In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota

Fermentation properties of oligosaccharides derived from lactulose (OsLu) and lactose (GOS) have been assessed in pH-controlled anaerobic batch cultures using lactulose and Vivinal-GOS as reference carbohydrates. Changes in gut bacterial populations and their metabolic activities were monitored over 24 h by fluorescent in situ hybridization (FISH) and by measurement of short-chain fatty acid (SCFA) production. Lactulose-derived oligosaccharides were selectively fermented by Bifidobacterium and lactic acid bacterial populations producing higher SCFA concentrations compared to GOS. The highest total SCFA production was from Vivinal-GOS > lactulose > OsLu > GOS. Longer incubation periods produced a selective fermentation of OsLu when they were used as a carbon source reaching the highest selective index scores. The new oligosaccharides may constitute a good alternative to lactulose, and they could belong to a new generation of prebiotics to be used as a functional ingredient for improving the composition of gut microflora.     

琼胶寡糖对黄瓜抗性及啶虫脒残留的影响

为探究琼胶寡糖作为激发子对植物的影响,本研究以碧翠18号黄瓜为试验材料,在其生长过程中喷施不同浓度琼胶寡糖,测定黄瓜的生长指标、抗性相关酶活和基因表达,以及农药啶虫脒残留量。结果表明,喷施琼胶寡糖可显著促进黄瓜植株生长,提高果实产量;提高黄瓜叶片超氧化物歧化酶、过氧化物酶、多酚氧化酶、过氧化氢酶活性,降低丙二醛含量;并上调几丁质酶与病程相关蛋白1基因的表达,最高表达量分别为对照组的10.4倍和14.5倍。此外,喷施琼胶寡糖有效降低了啶虫脒在黄瓜上的半衰期,加速其降解。浸果试验结果也显示,琼胶寡糖可显著降低农药残留,100 μg·mL^-1琼胶寡糖溶液可减少农残68.29%。因此,琼胶寡糖具有促进黄瓜植株生长、提高抗性及减少果实农药残留的效果,在农业生产中具有良好的开发价值。     

Straightforward process for removal of milk fat globule membranes and production of fat-free whey protein concentrate from cheese whey

A straightforward method for the separation of milk fat globule membrane (MFGM) and production of fat-free whey protein concentrate/isolate from cheese whey has been developed. Lowering of the conductivity of the whey from its initial value of about 5600 μS cm(-1) to about 2000-500 μS cm(-1) via diafiltration with water caused selective precipitation of MFGM when incubated for 30 min at pH 4.2 and 35 °C. The whey proteins remained soluble in the supernatant under these conditions. Experimental evidence suggested that precipitation of MFGM at pH 4.2 was not due to a nonspecific effect of lowering of the conductivity of the whey but due to the specific effect of removal of Ca2+ from the whey. The lipid content of whey protein isolate obtained by this process was <0.2%, and the protein loss was <14%. The method provides an industrially feasible process for the production of fat-free whey protein concentrate/isolate. The MFGM, which is reported to contain bioactive/nutraceutical lipids and proteins, is a valuable byproduct of the process.     

In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria

In this work, in vitro fermentation of alternansucrase raffinose-derived oligosaccharides, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10), was carried out using small-scale pH-controlled batch cultures at 37 °C under anaerobic conditions with human feces. Bifidogenic activity of oligosaccharides with DP4-6 similar to that of lactulose was observed; however, in general, a significant growth of lactic acid bacteria Bacteroides , Atopobium cluster, and Clostridium histolyticum group was not shown during incubation. Acetic acid was the main short chain fatty acid (SCFA) produced during the fermentation process; the highest levels of this acid were shown by alternansucrase raffinose acceptor pentasaccharides at 10 h (63.11 mM) and heptasaccharides at 24 h (54.71 mM). No significant differences between the gas volume produced by the mixture of raffinose-based oligosaccharides (DP5-DP10) and inulin after 24 h of incubation were detected, whereas lower gas volume was generated by DP4 oligosaccharides. These findings indicate that novel raffinose-derived oligosaccharides (DP4-DP10) could be a new source of prebiotic carbohydrates.     

