In vitro inhibitory effect of tetrahydrocurcuminoids on Fusarium proliferatum growth and fumonisin B1 biosynthesis

Many plant pathogens produce toxic metabolites when growing on food and feed. Some antioxidative components seem to prevent fungal growth and mycotoxin formation. Recently, we synthesized a new class of powerful antioxidative compounds, i.e. tetrahydrocurcuminoids, and its structure/antioxidant activity relationships have been established. The South West of France produces large amounts of corn, which can be infected by Fusarium species, particularly F. proliferatum. In this context, the efficiency of tetrahydrocurcuminoids, which can be obtained from natural curcuminoids, was investigated to control in vitro the growth of F. proliferatum and the production of its associated mycotoxin, fumonisin B₁. The relation between structure and antifungal activity was studied. Tetrahydrocurcumin (THC1), with two guaiacyl phenolic subunits, showed the highest inhibitory activity (measured as radial growth on agar medium) against the F. proliferatum development (67% inhibition at a concentration of 13.6?µmol?ml^?1). The efficiencies of THC2 (36% at a concentration of 11.5?µmol?ml^?1), which contains syringyl phenolic units, and THC3 (30% at a concentration of 13.6?µmol?ml^?1), which does not have any substituent on the aromatic rings, were relatively close. These results indicate that the simultaneous presence of guaiacyl phenols and the enolic function of the β-diketone moiety play an important role in the inhibition mechanisms. The importance of this combination was confirmed using n-propylguaiacol and acetylacetone as molecular models. Under the same conditions, ferulic acid and eugenol, other natural phenolic antioxidants, were less efficient in inhibiting fungal growth. THC1 also reduced fumonisin B₁ production in liquid medium by approximately 35, 50 and 75% at concentrations of 0.8, 1.3, and 1.9?µmol?ml^?1, respectively. These very low inhibitory concentrations show that tetrahydrocurcuminoids could be one of the most promising biobased molecules for the control of mycotoxinogen fungal strains.     

In vitro effect of some fungicides on growth and aflatoxins production by Aspergillus flavus isolated from Capsicum powder

The aim of this study was to determine the effect of some pre-harvest fungicides on growth and aflatoxin (AF) production of three Aspergillus flavus strains found in Capsicum powder. Each isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on yeast extract sucrose agar and on a 3% paprika extract agar medium supplemented with different fungicides and incubated at 20 and 30°C during 7 days. Growth measurements were obtained on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the effects of the factors (strain, medium, temperature, time and fungicides) and their interaction over colony diameter and AF production was determined. Temperature constrained the effectiveness of fungicides in reducing growth, the fungicides being most effective at 20°C. The efficacy of the fungicides over AF production depended on the medium used and temperature. The most effective fungicides in inhibiting growth and AF production, regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 80% applied at a concentration of 0.75 and 3.5?g?l^?1, respectively. Care should thus be taken in the choice of a suitable fungicide because their effectiveness may depend on intra-specific variation and temperature. Moreover, it is necessary to take into account that the most efficient fungicide in reducing growth is not always the best choice for pre-harvest treatments because it may promote AF production. Thus, the best fungicide is the one that can simultaneous prevent growth and AF production.     

In vitro effect of some fungicides on growth and aflatoxins production by Aspergillus flavus isolated from Capsicum powder

The aim of this study was to determine the effect of some pre-harvest fungicides on growth and aflatoxin (AF) production of three Aspergillus flavus strains found in Capsicum powder. Each isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on yeast extract sucrose agar and on a 3% paprika extract agar medium supplemented with different fungicides and incubated at 20 and 30°C during 7 days. Growth measurements were obtained on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the effects of the factors (strain, medium, temperature, time and fungicides) and their interaction over colony diameter and AF production was determined. Temperature constrained the effectiveness of fungicides in reducing growth, the fungicides being most effective at 20°C. The efficacy of the fungicides over AF production depended on the medium used and temperature. The most effective fungicides in inhibiting growth and AF production, regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 80% applied at a concentration of 0.75 and 3.5 g l(-1), respectively. Care should thus be taken in the choice of a suitable fungicide because their effectiveness may depend on intra-specific variation and temperature. Moreover, it is necessary to take into account that the most efficient fungicide in reducing growth is not always the best choice for pre-harvest treatments because it may promote AF production. Thus, the best fungicide is the one that can simultaneous prevent growth and AF production.     

