Interactions between beta-lactoglobulin and pectins during in vitro gastric hydrolysis

This paper deals with the influence of different levels of three pectins, low-methylated pectin (LMP), high-methylated pectin (HMP), and low-methylated and amidated pectin (LMA), on the in vitro gastric hydrolysis of beta-lactoglobulin (beta-lg). Proteolysis by pepsin consisted of a 2-h progressive reduction of pH. A turbidity measurement of beta-lg-pectin mixtures was carried out during the proteolysis. The influence of pectins on pepsin enzymatic activity was also evaluated. beta-Lg was resistant to peptic digestion. The presence of each of the three pectins at a concentration of 50 wt % increased the N release at all pH values considered, despite a significant inhibition of the pepsin enzymatic activity with the pectins. The turbidity of beta-lg solutions during proteolysis was reduced by the addition of pectins, because of the formation of electrostatic complexes between this protein and pectins. The increase of N release could be a false positive result due to the difficulty of precipitating protein by trichloroacetic acid because of the formation of electrostatic complexes demonstrated by the decrease of turbidity.     

Gelling properties of heat-denatured beta-lactoglobulin aggregates in a high-salt buffer

Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.     

Specificity of disulfide bond formation during thermal aggregation in solutions of beta-lactoglobulin B and kappa-casein A

Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.     

Steric effects governing disulfide bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and alpha-lactalbumin

Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.     

Aggregation of peptides during hydrolysis as a cause of reduced enzymatic extractability of soybean meal proteins

With the purpose of analyzing the size and composition of enzyme-unextractable proteins in differently heat-treated soybean meals, a selection of extractants was screened for their ability to extract these proteins from enzyme-unextractable residues. The largest effects were obtained with urea, urea plus beta-mercaptoethanol, and dilute alkali; the latter extracted up to 87% of the enzyme-unextractable protein. Gel permeation chromatography indicated that a large proportion of the extracted material was of high molecular weight. However, the combined results from gel electrophoresis, LC-MS, and MALDI-ToF MS showed that the extracted protein material was composed of aggregated peptides. The largest aggregates were observed in the enzymatic residues originating from meals heat-treated at high humidity. Extracted aggregates were fully degraded upon subsequent proteolytic treatment.     

Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides

The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.     

Application of respiration and FDA hydrolysis measurements for estimating microbial activity during composting processes

Olive-tree leaves (OL) were mixed with olive press cake (OPC) and extracted olive press cake (EPC) at 1:1 dw/dw ratios to prepare two composting mixtures (OL+OPC and OL+EPC). Both CO₂–C evolution and fluorescein diacetate (FDA) hydrolysis, determined as estimates of the microbial activity during composting, were related to temperature fluctuations in the compost piles, showing greater values at the temperature peaks, compared to the end, of each thermophilic phase. This, however, was only shown after handling and incubating samples at the temperatures of the compost mixtures at the sampling times and not at a low standard temperature. Incubating samples from thermophilic phases at low standard temperatures resulted in underestimation of the microbial activity occurring during composting. The effect of incubation temperature was less dramatic for FDA hydrolysis compared to CO₂–C evolution measurements, probably reflecting the reduced dependence of enzymes involved in FDA hydrolysis on the respective temperatures. However, FDA hydrolysis was a less sensitive indicator of microbial activity, probably due to extracellular cleavage of fluorescein by persistent esterases, at lowered microbial activity phases. Total microbial biomass, estimated by the fumigation–extraction method, was not consistently related to temperature fluctuations during composting and showed a clear increase at the end of composting, probably resulting from a large slow-growing mycelial community colonising the end products. Since high temperatures did not induce significant non-microbial CO₂–C release and FDA degradation, we propose the performance of microbial activity measurements during thermophilic composting phases at the actual temperatures evolving in the composts.     

beta-lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties of hydrolysate fractions formed by the action of plasmin.

beta-Lactoglobulin (betaLg) was hydrolyzed by plasmin to a degree of hydrolysis of 4%. The hydrolysate was fractionated by ion-exchange chromatography and subsequent hydrophobic-interaction chromatography. The betaLg peptide fraction consisting of smaller peptides (mostly <2 kDa) had poor foam- and emulsion-forming and -stabilizing properties. Most of the betaLg peptides were identified (in either the nonreduced or reduced form) by mass spectrometry on the basis of the known primary structure of the intact protein and the specificity of the enzyme. The peptides formed during betaLg/plasmin-hydrolysis were (1) peptides lacking a cysteyl residue, (2) peptides composed of a single amino acid chain containing intramolecular disulfide bonds, and (3) peptides composed of two amino acid chains linked by an intermolecular disulfide bond. It appeared that significant SH/SS-exchange had taken place during hydrolysis. Many of the peptides present in the peptide fraction that exhibited good functional properties were disulfide-linked fragments.     

Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting

The mycotoxin ochratoxin A is degraded by up to 90% during coffee roasting. In order to investigate this degradation, model heating experiments with ochratoxin A were carried out, and the reaction products were analyzed by HPLC-DAD and HPLC-MS/MS. Two ochratoxin A degradation products were identified, and their structure and absolute configuration were determined. As degradation reactions, the isomerization to 14-(R)-ochratoxin A and the decarboxylation to 14-decarboxy-ochratoxin A were identified. Subsequently, an analytical method for the determination of these compounds in roasted coffee was developed. Quantification was carried out by HPLC-MS/MS and the use of stable isotope dilution analysis. By using this method for the analysis of 15 coffee samples from the German market, it could be shown that, during coffee roasting, the ochratoxin A diastereomer 14-(R)-ochratoxin A was formed in amounts of up to 25.6% relative to ochratoxin A. The decarboxylation product was formed only in traces. For toxicity evaluations, first preliminary cell culture assays were performed with the two new substances. Both degradation products exhibited higher IC50 values and caused apoptotic effects with higher concentrations than ochratoxin A in cultured human kidney epithelial cells. Thus, these cell culture data suggest that the degradation products are less cytotoxic than ochratoxin A.     

beta-lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.

beta-Lactoglobulin (betaLg) was subjected to limited hydrolysis by trypsin, plasmin, and endoproteinase from Staphylococcus aureus V8 (S.aur.V8) to degrees of hydrolysis (DH) of 1, 2, and 4%. The several hydrolysates had different peptide compositions (determined by reversed-phase HPLC and gel-permeation chromatography [GPC]). GPC under nondenaturing, denaturing, and denaturing plus reducing conditions showed that the peptides formed were linked by hydrophobic interactions or by disulfide bonds or were not linked at all. At very low protein concentration, some differences in emulsion-forming properties were observed: only the plasmin hydrolysates could form emulsions with a uniform particle-size distribution. The emulsions formed with S.aur.V8 hydrolysates had poor emulsion-stabilizing properties. Some hydrolysates showed increased foam-forming properties in comparison with the intact protein. All foams formed were stable. Overall, the plasmin hydrolysate (DH4) contained relatively much larger molecules and/or hydrophobic molecules. Many molecules were disulfide-linked peptides. This hydrolysate also had the best functional properties.     

Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase

Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.     

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.     

Changes in microbial biomass and protease activities of soil associated with long-term sugar cane monoculture

 As part of a broader study, the aim of which is to identify soil factors that might be associated with yield decline of sugar cane, microbial biomass and protease activities were examined in soil samples collected from seven paired old and new land sites in three cane-growing districts of north Queensland. No consistent changes in soil protease activities were observed, although some sites exhibited specific effects, as a result of extensive periods of sugar cane monoculture. Soil microbial biomass, however, was significantly lower in those soils where sugar cane had been grown for extended periods. The implications of a lowering of soil microbial biomass on sugar cane yields and sustainability are discussed. Received: 24 June 1997     

ACE-inhibitory and radical-scavenging activity of peptides derived from beta-lactoglobulin f(19-25). Interactions with ascorbic acid

In this work, the angiotensin-converting enzyme (ACE)-inhibitory and radical-scavenging activities of the beta-lactoglobulin (beta-Lg)-derived peptides WY f(19-20), WYS f(19-21), WYSL f(19-22), WYSLA f(19-23), WYSLAM f(19-24), and WYSLAMA f(19-25) have been determined. The ACE-inhibitory activity (IC50) varied from 38.3 to 90.4 microM, with the exception of WYS (>500 microM). All beta-Lg-derived peptides also exhibited radical-scavenging activity (oxygen radical absorbance capacity (ORAC) values ranged from 4.45 to 7.67 micromol Trolox equivalents/micromol of peptide). The presence and position of amino acids Trp, Tyr, and Met were proposed to be responsible for the antioxidant activity. The equimolar amino acid mixtures of all the peptides showed ORAC values lower than those of the corresponding peptides, indicating that the peptidic bond or the structural conformation had a positive influence on this activity. Finally, positive antioxidant effects of WYS, WYSL, and WYLA with ascorbic acid were observed, whereas WY and WYSLAM showed negative effects, both cases for different molar ratio mixtures. These results should be taken into account in the development of new food ingredients on the basis of peptides from beta-Lg.     