Soil moisture and the rates of biodegradation of diallate and triallate

Degradation experiments were combined with biomass measurements and adsorption tests to determine how soil moisture content influences the rates of degradation of ⁴¹C-labelled diallate and triallate. In soils treated with 1 μg^?1 herbicide and incubated at constant temperature and moisture, degradation rates were regulated by two variables: the quantity of microbial biomass in the soil; and the quantity of herbicide dissolved in the soil solution. The quantity of biomass was influenced by soil water content and the duration of incubation. The amounts of herbicide in solution were determined by the amount of water present and the total quantity of herbicide in the soil. In all soil samples, the rates of degradation increased with increasing water content but decreased with prolonged incubation. The factors responsible for decrease with time were the loss of biomass during incubation and the decline in herbicide concentration in the soils as degradation proceeded.     

黑土环境中乙草胺的微生物降解特征研究

通过室内恒温(25℃)避光培养试验,研究了黑土环境中乙草胺的微生物降解特征。在适宜水分条件下,将土壤样品分别进行常规、灭菌、选择性抑菌剂加入等处理后培养并测定土壤乙草胺含量和土壤微生物量。结果显示:在灭菌土壤中乙草胺的残留量较未灭菌土壤显著增加,未灭菌土壤中乙草胺残留数量与微生物量变化密切相关,表明微生物活性是影响乙草胺降解的主要因素。适当的水分有益于土壤中微生物生长,从而促进了土壤中乙草胺的降解。加入青链霉素后乙草胺残留量远大于放线菌酮和常规培养,表明细菌比真菌具有更强的降解乙草胺的能力。随着乙草胺的施药量增加,初期微生物量显著降低,是导致乙草胺总降解率下降的主要原因。     

Degradation of PCP in soils

In laboratory experiments, the degradation of PCP in soil with regard to the relationship to soil properties was studied under upland and flooded conditions using gas-chromatographic techniques. The degradation products and their behavior were elucidated by using 10 diCferent soils collected from rice fields and adjacent upland fields and one sample of a subsoil from the forest. The results are as follows:

1) The degradation of PCP in soils was faster under flooded conditions than upland conditions.

2) The degradation under flooded conditiont was more rapid in soils collected from rice fields than in those from adjacent upland fields, Tbe reverse was true under upland conditions.

3) The degradation rate was highly correlated with the organic matter content of the soil. Almost 100% of the PCP remained in the subsoil sample even after 50 days of incubation. The rate was slightly correlated with the clay mineral composition, free iron content, phosphate absorption coefficient and C.E.C., but hardly at all with texture, clay content, degree of base saturation, soil pH and available phosphorus content.

4) As the degradation products of PCP, 3 tetrachlorophenols, 4 or 5 trichlorophenols and PCP methyl ether were detected, PCP methyl elher and 2, 3, 4, 5-tetrachlorophenol were the major products, but the amount of the latter varied greatly during the course of incubation.     

Characterization of alginase and elicitor-active oligosaccharides from Gracilibacillus A7 in alleviating salt stress for Brassica campestris L

Alginase was purified from Gracilibacillus A7 and evaluated for its ability to produce elicitor-active oligosaccharides. The optimum conditions for the alginase reaction are as follows: temperature, 40 °C; pH, 8.0; alginate content, 0.3-0.7%; and the presence of Na(+) and Mg(2+) metal ions. The degree of polymerization (DP) decreased as the reaction time of the alginase progressed, achieving values of 5.4 and 3.3 after 240 and 300 min, respectively. The relative root length (RRL) of the Brassica campestris L. increased with the addition of oligosaccharides with reduced DP values. The oligosaccharides with lower DP values are effective in reducing the effect of salt stress on the activity of the superoxide dismutase (SOD) and guaiacol peroxidase (POD), and oligosaccharides with moderate DP values can reduce the increase in lipid peroxidation activities (as malondialdehyde content) induced by salt stress. These results suggest that oligosaccharides may act as osmoprotective agents during the plant germination process.     

