Using regression and Multifactorial Analysis of Variance to assess the effect of ascorbic, citric, and malic acids on spores and activated spores of Alicyclobacillusacidoterrestris.

The type strain of the species Alicyclobacillus acidoterrestris (DSM 3922) and the strain CB1 (accession number: KP144333) were studied in this research to assess the effects of three weak acids (malic, citric, and ascorbic acids), pH (3 or 4), and spore status (spores, and activated spores). Acids were used to prepare 7 different blends, and the blends used to reduce the pH of Malt Extract broth to 3 and 4; then, media were inoculated with spores or activated spores, stored at 45 °C (optimal temperature for A. acidoterrestris growth), and analyzed immediately and after 2 and 7 days. Data were preliminary standardized as increase/reduction of microbial population, compared to the initial concentration, and modelled through two different statistical approaches (multifactorial ANOVA, and multiple regression). Finally, a binary code (0-no growth or reduction of viable count; 1-growth) was used to perform a multiple regression analysis on the growth probability of A. acidoterrestris. Generally, ascorbic acid was the most effective compound, but other acids (e.g., malic acid) could contribute to increase the inactivation ratio; concerning spore status, the highest sensitivity of activated spores suggests that acids probably act during the outgrowing phase. Finally, the two strains showed different trends at pH 3.0, being the type strain the most resistant one.

Biofilm formation by potentially probiotic Saccharomyces cerevisiae strains.

Four wild strains of Saccharomyces cerevisiae and the collection strain S. cerevisiae var. boulardii ATCC MYA-796 were used as test organisms to study the effect of some environmental conditions on the formation of biofilm by potentially probiotic yeasts. In a first step, the formation of biofilm was studied in four different media (YPD-Yeast Peptone Glucose; diluted YPD; 2% BP, a medium containing only bacteriological peptone; 2% GLC, a medium containing only glucose). Then, the dilution of YPD was combined with pH and temperature through a mixture design to assess the weight of the interaction of the variables; the experiments were done on S. boulardii and on S. cerevisiae strain 4. The dilution of nutrients generally determined an increased biofilm formation, whereas the effect of pH relied upon the strain. For S. cerevisiae strain 4, the highest level of sessile cells was found at pH 4-5, while S. boulardii experienced an enhanced biofilm formation at pH 6.0. Concerning temperature, the highest biofilm formation was found at 25-30 °C for both strains. The importance of this work lies in its extension of our knowledge of the effect of different environmental conditions on biofilm formation by potentially probiotic S. cerevisiae strains, as a better understanding of this trait could be an important screening tool into the selection of new multifunctional yeasts.

A low-power ultrasound attenuation improves the stability of biofilm and hydrophobicity of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 and Acidipropionibacterium jensenii DSM 20535.

The main topic of this paper was to study the effect of ultrasound-attenuation (US) on the surface properties of propionibacteria (Acidipropionibacterium jensenii DSM 20535 and Propionibacterium freudenreichii DSM 20271). A preliminary screening was done by using different power levels (40 and 60%) and treatment times (4, 6, and 8 min); immediately after sonication, acidification and viable count were tested. The best combinations to avoid post-acidification after 6 h were the following: A. jensenii DSM 20535: power, 40%; time, 8 min; P. freudenreichii subsp. freudenreichii DSM 20271: power, 60%; time, 4 min. Moreover, the effect of US on the growth patterns, surface properties (biofilm formation and hydrophobicity), resistance to some selected antibiotics, and release of intracellular components was evaluated; the experiments were done immediately after the treatment. US-treatment improved the stability of biofilm after 5-7 days, caused an increase of hydrophobicity (from 15 to 27%) immediately after sonication, and determined an increase of cell permeability, as suggested by the release of intracellular components within 24 h and by the increased sensitivity to some antibiotics. This paper is the first report on US-attenuation on propionibacteria and could the background for future researches to modulate the surface properties of these microorganisms.

Functional cream cheese supplemented with Bifidobacterium animalis subsp. lactis DSM 10140 and Lactobacillus reuteri DSM 20016 and prebiotics.

