Debittering and Hydrolysis of a Tryptic Hydrolysate of β-casein with Purified General and Proline Specific Aminopeptidases from Lactococcus lactis ssp. cremoris AM2

ABSTRACT: In this study, purified β-casein was hydrolysed with trypsin to produce a bitter substrate. The role of 3 aminopeptidases, a general aminopeptidase lysyl- para -nitroanilide hydrolase (KpNA-H), X-prolyl dipeptidyl aminopeptidase (Pep X) and aminopeptidase P (Pep P) each purified from Lactococcus lactis ssp. cremoris AM2, in the hydrolysis and debittering of the tryptic hydrolysate of β-casein, was then studied. The hydrolysates were analyzed for percentage degree of hydrolysis (DH%) and bitterness score. Results indicate that the hydrolysis and debittering potential of the general aminopeptidase (KpNA-H) is limited in the absence of proline specific aminopeptidases. Statistically significant (p < 0.001) reductions in bitterness were obtained following incubation of the tryptic digest of β-casein with specific combinations of the above aminopeptidases.     

THE DEGRADATION OF CHlTOSAN WITH THE AID OF LIPASE FROM RHIZOPUS JAPONICUS FOR THE PRODUCTION OF SOLUBLE CHlTOSAN

Lipase from Rhizopus japonicus degraded chitosan resulting in soluble chitosan hydrolysates with molecular weight of about 30–50 kDa as shown by size exclusion chromatography. Optimal temperature for the hydrolysis of chitosan was 40C. The chitosan degradation products were fractionated stepwise according to their molecular weights by ultrafiltration with the filtration range of over 0.1 μm, 0. l μm to 30 kDa, 30 kDa to 10 kDa, 10 to 3 kDa, and 3 to 0.2 kDa. These fractions exhibited molecular weights of 50, 41, 41, 35, and 30 kDa, respectively. The molecular weights did not coincide with the pore size of filter membranes. Chitosan hydrolysate exhibited almost the same structural composition in IR spectra as chitosan flakes, except the peak of 1550 nm^?1 that appeared to be the COO residue shifted from sodium acetate buffer to amine residue of chitosan. All fractions showed high solubility at neutral pH. The chitosan hydrolysates exhibiting molecular weights between 30 and 41 kDa were considered to be most suitable as a food additive or functional agent as demonstrated by sensory evaluation.     

微胶囊化蛋白酶在干酪制备中的应用及干酪成熟过程中电泳分析

将采用冷冻干燥工艺得到的明胶-阿拉伯胶-凝乳酶微胶囊用于干酪制备过程中,在排乳清之后向干酪中添加5.00 g微胶囊化蛋白酶,以促进干酪的成熟。在干酪成熟过程中通过三羟甲基氨基甘氨酸-尿素-十二烷基磺酸钠聚丙烯酰胺凝胶电泳以及毛细管电泳分析监测干酪中pH 4.6不溶性氮部分的变化情况。结果表明：干酪在成熟初期,αs1-酪蛋白较β-酪蛋白先水解,生成αs1-酪蛋白（f 102～199）和αs1-I-酪蛋白;干酪成熟21 d时,β-酪蛋白开始水解,αs1-酪蛋白继续保持水解,生成的大的水解产物同时也发生二次水解;干酪成熟90 d时干酪中β-酪蛋白比αs1-酪蛋白具有更广泛的水解程度,酪蛋白降解的同时,生成大量新的小分子肽。     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

啤酒废酵母蛋白水解物的制取工艺

利用啤酒废酵母资源,通过破壁预处理与复合酶降解相结合的工艺,制取特定相对分子质量范围的蛋白水解物.响应面分析结果表明,在温度55～60℃,pH6.6～6.8,β-葡聚糖酶用量15～20U/g(以酵母计)的条件下,有最高的肽提取率.酶解7h,蛋白质提取率大于82%,经HPLC测定,水解产物中65%组分的相对分子质量在210～635之间,处于二肽和三肽相对分子质量范围.     

