Protective role of lactic acid bacteria and yeasts as dietary carcinogen-binding agents - a review.

The importance of food contaminants in the link between diet and cancer has been widely demonstrated. Therefore, different physical and chemical strategies for the control of human exposure to such dietary carcinogens has been explored; however, most of these strategies are complex, costly, and have low efficiency which limited their applications. Hence, microbiological methods have been receiving more attention. Recent in vitro and in vivo studies have indicated that lactic acid bacteria (LAB) and yeast may act as dietary carcinogen-binding agents. This review describes the promising protective role of strains belonging mainly to the Lactobacillus, Bifidobacterium and Saccharomyces genera by acting as dietary carcinogen-binding agents. This property suggests that these microorganisms may have a protective role by reducing the bioaccessibility of dietary carcinogens, thereby decreasing their toxic effects. The mechanisms by which the binding process takes place have not been completely elucidated; thus, the possible underlying mechanisms and factors influencing carcinogens-binding will be addressed.

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes.

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

In Silico Prediction and In Vitro Assessment of Multifunctional Properties of Postbiotics Obtained From Two Probiotic Bacteria.

In this study, a global metabolite profile using Raman spectroscopy analysis was obtained in order to predict, by an in silico prediction of activity spectra for substance approach, the bioactivities of the intracellular content (IC) and cell wall (CW) fractions obtained from Lactobacillus casei CRL 431 and Bacillus coagulans GBI-30 strains. Additionally, multifunctional in vitro bioactivity of IC and CW fractions was also assessed. The metabolite profile revealed a variety of compounds (fatty acids, amino acids, coenzyme, protein, amino sugars), with significant probable activities (Pa > 0.7) as immune-stimulant, anti-inflammatory, neuroprotective, antiproliferative, immunomodulator, and antineoplastic, among others. Moreover, in vitro assays exhibited that both IC and CW fractions presented angiotensin-converting enzyme-inhibitory (> 90%), chelating (> 79%), and antioxidant (ca. 22-57 cellular antioxidant activity units) activities. Our findings based on in silico and in vitro analyses suggest that L. casei CRL 431 and B. coagulans GBI-30 strains appear to be promising sources of postbiotics and may impart health benefits by their multifunctional properties.

Modulatory Effect of the Intracellular Content of Lactobacillus casei CRL 431 Against the Aflatoxin B1-Induced Oxidative Stress in Rats.

It has been recognized that lactic acid bacteria exhibit antioxidant properties, which have been mainly endorsed to the intact viable bacteria. However, recent studies have shown that intracellular content (IC) may also be good sources of antioxidative metabolites, which may potentially contribute to oxidative homeostasis in vivo. Hence, the modulatory effect of the intracellular content of Lactobacillus casei CRL 431 (IC431) on aflatoxin B1 (AFB1)-induced oxidative stress in rats was evaluated on the basis of its influence on hepatic lipid peroxidation (LPO), antioxidant status-antioxidant capacity (TAC), catalase (CAT), and glutathione peroxidase (GPx) activities; and on the oxidative stress index (OSi). Results demonstrated that CAT and GPx activities, and TAC, determined in plasma samples, were significantly (P < 0.05) higher in rats treated with AFB1 plus IC431 (3.98 µM/min/mg protein, 1.88 µM/min/mg protein, and 238.7 µM Trolox equivalent, respectively) than AFB1-treated rats (3.47 µM/min/mg protein, 1.46 µM/min/mg protein, and 179.7 µM Trolox equivalent, respectively). Furthermore, plasma and liver tissue samples from rats treated with AFB1 plus IC431 showed significantly (P < 0.05) lower LPO values (52 and 51%, respectively) and OSi (59 and 51%, respectively) than AFB1-treated rats. Hence, our results proved that the intracellular content of Lact. casei CRL 431 contains metabolites that are capable to modulate the antioxidant defense systems in living organism, which may help to ameliorate the damage associated to AFB1-induced oxidative stress.

Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains.

Milk-derived bioactive peptides with a single activity (e.g., antioxidant, immunomodulatory, or antimicrobial) have been previously well documented; however, few studies describe multifunctional bioactive peptides, which may be preferred over single-activity peptides, as they can simultaneously trigger, modulate, or inhibit multiple physiological pathways. Hence, the aim of this study was to assess the anti-inflammatory, antihemolytic, antioxidant, antimutagenic, and antimicrobial activities of crude extracts (CE) and peptide fractions (<3 and 3-10 kDa) obtained from fermented milks with specific Lactobacillus plantarum strains. Overall, CE showed higher activity than both peptide fractions (<3 and 3-10 kDa) in most of the activities assessed. Furthermore, activity of <3 kDa was generally higher, or at least equal, to the 3 to 10 kDa peptide fractions. In particular, L. plantarum 55 crude extract or their fractions showed the higher anti-inflammatory (723.68-1,759.43µg/mL of diclofenac sodium equivalents), antihemolytic (36.65-74.45% of inhibition), and antioxidant activity [282.8-362.3µmol of Trolox (Sigma-Aldrich, St. Louis, MO) equivalents]. These results provide valuable evidence of multifunctional role of peptides derived of fermented milk by the action of specific L. plantarum strains. Thus, they may be considered for the development of biotechnological products to be used to reduce the risk of disease or to enhance a certain physiological function.

Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system.

The aim of this study was to evaluate the capability of Lactobacillus reuteri NRRL 14171 and Lactobacillus casei Shirota to remove dietary acrylamide (AA) under simulated gastrointestinal conditions using a dynamic system. The effects of different AA levels or bacteria concentration on toxin removal by Lactobacillus strains were assessed. Thereafter, AA-removing capability of bacteria strains under either fasting or postprandial simulated gastrointestinal conditions was evaluated. Commercial potato chips were analyzed for their AA content, and then used as a food model. Average AA content (34,162µg/kg) in potato chips exceeded by ca. 34-fold the indicative values recommended by the EU. Toxin removal ability was dependent on AA content and bacterial cell concentration. A reduction on bacterial viability was observed in the food model and at the end of both digestive processes evaluated. However, bacteria survived in enough concentrations to remove part of the toxin (32-73%). Both bacterial strains were able to remove AA under different simulated gastrointestinal conditions, being L. casei Shirota the most effective (ca. 70% removal). These findings confirmed the risk of potato chips as dietary AA exposure for consumers, and that strains of the genus Lactobacillus could be employed to reduce the bioavailability of dietary AA.

Screening of Lactobacillus strains for their ability to produce conjugated linoleic acid in milk and to adhere to the intestinal tract.

Conjugated linoleic acid (CLA) has been shown to provide beneficial effects on health; however, the amount consumed in food is far from that required for the desired effects. Thus, increasing the CLA content in dairy foods through milk fermentation with specific lactic acid bacteria (LAB) offers an interesting alternative. Moreover, some LAB may be able to adhere to the intestinal mucosa and produce CLA through endogenous synthesis. Therefore, the objective of this study was to screen LAB isolates for their ability to produce CLA in skim milk and in simulated gastrointestinal conditions. Additionally, the ability of selected CLA-producing LAB to adhere to the intestinal mucosa in a murine model was assessed. Results showed that of 13 strains of Lactobacillus tested, only 4 were able to produce CLA in skim milk supplemented with linoleic acid (13.44 ± 0.78 to 50.9 ± 0.26 µg/mL). Furthermore, these 4 Lactobacillus strains were able to survive and produce CLA in simulated gastrointestinal conditions and to adhere to the intestinal mucosa of Wistar rats after 7 d of oral inoculation with fluorescently labeled bacteria. Accordingly, these 4 Lactobacillus strains may be used to manufacture fermented dairy foods to increase CLA content, and consumption of these fermented milks may result in CLA produced endogenously by these LAB.