The aim of this study was to develop a functional fresh cream cheese with Bifidobacterium animalis subsp. lactis DSM 10140 or Lactobacillus reuteri DSM 20016 and prebiotics (inulin, FOS and lactulose). The research was divided into two steps: in vitro evaluation of the effects of prebiotic compounds; validation at laboratory level with production of functional cream mini-cheeses. Prebiotics showed a protective effect: B. animalis subsp. lactis DSM 10140 cultivability on Petri dishes was positively influenced by lactulose, whereas fructooligosaccharides (FOS) were the prebiotic compounds able to prolong Lb. reuteri DSM 20016 cultivability. At 30 °C, a prolongation of the death time (more than 300 days) was observed, while the controls showed death time values about 100 days. At 45 °C, death time values increased from 32.2 (control) to 33, 35, and 38 days in the samples added with FOS, inulin and lactulose, respectively. Lactulose and FOS were chosen to be added to cream mini-cheeses inoculated with B. animalis subsp. lactis DSM 10140 and Lb. reuteri DSM 20016, respectively; the proposed functional cream cheese resulted in a product with favourable conditions for the viability of both probiotics which maintained cultivable cells above the recommended level during 28 days of storage at 4 °C with good sensory characteristics.

Nonthermal Technologies for Fruit and Vegetable Juices and Beverages: Overview and Advances.

In recent years, there has been a growing interest in the design of novel nonthermal processing systems that minimally modify sensory, nutritional, and functional properties of fruit and vegetable juices and beverages. The benefits of nonthermal treatments are strongly dependent on the food matrix. Thus, an understanding of the effects that these technologies exert on the properties of juices and beverages is important to design and optimize technological parameters to produce value-added products. This review covers research on nonthermal electrical treatments, high pressure processing, ultrasound, radiation processing, inert gas treatments, cold plasma, and membrane processing. Advances towards optimization of processing conditions, and combined technologies approaches have been also extensively reviewed. This information could be useful to: (1) manage processing systems and optimize resources; (2) preserve nutritional value and organoleptic properties, and (3) provide processing conditions for validation of these technologies at the industrial scale.

Autochthonous lactic acid bacteria with probiotic aptitudes as starter cultures for fish-based products.

This study focused on the selection of lactic starters with probiotic properties for the production of fermented fish-products by the use of a multivariate approach (Cluster Analysis and Principal Component Analysis). Seventy-five isolates were recovered from fish intestinal microbiota and characterized by evaluating phenotypical, technological and probiotic traits; the most promising isolates were molecularly identified and then used into fish fermented sausage production. Namely, data from technological characterization were modelled through Growth Index and used as input to run a preliminary selection. Thus, 15 promising strains were selected and subjected to probiotic characterization; considering the results from probiotic tests, 3 promising strains were finally chosen (11, 68 and 69), identified as members of the genus Lactobacillus and used for the validation at laboratory level through the assessment of their performances for the production of fermented fish sausages. The results were promising as the use of the selected strains reduced the fermentation time (2 days) ensuring a good microbiological quality of the final product.

A possible approach to assess acidification of meat starter cultures: a case study from some wild strains of Lactobacillus plantarum.

BACKGROUND: The performances of four autochthonous isolates of Lactobacillus plantarum were assessed to study the most important variables acting on acidification and to propose a possible step-by-step approach for the validation at laboratory scale. This main topic was addressed through three intermediate steps: (1) evaluation of acidification in liquid and solid media, as a function of salt, nitrites, nitrates, lactose, pepper and temperature; (2) assessing acidification in a pork-meat preparation; and (3) designing a protocol to improve the performances at sub-optimal temperatures. The concentration of the ingredients and the temperature were combined through a 3k-p Fractional Factorial Design. Acidification and viable count were assessed and modelled through a multi-factorial ANOVA. RESULTS: In model systems acidification was affected by lactose and was maximum (ΔpH of ca. 2.8-3.0) in the combinations containing 0.4% lactose, 250 mg kg-1 nitrates or 150 mg kg-1 nitrites, 5% salt, and at 30 °C. Solid media caused a higher acidification. In the pork meat preparation, the effect of salt and nitrites was significant. At 10 °C the strains could not reduce pH, but this ability could be induced using an adaptation step. CONCLUSION: Acidification was affected by lactose in the model system, whereas in meat preparation the other variables were significant. In addition, a protocol to improve acidification at 10 °C was optimised. © 2016 Society of Chemical Industry.

Fungal bioremediation of olive mill wastewater: using a multi-step approach to model inhibition or stimulation.

