Establishment of conditions for green table olive fermentation at low temperature

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

Preparation of a Lactobacillus plantarum starter culture for cucumber fermentations that can meet kosher guidelines

A method is described for growth of a Lactobacillus plantarum starter culture in jars of commercially available pasteurized fresh-pack kosher dill cucumbers so that jars can be used to inoculate commercial scale cucumber fermentation tanks. A procedure is also described to transfer lactic acid bacteria from frozen storage in MRS broth into cucumber juice and commercial jars of kosher dill cucumbers so that a selected strain of lactic acid bacteria can be kosher certified for commercial fermentations in processing plants that operate under kosher certification. The strain of L. plantarum used in these experiments grew to maximum cell numbers in 4 d at 20 to 25 °C and then maintained viable cell numbers for 2 wk at >10(8) CFU/mL so the culture was suitable for inoculation of fermentation tanks. Refrigeration of jars of culture after they grow to maximum numbers minimizes die-off of cells sufficiently so that a pure culture can be maintained by aseptically transferring brine containing viable bacteria to a new pH-adjusted jar only once every 4 mo. PRACTICAL APPLICATION: This report describes a method to prepare a lactic acid bacteria starter culture suitable for kosher vegetable fermentations.     

Evaluation of malolactic-deficient strains of Lactobacillus plantarum for use in cucumber fermentations

In the fermentation of cucumbers, naturally occurring strains of Lactobacillus plantarum decarboxylate malic acid (MDC⁺) to form lactic acid and CO₂. Since CO₂ buildup in the brine contributes to bloater damage of the cucumbers, it is desirable to have strains of L. plantarum that do not decarboxylate malic acid (MDC^-1). Two MDC^- mutants, obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis of different MDC⁺ strains, and their parent strains of L. plantarum wre evaluated in laboratory fermentations of filter-sterilized cucumber juice and of whole cucumbers as to growth rate, end-products, residual malic acid, and competitiveness with the natural flora. Effects of temperature (15 to 40°C) and NaCl concentration (0-6%) on growth in cucumber juice were determined. One MDC^-1 strain, designated MOP3-M6, was selected for further development because of its relative dominance in cucumber ferementations, high residual malic acid concentration after fermentation and greater salt tolerance as compared to its closest rival mutant culture. Growth lag and generation times averaged 1·6 and 1·2 times greater, respectively, for the MOP3-M6 mutant than its parent. However, this mutant may still have application as a starter culture for cucumber fermentation, particularly under relatively aseptic conditions.     

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.     

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.     

Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chloride salt mixtures

NaCl plays an important role in table olive processing affecting the flavour and microbiological stability of the final product. However, consumers demand foods low in sodium, which makes necessary to decrease levels of this mineral in fruits. In this work, the effects of diverse mixtures of NaCl, CaCl₂ and KCl on the fermentation profiles of cracked directly brined Manzanilla-Aloreña olives, were studied by means of response surface methodology based in a simplex lattice mixture design with constrains. All salt combinations led to lactic acid processes. The growth of Enterobacteriaceae populations was always limited and partially inhibited by the presence of CaCl₂. Only time to reach half maximum populations and decline rates of yeasts, which were higher as concentrations of NaCl or KCl increased, were affected, and correspondingly modelled, as a function of salt mixtures. However, lactic acid bacteria growth parameters could not be related to initial environmental conditions. They had a longer lag phase, slower growth and higher population levels than yeasts. Overall, the presence of CaCl₂ led to a slower Enterobacteriaceae and lactic acid bacteria growth than the traditional NaCl brine but to higher yeast activity. The presence of CaCl₂ in the fermentation brines also led to higher water activity, lower pH and combined acidity as well as a faster acidification while NaCl and KCl had fairly similar behaviours. Apparently, NaCl may be substituted in diverse proportions with KCl or CaCl₂ without substantially disturbing water activity or the usual fermentation profiles while producing olives with lower salt content.     

植物乳杆菌-酿酒酵母混合预发酵对鲜湿面理化性质、贮藏特性及风味品质的影响

研究酿酒酵母、植物乳杆菌不同混合发酵方案对鲜湿面感官品质、质构特性、贮藏特性和风味品质的影响。将老面团中筛选的酿酒酵母和植物乳杆菌制成发酵菌剂,接种于面团中进行发酵用以生产鲜湿面,结果表明：混合发酵鲜湿面感官评分高于单一菌种发酵,酿酒酵母发酵可以改善鲜湿面的硬度和弹性,酿酒酵母、植物乳杆菌和混合发酵都可以有效提高鲜湿面中蛋白质含量,混合发酵组蛋白质质量分数达到（7.82±0.36）%;在120℃处理后,酿酒酵母发酵和混合发酵组鲜湿面质构品质和感官评分得到提高,且贮藏期内植物乳杆菌发酵、酿酒酵母发酵、混合发酵组的老化焓ΔH相比对照组出现下降,植物乳杆菌发酵和混合发酵组的色泽相比于对照组更亮（L*值更大）;相比对照组中的40种香气成分,植物乳杆菌发酵组检测到48种香气成分,酿酒酵母发酵组检测到49种香气成分,混合发酵组检测到53种香气成分,醛类、酯类、酸类为鲜湿面条的主要风味物质,发酵组3类物质相对含量都出现明显提高,说明混合发酵可以促进风味物质的产生。主成分分析表明,各组分香气成分具有明显区别。     

