Cytotoxic impact of phenolics from Lamiaceae species on human breast cancer cells

The aim of this study was to evaluate the cytotoxicity of dried aqueous extracts from Thymus serpyllum (ExTs), Thymus vulgaris (ExTv), Majorana hortensis (ExMh), and Mentha piperita (ExMp), and the phenolic compounds caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), luteolin-7-O-glucuronide (Lgr), luteolin-7-O-rutinoside (Lr), eriodictiol-7-O-rutinoside (Er), and arbutin (Ab), on two human breast cancer cell lines: Adriamycin-resistant MCF-7/Adr and wild-type MCF-7/wt. In the MTT assay, ExMh showed the highest cytotoxicity, especially against MCF-7/Adr, whereas ExMp was the least toxic; particularly against MCF-7/wt cells. RA and LA exhibited the strongest cytotoxicity against both MCF-7 cell lines, over 2-fold greater than CA and Lgr, around 3-fold greater than Er, and around 4- to 7-fold in comparison with Lr and Ab. Except for Lr and Ab, all other phytochemicals were more toxic against MCF-7/wt, and all extracts exhibited higher toxicity against MCF-7/Adr. It might be concluded that the tested phenolics exhibited more beneficial properties when they were applied in the form of extracts comprising their mixtures.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Bifidobacterium bifidum BF-1 suppresses Helicobacter pylori-induced genes in human epithelial cells

Helicobacter pylori infection alters gene expression in host cells. Specifically, inflammatory chemokines such as IL-8 are upregulated in the gastric mucosa during H. pylori infection. Although the mechanism by which H. pylori causes inflammation of the gastric mucosa is not yet understood, many studies have suggested that nuclear factor kappa B (NF-κB) plays a key regulatory role in host cells. We have shown that preincubation with Bifidobacterium bifidum strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses induction of IL-8 by the pathogen. To investigate how how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been preincubated with BF-1 and infected with H. pylori. We found that preincubation with BF-1 suppresses the expression of H. pylori-induced genes in human cells and that most of the affected genes are related to the NF-κB signaling pathways. These results suggest that BF-1 can affect the regulatory mechanism of the NF-κB signaling pathways.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.     

The methoxyflavones in Citrus reticulata Blanco cv. ponkan and their antiproliferative activity against cancer cells

The major polymethoxyflavones in the fruit (ponkan) peels of Citrus reticulata Blanco cv. ponkan were identified as isosinensetin, sinensetin, nobiletin and tetramethyl-o-scutellarein by a combined separation using high-speed countercurrent chromatography and preparative high performance liquid chromatography, and structure elucidation by electrospray ionisation mass spectrometry (ESI-MS) and ¹H and ¹³C nuclear magnetic resonance (NMR). The antiproliferative activity of the four compounds against four cancer cell lines (A549, HL-60, MCF-7 and HO8910) showed that isosinensetin had a lower IC₅₀ value for MCF-7 and HO8910 cancer cell lines. Determination of polymethoxyflavones in ponkan peels from different cultivation regions displayed relatively steady contents of the four compounds and a higher content of isosinensetin, which suggested that ponkan peels are excellent sources of functional polymethoxyflavones that may help prevent female cancers, such as ovarian cancer and breast cancer.     

Characterization of bacteriocins produced by two strains of Lactobacillus plantarum isolated from Beloura and Chouriço, traditional pork products from Portugal

Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chouriço, respectively, inhibited the growth of a number of Gram-positive and Gram-negative meat spoilage bacteria. According to trycine–SDS–PAGE, bacST202Ch and bacST216Ch are approximately 3.5 and 10.0 kDa in size, respectively. Maximal activity of bacST202Ch (25,600 AU/ml) was recorded after 27 h of and bacST216Ch (102,400 AU/ml) after 22 h of growth. The mode of activity, as determined against Enterococcus faecium HKLHS, is bactericidal. Both peptides adsorb to the surface of the producer cells, but at very low concentrations. Both peptides remained active after 120 min at 100 ^oC and after 2 h of incubation at pH 2.0–12.0. Treatment for 120 min at 121 ^oC did not affect bacST216Ch activity. Activity of bacST202Ch and bacST216Ch was not affected by 1% Triton X-100, Tween 80, Tween 20, SDS, NaCl, urea and EDTA. Bacteriocin ST216Ch was deactivated in the presence of 1% Triton X-114. The nucleotide sequence of a 1044 bp DNA fragment amplified from L. plantarum ST202Ch is identical to the structural gene encoding pediocin PA-1, suggesting that the two bacteriocins are identical. Based on the broad spectrum of antibacterial activity, strains ST202Ch and ST216Ch may be used as starter cultures in the fermentation of meat products.     

Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.     

Characterization of glucan-producing Leuconostoc strains isolated from sourdough

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by ¹H and ¹³C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1 → 2), α-(1 → 3) and α-(1 → 6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)₅ primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1 → 2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.     

Short communication: Conservation of Streptococcus uberis adhesion molecule and the sua gene in strains of Streptococcus uberis isolated from geographically diverse areas

The objective was to identify and sequence the sua gene (GenBank no. DQ232760; http://www.ncbi.nlm.nih.gov/genbank/) and detect Streptococcus uberis adhesion molecule (SUAM) expression by Western blot using serum from naturally S. uberis-infected cows in strains of S. uberis isolated in milk from cows with mastitis from geographically diverse areas of the world. All strains evaluated yielded a 4.4-kb sua-containing PCR fragment that was subsequently sequenced. Deduced SUAM AA sequences from those S. uberis strains evaluated shared >97% identity. The pepSUAM sequence located at the N terminus of SUAM was >99% identical among strains of S. uberis. Streptococcus uberis adhesion molecule expression was detected in all strains of S. uberis tested. These results suggest that sua is ubiquitous among strains of S. uberis isolated from diverse geographic locations and that SUAM is immunogenic.     

