Immunostimulatory effect of β-cryptoxanthin in vitro and in vivo

Beta-cryptoxanthin (β-Crp), a mono-hydroxylated β-carotene, is a major dietary provitamin A xanthophyll. The immunostimulatory effect of β-Crp was examined using human hybridoma HB4C5 cells and mouse primary lymphocytes in vitro and using mice in vivo. IgM production by the HB4C5 cells and both IgA and IgG productions by mouse primary lymphocytes from mesenteric lymph node and spleen were accelerated in vitro by the treatment of β-Crp in dose-dependent manners. Afterwards 6-week-old female BALB/c mice were administered with a low or high dose of β-Crp for 14 days, mesenteric lymph node and spleen were excised and the immunoglobulin production levels were evaluated using lymphocytes from mesenteric lymph node and spleen in vivo. Results exhibited that all of IgA, IgG, and IgM productions by lymphocytes from mesenteric lymph node were increased. Especially, the IgG production increased with statistically significant differences in both of the low and high dose groups against the control group. The immunostimulatory effect on splenocytes was also observed although not as clear as on lymphocytes from mesenteric lymph node. Overall results suggest that β-Crp may stimulate the humoral immunity in mammals. Hence, β-Crp might have a potentially significant impact on human health and on prevention of diseases.     

Diet high in oat β-glucan activates the gut-hypothalamic (PYY₃₋₃₆-NPY) axis and increases satiety in diet-induced obesity in mice

This study tested the effects of (1→3),(1→4) β‐D ‐glucan from oats, on activation of the gut‐hypothalamic (PYY_3–36‐NPY) axis, satiety, and weight loss in diet‐induced obesity (DIO) mice. DIO mice were fed standard lab chow diets or varied doses of β‐glucan for 6 weeks. Energy intake, satiety, body weight changes and peptide Y‐Y_3‐36 (PYY_3‐36) were measured together with a satiety test and measurement of neuropeptide Y (NPY) mRNA expression in the hypothalamic arcuate nucleus (Arc). The average energy intake (‐13%, p<0.05) and body weight gain was lower with increasing β‐glucan over 6 wk with acute suppression of energy intake over 4 h. The highest β‐glucan diet significantly increased plasma PYY_3‐36, with suppression of Arc NPY mRNA.     

Exogenous β-mannanase supplementation improved immunological and metabolic responses in lactating dairy cows

Exogenous enzymes have been used to improve nutrient utilization in several species of livestock, particularly swine and poultry. In addition, improved immunological and metabolic traits have been reported in nonruminants. The objective of this study was to determine the effects of β-mannanase supplementation on milk yield and composition, and immunological and metabolic responses in lactating Holstein dairy cows. Two weeks after calving, 20 Holstein cows (10 multiparous and 10 primiparous) were blocked by parity and assigned to 1 of 2 diets for 182 d. All cows were housed in the same environment and fed the same basal diet. The basal diet of the treatment group was supplemented with β-mannanase (CTCBio Inc., Seoul, South Korea) at 0.1% of concentrate dry matter. No differences were detected between the control and enzyme supplement groups in milk yield parameters or milk composition. Supplementation of β-mannanase enzyme reduced blood haptoglobin levels in supplemented multiparous cows compared with controls. Furthermore, nonesterified fatty acid concentration levels tended to be lower in cows fed β-mannanase, regardless of parity. Neither immunoglobulin G nor milk somatic cell count was affected by β-mannanase supplementation, regardless of parity. The number of insemination services tended to be lower in cows fed diets supplemented with β-mannanase. Results from this study suggest that supplementation of β-mannanase exogenous enzyme could help to reduce instances of systemic inflammation and decrease fat mobilization in lactating Holstein cows. Multiparous cows are considered susceptible to acute infections and inflammation; thus, the enzyme had a greater effect in multiparous cows.     

