Enzymatic Conditions of an In Vitro Method to Study Protein Digestion

An in vitro digestion method was proposed for studying protein digestion. Protein (casein) was submitted to peptic proteolysis in a closed system followed by hydrolysis with pancreatic enzymes in a “digestion cell” with continuous elimination of digested products by dialysis. The peptic digestion was performed with a pepsin source of high specific activity (3152 units/mg protein) with an enzyme:substrate (E:S) ratio of 1: 250. The second-step of proteolysis was carried out with pancreatin for 6 hr at an E:S ratio of 1:25. A 10 mM sodium phosphate buffer, pH 7.5, was used as the circulating dialysis buffer.     

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.     

Digestion kinetics of fiber: influence of in vitro buffer pH varied within observed physiological range.

In vitro buffer pH reflective of the diurnal variation in ruminal pH was evaluated for its impact on digestion kinetics of NDF from three forage sources. Alfalfa hay, bromegrass hay, and corn silage were incubated in phosphate-bicarbonate buffer solution adjusted to pH 6.8, 6.5, 6.2, 6.0, 5.8, or 5.5 using 1 M citric acid. Ash-free NDF was measured after 0, 6, 12, 18, 24, 48, 72, and 96 h of fermentation. The experiment was replicated three times; kinetic parameters of fiber digestion were estimated by logarithmic transformation, and by linear and nonlinear regression procedures. Lag in NDF digestion increased as pH fell from 6.8 to 6.5 and again when pH decreased below 6.0. Decreasing buffer pH below 6.2 dramatically reduced NDF digestion rate for alfalfa hay and corn silage but had no significant effect on NDF digestion rate of bromegrass hay. Lag in NDF digestion increased below pH 6.0 for both alfalfa and corn silage but increased only when pH fell below 6.2 for bromegrass. Results suggest that lowered pH exerts its negative effect on NDF digestion between pH 6.2 and 5.8, as evidenced by increased lag and decreased rate, and that critical pH and specific, affected component of digestion varied among forages.     

Optimisation of in vitro measurement of available iron from different fortificants in citric fruit juices

The percentage of dialyzable ferrous and total iron were studied in a citric juice (pineapple and passion fruit) fortified with ferrous sulphate, micronised dispersible ferric pyrophosphate and ferrous bis-glycinate in similar concentrations (49.2 mg Fe/l). The in vitro method of Kapsokefalou and Miller (1991) [Kapsokefalou, M., & Miller, D. D. (1991). Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. Journal of Food Science, 56, 352–355.] was optimised for this matrix using 0.15 N PIPES buffer (pH 8.5) to adjust pH during pancreatic digestion. We also studied different pH values of Hepes buffer used in the measurement of iron concentrations with Ferrozine (chromogen solution). The maximum absorbances were obtained with a Hepes buffer pH value of 8.5. Ferrous sulphate was used as a reference salt due to its high bioavailability, although novel compounds, such as ferrous bis-glycinate and micronised dispersible ferric pyrophosphate, showed a high relative iron availability in this juice. Taking into account that percentage of dialysable ferrous iron is considered to be the more available fraction of total iron, the iron fortificant ferrous bis-glycinate proved to be more adequate for fortifing citric juices, giving a 10.7% of dialyzable ferrous iron. Moreover, the percentage of dialyzable total iron from ferrous bis-glycinate (31.0%) was statistically higher than those from ferrous sulphate and micronised ferric pyrophosphate (28.4% and 28.2%, respectively).     

以不同体外模拟系统评价大豆蛋白的胃消化特性

采用静态消化模型和半动态消化模型,评价经透析处理和未经透析处理的大豆蛋白的胃消化特性。结果表明：在静态消化模型中,透析对大豆蛋白的胃消化过程没有影响;但在半动态消化模型中,未经透析的大豆蛋白pH值、排空内容物的干物质质量和游离氨基的含量与经透析处理的大豆蛋白存在一定差异,表明未经透析处理的大豆蛋白保留较多大豆中的成分,使其缓冲能力更强,胃排空滞后,蛋白质消化速率减慢。此外,静态消化模型和半动态消化模型有不同的优势,所以可以根据不同的研究目的采用不同的消化模型。这项工作将为大豆蛋白产品的开发和健康效应提供新的见解。     

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.     

