Changes in volatile compounds and related biochemical profile during controlled fermentation of cv. Conservolea green olives

The effect of controlled fermentation processes on the profile of volatile and other biochemical compounds of cv. Conservolea green olives processed by the Spanish method was studied. The different treatments included: (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a wild strain of Lactobacillus plantarum isolated from a previous fermentation, (c) uninoculated spontaneous process (control). Microbial growth, pH, titratable acidity, reducing sugars, organic acids and volatile compounds were monitored. Starter cultures were effective in establishing an accelerated fermentation process. Both were able to reduce the survival period of Enterobacteria by 7 days, minimizing thus the likelihood of spoilage. Higher acidification of the brines and faster pH drop was observed in inoculated processes, with L. pentosus presenting better performance than the wild strain of L. plantarum. Lactic and acetic were the major organic acids detected by HPLC, the concentration of which increased in the course of fermentation. Citric and malic acids were also present in the brines but they were degraded completely within the first 2 weeks of fermentation. Ethanol, methanol, acetaldehyde, ethyl acetate, isobutyric acid were the major volatile compounds identified by GC. Their concentration varied greatly among the fermentation processes, reflecting varying degrees of microbial activity in the brines.     

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.     

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.     

Biofilm formation on abiotic and biotic surfaces during Spanish style green table olive fermentation

In this work, the establishment of polymicrobial communities on the surfaces which come into contact with the brine during Spanish style Gordal cv. green olive fermentation when subjected to spontaneous or controlled processes (inoculated with Lactobacillus pentosus LPCO10 or 128/2) was studied. Scanning electron microscopy showed that L. pentosus and yeast populations were able to form mixed biofilms throughout the fermentation process on both abiotic (glass slide) and biotic (olive skin) surfaces. The biofilm architectures in both supports were completely different: on the glass slides only aggregates of L. pentosus and yeasts without any polymeric matrix surrounding them were found while on the skin of the fruits, true mature biofilms were observed. During fermentation, the lactic acid bacteria (LAB) population on the olives remained similar while that of yeasts increased progressively to reach similar levels at the end of the process (8-9 log CFU/cm(2)). Molecular analysis showed that different populations of L. pentosus and yeasts were the only microbial members of the biofilm formed during fermentation, regardless of inoculation. Hence, the green olive surface provides an appropriate environmental condition for the suitable development and formation of complex biofilms during controlled or natural table olive processing.     

Survival of Bacillus cereus vegetative cells during Spanish-style fermentation of conservolea green olives

The aim of this research was to investigate the survival of artificially inoculated Bacillus cereus during Spanish-style green olive fermentation. Olives were initially treated with lye and subjected to different fermentation procedures including (i) heat shock (85 degrees C for 10 min) and inoculation with Lactobacillus plantarum ACA-DC 287, (ii) heat shock and inoculation with L. plantarum ACA-DC 146, (iii) heat shock and inoculation with Lactobacillus pentosus isolated during previous studies, and (iv) fermentation by indigenous flora (control process). Microbial growth and survival, pH, titratable acidity, and organic acid evolution were monitored. Inactivation of B. cereus was observed during all processes. The pathogen population declined during all fermentations, but a tailing effect was observed in the brines when the population reached 2 log CFU/ml, at which point the pathogen does not pose a risk to human health. The rate of inactivation was higher in heat-shocked inoculated olives (mean of -2.21 log CFU/day) compared with control olives (-1.26 log/day), indicating an advantage of heat shock and inoculation over spontaneous fermentation. The production of organic acids (primarily lactic acid) during fermentation seemed to be the main factor that determined the behavior of the pathogen under stress conditions prevailing in the brine. Principal components analysis was useful for distinguishing among the different fermentation processes on the basis of the relevant organic acid profile.     

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.     