Administration of a β-glucan-enriched extract activates beneficial hepatic antioxidant and anti-inflammatory mechanisms in streptozotocin-induced diabetic rats

The beneficial effect of a commercially available β-glucan-enriched extract (BGEE) from cereal grain, against a diabetes-induced hepatic redox imbalance and inflammatory response in streptozotocin-induced diabetic rats was evaluated. Diabetic rats that were treated with BGEE exhibited lower hyperglycaemia and improved biochemical parameters of liver damage. BGEE attenuated hepatic oxidative stress, revealed by a decreased concentration of thiobarbituric acid reactive substances and a restored GSH/GSSG ratio. BGEE also exerted an anti-inflammatory effect on the liver, evidenced by the normalization of the serum concentrations of the “positive” and “negative” acute-phase proteins, α₂-macroglobulin and albumin, respectively, as well as by upregulation of anti-inflammatory cytokine IL-10 and IL-4 mRNA expression, and inhibition of RAGE/NF-κB signaling. These findings suggest that BGEE application exerts a beneficial effect in the liver of streptozotocin-induced diabetic rats via its antioxidant and anti-inflammatory activities, and that it therefore possesses an important potential in diabetes management.     

Allergenic and immunogenic potential of cow's milk β-lactoglobulin and caseins evidenced without adjuvant in germ-free mice

Scope: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ‐free (GF) mice exhibit Th2‐polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant. Methods and results: β‐lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ‐ and αS1‐caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG‐reactivation, IL‐4, IL‐5, IL‐13 and IFN‐γ productions by splenocytes were higher in GF mice than in CV mice. Heat‐treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat‐treatment increased protein immunogenicity and led to the production of anti‐BLG and anti‐κ‐casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL‐4, IL‐5 and IL‐13 in BLG‐reactivated splenocytes from CV mice. Conclusion: Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.     

Immunoreactive properties of α-casein and κ-casein: Ex vivo and in vivo studies

The aim of this study was to evaluate the ex vivo and in vivo studies immune potential of α- and κ-casein. Ex vivo, naïve mouse splenocytes were stimulated with α- or κ-casein. After 120 h of culture, the proliferation index (PI), determined by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, did not vary for either antigen, suggesting similar ex vivo immunogenic potential of both casein fractions. In vivo, BALB/ccmdb mice were sensitized with α- or κ-casein and then gavaged with primary antigen. Mice immunized with α-casein had higher levels of IgG (2^16.33) and IgA (2^10.22) in serum at the end of the experiment compared with mice immunized with κ-casein (2¹⁵ and 2^9.3 for IgG and IgA, respectively). The use of α-casein for mouse immunization and ex vivo lymphocyte stimulation resulted in higher concentrations of secreted cytokines (IL-4, IL-10) compared with κ-casein stimulation. This is consistent with increasing regulatory T cell (Treg) lymphocyte populations, independent of the antigen used for stimulation. In summary, the immunogenic potential of α- and κ-casein was similar. Humoral and cellular immune responses confirmed their strong, independent potential to induce B and T cells. We propose that the lymphocyte proliferation index be used as an initial screening for protein immunogenicity.     

Grape skin extract inhibits mammalian intestinal α-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice

Intestinal α-glucosidases are the key enzymes responsible for starch digestion and absorption and their inhibition has been proven effective in both preventing and treating diabetes through improvement of postprandial hyperglycaemia. This study, for the first time, identified that a Norton grape skin extract (GSE) significantly inhibited mammalian intestinal α-glucosidases but not other digestive enzymes including structurally relevant pancreatic α-amylase. Norton GSE inhibited rat intestinal α-glucosidases through a competitive mode with an IC₅₀ of 0.384 mg/ml. Further animal study revealed that the oral intake of Norton GSE (400 mg/kg) significantly reduced postprandial blood glucose by 30.9% in the streptozocin-treated male C57BL/6 J mice following starch challenge. These findings suggest that Norton GSE may have a unique property of suppressing postprandial blood glucose through a mechanism involving the inhibition of α-glucosidases, thereby providing a novel dietary opportunity for diabetes management.     

