Purification and characterisation of milk‐clotting and caseinolytic activities of pepsin isolated from adult sheep abomasa

Ovine pepsin was isolated and assessed for its milk‐clotting properties and caseinolytic activity in comparison with commercial chymosin. Ovine pepsin showed similar responses to variations in pH, temperature and CaCl₂ concentration of milk compared with chymosin, although its pH sensitivity was higher. SDS‐PAGE electrophoretic analysis of the casein fractions treated with ovine pepsin showed that alpha‐casein was more susceptible to proteolysis than beta‐casein, in contrast to chymosin. Curd‐firming properties of skim milk gels obtained with ovine pepsin and chymosin were evaluated by Gelograph under the same conditions. Curd produced using ovine pepsin was less firm than that made with chymosin.     

Diffuse Reflectance Profiles of Eight Milk-Clotting Enzyme Preparations

Eight milk-clotting enzyme prepartations were standardized to equal clot time and used to coagulate pasteurized whole milk. Diffuse reflectance profiles were monitored for 60-min using a fiber optic sensor sensitive to infrared light at 950 nm. Modified M. miehei and M. pusillus protease, recombinant chymosin and calf rennet produced similar profiles. Rates of increase in diffuse reflectance were E. parasitica recombinant chymosin > calf rennet > modified M. miehei, M. pusillus var. Lindt > 50:50 blend of calf rennet and bovine pepsin > unmodified M. miehei > pepsin. Monitoring milk coagulation as described may be useful during cheese making and allow setting optimal conditions for milk-clotting enzyme preparations.     

MILK CLOTTING AND CHEESE MAKING PROPERTIES OF A CHYMOSIN-LIKE ENZYME FROM HARP SEAL MUCOSA

Pepsin A was isolated from the gastric mucosa of two year old harp seal (Pagophilus groenlandicus). Seal pepsin A has a relatively high CU/PU ratio of 0.074, although it is lower than that of calf chymosin (0.170). Equivalent milk clotting units of chymosin and seal pepsin A catalyzed formation of the same amount of nonprotein nitrogen (NPN) when incubated with 2% casein and for both there was no appreciable increase in NPN formation in the second hour of incubation. Seal pepsin A was similar to chymosin in clotting milk substrate with respect to the influence of pH, dilution, calcium chloride and temperature. Cheddar cheeses prepared with either calf rennet or pepsin A as coagulating agent were similar in yield, chemical composition and taste as judged by sensory preference tests. Estimation of protein degradation in the aged cheeses by ultraviolet absorption of citrate-HC1 extracts, gel filtration chromatography of protein soluble in 6 N urea, and determination of amino acids indicated there was slightly more protein degradation in cheese prepared with calf rennet after 30 weeks aging. Sal pepsin A appears to make a major contribution to the excellent cheese-making characteristic of crude seal pepsin(s). However, other components of the seal mucosa extract are probably responsible for the accelerated aging of Cheddar cheese noted in previous reports.     

Purification and characterization of chymosin and pepsin from kid

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin 相似文献   

Interactions of digestive enzymes and milk proteins with tea catechins at gastric and intestinal pH

The interactions of digestive enzymes (pepsin, pancreatin) and milk proteins (β‐casein, β‐lactoglobulin (β‐Lg)) with (?)‐epigallocatechin gallate (EGCG), (?)‐epigallocatechin (EGC) and (?)‐epicatechin (EC) at gastric and intestinal pH were investigated by fluorescence spectroscopy. The results indicated that in the gastric environment, all three tea catechins showed binding affinities in descending order of strength with β‐casein first, followed by β‐Lg and then pepsin. The highest affinity was observed for EGCG–β‐casein, with a binding constant (K_A) of 2.502(±0.201) × 10⁵ m ^?1. In the intestinal environment, the binding strengths of the proteins with EGCG and EGC were in the order β‐Lg > pancreatin > β‐casein; for binding with EC, the strength order was β‐casein > β‐Lg > pancreatin. The combination EGCG–β‐Lg had the strongest binding affinity, with a K_A of 14.300(±0.997) × 10⁵ m ^?1. Thermodynamic analysis revealed that tea catechins complexed with milk proteins and digestive enzymes via different hydrophilic and hydrophobic interactions depending on the different digestion environments and types of catechins, proteins and enzymes.     

