Model studies on reactions of plant phenols with whey proteins

Whey proteins were modified by reaction with selected phenolic compounds (ferulic-, chlorogenic-, caffeic- and gallic acid) and related substances (quinic acid and p-quinone) as well as with extracts from coffee, tea, potato and pear at pH 9. The derivatives formed were characterized in terms of their physicochemical and digestion properties. The derivatization was accompanied by a reaction at the lysine and tryptophan side chains, whereby their content was decreased in comparison to that in the control whey proteins. Moreover, the solubility of the derivatives decreased over a broad pH range and the derivatization influenced the hydrophobe-hydrophile character of the whey proteins. The isoelectric points were shifted to lower pH values in the order of reactivity as follows: gallic acid > p-quinone > caffeic acid > chlorogenic acid. The other derivatives showed no or few changes compared to the control whey proteins. The formation of high molecular fractions was documented with SDS-PAGE. Especially the derivatives of chlorogenic-, caffeic-, gallic acid and p-quinone showed an increase in molecular weight of beta-lactoglobulin fraction from 18,300 to 20,000 Da. A dimer formation in molecular range 40,000 was also registered. MALDI-TOF-MS was applied to characterize the binding of the individual phenolic compounds or their oxidation products to the whey protein fractions, alpha-lactalbumin and beta-lactoglobulin. In vitro experiments showed that the digestion of the derivatized whey proteins with the enzymes of the gastrointestinal tract (trypsin, chymotrypsin, pepsin and pancreatin) was adversely effected. Similar results with regard to physicochemical characterization and digestion properties of the whey proteins treated with the applied extracts from plant beverages, fruit and vegetable were also documented. Coffee and tee were comparatively the most reactive extracts.     

Reactions of phenolic substances with lysozyme — physicochemical characterisation and proteolytic digestion of the derivatives

Lysozyme was modified by covalent attachment of selected phenolic compounds (m-, o-, p-dihydroxybenzenes, ferulic and gallic acid) at pH 9. The derivatives formed were characterized in terms of their physicochemical and digestion properties. The derivatization was accompanied by a reduction of lysine and tryptophan residues. Moreover, the solubility of the derivatives decreased over a broad pH range and the derivatization increased the hydrophobicity. The isoelectric points were shifted to lower pH values and formation of high molecular weight fractions occurred. In vitro experiments showed that, the peptic digestion of the derivatized lysozyme was adversely affected, whereas the tryptic, chymotryptic and pancreatic hydrolysis seemed to be favoured. The lytic activity of all the resulting lysozyme derivatives was reduced.     

Reactions of Chlorogenic Acid with Lysozyme: Physicochemical Characterization and Proteolytic Digestion of the Derivatives

ABSTRACT: The lysozyme-chlorogenic acid-derivatives formed at different pH were characterized in terms of their physicochemical and digestion properties, whereby pH 10 led to the highest derivatization. The results showed reduction of lysine residues and a distinctive decrease of the tryptophan fluorescence with increasing lysozyme derivatization. The solubility decreased over a broad pH range with a parallel increase in hydrophobicity of the derivatives. The isoelectric points were shifted to lower pH values and high molecular fractions were formed. The influence of the protein derivatization on the in vitro digestibility was also demonstrated. The peptic digestion of the derivatized lysozyme was adversely affected, whereas the tryptic and chymotryptic hydrolysis seemed to be favored.     