几种蛋白酶对文蛤肉的水解效果

在一定的酶解条件下,用Alcalase 2.4L、胰蛋白酶、复合风味酶、酸性蛋白酶、中性蛋白酶等几种蛋白酶对文蛤内进行水解,测定了这几种蛋白酶的水解度,以及其水解物的氨基态氮含量、可溶性蛋白含量和透明度,并用凝胶过滤色谱(Sephedex G-25)对水解物的分子质量分布进行了粗略分析。结果表明,Alcalase水解物分子质量的分布范围较广,适合制备具有一定肽链要求的短肽,酸性蛋白酶水解物分子质量较小,适合制备文蛤调味品,中性蛋白酶水解物分子质量主要集中在1 000 u左右与10 000u左右,胰蛋白酶和复合风味酶的水解物分子质量大部分在1 500 u以上。     

酶解中国毛虾制备低分子肽的研究

以中国毛虾为原料,采用酶法制备低分子肽。利用响应面法优化了Alcalase4L酶水解中国毛虾的工艺参数:温度57.1℃、pH8.0、加酶量为46mL/kg,酶解产物的平均肽链长从0.5h的14.02逐渐降低到8h的3.96。采用凝胶层析法测定了酶解产物的分子质量分布,不同时间酶解产物的SephadexG-15凝胶色谱图中均出现3个吸收峰,前2个峰的分子质量在18722～641u和641～35u之间。从峰值变化可以判断出,随着酶解的进行,低分子肽的量逐渐增加。     

干酪乳清酶解产物抗氧化肽的分离和纯化

干酪乳清Alcalase 2.4L酶解的最优条件,即酶解时间为2 h、pH 9.5、酶与底物比为4%、酶解温度为50 ℃。利用超滤、葡聚糖凝胶层析和三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（tricine-sodiumdodecyl sulfate-polyacrylamide gelelectrophoresis,tricine SDS-PAGE）等方法从干酪乳清中提取抗氧化性蛋白肽。分别考察操作压力、料液温度、料液pH值、操作时间对超滤膜膜通量的影响。乳清酶解物超滤的最佳条件为：压力0.25 MPa、温度30 ℃、时间120 min、初始pH 9.0。分子质量4 000～6 000 D肽的水解液脂质过氧化抑制率最高,达到47.28%。利用Sephadex G-50型葡聚糖凝胶进行纯化,将分离的组分进行Tricine-SDS-PAGE分析和脂质过氧化抑制率的测定。第34管洗脱液的脂质过氧化抑制率最高,分子质量范围为4 000～4 100 D。     

Proteolysis of αs1-Casein by Papain in a Complex Environment. Influence of Ionic Strength on the Reaction Products

The influence of NaCl on bovine α_s1-casein (α_s1-CN) hydrolysis by papain (EC 3.4.22.2) was studied at pH 6.5 and 40°C. The electrophoretic analysis in presence of sodium dodecyl sulfate (SDS), showed that α_s1-CN was sequentially hydrolyzed as the reaction time increased. The analysis by isoelectric focusing electrophoresis showed that after 1 min, the resulting peptides had their isoelectric point (pl) assessed between 4 and 9. With the increase of NaCl concentration, two peptides of apparent pl near pH 5 and 8.5 were progressively liberated. The chromatographic analysis by reverse phase HPLC showed that a major peak, observed after 1 min of reaction, persisted in the hydrolysates obtained in the presence of salt at all reaction times. It was concluded by the peptidic sequence determination that the hydrophobic C-terminal region of α^s1-CN was not hydrolyzed in the presence of NaCl.     

Purification of a histidine-containing peptide with calcium binding activity from shrimp processing byproducts hydrolysate

The shrimp processing byproducts were hydrolyzed by various proteases, and calcium binding activity of the hydrolysates was examined. Among the digests, trypsin digest showed the most potent calcium binding activity (0.294 mmol/g-protein) and highest degree of hydrolysis (18.4%). The trypsin hydrolysate was fractionated according to the molecular weights using ultrafiltration membrane system. The lowest molecular weight fraction (<1 kDa) showed the highest calcium binding activity. Then, the lowest molecular weight fraction was isolated and purified by ion-exchange chromatography, gel filtration, and ODS reversed high-performance liquid chromatography. The purified peptide showed the highest calcium binding activity of 2.70 mmol/g-protein, and its structure was identified as Thr-Cys-His by ESI/MS/MS. Therefore, these results suggested that the peptide derived from shrimp processing byproducts protein hydrolysates is responsible for higher calcium binding properties and may be as natural functional additive in food industry.     

