RADIOACTIVE METHIONINE INCORPORATION INTO PEPTIDE CHAINS BY ENZYMATIC MODIFICATION

Enzymatic peptide modification was carried out using L-methionine-S-methyl-¹⁴C methyl ester hydrochloride and L-^3H methionine ethyl ester hydrochloride in the reaction mixture. The experimental results revealed that part of the L-methionine-S-methyl-¹⁴ C methyl ester was bound as methionine to enzymatic hydrolysates of casein and serum albumin in the presence of α-chymotrypsin, interestingly in the highest molecular weight fraction of the product protein. A maximum curve was found to describe the relation between α -chymotrypsin-induced incorporation of methionine and the ratio of L-^3H methionine ethyl ester to protein hydrolysate. The covalent nature of amino acid incorporation was supported by SDS polyacrylamide gel electrophoresis in the presence of urea. The isoelectric focusing patterns of the products indicate that transpeptidation plays an essential role in the EPM reaction. These experimental findings suggest that enzymatic peptide modification is a suitable method for the production of proteins of designed amino acid composition.     

REDUCTION OF ALLERGENICITY AND INCREASING THE BIOLOGICAL VALUE OF BUFFALO'S MILK PROTEINS BY ENZYMATIC MODIFICATION

Buffalo milk proteins were covalently enriched with methionine (Met) during the enzymatic peptide modification (EPM) process. The maximal Met-enrichment occurred when the α-chymotryptic hydrolysate of buffalo's milk proteins was treated with methionine ethyl ester in the presence of α-chymotrypsin during the EPM. Methionine content of this product was more than three times (5.4%) higher than that of the original buffalo milk protein (1.5%). The electrophoretic patterns of the modified proteins showed that the enzymatic reaction was mainly characterized by transpeptidation. Molecular masses of the modified protein fractions were influenced by the concentration of L-methionine ethyl esters in the reaction mixture during the EPM. EPM products from the buffalo milk protein hydrolysates with α-chymotrypsin and trypsin or with pepsin and trypsin showed low allergenic activities compared with those when buffalo milk proteins were hydrolyzed with α-chymotrypsin or with pepsin alone.     

发酵豆粕生产大豆多肽研究

利用30L发酵罐发酵豆粕,对微生物法生产大豆多肽的工艺进行了研究。结果显示,随着发酵时间的延长,发酵液水解度不断上升,并在发酵后期达到30%左右;发酵液中的多肽含量先增后减,表明发酵进程是按大豆蛋白水解成多肽,多肽水解成游离氨基酸顺序进行。酶活分析表明发酵菌株Bacillus.subtilisSHZ3能同时分泌蛋白酶和羧肽酶,分别水解大豆蛋白和肽链末端的疏水性氨基酸,使大豆蛋白的水解和多肽的脱苦在发酵过程中一步完成,生产出不苦的大豆多肽。凝胶层析表明,随着发酵时间的延长,发酵液中肽链变短,24h发酵所得主要为300～1000Da具有潜在生物活性的短链肽。     

蚕蛹多肽制备工艺及其体外抗氧化活性

采用碱性蛋白酶、风味蛋白酶、复合蛋白酶、木瓜蛋白酶、胰蛋白酶水解蚕蛹蛋白,以水解度和清除DPPH·能力为指标对酶解过程进行分析,并研究水解产物多肽的体外抗氧化活性。结果表明,碱性蛋白酶对蚕蛹蛋白具有较好的水解效果,其水解产物有较高抗氧化活性,对DPPH·、超氧阴离子自由基(O2·)和羟自由基(·OH)都具有较强的清除能力。     

Determination of sulfur-containing amino acids in food products

To define the most accurate method for determination of sulfur-containing amino acids in foods, the possibility was tried of using a method suggested by Moore. The method is based on preliminary oxidation of cystine and methionine by performic acid and transformation of these substances to acid hydrolysis resistant cysteic acid and methionine sulphone. Model experiments and use of foods (wheat flour, grade II, and protein part of chicken egg) demonstrated that exposure of proteins to performic acid brings about oxidation of sulfur-containing amino acids to the hydrolysis-resistant forms which may be defined by chromatography with a greater accuracy. Experiments without preliminary oxidation by performic acid showed that foods experience an appreciable destruction (by 30-60%) of cystine and methionine. Therefore, it is advisable that during studies into amino acids contained by proteins, additional hydrolysis may be performed with preliminary oxidation of the samples by performic acid, which enables the determination of the content of methionine and cystine with a greater accuracy.     

