In vitro analysis of binding capacities of calcium to phytic acid in different food samples

The present work was performed to study the role which plays phytic acid in calcium binding, and to determine the calcium binding capacities in different foods, using in vitro extractions. Different food samples (soybeans, oats, chickpea, rice flour, and corn semolina) were extracted for 4 h at 37 °C using artificial simulated gastrointestinal juice (pepsin) at pH=2. The total calcium and phytic acid concentrations were determined by AAS and capillary electrophoresis, respectively, at pH=2 and pH=8 after neutralisation with a sodium hydroxide solution (3 M). Having determined the binding capacities of calcium in each food, we then use these results to estimate the fraction of calcium available for resorption during the process of digestion, when food moves from the acid pH of the stomach to the alkaline milieu of the intestines. The results obtained for the foods analysed show that the capacity of calcium to bind to phytic acid exhibits a clear pH dependence. The calculated calcium binding capacities, or the molar ratio of calcium to phytic acid in the in vitro extracted foods, varies from 3 mol calcium per mol phytic acid for soybean, chickpea and oats, to 2 mol calcium per mol phytic acid for rice, to1 mol calcium per mol phytic acid in corn semolina. Calcium may bind to one or more of the phosphate groups of phytic acid. Previous studies have demonstrated that phytic acid has the ability to bind minerals, proteins, and starch, and have then considered it as an inhibitor to the bioavailability of minerals and trace elements.     

水溶性黑木耳多糖体外结合胆酸盐能力的分析

本文通过水提醇沉提取黑木耳多糖,同时用除蛋白和不除蛋白两种方法处理,得到12种不同浓度醇沉水溶性混合多糖;并对12种多糖的体外结合胆酸盐能力进行分析。结果显示:70%乙醇醇沉得到的水溶性黑木耳多糖体外结合胆酸作用较强,并且除蛋白多糖比不除蛋白多糖体外结合甘氨酸胆酸钠的能力强。同时,多糖浓度0.8 mg/m L时,结合达到稳定,为最适宜加入量。实验结果为AAP在降血脂机制方面的进一步研究提供理论依据。      

In vivo and in vitro effects of the mycotoxins zearalenone and deoxynivalenol on different non-reproductive and reproductive organs in female pigs: a review.

This review summarizes the toxicological data on the effects of the mycotoxins zearalenone (ZON), its metabolites, and deoxynivalenol (DON) on different parameters relating to reproductive and non-reproductive organs in female pigs. In vivo, 22 mg ZON kg(-1) in the diet cause alterations in the reproductive tract of swine such as in the uterus, and affects follicular and embryo development. ZON and its metabolites have been shown to bind competitively to oestrogen receptors in an in vitro system. The feeding of pigs with a 9 mg DON kg(-1)-contaminated diet can act on protein synthesis, humoral and cellular immune response depending on dose, exposure and timing of functional immune assay, and affect liver and spleen cell structures. Beside these effects, reproductive alterations were observed in pigs, too. Both in vivo and in vitro exposure to DON decreased oocyte and embryo development. In vitro application of DON to uterine cells inhibits their proliferation rate and modulates the process of translation at a different molecular level when compared with the in vivo application. The histopathological results provide evidence of spleen and liver dysfunction in the absence of clinical signs, especially in pigs fed higher concentrations of Fusarium toxin-contaminated wheat. Prepuberal gilts react more sensitively to DON > ZON feeding compared with pregnant sows. In the liver, histopathological changes such as glycogen decrease and interlobular collagen uptake were only observed in prepuberal gilts, whereas enhancement of haemosiderin was found in both perpuberal gilts and pregnant sows. This review presents some of the current knowledge on the biological activities of ZON and DON in pig. Altogether, ZON affects reproduction of pigs most seriously because it possesses oestrogenic activity. However, DON affects reproduction in pigs via indirect effects such as reduced feed intake, resulting in reduced growth or impairment of function in vital organs such as liver and spleen.     