蓝莓汁乳酸菌的发酵特性

蓝莓汁中分别接种干酪乳杆菌、保加利亚乳杆菌、植物乳杆菌和嗜酸乳杆菌进行发酵,比较蓝莓汁发酵过程中乳酸菌活菌数、p H值、总滴定酸、总糖、还原糖、总花色苷、总酚、抗氧化能力和色泽等变化。结果表明,蓝莓汁营养丰富,上述各种乳酸菌均能在蓝莓汁中正常生长,发酵24 h,活菌数达8.10 lg cfu/mL以上;此外,植物乳杆菌和嗜酸乳杆菌产酸能力均强于其它两种乳酸菌,可滴定酸含量均超过10.29 g/L,而植物乳杆菌对蓝莓汁中糖的消耗能力最强,总糖的残留量为40.26%;在花色苷和总酚的保留率以及抗氧化能力等方面,植物乳杆菌和嗜酸乳杆菌也要优于其它两种乳酸菌。植物乳杆菌与嗜酸乳杆菌单独发酵和混合发酵时蓝莓汁营养品质的差异分析表明,单独发酵组与混合发酵组在抗氧化能力和总酚的保留上无明显差异,但混合发酵组在花色苷和色泽的保留方面明显优于单独发酵组。综上所述,蓝莓汁应以植物乳杆菌与嗜酸乳杆菌混合发酵为宜。     

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.     

植物乳杆菌和酿酒酵母发酵菠萝汁的性能比较及产物分析

采用植物乳杆菌（Lactobacillus plantarum）和酿酒酵母（Saccharomyces cerevisiae）分别进行单菌与混菌发酵菠萝汁,并比较分析不同接菌方式下菠萝汁的发酵性能及发酵体系中的产物。结果表明,相同接种量发酵72 h后,植物乳杆菌单独发酵活菌数可达108 CFU/mL,分别约为酿酒酵母单独发酵和二者混合发酵活菌数的3倍和10倍;植物乳杆菌的接入使发酵液pH降至3.21,而酿酒酵母的接入使菠萝汁中氨基酸态氮降至3.15×10-4 mg/L,糖类物质几乎被消耗殆尽。发酵产物分析结果发现,植物乳杆菌单独发酵产生大量乳酸（每升增加12.02 g/L）和少量乙酸（每升增加0.40 g/L）,影响了口感,而酿酒酵母单独发酵则使菠萝汁中原有的香气物质损失较多（4种）。综合分析,采用二者混合发酵,既可保有菠萝本身的营养物质和特有香气,又可赋予其一定的发酵风味,获得口感良好的菠萝发酵饮品。     

Malic Acid as a Source of Carbon Dioxide in Cucumber Juice Fermentations

The degradation of malic acid to lactic acid and CO₂ during fermentation of cucumber juice was investigated. This malolactic reaction was the major source of CO₂ when cucumber juice was fermented by Lactobacillus plantarum. It may also be an important CO₂ source in controlled cucumber fermentations. In addition to CO₂ production, the degradation of malate served to buffer the fermentation and increase sugar utilization. The pH after 7 days’fermentation was 2.8 when 13 mM malic acid was present in the juice and 4.1 with 135 mM malic acid. In the same fermentations, 52% of the sugars were degraded with the low malic acid concentration while complete sugar utilization was observed with the highest malic acid level.     

A study on the implications of NaCl reduction in the fermentation profile of Conservolea natural black olives

This study examined the impact of different mixtures of NaCl, KCl, and CaCl₂ on the fermentation profiles of Conservolea natural black olives. Five different combinations of chloride salts were investigated, namely (i) 8% NaCl (control treatment), (ii) 4% NaCl and 4% KCl, (iii) 4% NaCl and 4% CaCl₂, (iv) 4% KCl and 4% CaCl₂, and (v) 2.6% NaCl-2.6% KCl-2.6% CaCl₂. The changes in the microbial association (lactic acid bacteria, yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, volatile compounds, and mineral content in olive flesh were analyzed. Results demonstrated that all salt combinations led to vigorous lactic acid processes based on the obtained values of pH (3.9-4.2) and titratable acidity (0.70-0.86 g lactic acid per 100 ml brine). Organoleptic evaluation was a critical factor in the acceptability of the final product. Increasing concentrations of CaCl₂ or a combination of KCl and CaCl₂ rendered the product bitter with low acceptability by the taste panel. Only one combination of chloride salts (4% NaCl and 4% KCl) could finally produce olives with lower sodium content and good organoleptic attributes. The results of this study could be employed by the Greek table olive industry in an attempt to produce natural black olives with less sodium without affecting the traditional taste of fermented olives in order to meet consumers’ demand for low sodium dietary intake.     