BACKGROUND: Olive mill wastewaters (OMWWs) possess a strong environmental impact; the use of fungi as tools for bioremediation could be a promising method. RESULTS: Twenty-nine fungi were grown on minimal media supplemented with five different kinds of OMWWs (5-15%). Radial growth was assessed for 21 days and the data were modelled through the Dantigny-logistic like function to estimate τ, i.e. the time to attain half of the maximum diameter. Growth on potato dextrose agar and water agar (WA, minimal medium without supplementation) was used as reference. The differences in τ between PDA/WA and minimal media with OMWWs were modelled through a multi-factorial ANOVA, using the concentration of OMWW, the kind of wastes and fungi as categorical predictors. Finally, a principal component analysis was run to group and divide resistant and sensitive fungi. Some fungi experienced a positive Δτ, thus suggesting an inhibition by OMWW, whereas other isolates were enhanced. CONCLUSIONS: Some isolates (for example Aspergillus ochraceus) showed a promising trend and could be possible candidates for a validation on a real scale. © 2016 Society of Chemical Industry.

Thermal Treatments for Fruit and Vegetable Juices and Beverages: A Literature Overview.

Fruit and vegetable juices and beverages are generally preserved by thermal processing, currently being the most cost-effective means ensuring microbial safety and enzyme deactivation. However, thermal treatments may induce several chemical and physical changes that impair the organoleptic properties and may reduce the content or bioavailability of some nutrients; in most cases, these effects are strongly dependent on the food matrix. Moreover, the efficacy of treatments can also be affected by the complexity of the product and microorganisms. This review covers researches on this topic, with a particular emphasis on products derived from different botanical sources. Technologies presented include conventional and alternative thermal treatments. Advances toward hurdle-based technology approaches have been also reviewed.

Using physical approaches for the attenuation of lactic acid bacteria in an organic rice beverage.

A wild strain of Lactobacillus plantarum, isolated from an Italian sourdough, was inoculated in an organic rice drink; however, it caused a strong acidification. Thus, it was preliminary processed through homogenization (single or multiple passes) or sonication (US) and then inoculated in the beverage. The samples were stored at 4 °C and analyzed to assess pH, production of lactic acid, viable count and sensory scores. A US-2-step process (power, 80%) could control acidification; viability and sensory traits were never affected by sonication. This result was confirmed on two commercial probiotics (Lactobacillus casei LC01 and Bifidobacterium animalis subsp. lactis Bb12). In the 2nd step samples inoculated with attenuated strains were also stored under thermal abuse conditions (25 or 37 °C for 4 or 24 h, then at 4 °C) and the results showed that US could control acidification for a short thermal abuse. Finally, US-attenuated starter cultures were inoculated in the rice drink containing ß-glucans as healthy compounds; the targets did not cause any significant change of prebiotic.

Influence of prebiotics on Lactobacillus reuteri death kinetics under sub-optimal temperatures and pH.

Eaten foodstuffs are usually fortified with prebiotic ingredients, such as inulin and oligofructose (FOS). The main goal of this study was to evaluate the combined effects of inulin and FOS with either suboptimal pH or storage temperature on the viability of Lactobacillus reuteri DSM 20016. Data were modeled through Weibull equation for the evaluation of the microbiological shelf life and the survival time. Prebiotics enhanced the microbiological shelf life and enhanced the survival time of the target bacterium. The use of the factorial ANOVA highlighted that inulin and FOS exerted a different effect as a function of pH and temperature. Inulin prolonged survival time under acidic conditions, while the effect of glucose + FOS was significant at pH 8. Finally, temperature could act by increasing or decreasing the effect of prebiotics, as they could exert a protective effect at 30 °C but not at 44 °C. As the main output of this research, we could suggest that the effect of prebiotics on L. reuteri could be significantly affected by pH and temperature, thus pinpointing that the design of a symbiotic food should also rely on these factors.

Spore inactivation and DPA release in Alicyclobacillus acidoterrestris under different stress conditions.

