Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria

The effect of controlled fermentation processes on the microbial association and biochemical profile of cv. Conservolea naturally black olives processed by the traditional anaerobic method was studied. The different treatments included (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a strain of Lactobacillus plantarum isolated from a fermented cassava product and (c) uninoculated spontaneous process. Microbial growth, pH, titratable acidity, organic acids and volatile compounds were monitored throughout the fermentation. The initial microbiota consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all processes. Both starter cultures were effective in establishing an accelerated fermentation process and reduced the survival period of Gram-negative bacteria by 5 days compared with the spontaneous process, minimizing thus the likelihood of spoilage. Higher acidification of the brines was observed in inoculated processes without any significant difference between the two selected starter cultures (113.5 and 117.6mM for L. plantarum and L. pentosus, respectively). L. pentosus was also determined as the major species present during the whole process of spontaneous olive fermentation. It is characteristic that lactic acid fermentation was also initiated rapidly in the spontaneous process, as the conditions of fermentation, mainly the low salt level (6%, w/v) favored the dominance of lactic acid bacteria over yeasts. Lactic, acetic and propionic were the organic acids detected by HPLC in considerable amounts, whereas citric and malic acids were also present at low levels and degraded completely during the processes. Ethanol, methanol, acetaldehyde, ethyl acetate were the major volatile compounds identified by GC. Their concentrations varied among the different treatments, reflecting varying degrees of microbial activity in the brines. The results obtained from this study could help the Greek table olive industry to improve the existing processing schemes in order to increase product consistency and quality expanding the international market for naturally black olives.     

Occurrence of Listeria monocytogenes in green table olives

Microbiological safety of green table olives from different cultivars, prepared by both the Spanish-style and biological methods and fermented with starter cultures of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus pentosus), was investigated. The fermentation process was monitored by measuring pH values, titratable acidities, and growth of lactic acid bacteria over time. During fermentation, lactic acid bacteria and yeasts were major microbial populations. Microbiological safety was evaluated by analysis for Listeria monocytogenes with the use of an enrichment method during storage (from 55 days to 18 months). Results demonstrated that L. monocytogenes can survive and grow in green table olives. L. monocytogenes was found in one of the commercial (thermally treated) samples analyzed and in all samples older than 2 months, irrespective of olive cultivar, lactic acid bacteria starter used, pH and titratable acidity of brine samples, or treatment applied.     

Green olive fermentation using spontaneous and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> cultures

The aim of this work was to evaluate the performance of spontaneous and Lactobacillus cultures in Spanish style fermentation of olive fruits. A fermentation with Lactobacillus plantarum ATCC 8014 was carried out for comparison. Fermentation was performed at room temperature during 120 days in two different concentrations of NaCl (2.4–13.6 % w/v) and sucrose (0–0.82 % w/v) in the brines. The pH, chlorides, lactic acid, reducing sugar and lactic bacteria in selective medium were investigated during the olive fermentation. After 120 days of fermentation, the formulation with 12 % NaCl in the brine hampered the growth of lactic bacteria, avoiding the consumption of reducing sugars. However, low salt concentrations (≤4 %) promoted a decrease in pH, an increase of the lactic acid and the growth of lactic bacteria, providing best environmentally suitable for the fermentation process. Whereas using concentrations of 12 % NaCl and 0.7 % sucrose showed better chemical conditions, ensuring the safety of the product produced. Multivariate analysis showed that the olives with both cultures were in close relation to lactic acid and reducing sugar and different from lactic bacteria.     

Effect of calcium chloride on mechanical properties and microbiological characteristics of cv. Conservolea naturally black olives fermented at different sodium chloride levels

The effect of calcium chloride (CaCl₂)(5 gL^?1) and sodium chloride (NaCl) concentration (40, 60 and 8 gL^?1) on the microbiological and mechanical properties of naturally black olives of cv. Conservolea in brines was studied. In 40 and 60 g L^?1 brines the growth of lactic acid bacteria was favoured over that of yeasts, resulting in rather complete lactic acid fermentation as indicated by high free acidity (9.8–11.5 g lactic acid L^?1) and low pH (3.7–3.8). At 80 g L^?1 brine, yeasts were the dominant members of the microflora, rendering a product with lower acidity (8 g lactic acid L^?1) and higher pH (4.3–4.5). In the presence of CaCl₂ there was a consistent increase in the depth of the peripheral region in which cell wall breakage occurred. When cells separated, perforated walls were observed at sites associated with plasmodesmata. The flesh was strongest and stiffest when CaCl₂ was added to olives treated with 40 g L^?1 brine, consistent with cell wall breakage being the predominant mode of failure. The only observed effect on the mechanical properties of the skin was a stiffening at 60 g L^?1 brine on addition of CaCl₂. Copyright © 2007 Society of Chemical Industry     

