Determination of triterpenic acids in natural and alkaline-treated Greek table olives throughout the fermentation process

Maslinic and oleanolic acids are among the most abundant triterpenic acids found in olive fruits. Both acids are considered to be important bioactive compounds which can confer multiple beneficial health effects to the consumer. In the present work, we have monitored and quantified maslinic and oleanolic acids throughout processing in alkaline-treated green olives (Spanish-style) and in natural green olives of the Conservolea variety that is particularly popular in Greece. Our findings clearly demonstrate that the fast de-bittering process with NaOH treatment in Spanish-style olives has a profound negative effect on the concentration of both acids. This decrease of concentration was more prominent regarding maslinic acid when compared to oleanolic acid. In contrast, the slow de-bittering during natural fermentation of green olives had no effect on the content of maslinic or oleanolic acid. To verify the broad applicability of our observation we have also looked into the natural processing of the Kalamon variety (Greek-style). Our findings were consistent, since once again, natural fermentation did not influence the concentration of both acids.     

Modelling the inhibition of sorbic and benzoic acids on a native yeast cocktail from table olives

The single and combined effects, in a synthetic medium at selected pH values, of sorbic and benzoic acids on a yeast cocktail (Saccharomyces cerevisiae, Pichia anomala, Issatchenkia occidentalis, and Candida diddensiae, isolated from table olives) have been studied. Applying the checkerboard method the minimum inhibitory concentration (MIC) obtained for the respective individual preservatives (expressed as undissociated acid) were: sorbic acid, 5.94, 3.85 and 3.19 mM at pH of 4.5, 4.0, and 3.5, respectively; and benzoic acid, not detected (at total 20.5 mM), 10.40 and 6.83 mM, respectively, for the same pH levels. The estimated fractional inhibitory concentrations (FIC) indexes showed additive effects between inhibitors. Fractional area (fa), modelled by the (extended) Lambert and Lambert [2003. A model for the efficacy of combined inhibitors. J. Appl. Microbiol. 95, 734-743] equation (ELPM), also showed additives of both preservatives but different shapes in the dose-response curves; the individual MIC (as undissociated acid) deduced from this method were: 5.60, 3.31, and 3.26 mM for sorbic acid at pH of 4.5, 4.0, and 3.5, respectively; and 29.65 (extrapolated), 10.00, and 6.25 mM for benzoic acid at the same pH levels. Mixtures above the curves connecting the limits (MIC) at each pH were also inhibitory. There was agreement between MIC values from FIC and ELPM, although the last one provided further information on the inhibition behaviour. I. occidentalis was the most resistant yeast of the cocktail to sorbic and benzoic acids.     

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.     

Effect of storage temperature and gas permeability of packaging film on the growth of lactic acid bacteria and Brochothrix thermosphacta in cooked meat emulsions

The effect of gas permeability of packaging film on the growth of lactic acid bacteria and Brochothrix thermosphacta in cooked meat emulsions stored at 0, 8 and 15 °C was investigated. The estimated parameters from Gompertz equation for the assayed temperature–oxygen permeability combinations showed LAB development to be significantly greater than those of B. thermosphacta. The influence of the two sources of variation (oxygen permeability of packaging film and temperature) on the growth parameters of LAB and B. thermosphacta was analysed showing a significant effect (P<0.001) of the temperature on both bacterial population while the film permeability had only a significant influence (P<0.001) on B. thermosphacta growth. Under the conditions of this study the packaging film influenced the maximum counts and growth rates of both organisms. Since the inhibition of B. thermosphacta occurred when the meat product was vacuum-packaged in films possessing high oxygen permeability and the effect of pH was found not to be associated with the growth inhibition, accumulation of hydrogen peroxide produced by LAB may possibly be one of the main factors responsible for B. thermosphacta inhibition. Shelf-life of vacuum-packaged cooked meat emulsions in high oxygen transmission rate films will be guarantied and a temperature abuse will not result in an increase of spoilage by LAB.     

