Effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion

The effect of some polyphenols (resveratrol, catechin, epigallocatechin-3-gallate, and quercetin) and beverages (red wine and green tea) on the enzymatic activity of pepsin during the digestion of three different substrates (pork meat, insoluble azocasein, and denatured hemoglobin) has been investigated. The tested polyphenols and beverages increase the initial velocity of the reaction, and the activating effect is concentration dependent. The order of effectiveness of polyphenols in increasing the initial velocity of the reaction is resveratrol > or = quercetin > epigallocatechin-3-gallate > catechin. The kinetic data obtained with soluble denatured hemoglobin show that the K(m) for the substrate is not changed, whereas the V(max) of the reaction is increased. Pepsin activity follows a simple Michaelis-Menten kinetic suggesting that k(3) is increased by polyphenols. To the authors' knowledge, the present study is the first demonstration that some polyphenols and related beverages are able to enhance the enzymatic activity of pepsin.     

Growth and competitiveness of the New Zealand tree species Podocarpus cunninghamii is reduced by ex-agricultural AMF but enhanced by forest AMF

The composition of arbuscular mycorrhizal fungi (AMF) communities found in agricultural systems has been found to be very different to that of forest. The implications of this, if any, for the restoration of indigenous forest on ex-agricultural land is poorly understood. This study investigated the effect that AMF communities isolated from ex-agricultural and forest soils have on the growth of an indigenous New Zealand tree species (Podocarpus cunninghamii). The forest AMF community was isolated from a remnant stand of P. cunninghamii forest and the ex-agricultural AMF from a retired grazing grassland. In addition, the study examined how the two AMF communities affected the competitiveness of P. cunninghamii when grown in competition with an invasive grass species (Agrostis capillaris), which is frequently dominant on ex-agricultural land in New Zealand. P. cunninghamii growth was significantly decreased by inoculation with ex-agricultural AMF compared to forest AMF. Furthermore, the forest AMF community was able to significantly increase P. cunninghamii root production when in competition with A. capillaris. The findings suggest that when attempting to restore indigenous forest on ex-agricultural land, inoculation of tree seedlings with appropriate forest AMF may improve their growth and survival.     

Fungal solubilization of rock phosphate is enhanced by forming fungal-rhizobial biofilms

Biosolubilization of rock phosphate (RP) using a Penicillium spp., an Aspergillus spp., Pleurotus ostreatus, Bradyrhizobium elkanii SEMIA 5019 and their fungal-rhizobial biofilms was investigated. Eppawala Rock Phosphate (ERP, total P concentration 17.6%), a RP from a deposit in Sri Lanka was used. Penicillium spp.-B elkanii SEMIA 5019 biofilm released the highest amount of P from the ERP with the highest P release-to-P uptake ratio. The P release of Penicillium spp. alone was significantly lower than that of its biofilm. Similarly, P. ostreatus-B. elkanii SEMIA 5019 biofilm showed a higher P release than P. ostreatus alone. However, P. ostreatus alone or its biofilm showed lower P release-to-P uptake ratios indicating relatively higher P uptake compared to the P release. The Aspergillus spp., showed a moderate P release. Large bradyrhizobial cell clusters attached to the mycelial mat of Penicillium spp. and P. ostreatus were observed under light microscope after 12 and 25 days of incubation, respectively. The present study, identified an effective method of fungal-rhizobial biofilm mediated solubilization of RP.     

Ethyl ester formation is enhanced by ethanol addition in mini Swiss cheese with and without added propionibacteria

Esters are important contributors to cheese flavor, but their mechanisms of synthesis in cheese are largely unknown. This study aimed to determine whether ethanol concentration limits the formation of ethyl esters in cheese. Mini Swiss cheeses were manufactured with (E) or without (C) the addition of ethanol to cheese milk. Ethanol concentrations (enzymatic analysis) were 64 +/- 17 and 330 +/- 82 microg g(-1), respectively, in C and E cheeses. E cheeses also contained 5.4 +/- 2.3 times more of the five ethyl esters quantified than C cheeses, regardless of the concentrations of esters in C cheeses (range 1-128 ng g(-1)). Furthermore, the presence of propionibacteria added as acid-producing secondary starters was associated with greater concentrations of esters, due to the increase in acid concentrations that propionibacteria induced and/or to an involvement of propionibacteria enzymes in ester synthesis. This study demonstrates that ethanol is the limiting factor of ethyl ester synthesis in Swiss cheese.     