Bioavailability study using an in-vitro method of iodine and bromine in edible seaweed

Raw edible seaweed harvested in the Galician coast (Northwester Spain), including two red seaweed types (Dulse and Nori), three brown seaweed (Kombu, Wakame and Sea Spaghetti), one green seaweed (Sea Lettuce) and one microalgae (Spirulina platensis) were analysed for total iodine and total bromine, as well as for iodine and bromine bioavailability by in-vitro methods (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked seaweed sample (canned in brine) consisting of a mixture of two brown seaweed (Sea Spaghetti and Furbelows) and a derived product (agar–agar) from the red seaweed Gelidiumm sesquipedale, were also included in the study. All measurements were carried out by inductively coupled plasma–mass spectrometry using tellurium and yttrium as internal standards for iodine and bromine, respectively. An optimised microwave assisted alkaline (TMAH) digestion procedure was used as sample pre-treatment for total iodine and bromine determinations, as well as for the determination of both elements in the non-dialyzable fractions. PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for the intestinal digestion. Accuracy of the method (total bromine and iodine determinations) was assessed by analysing a NIES-09 certified reference material. The accuracy of the in-vitro procedure was established by a mass-balance study which led, after statistical evaluation (95% confidence interval), good accuracy of the whole in-vitro process. The highest dialyzability bromine percentages (36 ± 0.7% and 47 ± 3.0%) were obtained for red seaweed (Dulse and Nori), while higher dialyzability iodine was assessed for the brown seaweed (Kombu), around 17% ± 0.7%.     

皮革中镉含量的阳极溶出伏安极谱法测定

建立了微波消解法处理皮革样品、阳极溶出伏安极谱法测定样品溶液中镉含量的检测方法。在草酸铵-氯化铵-盐酸缓冲溶液(pH值为1.65)中加入样品溶液,富集时间为60s,氮气脱氧时间为300s,线性范围为0.2～100μg/L,检测限为0.1μg/L,相对标准偏差(n=8)为7.83%,回收率在97.3%～107.1%之间。结果表明,伏安极谱法测定皮革中镉含量简便可行。     

苦荞凝集素的稳定性及体外消化性

目的:从苦荞种子中分离得到一种凝集素(tartary buckwheat lectin,TBL),研究其酸碱、热稳定性及体外消化性。方法:采用酸性缓冲液浸提脱脂荞麦粉及阴离子交换层析纯化凝集素,TBL分别经沸水浴加热不同时间、不同pH处理、模仿胃液、模仿胰液消化后,采用SDS-PAGE及灰度扫描分析,研究其稳定性及体外消化性。结果表明:选用pH4.7浸提缓冲液可简化提取工艺,采用一步层析可得到电泳纯TBL。沸水浴加热60 min时,TBL仍可保留50%以上。在pH4~12条件处理30 min后,BTL的保留率均在80%以上。体外消化实验表明,TBL对人工模拟胃液有一定抗性,降解一半TBL所需时间为10 min,而TBL在人工模拟肠液中很难消化,即使作用时间为50 min时,TBL也未发生明显降解。将TBL沸水浴加热30 min后再进行体外消化实验,发现在模拟肠液及模拟胃液中,仅处理10 min时,SDS-PAGE结果中TBL蛋白条带完全消失,表明被TBL被消化酶完全降解。结论:天然TBL具有良好的稳定性,耐热、耐酸碱、耐胰蛋白酶消化,在胃蛋白酶液中降解也较为缓慢。预加热处理可明显提高TBL在模拟胃液及模拟肠液中的消化率。     

