Novel bifidobacteria strains isolated from nonconventional sources. Technological, antimicrobial and biological characterization for their use as probiotics.

AIM: To characterize four novel autochthonous bifidobacteria isolated from monkey faeces and a Bifidobacterium lactis strain isolated from chicken faeces by evaluating their technological and biological/functional potential to be used as probiotics. Different stressors, including food process parameters and storage, can affect their viability and functionality. METHODS AND RESULTS: The resistance to frozen storage, tolerance to lyophilization and viability during storage, thermal, acidic and simulated gastric resistance, surface hydrophobicity and antimicrobial activity against pathogens were studied. Bifidobacterium lactis Bb12 and INL1 were used as reference strains. The results obtained demonstrated that the new isolates presented strain-dependent behaviour. Good results were obtained for thermal resistance, frozen storage at -80°C and lyophilized powders maintained at 5°C. Cell viability during refrigerated storage was higher when the strains were resuspended in milk at pH 5·0 than at 4·5. The surface hydrophobicity ranged between 7 and 98% depending on the strain. The simulated gastric resistance was improved for the strains incorporated in cheese. Regarding antimicrobial activity, bifidobacteria isolated from monkey presented higher inhibitory capacity than the reference strains. CONCLUSION: This research provides a deeper insight into new strains of bifidobacteria isolated from primates and chicken that have not been previously characterized for their potential use in dairy products and confirm the most robust stress tolerance of B. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of expanding the available bifidobacteria with the potential to be added to a probiotic food necessarily implies characterizing them from different points of view, especially when considering unknown species. For monkey isolates (which showed higher antimicrobial activity against pathogens), more in-depth knowledge is needed before applying strategies to improve their performance. On the contrary, the chicken isolate B. lactis P32/1 showed similar behaviour to the references B. lactis strains; therefore, it could be considered as a potential probiotic candidate.

Transcriptomic approach and membrane fatty acid analysis to study the response mechanisms of Escherichia coli to thyme essential oil, carvacrol, 2-(E)-hexanal and citral exposure.

AIMS: The application of essential oils (EOs) and their components as food preservatives is promising but requires a deeper understanding of their mechanisms of action. This study aims to evaluate the effects of thyme EO, carvacrol, citral and 2-(E)-hexenal, on whole-genome gene expression (the transcriptome), as well as the fatty acid (FA) composition of the cell membranes of Escherichia coli K12. METHODS AND RESULTS: Therefore, we studied the response against 1 h of exposure to sublethal concentrations of natural antimicrobials, of exponentially growing E. coli K12, using DNA microarray technology and a gas chromatographic method. The results show that treatment with a sublethal concentration of the antimicrobials strongly affects global gene expression in E. coli for all antimicrobials used. Major changes in the expression of genes involved in metabolic pathways as well as in FA biosynthesis and protection against oxidative stress were evidenced. Moreover, the sublethal treatments resulted in increased levels of unsaturated and cyclic FAs as well as an increase in the chain length compared to the controls. CONCLUSIONS: The down-regulation of genes involved in aerobic metabolism indicates a shift from respiration to fermentative growth. Moreover, the results obtained suggest that the cytoplasmic membrane of E. coli is the major cellular target of EOs and their components. In addition, the key role of membrane unsaturated FAs in the response mechanisms of E. coli to natural antimicrobials has been confirmed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The transcriptomic data obtained signify a further step to understand the mechanisms of action of natural antimicrobials also when sublethal concentrations and short-term exposure. In addition, this research goes in deep correlating the transcriptomic modification with the changes in E. coli FA composition of cell membrane identified as the main target of the natural antimicrobials.

Effect of a sublethal high-pressure homogenization treatment on the fatty acid membrane composition of probiotic lactobacilli.