Preparation of betulinic acid nanoemulsions stabilized by ω-3 enriched phosphatidylcholine.

Bioactive compounds such as ω-3 fatty acids and terpenes, have been associated with beneficial health effects; however, their solubility in the gastrointestinal tract and its bioavailability in the body are low. Nanoemulsions offer a viable alternative to disperse lipophilic compounds and improve their dissolution, permeation, absorption and bioavailability. Enzyme modified phosphatidylcholine (PC) with ω-3 fatty acids was used as emulsifier to stabilize oil-in-water nanoemulsions generated using ultrasound device. These systems were used as carriers of betulinic acid, which has reported anti-carcinogenic activity. Phospholipase-catalyzed modification of PC allowed the incorporation of 50 mol% of ω-3 fatty acids. Formation variables such as oil type and ultrasound amplitude had effects on nanoemulsion characteristics. Incorporation of betulinic acid affected globule size; however, betulinic acid nanoemulsions below 200 nm could be prepared. The conditions under which betulinic acid nanoemulsions were obtained using the modified phosphatidylcholine with the smaller globule size (91 nm) were 10% PC, 25% glycerol, medium chain oil and 30% amplitude for 12 min in the sonicator. Storage temperature had an effect on the stability of the nanoemulsions, at 5°C we observed the smallest growth in globule size. The use of olive oil decreased the globule size growth during storage of the nanoemulsion stabilized with modified phosphatidylcholine, although globule size obtained was greater than 200 nm. Medium pH had a significant effect on the nanoemulsions; alkaline pH values improved storage stability. These results provide useful information for using this type of carrier system on the formulation of products in the pharmaceutical or food industry.

Antifungal activity of lactobacilli and its relationship with 3-phenyllactic acid production.

In this study, 13 lactic acid bacteria (LAB) strains (including 5 Lactobacillus casei, 2 Lactobacillus rhamnosus, 2 Lactobacillus fermentum, 1 Lactobacillus acidophilus, 1 Lactobacillus plantarum, 1 Lactobacillus sakei, and 1 Lactobacillus reuteri species) were assessed for both their antifungal activity against four food spoilage molds (Colletotrichum gloeosporioides, Botrytis cinerea, Penicillium expansum, and Aspergillus flavus) and their capability to produce the novel antimicrobial compound 3-phenyllactic acid (PLA). Results demonstrated that all molds were sensitive to varying degrees to the cell-free supernatants (CFS) from LAB fermentations (p<0.05), with growth inhibitions ranging from 2.65% to 66.82%. The inhibition ability of CFS was not affected by a heating treatment (121°C, 20 min); however, it declined markedly when the pH of CFS was adjusted to 6.5. With the exception of L. plantarum NRRL B-4496 and L. acidophilus ATCC-4495, all other LAB strains produced PLA ranging from 0.021 to 0.275 mM. The high minimum inhibitory concentration for commercial PLA (3.01-36.10mM) suggests that it cannot be considered the only compound related with the antifungal potential of studied LAB and that synergistic effects may exist among other metabolism products.

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 µg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 µg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides.

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 µg/mL). Various ACEI peptides derived from ß-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.

Colletotrichum gloeosporioides growth-no-growth interface after selected microwave treatments.

To study microwave heating for potential postharvest treatments against anthracnose disease, Colletotrichum gloeosporioides growth-no-growth response after selected microwave treatments (2,450 MHz) was fitted by using a logistic regression model. Evaluated variables were power level, exposure time, presence or absence of water in the medium during treatment, and incubation-observation time. Depending on the setting, the applied power ranged from 77.2 to 435.6 W. For the experiments on dry medium (mold spores over filter paper), exposure times were 1, 2, 3, or 4 min, whereas spores dispersed in potato dextrose agar, a wet medium, had exposure times of 3, 6, or 9 s. Growth (response = 1) or no growth (response = 0) was observed after two different incubation-observation times (4 or 10 days). As expected, high power levels and long exposure times resulted in complete inhibition of C. gloeosporioides spore germination. In a number of cases (such as low power levels and short treatment times), only a delay in mold growth was observed. Scanning electron micrographs showed signs of mycelia dehydration and structural collapse in the spores of the studied mold. Cell damage was attributed to heating during microwave exposure. Reduced logistic models included variables and interactions that significantly (P < 0.05) affected mold growth, and were able to predict the growth-no-growth response in at least 83% of the experimental conditions. Microwave treatments (4 min at any of the studied power levels in dry medium, and 9 s at power levels of 30% or more for wet medium) proved effective in the inhibition of C. gloeosporioides in model systems. These no-growth conditions will be tested further on fresh fruits in order to develop feasible postharvest microwave treatments.