Sensitivities to nitrogen and water addition vary among microbial groups within soil aggregates in a semiarid grassland

We investigated whether enhanced nitrogen (N) and water inputs would redistribute the microbial community within different soil aggregate size classes in a field manipulation experiment initiated in 2005. Distribution of microbial groups was monitored in large macroaggregates (>2000 μm), small macroaggregates (250–2000 μm), and microaggregates (<250 μm) in a semiarid grassland. Both arbuscular mycorrhizal (AM) fungi and saprophytic fungi were the most abundant in soil macroaggregates. The gram-negative bacteria were more abundant in soil microaggregates. Total phospholipid fatty acid (PLFA) concentration in general and actinomycetes in particular decreased with N addition under ambient precipitation but was unaffected by combined additions of N and water within the three soil aggregate fractions as compared to control plots. In contrast, the abundance of saprophytic fungi decreased with combined N and water addition, but it was not affected by N addition under ambient precipitation. The abundance of gram-positive bacteria increased with N addition under both ambient and elevated water conditions for all soil aggregate fractions. In summary, the higher short-term nutrient and water availabilities provoked a shift in soil microbial community composition and increased total PLFA abundance irrespectively of the level of soil aggregation. In the long term, this could destabilize soil carbon pools and influence the nutrient limitation of soil biota within different soil aggregate size classes under future global change scenarios.     

土壤团聚体氧化亚氮排放潜势与微生物学机制

氧化亚氮（N2O）是主要温室气体之一,土壤是N2O的重要排放源,其排放主要受N2O产生和还原的功能微生物影响。土壤团聚体是由原生颗粒（砂、粉、黏粒）、胶结物质和孔隙组成的土壤基本结构单元。土壤不同粒径团聚体之间因基质和孔隙差异形成特殊独立的微生境被视为N2O的生物化学反应器。在不同的微生境中,N2O产生和还原的功能微生物分布不同,因而土壤不同粒径团聚体N2O排放可能存在差异。目前在不同生态系统土壤全土N2O排放特征的报道较多,而对于不同粒径土壤团聚体N2O排放相对贡献尚不清楚、功能微生物分布还未知、N2O产生和还原热区尚未明确。本文综述了近年来国内外关于土壤团聚体对N2O产生和排放机制的研究,总结了土壤团聚体性状特征对N2O产生和还原的影响,阐述了不同粒径土壤团聚体对N2O排放影响的微生物学机制,进一步明确了今后需加强土壤团聚体N2O产生和还原的热区、环境因子阈值范围的确定、系列功能基因（酶）整体性的研究,以期为N2O模拟排放模型优化提供参考,为土壤N2O减排提供理论依据。     

免耕对潮土不同粒级团聚体有机碳含量及微生物碳代谢活性的影响

利用中国科学院封丘农业生态国家实验站玉米-小麦轮作免耕定位试验平台,研究不同耕作方式对潮土不同粒级团聚体的分布、有机碳含量及微生物群落碳代谢活性的影响。结果表明,与传统翻耕处理相比,免耕潮土中大于250μm粒级团聚体显著增加(p0.05),50～2μm粒级团聚体显著减少(p0.05),250～50μm和小于2μm两个粒级团聚体没有明显变化;大于250μm和250～50μm两个粒级团聚体有机碳含量显著升高(p0.05),50～2μm和小于2μm两个粒级团聚体有机碳含量没有明显变化,250～50μm粒级取代50～2μm粒级成为对土壤有机碳总量贡献最大的团聚体。BIOLOG测试结果表明,免耕和翻耕两种措施下不同粒级团聚体微生物群落碳代谢特征存在明显差异,且免耕处理250～50μm和小于2μm两个粒级团聚体微生物群落碳代谢活性显著低于翻耕处理(p0.05)。结果显示,免耕有利于小粒级团聚体向大粒级团聚体方向聚合,大于50μm粒级团聚体固持的有机碳增多,其中持碳最多的250～50μm粒级团聚体的微生物碳代谢活性下降,故土壤有机碳稳定性升高。