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.     

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.     

Use of a D-optimal mixture design to estimate the effects of diverse chloride salts on the growth parameters of Lactobacillus pentosus

The effects of NaCl, KCl, CaCl₂, and MgCl₂ and their mixtures on the ionic strength (IS) of the medium and the growth parameters of Lactobacillus pentosus were studied by means of a D-optimal mixture experimental design with constrains (total salt concentration ≤ 9.0%, wt/vol) and the generalized z-value. The IS was linearly related to the concentrations of the diverse salts and its increase, for similar concentrations of salts, followed the order MgCl₂ > CaCl₂ > NaCl > KCl. Within the experimental region, the lag phase duration (λ) was mainly affected by NaCl and CaCl₂ and the interaction KCl with MgCl₂. The maximum specific growth rate (μ_max) decreased as NaCl (the highest effect), CaCl₂, and MgCl₂ increased (regardless of the presence or not of previous NaCl); low KCl concentrations had a stimulating effect on μ_max, but its overall effect showed a similar trend to the other salts. The maximum population reached (N_max) was the least affected parameter and decreased as NaCl and CaCl₂ concentrations increased regardless of the presence of the other salts. The equations that expressed the growth parameters as a function of the diverse chloride salts, within the limits assayed, were developed and the corresponding z- and harmonic Z-values were estimated.     

Volatile organic compounds profile during milk fermentation by Lactobacillus pentosus and correlations between volatiles flavor and carbohydrate metabolism

Flavor, as one of the most important properties determining the acceptability and preference of fermented milks, is influenced by compositional and processing factors. In this study, we focused on the volatile organic compounds related to flavor during milk fermentation by Lactobacillus pentosus according to electronic nose analysis. Xylose (1% addition) metabolized by Lb. pentosus strongly affects the flavor of yogurt, with the potent volatile organic compounds of ethanol (3.08%), 2,3-butanedione (7.77%), and acetic acid (22.70%) detected using solid-phase microextraction coupled with gas chromatography-mass spectrometry analysis. Sensoryanalysis also showed skimmed yogurt fermented by Lb. pentosus with 1% xylose had the unique scores of sourness (acetic acid) and butter flavor (2,3-butanedione). Furthermore, α-acetolactate synthase and α-acetolactate decarboxylase in carbohydrate metabolism play important roles in milk fermentation. Under preferable conditions (pH 5.5, 42°C) for α-acetolactate synthase and α-acetolactate decarboxylase, the relative content of potent flavor compound 2,3-butanedione was 10.13%, which was 2.55% higher than common culture condition (pH 4.5, 37°C), revealing that xylose metabolized by Lb. pentosus has potential values for the milk product industry, such as the acceptability and preference of fermented milk product.     

A bacteriocin-like substance produced from Lactobacillus pentosus 39 is a natural antagonist for the control of Aeromonas hydrophila and Listeria monocytogenes in fresh salmon fillets

We studied the ability of Lactobacillus pentosus 39, a BLS (Bacteriocin-like substance)-producing strain, to control the growth of Aeromonas hydrophila ATCC 14715 and Listeria monocytogenes ATCC 19117 artificially added to fresh salmon fillets at refrigeration temperatures and under simulated cold-chain break conditions.At refrigeration temperatures, Lb. pentosus 39 protective culture and its putative bacteriocin significantly reduced A. hydrophila counts compared with the control (2.1 and 1.4 log CFU/g reductions, respectively). Similar behaviour was observed for L. monocytogenes (3.6 and 1.3 log CFU/g reductions, respectively).Under simulated cold-chain break conditions, an increase in temperature (30°C for 12h) produced an evident increase in the development of A. hydrophila, L. monocytogenes, but also of Lb. pentosus 39, with a consequent increase in BLS production. This condition resulted in a greater reduction of both pathogens compared with samples stored at 4°C throughout the experiment (2.8 log CFU/g reduction for A. hydrophila, 5.8 log CFU/g reduction for L. monocytogenes). In samples treated with the putative bacteriocin alone, a less marked decrease was observed.Our study demonstrates the capability of Lb. pentosus 39 to control the growth of psychrotrophic bacteria in an experimental seafood model system. A similar biopreservation technology could provide more prolonged shelf-life during storage of ready-to-eat seafood, ensuring safety, even under extreme conditions.     