荧光光谱法探究pH值对叶黄素与牛血清白蛋白相互作用的影响

采用荧光光谱法研究不同pH值（3.0、5.2、7.4）条件下叶黄素与牛血清白蛋白（bovine serum albumin,BSA）的相互作用,利用数学模型计算得到二者的猝灭常数和热力学参数等,分析叶黄素与BSA的荧光猝灭现象及蛋白构象的变化,最后通过位点竞争实验和分子模拟技术确定了二者的结合位点。荧光光谱表明,不同pH值条件下叶黄素对BSA均产生荧光猝灭效应,在298 K时,pH 7.4的猝灭常数最大,结合常数较大;热力学参数和分子对接结果表明,叶黄素与BSA间的作用力主要是疏水相互作用;同步荧光光谱表明BSA的构象在叶黄素与pH值的共同作用下发生改变;位点竞争性实验和分子对接实验表明,pH 7.4和pH 5.2时,二者的结合位点在亚结构域ⅡA和ⅢA之间,但更接近于Sudlow’s site Ⅱ;pH 3.0时,结合位点不在亚结构域ⅡA及ⅢA附近。结果表明,pH值对叶黄素和BSA的相互作用有影响,这为蛋白质与生物活性成分的相互作用提供一定的理论依据。     

Initial pH as a determinant of cellulose digestion rate by mixed ruminal microorganisms in vitro

In vitro fermentations of pure cellulose by mixed ruminal microorganisms were conducted under conditions in which pH declined within ranges similar to those observed in the rumen. At low cellulose concentrations (12.5 g/L), the first-order rate constants (k) of cellulose disappearance were successively lower at initial pH values of 6.86, 6.56, and 6.02, but in each case the value of k was maintained over a pH range of 0.3 to 1.2 units, as the fermentation progressed. Plots of k versus initial pH were linear, and k displayed a relative decrease of approximately 7% per 0.1 unit decrease in pH. At high cellulose concentration (50 g/L) and an initial pH of 6.8, cellulose digestion was initially zero order, the absolute rate of digestion declined with pH and digestion essentially ceased at pH 5.3 after only 30% of the added cellulose was digested. Further incubation resulted in a loss of bound N and P, suggesting that at low pH cells lysed or detached from the undigested fibers. Pure cultures of ruminal cellulolytic bacteria also were able to ferment cellulose to a minimum pH of 5.1 to 5.3, but the extent of fermentation was increased by coculture with noncellulolytic bacteria. A model is proposed in which the first-order rate constant of cellulose digestion is determined by the pH at which the fermentation is initiated, and end product ratios reflect greater activity of the noncellulolytic population as pH declines.     

Influence of buffer pH and raw corn starch addition on in vitro fiber digestion kinetics.

The impact was studied of buffer pH (5.8, 6.2, and 6.8) on in vitro digestion kinetics of NDF from alfalfa hay, bromegrass hay, corn silage, and alfalfa and bromegrass hays with raw corn starch added to approximate a ration containing 30% NDF. Ash-free NDF was determined at 0, 6, 12, 18, 24, 30, 36, 48, 72, and 96 h of fermentation. Kinetic parameters were estimated by nonlinear regression using an iteratively reweighted least squares technique. Addition of raw corn starch decreased fiber digestion rate for alfalfa hay and lag for bromegrass hay. Both rate and lag of NDF digestion of all substrates were affected negatively below pH 6.2. Predicted ruminal NDF digestibilities (as percentage of that at pH 6.8 treatment) declined below pH 6.2 for all forages; addition of starch decreased predicted ruminal NDF digestibility by 23% for both alfalfa and bromegrass hays, even at pH 6.8. Results suggest that low pH decreases fiber digestion rate and increases lag and that starch accentuates this effect for some substrates.     