Microbiological and physicochemical characterisation of green table olives of Halkidiki and Conservolea varieties processed by the Spanish method on industrial scale

The microbiological and physicochemical changes of industrially fermented Halkidiki and Conservolea green table olives were determined. Samples were analysed to monitor the population of lactic acid bacteria (LAB), yeasts and Enterobacteriaceae, together with changes in pH, acidity, salinity, colour, lactic acid, acetic acid and ethanol. LAB and yeast species diversity was evaluated at the beginning (1 day), middle (75 days) and final (135 days) stages of fermentation by RAPD-PCR genomic fingerprinting. Results revealed vigorous lactic acid processes as indicated by the dominance of LAB over yeasts. No Enterobacteriaceae could be detected after 30 days. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) dominated in the beginning of fermentation in both varieties. In the end, Lactiplantibacillus pentosus (formerly Lactobacillus pentosus) and Pediococcus ethanolidurans prevailed in Halkidiki and Conservolea varieties, respectively. As for yeasts, Kluyveromyces lactis/marxianus and Pichia manshurica prevailed at the onset of fermentation in Halkidiki and Conservolea varieties, whereas in the end Pichia membranifaciens dominated in both varieties.     

EFFECT OF THE USE OF DIFFERENT LACTIC STARTERS ON THE MICROBIOLOGICAL AND PHYSICOCHEMICAL CHARACTERISTICS OF NATURALLY BLACK TABLE OLIVES OF GEMLIK CULTIVAR

The effect of different lactic acid bacteria cultures on the physicochemical and microbiological characteristics of brined black olives of Gemlik cultivar at low fermentation temperature was studied. Fermentation was carried out according to the traditional Gemlik method with modifications like low salt concentration and lactic starter addition. The brines with 7% salt concentration were inoculated with lactic acid bacteria ( Lactobacillus brevis, Leuconostoc cremoris and L. paramesenteroides ), which were previously isolated from olive fruits at low temperatures and a commercial strain of Lb. plantarum. Fermentation procedures were carried out at controlled temperatures (between 10–12C). Lactic acid bacteria survival was accompanied by yeast development, no Pseudomonas and Enterobacter species were detected in all treatments during fermentation. The highest total titratable acidity, lowest pH and least yeast growth were determined at the brines and fermentation products, which were inoculated with L. cremoris.

PRACTICAL APPLICATIONS

The use of suitable starter cultures is necessary to improve the microbiological control of the naturally black table olive process, help to standardize the fermentation, increase the lactic acid yield and accordingly provide the production of olives with high quality. The requirements mentioned for starter cultures include a rapid and predominant growth, homofermentative metabolism, tolerance to salt, acid and polyphenols, and few growth factor requirements. Especially at the regions where olives were picked later when environmental temperatures are lower, the use of a starter culture that has the ability to grow at low temperatures may be necessary. Use of such starter cultures may help to increase acidification, to control some types of spoilage and to shorten the fermentation process.     

Effect of potassium sorbate and sodium benzoate on microbial population and fermentation of black olives

Black olive fermentation characteristics and diffusion of preservatives into olives were evaluated in brines containing 500 ppm potassium sorbate, 1000 ppm sodium benzoate, 500 ppm sodium benzoate + 250 ppm potassium sorbate and no preservative (control). Changes in brine pH, acidity and microbial population (lactic acid bacteria, yeasts, moulds) were followed during fermentation and storage. Results indicated that K‐sorbate when used at 500 ppm concentration in black olive fermentation had a slight stimulatory effect on the growth of lactic acid bacteria. The yeast counts of brines containing 500 ppm K‐sorbate and 1000 ppm Na‐benzoate were lower than for the brine containing 250 ppm K‐sorbate + 500 ppm Na‐benzoate and for the control with no preservative, while mould growth was completely inhibited in all treatments during fermentation. Mould counts stayed below the detection limit (<10 cfu g⁻¹) during 214 days of vacuum‐packaged storage. Yeast counts showed a progressive decline within 28 days of storage and then stayed relatively constant in all treatments thereafter. The level of yeast population was higher in the control sample than in the sample containing both preservatives during storage. The diffusion of Na‐benzoate and K‐sorbate into the olives after equilibrium was 64% and 80% during fermentation respectively. © 1999 Society of Chemical Industry     

Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chloride salt mixtures

NaCl plays an important role in table olive processing affecting the flavour and microbiological stability of the final product. However, consumers demand foods low in sodium, which makes necessary to decrease levels of this mineral in fruits. In this work, the effects of diverse mixtures of NaCl, CaCl₂ and KCl on the fermentation profiles of cracked directly brined Manzanilla-Aloreña olives, were studied by means of response surface methodology based in a simplex lattice mixture design with constrains. All salt combinations led to lactic acid processes. The growth of Enterobacteriaceae populations was always limited and partially inhibited by the presence of CaCl₂. Only time to reach half maximum populations and decline rates of yeasts, which were higher as concentrations of NaCl or KCl increased, were affected, and correspondingly modelled, as a function of salt mixtures. However, lactic acid bacteria growth parameters could not be related to initial environmental conditions. They had a longer lag phase, slower growth and higher population levels than yeasts. Overall, the presence of CaCl₂ led to a slower Enterobacteriaceae and lactic acid bacteria growth than the traditional NaCl brine but to higher yeast activity. The presence of CaCl₂ in the fermentation brines also led to higher water activity, lower pH and combined acidity as well as a faster acidification while NaCl and KCl had fairly similar behaviours. Apparently, NaCl may be substituted in diverse proportions with KCl or CaCl₂ without substantially disturbing water activity or the usual fermentation profiles while producing olives with lower salt content.     

Use of starter cultures of lactic acid bacteria and yeasts in the preparation of togwa,a Tanzanian fermented food

Starter cultures of lactic acid bacteria (Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus pentosaceus) and yeasts (Candida pelliculosa, Candida tropicalis, Issatchenkia orientalis and Saccharomyes cerevisiae) isolated from native togwa were tested singly or in combination for their ability to ferment maize-sorghum gruel to produce togwa. All species of bacteria showed an ability to ferment the gruel as judged by lowering the pH from 5.87 to 3.24-3.49 and increasing the titratable acidity from 0.08% to 0.30-0.44% (w/w, lactic acid) in 24 h. Yeasts used singly showed little activity within 12 h, but lowered the pH to 3.57-4.81 and increased the acidity to 0.11-0.21% in 24 h. Yeasts in co-culture with lactic acid bacteria (LAB) had a modest effect on the final acidity (P<0.05). The number of lactic acid bacteria and yeasts increased while the Enterobacteriaceae decreased with fermentation time. The pH was lowered and lactic acid produced significantly (P<0.05) fastest in natural togwa fermentation and in samples fermented by L. plantarum or L. plantarum in co-culture with I. orientalis. The content of fermentable sugars was reduced during fermentation. Most volatile flavour compounds were produced in samples from fermentation by P. pentosaceus and I. orientalis in co-culture with either L. plantarum or L. brevis.     

Aerobic industrial processing of Empeltre cv. natural black olives and product characterisation

The fermentation of natural black olives is currently carried out under anaerobic conditions. This work investigates the aerobic fermentation of this product in tanks containing 16 000 kg of Empeltre olives during two seasons. The microorganisms in brines were yeasts in all cases, whereas lactic acid bacteria growth was only observed in some tanks. Consequently, the pH during fermentation was in most cases higher than recommended 4.3 units. However, the Empeltre olive brines displayed bactericidal effect against Salmonella enterica, Sthapylococcus aureus, Escherichia coli and Listeria monocytogenes, with a 5 log population drop in one or 24 h depending on the brine age so that these olive brines are a harsh environment for the growth of these pathogens. It was found HyEDA as the major phenolic compound in both brine and olive pulp at the beginning of fermentation that slowly hydrolysed into hydroxytyrosol that reached a concentration up to 1500 mg kg⁻¹.     

Evaluation of a single and combined inoculation of a Lactobacillus pentosus starter for processing cv. Arbequina natural green olives

The production of Arbequina naturally green olives is a traditional and spontaneous process in which lactic acid bacteria (LAB) and yeasts are present. To better control the fermentation of olives, strains of LAB and yeasts that had been isolated from brines were used in this study.     