SECONDARY STRUCTURE OF SOME ASPARTYL PROTEINASES

A structural study of some aspartyl proteinases was undertaken. Secondary structure prediction methods indicate that chymosin, pepsin, penicillopepsin and Mucor miehei proteinase have relatively high proportions of β-sheet with active site aspartic acid residues located in β-turn regions. Secondary structure determination from far-UV CD spectral data support the above finding that the aspartyl proteinases have a high proportion of β-sheet. The proportion of β-sheet generally decreased at pH values greater than 6.3. More extensive unfolding occurred with pepsin and penicillopepsin than chymosin, Mucor miehei proteinase, Mucor pusillus proteinase and Endothia parasitica proteinase in the neutral to alkaline pH range. Results obtained from the near-UV CD spectra of the aspartyl proteinases indicate a change in spectra in the neutral to alkaline pH range which suggests the importance of aromatic groups to tertiary structure stability.     

不同凝乳酶生产干酪成熟期间氨基酸的变化

以鲜奶为原料,以进口的EZAL、MAOLL作发酵剂(由乳油连球菌和乳酸链球菌组成),以微生物酶、羔羊皱胃酶、小牛皱胃酶和猪胃蛋白酶、木瓜蛋白酶、无花果蛋白酶作为凝乳酶生产硬质干酪,研究不同凝乳酶对干酪成熟期间氨基酸变化的影响。结果表明:不同凝乳酶生产干酪成熟期间氨基酸(FAA)含量的变化趋势:羔羊皱胃酶>小牛皱胃酶>微生物凝乳酶>无花果蛋白酶>猪胃蛋白酶>木瓜蛋白酶。     

Factors influencing the gelation and rennetability of camel milk using camel chymosin

The effects of temperature, pH, concentration of camel chymosin and addition of CaCl₂ on the hydrolysis of κ-casein (κ−CN) and the coagulation kinetics of camel milk were investigated. The rate of κ−CN hydrolysis was higher at 40 °C than at 30 °C and with increasing addition of chymosin and decreasing pH. For all samples gelation was initiated at levels of camel milk κ−CN hydrolysis >95%. The gelation time (T_g) of camel milk was significantly reduced (from 717 to 526 s) at 30 °C when the concentration of chymosin was increased, but was independent of chymosin concentration at 40 °C. Reducing pH also reduced T_g. The gel firmness increased at 40 °C (58 Pa) compared with 30 °C (44 Pa) and effect of CaCl₂ addition on the gelation properties of camel milk was found to be dependent on pH; a significant improvement was only found at pH 6.3.     

混料设计优化牦牛“曲拉”凝乳酶干酪素的工艺及产品的性质分析

为提高牦牛产业的附加值,以牦牛乳提取酥油后进行凝固沉淀,再经自然发酵、风干而成的蛋白质含量丰富的产品“曲拉”为原料,采用胃蛋白酶、木瓜凝乳酶和酵母凝乳酶复配成混合凝乳酶,通过单因素实验和混料设计对凝乳干酪素的制备工艺进行研究,并对干酪素的理化性质、红外光谱特性、热力学性质进行分析。结果表明,混合酶质量分数1%(其中胃蛋白酶∶木瓜凝乳酶∶酵母凝乳酶的质量比为0.60∶0.18∶0.22),在pH 6.3、45℃、添加质量分数CaCl 21%条件下,凝乳30 min,出品率为80.35%。混合酶法制备干酪素的理化性质、红外光谱特性和热力学性质与小牛皱胃酶干酪素差异不显著,而且符合GB31638—2016要求。     

Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin

BACKGROUND: Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL^?1 in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. RESULTS: Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid‐containing and Ca²⁺‐deficient medium, but Ca²⁺ supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1‐N‐prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL^?1 in the K. lactis PMR1‐deficient mutant, sixfold higher than that in wild‐type K. lactis GG799. CONCLUSION: Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production. Copyright © 2010 Society of Chemical Industry     

Relative Proteolytic Action of Milk-Clotting Enzyme Preparations on Bovine α-, β- and β-casein

Concentrations of seven milk-clotting enzyme preparations were standardized to equal clot times. Portions of bovine α_s-, β- and κ-casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6-trinitrobenzene-sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on α_s- and β-caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β-casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on α_s- and β-caseins but was the least proteolytic on κ-casein.     

