C, yeast (Saccharomyces cerevisiae) and molds (Aspergillus tubingensis, Candida glaebosa, and Aspergillus niger) were noted. Denaturing gradient gel electrophoresis (DGGE) band patterns were almost constant regardless of the storage period. As for bacteria, Lactobacillus crustorum, L. brevis, and Microlaena stipoides were found in the makgeolli stored at 4°C, and L. crustorum, Lactobacillus sp., L. plantarum, L. brevis, L. rhamnosus, and L. similis were found in the makgeolli stored at 25°C. In particular, in the makgeolli stored at 25°C, L. crustorum and L. plantarum presented dark bands and were identified as the primary microorganisms that affected spoilage of fresh makgeolli.     

Lactobacilli in Ensiled High-Moisture Corn

Aerobic mesophilic bacteria, molds, yeasts, and Lactobacillus species were enumerated at various intervals of the ensiling process of high-moisture corn (HMC). A total of 466 isolates of Lactobacillus were identified to determine the species of Lactobacillus associated with ensiled HMC and remoistened corn. The numbers of aerobic bacteria in the ensiled HMC increased to 10(8) per gram within 1 week and remained at this level throughout the storage period. The numbers of aerobic bacteria in the remoistened corn increased to 10(9) per gram and remained at this level during the 60-day storage period, after which they increased rapidly. Yeasts were the predominant microflora of the ensiled HMC. Mold numbers were low in the silos, but were very high in the remoistened corn, especially toward the end of the 60-day storage period. The species of Lactobacillus most frequently isolated from the ensiled HMC were L. plantarum, 52% of the isolates; L. brevis, 19% of the isolates; L. brevis (atypical), 15%; L. fermenti, 11%; and L. buchneri, 3%. The lactic acid bacteria, therefore, are the predominant bacterial flora of ensiled HMC, as they are of other types of plant forages that undergo similar fermentations.     

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.     

Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.     

Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.     

Competitive Growth of Genetically Marked Malolactic-Deficient Lactobacillus plantarum in Cucumber Fermentations

Procedures were developed for the differential enumeration of an added strain of Lactobacillus plantarum and indigenous lactic acid bacteria (LAB) during the fermentation of brined cucumbers. The added strain was an N,N-nitrosoguanidine-generated mutant that lacked the ability to produce CO₂ from malic acid (MDC^-). The MDC^- phenotype is desirable because CO₂ production from malic acid decarboxylation has been shown to contribute to bloater formation in fermented cucumbers. A basal medium containing malic acid and adjusted to pH 4.0 permitted growth of indigenous LAB (predominantly MDC⁺), but not growth of the added MDC^- culture. Transformation of the MDC^- culture by electroporation with cloning vector pGK12 conferred chloramphenicol resistance, which permitted selective enumeration of this culture. The reversion frequency of the MDC^- mutation was determined by a fluctuation test to be less than 10^-10. The level of retention of plasmid pGK12 was greater than 90% after 10 generations in cucumber juice medium at 32°C. With the procedures developed, we were able to establish the ratio of MDC^- to MDC⁺ LAB that results in malic acid retention in fermentations of filter-sterilized cucumber juice and unsterilized whole cucumbers under specified conditions.     

Lactic Acid Bacterial Fermentation of Burong Dalag

The fermented food, burong dalag, prepared in many Filipino homes, was studied to determine the nature of the microbiological and chemical changes that occur during fermentation. This is a lactic acid bacterial fermentation in which the species Leuconostoc mesenteroides, Pediococcus cerevisiae, and Lactobacillus plantarum played the major acid-producing role. The pH was lowered to below 4.0, and about 0.9% acid as lactic acid was attained in 1 week. It was essential to keep the product covered well to exclude air and subsequent growth of yeasts and mold.