Interactions Leading to Formation of Casein Submicelles

Gel chromatography was employed in studies of interactions of soluble whole casein that was prepared by dissociation of casein micelles with ethylenedia-minetetraacetate. With increasing protein concentration at pH 6.6 and 37°C, components of whole casein associate to polymers that approach molecular radii with apparent upper limit of 9.4 ± .4 nm. With decreasing protein concentration, κ-casein dissociates from the other casein components. This was shown by analysis of the eluted protein boundary by gel electrophoresis and radial immunodiffusion. The peak maximum elution volume and the broad, skewed character of the separated κ-casein peak indicate that in whole casein κ-casein exists in a size distribution of disulfide bonded polymers. This apparently suggests that SH-κ-casein monomers aggregate independently of the other casein components in the growth of casein submicelles, and additional studies with the purified casein components support this concept. However, after disulfide bond reduction with dithiothreitol, chromatography of whole casein over the same concentration range did not result in separation of SH-κ-casein polymers, because all of the casein was eluted under one peak. These findings show that, in vivo, casein submicelles could be formed by interaction of SH-κ-casein monomers with those of α_s- and β-casein, followed by S-S-κ-casein polymer formation through oxidation after milking.     

Pepsin-induced changes in the size and molecular weight distribution of bovine casein during enzymatic hydrolysis

Bovine casein was digested with pepsin at pH 2.0 in a batch-stirred tank reactor. To investigate the effect of peptic digestion on the aggregate size and molecular weight distribution of bovine casein, the resulting hydrolysates were examined by size-exclusion chromatography coupled with multiangle laser light scattering and dynamic light scattering. Casein was resolved by size-exclusion chromatography into 2 major peaks corresponding to aggregates and monomers, both of which showed a continuous decrease as hydrolysis proceeded. However, the ratio of aggregates to monomers was maintained at almost 1 (2:2.5) during the initial 30-min hydrolysis, indicating that the caseins in solution were in a type of equilibrium between aggregates and monomers. Upon peptic hydrolysis, casein aggregates increased in size and molecular weight, and exhibited a decrease in intermolecular repulsion. This finding was confirmed by dynamic light scattering measurements, which traced the changes in the hydrodynamic radii and light scattering intensities of casein hydrolysates. In addition, the release kinetics of peptide fractions with different molecular weights was also examined. It was concluded that the increase in hydrophobic attraction and the reduction in intermicellar repulsion might promote the growth in aggregate size of bovine casein during the limited hydrolysis.     

Molecular and physical characteristics of squid (Todarodes pacificus) skin collagens and biological properties of their enzymatic hydrolysates

ABSTRACT:  The physicochemical properties of squid skin collagens and biological activity of their enzymatic hydrolysates were determined to produce more value-added materials. The amino acid compositions of the inner and outer squid skin collagens were similar, but distinct from that of bovine tendon collagen in respect to the higher levels of aspartic acid, arginine, threonine, and serine, and of the lower levels of alanine, proline, and hydroxyproline. SDS-PAGE patterns suggested that squid skin collagen consisted of at least 2 different polypeptides (α1 and α2 chains) and their cross-linked chains. The molecular weights of α1 and α2 chains of bovine tendon collagens were higher than those of the corresponding α1 and α2 chains of squid skin collagens. The denaturation temperatures of inner and outer skin collagens were 27.1 and 27.3 °C, respectively, which were about 9 °C lower than that of bovine tendon collagen. Water holding capacities of inner and outer squid skin collagens were relatively similar, but were significantly greater than that of bovine tendon collagen. The maximum hydrolysis of squid skin collagens was obtained as follows: for outer skin collagen, enzyme concentration, 3.5%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C, whereas for inner skin collagen, enzyme concentration, 3.2%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C. The enzymatic hydrolysates of outer and inner skin collagens were separated by Sephacryl S-100 column, resulting in the production of 3 fractions (F1, F2, and F3). F3 fraction exhibited higher antioxidant, tyrosinase inhibitory, and antielastase activities than the other fractions.     

Influence of Enzymatic Hydrolysis on Structure and Emulsifying Properties of Sardine (Sardina pilchardus) Protein Hydrolysates

The influence of enzymatic modification with Alcalase was studied on surface hydrophobicity, molecular weight distribution and emulsifying properties of ten hydrolysates from sardine differing in degree of hydrolysis. Surface hydrophobicity was evaluated fluorometrically using 1 anilino-8-naphthalenesulfonate (ANS); molecular weight distribution was determined by gel chromatography on a Superfine Sephadex G50. Emulsifying capacity and stability were evaluated as functions of pH values and product concentration, in comparison with sodium caseinate. Results showed emulsifying properties, surface hydrophobicity and the high molecular weight fraction decreased as degree of hydrolysis increased. Thus production of hydrolysates with desired molecular structures and emulsifying properties is possible.     

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.     