Reduction of tetrazolium salt XTT by aminoreductone formed during the Maillard reaction of lactose

Lactose, a reducing disaccharide abundant in milk, reacts extensively with the amino groups of protein through the Maillard reaction. The Maillard reaction products showed 3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzensulfonic acid hydrate (XTT) reducibility. The objective of this study was to clarify the Maillard reaction products responsible for the XTT reducibility. When lactose and butylamine were heated at 100 degrees C, the characteristic UV maximum at 320 nm was recognized and the relationship between the XTT reducibility and the compound with absorption maximum at 320 nm was investigated. The time course and the dependence on the heating temperature of the formation of the compound with absorption maximum at 320 nm were similar to those of the XTT reducibility. Their relationship showed a significant correlation (r = 0.967, n = 19). Furthermore, the spectrum change of the heated model solution by the addition of XTT suggested that the compound with absorption maximum at 320 nm would be involved in the reduction of XTT. Because the compound with absorption maximum at 320 nm was identified as an aminoreductone, 1-(butylamino)-1,2-dehydro-1,4-dideoxy-3-hexulose, by NMR analysis, it can be concluded that this was the main XTT-reducing substance.     

alpha-Dicarbonyl compounds formed by nonenzymatic browning during the dry heating of caseinate and lactose

A method using high-performance liquid chromatography with UV and electrospray ionization mass spectrometry detection was developed for monitoring the alpha-dicarbonyl compound profiles generated from nonenzymatic browning using o-phenylenediamine (OPD) as a trapping agent. The alpha-dicarbonyl compounds were generated by the "dry" reaction of sodium caseinate and lactose heated at various relative humidities (RHs). The proportions of alpha-dicarbonyls formed were different for samples heated at low, intermediate, and high RHs. This study shows that relatively large amounts of 3-deoxypentosulose and galactosyl 2-pentosulose are produced under high RHs, while galactosyl hexosulose and 1,4-dideoxyhexosulose are elevated under low RH conditions. Both caramelization and Maillard reaction pathways contributed to the generation of alpha-dicarbonyls.     

胃蛋白酶水解珠蛋白获得ACE抑制肽的工艺优化(简报)

以猪血中提取的珠蛋白为原料,水解度为指标,对胃蛋白酶水解珠蛋白的工艺条件进行优化,然后测定不同水解阶段产物对血管紧张素Ⅰ转换酶（ACE）抑制活性的影响,来筛选具有较高抑制ACE活性的生物功能活性肽。结果表明：在酶量为0.3%,初始pH 值2.6,蛋白浓度0.7%,温度37℃条件下,时间为12 h时,水解度值最大,为11.85%;在其他因素不变条件下,水解时间为4 h时,水解产物对ACE抑制率达到最大值,其ACE抑制率为93.2%,水解度为9.64%。     

Characterization of key chromophores formed by nonenzymatic browning of hexoses and L-alanine by using the color activity concept

Thermal treatment of an aqueous solution of D-glucose and L-alanine in the presence of the carbohydrate degradation product furan-2-aldehyde resulted in the formation of a variety of colored compounds, among which (Z)-2-[(2-furyl)methylidene]-5, 6-di(2-furyl)-6H-pyran-3-one (I), [E]- and [Z]-1, 2-bis(2-furyl)-1-pentene-3,4-dione (IIa/IIb), 4, 5-bis(2-furyl)-2-methyl-3H-furan-2-one (III), and (S,S)- and (S, R)-2-[4, 5-bis(2-furyl)-2-hydroxy-2-methyl-3(2H)-pyrrol-1-yl]propionic acid (IVa/IVb) as well as 2-[(2-furyl)methylidene]-4-hydroxy-5-[(E)-(2-furyl)methylidene]methyl -2H-furan-3-one (V) were successfully identified as the most intense by application of the color dilution analysis. To measure the contribution of these colorants to the overall color of the browned Maillard mixture, color activity values were calculated as the ratio of the concentration to the visual detection threshold of each colorant. By application of this color activity concept, 16.0% of the overall color of the Maillard mixture accounted for these five types of colorants, thus confirming them as key chromophores. On the basis of synthetic model experiments, the formation pathways leading to the chromophores IIa/IIb, III, and IVa/IVb were proposed.     

Aroma of fresh strawberries is enhanced by ripening over red versus black mulch.

