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.     

Influence of fruit ripeness and salt concentration on the microbial processing of Arbequina table olives

Arbequina table olives are processed as “naturally green olives”, they are directly placed in brine and fermentation starts spontaneously. Olives are harvested just before they change to ‘turning colour’. Different salt concentrations are used depending on the producer. The aim of the study was to evaluate how (i) the ripeness of the olive when it is harvested and (ii) the salt concentration of the brine influence the different microorganism populations in brine during the fermentation of Arbequina table olives.The results showed that the Enterobacteriaceae population lasted longer in black and turning colour olives than in green olives, whereas the growth of lactic acid bacteria was delayed in green olives. A higher salt concentration favoured the elimination of Enterobacteriaceae and hindered yeast growth. The main yeast species identified were Pichia anomala, Candida sorbosa and Candida boidinii, while Lactobacillus plantarum was the only lactic acid bacteria species involved in the process. In a sensory test, panellists preferred green olives and were not able to tell the laboratory-scale processed olives from a commercial sample, nor could they distinguish green olives from different brines.     

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.     

Effect of brine replacement and salt concentration on the fermentation of naturally black olives

This study was undertaken to modify the traditional fermentation of naturally black olives in order to obtain higher quality black table olives. After holding olives in tap water or low salt brine prior to fermentation, acidification of the cover brine and fermentation at two different salt concentrations (6 and 14 g/ 100 ml NaCl) were applied to Gemlik variety olives in three batches. Fermentations were monitored using physical, chemical and microbiological analyses and effects of the above changes to fermentation were investigated. Yeasts were the predominant microorganisms in all of the batches. Growth enhancement of lactic acid bacteria by brine replacement occurred in the fermentation with 6 g/ 100 ml NaCl cover brine but not with 14 g/100 ml NaCl brine. The highest level of titratable acidity (0.59 g/100 ml) formed during fermentation with 6 g/100 ml NaCl brine followed by fermentation with 6 g/100 ml NaCl brine (0.43 g/100 ml) where the brine was replaced after 48 days. The lowest level of titratable acidity (0.36 g/100 ml) was formed using a high salt brine. Reducing sugars were consumed effectively by microorganisms in all the batches and residual levels in the brines were 0.05–0.1 g/100 g ml. NaCl contents of the final products were 5.20 g/ 100 g (olives with high salt brine) and 3.27–3.58 g/100 g (olives with low salt brine). Moisture contents of the processed olives were in the range 49.58–50.79 g/ 100 g. The water activity (a_w) value of olives fermented with the high salt brine was found to be 0.947 while a_w values of olives fermented with the low salt brine were 0.962–0.964. Protein content of the olives ranged between 1.76–1.95 g/100 g and the ash contents were found to be 2.41–2.81 g/100 g in olives with low salt brine and 4.70 g/100 g in olives with high salt brine. According to sensory evaluations of the final products, no differences between the treatments were found (P > 0.05).     

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 processing techniques on antioxidative enzyme activities, antioxidant capacity, phenolic compounds, and fatty acids of table olives

In this study, olive fruits (Olea europeae cv. Gemlik) of the most common sources of table olives in Turkey were used. Total polyphenol content (TPC), antioxidant capacity (AC), and antioxidant enzymes (catalase, CAT; ascorbate peroxidase, APX; and glutathione reductase, GR) of table olives were compared by 4 different methods of ripe table olive processing. Results revealed that TPC of the processed olives ranged from 117.44 to 418.69 mg gallic acid equivalents/g fresh weight (f.w.). The highest AC as mg Trolox equivalents of 189.58/g f.w. was obtained from unprocessed black olives. CAT, APX, and GR activities of unprocessed olives were higher than those obtained in all processed olives. In conclusion, TPC, AC, and antioxidant enzyme activities are strongly affected by fruit ripening and processing in table olives of ‘Gemlik’ cultivar. In addition, the best processing technique is untreated black olives in brine for antioxidant properties.     

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.     

Biogenic amine formation and "zapatera" spoilage of fermented green olives: effect of storage temperature and debittering process

The effects of temperature and the debittering process on amine formation and other chemical changes related to "zapatera" spoilage of fermented green table olives during storage, without any chemical correction, were studied. Unwashed olive brines were more concentrated in all analyzed compounds, except NaCl. No changes in formic, acetic, and succinic acids or in ethanol, hydroxytyrosol, or tyrosol were observed in the olive brines during storage. The concentration of putrescine in the brine at the beginning of storage and end of fermentation was about 38 mg/liter, and it did not change during storage. This amine only seems to be produced during the active fermentation phase. The effects of temperature and the type of debittering process and time and its interactions (except the time x temperature x debittering process on pH) had significant effects on the production of cadaverine and tyramine, as well as on changes of pH and lactic and propionic acids. Storage at 15 degrees C produced a complete stabilization of the fermented olives. However, storage of washed olives at 20 and 28 degrees C produced a gradual decrease of lactic acid content, an increase in pH, production of propionic acid, and formation of cadaverine and tyramine, the effect becoming greater as the temperature rose. It appears that formation of cadaverine and tyramine only occurs during storage and might be related to zapatera spoilage. Changes were always significantly lower in unwashed olives, which leads to a practical stabilization of the product.     