Novel oxidative metabolites of the mycoestrogen zearalenone in vitro

The estrogenic mycotoxin zearalenone (ZEN) is known to get metabolized to the alpha-and beta-isomers of zearalenol, but no hydroxylation products of ZEN have yet been reported as metabolites in animals or humans. We have therefore incubated ZEN with microsomes from rat liver in the presence of a nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-regenerating system and analyzed the extracted metabolites with HPLC and GC-MS after trimethylsilylation. A total of 17 in vitro metabolites were observed. The two major metabolites were tentatively identified as monohydroxylated ZEN with the newly introduced hydroxyl group localized in the aliphatic macrocyclic ring. According to the GC-MS analysis, other six monohydroxylation products of ZEN were formed as minor metabolites, together with alpha-and beta-zearalenol and monohydroxylated zearalenols. Thus, ZEN has a considerable propensity for undergoing metabolic hydroxylation reactions in vitro, and the in vivo formation and biological properties of such oxidative metabolites should now be studied.     

In vitro binding of mutagenic pyrolyzates to lactic acid bacterial cells in human gastric juice

The binding of mutagenic pyrolyzates to freeze-dried cells of Streptococcus cremoris Z-25 was investigated in human gastric juice and compared with the ability of these pyrolyzates to bind in distilled water (pH 6.30). The pH of donated gastric juice ranged from 1.2 to 7.8. Binding that occurred in gastric juice was pH dependent, and binding was affected by NaCl, CaCl2, and MgCl2. Les sof Trp-P-2 was bound in gastric juice at low pH (1.2, 1.48, and 2.1) than at pH 4 to 8. The strain bound simultaneously Trp-P-1, Trp-P-2, and Glu-P-1. The maximum amounts of Trp-P-2 bound to S. cremoris Z-25 were approximately 47.50 micrograms/mg of cells. Freeze-dried cells treated at 120 degrees C for 15 min or 80 degrees C for 3 h showed increased binding of Trp-P-2. However, binding decreased 10% after heating at 120 degrees C for 15 min. Cells nonthermally killed by gastric juice (pH 1.5) had the same Trp-P-2 binding capacity as viable cells. Lactobacillus acidophilus IFO 13951 and Bifidobacterium bifidum IFO 14252 representation of the intestinal bacteria nonthermally killed by gastric juice also showed larger binding of Trp-P-2.     

拐枣不同提取物的体外抗氧化作用

对拐枣子、拐枣和拐枣枝体积分数为80%的乙醇溶液浸提物抗氧化性进行比较,再选取拐枣为试验材料,经体积分数为80%的乙醇浸提浸膏分别用石油醚、乙酸乙酯、正丁醇、甲醇萃取,福林酚法测定不同极性部位多酚含量,通过测定DPPH自由基、ABTS自由基的清除率,研究拐枣萃取物的抗氧化作用。结果表明,拐枣对DPPH和ABTS自由基清除率分别为87.07%和95.67%,其抗氧化性高于拐枣子及拐枣枝;拐枣乙醇提取物浸膏不同极性萃取物中,乙酸乙酯萃取物的抗氧化能力最强,其多酚含量为202.8 mg/g,对DPPH和ABTS自由基清除作用的IC50分别为182 μg/mL和60 μg/mL,最大清除效率分别为94.0%和98.57%。试验表明拐枣具有较好的抗氧化能力,具有开发利用的价值。     

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.     