大豆蛋白和鱼肉复合挤压过程中水分状态变化研究

大豆蛋白含有丰富的氨基酸,同时鱼肉的脂肪比较丰富,鱼糜相关制品在我国的开发存在很大的发展空间。以大豆蛋白和鱼糜为原料,采用双螺杆挤压机对其进行复合挤压组织化,研制出一种新型的复合蛋白产品。利用低场核磁共振分析测得不同加工工艺条件下的复合蛋白产品自由水与结合水的相对变化情况,并对其变化规律进行分析。同时,对产品和原料的常规理化指标进行了检测与比较。结果表明:新型复合蛋白产品的保水性较好,自由水与结合水含量相对合理,蛋白质含量与脂肪含量等理化指标符合健康食品的标准。     

AMINO ACID COMPOSITION AND NUTRITIONAL IMPLICATIONS OF MILK FERMENTED BY VARIOUS LACTIC CULTURES

A total of nine milk products were analyzed for total and free amino acids and total and nonprotein nitrogen. The products were: (1) whole milk (WM), (2) skim milk (SM), (3) WM fermented by Streptococcus thermophilus, (4) SM fermented by S. thermophilus, (5) WM fermented by Lactobacillus bulgaricus, (6) SM fermented by L. bulgaricus, (7) WM fermented by L. acidophilus, (8) SM fermented by L. acidophilus and (9) commercially prepared buttermilk. Essential amino acid content of the fermented milk was lower than that of the whole or skim milk. Fermentation of milk by lactic cultures decreased the total lysine (except butter milk) and methionine contents of all the samples. Since these two amino acids are limiting amino acids in protein malnourished areas, lactic fermentation of milk may be undesirable based on the chemical scores of the protein. However, the free amino acid content, including that of lysine and methionine, increases significantly upon lactic fermentation of milk. This predigestion of protein might result in an increased availability of the amino acids to offset the reduction in amino acids during fermentation and may even result in a better quality protein. Biological evaluation of the fermented milk proteins is necessary to evaluate their nutritional quality. L. bulgaricus was the most proteolytic of all the organisms used.     

Effect of preliminary and culinary processing on amino acid content and protein quality in frozen French beans

The aim of the study was to evaluate the content of amino acids and protein quality of French bean pods. The investigated material consisted of the raw vegetable, fresh pods cooked to consumption consistency, and two kinds of frozen products stored for 12 months at ?20 °C and then prepared for consumption: frozen beans obtained using the traditional method (blanching before freezing) and frozen products of the ready‐to‐eat type (cooking before freezing). A comparison of the amino acid content in the product prepared for consumption showed that the lowest quantities were found in French beans obtained using the traditional method; products obtained using the modified technology and beans cooked directly after harvest had similar levels of most amino acids. The content of amino acids in 16 g N was less varied than in 100 g of the product. The protein in all the three products prepared for consumption as well as that in fresh bean pods hardly differs, as confirmed by the values in the essential amino acid index (EAA). The first limiting amino acid was methionine with cystine and the second was lysine.     