Strawberry (Fragaria ananassa Duch) is a high-value food crop, the aroma of which is important in consumer satisfaction. We hypothesized that the concentration of fresh strawberry aroma compounds could be enhanced by growing the berries over a red plastic mulch that was formulated to reflect more far-red (FR) and red light (R) and a higher FR/R photon ratio than is reflected by standard black plastic mulch. Strawberries of the cultivars "Chandler" and "Sweet Charlie" were grown in trickle-irrigated field plots over the two colors of mulch. The berries were harvested when ripe, and aroma compounds were isolated by dynamic headspace sampling. Entrained compounds were quantified by gas chromatography and identified by gas chromatography/mass spectroscopy. Twenty-three compounds were identified, and most of them were aliphatic esters. Strawberries that ripened over the red mulch during periods of sunny weather had significantly higher concentrations of aroma compounds than berries grown over the black plastic mulch. Total levels of aroma compounds from berries grown over red plastic mulch were higher for both Chandler and Sweet Charlie. We postulate that FR and R in light reflected from the red mulch acted through the natural phytochrome system to modify gene expression enough to result in greater concentration of aroma compounds in fresh strawberries.     

Synthesis of volatile compounds of virgin olive oil is limited by the lipoxygenase activity load during the oil extraction process

The aim of this work was to determine whether the lipoxygenase (LOX) activity is a limiting factor for the biosynthesis of virgin olive oil (VOO) volatile compounds during the oil extraction process. For this purpose, LOX activity load was modified during this process using exogenous LOX activity and specific LOX inhibitors on olive cultivars producing oils with different volatile profiles (Arbequina and Picual). Experimental data suggest that LOX activity is a limiting factor for the synthesis of the oil volatile fraction, this limitation being significantly higher in Picual cultivar than in Arbequina, in line with the lowest content of volatile compounds in the oils obtained from the former. Moreover, there is evidence that this limitation of LOX activity takes place mostly during the milling step in the process of olive oil extraction.     

Microbial activity in the rhizosphere soil of six herbaceous species cultivated in a greenhouse is correlated with shoot biomass and root C concentrations

The stimulation of rhizosphere microorganisms by exudates released from roots is important for nutrient cycling and differs between plant species. The reasons for this between-species variability are poorly understood. We studied correlations between shoot biomass, soluble and non-soluble root C concentrations and rhizosphere bacterial abundance (CFU: colony forming units) and an index of microbial activity (in vitro utilization of [U-¹⁴C]glucose by soil microorganisms). We studied Briza media and Rumex acetosella (nutrient-poor habitats), Epilobium hirsutum, Eupatorium cannabinum, Rumex obtusifolius and Urtica dioica (nutrient rich habitats) cultivated in a greenhouse for 5 weeks in a forest soil. We found significant differences among species for the bacterial abundance and microbial activity in the rhizosphere. These differences poorly reflected the nutrient richness of the common habitats for these species, possibly because the soil conditions were not optimal. Nevertheless, microbial activity was positively correlated with root soluble C concentration and shoot biomass and negatively correlated with the concentration of non-soluble C in roots. These preliminary results suggest that the carbon economy could be an important control of the between-species variability of microbial activity in the rhizosphere.     

基于状态向量增强ByteTrack的新生羔羊活动量自动计算方法

为评价母羊繁殖性能并及时发现分娩栏中的弱活力羔羊,该研究提出一种基于状态向量增强ByteTrack的新生羔羊活动量自动计算方法。针对传统ByteTrack算法在跟踪目标被遮挡时易发生身份切换的问题,引入置信度信息增强的状态向量,提高跟踪算法区分遮挡与被遮挡羔羊的能力。针对跟踪目标丢失导致轨迹预测不准确的问题,构建目标丢失期间的虚拟轨迹并重更新轨迹状态向量,以纠正轨迹误差。在获取各羔羊活动轨迹后,计算各羔羊帧间移动距离统计羔羊活动量。在江苏海门山羊研发中心采集的新生羔羊活动视频数据集上,测试状态向量增强的ByteTrack多目标跟踪算法性能。测试结果表明,研究提出的多目标跟踪方法在高阶跟踪精度、多目标跟踪精度、多目标跟踪准确度、IDF1得分上分别达到80.8%、86.1%、84.5%和92.2%,相较于现有算法的最高精度,分别提高2.7、0.2、2.3和3.9个百分点。该研究所提方法能够实现同窝多只新生羔羊的稳定跟踪,为新生羔羊活动量的自动计算、母羊繁殖性能的自动评估提供技术支撑。     

The Cd‐tolerant rice mutant cadH‐5 is a high Cd accumulator and shows enhanced antioxidant activity