褐土中铁的氧化还原与碳素转化

采用泥浆厌氧恒温培养的方法,研究了光照对旱作褐土中氧化铁的厌氧还原过程的影响,探讨了土壤中铁氧化物的还原-氧化过程与碳素转化的关系。结果表明,旱作褐土中游离铁氧化物的55.31%可在厌氧避光条件下发生还原,厌氧光照条件下游离铁氧化物的还原率最大仅为38.90%,还原产生的Fe(Ⅱ)可能被蓝细菌中的鱼腥蓝细菌属光合过程产生的氧氧化,40 d培养后其游离铁氧化物还原率低至7.95%。厌氧避光条件下培养40 d后土壤中水溶性总碳、无机碳含量分别增加了69%和246%,厌氧光照条件下水溶性总碳和无机碳则呈现先增加后降低的趋势,40 d培养后仅为反应前的47%和70%。水溶性总碳和无机碳含量分别与Fe(T)和Fe(Ⅱ)含量呈极显著线性正相关关系。     

Enzymatic production and characterization of konjac glucomannan oligosaccharides

Enzymes from a balanced human gut flora are promising tools to design prebiotic oligosaccharides. In this study, we investigated the action of enzymes from fecal bacteria on the complex polysaccharide konjac glucomannan (KGM). The oligosaccharides produced were compared to oligosaccharides from KGM digests with fungal endo-β-(1,4)-glucanase (EG) or endo-β-(1,4)-mannanase (EM). For this purpose, the oligosaccharides from the different digests were first studied for their structural characteristics like monosugar composition and exo-enzymatic degradability, as monitored by capillary electrophoresis with laser-induced fluorescence detection. Whereas the oligosaccharides produced by EG and EM were characteristic for the selectivity of the respective enzyme in cleaving the mannose-/glucose-sugar linkages of KGM, oligosaccharides produced by the fecal enzymes did not point to a sugar-selective degradation. The oligosaccharide fragments from the different digests indicated the KGM polysaccharide to be composed of a backbone composed of short mannose and glucose sequences, to which branches rich in mannose are attached.     

玉米草（Zea Mexicana）与海藻寡糖联合修复石油烃污染土壤的研究

采用玉米草及海藻寡糖联合修复技术研究了石油污染土壤的修复效果,对修复过程中酶活性变化进行了测定,并采用变性梯度凝胶电泳（DGGE）技术测定了土壤中微生物群落的变化。结果表明,种植玉米草可以有效提高土壤中石油烃的降解,与对照相比石油烃降解率增加了11%;加入不同浓度海藻寡糖进一步增加了石油烃的降解效果,降解率最高达到28.6%。种植植物及加入海藻寡糖可以有效提高多酚氧化酶、脱氢酶及尿酶的活性。PCR-DGGE结果表明植物种植及海藻寡糖的加入增加了土壤中微生物数量,其微生物群落结构与未种植植物及修复前土壤相比发生了较大的变化。     

Determination of total dietary fiber in foods and food products: collaborative study

A collaborative study was conducted to determine the total dietary fiber (TDF) content of food and food products, using a combination of enzymatic and gravimetric procedures. The method was basically the same as published earlier (J. Assoc. Off. Anal. Chem. (1984) 67, 1044-1052), with changes in the concentration of alcohol and buffers, time of incubation, sample preparation, and some explanatory notes, all with the intent of decreasing the coefficient of variation (CV) of the method. Duplicate blind samples of soy isolate, white wheat flour, rye bread, potatoes, rice, wheat bran, oats, corn bran, and whole wheat flour were analyzed by 9 collaborators. TDF was calculated as the weight of the residue minus the weight of protein and ash. CV values of the data from all laboratories for 7 of the samples ranged from 1.56 to 9.80%. The rice and soy isolate samples had CV values of 53.71% and 66.25%, respectively; however, each sample contained only about 1% TDF. The enzymatic-gravimetric method for determining TDF has been adopted official first action.     

Utilization of monolinolein as a substrate for conjugated linoleic acid production by Bifidobacterium breve LMC 520 of human neonatal origin

This study was designed to isolate bifidobacteria from human intestines that efficiently converts monolinolein, a monoglyceride form of linoleic acid, into conjugated linoleic acid (CLA), as well as to optimize culture conditions for improving CLA production during milk fermentation. Among 150 screened neonatal bifidobacteria, Bifidobacterium breve LMC 520 showed the highest CLA-producing ability and was tested with different types of fat substrates at various concentrations to determine the optimal conditions for CLA production. Monolinolein was tested as a substrate for CLA production. The incubation time optimized for CLA production was 24 h, and CLA production was proportionally increased with monolinolein concentration. The incubation of LMC 520 with commercial starter strains caused minimal reduction in CLA production. Our results demonstrate that the CLA-producing ability of B. breve LMC 520 could offer beneficial effects when utilized as a starter culture for the development of functional dairy products.     