γ-Aminobutyric acid treatment reduces chilling injury and activates the defence response of peach fruit

Peach fruit were immersed in 5 mM γ-aminobutyric acid (GABA) solution for 10 min at 20 °C and then stored at 1 °C for 5 weeks to investigate the effect of GABA treatment on chilling injury (CI), antioxidant enzymes and energy status in peach fruit. The results showed that GABA treatment significantly inhibited CI incidence of peaches and enhanced activities of antioxidant enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, glutathione S-transferase, monodehydroascorbate reductase and dehydroascorbate reductase. The treatment also increased contents of adenosine triphosphate and adenosine diphosphate, but lowered adenosine monophosphate content, which resulted in a higher level of energy charge in treated fruit. These results indicated that GABA increased chilling tolerance of peach fruit through enhancing its enzymatic antioxidant system and maintaining energy status in peach fruit.     

Growth inhibitory efficacy of lycopene and β-carotene against androgen-independent prostate tumor cells xenografted in nude mice

Scope : In this study, we evaluated the efficacy of lycopene against the growth of prostate cancer in vivo. Methods and results : Athymic nude mice were implanted subcutaneously with human androgen‐independent prostate carcinoma PC‐3 cells. They were supplemented with a low or a high dose of lycopene (4 and 16 mg/kg) and a single dose of β‐carotene (16 mg/kg) twice a week for 7 wk. At the end of the experiment, both lycopene and β‐carotene strongly inhibited the tumor growth, as evidenced by the decrease in tumor volume and tumor weight. High‐dosage lycopene and β‐carotene significantly decreased the expression of proliferating cell nuclear antigen in tumor tissues and increased the levels of insulin‐like growth factor‐binding protein‐3 in plasma. In addition, high‐dosage lycopene supplementation significantly decreased the vascular endothelial growth factor (VEGF) levels in plasma. In contrast, β‐carotene supplementation significantly increased the VEGF levels, as compared with tumor control group. Conclusion : Lycopene and β‐carotene supplementation suppressed the growth of prostate tumor cells, and the effects are likely associated with reduction of proliferation (attenuation of proliferating cell nuclear antigen expression) and with interference of the insulin‐like growth factor 1 signaling (increased plasma insulin‐like growth factor‐binding protein‐3 levels). Furthermore, the inhibition of VEGF by lycopene suggests that the antitumor mechanisms of lycopene also involve anti‐angiogenesis.     

Variants of β-casofensin,a bioactive milk peptide,differently modulate the intestinal barrier: In vivo and ex vivo studies in rats

β-Casofensin is a bioactive milk peptide that modulates the intestinal barrier, particularly through its action on goblet cells. β-Casofensin corresponds to fragment (f) 94–123 of the bovine β-casein (β-CN) A2 variant. Fifteen genetic variants of bovine β-CN (A1–3, B–G, H1–2, I–L) are known, of which the A2, A1, and B forms are the most common. These variants differ from each other by the substitution of one or more amino acids, some of which are localized in f94 to 123. The aim of our study was to compare the intestinal effects of β-casofensin A2 and its 3 main variants: A1, A3, and B. For this purpose, a solution (0.1 µM; 10 μL/g of body weight, postnatal d 10–20) containing β-casofensin A2, one of its variants (A1, A3, or B), or drinking water (control; CT) was administered to rat pups orally. After euthanasia (postnatal d 20), intestinal segments were collected for biochemical and histochemical analysis and also used to determine paracellular permeability to fluorescein isothiocyanate-labeled 4-kDa dextran in an Ussing chamber. We also studied the direct effects of β-casofensin A2 and its A1 variant on the paracellular permeability of jejunum segments of adult rats. β-Casofensin A2 and its B variant significantly increased the population of goblet cells compared with the CT, A1, and A3 groups. The mucin 2 mRNA level was significantly higher in the β-casofensin A2 group than in the CT, A3, and B groups. Our results also revealed that the protein expression of zonula occludens-1 and occludin was reduced in the jejunum of rats in the A1, A3, and B groups compared with the CT group. However, the A1 variant was the only peptide to decrease jejunal permeability compared with the CT group. This variant, tested directly in the apical compartment of an Ussing chamber at a concentration of 0.1 nM, also reduced jejunal permeability. In conclusion, the substitution of a single amino acid alters the effect of β-CN sequence f94 to 123 on goblet cells and on intestinal permeability. A genetic polymorphism of β-CN can affect the biological activity of peptides derived from this protein. These data should be taken into account in the production of bioactive foods.     