Naturally fermented black olives: Effect on cell wall polysaccharides and on enzyme activities of Taggiasca and Conservolea varieties

Black olives of Taggiasca (Ta) and Conservolea (Co) varieties were processed according to the Greek style method in order to investigate the effect of this type of table olive processing on cell wall composition. Naturally black processing involves the storage in brine of fully ripe olives for several months, allowing a spontaneous fermentation by a mixed flora followed by fermentation by the lactic acid bacteria and yeasts. The smaller fruits of Ta variety are richer in pectic polysaccharides, accounting for half of total cell wall polysaccharides (12 mg/fruit), whereas in Co they accounted for one third (23 mg/fruit). Fresh Co olives had higher proportion of glucuronoxylans and xyloglucans (33%), whereas these polysaccharides accounted for 22% in Ta. The processing did not cause significant variations in the cell wall polysaccharide composition of Ta fruits, although pectic polysaccharides became more soluble in aqueous solutions. Conversely, processed Co olives had slightly higher amounts of galacturonan-rich pectic polysaccharides than the unprocessed fruits, suggesting that the long stage in brine might have contributed to the stabilisation and/or the biosynthesis of new polysaccharides. The changes caused by processing on cell wall polysaccharides appear to be closely related to the activity and availability of cell wall degrading enzymes.     

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.     

Antioxidant phenolic compounds loss during the fermentation of Chétoui olives

Evolution of phenolic compounds was studied during spontaneous and controlled fermentations of “Chétoui” cultivar olives at three degree of ripeness. Both spontaneous and controlled fermentations led to an important loss of total phenolic compounds with a reduction rate of 32–58%. Consequently, the antioxidant activity decreased by 50–72%. After fermentations, hydroxytyrosol and caffeic acid concentrations increased, whilst protocatechuic acid, ferulic acid and oleuropein concentrations decreased. The hydroxytyrosol concentration in black olives increased from 165 mg/100 g dry weight to 312 and 380 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. The oleuropein concentration in green olives decreased from 266 mg/100 g dry weight to 30.7 and 16.1 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. During olive fermentation, phenolic loss is essentially due to the diffusion of these compounds into the brine; the main phenolic compound identified and quantified in the different brines was hydroxytyrosol. To preserve antioxidant quality of table olives it is necessary to use a controlled process to minimise phenolic compound loss.     

Content of biogenic amines in table olives

Content of biogenic amines in flesh and brines of table olives was determined by high-pressure liquid chromatography analysis of their benzoyl derivatives. No biogenic amines were found in the flesh of fresh fruits at any stage of ripeness. Contents of biogenic amines in Spanish-style green or stored olives increased throughout the brining period but were always higher in the former. Putrescine was the amine found in the highest concentration. Small quantities of cadaverine were found in the samples taken after 3 months of brining. This compound and histamine, tyramine, and tryptamine were also found in samples taken after 12 months. Gordal cultivar showed the highest contents, followed by Manzanilla and Hojiblanca. No relationship was found between contents of biogenic amines and lactic acid production or table olive spoilages, although zapatera olives had considerably higher amounts than those brines that had undergone a normal process. Concentrations in directly brined olives were markedly lower than contents in Spanish-style olives. With respect to partition between flesh and brine, there was equilibrium between both media in the case of Spanish-style olives, whereas the contents in directly brined olives were higher in flesh than brine.     

Identification and characterization of yeast isolated from the elaboration of seasoned green table olives

The purpose of this study was to investigate the yeast population during the processing of green table olives. In the fresh olives, yeast were found at concentrations of around 3.0 log cfu/g, with Cryptococcus spp. being predominant. In the brine, the yeast concentrations were greater than 4.9 log cfu/ml, with Pichia anomala, Kluyveromyces marxianus, and Saccharomyces cerevisiae being the predominant species. Unlike the yeast isolated from the fresh olives, the strains obtained from the olive brine mostly showed low pectolytic but high catalase activities. Some of these strains also exhibited other biochemical desirable properties for the fermentation of green table olives, including their lipolytic activities and their assimilation or production of organic acids in the brine. Seven strains in particular of P. anomala, K. marxianus, S. cerevisiae, and Candida maris showed the best properties for use in trials as starter culture in pilot fermenters.     