金花茶多糖的体外消化及酵解特性研究

采用水提醇沉法提取金花茶多糖,并通过DEAE-52纤维素柱层析对其分级纯化;通过模拟人体胃肠道消化模型对金花茶多糖进行体外消化,并检测消化过程中多糖分子量及消化产物中还原糖含量和游离单糖,探索金花茶多糖的消化特性;通过建立人体粪便酵解模型对金花茶多糖进行体外酵解,并测定酵解过程中酵解产物的pH、总糖和还原糖含量、SCFAs含量及乳酸杆菌、双歧杆菌、大肠杆菌的菌落总数,探索金花茶多糖的酵解特性。结果表明:金花茶多糖经DEAE-52纤维素柱层析纯化后,得到多糖级分TPS1、TPS2和TPS3。体外消化过程中,模拟唾液、模拟肠液没有改变TPS1、TPS2和TPS3的分子量,消化产物中还原糖含量也未见显著变化;而模拟胃液致使TPS1、TPS2和TPS3的分子质量略微降低,且还原糖含量由0.129±0.016、0.155±0.026、0.147±0.017 mmol/L增加为0.223±0.018、0.319±0.013、0.294±0.030 mmol/L;体外消化结束后未检测到游离单糖产生。体外酵解过程中,添加了TPS1、TPS2和TPS3的酵解液组的pH相较空白对照组均明显降低,总糖消耗率分别为91.4%、89.0%、94.5%,且还原糖含量在0~18 h期间升高,18~8 h期间降低;乙酸、丙酸、丁酸的浓度相较对照组都得到明显提升,增加倍数TPS3组>TPS2组>TPS1组;另外,TPS1、TPS2和TPS3均能够促进乳酸杆菌、双歧杆菌的增殖,并抑制大肠杆菌的生长,且益生效果TPS3>TPS2>TPS1。综上,金花茶多糖在体外消化模型中不易被消化,并能够被人体肠道菌群分解利用,产生大量乙酸、丙酸、丁酸等,具有潜在的益生作用,且TPS3较TPS2、TPS1具有更好的益生效果。     

玉米低聚肽硒螯合物的理化性质及稳定性

以玉米低聚肽和亚硒酸钠为原料制备玉米低聚肽硒螯合物,对其水分、酸溶蛋白、总氮(粗蛋白)、分子量分布进行了考察,然后以螯合态的硒含量和分子量分布为指标,研究了螯合物对温度、pH、消化方式的稳定性,结果表明:玉米低聚肽硒螯合物的螯合率为54.25%±0.24%,得率为53.02%±0.17%,水分含量为7.46%±0.07%,酸溶蛋白含量为16.38%±0.03%,总氮含量为21.17%±0.35%,分子量低于1000 u的比例为85.1512%。在25~100℃环境下,分子量低于1000 u的比例变化不超过7%,硒含量最低不小于70%;在pH=2~12的环境下,分子量低于1000 u的比例变化最高不超过16%,硒含量最低仍在75%以上;在经过胃蛋白酶、胰蛋白酶和两种酶的消化后,分子量低于1000 u的比例分别增加5%、7%和8%,硒含量最低仍在70%以上。这说明玉米低聚肽硒螯合物具有一定的热稳定性、酸碱稳定性和消化稳定性。     

In vitro inhibition of α‐chymotryptic activity by phenolic compounds

α‐Chymotrypsin was modified by covalent attachment of selected phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, quinic acid, m‐/o‐/p‐dihydroxybenzene and p‐benzoquinone) at pH 9. The derivatives formed were characterised in terms of their activity and selected physicochemical properties. In vitro experiments showed that the proteolytic digestion of food proteins with α‐chymotrypsin derivatives was adversely affected. This decrease depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The derivatisation was accompanied by a reduction in the amount of free lysine and tryptophan residues. Moreover, the solubility of the derivatives decreased over a broad pH range, with a parallel increase in the hydrophobicity. The isoelectric point was shifted to a lower pH value, and formation of high‐molecular‐weight fractions was documented by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). © 2001 Society of Chemical Industry     

Effect of Lectins and the Mixing of Proteins on Rate of Protein Digestibility

The rate of digestibility of protein in raw bean extract (RBE), heat-treated bean extract (HBE), casein and bovine serum albumin (BSA) was determined. The pepsin and/or pancreatin hydrolysis of RBE which contains lectins or hemagglutinins was less than that of other proteins. Addition of lectins at the same concentration present in RBE decreased the rate of digestion of HBE, casein and BSA to levels close to that of RBE. In comparison with the respective single proteins, mixtures of RBE or HBE with casein have lower digestibilities while a mixture of casein and BSA, higher digestibilities. The results suggest that lectins can affect the activity of digestive enzymes and that mixing of proteins has an effect on digestibility which is unpredicted by amino acid composition.     