团头鲂不同生长阶段肌肉营养成分分析及评价

分析团头鲂（Megalobrama amblycephala）不同生长阶段,肌肉中水解及游离氨基酸组成和含量,并对其营养价值和产品风味进行评价。结果表明：团头鲂肌肉一般营养成分（水分、灰分、粗蛋白、脂肪）随着生长阶段不同并未表现出显著差异,各成分含量基本保持恒定。水解及游离氨基酸含量分别为（7.77±0.79）g/100 g（湿质量）和（0.98±0.10）g/100 g（湿质量）;不同生长阶段未表现出显著性差异。必需氨基酸组成齐全,必需氨基酸占总氨基酸比值为34.62%～38.77%,必需氨基酸与非必需氨基酸比值为70.75%～85.99%。氨基酸评分和化学评分都表明其第一限制氨基酸为甲硫氨酸和胱氨酸。不同生长阶段,团头鲂肌肉中游离氨基酸及其衍生物组成和含量无显著差异,其中牛磺酸含量最高,占游离氨基酸总量的32.04%。     

牛骨股原蛋百肽体外清除自由基活性的研究

对经碱性蛋白酶Alcalase 2.4L水解的牛骨胶原蛋白肽清除自由基活性进行了研究。根据对羟基自由基、超氧阴离子自由基、二苯代苦味酰基自由清除能力的测定,以及还原能力、脂质抗氧化能力等抗氧化活性指标的测定,确定了水解度为7.9%左右的胶原蛋白肽清除自由基的综合效果最好,此时酶解产物的平均分子量为2025Da,含15个左右的氨基酸。在此条件下,超氧阴离子(O2-·)的清除率为63.86%,羟基自由基(·OH)的清除率为99.55%,二苯代苦味酰基自由基(DPPH·)的清除率为27.03%,对大豆卵磷脂过氧化作用的抑制率为74.78%。     

CHANGES IN NITROGEN FRACTIONS AND FREE AMINO ACIDS DURING RIPENING OF SPANISH DRIED BEEF "CECINA"

The changes in nitrogen fractions (total nonprotein nitrogen -NPN-, amino acid nitrogen, peptide nitrogen and volatile basic nitrogen) and free amino acids during the ripening of "cecina" (a Spanish dried beef product) were tracked. The amount of total NPN, amino acid nitrogen and volatile basic nitrogen increased during processing, while peptide nitrogen decreased in the last stage. An increase was observed in all amino acids except taurine. In the final product, the most abundant free amino acids were leucine-isoleucine mixture, lysine and proline-methionine mixture. Taking into account the concentrations of the most abundant amino acids detected in cecina and the taste thresholds described for L-amino acids in water, it appears that several amino acids i.e. lysine, alanine, valine and glutamic acid, could contribute to the flavor of this meat product.     

复合酶水解蚕蛹蛋白制备功能性寡肽的工艺研究

为了制备分子量低、肽含量高的蚕蛹功能性寡肽,本文采用单素实验与响应面分析法,对不同蛋白酶及其不同组合、温度、pH值、底物浓度、酶的添加量等因素对蚕蛹分离蛋白水解工艺的影响进行了研究.研究结果表明,多酶复合水解可显著提高蚕蛹蛋白的水解度和寡肽得率,其中胰酶、风味蛋白酶与中性蛋白酶是最佳多酶组合,其最佳水解条件为底物浓度7.33％、酶添加量[E]/[S]3.62％、pH7.38、水解温度54.6℃、水解时间6h.在此条件下,蚕蛹蛋白的水解度高达29.2％,寡肽得率高达为81.14％.酶解后产品数均分子量为665.5 Da,重均分子量为726.9 Da,肽含量高达74.6％,而游离氨基酸含量仅为7.33％,表明复合酶解虽然提高了蚕蛹蛋白的水解度,但主要产物仍然是低分子寡肽,并没有大量生成游离氨基酸.     