大豆磷脂克拉霉素脂质体制备及性质研究

采用薄膜冷冻法制备了克拉霉素脂质体,以高效液相色谱法为分析手段,采用反透析法测定克拉霉素脂质体的包封率,并通过正交实验确定了最佳制备条件为:水浴温度为45℃,m(克拉霉素):m(磷脂)=1:35,m(磷脂):m(胆固醇)=4:1,m(有机相):m(水相)=4:1,水合介质为pH6.8的磷酸盐缓冲溶液。在此条件下,克拉霉素脂质体的包封率达到81%,并从形态、粒径、渗漏率等方面对克拉霉素脂质体进行了质量评价。     

Separate determination of ethylenediaminetetraacetic acid and its calcium chelate in foods by colorimetry

A simple colorimetric method is described for the separate determination of ethylenediaminetetra acetic acid (free EDTA) and its calcium chelate (Ca-EDTA) in commercial foods. The underlying principle of the method is that free EDTA is adsorbed by a cation exchange column, but Ca-EDTA is not under weak alkaline conditions. The sample was homogenized with 0,1 n-NaOH and then subjected to equilibrium dialysis against 0,02 n-NaOH at ambient temperature for a definite time (not less than 12 h). An aliquot of the dialysate was measured into a beaker. After the pH of the solution had been adjusted to 8.5 it was applied to a pre-packed cation-exchange column (Amino-form). Free EDTA was adsorbed by this column, whilst Ca-EDTA passed through it without any loss. For assay of Ca-EDTA, the pH of the eluate was adjusted to 2.5, followed by the addition of an excess amount of CuSO4 to convert Ca-EDTA into Cu-EDTA. This solution was then divided into two portions of equal volume. One portion was subjected to the assay of free copper ions, and the other to the assay of total (the sum of free and chelated) copper ions. The difference between the two values is a measure of Ca-EDTA. For the assay of free EDTA, EDTA adsorbed by the column was eluted with 0.5 n-acetate buffer (pH 5.0), followed by the treatment in the same manner as in the case of Ca-EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apple Polyphenol Oxidase Inhibitor(s) from Common Housefly (Musca Domestica L.)

ABSTRACT: A new natural apple polyphenol oxidase (PPO) inhibitor(s) from housefly ( Musca domestica L.) was discovered. Crude inhibitor (s) isolated by buffer extraction, heat treatment, and dialysis from housefly pupae inhibited the activity of apple PPO up to 90% at pH values above 5.0. Inhibition was strictly pH-dependent. The inhibitor(s) was further characterized by employing heat, freezing and thawing, irradiation, pH adjustment, and ultrafiltration studies. The potential PPO inhibitor(s) was stable to heating at 100 °C for 1 h, repeated freezing and thawing, and irradiation. The inhibitor(s) was most stable at pH around 5.0 and least stable at alkaline pH. The PPO inhibition profile of housefly during metamorphosis also was evaluated.     

TOMATO POLYGALACTURONASE EXTRACTABILITY

The extractability of polygalacturonase (PG) activity from alcohol-insoluble solids (AIS) prepared from red-ripe tomato pericarp tissue was investigated using acetate (pH 4.5), citrate (pH 4.5) and MES (pH 6.0) buffers. The acetate buffer was also investigated with increasing concentrations of NaCl, CaCl₂, MgCl₂, ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethylether)-N,N'-tetraacetic acid (EGTA). Heat treatment of AIS to prepare control material suppressed PG extractability but not in situ PG activity, as evidenced by the release of significant quantities of soluble uronides into extraction media. With only a single extraction, acetate buffer containing > 1 M NaCl yielded the greatest amount of PG activity (> 50% of maximum extractability), with MES buffer being only marginally less efficient. However, 3 successive extractions over a 2 h period yielded significantly greater amounts of PG activity. The greatest yields of PG activity were obtained by successive extraction with parent MES and acetate buffers. There appeared to be little benefit in adding NaCl or a chelating agent to the extraction medium. Use of these extractants is suggested to have led to losses of PG activity during dialysis, via coprecipitation of PG protein with otherwise soluble uronide material that was released in greater quantities when these extractants were used. Increasing CaCl₂ and MgCl₂ concentrations reduced the amount of extracted PG activity similarly, to about 50% of maximum levels with successive extractions.     