Effect of post-fermentation storage on Spanish-style green Manzanilla olives

The work studies the physicochemical, microbiological, and sensory changes which take place during the post-fermentation storage phase of Spanish-style green Manzanilla olives, achieved mainly by increasing NaCl to up to ∼90 g/L. The storage caused: i) an overall significant titratable acidity decrease and combined acidity increase, ii) a degradation of the texture of the fruits, iii) a considerable decrease in the lactic acid bacteria population, and iv) non significant changes in the yeast population. The later packaging of olives improved the colour index of the fruits, but did not prevent texture degradation. The multivariate statistical analysis of their sensory scores showed that the fermentation profiles of olives may persist even after the post-fermentation storage.     

Effects of ascorbic acid, sodium metabisulfite and sodium chloride on freshness retention and microbial growth during the storage of Manzanilla-Aloreña cracked table olives

The effect of the mixture of sodium metabisulfite (SM), ascorbic acid (AA), and NaCl on the surface colour of fruits and microbial growth during the storage of cracked Manzanilla-Aloreña table olives was studied. During storage, the green colour of fruits, measured by the hue angle (tang⁻¹(b/a)), decreased rapidly, indicating a degradation in the green and a change towards more reddish tones. The degreening rate (k_c) was lower for the highest AA concentration, while the highest final hue angle was found for the highest SM level, indicating different antioxidant mechanisms. Enterobacteriaceae were not enumerated after 20 days of brining. AA, SM, and NaCl, within the concentrations essayed, did not affect yeast growth; however, lactic acid bacteria (LAB) were partially inhibited by the presence of SM and AA, showing the highest growth rate in the absence of both. The maximum population of LAB was reached at levels of SM below 0.75 g/l, intermediate AA (7.5 g/l) and NaCl (70 g/l) concentrations. Appropriate storage conditions could be obtained at the maximum levels of AA (15.0 g/l) and SM (1.5 g/l) and the lowest level of NaCl (60 g/l).     

Modeling the growth of lactic acid bacteria in sliced ham processed by high hydrostatic pressure

The main responsible for the spoilage of cooked cured meat products stored under refrigerated and anaerobic conditions are lactic acid bacteria. The application of high hydrostatic pressure (HHP) reduces the lactic acid bacterial growth extending the product shelf-life and preserving natural taste, texture, color and vitamin content. This work studied the influence of pressure level and holding time on the lactic acid bacterial growth in vacuum-packaged sliced ham. Modified Gompertz and Logistic models were used to fit experimental data obtained from post-treatment microbial counts carried out along the product storage. Samples of sliced vacuum-packaged ham treated by HHP and control samples (non-treated) were stored at 8 °C until the microorganism population reached 10⁷ CFU/g. An experimental planning 2² with triplicate at the central point was designed to determine the influence of pressure level (200, 300, and 400 MPa) and holding time (5, 10, and 15 min) on the product shelf-life. The results have shown that the pressure intensity and the holding time significantly influenced microbial population over the product storage. Shelf-life of ham treated at 400 MPa for 15 min was extended from 19 (control samples) to 85 days.     

The sourdough microflora Microbiological, biochemical and breadmaking characteristics of doughs fermented with freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters

Freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters made withLactobacillus sanfrancisco CBI,L. plantarum DC400 andSaccharomyces cerevisiae 141 and/orS. exiguus M14 were used for leavening wheat doughs, and their microbiological, biochemical and breadmaking characteristics were compared with those of Italian traditional doughs produced by baker's yeast. All the doughs fermented with starters had more balanced microbiological and biochemical characteristics than dough started with baker's yeast in which alcoholic fermentation end-products largely predominated. By using starters, the greatest lactic acid bacteria cell number and acetic acid production, were achieved, along with more complete profiles of volatile compounds and greater structural stability of fermented doughs. Fresh-cell starters showed higher microbial functionality and represented the only way to enrich the doughs withS. exiguus M14, some of which survived the freeze-drying process. No differences were detected between the two different types of freeze-dried starters and the subsequent use (10 times) of doughs initially produced with freezedried starters eliminated initial differences in the microbial functionality with respect to fresh-cell starters.     