甘薯交联抗性淀粉热力学性质与消化性研究

根据膨胀度、糊化度及差示扫描量热仪(DSC)测得热力学参数,综合分析甘薯交联抗性淀粉和原淀粉热力学性质,并采用Jenkins提出In–vitro模型测定淀粉体外消化性。结果表明:在同一温度下,甘薯交联抗性淀粉膨胀度和糊化度均较原淀粉低,且交联剂用量越高,淀粉膨胀度和糊化度越小;DSC测试结果显示,甘薯交联抗性淀粉相转变温度To、Tp、Tc随交联剂用量增加而升高,Tc–To和△H均比原淀粉低。In–vitro消化模拟实验表明,甘薯交联抗性淀粉消化性比原淀粉低,并随交联剂含量增加,消化产物量减少,消化速度降低。     

草鱼鱼鳞酶溶性胶原蛋白粘度特性及变性温度研究

采用旋转流变仪系统考察了浓度、pH、剪切速率、NaCl、CaCl2、丙三醇、乙醇和保温时间对草鱼鱼鳞酶溶性胶原蛋白(PSC)粘度的影响。结果显示,PSC溶液粘度随浓度增大呈指数增加;pH 3时粘度最大,pH 4和pH6~9时粘度下降,pH 10时粘度又陡然回升;粘度随剪切速率的增大呈对数下降;NaCl和CaCl2的添加都会使PSC溶液粘度下降;丙三醇和乙醇添加量在10%以内,其添加浓度与粘度成正相关;保温时间长短对PSC溶液的粘度影响不大。通过粘度变化考察了不同处理下的变性温度,结果表明,随温度升高,PSC乙酸溶液粘度降低,在28.0℃左右粘度急剧下降,胶原蛋白变性,这与乌氏粘度计测定结果(胶原蛋白乙酸溶液的变性温度为32.0℃)存在较大差异。添加4%丙三醇或4%乙醇对PSC溶液的变性温度基本没有影响,而添加1.5%NaCl或2%CaCl2时,都使变性温度下降到22.0℃左右;PSC水溶液粘度明显大于其柠檬酸和乙酸溶液,且变性温度(32.0℃)高于其柠檬酸(26.0℃)和乙酸(28.0℃)溶液。     

Bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns

To assess bioaccessible and dialysable apple polyphenols available for potential uptake by intestinal epithelial cells, an in vitro gastrointestinal (GI) digestion method was developed and main polyphenols investigated by UPLC. Polyphenolic profiles in the gastric medium were similar to those natively occurring in apples; however, bioaccessible polyphenols were at lower concentrations than those in the apples. The polyphenolic profile was altered during intestinal digestion, with a considerable decrease of total polyphenols. Flavan-3-ols were completely unstable in the intestinal medium, owing to their pH sensitivity. In addition, 41-77% of bioaccessible chlorogenic acid, the major abundant hydroxycinnamic acid in apples, was degraded during intestinal digestion, with partial isomerisation to cryptochlorogenic acid and neochlorogenic acid. All polyphenols found in the intestinal medium were dialysable, but were present at lower concentrations, suggesting that dialysable polyphenols can potentially be taken up by the enterocytes. These results highlight that GI digestion may substantially affect native apple-derived polyphenolic patterns and concentrations.     

不同来源胶原蛋白抗冻活性的研究

采用酶法制备不同来源的胶原蛋白,并对其纯度及氨基酸组成进行鉴定。在此基础上,利用差示扫描量热仪及低温显微镜对其热滞活性（thermal hysteresis activity,THA）和重结晶抑制效应（ice recrystallizationinhibition,IRI）进行了研究。结果表明：所制备的胶原蛋白均为典型的Ⅰ型胶原,纯度较高,其分子质量约为330 kD;相比于牛血清白蛋白,不同来源的胶原蛋白中,猪皮胶原具有较高的热滞活性,即当保留温度为－0.2 ℃,体系冰晶含量φ≤5% 时,THA为0.52 ℃;在猪皮胶原蛋白体系中形成的冰晶为不规则的圆球形,不易对细胞及组织造成伤害,且经过一次循环后,冰晶无显著增大。     