Mixed Culture Fermentation of Cucumber Juice with Lactobacillus plantarum and Yeasts

Saccharomyces cerevisiae and Saccharomyces rosei were tested for use in mixed culture fermentation of cucumber juice with Lactobacillus plantarum. Extent of sugar utilization and the ratio of products formed (lactic acid:ethanol) were influenced by time of inoculation and cell numbers of each microorganism. Sugar fermentation was complete within 6 days at 28°C when similar numbers of bacteria and yeasts were added simultaneously, or when yeasts were added before the bacteria. Sugar remained when only L. plantarum was added, or when yeasts were added in low numbers. Glycerol was produced when yeasts were present, the concentration being directly related to NaCl concentration. Results suggest advantageous uses of yeasts to complete fermentation and to modify acidity.     

A study on the implications of NaCl reduction in the fermentation profile of Conservolea natural black olives

This study examined the impact of different mixtures of NaCl, KCl, and CaCl₂ on the fermentation profiles of Conservolea natural black olives. Five different combinations of chloride salts were investigated, namely (i) 8% NaCl (control treatment), (ii) 4% NaCl and 4% KCl, (iii) 4% NaCl and 4% CaCl₂, (iv) 4% KCl and 4% CaCl₂, and (v) 2.6% NaCl-2.6% KCl-2.6% CaCl₂. The changes in the microbial association (lactic acid bacteria, yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, volatile compounds, and mineral content in olive flesh were analyzed. Results demonstrated that all salt combinations led to vigorous lactic acid processes based on the obtained values of pH (3.9-4.2) and titratable acidity (0.70-0.86 g lactic acid per 100 ml brine). Organoleptic evaluation was a critical factor in the acceptability of the final product. Increasing concentrations of CaCl₂ or a combination of KCl and CaCl₂ rendered the product bitter with low acceptability by the taste panel. Only one combination of chloride salts (4% NaCl and 4% KCl) could finally produce olives with lower sodium content and good organoleptic attributes. The results of this study could be employed by the Greek table olive industry in an attempt to produce natural black olives with less sodium without affecting the traditional taste of fermented olives in order to meet consumers’ demand for low sodium dietary intake.     

Efficacy of natamycin to control fungal growth in natural black olive fermentation

The effect of natamycin as a fungal control agent in natural black olive fermentation according to the traditional anaerobic system was studied. Black Conservolea olives were subjected to spontaneous fermentation in 8% (w/v) NaCl brine solution (control treatment) or brine supplemented with 0.01% (w/v) natamycin (active compound) for an overall period of 60 days. The changes in the microbial association (lactic acid bacteria, yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, and volatile compounds were monitored throughout fermentation. The initial microbiota consisted of lactic acid bacteria, yeasts, and Enterobacteriaceae. The addition of natamycin in the brine inhibited the growth of yeasts, without affecting the population dynamics of bacteria, resulting in a more vigorous fermentation with higher titratable acidity compared to spontaneous control process. Moreover, the presence of natamycin inhibited mould spoilage caused by the development of fungal mycelium on the surface of the brine during the traditional anaerobic fermentation system employed widely in Greece in natural black olive processing. Natamycin could be an important component of a processing system to control fungal growth in natural black olive fermentation and at the same time it has the potential to enhance the process by favouring the growth of the indigenous population of lactic acid bacteria against other competing microorganisms.     

Pickling and storage of caperberries (Capparis spp.)

 Caperberries (0.7–1.9 cm diameter), collected in June 1996, were pickled for 1 month in 5% and 10% brine. Some physical, chemical and microbiological analyses were made during their fermentation and storage. While lactic acid bacteria (LAB) increased during fermentation in 5% brine, they did not increase after 15 days in 10% brine. In some cases, total bacterial (TB) growth decreased. In both brines, yeasts/moulds and coliforms (except for 5% brine on day 15) were not present after 15 and 20 days, respectively. For both caper species, the most suitable salt concentration, for LAB activity was 5–10% NaCl, and the best length of fermentation with respect to product colour, flavour, acidity, pH and LAB activity in brine was 20–25 days. The texture was maintained well during storage. Acidity and pH were markedly affected by the concentration of brine. The quality of all samples was maintained with a 15% concentration of old or fresh brine during storage. The colour of the samples, especially those of C. ovata, stored with old brine was darker that of fresh berries. As no "off" flavour was observed, it was concluded that pickled caper berries can be stored in fresh brine with a 15% NaCl concentration.     

