Debittering of a Tryptic Digest of Bovine p-casein Using Porcine Kidney General Aminopeptidase and X-Prolydipeptidyl Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

ABSTRACT: The role of leucine aminopeptidase (LAP) from pig kidney cytosol and X-prolyldipeptidyl aminopeptidase (Pep X) from Lactococcus lactis subsp. cremoris AM2 in the hydrolysis and debittering of a tryptic digest of β-casein was studied. Hydrolysis was monitored by quantifying the release of primary amino groups and bitterness by use of a trained sensory panel. Sequential incubation of the bitter tryptic hydrolysate with LAP, Pep X and LAP resulted in higher levels of hydrolysis and significantly (P < 0.001) lower levels of bitterness than incubation with LAP alone. The results demonstrate the central role proline-specific aminopeptidases can play in the hydrolysis and debittering of food protein hydrolysates.     

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Method for the Quantification of β-Casein Fragment (f 193-209)

ABSTRACT: Bitter cheese extract was incubated with cell-free extract of Lactobacillus helveticus strain WSU19. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis showed an intense peak at m/z-1880 that was markedly reduced after a 24-h incubation, as was bitterness. The m/z-1880 peptide was identified as β-casein (f193-209). Synthetic β-casein (f193-209) was mixed with internal standards, asparagine or deuterated glutamine substitution in β-casein (f193-209). Concentration ratios of β-casein (f193-209) to internal standard were plotted against signal ratios. Plots were linear for both internal standards. This study shows that MALDI-TOF is a rapid method for measuring a specific peptide in cheese and may be useful for assessing cheese ripening and screening cultures for debittering activity.     

Debittering of enzymatic hydrolysates using an aminopeptidase from the edible basidiomycete Grifola frondosa

Bitter peptide solutions, prepared by the enzymatic hydrolysis of soy protein and milk casein, were treated with an aminopeptidase from the edible basidiomycete Grifola frondosa. As the incubation time elapsed, the amount of free amino acids released increased and the bitterness of the enzyme reaction mixtures decreased. However, the debittering of the milk casein hydrolysate by the aminopeptidase was less effective than that observed for the soy protein hydrolysate. Hydrophobic amino acids such as valine, leucine, phenylalanine, tyrosine, and isoleucine were preferentially released from the bitter solutions by the action of the aminopeptidase.     

毛霉AS3.2778脯氨酸氨肽酶的部分纯化及性质研究

毛霉蛋白酶对大豆蛋白有较高的水解效率并对蛋白水解物有良好的脱苦效果,因此在大豆多肽的制备方面显示出很好的应用前景。为了开发这一蛋白酶系,实验中P采用硫酸铵盐析、离子交换层析、疏水层析及凝胶层析等方法对其进行了分离纯化,从雅致放射毛霉AS3.2778的发酵麸曲中部分纯化得到一氨肽酶组分,并对其性质进行了探讨。纯化的毛霉氨肽酶是一典型的脯氨酸氨肽酶,它对小肽N端的脯氨酸有非常强的水解能力;该氨肽酶在40～45℃、pH6.5有最大催化活性,在30℃以内,pH5.0～8.0有很好的稳定性;在所试验的几种蛋白酶抑制剂中,仅1 mmol/L的苯甲基磺酰氟(PMSF)对毛霉氨肽酶有抑制作用,由此说明纯化的毛霉氨肽酶可能是一种丝氨酸蛋白酶;常见金属离子对该氨肽酶活性的影响不明显;脱苦实验结果表明,纯化的毛霉氨肽酶对于大豆蛋白水解物的苦味有明显的去除效果。     

