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.     

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.     

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.     

Evidence for the involvement of lipoxygenase in the oxidative processes associated with the appearance of green staining alteration in the Gordal olive

Lipoxygenase (LOX) activity and chloroplast pigment content were monitored during fruit growth in Gordal and Manzanilla olive varieties (Olea europaea regalis and Olea europaea pomiformis respectively). At all growth stages, LOX activity was greater in Gordal than in Manzanilla, and in both varieties, enzymatic activity peaks coincided with the maximum presence of oxidised chlorophyll pigments in the fruits. The higher lipid peroxidation potential measured directly in vitro and indirectly in vivo in the Gordal variety and its correspondence with higher contents of oxidised catabolites of chlorophyll suggested a greater tendency and sensitivity of this variety to oxidative processes. This could also explain the high organelle disorganisation levels reached during industrial processing of the fruit, allowing the formation of copper–chlorophyll complexes associated with the green staining alteration that affects Gordal olives. Copyright © 2003 Society of Chemical Industry     

Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.     

Changes of physicochemical and sensory characteristics of packed ripe table olives from Spanish cultivars during shelf‐life

Changes in the physicochemical and sensory characteristics of commercial plain (whole) and pitted ripe olives of the Gordal, Manzanilla, Hojiblanca and Cacereña cultivars were studied during a three‐year period in conditions that mimicking the storage of such products in real life. No spoilage was developed during this period. Throughout the shelf‐life, the pH of the cover brines had a marked valley decrease at the beginning of storage followed by a progressive further decline, the surface colour (measured instrumentally) and firmness of the olives followed a first‐order kinetic decay, mitigated according to the iron and calcium contents. Also, a slight browning of the cover brines at the beginning of the storage was observed. No significant changes in most of the sensory characteristics were observed by the panel test during shelf‐life except for a limited change in olive surface colour. At the end of the shelf‐life, most of the samples were classified as ‘extra’ category according to the IOC sensory evaluation method and only plain Gordal presentation was classified as ‘first, choice or select’.     

Regeneration of Spanish Style Green Table Olive Brines by Ultrafiltration

Ultrafiltration was applied to regenerate brines from green table olive fermentations. Experiments were conducted to select optimum membrane pore size and working conditions, as well as effects on characteristics of brines and olives packed with these regenerated brines. According to the results, good decoloration of such solutions was achieved with polysulfonate membranes of 1,000 daltons pore size. Best working conditions were : 18 bars, room temperature, room temperature (25°C), and without any previous filtration. Very low reduction of NaCl and slight decrease of lactic acid were noted. No effect on the quality of packed olives with the treated brines was detected.     

Spanish‐style green table olive shelf‐life

Changes in the physicochemical and sensory characteristics of commercial whole, pitted and stuffed with pepper, garlic or anchovy paste Spanish‐style green table olives of the Gordal, Manzanilla and Hojiblanca cultivars were studied during a three‐year period. No olives developed any spoilage during this period, and at the end of it, all presentations were classified as ‘extra’ according to the sensory evaluation methodology issued by the International Olive Council. Throughout the shelf‐life, surface colour index and firmness of the olives degraded in accordance with a first‐order kinetics, while a gradual browning of the cover brines was observed; however, products added with ascorbic acid maintained a stable colour, while those with calcium content showed reduced firmness degradation. No significant changes in most of the sensory characteristics were observed by the panel test during shelf‐life and only slight changes in firmness, and surface colour were noticed in samples with significant differences in these attributes when measured instrumentally.     

Influence of Yeasts on the Oil Quality Indexes of Table Olives

After moisture, fat is the major constituent of table olives. However, scarce studies have been carried out to determine the influence of microorganisms and type of processing on the modification of their quality indexes. The present survey studies the influence of lipolytic (Candida boidinii TOMC Y5 and Wickerhamomyces anomalus TOMC Y10) and nonlipolytic (Debaryomyces etchellsii TOMC Y9 and Pichia galeiformis TOMC Y27) yeasts on the oil quality indexes of Manzanilla and Hojiblanca green fruits processed as directly brined and lye‐treated table olives. Overall, the inocula scarcely used available sugars, except the lipolytic C. boidinii strain in lye‐treated olives. Acetic acid production was limited in all conditions, except for the D. etchellsii strain in directly brined Manzanilla fruits. Ethanol formation was also reduced, although the W. anomalus (in both types of elaboration) and the C. boidinii (in lye‐treated olives) strains produced significantly higher proportions. Apparently, changes in the oil quality indexes of processed olives were not related to the presence of yeasts, and hence, could have been caused by the endogenous activity of the fruits. A principal component analysis using the microbiological, physicochemical, and oil quality data supported this hypothesis, grouping treatments according to olive variety and type of elaboration, while segregation due to yeast inocula was not observed.     

Triterpenic acids in table olives

An experimental investigation was carried out for the first time on the triterpenic acids in table olives. Maslinic acid was found in a higher concentration than oleanolic acid in the flesh of 17, unprocessed olive varieties, with the Picual and the Manzanilla varieties showing the highest and almost the lowest contents, respectively. The level of triterpenic acids in several types of commercial black and green olives ranged from 460 to 1470 mg/kg fruit, which represents a much higher value than reported for virgin olive oils. In fact, the NaOH treatment employed to debitter black and green olives reduced the concentrations of these substances in the flesh because of their solubilisation into alkaline solutions. Thus, natural black olives, which are not treated with NaOH, showed a higher concentration than 2000 mg/kg in the olive flesh. These results will contribute to the reevaluation of table olives from a nutritional and functional point of view because of the promising bioactivity properties attributed to olive triterpenic acids.     

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.     