COLLAGEN TYPES IN MECHANICALLY DEBONED CHICKEN MEAT

Mechanically deboned chicken meat (MDCM) from neck parts was digested with pepsin for 24 h at 4C. The solubilized collagens were subjected to salt fractionation both at acidic and neutral pH. The precipitates obtained after centrifugation, dialysis and lyophilization were quantitatively evaluated and collagen types were identified. The precipitate formed at 0.7 M NaCl pH 2.5, was dissolved in 1.0 M NaCl, 0.05 M-Tris-HCl, pH 7.5 buffer and further fractionated by sequential salt precipitation at 1.8, 2.5 and 4.5 M. Collagen types I, II, III and V were detected by electrophoresis techniques. Type I collagen was the major component. The presence of type II collagen indicated MDCM contained cartilaginous tissues.     

Kinetics of an Acid-Base Catalyzed Reaction (Aspartame Degradation) as Affected by Polyol-Induced Changes in Buffer pH and pKa Values

ABSTRACT:  The kinetics of an acid-base catalyzed reaction, aspartame degradation, were examined as affected by the changes in pH and pK_a values caused by adding polyols (sucrose, glycerol) to phosphate buffer. Sucrose-containing phosphate buffer solutions had a lower pH than that of phosphate buffer alone, which contributed, in part, to reduced aspartame reactivity. A kinetic model was introduced for aspartame degradation that encompassed pH and buffer salt concentrations, both of which change with a shift in the apparent pK_a value. Aspartame degradation rate constants in sucrose-containing solutions were successfully predicted using this model when corrections (that is, lower pH, lower apparent pK_a value, buffer dilution from the polyol) were applied. The change in buffer properties (pH, pK_a) from adding sucrose to phosphate buffer does impact food chemical stability. These effects can be successfully incorporated into predictive kinetic models. Therefore, pH and pK_a changes from adding polyols to buffer should be considered during food product development.     

破壁灵芝孢子的体外模拟消化特性研究

为探究破壁灵芝孢子在口腔和胃肠道中的消化吸收情况以及对肠道环境的影响。以机械碾压法、微波法和超声法破壁的灵芝孢子（Ganoderma lucidum spore,GLS）为研究对象,经体外模拟人体口腔、胃、小肠消化系统及透析模型,随后将消化后的底物进行体外发酵。测定消化各个阶段的灵芝孢子失质量率、多糖和三萜化合物释放量、生物可接受率和透析率以及小肠消化底物在0、6、12、24、48 h体外发酵过程中pH变化。结果显示,胃和小肠是灵芝孢子的主要消化场所,破壁组平均失质量率达到23.84%,其中机械碾压组更易被消化,其失质量率为29.46%。97%的多糖在胃肠液中溶出,机械碾压组多糖的生物可接受率具有最高水平,为87.33%。三萜只在破壁组小肠模拟消化中有微量溶出,平均约1.50±0.04 mg/g,95.2%的三萜随沉淀进入结肠。体外发酵结果显示,发酵液pH在0~12 h持续下降,并在12 h基本到达发酵终点。对比三种破壁灵芝孢子的消化特性,机械碾压法破壁的品质可观,不失为企业规模化破壁生产的优选。综上所述,破壁有利于提高灵芝孢子的有效成分在人体消化环境内释放量和生物可接受率,促进结肠发酵产酸,有助于调节人体肠道环境。     

Comparison of Methods to Remove Endogenous Iron from Soy Protein Isolates

The relative effectiveness of ion exchange, dialysis, heating and partial enzyme digestion treatments and the use of EDTA, oxalate, and ascorbic acid was evaluated for removing endogenous Fe from soy protein isolate. The pH 2 ascorbic acid dialysis treatment removed about 60% of the endogenous Fe from soy isolate, but the neutral to mildly alkaline treatments generally failed to remove > 30% of the endogenous Fe. The results of this study were interpreted to indicate that factors other than the proposed phytate-Fe-protein complex were responsible in large measure for the incomplete removal of endogenous Fe from soy protein isolate.     