Reduction of fumonisin B₁ in corn grits by twin-screw extrusion

Abstract: This study was designed to investigate the fate of fumonisins in flaking corn grits during twin‐screw extrusion by measuring fumonisin B₁ (FB₁) and its analogs with a mass balance approach. Food grade corn grits and 2 batches of grits contaminated with FB₁ at 10 and 50 μg/g by Fusarium verticillioides M‐2552 were processed with or without glucose supplementation (10%, w/w) with a twin‐screw extruder. Extrusion reduced FB₁ in contaminated grits by 64% to 72% without glucose and 89% to 94% with added glucose. In addition, extrusion alone resulted in 26% to 73% reduction in the levels of fumonisin B₂ and fumonisin B₃, while levels of both mycotoxins were reduced by >89% in extruded corn grits containing 10% glucose. Mass balance analysis showed that 38% to 46% of the FB₁ species detected in corn extruded with glucose was N‐(deoxy‐D‐fructos‐1‐yl)‐FB₁, while 23% to 37% of FB₁ species detected in extruded corn grits with and without added glucose was bound to the matrix. It was also found that the hydrolyzed form of FB₁ was a minor species in extruded corn grits with or without added glucose, representing <15% of the total FB₁ species present. Less than 46% of FB₁ originally present in corn grits could be detected in the fumonisin analogues measured in this study. Research is needed to identify the reaction products resulting from extrusion processing of fumonisin‐contaminated corn products. Practical Application: Twin‐screw extrusion is widely used in food industry for its versatility. This technology may reduce the level of fumonisins in corn particularly with added glucose.     

Calcium montmorillonite clay reduces urinary biomarkers of fumonisin B₁ exposure in rats and humans

Fumonisin B? (FB?) is often a co-contaminant with aflatoxin (AF) in grains and may enhance AF's carcinogenicity by acting as a cancer promoter. Calcium montmorillonite (i.e. NovaSil, NS) is a possible dietary intervention to help decrease chronic aflatoxin exposure where populations are at risk. Previous studies show that an oral dose of NS clay was able to reduce AF exposure in a Ghanaian population. In vitro analyses from our laboratory indicated that FB? (like aflatoxin) could also be sorbed onto the surfaces of NS. Hence, our objectives were to evaluate the efficacy of NS clay to reduce urinary FB? in a rodent model and then in a human population highly exposed to AF. In the rodent model, male Fisher rats were randomly assigned to either FB? control, FB??+?2% NS or absolute control group. FB? alone or with clay was given as a single dose by gavage. For the human trial, participants received NS (1.5 or 3?g?day?1) or placebo (1.5?g?day?1) for 3 months. Urines from weeks 8 and 10 were collected from the study participants for analysis. In rats, NS significantly reduced urinary FB? biomarker by 20% in 24?h and 50% after 48?h compared to controls. In the humans, 56% of the urine samples analysed (n?=?186) had detectable levels of FB?. Median urinary FB? levels were significantly (p?90% in the high dose NS group (3?g?day?1) compared to the placebo. This work indicates that our study participants in Ghana were exposed to FB? (in addition to AFs) from the diet. Moreover, earlier studies have shown conclusively that NS reduces the bioavailability of AF and the findings from this study suggest that NS clay also reduces the bioavailability FB?. This is important since AF is a proven dietary risk factor for hepatocellular carcinoma (HCC) in humans and FB? is suspected to be a dietary risk factor for HCC and oesophageal cancer in humans.     

Identification of the first fumonisin mycotoxins with three acyl groups by ESI-ITMS and ESI-TOFMS following RP-HPLC separation: palmitoyl, linoleoyl and oleoyl EFB₁ fumonisin isomers from a solid culture of Fusarium verticillioides