A novel Lactobacillus pentosus-paired starter culture for Spanish-style green olive fermentation

A new starter culture consisting of two Lactobacillus pentosus strains was developed and successfully used for Spanish-style green olive fermentations in an industrial study. The inoculum, consisting of L. pentosus LP RJL2 and LP RJL3 strains, was inoculated in 10,000 kg glass fiber containers at 10⁶ CFU/ml and 10⁵ CFU/ml, final concentration respectively, in five different olive processing plants in the south of Spain. As a control, uninoculated fermentors were also used. In all inoculated fermentors, the paired starter rapidly colonized the brines to dominate the natural microbiota and persisted throughout fermentation. A decline in pH to reach about 5.0 was achieved in the first 15–20 days, reaching about 4.0 at the end of the process. The lactic acid concentration in brines increased rapidly in the first 20 days of fermentation (0.3–0.4 g/100 ml) to give values higher than 0.8 g/100 ml at the end of the process. In contrast, increasing lactic acid concentration was slower in uninoculated than in the inoculated brines, and the final concentrations were lower. Although reaching similar values at the end of the process, the decline in pH in uninoculated fermentors was slower than in the inoculated ones. These results show the efficacy of the new starter culture to control the lactic acid fermentation of Spanish-style green olives.     

Use of selected autochthonous lactic acid bacteria for Spanish-style table olive fermentation

The present work presents a successful attempt to achieve an enhanced and more predictable fermentation process in Spanish-style green olive technology by selection and use of autochthonous starter cultures. During the first phase of this work, two Spanish-like fermentations of green table olives of cultivar (cv) "Nocellara del Belice", coming from irrigated and not irrigated fields, were monitored, in order to highlight the best agricultural conditions for drupe production and to isolate lactic acid bacteria strains with relevant technological properties. Among 88 identified isolates, one Lactobacillus pentosus strain showed remarkable biochemical features and high acidification rate in synthetic brine. In the second phase, the selected strain was used as starter culture in three different trials to establish the best conditions for its use. Microbial counting, as well as starter tracking by M13 RAPD-PCR, reflected the optimal adaptation of the strain to the environment. Spontaneous fermentation needed a 14-day long lag phase to reach the same population as the inoculated trials. Moreover, sensory traits of table olives obtained with adjunct culture showed better characteristics compared to those processed in the other trials, in particular concerning the presence of off-odours.     

剁辣椒发酵过程中菌群与有机酸变化规律分析

以自然发酵为对照,以植物乳杆菌W-4进行剁辣椒纯种发酵。研究结果表明：在发酵过程中,接种发酵能使乳酸菌快速成为优势菌,较快地降低剁辣椒的pH值。接种发酵具有较高数量的菌落总数和乳酸菌,酵母菌在自然发酵和接种发酵中数量相当,变化趋势基本一致,而大肠菌群只在发酵前期出现,而后消失。有机酸测定结果表明接种发酵具有较高含量的乳酸,而自然发酵具有较高含量的柠檬酸和苹果酸,而乙酸只在自然发酵的前期出现。     

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.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

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.     

Olive Fermentation and Processing: Scientific and Technological Challenges

ABSTRACT: The 3 main commercial table olive preparations are the Spanish-style green olives, the Greek-style naturally black olives, and the Californian-style black ripe olives. In all cases, fruits undergo fermentation in brine solution, which preserves them and increases palatability. Lactic acid bacteria dominate in brines of green olives, while mainly fermentative yeasts are found in brines of black ones. The fermentation is spontaneous and begins as soon as olives are put into brine. Research on table olives has been going on for 100 years, but many questions remain. Previous work has focused on the efficient utilization of starter cultures in order to control the fermentation, to guarantee the absence of spoilage, and to govern the relationship between olive flavor and microorganisms. Nowadays, there is concern over environmental problems that the fermentation solutions represent, in particular their high NaCl content. Fermentation or simply storing olives in water without salt presents some problems. The high concentration of sodium benzoate used for storing black ripe olives by the American industries is not advisable for European processes due to the residual amount of preservative in the final product. Fermentation of green olive wash waters, in order to obtain valuable products, is being pursued, but polymeric inhibitors are problematic and undesirable microorganisms grow. The consumption of organic foods is increasing and black olives fit the definition since they are processed without chemical treatment. However, the hydrolysis of the bitter compound oleuropein, either by microorganisms or acids, needs to be studied.     