分步酶解小麦面筋蛋白制备低苦味肽粉的研究

目的 研究分步酶解小麦面筋蛋白(wheat gluten, WG)制备低苦味肽粉的工艺。方法 选用中性蛋白酶、木瓜蛋白酶、胃蛋白酶水解WG至8%水解度,接着用风味蛋白酶对水解产物进行脱苦处理,对不同酶解产物中苦味肽的特性进行系统研究,探究苦味肽含量、氨基酸组成、分子量分布、表面疏水性等指标变化对WG酶解物苦味值的影响,对比风味蛋白酶对不同单酶酶解物的脱苦效果差异,分析风味蛋白酶对WG酶解物脱苦的内在机理,进而确定制备低苦味小麦蛋白肽粉的最佳酶解工艺。结果 中性蛋白酶的酶解产物经风味蛋白酶作用后,脱苦效果最显著,苦味肽苦味值从4.08降至2.25,酶解产物的苦味值可下降56.42%。木瓜蛋白酶的酶解产物经风味蛋白酶作用4 h后,酶解产物的苦味值最低,制备出苦味值为1.28的WG低苦味肽粉。结论 经分步酶解作用后,酶解产物中苦味肽的含量下降;疏水性氨基酸比例的下降和游离氨基酸含量的升高引起苦味肽苦味阈值的增大,共同导致酶解产物苦味值显著降低,该研究为酶解脱苦技术的快速发展和WG活性肽工业化生产提供新的参考。     

Hydrolysis of β-casein (193-209) Fragment by Whole Cells and Fractions of Lactobacillus casei and Lactococcus lactis

Whole cells and fractions of Lactococcus lactis subsp. lactis IFPL 359 and Lactobacillus casei subsp. casei IFPL731 were studied. Hydrolysis products were separated by reversed-phase, high-performance liquid chromatography (RPHPLC). Under conditions, pH 5.2 and 3% NaCl, L. casei IFPL 731 was more active in hydrolysis of the b-casein (f193-209) peptide than was L. lactis IFPL 359. This hydrolyzing activity was attributed for L. casei IFPL 731 by the cell-wall proteinase. Hydrolysis of the peptide by the intracellular extract of L. casei IFPL731 was mainly located in the fraction that contained endopeptidase and Pep N aminopeptidase activities. Results may help provide approaches and treatments to control bitterness in cheese products.     

酶解制备金乌贼抗氧化产物的工艺优化及营养评价

目的:研究金乌贼蛋白抗氧化酶解产物(antioxidant enzymatic hydrolysate,AEH)的制备和脱苦工艺,并对其氨基酸组成和营养价值进行评价.方法:分别以·OH清除率和苦味值为指标,利用正交试验设计对木瓜蛋白酶制备AEH的水解条件及活性炭脱苦工艺进行优化;利用氨基酸自动分析仪测定AEH的氨基酸组...     

氨肽酶脱苦效果的研究

目的研究从豆腐乳中筛选到的一种枯草芽孢杆菌所产的氨肽酶脱除大豆蛋白质水解物苦味的效果。方法用高效液相色谱法和感官评定。结果1％的大豆蛋白质溶液经胰蛋白酶水解，水解液呈苦味，添加氨肽酶可较好地脱苦，水解液中游离氨基酸的总量增加了28．8％，疏水氨基酸的量也明显增加。结论枯草芽孢杆菌氨肽酶具有较好的脱苦效果。     

SOLUBILITY AND EMULSIFYING PROPERTIES OF BETACASEIN MODIFIED ENZYMATICALLY BY TRYPSIN

Beta A1-casein was treated with TPCK-trypsin to give 3.2, 5.0, 5.8 and 7.4% hydrolysis of the peptide bonds. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the resulting peptides had apparent molecular weights in the range 8,000–4,000. Size-exclusion chromatography of hydrolyzed samples showed four major peaks near 15,000, 5,500, 3,500 and 2,500 molecular weights, representing 17, 15, 7 and 14% of the material, respectively, after 3.2% hydrolysis and 9, 6, 14 and 52% of the material, respectively, after 7.4% hydrolysis. Between the extremes 3.2% and 7.4% hydrolysis, a peak near 8,500 molecular weight was present until 5.8% hydrolysis then disappeared after 7.4% hydrolysis to be replaced by a peak near 12,000 molecular weight. Peptides recovered from reversed-phase high performance liquid chromatography were analyzed by determination of their amino acid composition and identified in the sequence of β-casein. After trypsin treatment, the solubility of β-casein hydrolysates was largely increased at pH 4.0–7.5. The emulsifying activity of the hydrolysates was higher than that of β-casein in the range of pH 1.5–3.5 and 6.5–10.0, but all the emulsions obtained with trypsin-treated β-casein were less stable than those obtained with original β-casein.     