The aim of this study was to apply RP-HPLC/ESI-ITMS and RP-HPLC/ESI-TOFMS to investigate and characterise six new higher molecular weight fumonisins (three pairs of isomers) extracted from a Fusarium verticillioides-infected solid rice culture. The ITMS and ITMS2 spectra clearly indicated the m/z values (960, 984 and 986) of the protonated molecules and the FB? toxin-like structures of these compounds, respectively. Moreover, the data evaluation software of the TOFMS equipment unambiguously demonstrated the exact masses of the protonated molecules and the suggested empirical formulae (C??H??NO??, C??H??NO?? and C??H??NO??) of the new fumonisins, with mass accuracy in the range between 0.1 and -1.1 ppm. Subtraction of the empirical formula of FB? toxin (C??H??NO??) from these formulae and correction for the mass of water split-off from the fumonisin molecule during ester formation resulted in the empirical formulae of the fumonisin backbone esterifying agents (fatty acids): C??H??O? (palmitic acid, PA), C??H??O? (linoleic acid, LA) and C??H??O? (oleic acid, OA). We denoted the new compounds as esterified FB? (EFB?) toxins, with the suggested names EFB? PA, iso-EFB? PA, EFB? LA, iso-EFB? LA, EFB? OA and iso-EFB? OA. The total amount of these new compounds comprised 0.1% of the FB? concentration, which may be rated as significant when it is considered that these new components are significantly more apolar than earlier-described fumonisins, and their uptake into and toxicity elicited in the various tissues of living organisms may therefore also be significantly different from those of other fumonisins.     

In vitro α-glucosidase and pancreatic lipase inhibitory activities and antioxidants of Samnamul (Aruncus dioicus) during rehydration and cooking

In vitro α-glucosidase and pancreatic lipase inhibitory activities of ethanol extracts of samnamul (dried young shoots of Aruncus dioicus) during rehydration (soaking, 30 min boiling, and re-soaking in water) and subsequent cooking at 180°C with/without perilla oil were determined for evaluation of its anti-diabetic and antiobesity potential. Polyphenol and flavonoid contents were monitored by spectrophotometry. Rehydration and boiling of samnamul reduced α-glucosidase and pancreatic lipase inhibitory activities as well as polyphenol and flavonoid contents, whereas soaking time did not have a significant effect. Of the rehydration and cooking processes, 30 min boiling was the most critical in reducing anti-diabetic and anti-obesity activities as well as antioxidant contents. Perilla oil addition improved α-glucosidase inhibitory activity, possibly through polyphenol addition. These results suggest that dietary intake of samnamul can reduce the risks of diabetes and obesity, and perilla oil can additionally improve its health benefits.     

Evaluating the effect of temperature on microbial growth rate--the Ratkowsky and a Bělehrádek-type models

The objective of this paper to conduct a parallel comparison of a new Bělehrádek-type growth rate (with an exponent of 1.5, or the Huang model), Ratkowsky square-root, and Ratkowsky square equations as secondary models for evaluating the effect of temperature on the growth of microorganisms. Growth rates of psychrotrophs and mesophiles were selected from the literature, and independently analyzed with the 3 models using nonlinear regression. Analysis of variance (ANOVA) was used to compare the means of growth rate (μ), estimated minimum temperature (T(min) ), approximate standard errors (SE) of T(min) , model mean square errors (MSE), accuracy factor (A(f) ), bias factor (B(f) ), relative residual errors (δ), Akaike information criterion (AICc), and Bayesian information criterion (BIC). Based on the estimated T(min) values, the Huang model distinctively classified the bacteria into 2 groups (psychrotrophs and mesophiles). No significant difference (P > 0.05) was observed among the means of the μ values reported in the literature or estimated by the 3 models, suggesting that all 3 models were suitable for curve fitting. Nor was there any significant difference in MSE, SE, δ, A(f) , B(f) , AICc, and BIC. The T(min) values estimated by the Huang model were significantly higher than those estimated by the Ratkowsky models, but were in closer agreement with the biological minimum temperatures for both psychrotrophs and mesophiles. The T(min) values estimated by the Ratkowsky models systematically underestimated the minimum growth temperatures. In addition, statistical estimation showed that the mean exponent for the new Bělehrádek-type growth rate model may indeed be 1.5, further supporting the validity of the Huang model.     

The effect of pectin concentration and degree of methyl-esterification on the in vitro bioaccessibility of β-carotene-enriched emulsions