In vitro proteolysis of myofibrillar proteins from beef skeletal muscle by caspase-3 and caspase-6

The objective of the study was to investigate in vitro degradation of myofibrils by caspase-3 or -6. Myofibrillar proteins prepared from beef skeletal muscle were incubated with caspase-3 or -6 at 30 °C for 2 or 12 h, and subsequently, protein degradation was detected. Results showed that caspase-3 and -6 reproduced the degradation patterns of titin and nebulin observed during normal postmortem (PM) aging; however, they only reproduced the 28 kDa fragment derived from troponin-T. Caspase-3 induced only minor degradation of desmin. However, caspase-6 caused increasing degradation of desmin with extended incubation time and produced three degradation fragments (45, 29, and 27 kDa) of which only the 45 kDa fragment has been reported in aged beef. Therefore, caspase-3 or -6 could only reproduce a part of myofibrillar protein degradation or degradation fragments observed in naturally aged meat and may be involved in PM proteolysis of muscle proteins together with other endogenous proteases.     

Plant regulation of microbial enzyme production in situ

Soil extracellular enzymes regulate the rate at which complex organic forms of nitrogen (N) become bio-available. Much research has focused on the limitations to heterotrophic enzyme production via lab incubations, but little has been done to understand the limitations to enzyme production in situ. We created root and symbiotic mycelia exclusion treatments using mesh in-growth bags in the field to isolate the effect of roots and other portions of the microbial community on enzyme production. When fertilized with complex protein N we found increases in N-degrading enzyme concentrations only when root in-growth was allowed. No response was observed when complex N was added to root-free treatments. Expanding on economic rules of microbial element limitation theory developed from lab incubation data, we suggest this is due to active transport of labile carbon (C) from roots to associated microbial communities in root bags. Roots alleviate C limitation of microbial enzyme synthesis, representing a tradeoff between plants and microbes–plant C for microbially-derived N.     

Effects of soil storage on the microbial community and degradation of metsulfuron-methyl

The effect storage had on the microbial biomass in two soils (Trevino and Fargo) was compared to the effect storage had on each soil's capacity to degrade metsulfuron-methyl. Soils were collected from the field and used fresh (<3 weeks old) or stored at 20 and 4 degrees C for 3 or 6 months. The phospholipid fatty acid content of the soils was used to monitor changes in the microbial biomass during storage and incubation in a flow-through apparatus. In both soils, [phenyl-U-14C]metsulfuron-methyl was used to monitor changes in the route and rate of degradation along with 14CO2 evolution (mineralization). Total microbial biomasses in both soils were significantly reduced for soils incubated in the flow-through apparatus, whereas only the Trevino soil's microbial biomass was significantly reduced as a result of storage. The microbial communities of both soils were significantly different as a result of storage as shown by discriminant analysis. In both soils, degradation rate, pathway of degradation, and mineralization of metsulfuron-methyl were significantly affected by storage compared to fresh soil. The half-life of metsulfuron-methyl increased significantly (P < 0.05) in the Trevino soil from 45 days (fresh) to 63 days (stored soil), whereas in the Fargo soil half-lives increased significantly (P < 0.05) from 23 days (fresh) to 29 days (soils stored for 6 months). In both soils, mineralization of [14C]metsulfuron-methyl was significantly (P < 0.05) higher in fresh soils compared to stored soils. The degradation pathways of metsulfuron-methyl changed with storage as evidenced by the loss of formation of one biologically derived metabolite (degradate) in stored soils compared to fresh soils.     