Angiotensin I‐converting enzyme inhibitory peptides in skim milk fermented with Lactobacillus helveticus 130B4 from camel milk in Inner Mongolia,China

BACKGROUND: Angiotensin I‐converting enzyme (ACE) is a dipeptidyl carboxypeptidase associated with the regulation of blood pressure. ACE inhibition results in a lowering of blood pressure. Lactic acid bacteria are known to produce ACE inhibitors during fermentation. Fermented camel milk is the main traditionally fermented dairy food for desert nomads. The beneficial effects of fermented camel milk, which include the prevention of such diseases and conditions as gastroenteritis, tuberculosis and hypertension, have been demonstrated experimentally. RESULTS: ACE inhibitory activity was observed in fermented milk containing Lactobacillus helveticus 130B4, a strain isolated from traditionally fermented camel milk. The peptide that inhibited ACE was purified from the fermented milk by reverse‐phase high‐performance liquid chromatography. The amino acid sequence of the peptide was identified as Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp (IC₅₀ = 19.9 µmol L^?1). The same Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp sequence was found in κ‐casein (κ‐CN) f107–115 from milk. The inhibitory activity of this nonapeptide (κ‐CN f107–115) was almost preserved even after successive digestion with pepsin, trypsin and chymotrypsin. Furthermore, the inhibitory activity of the purified peptide was completely preserved after heat treatment at 100 °C for 20 min. CONCLUSION: The fermented milk prepared with Lactobacillus helveticus 130B4 contained an ACE inhibitory peptide, κ‐CN 107–115. This fermented milk was expected to have anti‐hypertensive effect after ingestion because the peptide was stable to digestive protease and heat treatment in vitro. Copyright © 2008 Society of Chemical Industry     

Casein breakdown during ripening of Idiazabal cheese: influence of starter and rennet type

The objective of this work was to determine the effect of starter and rennet type on casein breakdown during Idiazabal cheese ripening. Four batches of cheeses were manufactured with two rennets, commercial calf rennet and artisanal lamb rennet, and the use of natural flora or a commercial starter. Electrophoretic analysis of cheese samples showed six bands identified as α_s1‐, α_s2 + β‐, α_s1‐I‐, γ₁‐, β‐I‐ and para‐κ‐casein. As expected, the casein breakdown during cheese ripening was considerably affected by rennet type and the use of a commercial starter. The artisanal lamb rennet produced a higher hydrolysis of casein fractions than the commercial calf rennet, probably owing to its high percentage of chymosin (around 78%). The effect of addition of starter on proteolysis was dependent on the casein fractions generated by artisanal lamb rennet or commercial calf rennet. © 2000 Society of Chemical Industry     

Heat-Stability of Milk-Clotting Enzymes in Conditions Encountered in Swiss Cheese Making

Chymosin is inactivated in gelled milk at pH 6.50 at 53°C according to a biphasic kinetic. Both phases appear to follow first-order kinetics. The D-value for the first phase is 30 min. In the same medium, the Mucor miehei and Mucor pusillus proteases are much more stable (D_53°C > 100 min) while bovine pepsin, a heat-labile Mucor miehei protease and the Endothia parasitica protease are rapidly inactivated (D_53°C < 10 min). pH appears to be the most important parameter for heat stability. Protein and calcium concentrations affect the resistance to heat treatment. A residual activity of chymosin in Swiss-type curd will be very weak at the most unless the curd is cooked at pH < 6.50.     

The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological quality

The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentrations ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly regarding their sensory properties and had an acceptable microbiological quality. This study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal.     

Effects of Enzyme Choice and Fractionation of Commercial Enzyme Preparations on Protein Recovery in Curd

Five different commercial milk clotting preparations (bovine rennet, calf rennet, calf rennet-porcine pepsin mixture, Mucor miebei protease, and modified Mucor miehei protease) were adjusted to equivalent milk clotting activities and then used to clot milk. Percentages of protein in the resulting wheys were compared. Calf rennet, bovine rennet, or modified Mucor miehei protease caused less loss of protein to the whey than Mucor miehei protease or calf rennet-porcine pepsin mixture. The five enzyme preparations were then fractionated by gel filtration. Fractions with milk clotting activity were pooled. Original enzyme preparations and the pooled fractions made from them were standardized to the same clotting activity, then used to coagulated milk to compare their effect on protein loss to the whey. Fractionation significantly improved protein recovery when bovine rennet and calf rennet-porcine pepsin mixture were used as coagulants but not when calf rennet, Mucor miehei protease, or modified Mucor miehei protease were used.     