Soluble fibers, like pectin, are known to influence the physicochemical processes during the digestion of dietary fat and may therefore affect the absorption of lipophilic micronutrients such as carotenoids. The objective of the current work was to investigate whether the pectin concentration and degree of methyl-esterification (DM) influence the bioaccessibility of carotenoids loaded in the oil phase of oil-in-water emulsions. The in vitro β-carotene bioaccessibility was determined for different oil-in-water emulsions in which 1 or 2% citrus pectin with a DM of 99%, 66% and 14% was present. Results show that pectin concentration and DM influence the initial emulsion properties. The most stable emulsions with the smallest oil droplets (D(v,0.9) of 15–16 μm) were obtained when medium or high methyl-esterified pectin was present in a 2% concentration while gel-like pectin structures (D(v,0.9) of 114 μm), entrapping oil droplets, were observed in the case where low methyl-esterified pectin was present in the aqueous emulsion phase. During in vitro stomach digestion, these gel-like structures, entrapping β-carotene loaded oil droplets, significantly enlarged (D(v,0.9) of 738 μm), whereas the emulsion structure could be preserved when the medium or high methyl-esterified pectin was present. Initial emulsion viscosity differences, due to pectin concentration and especially due to pectin DM, largely disappeared during in vitro digestion, but were still significant after the stomach digestion phase. The observed differences in emulsion structure before and during in vitro digestion only resulted in a significant difference between emulsions containing low methyl-esterified pectin (β-carotene bioaccessibility of 33–37%) and medium/high methyl-esterified pectin (β-carotene bioaccessibility of 56–62%).     

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.     

A new mechanistic growth model for simultaneous determination of lag phase duration and exponential growth rate and a new Bĕlehdrádek-type model for evaluating the effect of temperature on growth rate

A new mechanistic growth model was developed to describe microbial growth under isothermal conditions. The new mathematical model was derived from the basic observation of bacterial growth that may include lag, exponential, and stationary phases. With this model, the lag phase duration and exponential growth rate of a growth curve were simultaneously determined by nonlinear regression. The new model was validated using Listeria monocytogenes and Escherichia coli O157:H7 in broth or meat. Statistical results suggested that both bias factor (B_f) and accuracy factor (A_f) of the new model were very close to 1.0. A new B?lehdrádek-type rate model and the Ratkowsky square-root model were used to describe the temperature dependence of bacterial growth rate. It was observed that the maximum and minimum temperatures were more accurately estimated by a new B?lehdrádek-type rate model. Further, the inverse of square-roots of lag phases was found proportional to temperature, making it possible to estimate the lag phase duration from the growth temperature.     

Yeast cell based feed additives: studies on aflatoxin B₁ and zearalenone

Thirty commercially available yeast cell wall products and two reference bentonites were tested for their ability to bind aflatoxin B(1) (AFB1) and zearalenone (ZON) in buffer solutions at pH 3 and pH 6.5 as well as in real gastric juice. For most products, the binding efficacy of AFB1 correlated with the ash content, which was between 2.6 and 89%, and constituted the inorganic non-volatile components, such as mineral clays, of the samples. Samples with smectite as the main ash component showed the highest binding efficacy; yet, a correlation with the content of mannanooligosaccharides (MOS) and β-glucans from yeast cell walls was not observed. Products containing >30% ash showed AFB1 adsorption values >90% at least in one of the investigated media whereas most products with <10% ash did not exceed adsorption rates of 20%. In the case of ZON, adsorption efficiency ranged between 10 and 60%. It tended to be lowest for products with MOS and β-glucan contents <10% and greatest for products with MOS and β-glucan contents >50%. However, there was no general correlation between the adsorption of ZON and the concentration of MOS and β-glucans. Different products of one brand sold in different countries were observed to bind AFB1 to different degrees, which was explained by the difference in ash contents and mineral composition. In the case of ZON, differences in adsorption between products of the same brand were less pronounced.     

In vitro exposure of Acer negundo pollen to atmospheric levels of SO₂ and NO₂: effects on allergenicity and germination

In the last years, a rising trend of pollen allergies in urban areas has been attributed to atmospheric pollution. In this work, we investigated the effects of SO(2) and NO(2) on the protein content, allergenicity, and germination rate of Acer negundo pollen. A novel environmental chamber was assembled to exposure pollen samples with SO(2) or NO(2) at two different levels: just below and two times the atmospheric hour-limit value acceptable for human health protection in Europe. Results showed that protein content was lower in SO(2)-exposed pollen samples and slightly higher in NO(2)-exposed pollen compared to the control sample. No different polypeptide profiles were revealed by SDS-PAGE between exposed and nonexposed pollen, but the immunodetection assays indicated higher IgE recognition by all sera of sensitized patients to Acer negundo pollen extracts in all exposed samples in comparison to the nonexposed samples. A decrease in the germination rate of exposed in contrast to nonexposed pollen was verified, which was more pronounced for NO(2)-exposed samples. Our results indicated that in urban areas, concentrations of SO(2) and NO(2) below the limits established for human protection can indirectly aggravate pollen allergy on predisposed individuals and affect plant reproduction.     