Changes in properties of soil-derived dissolved organic matter induced by biodegradation

Properties of dissolved organic matter (DOM) determine its biodegradation. In turn, biodegradation changes the properties of the remaining DOM, which may be decisive for the formation of stable organic carbon in soil. To gain information on both mechanisms and controlling factors of DOM biodegradation and the properties of biodegraded DOM, we investigated changes in the composition of 13 different DOM samples extracted from maize straw, forest floors, peats, and agricultural soils during a 90-day incubation using UV absorbance, fluorescence emission spectroscopy, FTIR-spectroscopy, ¹H-NMR spectroscopy, pyrolysis-field ionization mass spectroscopy (Py-FIMS), and ¹³C natural abundance before and after incubation. Changes in the DOM properties were related to the extent of biodegradation determined by the release of CO₂. Increasing UV absorption and humification indices deduced from fluorescence emission spectra, and increasing portions of aromatic H indicated relative enrichment of aromatic compounds during biodegradation. This enrichment significantly correlated with the amount of DOC mineralized suggesting that aromatic compounds were relatively stable and slowly mineralized. ¹³C depletion during the incubation of highly degradable DOM solutions indicated an enrichment of lignin-derived aromatic compounds. Py-FI mass spectra indicated increasing contents of phenols and lignin monomers at the expense of lignin dimers and alkylaromatics during incubation. This partial degradation of higher-molecular, lignin-derived DOM compounds was accompanied by relative increases in the proportions of lower-molecular degradation products and microbial metabolites. Carbohydrates, especially when abundant at high initial contents, seem to be the preferred substrate for microorganisms. However, four independent methods suggested also some microbial production of carbohydrates and peptides during DOM degradation. After incubation, the composition of highly degradable DOM samples became similar to relatively stable DOM samples with respect to aromaticity, carbohydrate content, and thermal stability. We conclude that DOM biodegradation seems to result in organic matter properties being a precondition for the formation of stable carbon. These structural changes induced by DOM biodegradation should also result in stronger DOM sorption to the soil matrix additionally affecting DOM stabilization.     

Storage stability of ascorbic acid incorporated in edible whey protein films

The stability of ascorbic acid (AA) incorporated in whey protein isolate (WPI) film and the related color changes during storage were studied. No significant loss of AA content was found in any films prepared from pH 2.0 casting solution stored at 30% relative humidity (RH) and 22 °C over 84 days. Total visible color difference (ΔE*(ab)) of all films slowly increased over storage time. The ΔE*(ab) values of pH 3.5 films were significantly higher than those of pH 2.0 films. The stability of AA-WPI films was found to be mainly affected by the pH of the film-forming solution and storage temperature. Oxidative degradation of AA-WPI films followed Arrhenius behavior. Reduction of the casting solution pH to below the pK(a1) (4.04 at 25 °C) of AA effectively maintained AA-WPI storage stability by greatly reducing oxidative degradation, whereas anaerobic and nonenzymatic browning were insignificant. The half-life of pH 2.0 AA-WPI film at 30% RH and 22 °C was 520 days.     

电渗析技术在大豆低聚糖溶液脱盐上的应用

从甜浆中提取的大豆低聚糖粗提液中含有很多的盐类,影响糖液的纯度。该试验通过离子交换膜辅助的电渗析法对大豆低聚糖模拟溶液进行脱盐处理,确定了较好的试验操作参数。试验结果表明:电渗析法对大豆低聚糖模拟液进行脱盐是可行的,且脱盐效果较好,脱盐率达到96.07%;该试验条件下较佳的工作电压为20 V,流量为60 L/h,样品稀释倍数为15倍。通过以上条件处理后,大豆低聚糖的保留率达到83.82%。     

Plant regulation of microbial enzyme production in situ

Soil extracellular enzymes regulate the rate at which complex organic forms of nitrogen (N) become bio-available. Much research has focused on the limitations to heterotrophic enzyme production via lab incubations, but little has been done to understand the limitations to enzyme production in situ. We created root and symbiotic mycelia exclusion treatments using mesh in-growth bags in the field to isolate the effect of roots and other portions of the microbial community on enzyme production. When fertilized with complex protein N we found increases in N-degrading enzyme concentrations only when root in-growth was allowed. No response was observed when complex N was added to root-free treatments. Expanding on economic rules of microbial element limitation theory developed from lab incubation data, we suggest this is due to active transport of labile carbon (C) from roots to associated microbial communities in root bags. Roots alleviate C limitation of microbial enzyme synthesis, representing a tradeoff between plants and microbes–plant C for microbially-derived N.