UNLABELLED: High-pressure homogenization (HPH) has been proposed to be applied directly to lactic acid bacterial cells at sublethal levels to enhance some functional properties. As the principal target of HPH are the cell surface envelope structures, the aim of this work was to study the effect of a HPH treatment, applied at 50 MPa, on cell membrane stress responses of already-known functional strains, isolated from Argentinean products. Specifically, the membrane fatty acid composition of cells before and after the sublethal treatment was investigated, and the results showed that plasma membranes, their level of unsaturation and their composition are involved in response mechanisms adopted by microbial cells when subjected to a sublethal HPH stress. In fact, the data obtained demonstrated that the treatment was able to modify the fatty acid profile of the different strains, although a uniform response was not observed. Further studies are necessary both to elucidate the role of each fatty acid in the cell response mechanisms and to clarify the changes in membrane compositions induced by HPH treatment also in relation to the applicative potential of this technique. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to understand the response mechanisms activated in cells exposed to pressure stress. It has been demonstrated that high-pressure homogenization (HPH) treatments, conducted at sublethal levels, could increase some important functional and technological characteristics of nonintestinal probiotic strains. The findings of this paper can contribute to elucidate the mechanisms through which these treatments can modify these strain probiotic properties that are related to outermost cell structures, also principal target of HPH.

Resistance of two temperate Lactobacillus paracasei bacteriophages to high pressure homogenization, thermal treatments and chemical biocides of industrial application.

Temperate bacteriophages ф iLp84 and ф iLp1308, previously isolated from mitomycin C-induction of Lactobacillus paracasei strains 84 and CNRZ1308, respectively, were tested for their resistance to several physical and chemical treatments applied in dairy industry. Long-term survival at 4 °C, -20 °C and -80 °C, resistance to either thermal treatments of 63 °C, 72 °C and 90 °C, high pressure homogenization (HPH, 100 MPa) or classic (ethanol, sodium hypochlorite and peracetic acid) and new commercial sanitizers, namely A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid), were determined. Phages were almost completely inactivated after eight months of storage at 25 °C, but viability was not affected at 4 °C, -20 °C or -80 °C. Both phages tolerated well HPH treatments. Phage iLp1308 showed higher thermal resistance than ф iLp84, but neither resisted 90 °C for 2 min. Best chemical inactivation was accomplished using peracetic acid or biocides A, C and E, whereas biocides B and D were completely ineffective. These results help to improve selection of chemical agents and physical treatments to effectively fight against phage infections in dairy plants.

Probiotic Crescenza cheese containing Lactobacillus casei and Lactobacillus acidophilus manufactured with high-pressure homogenized milk.

High-pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment of food preservation and diversification. Its effectiveness on the deactivation of pathogenic and spoilage microorganisms in model systems and real food is well documented. To evaluate the potential of milk treated by HPH for the production of Crescenza cheese with commercial probiotic lactobacilli added, 4 types of cheeses were made: HPH (from HPH-treated milk), P (from pasteurized milk), HPH-P (HPH-treated milk plus probiotics), and P-P (pasteurized milk plus probiotics) cheeses. A strain of Streptococcus thermophilus was used as starter culture for cheese production. Compositional, microbiological, physicochemical, and organoleptic analyses were carried out at 1, 5, 8, and 12 d of refrigerated storage (4 degrees C). According to results obtained, no significant differences among the 4 cheese types were observed for gross composition (protein, fat, moisture) and pH. Differently, the HPH treatment of milk increased the cheese yield about 1% and positively affected the viability during the refrigerated storage of the probiotic bacteria. In fact, after 12 d of storage, the Lactobacillus paracasei A13 cell loads were 8 log cfu/ g, whereas Lactobacillus acidophilus H5 exhibited, in P-P cheese, a cell load decrease of about 1 log cfu/g with respect to the HPH-P cheese. The hyperbaric treatment had a significant positive effect on free fatty acids release and cheese proteolysis. Also, probiotic cultures affected proteolytic and lipolytic cheese patterns. No significant differences were found for the sensory descriptors salty and creamy among HPH and P cheeses as well as for acid, piquant, sweet, milky, salty, creamy, and overall acceptance among HPH, HPH-P, and P-P Crescenza cheeses.

