Z. Ustunol

Antimicrobial Edible Films and Coatings

Increasing consumer demand for microbiologically safer foods, greater convenience, smaller packages, and longer product shelf life is forcing the industry to develop new food-processing, cooking, handling, and packaging strategies. Nonfluid ready-to-eat foods are frequently exposed to postprocess surface contamination, leading to a reduction in shelf life. The food industry has at its disposal a wide range of nonedible polypropylene- and polyethylene-based packaging materials and various biodegradable protein- and polysaccharide-based edible films that can potentially serve as packaging materials. Research on the use of edible films as packaging materials continues because of the potential for these films to enhance food quality, food safety, and product shelf life. Besides acting as a barrier against mass diffusion (moisture, gases, and volatiles), edible films can serve as carriers for a wide range of food additives, including flavoring agents, antioxidants, vitamins, and colorants. When antimicrobial agents such as benzoic acid, sorbic acid, propionic acid, lactic acid, nisin, and lysozyme have been incorporated into edible films, such films retarded surface growth of bacteria, yeasts, and molds on a wide range of products, including meats and cheeses. Various antimicrobial edible films have been developed to minimize growth of spoilage and pathogenic microorganisms, including Listeria monocytogenes, which may contaminate the surface of cooked ready-to-eat foods after processing. Here, we review the various types of protein-based (wheat gluten, collagen, corn zein, soy, casein, and whey protein), polysaccharide-based (cellulose, chitosan, alginate, starch, pectin, and dextrin), and lipid-based (waxes, acylglycerols, and fatty acids) edible films and a wide range of antimicrobial agents that have been or could potentially be incorporated into such films during manufacture to enhance the safety and shelf life of ready-to-eat foods.

Conjugated linoleic acid concentration in processed cheese

The conjugated linoleic acid (CLA) concentration of a variety of processed cheese products ranged between 3.2 to 8.9 mg/g fat. Processing cheddar cheese at temperatures of 80°C and 90°C under atmospheric conditions increased (p < 0.05) CLA content, while processing under nitrogen (70°C, 85°C) had no effect. Increasing concentrations of whey protein concentrate (WPC) and its low molecular weight (LMW) fraction from 0 to 6% increased CLA formation. Six percent WPC and LMW fraction produced a 35% and 19% increase in CLA concentration, respectively, compared to processed cheese. The high molecular weight fraction of WPC did not increase CLA concentration. These results suggest that processing conditions and whey components play a role in CLA formation in processed cheese.

Stimulation of cytokine production in clonal macrophage and T-cell models by Streptococcus thermophilus: Comparison with Bifidobacterium sp. and Lactobacillus bulgaricus

The effects of four commercial strains of Streptococcus thermophilus used in yogurt manufacturing on cytokine production were evaluated by using a macrophage model (RAW 264.7 cells) and a T-helper-cell model (EL4.IL-2 thymoma cells) and compared to immunologically active strains of Lactobacillus bulgaricus, Bifidobacterium adolescentis, and Bifidobacterium bifidum. All cytokines (TNF-alpha and IL-6 in RAW 264.7 cells and IL-2 and IL-5 in EL4.IL-2 cells) were affected by heat-killed S. thermophilus in a strain- and dose-dependent fashion. Organisms of all three genera induced significant increases in IL-6 production by the macrophage line ranging from 31- to 192-fold, with S. thermophilus St 133 showing the greatest activity. The four S. thermophilus strains also strongly induced TNF-alpha production (from 135- to 176-fold). IL-6 and, to a lesser extent, TNF-alpha production were also increased when the macrophages were costimulated with lipopolysaccharide and cells of the three groups of lactic acid bacteria. Upon concurrent stimulation of EL4.IL-2 cells with phorbol 12-myristate-13-acetate, seven of the eight strains displayed significant enhancement of IL-2 and IL-5 production, with S. thermophilus being most effective. Taken together, the S. thermophilus strains stimulated macrophage and T-cell cytokine production to a similar or greater extent than did the species of Bifidobacterium and Lactobacillus. These and previous results lend further support to the contention that lactic acid bacteria, in a concentration-dependent manner, can differentially induce cytokine production in macrophages, but that the effects on T cells required a costimulatory signal and were less remarkable.