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.     

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.     

植物乳杆菌LPb1耐受性及发酵乳制备条件的优化

模拟人体消化道环境,即在人工胃液、肠液的环境下对植物乳杆菌LPb1的耐受性进行研究,结果表明：在37℃条件下,用人工胃液处理3h,收集的菌体再转入人工肠液处理3h,最后测得菌株存活率仍达88.6%。LPb1与一株嗜热链球菌混合发酵脱脂乳,与单菌发酵相比,混合菌产酸效果较好,发酵乳制品具有良好的持水性能,且发酵完成后,4℃冰箱贮存后酸化程度缓慢。通过单因素和响应面分析,混合发酵最佳发酵条件为：接种量4.45%、发酵时间8.53h、发酵温度40.40℃。     

Role of yeasts in table olive production

Table olives are a traditional fermented vegetable of the Mediterranean countries, but their production and consumption are now spread all around the world. Yeasts can play a double role in this food. They are present throughout the fermentative process and it is generally accepted that they can produce compounds with important organoleptic attributes determining the quality and flavour of the final product. However, yeasts can also be spoilage microorganisms in olive fermentation/storage and packing causing gas pockets, swollen containers, cloudy brines and off-flavours and off-odours. Candida boidinii, Debaryomyces hansenii, Pichia anomala, P. membranifaciens, Rhodotorula glutinis and Saccharomyces cerevisiae are species isolated with a high frequency from olive processes. Scarce information is still available about the ecology, biochemistry and molecular biology of these important microorganisms in table olives. A wider knowledge about these aspects could facilitate the possible application of yeasts as a starter culture, due to their ability to produce aromatic compounds, antioxidants, enzymes, and improve the growth of lactic acid bacteria.     

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.     

植物乳杆菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

在高粱汁培养基内同时接种植物乳杆菌（Lactobacillus plantarum）和高产酯酿酒酵母（Saccharomyces cerevisiae）,探究植物乳杆菌及其代谢产物对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。结果表明：植物乳杆菌对高产酯酿酒酵母生长及酒精发酵的影响不大,发酵结束后残糖量均＜5 g/L,乙醇含量为74～78 g/L;植物乳杆菌使高产酯酿酒酵母乙酸乙酯和高级醇产量下降,分别最多下降了15.31%、36.14%;培养基内不同乳酸质量浓度使酿酒酵母乙酸乙酯产量提高,乳酸质量浓度为7.1 g/L时,乙酸乙酯最多提高了39.84%;培养基内乳酸质量浓度在1.8～7.1 g/L时,高产酯酿酒酵母高级醇的产量明显降低,特别是苯乙醇的产量显著下降,下降了25.05%～75.64%。     

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.     

Use of Lactobacillus plantarum and glucose to control the fermentation of "Bella di Cerignola" table olives, a traditional variety of Apulian region (Southern Italy)

The purpose of this study was to evaluate the use of Lactobacillus plantarum, isolated from table olives "Bella di Cerignola," a traditional variety of Apulian region (Southern Italy), as a starter for this kind of food. We focused on the interaction of the starter with the natural occurring microflora, the quantitative/qualitative composition of yeast population, the decrease of pH, and the content of organic acids. After a preliminary characterization, 3 strains of Lb. plantarum, selected for their probiotic and technological performances, were used as a multiple-strain starter and inoculated (approximately 2%) in olives, processed according to Spanish style, brined at 8% and 10% of NaCl and added with 0.5% of glucose. The combination of the starter and glucose assured a correct fermentation course, decreasing the pH up to a safe value (4.3 to 4.5) and controlled the growth of yeasts. The concentrations of both L- and D-lactic acids increased throughout the fermentation, while citric and malic acids (both the isomers D and L) remained at low levels (0.2 to 0.4 g/L). Concerning yeast species, Candida guilliermondii was mainly isolated at the beginning (7 to 14 d), while C. famata prevailed at the end of fermentation. PRACTICAL APPLICATIONS: To the question "How to standardize and maintain quality of "Bella di Cerignola" olives (Southern Italy)" we can suggest the following answer: use Lb. plantarum and a low amount of glucose (0.5%). The result is a decrease of the pH below the safety break point.