Lactational and Systemic Responses to the Supplementation of Protected Methionine in Soybean Meal Diets

Twenty-seven Holstein cows (14 primiparous and 13 multiparous) were randomly assigned to diets containing soybean meal without or with 15 g of added DL-methionine daily, provided as 50 g of ruminally protected methionine product, during wk 4 through 16 postpartum. Cows were fed a 15.3% crude protein total mixed diet of (dry matter basis) 30% corn silage, 15% alfalfa hay, and 55% concentrate mix. Yields of milk (32.9 and 35.2 kg/d), 4% fat-corrected milk (27.8 and 29.5 kg/d), and solids-corrected milk (28.5 and 30.1 kg/d) were higher for cows fed supplemental methionine. Milk protein percentage (2.99 and 3.06) was increased with supplemental methionine, while the percentage of fat (2.96 and 3.00), solids-not-fat (8.69 and 8.73), and total solids (11.67 and 11.71) were similar among diets. Dry matter intake (19.3 and 21.3 kg/d) was higher with methionine supplementation. Ruminal pH, volatile fatty acids, ammonia, and serum urea were generally unaffected by methionine supplementation. Concentrations of methionine in arterial and venous serum were elevated slightly by methionine supplementation, but the first-limiting amino acid for milk production, as calculated by several methods, was not changed.     

基于乙醇注入-高压均质的蛋清肽脂质体制备及体内外缓释效果

为提高蛋清肽的稳定性,本实验采用高压均质辅助乙醇注入法制备蛋清肽脂质体。通过单因素试验确定脂质体制备的最佳工艺条件：2 mg/mL胆固醇、10 mg/mL蛋黄卵磷脂、50 mg/mL蛋清肽溶液、芯壁体积比3∶5、40 MPa高压均质7 min、100 W超声30 s,该条件下得到包埋率为（65.7±3.6）%的蛋清肽脂质体。选择1%吐温-80作为稳定剂,可以保证脂质体在30 d内具有良好的贮藏稳定性。此外,体外、体内释放实验结果表明,脂质体的包埋对蛋清肽有明显的缓释效果,并且小鼠血清中6 种必需氨基酸最高质量浓度明显提高;其中,色氨酸延后3.5 h释放,苯丙氨酸、缬氨酸、蛋氨酸延后2 h释放,脂质体包埋后赖氨酸最高质量浓度增加最多（523.53%）,蛋氨酸质量浓度增加最少（109.31%）。本研究获得了一种稳定性较强、包埋率较高、缓释效果较好的蛋清肽脂质体,可以有效实现氨基酸持续补充,为蛋清肽相关功能性产品的开发提供一定技术支持。     

Hydrolysis degree,peptide profile and phenylalanine removal from whey protein concentrate hydrolysates obtained by various proteases

The action of various proteases was tested for preparing whey protein concentrate (WPC) hydrolysates with high degree of hydrolysis (DH), appropriate peptide profiles and reduced phenylalanine (Phe) content. The peptide profile analysis included the fractionation of hydrolysates by size‐exclusion HPLC. The rapid correct fraction area method was used to quantify the components of the chromatographic fractions. Activated carbon (AC) was used to remove Phe, and its efficiency was evaluated by measuring the amount of Phe by second‐derivative spectrophotometry. The results showed that the DH of WPC hydrolysates increased and that the protease from Aspergillus oryzae yielded the highest DH value. This protease also produced the best peptide profile, that is, the highest di‐ and tripeptide content (16.14%), the highest amounts of free amino acids (18.43%) and the lowest amount of large peptides (18.76%). The proteases from both A. oryzae and Bacillus subtilis produced the highest Phe removals (79.0 and 77.8%, respectively).     

牙鲆鱼片冷藏过程中蛋白质降解规律及预测模型的构建

研究了牙鲆鱼片在冷藏过程中蛋白质的降解规律,测定了不同贮藏温度下鱼片的TVB-N值、TCA-可溶性肽、游离氨基酸、菌落总数变化及进行感官评价,建立基于蛋白质降解指标的品质预测模型。结果表明:随着贮藏时间的延长,牙鲆鱼片的TVB-N、TCA-可溶性肽、游离氨基酸含量及菌落总数值均呈现上升趋势,且温度越高蛋白质降解指标变化越显著。TVB-N值、TCA-可溶性肽含量、菌落总数与一级化学反应动力学模型有很好的拟合效果(R2>0.9),游离氨基酸含量与一级化学反应动力学模型有较好的拟合效果(R2>0.82);用Arrhenius方程构建蛋白质降解指标与贮藏温度、贮藏时间的动力学模型,具有较高的拟合精度(R2>0.9)。经验证,蛋白质降解指标的预测值与实际值的相对误差均在±10%内,该模型能很好地预测-2~10℃冷藏温度下牙鲆鱼片的品质变化。     

