Ramakrishna Nannapaneni

Human Colon and Liver Cancer Cell Proliferation Inhibition by Peptide Hydrolysates Derived from Heat-Stabilized Defatted Rice Bran

Rice bran, an economical, underutilized coproduct of rough rice milling, was used to produce peptide hydrolysates, which were investigated for anticancer activity. Protein hydrolysates prepared by Alcalase hydrolysis under optimized conditions were treated further to obtain gastrointestinal (GI)-resistant peptide hydrolysates. They were fractionated into >50, 10-50, 5-10, and <5 kDa sizes and evaluated for inhibitory activity on proliferation of human colon (Caco-2) and liver (HepG2) cancer cell lines by Trypan blue dye exclusion assay. GI-resistant <5 and 5-10 kDa sized peptide fractions inhibited growth of Caco-2 cells by 80%, and the <5 kDa fraction inhibited growth of HepG2 cells by approximately 50% compared to controls and nonresistant fractions. An MTS cell titer assay confirmed antiproliferative effects of the peptide fractions. The results demonstrated that 5-10 and <5 kDa sized GI-resistant fractions promoted significant (p < 0.05) inhibitory activities on both cancer cell lines compared to controls. More investigations are needed to show such value-added effects on the technofunctional and sensorial properties of the food protein and peptide matrices.

Reduction of Listeria monocytogenes on the Surface of Fresh Channel Catfish Fillets by Bacteriophage Listex P100

Bacteriophage Listex P100 (phage P100) was approved by the U.S. Food and Drug Administration and U.S. Department of Agricultures Food Safety and Inspection Service for Listeria monocytogenes control on both raw and ready-to-eat food products. In this article, we present the proof of concept on the influence of phage dose, phage contact time, and storage temperature on the listericidal activity of phage P100 in reducing the L. monocytogenes loads on the surface of fresh channel catfish fillet. The fresh catfish fillet samples were surface inoculated with approximately 4.3 log(10) colony forming units (CFU)/g of a two serotype mix (1/2a and 4b) of L. monocytogenes cells and then surface treated with phage P100. L. monocytogenes reduction was influenced by phage contact time and phage dose regardless of higher or lower temperature regimes tested on catfish fillet. The reduction in L. monocytogenes loads (p < 0.05) with the phage P100 dose of 2 x 10(7) plaque forming units (PFU)/g (7.3 log(10) PFU/g) was 1.4-2.0 log(10) CFU/g at 4 degrees C, 1.7-2.1 log(10) CFU/g at 10 degrees C, and 1.6-2.3 log(10) CFU/g at room temperature (22 degrees C) on raw catfish fillet. The phage contact time of 30 min was adequate to yield greater than 1 log(10) CFU/g reduction in L. monocytogenes, whereas 15 min contact time with phage yielded less than 1 log(10) CFU/g reduction in L. monocytogenes loads on catfish fillet. Phage P100 titer was stable on catfish fillet samples, and overall reductions in L. monocytogenes counts were still maintained over a 10-day shelf life at 4 degrees C or 10 degrees C by phage P100 treatment. These findings illustrate the effectiveness of an alternative generally recognized as safe antimicrobial such as bacteriophage Listex P100 in quantitatively reducing L. monocytogenes from fresh catfish fillet surfaces.

Bacteriophage significantly reduces Listeria monocytogenes on raw salmon fillet tissue.

We have demonstrated the antilisterial activity of generally recognized as safe (GRAS) bacteriophage LISTEX P100 (phage P100) on the surface of raw salmon fillet tissue against Listeria monocytogenes serotypes 1/2a and 4b. In a broth model system, phage P100 completely inhibited L. monocytogenes growth at 4 degrees Celsius for 12 days, at 10 degrees Celsius for 8 days, and at 30 degrees Celsius for 4 days, at all three phage concentrations of 10(4), 10(6), and 10(8) PFU/ml. On raw salmon fillet tissue, a higher phage concentration of 10(8) PFU/g was required to yield 1.8-, 2.5-, and 3.5-log CFU/g reductions of L. monocytogenes from its initial loads of 2, 3, and 4.5 log CFU/g at 4 or 22 degrees Celsius. Over the 10 days of storage at 4 degrees Celsius, L. monocytogenes growth was inhibited by phage P100 on the raw salmon fillet tissue to as low as 0.3 log CFU/g versus normal growth of 2.6 log CFU/g in the absence of phage. Phage P100 remained stable on the raw salmon fillet tissue over a 10-day storage period, with only a marginal loss of 0.6 log PFU/g from an initial phage treatment of 8 log PFU/g. These findings illustrate that the GRAS bacteriophage LISTEX P100 is listericidal on raw salmon fillets and is useful in quantitatively reducing L. monocytogenes.