To investigate the mechanism of cadmium (Cd) detoxification in rice (Oryza sativa L.), a Cd‐tolerant mutant cadH‐5, obtained by an Agrobacterium tumefaciens‐based gene‐delivery system, was used for a Cd‐tolerance and accumulation study. After 15 d of exposure to 0.75 mM CdCl₂, significant phenotypic differences were observed between the wild type (WT) and cadH‐5. When exposed to 0.5 mM CdCl₂, higher Cd levels were accumulated in cadH‐5 root cell wall, root cytosol, and membranes than those in WT. However, Cd concentrations in root tissues varied in both WT and cadH5. No significant difference of hydrogen peroxide (H₂O₂) concentrations was observed between WT and cadH‐5, while contents of cell‐wall polysaccharides and phytochelatins (PCs) in the mutant were higher compared to WT. The ratios of reduced glutathione to oxidized glutathione (GSH : GSSG) and ascorbate to dehydroascorbate (ASC : DHA) were lower in WT than in cadH‐5, while the NADPH : NADP⁺ ratio was different to the ratios of GSH : GSSG and ASC : DHA; the ascorbate peroxidase (APX, EC 1.11.1.11), glutathione peroxidase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities were lower in WT compared to cadH‐5. Our results indicate that under long‐term Cd stress, cadH‐5 plants can accumulate more Cd with more PC. Also, the redox status of ASC‐GSH cycle was more inhibited in WT than in cadH‐5 plants, rendering WT less able to scavenge reactive oxygen species (ROS). The cadH‐5 mutant maintains relatively high ASC, GSH, and NADPH concentrations, ratios of ASC : DHA, GSH : GSSG, and NADPH : NADP⁺, as well as antioxidative enzymatic activities and PC concentrations. Thus, it is tolerant of relatively high Cd accumulation.     

Soil microbial activity and biomass is stimulated by leguminous cover crops

The leguminous cover crops Atylosia scarabaeoides (L.) Benth., Centrosema pubescens Benth., and Pueraria phaseoloides (Roxb.) Benth., were grown in the interspaces of a 19 y–old coconut plantation and incorporated into the soil at the end of the monsoon season every year. At the end of the 12th year, soils from different depths were collected and analyzed for various microbial indices and their interrelationships. The objectives were to assess the effects of long‐term cover cropping on microbial biomass and microbial‐community structure successively down the soil profile. In general, total N (TN), organic C (OC), inorganic N, extractable P, and the levels of biological substrates viz., dissolved organic C (DOC) and N (DON), labile organic N (LON), and light‐fraction organic matter (LFOM) C and N decreased with depth at all the sites. Among sites, the cover‐cropped (CC) sites possessed significantly greater levels of TN, OC, DOC, DON, and LON compared to the control. Consequently, microbial biomass C (MBC), N (MBN), and P (MBP), CO₂ evolution, and ATP levels, in general, decreased with depth at all sites and were also significantly higher in the CC sites. Among the ratios of various microbial indices, the ratio of MBC to OC and metabolic quotient (qCO₂) declined with depth. Higher MBC‐to‐OC ratios and large qCO₂ levels in the surface soils could be ascribed to greater levels of readily degradable C content and indicated short turnover times of the microbial biomass. In contrast, the ratios of MBC to MBN and MBC to MBP increased with depth due to low N/P availability and relatively higher C availability in the subsoils. Cover cropping tended to enhance the ratios of MBC to OC, MBC to MBN, MBC to MBP, and ergosterol to MBC and decreased the ATP‐to‐MBC ratio at all depths. The relatively lower ATP‐to‐MBC ratios in the CC site, especially in the subsoil indicated microbial‐community structure possibly dominated by fungi. By converting the ergosterol content to fungal biomass, it was observed that fungi constituted 52%–63% of total biomass C at the CC site, but only 33%–40% of total biomass C at the control site. Overall, the study indicated that leguminous cover crops like P. phaseoloides or A. scarabaeoides significantly enhanced the levels of OC, N and microbial activity in the soils, even down to 50 cm soil depth.     

Distinctive fungal and bacterial communities are associated with mats formed by ectomycorrhizal fungi

The distinct rhizomorphic mats formed by ectomycorrhizal Piloderma fungi are common features of the organic soil horizons of coniferous forests of the Pacific Northwest. These mats have been found to cover 25-40% of the forest floor in some Douglas-fir stands, and are associated with physical and biochemical properties that distinguish them from the surrounding non-mat soils. In this study, we examined the fungal and bacterial communities associated with Piloderma mat and non-mat soils. Each mat and non-mat area was repeatedly sampled at four times throughout the year. Characterization of the mat activity and community was achieved using a combination of N-acetylglucosaminidase (NAGase) enzyme assays, and molecular analysis of fungal and bacterial communities using T-RFLP profiles, clone libraries, and quantitative PCR. Piloderma mats had consistently greater NAGase activity across all dates, although the magnitude of the difference varied by season. Furthermore, we found distinct fungal and bacterial communities associated with the Piloderma mats, yet the size of the microbial populations differed little between the mat and non-mat soils. Significant temporal variation was seen in the NAGase activity and in the sizes of the fungal and bacterial populations, but the community composition remained stable through time. Our results demonstrate the presence of two distinct microbial communities occupying the forest floor of Douglas-fir stands, whose populations and activities fluctuate seasonally but with little change in composition, which appears to be related to the physiochemical nature of mat and non-mat habitats.     