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1.

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B1 in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B1 binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB1 binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 microg/mL). In general, the human isolates bound the most aflatoxin B1 and the cheese isolates the least. Stability of the bacterial-aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6-9.2% release) reversible; the L. casei 7R1-aflatoxin B1 complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B1 binding and the differences between the strains was reduced.

Preparation of mono- and diacylglycerols by enzymatic esterification of glycerol with conjugated linoleic acid in hexane.

Esterification of glycerol with conjugated linoleic acid (CLA) was carried out in hexane. Lipase from Rhizomucor miehei provided a high degree of esterification (80%) in 8 h at 50 degrees C when used at 15% (w/w) in a system containing a 1:2 molar ratio of glycerol to free fatty acids. Esterification levels >80% were obtained in 8 h at 40 degrees C with 15% (w/w) lipase from Candida antarctica at the same molar ratio of reactants. The extent of esterification of CLA was >90% after 4 h of reaction at 50 degrees C with a 5% (w/w) loading of either R. miehei or C. antarctica lipase, together with a 1:1 molar ratio of substrates. Both enzymes incorporated the original CLA as acylglycerol residues in primarily 1,3-diacylglycerol and 1-monoacylglycerol. The CLA-rich acylglycerols can be employed as emulsifiers or as substitutes for natural fats and oils.

Potentialization of the lactoperoxidase system for preservation of raw milk in the tropics.

The antimicrobial ability of the lactoperoxidase system was increased by the addition of larger amounts of the thiocyanate and hydrogen peroxide at levels above those suggested by other authors. Results of laboratory and field trials revealed that the potentialized system was able to preserve poor-quality raw milk for longer periods of time, at "tropical" temperatures, than when used as recommended previously. It was possible to preserve some milks at 20 degrees C for more than one day, without diminishing their overall quality. At 36 degrees C, the milks did not show acidity development for about 10 hours. Tests conducted under real collection and transportation conditions validated these findings. It was therefore proved that the system can be used practically and that its bactericidal/bacteriostatic effect on the spoilage flora of milk can be increased in order to overcome the particularly adverse conditions of milk handling in the tropics.

Potentialization of the lactoperoxidase system for preservation of raw milk in the tropics

Se aumentó la capacidad antimicrobiana del sistema lactoperoxidas, mediante la adición de tiocianato y peróxido de hidrógeno en cantidades mayores a las sugeridas por otros autores. Los resultados de laboratorio y las pruebas de campo revelaron que por otros autoes. Los resultados de laboratorio y las pruebas de campo revelaron que el sistema potencializado pudo preservar leches de baja calidad microbiológica, a temperaturas "tropicales" por períodos más largos que al usarlo como se recomienda en la literatura. Se pudo conservar leches a 20-C por más de un día, sin menoscabo de su calidad general. A 36-C, las leches no acusaron desarrollo de acidez durante el término de 10 horas. Las pruebas realizadas en condiciones reales de recolección y transporte validaron los resultados de laboratorio. Se logró así probar que el sistema lactoperoxidasa es viable de uso en la práctica, y que su poder bactericida/bacteriostático sobre la flora deterioradora de la leche puede aumentarse a fin de superar las condiciones especialmente adversas que involucra el menejo de la leche en los trópicos