Effect of digestive enzymes on the activity of camel‐milk insulin

This study investigated the effect of digestive enzymes on the activity of camel‐milk insulin. The digestion was performed using the sequential action of pepsin and pancreatin. Proteolysis degree was estimated using the O‐phthaldialdehyde method. Insulin concentration was determined using an enzyme‐linked immunosorbent assay (ELISA). Results revealed that milk proteins were partially digested by pepsin alone and the degradation was increased during the pepsin–pancreatin digestion as compared to control. Insulin lost its activity after 30 min of pepsin digestion, and it was not detected by ELISA. This study strongly suggests that insulin is not responsible for the antidiabetic action of camel's milk.     

Concentrations of Volatile 4‐Alkyl‐Branched Fatty Acids in Sheep and Goat Milk and Dairy Products

Goat and sheep milk and dairy products thereof are characterized by a strong and unique flavor. In this context, the volatile minor fatty acid 4‐ethyloctanoic acid plays a prominent role along with 4‐methyloctanoic acid when both are present in free form. Using a novel GC/MS method in the selected ion‐monitoring mode, previously developed for sheep subcutaneous adipose tissue, we were able to analyze the total concentrations of these flavor‐relevant minor fatty acids as methyl esters in goat and sheep milk as well as in their products. Differences between the concentrations and ratios of 4‐methyloctanoic acid and 4‐ethyloctanoic acid in goat milk (n = 4), goat cheese (n = 4), sheep milk (n = 2), and sheep cheese (n = 4) were observed. Goat milk and cheese resulted in higher concentrations for both fatty acids (190 to 480 μg/g milk fat) and smaller 4‐Me‐8:0 to 4‐Et‐8:0 ratios (1.4 to 2.7) compared to sheep milk and cheese (78 to 220 μg/g milk fat; 4‐Me‐8:0 to 4‐Et‐8:0 ratio: 15 to 42). In all samples, the concentration of 4‐Me‐8:0 exceeded the one of 4‐Et‐8:0. However, due to its lower flavor threshold value the contribution of 4‐Et‐8:0 to the flavor was generally >76%. The calculated flavor values were >1400 for goat milk and cheeses and >200 for sheep milk and cheeses. In goat milk and its products, only a proportion of <0.1% 4‐alkyl‐branched fatty acids present in free form in the goat milk and <0.5% in the sheep samples would be sufficient to generate the characteristic goaty flavor. Parameters that promote or prevent the release of 4‐Me‐8:0, and especially 4‐Et‐8:0, will be decisive for the flavor in the resulting dairy product.     

Effect of Goat and Cow Milk Ratios on the Physicochemical,Rheological, and Sensory Properties of a Fresh Panela Cheese

Fresh cheeses, Panela type, were manufactured from cow milk and with goat milk incorporation, constituting 4 blends of milks (G10:C90, G20:C80, G30:C70, G40:C60, v/v). The cheeses were analyzed to determine the effect of the different goat milk ratios on the physicochemical, textural, rheological, and sensory properties over 15 d of storage. Significant differences in protein (14.6% to 18.5%), fat (13.0% to 19.4%), and moisture contents (51.7% to 61.3%), pH (6.38 to 6.67), color (L_h > 64.4, a_h > 1.06, b_h > 5.14), textural (σ_f > 14.8 kPa, ε_C: 0.77 to 0.79, elasticity modulus > 13.5 kPa), and rheological parameters (G′ > G′′, G′: 10.6 to 31.9 kPa, G′′: 2.39 to 7.31 kPa, tan δ: 0.21 to 0.24) were detected as a function of the milks ratio, as well as a function of the storage time. The incorporation of goat milk improved the overall quality in the formulation of Panela cheese, enhancing the texture, flavor and aroma, commonly associated with hand‐crafted cheeses when they are used in the proper ratio. Furthermore, the nutritional value of the cheese is increased with the incorporation of goat milk, which can contribute to a better consumer health.     

Natural heterogeneity of chymosin and pepsin in extracts of bovine stomachs

The two main components, chymosin and pepsin, in calf and adult bovine rennet show heterogeneity with respect to their charge and activity. A large number of single stomachs have been extracted and their isoenzymes have been analysed by HPLC. The results showed three genetic forms of chymosin A, B and C and these are present in individual stomachs in the following combinations: AA, BB, CC, AB, AC and BC. The C variant, which can be distinguished from the degradation product of chymosin A, has never been characterised before. All three chymosins have been purified and partially characterised. Also bovine pepsins show a natural variability: the ratio between the main pepsin fractions varies according to the age of the animal and it therefore provides a criterion to distinguish between pepsin from calf and adult bovine animals.