Effect of microencapsulated ferrous sulfate particle size on Cheddar cheese composition and quality.

Iron-fortified Cheddar cheese was manufactured with large microencapsulated ferrous sulfate (LMFS; 700-1,000 µm in diameter) or small microencapsulated ferrous sulfate (SMFS; 220-422 µm in diameter). Cheeses were aged 90 d. Compositional, chemical, and sensory characteristics were compared with control cheeses, which had no ferrous sulfate added. Compositional analysis included fat, protein, ash, moisture, as well as divalent cations iron, calcium, magnesium, and zinc. Thiobarbituric acid reactive species assay was conducted to determine lipid oxidation. A consumer panel consisting of 101 participants evaluated the cheeses for flavor, texture, appearance, and overall acceptability using a 9-point hedonic scale. Results showed 66.0% iron recovery for LMFS and 91.0% iron recovery for SMFS. Iron content was significantly increased from 0.030 mg of Fe/g in control cheeses to 0.134 mg of Fe/g of cheese for LMFS and 0.174 mg of Fe/g of cheese for SMFS. Fat, protein, ash, moisture, magnesium, zinc, and calcium contents were not significantly different when comparing iron-fortified cheeses with the control. Iron fortification did not increase lipid oxidation; however, iron fortification negatively affected Cheddar cheese sensory attributes, particularly the LMFS fortified cheese. Microencapsulation of ferrous sulfate failed to mask iron's distinct taste, color, and odor. Overall, SMFS showed better results compared with LMFS for iron retention and sensory evaluation in Cheddar cheese. Results of this study show that size of the microencapsulated particle is important in the retention of the iron in the cheese and its sensory attributes. This study provides new information on the importance of particle size with microencapsulated nutrients.

Effect of zinc fortification on Cheddar cheese quality.

Zinc-fortified Cheddar cheese containing 228 mg of zinc/kg of cheese was manufactured from milk that had 16 mg/kg food-grade zinc sulfate added. Cheeses were aged for 2 mo. Culture activity during cheese making and ripening, and compositional, chemical, texture, and sensory characteristics were compared with control cheese with no zinc sulfate added to the cheese milk. Compositional analysis included fat, protein, ash, moisture, zinc, and calcium determinations. The thiobarbituric acid (TBA) assay was conducted to determine lipid oxidation during aging. Texture was analyzed by a texture analyzer. An untrained consumer panel of 60 subjects evaluated the cheeses for hardness, off-flavors, appearance, and overall preference using a 9-point hedonic scale. Almost 100% of the zinc added to cheese milk was recovered in the zinc-fortified cheese. Zinc-fortified Cheddar cheese had 5 times more zinc compared with control cheese. Zinc-fortified cheese had higher protein and slightly higher fat and ash contents, whereas moisture was similar for both cheeses. Zinc fortification did not affect culture activity during cheese making or during the 2-mo aging period. The TBA value of control cheese was higher than that of zinc-fortified cheese at the end of ripening. Although zinc-fortified cheese was harder as determined by the texture analyzer, the untrained consumer panel did not detect differences in the sensory attributes and overall quality of the cheeses. Fortification of 16 mg/kg zinc sulfate in cheese milk is a suitable approach to fortifying Cheddar cheese without changing the quality of Cheddar cheese.

Effect of nonfat dry milk and major whey components on interleukin-6 and interleukin-8 production in human intestinal epithelial-like Caco-2 cells.

Bovine nonfat dry milk (NDM) and major whey components (lactose, alpha-lactalbumin, and beta-lactoglobulin) were evaluated for their effects on IL-6 and IL-8 production in human intestinal-like Caco-2 cells unstimulated or stimulated with IL-1beta. All the whey components investigated and NDM induced IL-6 production by Caco-2 cells; the most significant increase was observed with beta-lactoglobulin. In the case of IL-1beta-stimulated cells, neither NDM nor the major whey components investigated contributed to the induction of IL-6 production after they were stimulated. Induction of IL-8 production by both alpha-lactalbumin and beta-lactoglobulin was higher than that by lactose and NDM; alpha-lactalbumin was a more potent inducer of IL-8 than beta-lactoglobulin and IL-1beta alone in both unstimulated and stimulated cells. In Caco-2 cells that were stimulated with IL1-beta, NDM and all the major whey components investigated had a synergistic effect on induction of IL-8 production, indicating that IL-8 induction was amplified by prior stimulation of cells by IL-1beta. This synergistic effect was not observed with IL-6. Our results suggest that immunomodulatory properties of milk components may be affected by other complex events in the gut.