This paper reports on the inactivation of spores of 5 strains of Alicyclobacillus acidoterrestris under different stress conditions (acidic and alkaline pH, high temperature, addition of lysozyme, hydrogen peroxide and p-coumaric acid). The research was divided into two different steps; first, each stress was studied alone, thus pointing out a partial uncoupling between spore inactivation and DPA release, as H2O2 reduced spore level below the detection but it did not cause the release of DPA. A partial correlation was found only for acidic and alkaline pH. 2nd step was focused on the combination of pH, temperature and H2O2 through a factorial design; experiments were performed on both fresh and 4 month-old spores and pinpointed a different trend for DPA release as a function of spore age.

Screening of Propionibacterium spp. for potential probiotic properties.

The main topic of this paper is the evaluation of adhesion of propionibacteria to IPEC-J2 cells and the survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and antibiotic resistance of Propionibacterium freudenreichii subsp. shermanii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium thoenii. Adhesion to IPEC-J2 cell lines was ca. 25-35% and significantly increased with CaCl2. Moreover, propionibacteria showed a reduction of cell count of ca. 0.5% at pH 2.5 after 3 h, whereas cell count increased after 24 h with bile salts; finally, they significantly inhibited Escherichia coli O157:H7.

Use of central composite design in food microbiology: a case study on the effects of secondary phenols on lactic acid bacteria from olives.

This paper focuses on the use of statistical Design of Experiments (DoE) to investigate the effects of two anti-lactic acid bacteria compounds on growth and metabolism of lactobacilli isolated from Italian table olives. p-Coumaric and vanillic acids (0.0-0.4%) were used as phenolic compounds, which were combined with salt (0.0-6.0%) and glucose (0.0-4.0%) through a Central Composite Design. Three strains of Lactobacillus plantarum (5 log cfu/ml) were used as test organisms, samples were stored at 37 °C, and cell counts and pH were evaluated periodically. The growth of lactobacilli was affected in a significant way by salt, p-coumaric and vanillic acids, being the salt the most significant factor after 24 h (short storage time), then replaced by p-coumaric acid. p-Coumaric acid also played a significant role on the acidifying ability, expressed as decrease of pH of the medium: microbial metabolism, in fact, appeared as completely inhibited at 0.2% of p-coumaric acid.

Biotechnological innovations for table olives.

The production of table olives is based on a relatively simple flow chart, including debittering and lactic fermentation. Producers' demand for innovation was the background to design and test some innovative and alternative approaches, i.e. the selection of suitable starter cultures (both lactic acid bacteria and yeasts), the production of functional olives inoculated with probiotic strains, the use of bioremediation (i.e. degradation of oleuropein by bacteria or yeasts), as well as the inoculation of functional starter cultures with a strong biopreservative effect. This article reports a brief description of the most important innovations, with a special focus on the role of traditional and innovative starter cultures.

Selection of autochthonous strains as promising starter cultures for Fior di Latte, a traditional cheese of southern Italy.

BACKGROUND: This paper describes the selection of promising strains intended as starter cultures from the autochthonous lactic acid bacteria of Fior di Latte cheese (Apulia region, southern Italy). RESULTS: Ninety-five isolates were randomly selected from whey and Fior di Latte. After preliminary characterization based upon Gram staining, deamination of arginine, hydrolysis of esculine and production of CO2 from glucose and citrate, the isolates were studied for their growth at different temperatures (10, 15 and 45 °C), with salt addition (20, 40 and 65 g L(-1) ) and at pH 4.4 and their acidification score in MRS broth after 6 and 24 h. Data were modeled through the growth index and used as input to run a preliminary cluster analysis and a principal component analysis. In this way, nine promising strains were selected and used for validation at laboratory level, to study the acidification score in milk and propose some possible microbial mixtures. CONCLUSION: This paper reports the first research focusing on the design of a lactic starter for the production of Fior di Latte cheese, using a quantitative approach based on the evaluation of growth index and acidification score as well as on the use of a multivariate approach to select the most promising nine strains.

Decontamination of ochratoxin A by yeasts: possible approaches and factors leading to toxin removal in wine.

Biological decontamination of mycotoxins using microorganisms is one of the well-known strategies for the management of mycotoxins in foods and feeds. Yeasts are an efficient biosorbant, used in winemaking to reduce the concentration of harmful substances from the must which affect alcoholic fermentation (medium-chain fatty acids) or which affect wine quality in a negative way (ethyl phenols and sulphur products). In recent years, several studies have demonstrated the ability of yeasts to remove ochratoxin A (OTA) by live cells, cell walls and cell wall extracts, yeast lees. In spite of the physical and chemical methods applied to remove the toxin, the biological removal is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food. In addition, adsorption is recognized as economically viable, technically feasible and socially acceptable. This paper intends to review the current achievements of OTA removal mediated by yeasts, the recent updates in the selection of strains acting at the same time as starters and as biological tools to remove OTA and the factors affecting the removal process.