Antioxidant activity of ovine casein hydrolysates: identification of active peptides by HPLC–MS/MS

This paper shows the potential role of different ovine casein fractions and their hydrolysates to exert antioxidant activity. ABTS^·+ decolorization assay was used to evaluate the antioxidant activity of the casein fractions (β-, κ- and α_s-caseins) before and after their hydrolysis by pepsin, trypsin and chymotrypsin. Although the antioxidant activity increased in all the fractions after hydrolysis, the effect was particularly remarkable in the κ-casein fraction, which increased its antioxidant activity almost threefold. Further assays in a linoleic acid oxidation system showed that κ-casein hydrolysate inhibited lipid peroxidation. Analysis of the ovine κ-casein hydrolysate by RP-HPLC–MS/MS allowed the identification of 12 peptide sequences with potential antioxidant properties. One of the most abundant peptides, the fragment HPHPHLSF [f(98–105)] was chemically synthesized. Results showed that this κ-casein-derived peptide was a potent inhibitor of linoleic acid oxidation with an activity similar to that obtained with the synthetic antioxidant BHT. Although other peptides might also contribute, HPHPHLSF was the one most likely to be responsible for the activity found in the κ-casein hydrolysate.     

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.     

A study of the soluble complexes formed during calcium binding by soybean protein hydrolysates

ABSTRACT:  The soluble complexes formed between hydrolyzed soybean protein and calcium at pH 7.4 were investigated using dialysis, gel chromatography, and Fourier transform infrared spectrometry (FTIR). The results demonstrate that the amount of calcium bound was significantly different for soybean protein hydrolysates obtained using the proteases neutrase, flavourzyme, protease M, and pepsin. Maximum levels of calcium binding (66.9 mg/g) occurred with hydrolysates produced using protease M. Peptide fragments exhibiting high calcium binding capacity had molecular weights of either 14.4 kDa or 8 to 9 kDa, and the calcium binding capacity was linearly correlated with carboxyl group content ( R ²= 0.8204). FTIR experiments revealed that upon binding calcium, the amide I band underwent a shift to lower wave numbers. A wide, intense Ca–O absorption band also appeared between 400 and 100 cm⁻¹ in the far-infrared spectrum. The width and intensity of this band increased after treatment of samples with glutaminase. The amount of bound calcium was related to both the molecular weight of the peptides and to the carboxyl group content, and the most likely sites for calcium binding are the carboxyl groups of Asp and Glu.     

Characterization of hydrolysates produced by mild-acid treatment and enzymatic hydrolysis of defatted soybean flour

Acid (0.05–0.2 N HCl) pre-treatments and subsequent enzymatic hydrolysis (Alcalase™ and Flavourzyme™) of defatted soybean flour (DSF) were performed under aseptic conditions. The acid pre-treatment facilitated enzymatic hydrolysis of the protein in DSF by increasing the nitrogen solubility index. Protein was hydrolyzed primarily during the first 5 h of enzymatic hydrolysis. The degree of hydrolysis and α-amino nitrogen contents of the hydrolysates increased after acid pretreatment. The average peptide chain lengths were estimated at 7∼8 amino acid units after 3 h hydrolyzation by Alcalase, and 3–5 amino acid units after 21 h by Flavourzyme/Alcalase mixture. Gel permeation chromatography provided molecular size distribution to determine the molecular weights of the corresponding hydrolysates. At the end of 24 h enzymatic hydrolysis, the amounts of free amino acid, dipeptide and tripeptide accounted for almost half of the proteins in the hydrolysate, while the oligopeptides constituted 40%.     

Production of High-protein Hydrolysate from Poultry Industry Residue and their Molecular Profiles

The use of mechanically deboned poultry meat (MDPM) as a source of raw protein for the enzymatic preparation of hydrolysates was investigated using commercial proteases Alcalase and Flavourzyme. Hydrolysis conditions were optimized by response surface methodology, and the influence of temperature, pH, and enzyme concentration were evaluated to determine the hydrolysis index. For Alcalase, optimal values of temperature, pH, and enzyme concentration were found to be 50°C, 7.5%, and 2.5%, respectively, while for Flavourzyme the optimized variables were at 50°C, 6.0%, and 3.5%, respectively. Compared to raw MDPM, Alcalase produced a very high recovery of 89% of the nitrogen from the hydrolysates, while for Flavourzyme recovery was around 67%. Alcalase was used to produce a spray-dried hydrolysate containing 62% of total protein with molecular weights ranging from 5,807 to 12,000 Da, and had excellent microbiological quality.