Removal of Listeria monocytogenes biofilms with bacteriophage P100.

Listeria monocytogenes is an important foodborne pathogen with a persistent ability to form biofilm matrices in the food processing environments. In this study, we have determined the ability of bacteriophage P100 to reduce L. monocytogenes cell populations under biofilm conditions by using 21 L. monocytogenes strains representing 13 different serotypes. There were considerable differences in the ability of various strains of L. monocytogenes to form biofilms, with strains of serotype 1/2a showing maximum biofilm formation. Irrespective of the serotype, growth conditions, or biofilm levels, the phage P100 treatment significantly reduced L. monocytogenes cell populations under biofilm conditions. On the stainless steel coupon surface, there was a 3.5- to 5.4-log/cm2 reduction in L. monocytogenes cells by phage treatment. These findings illustrate that phage P100 is active against a wide range of L. monocytogenes strains in biofilm conditions.

Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions.

Seven orange oil fractions were screened for their ability to inhibit the growth of selected Campylobacter and Arcobacter spp. using the standard agar-disk diffusion assay. Cold pressed (CP) terpeneless Valencia orange oil was found to be the most inhibitory to both Campylobacter jejuni and Campylobacter coli, exhibiting maximum zones of inhibition up to 80+/-0.0 mm. Five-fold concentrated Valencia oil and distilled d-limonene resulted in Campylobacter inhibition zones ranging from 11.0+/-1.4 to 44+/-1.4 mm against both C. jejuni and C. coli. No inhibition of Arcobacter spp. was detected by 6 out of 7 orange fractions except CP terpeneless Valencia orange oil which produced inhibition zones varying from 9.5+/-0.7 to 29+/-1.4 mm. Naturally occurring C. jejuni UAF 244 was isolated from a whole retail chicken, confirmed by hippuricase gene PCR assay, and used to determine antimicrobial capacities of the CP terpeneless Valencia orange oil and limonene when applied on chicken legs and thighs. The two types of chicken parts did not influence the antimicrobial strength of both orange fractions. While the observed reduction of C. jejuni cells attached to the skin varied approximately 1.5 to 2 logarithms compared to the control, the growth inhibition of the bacterial cells by limonene in the rinse increased by 6-fold and complete inhibition without recovery of detectable viable cells occurred when CP Valencia orange oil was applied. The study demonstrated the potential of the selected commercial orange oil fractions to serve as natural antimicrobials against C. jejuni, C. coli, and Arcobacter spp.

Identification and characterization of two bacteriocin-producing bacteria isolated from garlic and ginger root.

Two bacteriocin-producing bacterial strains were isolated from garlic and ginger root by the agar overlay method. The bacteria were identified by 16S rRNA sequence analyses and fermentation patterns as Leuconostoc mesenteroides (garlic isolate) and Lactococcus lactis (ginger isolate). The bacteriocins were assigned the names leucocin BC2 and lactocin GI3, respectively. Physiochemical properties and antimicrobial spectra of the bacteriocins were determined by the spot-on-lawn method. Both bacteriocins were inhibited by proteolytic enzymes. Leucocin BC2 exhibited a narrow antimicrobial spectrum, inhibiting only Bacillus, Enterococcus, and Listeria species. Lactocin GI3 had a broader spectrum, inhibiting Bacillus, Clostridium, Listeria, Enterococcus, Leuconostoc, Pediococcus, and Staphylococcus species. Both bacteriocins remained active when heated at 90 degrees C for 15 min or 120 degrees C for 20 min. Leucocin BC2 assayed at 37 degrees C showed an inhibitory activity of 1,600 AU/ml, whereas at 8 degrees C the activity was 12,800 AU/ml. Conversely, lactocin GI3 activity was the same at both assay temperatures. Both bacteriocins remained active over a pH range of 2.0 to 9.0 and in various organic solvents. The activity of leucocin BC2 was increased when treated with 0.5% acetic acid and 0.5% lactic acid, whereas lactocin GI3 activity was decreased with either acid. The molecular mass values were 3.7 kDa for leucocin BC2 and 3.9 kDa for lactocin GI3. These results show that the inhibitory substances produced by the bacteria isolated from garlic and ginger are bacteriocins that appear to be different in some characteristics from previously reported bacteriocins.