Acid-induced unfolding of flounder hemoglobin: evidence for a molten globular state with enhanced pro-oxidative activity

The acid-induced unfolding of flounder oxyhemoglobin was investigated and the effect on pro-oxidative activity assessed. Hemoglobin exhibited multistep unfolding transitions as pH was lowered, with the major transition between pH 3.5 and 4 5. The protein was maximally acid-unfolded (but not fully unfolded) at approximately pH 2.5, and further titration with HCl led to a partially refolded protein due to a stabilizing effect of Cl(-) anions. At low pH, the protein retained a sizable amount of secondary structure and had increased ANS binding, suggesting a molten globular form at low pH. Dramatic changes in the heme environment occurred concurrently with the changes in protein conformation. These changes resulted in an enhancement in the pro-oxidative activity of the protein. The results show that an increase in flounder hemoglobin pro-oxidation was correlated with the extent of its unfolding, and they provide useful insight into what may occur with hemoglobin in processes where highly acidic conditions are employed.     

Interactions of whey proteins during heat treatment of oil-in-water emulsions formed with whey protein isolate and hydroxylated lecithin

The interactions of proteins during the heat treatment of whey-protein-isolate (WPI)-based oil-in-water emulsions with and without added hydroxylated lecithin were studied by examining the changes in droplet size distribution and the quantity and type of adsorbed and unadsorbed proteins. Heat treatment at 90 degrees C of WPI emulsions resulted in an increase in total adsorbed protein; unadsorbed beta-lactoglobulin (beta-lg) was the main protein interacting with the adsorbed proteins during the first 10 min of heating, but after this time, unadsorbed alpha-lactalbumin (alpha-la) also associated with the adsorbed protein. In emulsions containing hydroxylated lecithin, the increase in total adsorbed protein during heat treatment was much lower and the unadsorbed beta-lg did not appear to interact with the adsorbed proteins during heating. However, the behavior of alpha-la during heat treatment of these emulsions was similar to that observed in the emulsions containing no hydroxylated lecithin. In the presence of NaCl, the particle size of the emulsion droplets and the quantities of adsorbed protein increased markedly during heating. Emulsions containing hydroxylated lecithin were less sensitive to the addition of NaCl. These results suggest that the binding of hydroxylated lecithin to unfolded monomers or intermediate products of beta-lg reduces the extent of heat-induced aggregation of beta-lg and consequently decreases the interactions between unadsorbed beta-lg and adsorbed protein. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated whey protein and hydroxylated lecithin solutions.     

Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola

The role of soil organisms as possible driver of flowering has never been investigated. We hypothesized that Collembola (microarthropods) will change plant allocation to reproductive modes by changing soil nutrient availability. Individual seedlings of Poa annua were planted in microcosms, in the presence or absence of Collembola. Collembola affected biotic (fungal biomass) and abiotic ($\text{N}-{{\text{NO}}_3}^{-}$, P₂O₅) soil properties and some morphological (number of leaves, root biomass) and chemical (C:N, K, Mg, N) traits of P. annua. As a result, flowering of P. annua was promoted by the presence of Collembola. This provides experimental evidence that soil microarthropods can affect the reproduction strategy and phenology of a plant.     

Denitrification enzyme activity is limited by soil aeration in a wastewater-irrigated forest soil

 In land-based wastewater treatment systems (LTS), denitrification is an important nitrogen removal process. We investigated the factors limiting the denitrifying population in a forested LTS, by studying the individual and combined effects of soil aeration, water content, nitrate and carbon on denitrification enzyme activity (DEA). The size of the soil denitrifying population in the LTS appeared to be limited by soil aeration, and limiting oxygen availability increased the denitrifying population above that observed in the field. Furthermore, we found that wastewater irrigation altered the short-term response of denitrifiers to anaerobic soil conditions. Under low oxygen conditions, denitrifiers in the wastewater-irrigated soils produced enzymes sooner and at a greater rate than soils without a history of wastewater irrigation. We propose that the size of the denitrifying population cannot be expected to be large in free-draining, coarsely textured soils even when provided with additional nitrogen and water inputs. Received: 11 October 1999     

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.