Impact of milk protein type on the viability and storage stability of microencapsulated Lactobacillus acidophilus using spray drying

Three different milk proteins — skim milk powder (SMP), sodium caseinate (SC) and whey protein concentrate (WPC) — were tested for their ability to stabilize microencapsulated L. acidophilus produced using spray drying. Maltodextrin (MD) was used as the primary wall material in all samples, milk protein as the secondary wall material (7:3 MD/milk protein ratio) and the simple sugars, d-glucose and trehalose were used as tertiary wall materials (8:2:2 MD/protein/sugar ratio) combinations of all wall materials were tested for their ability to enhance the microbial and techno-functional stability of microencapsulated powders. Of the optional secondary wall materials, WPC improved L. acidophilus viability, up to 70 % during drying; SMP enhanced stability by up to 59 % and SC up to 6 %. Lactose and whey protein content enhanced thermoprotection; this is possibly due to their ability to depress the glass transition and melting temperatures and to release antioxidants. The resultant L. acidophilus powders were stored for 90 days at 4 °C, 25 °C and 35 °C and the loss of viability calculated. The highest survival rates were obtained at 4 °C, inactivation rates for storage were dependent on the carrier wall material and the SMP/d-glucose powders had the lowest inactivation rates (0.013 day−1) whilst the highest was observed for the control containing only MD (0.041 day−1) and the SC-based system (0.030 day−1). Further increase in storage temperature (25 °C and 35 °C) was accompanied by increase of the inactivation rates of L. acidophilus that followed Arrhenius kinetics. In general, SMP-based formulations exhibited the highest temperature dependency whilst WPC the lowest. d-Glucose addition improved the storage stability of the probiotic powders although it was accompanied by an increase of the residual moisture, water activity and hygroscopicity, and a reduction of the glass transition temperature in the tested systems

Oral Serum-Derived Bovine Immunoglobulin/Protein Isolate Has Immunomodulatory Effects on the Colon of Mice that Spontaneously Develop Colitis

Dietary immunoglobulin concentrates prepared from animal plasma can modulate the immune response of gut-associated lymphoid tissue (GALT). Previous studies have revealed that supplementation with serum-derived bovine immunoglobulin/protein isolate (SBI) ameliorates colonic barrier alterations in the mdr1a-/- genetic mouse model of IBD. Here, we examine the effects of SBI on mucosal inflammation in mdr1a-/- mice that spontaneously develop colitis. Wild type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% w/w) or milk proteins (Control diet), from day 21 (weaning) until day 56. Leucocytes in mesenteric lymph nodes (MLN) and in lamina propria were determined, as was mucosal cytokine production. Neutrophil recruitment and activation in MLN and lamina propria of KO mice were increased, but were significantly reduced in both by SBI supplementation (p < 0.05). The increased neutrophil recruitment and activation observed in KO mice correlated with increased colon oxidative stress (p < 0.05) and SBI supplementation reduced this variable (p < 0.05). The Tact/Treg lymphocyte ratios in MLN and lamina propria were also increased in KO animals, but SBI prevented these changes (both p < 0.05). In the colon of KO mice, there was an increased production of mucosal proinflammatory cytokines such as IL-2 (2-fold), IL-6 (26-fold) and IL-17 (19-fold), and of chemokines MIP-1β (4.5-fold) and MCP-1 (7.2-fold). These effects were significantly prevented by SBI (p < 0.05). SBI also significantly increased TGF-β secretion in the colon mucosa, suggesting a role of this anti-inflammatory cytokine in the modulation of GALT and the reduction of the severity of the inflammatory response during the onset of colitis

Effects of n-6 and n-3 polyunsaturated fatty acids on colorectal carcinogenesis

In vivo studies have demonstrated that high fat fish oil (HFFO) diets with high levels ofn-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA; 20:5n-3) can inhibit the formation of chemically-induced colon tumors during both the initiation and post-initiation phases of colorectal carcinogenesis compared with high fat corn oil (HFCO) diets which are rich in n-6 PUFAs such as linoleic acid (LA: 18:2n-6).Studies described in this thesis show that HFFO diets also protect against the initiation of aberrant crypt foci (ACF; precursor lesions of colon cancer) in F344 rats compared to HFCO diets. Furthermore, EPA also inhibited the proliferation of human colon adenocarcinoma Caco-2 cells compared to LA. The mechanism responsible for the inhibitory effects of n-3 PUFAs such as EPA on colorectal tumors may partly be related to inhibition of PGE 2 synthesis from arachidonic acid (AA; 20:4n-6). Plasma levels of PGE 2 were indeed lower in HFFO fed rats compared to HFCO fed rats. However, reductions in PGE 2 synthesis by EPA compared to AA in Caco-2 cells did not lead to differential effects on cell proliferation, which suggests that PGE 2 is not directly involved in regulation of cell proliferation in colon cancer cells by n-6 and n-3 PUFAs.Our results suggest that lipid peroxidation-induced oxidative stress might be an important mechanism by which n-3 PUFAs possess anti-carcinogenic effects. This is supported by the fact that HFFO diets with a high amount of EPA increased the amount of lipid peroxidation in F344 rats compared to HFCO diets with a high amount of LA. Levels of malondialdehyde, which is an endproduct of lipid peroxidation were also increased after incubation of Caco-2 cells with EPA. Furthermore, transcription of genes involved in oxidative stress is increased in HFFO fed rats, whereas addition of antioxidants diminishes the anticancer effects of n-3 PUFAs in Caco-2 cells, which also suggests that oxidation of n-3 PUFAs underlies their anticancer effects. Overall, the results might imply that n-3 PUFAs protect against colon carcinogenesis via increased oxidative stress that can ultimately lead to inhibition of cell proliferation to prevent clonal expansion or induction of apoptosis to stimulate disposal of damaged colonic epithelial cells. Diets enriched with high levels of n-3 PUFAs may thus have beneficial colon cancer inhibiting effects, which may be reduced by high levels of dietary antioxidants