Effect of processing on the amino acid content of New Zealand spinach (Tetragonia tetragonioides Pall. Kuntze)

The aim of this work was to evaluate the effect of processing on the amino acid content and protein quality of New Zealand spinach (Tetragonia tetragonioides Pall. Kuntze). In this research, fresh and cooked New Zealand spinach as well as two frozen products prepared for consumption, one obtained using the traditional method (blanching–freezing–storage–cooking) and the other a convenience food product obtained using a modified process (cooking–freezing–storage–defrosting and heating in a microwave oven), were analysed. Glutamic acid was the dominant amino acid in fresh New Zealand spinach, and the limiting amino acids were cystine and methionine. Technological and culinary processing caused a significant increase in amino acid content in 100 g of edible portion, with the exception of methionine and cystine in frozen products prepared for eating. Changes in amino acid content expressed in g/16 g of N (which corresponded to 100 g tissue protein) were not significant, with the exception of the lower glutamic acid content in the frozen product obtained using traditional processing method.     

Amino acid profile of raw and as-eaten products of spinach (Spinacia oleracea L.)

The aim of the work was to evaluate the amino acid composition of fresh spinach and spinach products prepared for consumption. The investigation included fresh and cooked spinach and two kinds of frozen product: one obtained using the traditional method (blanching-freezing-refrigerated storage) and then cooked; and the other, of the ready-to-eat type, obtained using the modified method (cooking-freezing-refrigerated storage) and then prepared for consumption in a microwave oven. In 100 g of edible parts of the as-eaten products, the content of amino acids exceeded that in the raw material from which they were obtained, except for sulphur amino acids and tyrosine. In the as-eaten products, the content of amino acids in protein calculated in 16 g N was similar to that in the raw material, except for lower tyrosine and higher arginine content in the traditional frozen product prepared for consumption. Cystine with methionine was the amino acid limiting the quality of protein in the investigated samples, except for the traditional frozen product prepared for consumption, where the Chemical Score (CS) index was 100.     

玉米蛋白复合风味蛋白酶水解物性质的研究

以玉米蛋白为原料,主要研究了通过复合风味蛋白酶水解制备的玉米蛋白水解物的功能特性。结果表明:玉米蛋白经复合风味蛋白酶水解后水解度为11.43%,体外实验该水解度下的玉米蛋白水解物结合胆汁酸的能力较强,相对分子质量分布在10 000～180 Da之间,水解物中有高含量的疏水性氨基酸残留。玉米蛋白水解物持水性为2.95 mL/g,持油性为2.13 mL/g,乳化性为49.96 mL/g,起泡性为126%。玉米蛋白水解物经Sephadex G-15、RP-HPLC分离纯化,对含量最高的组分F2,用MALDI-TOF/TOF MS/MS方法进行鉴定,其相对分子质量为244.2 Da,氨基酸组成为脯氨酸-谷氨酰胺。     

Elucidation of the Role of Nitrogenous Wort Components in Yeast Fermentation

The Free Amino Nitrogen (FAN) content of wort prescribes efficient yeast cell growth and fermentation performance. FAN consists of the individual amino acids, small peptides and ammonia ions formed during malting, the relative amounts of which vary. In this paper, the individual constituents of FAN were dissected and their effect on both ale and lager fermentations determined. The patterns of amino acid and small peptide uptake and the changes in extracellular protease activity revealed the dynamic environment that develops during fermentation. Lysine and methionine, previously identified as key amino acids in wort fermentation, were investigated further.