Effect of high-pressure homogenization, nonfat milk solids, and milkfat on the technological performance of a functional strain for the production of probiotic fermented milks.

The aim of this research was the evaluation of the effects of milkfat content, nonfat milk solids content, and high-pressure homogenization on 1) fermentation rates of the probiotic strain Lactobacillus paracasei BFE 5264 inoculated in milk; 2) viability loss of this strain during refrigerated storage; and 3) texture parameters, volatile compounds, and sensorial properties of the coagula obtained. The data achieved suggested a very strong effect of the independent variables on the measured attributes of fermented milks. In fact, the coagulation times were significantly affected by pressure and added milkfat, and the rheological parameters of the fermented milk increased with the pressure applied to the milk for added nonfat milk solids concentrations lower than 3%. Moreover, the polynomial models and the relative response surfaces obtained permitted us to identify the levels of the 3 independent variables that minimized the viability loss of the probiotic strain used during refrigerated storage.

Biogenic amines and ethyl carbamate in primitivo wine: survey of their concentrations in commercial products and relationship with the use of malolactic starter.

This research was conducted to determine the biogenic amine (BA) and ethyl carbamate (EC) concentrations in commercial Primitivo wine samples and the influence of the use of malolactic starter culture on concentrations of these potentially hazardous compounds in this wine. One hundred sixty bottles of wine from eight producers in the Apulia region of southern Italy were purchased at retail and analyzed. The most common BAs were histamine, 2-phenylethylamine, tyramine, cadaverine, putrescine, spermine, and spermidine. Putrescine (derived from ornithine) was the most abundant BA in all commercial Primitivo wines (5.41 to 9.51 mg/liter), 2-phenylethylamine was detected in only two commercial wines (at less than 2.12 mg/liter), and histamine was found at concentrations of 1.49 to 16.34 mg/liter. The concentration of EC in commercial Primitivo wine was 6.81 to 15.62 ppb, which is not considered dangerous for human health. Malolactic fermentation (MLF) affected the concentrations of BAs and EC differently. For EC, no significant differences were detected between samples of wine produced by spontaneous fermentation and wine that was inoculated malolactic starter. Mean EC concentrations were 12 and 14 ppb in two batches tested (always 18 ppb or less), regardless of whether the malolactic starter was added. Although present at trace levels in wine before the MLF, histamine accumulated during the MLF process, regardless of whether the malolactic starter was added. However, the increase in histamine was higher in wines without the malolactic starter. The concentrations of putrescine and cadaverine increased after MLF, especially in the wine that spontaneously fermented. The use of a selected malolactic starter resulted in reductions in BA concentrations in wine produced by this guided MLF compared with wine produced by spontaneous MLF.

High pressure homogenization versus heat treatment: effect on survival, growth, and metabolism of dairy Leuconostoc strains.

The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product.

Antimicrobial efficacy of citron essential oil on spoilage and pathogenic microorganisms in fruit-based salads.

The aim of this study was the evaluation of the effects of pure citral and citron essential oil on microbial spoilage and growth and survival of pathogenic microorganisms during storage. They were added in the syrup of industrial ready-to-eat fruit salads stored at 9 degrees C. Both citral (25 to 125 ppm) and citron essential oil (300, 600, 900 ppm) were able to prolong the microbial shelf life of the fruit-based salads. The essential oil gave excellent results, avoiding the undesirable effects attributable to the cytotoxicity of citral. Citron essential oil doubled the time needed for the wild microflora to reach concentrations able to produce a perceivable spoilage in condition of thermal abuse (9 degrees C). The same essential oil had reduced effects on the survival of Gram-negative species Salmonella Enteritidis and Escherichia coli, but showed a strong inhibition toward the Gram-positive pathogen Listeria monocytogenes.