Specific release of Albutensins from bovine and human serum albumin

Bovine Albutensin (BA) and human Albutensin (HA) represent f(208–216) of bovine serum albumin (BSA) and f(210–218) of human serum albumin (HSA), respectively. The release of BA and HA was studied during tryptic digestion of serum albumins in solutions at different pH values. Specific release of either BA or HA was achieved by tryptic hydrolysis of serum albumins in 0.1 m NaH₂PO₄ solution adjusted to pH 3.5. Under these conditions, about 29–50% of the theoretical maximum of BA and 18–23% of the theoretical maximum of HA were released at high purity after 18 h digestion. Heat treatment of BSA (90 °C for 1–60 min) prior to tryptic hydrolysis (pH 3.5) did not improve the release of Albutensins. Under the same heating conditions, Albutensin from either BSA or HSA proved to be stable. Tryptic hydrolysates at pH 3.5 of serum albumins were submitted to simulated gastro-intestinal digestion. BA and HA were stable under peptic hydrolysis whilst they were almost completely hydrolysed by Corolase PP™.     

粳米鲊海椒的抗氧化活性及其对小鼠肝脏脂质过氧化作用的影响

粳米鲊海椒是以新鲜红辣椒经过破碎后与米粉等配料以一定比例混合并添加适量盐后厌氧发酵一定时间而成的一种中国西南地区地方特色的自然乳酸发酵辣椒制品。由于辣椒富含多酚,以及发酵过程中结合型多酚可能会释放等,因此研究了鲊海椒化学抗氧化活性及对肝脏脂质过氧化作用的影响。对不同发酵时段鲊海椒研究结果显示,总还原力和氧自由基吸收能力（oxygen radical absorbance capacity,ORAC）在发酵15 d后即达到较高水平,其峰值分别出现在发酵15 d和发酵60 d。进一步抗氧化活性分析显示,发酵15 d样品清除1,1-二苯基-2-三硝基苯肼自由基的能力强于发酵60 d样品,但无显著差异（P＞0.05）。细胞抗氧化作用研究显示,发酵60 d样品羰基含量显著低于对照＋偶氮二异丁脒盐酸盐组（P＜0.05）,对牛血清白蛋白氧化抑制率是发酵15 d样品的2.38 倍;红细胞溶血延迟时间随处理质量浓度增加快速下降,发酵15 d样品下降速率快于发酵60 d样品,质量浓度为0.5 mg/mL时溶血延迟时间相对最长,发酵15 d和60 d样品差异不显著（P＞0.5）;发酵60 d样品对肝脏脂质过氧化的抑制作用显著强于发酵15 d样品（P＜0.05）,表明延长发酵时间有利于提升细胞抗氧化能力。相关性分析结果显示,不能用多酚含量判断样品的细胞抗氧化作用。本研究表明不能单独用化学抗氧化方法评价抗氧化能力,通过建立细胞模型综合评价食物的抗氧化性能更客观判断食物的抗氧化能力。     

Development of buffer systems for pH control and evaluation of pH effects on fiber digestion in vitro.

An in vitro buffering system capable of pH control between pH 5.8 and 6.8 was developed to examine the effect of media pH on disappearance of NDF at various times of fermentation and to assess initially the effect of media pH on kinetics of NDF digestion. The pH conditions selected for evaluation of these buffer systems were 5.8, 6.2, and 6.8. Use of McIlvaine's solution with sodium bicarbonate was not successful because of rapid drifting of pH downward during fermentation. To evaluate the effectiveness of citric or phosphoric acids as components of phosphate-bicarbonate buffer systems, alfalfa silage and a mixture of alfalfa silage and corn grain (1:1 mixture, dry basis) were fermented for 0, 12, 24, 48, and 72 h. The pH of each flask was measured at 0, 4, 12, 24, 48, and 72 h postinoculation, and pH was readjusted with bicarbonate solution when necessary. Drifting of media pH downward was more noticeable when phosphoric acid was used to adjust the buffer pH than with citric acid. Citric acid had no adverse effects on NDF digestion compared with phosphoric acid when used to adjust a phosphate-bicarbonate buffer system. Alfalfa hay, bromegrass hay, and corn silage were incubated for 0, 12, 24, 48, 72, or 96 h at pH 5.8 or 6.8 using the phosphate-bicarbonate buffer system adjusted with citric acid. Estimation of kinetics of NDF digestion indicated that a decrease in media pH from 6.8 to 5.8 resulted in a marked reduction in NDF digestion; the largest apparent difference was extended digestion lag time.     