Microbiological and physicochemical changes of naturally black olives fermented at different temperatures and NaCl levels in the brines

The effect of different temperatures (25°C, 18°C and ambient temperature) and NaCl levels in brines (4%, 6% and 8%) on the microbiological and physicochemical characteristics of naturally black olives of Conservolea variety was studied for up to 190 days. Fermentation was carried out according to the traditional anaerobic method. The initial microflora consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all fermentations. The prevailing micro-organisms were lactic acid bacteria and yeasts, the association of which was dependent on the conditions of fermentation. At 25°C and 18°C in brines containing 4% and 6% NaCl, the growth of lactic acid bacteria was favoured resulting in a lactic acid fermentation, as indicated by the high free acidity levels and low pH values in the brines. On the contrary, 8% NaCl concentration affected the growth of lactic acid bacteria and enhanced the activity of fermentative yeasts, producing a final product with lower free acidity and higher pH value. At ambient temperature, the counts of lactic acid bacteria followed the fluctuation of temperature regardless of salt concentration, while yeasts did not seem to be affected. The lactic acid bacteria identified belonged to the species Lactobacillus mesenteroides, Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus pentosus. The best conditions for fermentation were at 25°C and 6% NaCl, developing free acidity of 142 mM (1·28% w/v) lactic acid and pH value of 3·8. After 5 months of brining, olives fermented at 25°C were judged by panelists as being debittered and ready to eat. No off-odour development was detected in any case due to anomalous fermentation. The HPLC analysis revealed that citric, malic, tartaric, succinic, lactic and acetic were among the end products of fermentation.     

Degradation of ascorbic acid and potassium sorbate by different Lactobacillus species isolated from packed green olives

The aim of this research was to ascertain the lactic acid bacteria responsible for the degradation of ascorbic acid and/or potassium sorbate, isolated from packed green olives where these additives had diminished. A total of 14 isolates were recovered from samples of different green olive containers. According to partial sequencing of the 16S rRNA coding gene, Lactobacillus parafarraginis, Lactobacillus rapi, Lactobacillus pentosus, Lactobacillus paracollinoides, and Pediococcus ethanolidurans were identified. With the exception of L. pentosus and L. paracollinoides, the other species had not been mentioned in table olives before this study. Only three of the 14 isolates metabolized ascorbic acid in MRS broth, and the products from ascorbic acid in modified MRS broth without carbon sources were acetic and lactic acids. Except for the two L. rapi and the two P. ethanolidurans strains, the remaining 10 isolates depleted potassium sorbate added into MRS broth to some extent. The product generated by three of these strains was confirmed to be trans-4-hexenoic acid. The degradation of ascorbate or sorbate by lactic acid bacteria should be taken into account when these additives are used in food products where this group of bacteria may be present.     

Induced lactic acid fermentation of untreated green olives of the Conservolea cultivar by Lactobacillus pentosus

Inoculation of untreated green olives of the Conservolea cultivar (Olea europea media rotunda) with a commercial strain of Lactobacillus pentosus with/without glucose supplement was studied. Despite an initial loss in viability of 0.5 log cycles on average, due to lack of adaptation of the starter to the saline environment of the brine, cultures grew well and initiated an accelerated fermentation process. Inoculation reduced the survival period of Enterobacteriaceae, and consequently potential spoilage, and caused a quicker acidification of brines and decrease in pH compared with control uninoculated processes. The diffusion of phenolic compounds was slow, hindered by the epidermis of the fruits, and had no effect on the growth of the starter. HPLC analysis revealed that lactic and acetic acids were among the end‐products during the fermentation of green olives. Citric, tartaric and malic acids were also detected but at much lower concentrations. Results obtained from this work can be applied directly to industry for the effective use of starter cultures on natural fermentation processes of green olives without prior lye treatment. © 2003 Society of Chemical Industry     