Invited review: Advances in starter cultures and cultured foods.

With 2005 retail sales close to $4.8 million, cultured dairy products are driving the growth of dairy foods consumption. Starter cultures are of great industrial significance in that they play a vital role in the manufacturing, flavor, and texture development of fermented dairy foods. Furthermore, additional interest in starter bacteria has been generated because of the data accumulating on the potential health benefits of these organisms. Today, starter cultures for fermented foods are developed mainly by design rather than by the traditional screening methods and trial and error. Advances in genetics and molecular biology have provided opportunities for genomic studies of these economically significant organisms and engineering of cultures that focuses on rational improvement of the industrially useful strain. Furthermore, much research has been published on the health benefits associated with ingesting cultured dairy foods and probiotics, particularly their role in modulating immune function. The aim of this review is to describe some of the major scientific advances made in starter and non-starter lactic acid bacteria during the past 10 yr, including genomic studies on dairy starter cultures, engineering of culture attributes, advances in phage control, developments in methods to enumerate lactic acid bacteria and probiotics in dairy foods, and the potential role of cultured dairy foods in modulation of immune function.

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.

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.

Growth and viability of commercial Bifidobacterium spp. in honey-sweetened skim milk.

Two commercial Bifidobacterium bifidum (Bf-1 and Bf-6) were cultured in 12% (wt/vol) reconstituted nonfat dry milk (NDM) containing 5% (wt/wt) honey, sucrose, fructose, or glucose. Inoculated samples were incubated anaerobically at 37 degrees C for 48 h. Samples were collected at 12-h intervals and examined for (i) specific growth rate, (ii) pH, and (iii) levels of fermentation end products (lactic and acetic acids) as measured by high-performance liquid chromatography (HPLC). Viability of the organisms during 28 days of refrigerated storage at 4 degrees C was also assessed at 7-day intervals. Growth promotion and acid production were greatest when Bf-1 and Bf-6 were grown in the presence of honey. For both Bf-1 and Bf-6, retention of viability was greatest up to 14 days of refrigerated storageat 4 degrees C when they were grown and stored in the presence of honey compared to other sweeteners.

Solubility and moisture sorption isotherms of whey-protein-based edible films as influenced by lipid and plasticizer incorporation.

Plasticized whey-protein and whey-protein emulsion films were produced using sorbitol and glycerol as plasticizers and butterfat and candelilla wax as lipids. Protein, plasticizer, and lipid ratios were optimized to obtain acceptable free-standing flexible films. Water solubility (20 degrees C, 24 h) and moisture sorption isotherms (0.18-0.90 a(w), 25 degrees C) of the films were determined. The experimental moisture sorption isotherm values were fitted using the Guggenheim-Anderson-DeBoer (GAB) model. Solubility and equilibrium moisture contents (EMC) of the films were influenced by plasticizer and lipid incorporation. EMCs of all films increased rapidly at a(w) > or = 0.65. Incorporation of lipids reduced solubilities and EMCs of sorbitol- and glycerol-plasticized films. The effects of plasticizer and lipid type on GAB constants were also determined.

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 Peyer's 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 Peyer's 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 Peyer's 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 Peyer's 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.

Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase.

Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.

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.

Ingestion of yogurt containing Lactobacillus acidophilus and Bifidobacterium to potentiate immunoglobulin A responses to cholera toxin in mice.

Lactic acid bacteria have been reported to have benefits for the prevention and treatment of some forms of diarrhea and related conditions. To determine whether these effects might involve direct stimulation of the gastrointestinal immune response, we administered yogurt to try to enhance mucosal and systemic antibodies against an orally presented immunogen, cholera toxin. Yogurts were manufactured with starter cultures containing different species and strains of lactic acid bacteria. Mice were fed these yogurts for 3 wk, during which they were also orally immunized twice with 10 micrograms of cholera toxin. Blood was collected on d 0 and 21, and fecal pellets were collected weekly. Mice that were immunized orally with cholera toxin responded by producing specific intestinal and serum immunoglobulin (Ig)A anti-cholera toxin. Antibody responses of the IgA isotype were significantly increased in mice fed yogurts made with starters containing the conventional yogurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus supplemented with Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium infantis. Yogurt that was manufactured with starters containing only conventional yogurt bacteria produced less IgA anti-cholera toxin than did the control group fed nonfat dry milk. Although strong responses were also observed for IgG anti-cholera toxin in serum, the responses did not differ among groups. Thus, administration of yogurt supplemented with L. acidophilus and Bifidobacterium spp. enhanced mucosal and systemic IgA responses to the cholera toxin immunogen.