Volatile compounds in uninoculated and inoculated table olives with Lactobacillus plantarum (Olea europaea L., cv. Moresca and Kalamata)

Table olives' flavour plays an important role in consumer's acceptability and it depends on various factors such as varieties, intrinsic characteristics, ripening of fruit and processing technologies. Flavour biogenesis is also influenced by addition in brine medium of lactic acid bacteria as inoculants, which reduce spoilage risks and improve sensory characteristics. In this work, flavour profiles of uninoculated and inoculated table olives with Lactobacillus plantarum (cv. Moresca and Kalamata) have been analytically characterised and compared. Twenty-one volatile compounds comprise alcohols, aldehydes, ketones, esters as well as acids formed during Greek-style olive fermentation (3 months brining after) have been characterised by gas chromatography and GC/mass spectrometry. Very high contents of ethanol and appreciable amounts of ethyl acetate, isobutanol, 2-butanone, 1-propanol and 1-hexanol were revealed in all samples with a significant increase in inoculated samples with respect to uninoculated ones. Also 1-butanol, 3-pentanol, 3-hydroxy-2-butanone, cis-3-hexen-1-ol and 2-butanol which were present in lower amounts, disclosed a meaningful increase in inoculated samples of both varieties, especially in Moresca inoculated sample. Acetic acid, isopentanol, 2-pentanol, propyl acetate, ethyl propanoate and 4-penten-1-ol showed a significant increase in inoculated Kalamata sample. These results showed that inoculation of brine medium with lactic acid bacteria starters significantly influenced aroma profiles of both varieties, in particular an increase in concentration of various flavour compounds has been revealed in inoculated table olives.     

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.     

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.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Lactic acid bacteria from fermented table olives

Table olives are one of the main fermented vegetables in the world. Olives can be processed as treated or natural. Both have to be fermented but treated green olives have to undergo an alkaline treatment before they are placed in brine to start their fermentation. It has been generally established that lactic acid bacteria (LAB) are responsible for the fermentation of treated olives. However, LAB and yeasts compete for the fermentation of natural olives. Yeasts play a minor role in some cases, contributing to the flavour and aroma of table olives and in LAB development.     

Selection of Lactobacillus plantarum strains to use as starters in fermented table olives: Oleuropeinase activity and phage sensitivity

Fermented table olives (Olea europaea L.) are largely diffused in the Mediterranean area. Olives are picked at different stages of maturity and after harvesting, processed to eliminate the characteristic bitterness caused by the presence of the oleuropein glucoside and to become suitable for human consumption. The spontaneous fermentation of table olives mainly depends on lactic acid bacteria (LAB), and in particular on Lactobacillus plantarum which plays an important role in the degradation of oleuropein. The hydrolysis of oleuropein is attributed to the β-glucosidase and esterase activities of the indigenous LAB microflora. This study investigated the potential of L. plantarum strains isolated from dairy products and olives to be used as starters for fermented table olives. Forty-nine strains were typed by RAPD-PCR and investigated for the presence of the β-glucosidase (bglH) gene. The full sequence of the bglH gene was carried out. All the 49 L. plantarum strains were also tested for phage resistance. A total of six strains were selected on the basis of genotypic polymorphism, bglH gene sequence analysis, and phage resistance profile. These strains were further characterized to assess the acidifying capability, the growth at different temperatures, the tolerance to different NaCl concentrations, and the oleuropeinolytic activity. Although further characterizations are required, especially concerning the influence on sensory properties, L. plantarum proved to have the potential to be used as a debittering and fermentative agent in starter culture for fermented table olives.     

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.     

Evolution of aroma volatiles during storage of sourdough breads made by mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus

Two mixed starter cultures were used for sourdough bread making to evaluate their ability to improve quality and increase bread shelf-life: Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus mixed with the lactose fermenting yeast Kluyveromyces marxianus as alternative baker’s yeast. Control sourdough breads (K. marxianus) without the addition of bacteria, were also prepared. The changes on the headspace aroma volatiles during storage were assessed using solid-phase microextraction (SPME) GC–MS analysis. The effect of these changes on bread flavour was evaluated by consumer preference evaluations and the results were co-evaluated with those from the GC–MS analysis. The obtained results showed differences in the volatile composition of the different types of breads examined, as well as dramatic decreases of the number and the amount of volatiles after five days of storage. The sourdough breads made with K. marxianus and L. bulgaricus, had a more complex aroma profile, longer shelf-life and achieved the highest scores in the sensory tests.     

Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses

An implemented GC method to separate and quantify the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) was used to study the adaptive response to sublethal acid and cold stresses in Lactobacillus helveticus and Lactobacillus sanfranciscensis. The comparison of the composition of cellular fatty acids of the two strains and their changes after 2 h of stress exposure under micro-aerobic and anaerobic conditions indicated that the aerobic biosynthetic pathway for unsaturated fatty acids is prevalent in L. sanfranciscensis, while the anaerobic pathway is prevalent in L. helveticus. Indeed in the latter strain, in the presence of a source of oleic acid and under micro-aerobic conditions, C18:1n11 and its post-synthetic derivative C19cyc11 accounted for overall proportion ranging from 52 to 28% of the total FAs. On the other hand L. sanfranciscensis synthesizes by aerobic pathway C18:1n9 and transforms it to C19cyc9. However in this species the cumulative level of these two FAs did not exceed 30%. The relevant proportion of dodecanoic acid in the latter species suggests that carbon chain shortening is the principal strategy of L. sanfranciscensis to modulate fluidity or chemico-physical properties of the membranes.     