Ex vivo effects of lactobacilli, streptococci, and bifidobacteria ingestion on cytokine and nitric oxide production in a murine model

Increasing numbers of functional foods and pharmaceutical preparations are being promoted with health claims based on the potential probiotic characteristics of lactic acid bacteria and on their capacity for stimulating the host immune system. However, the specific immune effects of oral administration of these microbes still remains undefined. In this study, we tested the hypothesis that production of immunologic mediators by leukocytes in mice is affected by orally administered lactic acid bacteria. The specific objectives of this study were to evaluate the effects of exposure to eight different lactic acid bacteria in mice on ex vivo cytokine and nitric oxide production in leukocyte cultures. Mice were gavaged with 1 X 10(9) viable bacteria and peritoneal, Peyers patch and splenic leukocytes were isolated 8 h later. These were cultured for 2 or 5 days in the presence or absence of mitogens and then interleukin (IL)-6, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and nitric oxide production was measured. The results revealed that Lactobacillus acidophilus and L. casei potentiated IL-6 and IL-12 production by peritoneal cells whereas L. acidophilus upregulated IFN-gamma and nitric oxide. In contrast, L. helveticus, L. gasseri, L. reuteri, and Bifidobacterium attenuated the production of IL-6, IFN-gamma, and nitric oxide by peritoneal cells. TNF-alpha was not detectable in peritoneal cultures. None of the bacteria altered ex vivo production of cytokines or nitric oxide by Peyers patch or spleen cell cultures. Taken together, the results suggest that prior oral exposure to lactic acid bacteria could differentially potentiate or attenuate subsequent cytokine and nitric oxide production by peritoneal cells.

Viability of bifidobacteria in commercial dairy products during refrigerated storage

Commercial milk and two brands of yogurt containing bifidobacteria were obtained from retail outlets. All products were evaluated for viability of bifidobacteria and lactic acid bacteria during refrigerated storage at 4 degrees C. Milk was evaluated at 9, 6, and 3 days prior and past its expiration date. The yogurts were evaluated at 3, 2, and 1 week prior and past their expiration. Viability of bifidobacteria and lactic acid bacteria in milk and yogurt remained above 10(6) CFU/ml or g until the expiration date of the respective products. This microbial concentration is the recommended minimum dose to receive the health benefits of these organisms.

Potentiation of hydrogen peroxide, nitric oxide, and cytokine production in RAW 264.7 macrophage cells exposed to human and commercial isolates of Bifidobacterium

Bifidobacteria have been previously shown to stimulate immune function and this may be mediated by macrophages. The RAW 264.7 cell line was used here as a macrophage model to assess the effects of human and commercial Bifidobacterium isolates on the production nitric oxide (NO), hydrogen peroxide (H2O2) and the cytokines IL-6 and tumor necrosis factor (TNF)-alpha. Thirty three Bifidobacterium strains differentially stimulated the production of H2O2 NO, TNF-alpha, and IL-6 in a dose-dependent manner in 24-h cultures. In the presence of lipopolysaccharide (LPS) the effects of bifidobacteria on NO and H2O2 were masked and were less pronounced at the later stage of incubation. Co-stimulation of macrophages with both LPS and Bifidobacterium increased the production of IL-6 synergistically. In contrast, LPS reduced the ability of the bifidobacteria-induced macrophages to produce TNF-alpha. Our results demonstrated that both human and commercial Bifidobacterium strains can stimulate H2O2, NO, TNF-alpha, and IL-6 production, and this effect was strain-dependent. The in vitro approaches employed here should be useful in further characterization of the effects of bifidobacteria on gastrointestinal and systemic immunity.

Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: An in vitro comparison with commercial oligosaccharides and inulin

Five human intestinal Bifidobacterium spp., B. longum, B. adolescentis, B. breve, B. bifidum, and B. infantis, were cultured in reinforced clostridial medium (control) and in reinforced clostridial medium supplemented with 5% (wt/vol) honey, fructooligosaccharide (FOS), galactooligosaccharide (GOS), and inulin. Inoculated samples were incubated anaerobically at 37degrees C for 48 h. Samples were collected at 12-h intervals and examined for specific growth rate. Levels of fermentation end products (lactic and acetic acids) were measured by high-pressure liquid chromatography. Honey enhanced the growth of the five cultures much like FOS, GOS, and inulin did. Honey, FOS, GOS, and inulin were especially effective (P < 0.05) in sustaining the growth of these cultures after 24 h of incubation as compared with the control treatment. Overall, the effects of honey on lactic and acetic acid production by intestinal Bifidobacterium spp. were similar to those of FOS, GOS, and inulin.