Reduction of Campylobacter jejuni in a simulated chicken digestive tract by Lactobacilli cultures

Studies were conducted to investigate the impact of a selected lactobacilli mixed culture on Campylobacter jejuni in simulated chicken digestive tract models. Veronal buffer solutions corresponding to the pH of successive segments of the chicken digestive tract were prepared. The lactobacilli mixtures were prepared by mixing four fresh lactobacilli cultures, including Lactobacillus acidophilus, Lactobacillus fermenentum, Lactobacillus crispatus, and Lactobacillus brevis. The C. jejuni and lactobacilli mixture were mixed with sterile poultry feed, and the previously prepared veronal buffer solutions were then added separately. The mixture was incubated at 41.1 degrees C for various lengths of time with periodic agitation. The feed passage time for five segments of the digestive tract were adopted: crop (pH 4.5), 30 min; proventriculus (pH 4.4), 15 min; gizzard (pH 2.6), 90 min; small intestine (pH 6.2), 90 min; and large intestine (pH 6.3), 15 min. The Campylobacter and lactobacilli were enumerated. An antagonistic effect on C. jejuni by the tested lactobacilli spp. was found in individual sections and the complete simulated digestive tract models. In the simulated complete chicken digestion system, no C. jejuni were found during the final incubation period when a lactobacilli mixture was present. The results of this in vitro study indicate the potential value of future in vivo studies.     

富硒螺旋藻蛋白水解多肽的制备及其对ACE活性的抑制作用

从富硒螺旋藻(SeSP)中提取含硒总蛋白(SeSP-TP),建立蛋白酶水解制备含硒多肽(SeSP-PP)的最佳条件,体外检测SeSP-PP对血管紧张素转化酶(ACE)活性的抑制作用,采用ICP法快速测定藻粉及总蛋白中的硒含量。结果发现,5种常见蛋白酶对SeSP-TP的水解度趋势为:胃蛋白酶-胰蛋白酶-胰凝乳蛋白酶联合酶>碱性蛋白酶>木瓜蛋白酶>胰凝乳蛋白酶>胰蛋白酶>胃蛋白酶。利用胃蛋白酶-胰蛋白酶-胰凝乳蛋白酶联合酶水解,可显著提高蛋白水解度,达到87.94%,且联合水解的多肽对ACE抑制效率最高(89.47%)。碱性蛋白酶水解得到的多肽对ACE的抑制率也较高。当荧光检测出的含硒多肽高于50μg/mL时,对NO的合成有明显的促进效应,而含硒蛋白效果不明显。水溶性和非水溶性组分中测定的硒含量之和占总硒的95.47%,可能在提取制备SeSP-TP过程中,部分含硒蛋白质和含硒小分子会在沉淀、复溶和透析等步骤中丢失。     

Lipoxygenase in Sweet Corn Germ: Isolation and Physicochemical Properties

Off-flavor and off-aroma development, which may be catalyzed by lipoxygenase (LPO), are common in frozen stored sweet corn. Lipoxygenase activity in the germ fraction of sweet com (Zea nruys L. cv. Jubilee) was determined and compared with that in the degermed fraction. Lipoxygenase activity/g germ was about three times greater than that of the degermed fraction, Optimized procedures for isolation of lipoxygenase from the germ fraction were developed. Lipoxygenase was isolated by preparation as an acetone powder, extraction with 0.2M TrisHCI, pH 8.0 (4°C), fractionation with 40–60% saturated ammonium sulfate and dialysis. Optimum pH was 6–7 and temperature 50°C for activity of partially purified lipoxygenase. The enzyme appeared stable at pH 5–8 and ～90% of original activity was inactivated after heating in pH 7 buffer at 70°C for 3 min.