Technological characterization and probiotic traits of yeasts isolated from Altamura sourdough to select promising microorganisms as functional starter cultures for cereal-based products.

The main topic of this research was to select some suitable functional starter cultures for cereal-based food or beverages. This aim was achieved through a step-by step approach focused on the technological characterization, as well as on the evaluation of the probiotic traits of yeasts; the technological characterization relied on the assessment of enzymatic activities (catalase, urease, ß-glucosidase), growth under various conditions (pH, temperature, addition of salt, lactic and acetic acids) and leavening ability. The results of this step were used as input data for a Principal Component Analysis; thus, the most technologically relevant 18 isolates underwent a second selection for their probiotic traits (survival at pH 2.5 and with bile salts added, antibiotic resistance, antimicrobial activity towards foodborne pathogens, hydrophobic properties and biofilm production) and were identified through genotyping. Two isolates (Saccharomyces cerevisiae strain 2 and S. cerevisiae strain 4) were selected and analyzed in the last step for the simulation of the gastric transit; these isolates showed a trend similar to S. cerevisiae var. boulardii ATCC MYA-796, a commercial probiotic yeast used as control.

Study on bioactivity of cell-free filtrates from dairy propionibacteria.

In this paper, the bioactivity of cell-free extracts obtained by dairy propionibacteria strains was investigated. Probiotic bifidobacteria and lactobacilli were used as microbial targets. The extracellular filtrates were added as ingredient (1% v/v) into the growth medium; the effect of cell-free filtrates was evaluated through viable count of microbial targets on appropriate media, monitoring the response of target microorganisms both in growth and death phase. The Gompertz equation was used to model the experimental data. Kinetics and time parameters were estimated in order to quantify the effectiveness of cell-free filtrates effect. To emphasize the results about the bioactivity of cell-free extract, supporting a complete picture of response, a new approach was developed: Probiotic Stability Time was calculated. This temporal parameter, defined as the time over that the cell load preserve a living value upper than 10(7) cfu ml(-1), was very useful to evaluate the probiotic capability and effectiveness. A stimulant effect was registered on growth and a positive one was recordered on survival of both bifidobacteria and lactobacilli strains, and the results obtained suggest that a prebiotic activity by dairy propionibacteria cell-free filtrates could be supposed. The cell-free filtrate obtained from Propionibacterium freudenreichii subsp. shermanii was the most effective, in our experimental conditions. Although bifidobacteria were the most sensitive to the effect of cell-free filtrates, lactobacilli have been showed a similar probiotic stability time, showing a high sensitivity to the filtrates. This paper is the first report of a positive bioactivity by propionibacteria cell-free filtrates on lactobacilli.

Selection of promising lactic acid bacteria as starter cultures for sourdough: using a step-by-step approach through quantitative analyses and statistics.

BACKGROUND: The main goal of this research was to show how to use a qualitative assessment of some technological properties of lactic acid bacteria (LAB), combined with the evaluation of the growth index (GI), to select promising starter cultures for sourdough. RESULTS: Fifty-four strains of LAB were isolated from a single factory, identified by molecular tools and studied for their growth as a function of NaCl (20, 40 and 65 g L(-1)), temperature (45, 15 and 10 °C), pH 9.2 and acidification in MRS broth. The growth was evaluated through absorbance and data were modelled as GI. GIs were used to build frequency histograms and to run a principal component analysis (PCA). In this way, six strains, identified as Lactobacillus plantarum and able to grow in a wide range of conditions (temperature, pH and salt) and/or able to decrease the pH by 1.77-2.0 units, were selected and tested in a model system (flour and water) to study the acidification after 24 h and their viability after 14 days. CONCLUSION: The main result of this paper was to show how a simple step-by-step approach could be a useful tool to select promising starter cultures for sourdough. The method was based on (1) strain identification, (2) assessment of some traits through the GI, combined with simple statistical approaches (frequency histograms and PCA), and (3) preliminary validation in model systems.