Effects of lactic acid bacteria ingestion of 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 (Peyer's 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.

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, Peyer's 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 Peyer's 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.

Effects of yogurt ingestion on mucosal and systemic cytokine gene expression in the mouse.

To assess the potential for ingestion of yogurt to modulate immunity, its effects on basal gene expression of cytokines in systemic and mucosal sites were determined in mice. Yogurts were manufactured from pasteurized nonfat dry milk using five commercial starter cultures with or without Bifidobacterium sp. and Lactobacillus acidophilus. Treatment mice were fed the AIN-93G diet mixed 1:1 with unheated yogurt or heat-treated yogurt (wt/wt) for 2 and 4 weeks, and control mice were fed the AIN-93G diet mixed 1:1 (wt/wt) with nonfat dry milk. The viability of the various bacterial groups in unheated yogurts was maintained above 10(6) CFU/g throughout the feeding period. The yogurt-feeding regimens did not significantly affect weight gain. Relative mRNA levels in spleen, mesenteric lymph nodes, or Peyer's patches for the cytokines interferon-gamma, tumor necrosis factor-alpha, interleukin-2, -4, and -6, and the "housekeeping gene" beta2-microglobulin were determined by reverse transcriptase-polymerase chain reaction in conjunction with hybridization analysis. Prolonged feeding of some yogurts decreased expression of several cytokine mRNAs, the depression of tumor necrosis factor-alpha mRNA in the spleen being the most prominent effect. Heat-treated yogurts were more effective in altering cytokine mRNA expression than were unheated yogurts containing viable organisms. Generally, yogurts either had no effect or decreased specific cytokine mRNA in the test organs, regardless of whether they contained Bifidobacterium sp. and L. acidophilus. These results suggest that, in contrast with previous studies in vitro, some yogurt formulations may reduce rather than stimulate basal cytokine expression and that these effects are most prominent in the systemic compartment.

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.

Differential cytokine production in clonal macrophage and T-cell lines cultured with bifidobacteria.

When used in commercial fermented dairy products, bifidobacteria may enhance immunity by stimulating cytokine secretion by leukocytes. To assess whether interaction between bifidobacteria and leukocytes promote cytokine production, we cultured RAW 264.7 cells (macrophage model) and EL-4.IL-2 thymoma cells (helper T-cell model) in the presence of 14 representative strains of heat-killed bifidobacteria. In unstimulated RAW 264.7 cells, all bifidobacteria induced pronounced increases (up to several hundred-fold) in the production of tumor necrosis factor-alpha compared with that of controls. Interleukin-6 production by unstimulated cells also increased significantly, but less than did tumor necrosis factor-alpha. Upon concurrent stimulation of RAW 264.7 cells with lipopolysaccharide, production of tumor necrosis factor-alpha and interleukin-6 were both enhanced between 1.5- to 5.8-fold and 4.7- to 7.9-fold, respectively, when cultured with 10(8) bifidobacteria/ml. In unstimulated EL-4.IL-2 cells, bifidobacteria had no effect on the production of interleukin-2 or interleukin-5. Upon stimulation of EL-4.IL-2 with phorbol-12-myristate-13-acetate, there were variable increases in interleukin-2 secretion (up to 2.4-fold for 10(6) Bifidobacterium Bf-1/ml) and interleukin-5 secretion (up to 4.6-fold for 10(8) B. adolescentis M101-4). The results indicated that, even when variations among strains were considered, direct interaction of most bifidobacteria with macrophages enhanced cytokine production, but the effects on cytokine production by the T-cell model were less marked. Interestingly, the 4 bifidobacteria strains used commercially for diary foods showed the greatest capacity for cytokine stimulation. The in vitro approaches employed here should be useful in future characterization of the effects of bifidobacteria on gastrointestinal and systemic immunity.