Nutrigenomics applied to an animal model of Inflammatory Bowel Diseases: Transcriptomic analysis of the effects of eicosapentaenoic acid-and arachidonic acid-enriched diets

In vivo models of Inflammatory Bowel Diseases (IBD) elucidate important mechanisms of chronic inflammation. Complex intestinal responses to food components create a unique “fingerprint” discriminating health from disease. Five-week-old IL10−/− and C57BL/6J (C57; control) mice were inoculated orally with complex intestinal microflora (CIF) and/or pure cultures of Enterococcus faecalis and E. faecalis (EF) aiming for more consistent inflammation of the intestinal mucosa. Inoculation treatments were compared to non-inoculated IL10−/− and C57 mice, either kept in specific pathogen free (SPF) or conventional conditions (2×5 factorial design). At 12 weeks of age, mice were sacrificed for intestinal histological (HIS) and transcriptomic analysis using limma and Ingenuity Pathway Analysis Software. Colonic HIS was significantly affected (P \u3c 0.05) in inoculated IL10−/− mice and accounted for approximately 60% of total intestinal HIS. Inoculation showed a strong effect on colonic gene expression, with more than 2000 genes differentially expressed in EF·CIF-inoculated IL10−/− mice. Immune response gene expression was altered (P \u3c 0.05) in these mice. The second study investigated the effect of arachidonic (AA) and eicosapentaenoic acid (EPA) on colonic HIS and gene expression to test whether EPA, contrary to AA, diminished intestinal inflammation in EF·CIF IL10−/− mice (2×4 factorial design). AIN-76A (5% corn oil) and AIN-76A (fat-free) +1% corn oil supplemented with either 3.7% oleic acid (OA), AA or EPA were used. IL10−/− mice fed EPA- and AA-enriched diets had at least 40% lower colonic HIS (P \u3c 0.05) than those fed control diets (AIN-76A and OA diets). The expression of immune response and ‘inflammatory disease’ genes (down-regulated: TNF, IL6, S100A8, FGF7, PTGS2; up-regulated: PPAR, MGLL, MYLK, PPSS23, ABCB4 with EPA and/or AA) was affected in IL10−/−mice fed EPA- and AA-enriched diets, compared to those fed AIN-76A diet

Folic Acid study in HT-29 cells

Low intracellular folate levels diminish the growth rate of HT-29 human colon cancer cells. This is accompanied by a metabolic shift from cytosolic glycolysis towards mitochondrial oxidative phosphorylation, as demonstrated by a lower lactate production and an increased mitochondrial oxygen consumption rate. To obtain insight in the molecular effects underlying these changes, the steady state gene expression profiles of HT-29 cells with different intracellular folate concentrations were compared. The gene expression profile of HT-29 cells with low intracellular folate levels (grown for 3 weeks in 10 ng/ml folic acid (PGA)) was clearly distinct from that of the other exposure conditions, which provide sufficient intracellular folate levels (100 ng/ml PGA, 10 ng/ml methyltetrahydrofolate (MTHF) or 100 ng/ml MTHF). Intracellular folate deficiency, contrary to expectation, did not lead to major changes in expression of genes involved in energy metabolism. This suggests that the shift towards mitochondrial oxidative phosphorylation is not mediated at the transcription level. Furthermore, only minor changes in the expression of folate metabolism related genes were observed. The changes that were observed were consistent with nucleotide salvage and in agreement with nucleotide need of the slow-growing folate-deficient HT-29 cells. The major observed effects were on cell cycle related gene expression, which was increased and interferon-responsive gene expression, which was reduced. The increase in cell cycle related gene expression seems compensatory to the reduced cell growth. Down-regulation of the interferon-response may be explained by decreased expression of signal transducer and activator of transcription 1 upon folate deficiency

Folic Acid study in HT-29 cells

Low intracellular folate levels diminish the growth rate of HT-29 human colon cancer cells. This is accompanied by a metabolic shift from cytosolic glycolysis towards mitochondrial oxidative phosphorylation, as demonstrated by a lower lactate production and an increased mitochondrial oxygen consumption rate. To obtain insight in the molecular effects underlying these changes, the steady state gene expression profiles of HT-29 cells with different intracellular folate concentrations were compared. The gene expression profile of HT-29 cells with low intracellular folate levels (grown for 3 weeks in 10 ng/ml folic acid (PGA)) was clearly distinct from that of the other exposure conditions, which provide sufficient intracellular folate levels (100 ng/ml PGA, 10 ng/ml methyltetrahydrofolate (MTHF) or 100 ng/ml MTHF). Intracellular folate deficiency, contrary to expectation, did not lead to major changes in expression of genes involved in energy metabolism. This suggests that the shift towards mitochondrial oxidative phosphorylation is not mediated at the transcription level. Furthermore, only minor changes in the expression of folate metabolism related genes were observed. The changes that were observed were consistent with nucleotide salvage and in agreement with nucleotide need of the slow-growing folate-deficient HT-29 cells. The major observed effects were on cell cycle related gene expression, which was increased and interferon-responsive gene expression, which was reduced. The increase in cell cycle related gene expression seems compensatory to the reduced cell growth. Down-regulation of the interferon-response may be explained by decreased expression of signal transducer and activator of transcription 1 upon folate deficiency