The Contribution of Transcriptomic and Proteomic Analysis in Elucidating Stress Adaptation Responses of Listeria monocytogenes

The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecular stress adaptation responses in L. monocytogenes cells as revealed through transcriptome, proteome, genetic, and physiological analysis. The adaptation of L. monocytogenes to stress exposure is achieved through global expression changes in a large number of cellular components. In addition, the cross-protection of L. monocytogenes exposed to different stress environments might be conferred through various cellular machineries that seem to be commonly activated by the different stresses. To assist in designing L. monocytogenes mitigation strategies for ready-to-eat food products, further experiments are warranted to specifically evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of L. monocytogenes cellular components in response to specific stresses.

Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains.

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.

Inhibition and inactivation of Salmonella typhimurium biofilms from polystyrene and stainless steel surfaces by essential oils and phenolic constituent carvacrol.

Persistence of Salmonella biofilms within food processing environments is an important source of Salmonella contamination in the food chain. In this study, essential oils of thyme and oregano and their antimicrobial phenolic constituent carvacrol were evaluated for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms. A crystal violet staining assay and CFU measurements were utilized to quantify biofilm cell mass, with evaluating factors such as strain variation, essential oil type, their concentrations, exposure time, as well as biofilm formation surface. Of the three Salmonella strains, Salmonella Typhimurium ATCC 23564 and Salmonella Typhimurium ATCC 19585 produced stronger biofilms than Salmonella Typhimurium ATCC 14028. Biofilm formation by different Salmonella strains was 1.5- to 2-fold higher at 22°C than at 30 or 37°C. The presence of nonbiocidal concentrations of thyme oil, oregano oil, and phenolic carvacrol at 0.006 to 0.012% suppressed Salmonella spp. biofilm formation 2- to 4-fold, but could not completely eliminate biofilm formation. There was high correlation in terms of biofilm inactivation, as determined by the crystal violet-stained optical density (at a 562-nm wavelength) readings and the viable CFU counts. Reduction of biofilm cell mass was dependent on antimicrobial concentration. A minimum concentration of 0.05 to 0.1% of these antimicrobial agents was needed to reduce a 7-log CFU biofilm mass to a nondetectable level on both polystyrene and stainless steel surfaces within 1 h of exposure time.

Reduction of Listeria monocytogenes Biofilms on Stainless Steel and Polystyrene Surfaces by Essential Oils

Plant-derived essential oils were tested for their ability to eliminate biofilms of Listeria monocytogenes on polystyrene and stainless steel surfaces. Various concentrations of essential oils were tested with different contact times on biofilms of various ages. Preliminarily screening of nine essential oils and related phenolic compounds in a disk diffusion assay revealed that thyme oil, oregano oil, and carvacrol had the highest antimicrobial activity. Further screening of these three compounds against 21 L. monocytogenes strains representing all 13 serotypes indicated some strain-specific variations in antimicrobial activity. For 1-day-old biofilms of mixed L. monocytogenes strains produced at 22°C on polystyrene microtiter plates, only 0.1% concentrations of thyme oil, oregano oil, and carvacrol were needed to eliminate 7 log CFU per well. On the stainless steel coupons, a 0.5% concentration of these compounds was adequate to completely eliminate 4-day-old biofilms at 7 log CFU per coupon. Our findings indicate that these compounds are potential candidates for elimination of L. monocytogenes biofilms on stainless steel and polystyrene surfaces.