Calcium Chloride and Potassium Sorbate Reduce Sodium Chloride used during Natural Cucumber Fermentation and Storage

Cucumber fermentation characteristics and pickle quality were evaluated in brines containing equilibrium concentrations of 0-0.4% CaCl₂, 0-0.4% potassium sorbate and 0-10% NaCl. Changes in brine pH and acidity, cucumber texture and color, coliforms, lactic acid and total bacteria, yeasts and molds were followed over time. Results indicated that cucumber spoilage would eventually take place if NaCl or potassium sorbate were not present in the brine. The presence of CaCl₂ helped maintain cucumber firmness. A synergistic action between NaCl, CaCl₂ and potassium sorbate was seen, which allowed good quality pickles to be produced when moderate amounts of all three components were present in the brine (5% NaCl, 0.2% CaCl₂, 0.2% potassium sorbate).     

Study of the anti-lactic acid bacteria compounds in table olives

The analysis and formation of anti-lactic acid bacteria compounds in olive brines was performed for the main worldwide olive varieties intended for table olives. The results demonstrated that the growth of lactic acid bacteria in the brines of olives non-treated with NaOH is, in some way, variety dependant. Likewise, the most active antimicrobial compound, the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol, was not detected in fresh fruits but it was formed during brining from the hydrolysis of oleuropein and this reaction was enzymatically catalysed. Thus, the inactivation of the enzyme by heating the olives produced (i) an accumulation of oleuropein in olives and brines, (ii) the inhibition of the formation of antimicrobials and (iii) the growth of Lactobacillus pentosus in olive brines. These results provide tools for a full understanding of the growth or inhibition of lactic acid bacteria during fermentation of table olives.     

Main variables affecting the lactic acid fermentation of table olives

Among the olive varieties, Manzanilla is the most employed as table olives worldwide. Inoculation of the brines of Manzanilla and Gordal olives, which were not treated with alkali, with a strain of Lactobacillus pentosus was carried out and results indicated that variety is one of the main factors to be considered when fermenting olives, even more important than the salt level. This conclusion was based on the presence of a high concentration of antimicrobial compounds, namely the dialdehydic form of decarboxymethyl elenolic acid, either free or linked to hydroxytyrosol in the brines of the Manzanilla variety, as compared to the low level of these substances in the Gordal brines. Likewise, it was also observed that the inhibitory activity of Manzanilla olives against lactic acid bacteria could be counteracted by the addition of nitrogenous supplements such as MRS and yeast extract to their brines. Results obtained from this work are of great interest for the table olive industry in order to develop lactic acid fermentation in olives non‐treated with alkali.     

Inhibitors of lactic acid fermentation in Spanish-style green olive brines of the Manzanilla variety

Frequently, a delay or lack of lactic acid fermentation occurs during the processing of Spanish-style green olives, in particular of the Manzanilla variety. Many variables can affect the progress of fermentation such as temperature, nutrients, salt concentration, antimicrobials in brines, and others. In this study, it was demonstrated that an inappropriate alkaline treatment (low NaOH strength and insufficient alkali penetration) allowed for the presence of several antimicrobial compounds in brines, which inhibited the growth of Lactobacillus pentosus. These substances were the dialdehydic form of decarboxymethyl elenolic acid either free or linked to hydroxytyrosol and an isomer of oleoside 11-methyl ester. Olive brines, from olives treated with a NaOH solution of low concentration up to 1/2 the distance to the pit, contained these antimicrobials, and no lactic acid fermentation took place in them. By contrast, a more intense alkaline treatment (2/3 lye depth penetration) gave rise to an abundant growth of lactic acid bacteria without any antimicrobial in brines. Therefore, the precise cause of stuck fermentation in Manzanilla olive brines was demonstrated for the first time and this finding will contribute to better understand the table olive fermentation process.     

Ozonation process for the regeneration and recycling of Spanish green table olive fermentation brines

Treatment with ozone broke down the most characteristic polyphenols present in the fermentation brines of green table olives. The ozone required for their complete elimination was 15 and 7 mg/L in acidic solutions (pH 4.0) and alkaline conditions (pH 10.0), respectively. The usual microbial population of brines, lactic acid bacteria and yeasts, was simultaneously eliminated in parallel but needed higher ozone levels in acidic conditions. The ozone treated brines were then filtered through a 0.45 μm pore size and diluted (1:1) and re-used as cover brine for plain and pitted olive packing. After 3 months equilibrium, the cover brines from regenerated brines had slighter poorer colour and slightly higher pH. Fruits packed with diluted (1:1) ozonated brines in alkaline conditions had higher firmness and similar organoleptic characteristics to those using fresh brine. The partial regeneration and reuse of fermentation brines may be a cheap and technologically simple alternative for reducing the environmental problems derived from the high polluting charge of green olive fermentation brines.