氨肽酶脱苦效果的研究

目的研究从豆腐乳中筛选到的一种枯草芽孢杆菌所产的氨肽酶脱除大豆蛋白质水解物苦味的效果。方法用高效液相色谱法和感官评定。结果1%的大豆蛋白质溶液经胰蛋白酶水解,水解液呈苦味,添加氨肽酶可较好地脱苦,水解液中游离氨基酸的总量增加了28.8%,疏水氨基酸的量也明显增加。结论枯草芽孢杆菌氨肽酶具有较好的脱苦效果。     

EFFECT OF ADDITION OF α-, β-, AND κ-CASEIN, AND Na-CASEINATE ON VISCOELASTIC PROPERTIES OF SKIM MILK CURD

Creep compliance was used to determine the effects of the addition of α-, β-, and κ-casein, and Na-caseinate on the viscoelastic properties of skim milk curd. The results of all measurements can be represented by a six-element mechanical model. Addition of α- and β-casein, and Na-caseinate (1.80g/L) to raw skim milk reduced the instantaneous modulus of rigidity and final viscosity of the curd, while κ-casein addition at the same level increased both viscoelastic parameters. Shielding of κ-casein and depletion of serum Ca⁺⁺ ions by α- and β-casein is thought to have caused the reduction of curd rigidity and viscosity. Subsequent experiments indicated that the addition of β-casein before and after rennet hydrolysis produced different curd strength with the latter producing a stronger curd.     

Purification and Characterization of Aminopeptidase from Chicken Intestine

ABSTRACT: An aminopeptidase was purified 839-fold with 15% recovery from chicken intestine by a procedure involving ion exchange, gel filtration, hydrophobic interaction and affinity chromatography. The enzyme is a heteridimer of subunits 94000 Da and 66000 Da. The substrate specificity of the enzyme was in the order ala > arg > leu-β-naphthylamide. The enzyme was strongly inhibited by bestatin, puromycin, and 1, 10-phenanthroline. The aminopeptidase showed maximum activity at pH 6 and 40 to 50 °C. The enzyme significantly differs from the hitherto known major classes of aminopeptidases from chicken tissues in terms of molecular weight and biochemical characteristics.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.     

Debittering Casein Hydrolysates with Octadecyl-Siloxane (C18) Columns

Three casein hydrolysates (degree of hydrolysis (DH) of 23, 47, and 65.5 (%)) were debittered using three different hydrophobic adsorption columns, C18, C8, and phenolic resin (PR). Higher DH hydrolysates resulted in higher total nitrogen yields through the debittering process. The C18 and PR columns had more effective debittering than the C8 column, which had the highest processing yield. The PR column had much lower processing yield than the C18 column. Q-value correlated with bitterness in the hydrolysates. Antigenicity of treated hydrolysates was reduced by the debittering process.     

采用模糊综合评价法比较黑豆多肽的脱苦工艺

采用模糊数学综合评判法,分析比较了β-环糊精包埋脱苦和枯草杆菌氨肽酶水解脱苦等对黑豆多肽苦味值的影响,确定最佳脱苦工艺条件。结果表明,枯草杆菌氨肽酶的脱苦效果优于β-环糊精,枯草杆菌氨肽酶脱苦的最佳工艺条件为:加酶量1500LAPU、pH8.5、温度50℃、时间4h。     