Volatile profile of Spanish-style green table olives prepared from different cultivars grown at different locations

The volatile profiles of Spanish-style green table olives elaborated with Manzanilla, Gordal and Hojiblanca cultivars grown at different locations in Spain were established by solid phase micro-extraction (SPME) and gas chromatography coupled to mass spectrometry (GC–MS). A total of 102 volatile compounds were identified, belonging to distinct chemical classes, and 20 of them are reported for the first time in table olives. The headspace profile was predominated by alcohols and phenols, followed by acids and esters, whereas the relative amounts of the remaining classes were quite lower (< 5% in general). The principal compounds characterizing the headspace for most samples were p-creosol, phenylethyl alcohol, acetic acid, ethanol, benzyl alcohol, ethyl acetate, and (Z)-3-hexen-1-ol. Significant differences in the proportions of volatile compounds between samples from the Gordal cultivar and those from Manzanilla and Hojiblanca cultivars were detected and statistically visualized by principal component analysis (PCA). Among all the identified compounds, only (E)-2-decenal showed significant differences between the three cultivars without being significantly affected by locations where the fruits were grown.     

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.     

Utilization of Enterococcus casseliflavus and Lactobacillus pentosus as starter cultures for Spanish-style green olive fermentation

Green olive were fermentated with starter cultures of Enterococcus casseliflavus cc45 andLactobacillus pentosus CECT 5138. A total of 32 strains of enterococci isolated from brines at high pH were characterized and identified by phenotypic methods. The strain cc45 was selected after a preliminary screening and applied together with thelactobacillus culture the first day after brining, comparing the results with those obtained when both cultures were used sequentially, enterococci at day 1 and lactobacilli at day 2, and with uninoculated controls. Microbial growth, substrate and product evolution, and flavour of the fermented olives were studied. Inoculation produced a quicker acidification of brines, consumption of carbohydrates and decrease of pH. The best results were attained when both cultures of lactic acid bacteria were applied sequentially with 1day delay. This is the first report dealing with the presence of E. casseliflavus in table olive brines and their utilization as starter culture.     

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.     

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.     

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.     

Preservation of Manzanilla Aloreña cracked green table olives by high hydrostatic pressure treatments singly or in combination with natural antimicrobials

High hydrostatic pressure (HHP) treatments (singly or in combination with natural antimicrobials) were tested for stabilization of Manzanilla Aloreña seasoned olives stored at 25 °C. HHP (5 min) was highly effective on yeast populations at 300 MPa or higher. No viable yeasts were detected in samples treated at 400 MPa for up to three months. Low levels of endospore-forming bacteria were always detected after HHP treatments. Addition of nisin to the brines reduced bacterial counts by 1.4 log cycles but it had no effect on yeasts when tested singly or in combination with HHP treatment (400 MPa, 5 min). Thyme oil had almost no effect on yeast concentrations, but rosemary oil reduced yeast viable counts progressively during storage. Essential oils in combination with HHP (400 MPa, 5 min) significantly (p < 0.05) reduced the concentrations of aerobic mesophilic bacteria. Low-salt brined olives purged with N₂ or supplemented with ascorbic acid and then pressurized for 5 min at 450 or 550 MPa were preserved for up to 5 months without spoilage, suggesting that the NaCl content in brines of packed Manzanilla Aloreña table olives could be reduced considerably by application of HHP as a stabilization treatment.     

Profile of anti-lactic acid bacteria compounds during the storage of olives which are not treated with alkali

The objectives of this study were: (1) to characterize the phenolic and oleosidic compounds in the preservation solutions of black ripe olives, particularly those with antimicrobial activity and (2) to explain the growth of lactic acid bacteria (LAB) in the olive brines. Fruits were put into industrial tanks and preserved in acidified solutions under aerobic or anaerobic conditions. Lactic acid was analyzed as a marker of LAB growth in the olive brines. Hydroxytyrosol was the most concentrated phenolic compound in these solutions followed by hydroxytyrosol 4-glucoside, hydroxytyrosol 1-glucoside, tyrosol and salidroside. Among the oleosidic compounds, secoxyloganin and oleoside 11-methyl ester were found in a concentration between 0.3 and 3 mM, whereas secologanoside and oleoside were always detected at a concentration below 0.2 mM. Likewise, a notable concentration of the dialdehydic form of decarboxymethyl elenolic acid either free or linked to hydroxytyrosol was found at the beginning of preservation, which was high enough to inhibit LAB growth. However, an acidic degradation of these antimicrobials occurred with time, particularly in the summer, and it allowed for the growth of lactobacilli and the formation of lactic acid. The results of this study are a contribution to explain the microbial fermentation in acidified solutions of olives which are not treated with NaOH on an industrial scale.     

Effects of salt mixtures on Spanish green table olive fermentation performance

This work investigates the effect of sodium, potassium, and calcium chloride salts on the performance of Spanish green table olive fermentation using a simple centroid mixture design. The presence of calcium chloride hindered the diffusion of all sugars and delayed the period required to reach their respective maximum concentrations in brines. Such effects can prevent tumultuous processes and gas pocket spoilage. Acetic acid was present at low concentrations in all treatments but was not generated during fermentation. The production rate of lactic acid was either decreased or delayed by the presence of calcium chloride but adequate final conditions were always reached. The chemometric analysis classified treatments into groups according to the presence of calcium chloride and disclosed a stimulating effect of potassium on lactic acid production. Therefore, these techniques can be a useful tool to investigate olive fermentation performance. According to the results, acceptable Spanish green table olives can be produced using salt mixtures, with the subsequent reduced sodium content in the final products. The results obtained in this study could also be of interest for other fermented vegetables.