In vitro binding of calcium,iron and zinc by non-starch polysaccharides

The in vitro binding capacity of eight non-starch polysaccharides (agar, κ-carrageenan, gum xanthan, gum arabic, gum karaya, gum tragacanth, pectin and gum guar) was measured by equilibrium dialysis in neutral and acidic (0.1M HCl) solutions in the presence of divalent cations (Ca²⁺, Zn²⁺). No significant binding was observed in acidic conditions while, in neutral solutions, the extent of binding was correlated (P<0.1) to the cation-exchange capacity of the polysaccharides. It is apparent that the interactions are essentially electrostatic in nature, due to the presence of ionised carboxyl (uronic/pyruvic acids) and sulphated groups, in polyanionic polysaccharides. By contrast, significant binding occurs with Fe³⁺ in acidic conditions, presumably due to complexation (chelation). These data provide a clear insight into how non-starch polysaccharides interact with minerals and the potential nutritional consequence in terms of bioavailability.     

In vitro fermentation of rye carbohydrates including arabinoxylans of different structure

Freeze‐dried ileal effluent from cannulated pigs fed rye bread diets based on either whole rye or one of three rye milling fractions enriched in pericarp/testa, aleurone and endosperm was incubated in an in vitro fermentation system with faecal inocula from pigs fed a whole grain rye diet. Three and 48 hours of in vitro degradation of arabinoxylans were comparable to the caecal and faecal degradation respectively. The productions of short‐chain fatty acids were highest from the ileum samples of the diets high in fermentable carbohydrates (whole rye, aleurone and endosperm). However, fermentation of the pericarp/testa ileum sample produced half the amount of short‐chain fatty acids resulting from the other ileum samples despite the fact that the pericarp/testa carbohydrates (CHO) were not fermented at all. Treating the pericarp/testa with alkali prior to the incubation increased fermentability considerably (from 0 to 49% of total arabinoxylans) and suggested that, at least in lignified tissue, alkali‐labile cross‐links were more important determinants of arabinoxylan (AX) degradability than AX structure itself. Oxidative delignification of the pericarp/testa ileal effluent increased fermentability too, but to a much lower degree, which indicates that the type and extent of links between polymers and lignin rather than the amount of lignin are important in determining degradability. © 2000 Society of Chemical Industry     

不同饱和度脂肪酸豆甾醇酯的体外生物可给率北大核心CSCD

为了探究脂肪酸饱和度对其植物甾醇酯生物可给率的影响,采用体外模拟消化静态模型,系统研究了油酸豆甾醇酯、亚油酸豆甾醇酯和亚麻酸豆甾醇酯的体外模拟消化规律。结果表明:体外模拟消化结束后(120 min),亚油酸豆甾醇酯游离脂肪酸(FFA)释放率最高,为78.51%,其次是亚麻酸豆甾醇酯,为74.21%,油酸豆甾醇酯FFA释放率最低,为69.15%;3种豆甾醇酯的水解速率由大到小为亚麻酸豆甾醇酯>亚油酸豆甾醇酯>油酸豆甾醇酯;3种豆甾醇酯的水解度由大到小为亚油酸豆甾醇酯>亚麻酸豆甾醇酯>油酸豆甾醇酯;油酸豆甾醇酯、亚油酸豆甾醇酯和亚麻酸豆甾醇酯生物可给率分别为14.01%、11.11%和10.19%,总体差异较小。综上,豆甾醇酯中结合态的脂肪酸(相同碳链长度)饱和度影响其水解速率,但对豆甾醇酯生物可给率影响较小。     

In vitro binding of bile acids by lupin protein isolates and their hydrolysates

This study investigates the in vitro binding of bile acids by lupin, lupin protein isolates, and their hydrolysates compared to soybean products and cholestyramine. Sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glycocholate and sodium taurocholate were individually tested and analyzed spectrophotometrically by enzymatic reaction. A degree of hydrolysis of up to 20% did not affect the bile-acid binding capacity. De-oiled lupin and its hydrolysate bound all the bile acids to a significantly greater extent than de-oiled soy and its hydrolysate. Acid-soluble protein isolate from lupin showed a greater bile-acid binding capacity than acid-insoluble protein isolate. The amount of bile acid bound by acid-soluble lupin protein isolate was sometimes greater than the amount of bile acid bound by cholestyramine, which is well known as a cholesterol-reducing agent. There was no selective binding of particular types of bile acids. It can be concluded from these results that acid-soluble protein isolate from lupin may have potential application as a cholesterol-reducing agent for hypercholesterolemic patients.     