Effects of Lactic Acid Bacteria Ingestion on Basal Cytokine mRNA and Immunoglobulin Levels in the Mouse

An increasing number of functional foods and pharmaceutical preparations containing lactic acid bacteria are being promoted with health claims based on the potential probiotic characteristics and on their capacity for stimulating the host immune system. However, the specific immune effects of oral administration of these microbes remain undefined. In this study, we tested the hypothesis that basal gastrointestinal immune status in mice is affected by orally administered lactic acid bacteria. The specific objective of this research was to evaluate the effects of repeated oral exposure to viable and nonviable lactic acid bacteria (Lactobacillus acidophilus, L. bulgaricus, L. casei, and Streptococcus thermophilus) in mice on basal cytokine mRNA expression in mucosal (Peyers patches), systemic (spleen), and lymphoid tissue and on immunoglobulin levels. The results indicated that oral exposure to 10(9) CFU/day for up to 14 days did not significantly affect basal interferon-gamma, tumor necrosis factor-alpha, or interleukin-6 mRNA expression or total serum and intestinal immunoglobulins.

Effects of ingestion of yogurts containing Bifidobacterium and Lactobacillus acidophilus on spleen and Peyer's patch lymphocyte populations in the mouse.

Certain probiotic lactic acid bacteria have been reported to improve immune system function. Here, the effects of ingesting yogurts on lymphocyte populations in the spleens and Peyers patches were determined in mice. Three probiotic-supplemented yogurts containing Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium, and Lactobacillus acidophilus and one conventional yogurt containing only S. thermophilus and L. bulgaricus were prepared from commercial starter cultures and used in the study. B6C3F1 female mice were fed the four different types of yogurts mixed with an AIN-93G diet in a 50:50 (wt/wt) ratio. Nonfat dry milk mixed at a 50:50 (wt/wt) ratio with AIN-93G diet was used as the control. After a 14-day feeding period, spleen and Peyers patches were removed and lymphocytes subjected to phenotype analysis by flow cytometry. Ingestion of the four yogurts had no effect on percentages of CD8+ (cytotoxic T cells), B220+ (B cells), IgA+, or IgM+ cells in spleen or Peyers patches. The percentage of CD4+ (T helper) cells was significantly increased in the spleens from one group of mice fed a yogurt containing Bifidobacterium and L. acidophilus, and a similar trend was found in the remaining two probiotic-supplemented yogurts. Effects on CD4+ populations were not observed in spleens of mice fed conventional yogurt or in the Peyers patches of any of the four yogurt groups. In total, the results suggested that ingestion of conventional or probiotic-supplemented yogurts for 2 weeks had very little effect on lymphocyte distribution in the systemic or mucosal immune compartments.

Mode of inactivation of probiotic bacteria affects interleukin 6 and interleukin 8 production in human intestinal epithelial-like Caco-2 cells.

Five lactic acid bacteria and two bifidobacteria strains were heat or irradiation inactivated. Inactivated cultures were evaluated for their effects on cytokines interleukin (IL) 6 and IL-8 production in human intestinal-like Caco-2 cells. For both heat- and irradiation-inactivated cultures, production of IL-6 and IL-8 was dependent on the specific microorganism. However, with all of the cultures, both IL-6 and IL-8 production was significantly higher (P < 0.05) in Caco-2 cells that were treated with heat-inactivated probiotic bacteria compare to the irradiation-inactivated bacteria. In the majority of the cases, heat-inactivated bacteria induced IL-6 and IL-8 production, whereas irradiation-inactivated bacteria attenuated both cytokine production. Our results indicate that the same probiotic bacteria used in the same cell culture could provide opposite cytokine production and immune modulation results based on its mode of inactivation; therefore, it is important to describe inactivation methods and conditions in detail when characterizing probiotic effects.