Molecular characterization of Prototheca strains isolated from Italian dairy herds

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.     

Antioxidant activity and genotoxic effect on HeLa cells of phenolic compounds from infusions of Quercus resinosa leaves

Water infusions of mature and fresh Quercus resinosa leaves were evaluated for antioxidant activity and genotoxic effects on HeLa cells. Native Mexicans used to drink Q. resinosa leaves tea as a refreshing beverage. The air dried leaves were pulverised and boiled in water, then their phenolic content and condensed tannins were determined. The chromatographic profile of 15 phenolic components in Quercus leaves infusions was also determined by HPLC. In vitro analysis of antioxidant capacity of leaves infusion extracts were performed by the DPPH method and the deoxyribose assay. The genotoxicity of Q. resinosa leaves extracts was evaluated on HeLa cells as well as its underlying mechanism by the single-cell electrophoresis assay (comet assay). Results show that fresh leaves infusions increase the oxidative process and other damage to DNA in transformed human cells. Fresh leaves from Q. resinosa may serve as a potential source of phenolics with anticancer activity.     

Degradation of ascorbic acid and potassium sorbate by different Lactobacillus species isolated from packed green olives

The aim of this research was to ascertain the lactic acid bacteria responsible for the degradation of ascorbic acid and/or potassium sorbate, isolated from packed green olives where these additives had diminished. A total of 14 isolates were recovered from samples of different green olive containers. According to partial sequencing of the 16S rRNA coding gene, Lactobacillus parafarraginis, Lactobacillus rapi, Lactobacillus pentosus, Lactobacillus paracollinoides, and Pediococcus ethanolidurans were identified. With the exception of L. pentosus and L. paracollinoides, the other species had not been mentioned in table olives before this study. Only three of the 14 isolates metabolized ascorbic acid in MRS broth, and the products from ascorbic acid in modified MRS broth without carbon sources were acetic and lactic acids. Except for the two L. rapi and the two P. ethanolidurans strains, the remaining 10 isolates depleted potassium sorbate added into MRS broth to some extent. The product generated by three of these strains was confirmed to be trans-4-hexenoic acid. The degradation of ascorbate or sorbate by lactic acid bacteria should be taken into account when these additives are used in food products where this group of bacteria may be present.     

Effect of Capsicum carotenoids on growth and aflatoxins production by Aspergillus flavus isolated from paprika and chilli

The aim of this study was to determine the effect of a carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and aflatoxins (AF) production of AF-producing Aspergillus flavus isolates.     

Antimetastatic activity of polyphenol-rich extract of Ecklonia cava through the inhibition of the Akt pathway in A549 human lung cancer cells

An ethyl acetate extract (ECE) of a brown alga, Ecklonia cava, was examined for its anti-metastatic effect, using A549 human lung carcinoma cells. ECE treatment significantly suppressed the migration and invasion of A549 cells in a concentration-dependent manner. It also strongly down-regulated the matrix metalloproteinase (MMP)-2 activity of the cancer cells by gelatin zymography assay. For elucidating its mechanism of action in cancer cell metastasis, ECE was further investigated for various cell signalling pathways, including JNK, ERK, p38, and Akt. In this, ECE showed an anti-metastatic effect in a concentration- and time-dependent manner by the mechanism of suppression of Akt and p38, but not JNK and ERK. These results, for the first time, suggest that ECE (a polyphenol-enriched, highly anti-oxidative fraction of brown alga, E. cava) may have therapeutic potential in metastatic lung cancer, based on its strong inhibitory effects on the migration and invasiveness of A549 human lung adenocarcinoma cells.     

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1–50 mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100 mg L⁻¹. Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.     

Antimicrobial activity of catechol isolated from Diospyros kaki Thunb. roots and its derivatives toward intestinal bacteria

The growth-inhibiting activities of the methanol extract of Diospyros kaki Thunb. roots were examined on the growth of Bifidobacterium breve, B. longum, Clostridium difficile, Clostridium perfringes, Escherichia coli, and Lactobacillus casei. In addition, the biologically active component of D. kaki roots was purified using silica gel column chromatography and HPLC. The active component was characterised as catechol by spectroscopic analyses. The antimicrobial activity of the isolated catechol varied according to the dose and bacterial strains tested. Catechol significantly (++++) inhibited the growth of C. perfringens at 2.0 mg/disc, and moderately (++) inhibited its growth at 0.25 mg/disc. At a dose of 5.0 mg/disc, catechol significantly inhibited the growth of C. difficile and moderately inhibited the growth of E. coli. However, this isolate did not inhibit the growth of bifidobacteria and lactobacilli. When various functional groups were added to the catechol, selective growth-inhibiting activity against harmful intestinal bacteria was observed in response to treatment with low concentrations. Taken together, these findings indicate that D. kaki root-isolated catechol and its derivatives (4-nitrocatechol, 4-tert-butylcatechol, tetrabromocatechol) could be useful as preventive agents against diseases caused by harmful intestinal bacteria.