Establishment of conditions for green table olive fermentation at low temperature

Four Lactobacillus plantarum strains were isolated from table olive cold fermentation brines. Their specific growth rate and acidification in MRS broth and in green table olive brines were studied by means of a mixed 2 pH (4.5 and 5.0) x 3 salt (3, 4 and 5%, w/v, NaCl) x 3 incubation temperature (9, 12 and 15 degrees C) levels factorial design. In MRS broth, the greatest effect (linear) on acidification was due to temperature. In brine, the effects were considerably less, pH (linear) being the most important for specific growth rate, and temperature (linear) for acidification. In both media, an initial pH of 5.0 led to good acidification at 12-15 degrees C. The effectiveness of the conditions found (initial pH of 5.0; 3%,w/v, NaCl; and incubation at 12 degrees C) was confirmed in simulated green olive fermentations with three of the strains, which proved especially robust. Behaviour in terms of growth and acidification rates was similar for these strains, and comparable to that observed in traditional processes, although mannitol and sucrose were not metabolised and fructose was only partially used. This leads to the possibility of obtaining normal fermentation processes of table olives in cold regions when appropriate initial conditions and starter cultures are used.     

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.     

Diversity of bacterial population of table olives assessed by PCR-DGGE analysis

Nocellara Etnea and Geracese table olives are produced according to traditional process, in which lactic acid bacteria (LAB) and yeasts are the dominant microorganisms. With the aim to evaluate the effect of selected starter cultures on dynamics of bacterial population during fermentation and on growth/survival of Listeria spp. artificially inoculated into the olive brine, a polyphasic approach based on the combination of culturing and PCR-DGGE analysis was applied. Plating results showed a different concentration of the major bacterial groups considered among cultivars and the beneficial effect of LAB starters, which clearly inhibited Enterobacteriaceae. Moreover, results indicated that the brine conditions applied did not support the growth/survival of Listeria monocytogenes strain, artificially inoculated, highlighting the importance of selecting right fermentation parameters for assuring microbiological safety of the final products. Comparison of DGGE profile of Nocellara Etnea and Geracese table olives, displayed a great difference among cultivars, revealing a wide biodiversity within Lactobacillus population during Geracese olives fermentation. Based on cloning and sequencing of the most dominant amplicons, the presence, among others, of Lactobacillus paracollinoides and Lactobacillus coryniformis in Geracese table olives was revealed in table olives for the first time.     

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     

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.     

Olive glutaraldehyde-like compounds against plant pathogenic bacteria and fungi

Aqueous solutions from table olives constitute a major environmental problem because of their high mineral and organic contamination but they also contain compounds with antimicrobial properties. In this study, the bactericidal and fungicidal activities of salt-free table olive solutions against several phytopathogenic micro-organisms were examined. The storage solutions of black ripe olives showed noticeable bactericidal activity against species of Erwinia, Clavibacter, Agrobacterium and Pseudomonas, although the washing waters from Spanish-style green olives were less effective. In fact, black olive storage solutions diluted to 20–30% were able to reduce the inoculated population of Erwinia amylovora and Pseudomonas syringae up to 6–7 log. This bactericidal activity was correlated with the presence of olive glutaraldehyde-like compounds such as the dialdehydic form of decarboxymethyl elenolic acid in these solutions, either free or linked to hydroxytyrosol. Experiments in vitro demonstrated that the bactericidal activity of these substances could explain most of the activity of the black olive storage solutions. Moreover, these salt-free table olive solutions also showed fungicidal activity against species of Phytophthora, Colletotrichum, Alternaria, Botrytis and Pestalotiopsis. The black olive storage solutions were more fungicidal than the washing solutions from Spanish-style green olives, with mycelial growth inhibition of up to 100% being attained, in some instances. These findings open the possibility of using table olive wastewaters in agriculture for pest management.     

Industrial implementation of black ripe olive storage under acid conditions

Restrictions on the discharge of chloride in wastewater streams have recently increased, and processors of black ripe olives have to look for alternative storage systems to the traditional brines. Ripe olives of the Hojiblanca variety were stored under aerobic and anaerobic conditions in industrial underground tanks for one year. The aerobic systems assayed with or without NaCl produced a continuous consumption of sugars as they diffused from the fruits to the surrounding liquid. At the same time sugars accumulated in the liquid for months when anaerobic conditions were employed and a high concentration of acetic acid was used. In the end, glucose was consumed with time as well and, in addition to yeasts, acetic acid and lactic acid bacteria grew in the cover solutions. The assessment of olives stored under the different systems and processed as black ripe olives revealed that the traditional aerobic brines gave rise to darker and firmer fruits. However, olive industries must eliminate chlorides from their waste streams. Promising new storage systems were (i) anaerobic preservation of olives in water with an initially high concentration of acetic acid, and (ii) aerobic preservation in water with an initially low concentration of acetic acid. Both systems produced a good quality product, free of microbial spoilage and organoleptic defects.