Heat-Induced Changes in the Proteins of Whey Protein Concentrate

Three-level fractional factorial experiments were used to study effects of heating conditions (pH, time, temperature, solids content, calcium addition) on whey protein concentrate. Increasing pH and temperature led to lower solubility at pH 4.6 and 7.0, lower sulfhydryl content, higher hydroxymethylfurfural, generally darker color, lower DNBS-available lysine and altered pepsin pancreatin digestion profiles. Mercaptoethanol and SDS demonstrated relative importance of disulfide and hydrophobic bonds on solubility loss. Polyacrylamide gel electrophoresis indicated heat stability of proteose peptones; susceptibility was greatest at pH 8.0, 95°C for β-lactoglobulin and α-lactalbumin, and pH 4.6, 95°C for bovine serum albumin. HPLC gel filtration showed that heating rendered a high molecular weight fraction undissociable by mercaptoethanol.     

自然发酵酸浆中一株高产酸菌的分离与鉴定

为明确自然发酵酸浆中的优势产酸菌种并为酸浆豆腐工业化生产奠定基础,以实验室自制黄浆水为培养基,采用平板涂布法,以代谢产酸量和体系pH为指标,从自然发酵酸浆中分离筛选出一株高产酸菌(植物乳杆菌T-02)。该菌株在37 ℃下发酵24 h后pH下降至3.6,总产酸量达35.3 g/L;发酵48 h后pH下降至3.6以下,总产酸量为40.8 g/L,具有较强的产酸能力,可作为酸浆豆腐的纯菌种发酵菌株。采用形态学结合生理生化试验及分子生物学方法鉴定该菌株为植物乳杆菌。     

Effects of pH and pH fluctuations on microbial fermentation and nutrient flow from a dual-flow continuous culture system

Eight dual-flow continuous culture fermenters (1400 ml) were used in two consecutive periods to study the effects of pH and pH fluctuations on microbial fermentation and nutrient flow. Fermenters were maintained at 39 degrees C, with solid and liquid dilution rates of 5 and 10%/h, respectively, and fed continuously a 60% alfalfa hay and 40% concentrate diet (18.9% crude protein, 36.6% neutral detergent fiber, 17.6% acid detergent fiber). Treatments were high pH (constant at 6.4); low pH (constant at 5.7); cycles of 4 h at pH 6.4 and 4 h at pH 5.7; and pH constant at 6.4, except for two 30-min drops per day to pH 5.7, followed by a 3-h slow recovery to pH 6.4. The low pH (constant at 5.7) produced lower apparent dry matter, neutral detergent fiber and acid detergent fiber digestion, lower total and branch-chained volatile fatty acid concentrations, and lower acetate and higher propionate proportions than high pH (constant at 6.4). There were no differences in these estimates between constant high pH and the two treatments that alternated high pH and low pH. The constant low pH reduced protein degradation and increased nonammonia N and dietary N flow compared with constant high pH. The pH treatments had no effect on bacterial N flow or efficiency of microbial protein synthesis. Flow of essential amino acids was highest for constant low pH and lowest for constant high pH. Results indicate that constant low pH reduced fiber and protein digestion and increased the flow of total and some individual amino acids. However, the effects of transitory decreases of pH were either small or insignificant with the conditions tested in this study.