Influence of addition of Raftiline HP on the growth, proteolytic, ACE- and α-glucosidase inhibitory activities of selected lactic acid bacteria and Bifidobacterium

This study examined the influence of Raftiline HP^®, added at the rate of 1, 2 and 3 g 100 mL⁻¹ to reconstituted skim milk, on the growth and biochemical activities of selected strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium longum. The growth of B. longum and S. thermophilus was improved in reconstituted skim milk (RSM) containing 1 g 100 mL⁻¹ Raftiline HP^®. All the organisms except for S. thermophilus produced more lactic acid and acetic acid in the presence of Raftiline HP^® than in the control. L. acidophilus and B. longum showed improvement in the proteolytic capabilities at all the three levels of Raftiline HP^® addition. L. delbrueckii ssp. bulgaricus showed maximum percent ACE inhibition in RSM containing 2 g 100 mL⁻¹ Raftiline HP^® while B. longum exhibited this potential in RSM containing 3 g 100 mL⁻¹ Raftiline HP^®. All organisms, except L. delbrueckii ssp. bulgaricus, however, showed improvement in the α-glucosidase inhibitory activity in RSM containing Raftiline HP^®.     

In vitro iron absorption of α-lactalbumin hydrolysate-iron and β-lactoglobulin hydrolysate-iron complexes

To study the feasibility of promoting iron absorption by peptides derived from α-lactalbumin and β-lactoglobulin, the present work examined the transport of iron across Caco-2 monolayer cell as in vitro model. Caco-2 cells were seeded in bicameral chambers with α-lactalbumin hydrolysate-Fe (α-LAH-Fe) complex and β-lactoglobulin hydrolysate-Fe (β-LGH-Fe) complex, α-LAH and iron mixture, β-LGH and iron mixture, FeSO₄ and ascorbic acid mixture, and FeSO₄. In addition, the cytotoxicity of α-LAH-Fe and β-LGH-Fe complexes were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The iron absorption and ferritin content were assessed using the coupled in vitro digestion/Caco-2 cell model. Results support that peptide-iron complexes can promote ferritin formation and it is possible to apply β-LGH-Fe complexes as iron-fortified supplements with high iron absorbability.     

The effect of glycation on foam and structural properties of β-lactoglobulin

The goal of the present work was to evaluate whether the Maillard reaction, with glucose and lactose as substrates, improves the foaming properties of β-lactoglobulin. Lactose led to the lowest degree of modification without significant differences by reaction time and by protein:sugar molar ratio. However, in the case of glucose, the degree of glycation increases with reaction time and molar ratio. The results obtained by UV fluorescence, surface hydrophobicity and differential scanning calorimetry clearly showed differences in the degree of folding of β-lactoglobulin upon modification with different sugars or thermal treatment, with changes in the foaming capacity of β-lactoglobulin. All the modified samples exhibited a significant increase (α ? 0.05) in draining stability (Kg) as compared to the non-thermally treated sample. In addition, foams formed by lactose-glycated samples were more stable than those formed by glucose-glycated ones. A significant increase (α ? 0.05) of foam stability with reaction time was also detected, particularly in glucose-glycated samples.     

Collagen production ability of milk basic protein is dependent on stimulatory effect of transforming growth factor-β1 and β2

Milk basic protein (MBP), a mixture of positively charged whey proteins extracted from cows' milk, regulates bone-turnover and stimulates collagen production in osteoblasts. Whey contains growth factors, including transforming growth factor (TGF)-β1, which enhances collagen production in fibroblasts and osteoblasts. However, the extent to which bovine TGF-β1 and TGF-β2 in MBP contribute to collagen production in human mesenchymal cells has not been clarified. The separated MBP fractions increased the concentration of procollagen C-terminal peptide in human dermal fibroblasts in correlation to the bovine TGF-β1 and TGF-β2 concentrations. A similar trend was observed with respect to bovine TGF-β2 and human osteoblasts. These stimulatory effects were inhibited by ALK5 inhibitor II as a TGF-β receptor I signal inhibitor. Accordingly, bovine TGF-β1 and TGF-β2 in MBP could induce collagen production in human mesenchymal lineages, such as fibroblasts and osteoblasts. These results may extend the use of MBP as a functional food ingredient.