体外模拟发酵探究KGM和KOS与不同胃肠段微环境的影响

不同胃肠段微环境对机体健康的影响及其与膳食纤维的相互作用是目前研究的热点。该文为探究魔芋葡甘聚糖(konjacglucomannan,KGM)和魔芋葡甘低聚糖(konjac oligosaccharides,KOS)与不同胃肠段微环境的影响,分别以KGM和KOS为碳源,采用YCFA培养基培养不同胃肠段微生物,同时做葡萄糖(glucose)和不加碳源的空白对照,测定不同发酵液pH值、黏度、菌落总数、短链脂肪酸(short-chain datty acids,SCFAs)、总糖及还原糖含量。与对照相比,随着发酵时间的延长,添加KGM和KOS的发酵液pH降低,SCFAs含量增加,菌落总数增加;KGM显著增加胃及小肠发酵液黏度,总糖和还原糖含量略有降低,大肠发酵液中还原糖含量先增加后减少。KGM和KOS可调节不同胃肠段发酵液环境,增加微生物数量及代谢产物含量,从而更有利于机体健康,为KGM和KOS的应用提供新思路。     

In vitro fermentation of different types of α-amylase resistant corn starches

α -amylase resistant corn starches were in vitro fermented (72 h) using cecal content of rat as inoculum. Resistant starches were prepared from native (NCS), retrograded (RS_A ), and retrograded and boiled (RS_B ) corn starch by treatment with α-amylase to remove digestible starch. Short-chain fatty acid (SCFA) production, molar ratios (acetate: propionate: butyrate), fermentability with respect to lactulose and dry matter disappearance were measured. SCFA production was significantly correlated with non-fermented residue (P <0.001). The three starches showed a similar fermentability, although the rate of SCFA production and disappearance of substrate were slower for retrograded starches. NCS was fermented early while RS_A and RS_B prolonged their degradation to 72 h. There were slight differences in the molar ratios of acetate, propionate and butyrate. Structural changes in the materials were observed by scanning electron microscopy (SEM). In summary, commonly utilised domestic cooking and technological treatments modified the in vitro fermentative characteristics of resistant corn starches probably due to structural changes. Received: 15 November 1999 / Revised version: 27 January 2000     

In vitro digestion of fresh alfalfa under different conditions of ruminal pH

BACKGROUND: Relatively low ruminal pH values have been frequently registered in dairy cows grazing alfalfa, which can be involved in reducing feed digestion. An in vitro experiment was carried out to study the effect of ruminal pH (6.4, 6.1, 5.8 and 5.5) on the digestion of fresh alfalfa. RESULTS: Decreasing the pH, in vitro gas production (ivGP) decreased (P < 0.05). The lowest ivGP was registered at pH 5.5 and it was product of a higher lag time and a lower digestion rate. Dry matter disappearance (DMD) was not affected by pH at 48 h (P > 0.05). Neutral detergent disappearance (NDFD) at 48 h decreased below pH 6.1. The NDFD was reduced by 62% at pH 5.5 with respect to results at pH 6.4 and 6.1 (where the highest DMD and NDFD were observed). CONCLUSION: As expected, low rumen pH decreased alfalfa digestion. However, limits to ruminal digestion activity differed from those usually proposed for TMR diets. It is apparent that different relationships between rumen pH and NDFD exist when cows graze fresh alfalfa or grasses. Moreover, our results suggest the convenience to complement the data obtained through ivGP, DMD and NDFD. While ivGP and DMD seem to be more useful at early digestion times, NDFD may be a good predictor of final digestion. Copyright © 2009 Society of Chemical Industry     