Identification of bioactive peptides and quantification of β-casomorphin-7 from bovine β-casein A1, A2 and I after ex vivo gastrointestinal digestion

This study investigated whether different genetic variants of β-casein (β-CN) give rise to different bioactive peptides during digestion. β-CN was purified from bovine milk of genetic variants A1, A2 and I, and digested with human gastrointestinal juices in a static ex vivo model. Mass spectrometry analyses revealed that the peptide ⁶⁰YPFPGPIPN⁶⁸ was exclusively identified from variants containing proline at position 67. Most strikingly, the opioid peptide β-casomorphin-7, ⁶⁰YPFPGPI⁶⁶, was identified from both variants A1 and A2 after simulated digestion, though with concentration being somewhat higher after digestion of the variant A1, compared with variants A2 and I. The peptides ¹³⁴HLPLP¹³⁸ and ¹³³LHLPLP¹³⁸ were both identified after initial 5 min of duodenal digestion. In conclusion, genetic variation of β-CN may affect proteolysis during digestion; however, the release of β-casomorphin-7 (BCM7) does not seem to be linked solely to variant A1, as earlier suggested by relevant published literature on in vitro digestion.     

Effect of milk protein composition and amount of β-casein on growth performance,gut hormones,and inflammatory cytokines in an in vivo piglet model

The objective of this work was to better understand the effect of differences in milk protein composition, and specifically, a change in β-casein to total casein in a milk-based matrix, on growth performance and metabolic and inflammatory responses using a piglet model. Three formulas were optimized for piglets, with similar metabolizable energy, total protein content, and other essential nutrients. Only the protein type and ratio varied between the treatments: the protein fraction of the control diet contained only whey proteins, whereas 2 other matrices contained a whey protein to casein ratio of 60:40, and differed in the amount of β-casein (12.5 and 17.1% of total protein). Piglets fed formula containing whey proteins and caseins, regardless of the concentration of β-casein, showed a significantly higher average daily gain, average daily feed intake, and feed efficiency compared with piglets consuming the formula with only whey protein. Consumption of the formula containing only whey protein showed higher levels of plasma glucagon-like peptide-1 and ghrelin compared with the consumption of formula containing casein and whey protein. A positive correlation was observed between postprandial time and glucagon-like peptide-1 response. The intestinal pro-inflammatory cytokine tumor necrosis factor α increased significantly in piglets fed the whey protein/casein diet compared with those fed whey protein formula. All formula-fed piglets showed a lower level of IL-6 cytokine compared with the ad libitum sow-fed piglets, regardless of composition. No significant differences in the anti-inflammatory IL-10 concentration were observed between treatment groups. Milk protein composition contributed to the regulation of piglets‘ metabolic and physiological responses, with whey protein/casein formula promoting growth performance and a different immune regulatory balance compared with a formula containing only whey protein. Results indicated no differences between treatments containing different levels of β-casein.     

Effect of oral administration of β-glucans derived from Aureobasidium pullulans SM-2001 in model mice and rat with atopic dermatitis-like phenotypes

Food Science and Biotechnology - In this study, we investigated the anti-atopic dermatitis (AD) activity of β-glucans derived from Aureobasidium pullulans SM-2001 (βGdAP). βGdAP was...     

Simultaneous and confirmative detection of multi-residues of β2-agonists and β-blockers in urine using LC-MS/MS/MS coupled with β-receptor molecular imprinted polymer SPE clean-up

A liquid chromatography–linear ion-trap spectrometry (LC-MS³) method using β-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 β₂-agonists and 21 β-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by β-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using β-receptor MIP SPE. A Supelco Ascentis^® express Rp-Amide column was used to separate the analytes, and MS³ detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 μg l^–1. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02–0.18 and 0.05–0.60 μg l^–1, respectively. As a result of the selective clean-up by MIP SPE and MS³ detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring β₂-agonist and β-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.