Alcalase 2.4L碱性内切酶和Flavourzyme风味酶酶解大豆分离蛋白及脱苦工艺优化

以大豆分离蛋白为原料,选用Alcalase 2.4L碱性内切酶和Flavourzyme风味蛋白酶对大豆分离蛋白进行酶法水解及脱苦工艺研究。以水解度和苦味分值为考察值,对酶解工艺进行优化,确定最佳条件。结果表明:Alcalase2.4L碱性内切酶最佳酶解条件为加酶量14 000 U/g、酶解温度60℃、酶解pH8.5、底物质量分数5%,酶解时间2h,最终水解度为45.34%,此时水解液苦味值为4。Flavourzyme风味蛋白酶对水解液进行二次水解的最优酶解条件为加酶量300 U/g、酶解温度55℃、酶解pH 7.0、酶解时间3 h,此条件下大豆分离蛋白水解液苦味值最低为1.2。Alcalase2.4L碱性内切酶和Flavourzyme风味蛋白酶水解大豆分离蛋白使水解度得到较大提高的同时也解决了水解液的苦味问题。     

Debittering Mechanism in Bitter Peptides of Enzymatic Hydrolysates from Milk Casein by Aminopeptidase T

The bitter peptide fraction present in casein hydrolysates obtained by using three proteases (subtilisin, papain and trypsin) was treated with aminopeptidase T from Thermus aquaticus YT-1. The bitterness of the bitter peptide fraction could be decreased, and it sometimes disappeared completely, with an increase in free amino acids. The percentages of total free amino acids released from each bitter peptide fraction (subtilisin, papain and trypsin) by aminopeptidase digestion for 20 hr were approximately 11%, 8.7%, and 6.5%, respectively. Bitter peptide (α_s1-CN f91-100) was isolated from a tryptic hydrolysate of casein by HPLC, its threshold value of bitterness being 2.9 ppm (w/v). The peptide (α_s1-CN f96-100) obtained from the amino peptidase digestion of this bitter peptide showed no bitterness.     

Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.     

The hepatopancreas enzymes of the crustaceans Munida and their potential application in cheese biotechnology

Proteolytic and peptidase activities were extracted from the hepatopancreas of the crustaceans Munida and characterized by enzymatic assay, 2D zymography and mass spectrometry. Results showed the presence of several isotrypsin-like and isochymotrypsin-like enzymes, aminopeptidases and carboxypeptidases A and B. Six different acidic forms of trypsin were detected using specific inhibitors and 2D zymography. Trypsin-like activity was higher than chymotrypsin-like activity. On the basis of previous evidences in food biotechnology and cheese production, the digestive enzymes of the crustaceans Munida were tested for their ability to degrade casein, a process involved in cheese production. As a result, the Munida enzymes were found to degrade the chymosin-derived β-casein fragment f193-209, one of the peptides associated with bitterness in cheese, revealing their possible application in cheese technology to lower the unpleasant bitter flavour in some cheeses.     

Purification and properties of two intracellular aminopeptidases produced by Brevibacterium linens SR3

Two aminopeptidases, designated as aminopeptidase I (API) and aminopeptidase II (APII), were isolated from the disrupted cells of Brevibacterium linens SR3 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, Q-Sepharose and Sepharose CL-6B chromatography. Optimal temperature for enzymatic activity was 45°C for API and 37°C for APII, and the pH optimum was found to be 8.5 for API and 8.0 for APII. Divalent metals ions like Co²⁺ and Ni²⁺ increased enzymatic activity while Ca²⁺, Cu²⁺ and Fe²⁺ had an inhibitory effect. The enzymes were strongly inhibited by EDTA, 1,10-Phenantroline and cysteine, indicating that both aminopeptidases were metalloenzymes. The K_m values of API and APII for l-leucine-p-nitroanilide were 1.0 and 0.25 mm, respectively. Native molecular masses of aminopeptidases I and II were 80 and 220 kDa after gel filtration chromatography while molecular masses of 14 and 18 kDa, were seen, after SDS-polyacrylamide gel electrophoresis.