In vitro affinity for nicotine of soft contact lenses of different materials

Smoking is a risk factor for the development of microbial keratitis and corneal infiltrates in contact lens (CL) wearers. It is still unknown if this risk is directly associated with the presence of nicotine in the eye and if adherence of nicotine on the CL can enhance these effects. A better understanding of the interaction between nicotine and CL materials could offer insights to explain this risk associated with smoking. The aim of this work was to compare the affinity of nicotine to different soft CL materials. CLs from FDA groups I, II, IV, and V were incubated in a 2-mM nicotine solution for 24 h and then in a 0.9% saline solution for the next 24 h. The amount of absorbed and released nicotine per CL was deduced as a function of time (t) by ultraviolet (UV) spectrophotometry and normalised to the mass of the hydrated CL. The data were described by the equation y = b –a t^?1, where a and b are constants, and b represents the mass reached at the plateau after ~ 10 min of exposure. Groups IV and V displayed the highest (0.80 ± 0.09 µg) and lowest (0.27 ± 0.08 µg) nicotine absorption per mg of hydrated CL, respectively. The CL affinity for nicotine could be ascribed to the interaction between the positive charge of nicotine pyrrolidine nitrogen and the negative charges of the CLs, especially for the ionic IV group.     

Vomitoxin and zearalenone content of soft wheat flour milled by different methods

Given the prominence and the growing importance of mycotoxins in human and animal health, and particularly of vomitoxin and zearalenone in people who use wheat and wheat products as their staple diet, we investigated two different types of wheat milling. Wheat produced according to good manufacturing practice related to mycotoxin risks (from sowing to harvesting) was used to compare the vomitoxin and zearalenone content of soft wheat flour, following the use of two different types of milling, traditional milling with a stone mill and modern milling with a roller mill. Moreover, the vomitoxin and zearalenone content was also evaluated in commercial stone-milled and roller-milled flours. Our results show that stone milling reduced vomitoxin and zearalenone content in flours, compared with the use of the roller-mill system.     

In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype

Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.     

Production of zearalenone in vitro and in corn grains stored under modified atmospheres

The production of zearalenone by an isolate of Fusarium equiseti was studied in chemically defined medium and in corn grains stored under modified atmospheres. An increase in the concentrations of sucrose or xylose in Czapek's medium resulted in increased toxin production, while no toxin was produced when lactose was present in the medium. Methionine (10(-2) and 10(-3) M) and cystine (10(-3) M) added to Czapek's medium inhibited zearalenone production. When amino acids or nitrogen salts were added as the sole nitrogen source, only alanine, tryptophan and NH4Cl totally inhibited zearalenone production. Zearalenone production was inhibited almost completely in high-moisture corn grains (27%) kept under atmospheres enriched with high CO2 levels (60%, 40% or 20%) with either 20% or 5% O2. However, a lower amount of CO2 was needed to inhibit fungal development and toxin formation when a reduced O2 level was applied.     

Aromatic hydroxylation is a major metabolic pathway of the mycotoxin zearalenone in vitro

Zearalenone (ZEN) is a common mycotoxin, for which only reductive metabolites have been identified so far. We now report that ZEN is extensively monohydroxylated by microsomes from human liver in vitro. Two of the major oxidative metabolites arise through aromatic hydroxylation and are catechols. Their chemical structures have been unambiguously determined by using deuterium‐labeled ZEN and by comparison with authentic reference compounds. Moreover, both catechol metabolites of ZEN were substrates of the enzyme catechol‐O‐methyl transferase. One of the monomethyl ethers represented the major metabolite when ZEN was incubated with rat liver slices, thus demonstrating that catechol formation also takes place under in vivo‐like conditions. Out of ten major human cytochrome P450 (hCYP) isoforms only hCYP1A2 was able to hydroxylate ZEN to its catechols with high activity. Catechol formation represents a novel pathway in the metabolism of ZEN and may be of toxicological relevance.