Isolation and characterization of the microbial population of different South African kefir grains

Kefir, a slightly acidic fermented milk, is produced by adding lactic acid bacteria and yeasts, in the form of grains, to milk. The bacteria and yeasts present in the kefir grains are known to vary widely. Selective growth media and morphological and biochemical characteristics were used for the isolation and identification of the microbes present in the grains from eight different sources in South Africa. The kefir grains were activated in milk for only 24 h to prevent any changes in the microbial population of the grains. The microbial numbers varied between 6.4 × 10⁴ and 8.5 × 10 ⁸  cfu/g on the media selective for the bacterial species and between 1.5 × 10 ⁵ and 3.7 × 10 ⁸   cfu/g on the media selective for the yeast species. The bacterial genera that were identified included Lactobacillus , Leuconostoc and Lactococcus and the yeast genera included Zygosaccharomyces , Candida and Saccharomyces . The distribution frequencies of the microbes in the different grains were determined and most of the grains were dominated by two microbial species. No pediococci, acetic acid bacteria or propionibacteria were detected.     

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 °C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 °C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability.     

培养温度pH值对保加利亚杆菌和嗜热链球菌混合生长的影响

本文研究了培养温度（３８℃、４１℃、４４℃）及控制ＰＨ值（５－５．５、６．０－６．５）对保加利亚杆菌嗜热链球菌混合培养的影响。结果表明，较高温度（４４℃）有利于中利亚杆菌生长，且混合培养的总菌数多；较低ＰＨ值（５．０－５．５）对嗜热链球菌有一定抑制作用，且混合培养的总功数低于ＰＨ值６．０－６．５或对照组。因此，高温度（４４℃）、低ＰＨ值（５．０－５．５）可以促进混合培养中保加利亚杆菌和嗜热链球菌快     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

Assessment of the probiotic potential of lactic acid bacteria isolated from Minas artisanal cheese produced in the Campo das Vertentes region,Brazil

The microbiota of the Brazilian Minas artisanal cheese, made from raw milk, is not well known and may include probiotic bacteria. This study aimed to assess the in vitro and in vivo probiotic properties of the lactic acid bacteria isolated from these cheeses. Thirty‐six samples of the Lactobacillus/Pediococcus group were selected for in vitro investigation. Pediococcus acidilactici (PA2) and Lactobacillus plantarum (LP4) showed the best results and were tested for their ability to protect Salmonella Typhimurium orally infected mice. LP4 showed better probiotic potential than PA2 and allowed higher values of weight gain (P < 0.05). Thus, Lb. plantarumLP4 may have potential use as a probiotic in foods after future technological screening.     

Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by culture-dependent techniques and PCR-DGGE

The microbial ecology of 22 samples of commercially available Ciauscolo salami were investigated using a polyphasic approach, based on culture-dependent and -independent techniques. The viable counts of pathogen and hygiene indicator microorganisms highlighted the adequate application of good manufacturing practices, while the viable counts of the lactic acid bacteria, coagulase negative cocci, and yeasts showed dominance of the first of these microbial groups. Bacterial and fungal DNA were extracted directly from the salami and amplified by PCR, using two primer sets targeting the 16S and 28S rRNA genes, respectively. Denaturing gradient gel electrophoresis (DGGE) and sequencing of selected bands were used to investigate the microbial ecology of these Ciauscolo salami. The most frequently found bacterial species were Lactobacillus sakei and Lb. curvatus, while Debaryomyces hansenii was the prevalent yeast species detected. Cluster analysis of the DGGE profiles and calculation of biodiversity indices allowed the degree of microbial similarity across these salami to be determined.     

Effects of pectinolytic yeast on the microbial composition and spoilage of olive fermentations

This study resulted in the identification of pectinolytic yeasts in directly brined Sicilian-style green olive fermentations and examination of the influence of those yeasts on the microbial composition and quality of fermented olives. Firstly, defective olives processed in Northern California from 2007 to 2008 and characterized by high levels of mesocarp tissue degradation were found to contain distinct yeast and bacterial populations according to DNA sequence-based analyses. Strains of (pectinolytic) Saccharomyces cerevisiae, Pichia manshurica, Pichia kudriavzevii, and Candida boidinii isolated from directly brined olives were then inoculated into laboratory-scale olive fermentations to quantify the effects of individual yeast strains on the olives. The pH, titratable acidity, and numbers of lactic acid bacteria (LAB) and yeasts varied between the fermentations and fermentations inoculated with P. kudriavzevii and C. boidinii promoted the development of LAB populations. Olive tissue structural integrity declined significantly within 30, 74, and 192 days after the inoculation of pectinolytic S. cerevisiae, P. manshurica and C. boidinii, respectively. In comparison, tissue integrity of olives in control fermentations remained intact although pectinolytic yeasts were present. Notably, pectinolytic yeasts were not found in fermentations inoculated with (non-pectinolytic) P. kudriavzevii and olives exposed to a 1:1 ratio of P. kudriavzevii and P. manshurica exhibited no significant tissue defects. This study showed that pectinolytic yeast are important components of directly brined green olive fermentations and damage caused by pectinolytic yeasts might be prevented by other microbial colonists of the olives.     

Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava and use of competitive strains as starters

The adaptability of lactic acid bacteria (LAB) and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava was investigated using PCR-DGGE and bacteriological culture combined with rRNA gene sequence analysis. Sourdoughs were prepared either from flours of the cereals wheat, rye, oat, barley, rice, maize, and millet, or from the pseudocereals amaranth, quinoa, and buckwheat, or from cassava, using a starter consisting of various species of LAB and yeasts. Doughs were propagated until a stable microbiota was established. The dominant LAB and yeast species were Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus spicheri, Issatchenkia orientalis and Saccharomyces cerevisiae. The proportion of the species within the microbiota varied. L. paralimentarius dominated in the pseudocereal sourdoughs, L. fermentum, L. plantarum and L. spicheri in the cassava sourdough, and L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs. S. cerevisiae constituted the dominating yeast, except for quinoa sourdough, where I. orientalis also reached similar counts, and buckwheat and oat sourdoughs, where no yeasts could be detected. To assess the usefulness of competitive LAB and yeasts as starters, the fermentations were repeated using flours from rice, maize, millet and the pseudocereals, and by starting the dough fermentation with selected dominant strains. At the end of fermentation, most of starter strains belonged to the dominating microbiota. For the rice, millet and quinoa sourdoughs the species composition was similar to that of the prior fermentation, whereas in the other sourdoughs, the composition differed.     

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.     

Inhibitory effects of lactic acid and lauricidin on spoilage organisms of chicken breast during storage at chilled temperature

Different concentrations of lauricidin (LU, containing 1% lactic acid) and lactic acid alone (LA) were evaluated for their effectiveness in reducing naturally occurring microflora of raw chicken breasts. Chicken breasts were dipped in 0 (control), 0.5, 1.0, 1.5, and 2.0% solutions of LU (w/v) or LA (v/v) for 10, 20, and 30 min and stored at 4 °C for 14 d. Total Plate Counts (TPC) and populations of Pseudomonas spp. and Enterobacteriaceae were determined before and after dipping and after storing for 1, 3, 7, 10, and 14 d. Additionally, Hunter L, a, and b values and pH of the chicken breast were also determined. From the obtained results, TPC on chicken breast treated with LU was found to be decreased by 0.92 to 1.2 log CFU/g from a mean initial log 5.69 CFU/g, while those dipped in LA decreased by 0.53 to 2.36 log CFU/g. Pseudomonas population on chicken breast dipped in LU decreased by 0.79 to 1.77 log CFU/g from an initial 3.90 log CFU/g, while in LA treated it decreased by 0.39 to 1.82 log CFU/g. Enterobacteriaceae counts were also found to be reduced by 0.14 to 1.14 log CFU/g on chicken breast dipped in LU, while the reduction was from 0.59 to 2.18 log CFU/g in chicken breast dipped in LA. The major bacterial types isolated from LU treated chicken breast belonged to the Enterobacteriaceae group, which included: Enterobacter, E. coli and Citrobacter. Whereas, in the LA treated breast it belonged to: Pseudomonas, E. coli, and Kocuria rhizophila (formerly Micrococcus luteus). Dipping chicken breast in LU and LA caused a significant decrease (p ≤ 0.05) in their pH values. Also, treatment with LU and LA caused a slight darkening in color (decreased Hunter L value), increase in redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Based on the results obtained in the present study, Lactic acid and Lauricidin showed high potential to be used as a sanitizer in reducing the population of spoilage microorganisms naturally occurring on raw chicken, and can be explored commercially for extension of their shelf life.