Vapor Analysis of Fermented Spanish-type Green Olives by Gas Chromatography

SUMMARY: Five major components were detected gas chromatographically in the head-space vapor (HSV) of Spanish-type green olives fermented by pure cultures of Lactobacillus plantarum, Pediococcus cerevisiae and Leuconostoc mesenteroides. Three of these compounds were identified as acetaldehyde, methyl sulfide, and ethanol. The same compounds were present in unfermented olives but in different amounts. Olives that had undergone a natural fermentation contained the above five compounds, and, in addition, a varying number of other compounds. These results indicated that HSV analysis may be a rapid method for detecting volatile end products resulting from the metabolism of various microorganisms. A high ethanol content was found in olive brines that contained a predominance of yeasts. Abnormal fermentations gave unique HSV profiles, one of which indicated a high level of 2-butanol. Methyl sulfide was found to be a major odor component of fermented as well as unfermented olives. Acetaldehyde and ethanol contributed secondarily to the odor. Primary contributions of fermentation by the above lactic acid bacteria to the flavor of olives were: (1) production of a desirable level of acidity, and (2) utilization of fermentable sugars to the exclusion of microorganisms which produce metabolic end products with undesirable flavor characteristics.     

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.     

乳杆菌发酵剂对酸肉挥发性风味成分的影响

分别采用植物乳杆菌、清酒乳杆菌和类植物乳杆菌作为发酵剂对酸肉发酵过程中挥发性风味成分的影响。 电子鼻分析结果表明,酸肉的整体风味随发酵时间的增加差异显著;利用固相微萃取-气相色谱-质谱技术对酸肉发 酵过程中的挥发性风味物质进行检测,结果表明,酯类物质的相对含量显著增加,且增加的主要是乙酯类物质,发 酵结束时,添加植物乳杆菌、类植物乳杆菌和清酒乳杆菌组酸肉的乙酯类物质相对含量（56%、53%和60%）均显 著高于对照组（47%）,说明乳杆菌发酵剂的添加有利于酸肉中乙酯类物质的形成;通过Heatmap聚类分析发现, 发酵结束时,添加乳杆菌发酵剂的3 组酸肉整体风味比较接近,但与对照组差异较大,说明乳杆菌发酵剂的添加影 响了酸肉的风味品质。此外,发酵过程中添加发酵剂的酸肉中乳酸菌数量增加较快,肠杆菌数量显著降低,这保证 了酸肉的食用安全性。     

蔬菜发酵菌种的筛选及发酵特性

为了选择适合蔬菜发酵的发酵菌种,本研究从四川泡菜老汤中分离的6 种乳酸菌Lactobacillus plantarum、Lactobacillus fermentum、Lactobacillus brevi、Lactobacillus pentosus、Leuconostoc mesenteroides、Lactobacillus lactics筛选发酵菌种。6 种乳酸菌接入亚硝酸盐的MRS培养液中,6 种乳酸菌还原亚硝酸盐的大小顺序为Lactobacilluspentosus＞Lactobacillus plantarum＞Leuconostoc mesenteroides＞Lactobacillus fermentum＞Lactobacillus lactics＞Lactobacillus brevis。以pH值降低的速率为发酵速率,Lactobacillus plantarum、Lactobacillus pentosus、Leuconostocmesenteroides这3 种发酵剂的发酵速率较其他3 种快,并且通过乳酸菌的全细胞蛋白电泳实验得出此3 种乳酸菌在甘蓝发酵过程中能够成为优势菌。从乳酸菌还原亚硝酸盐的能力、发酵速率、发酵菌种的生存能力（是否能成为优势菌）的实验结果表明Lactobacillus plantarum、Lactobacillus pentosus、Leuconostoc mesenteroides这3 种乳酸菌作为蔬菜发酵的发酵剂。通过对3 种发酵菌种发酵特性的研究可知,Leuconostoc mesenteroides较另两种生长周期短,稳定期维持时间短,很快进入衰退期。Lactobacillus plantarum菌较Lactobacillus pentosus、Leuconostoc mesenteroides耐酸,Leuconostoc mesenteroides对酸敏感。Leuconostoc mesenteroides最适生长温度为30 ℃,Lactobacillus plantarum、Lactobacillus plantarum两种菌的最适生长温度是37 ℃。15 ℃条件下Leuconostoc mesenteroides的光密度（OD600 nm）值很低,说明Leuconostoc mesenteroides较Lactobacillus plantarum、Lactobacillus pentosus对低温敏感。