Selenium enriched green tea increase stability of Lactobacillus casei and Lactobacillus plantarum in chitosan coated alginate microcapsules during exposure to simulated gastrointestinal and refrigerated conditions

The effects of selenium enriched green tea (SGT; 85.8–96 mg/kg) in different concentrations of 1 g and 2 g/100 mL, on the in vitro exposure to simulated gastrointestinal juice and refrigerated storage of encapsulated Lactobacillus casei and Lactobacillus plantarum were investigated in chitosan coated alginate beads. The encapsulation yield of viable cells in chitosan coated alginate beads with and without SGT was not significantly different (P < 0.05). These results together with the study about the survival of probiotic bacteria in microspheres with SGT during storage at 4 °C, demonstrated significantly higher number (P < 0.05) of survival bacteria in microcapsules with SGT 2 g/100 mL. Microencapsulated L. casei and L. plantarum with SGT 1 g and 2 g/100 mL were resistant to simulated gastric conditions (pH 2.0, 2 h) and bile solution (3 g/100 mL, 2 h) resulting in significantly (P < 0.05) improved survival when compared with microencapsulation without SGT addition.     

Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures

Lactic acid bacteria (19 isolates) from Bella di Cerignola Italian table olives were investigated for their technological and probiotic properties for the selection of multifunctional starter cultures for table olives. The bacteria were first identified by phenotyping and genotyping, then characterized for the production of biogenic amines, growth at different pH, NaCl concentrations, and temperatures. The potentiality of the bacteria to have some probiotic properties (antimicrobial activity against foodborne pathogens, survival in low pH and in the presence of bile salts, ability to adhere to the mammalian cells model IPEC-J2) was also investigated. Eighteen of the studied isolates were identified as Lactobacillus plantarum and one as Enterococcus faecalis. All bacteria were able to grow at a range of pH between 4.0 and 10.0 as well as in media supplemented with 2.5 to 7.5% of NaCl and 0.3% bile salts and survived in MRS broth acidified at pH 2.5; moreover, they inhibited significantly Escherichia coli O157:H7. The adhesion to IPEC-J2 cells was in general low to moderate (5.3 to 8.3%); however, 2 isolates of L. plantarum (c16 and c19) showed interesting higher adhesion values (up to 16%). Our results suggest that at least 3 isolates could be possible multifunctional starters for Bella di Cerignola olives: L. plantarum 16 and 19 for mainly their probiotic properties and L. plantarum 10 for mainly its technological characteristics. Practical Application: A functional starter is a microorganism exerting benefits on human health (probiotic) and able to guide a fermentation (starter). The main goal of this article was to select a functional starter for table olives.     

Volatile compounds in table olives (Olea Europaea L., Nocellara del Belice cultivar)

Table olive quality depends on a number of factors, including fruit characteristics, ripening and processing technologies. Volatile and semi-volatile organic compounds, present both in the sample matrix and in the headspace aroma, are responsible for the olive fruit flavour influencing the consumer’s preference.     

Lactobacillus salivarius: Bacteriocin and probiotic activity

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.     

pH and dose-dependent effects of quercetin on the fermentation capacity of Lactobacillus plantarum

This article reports a study of how quercetin affects the capacity of Lactobacillus plantarum RM71 to ferment different media, including a chemically defined medium (CDM) and media relevant for practical fermentation processes. It is shown for the first time that quercetin exerts pH- and dose-dependent effects on the fermentation performance of L. plantarum. At an initial pH of 5.5, quercetin promoted quicker growth upon inoculation at increased quercetin concentrations, while a detrimental dose-dependent lengthening of the lag phase was observed at an initial pH of 6.5. The time course of sugar consumption and lactic acid production data tracking in pH 5.5 CDM showed that quercetin promoted quicker sugar consumption as a result of earlier sugar uptake and lactic acid production than in the control. A model wine and a similar medium with modified sugar composition were fermented with L. plantarum RM71 on quercetin. Quercetin improved several key fermentation traits for the performance of L. plantarum in food production, including accelerated fermentation of various sugars, and accelerated malolactic fermentation and lactic acid production. Quercetin was not catabolized by L. plantarum in the fermentations, so the antioxidant properties of the flavonol were protected against degradation while the bacterium improved its growth performance.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.