Sarcoplasmic Protein Extracts Research Articles

Hydrolysis of Beef Sarcoplasmic Protein by Dry-Aged Beef-Isolated Penicillium oxalicum and Its Associated Metabolic Pathways.

Yanbian cattle have a unique meat flavor, and high-grade meat is in short supply. Therefore, in this study, we aimed to improve the added value of Yanbian cattle low-fat meat and provide a theoretical reference for the subsequent development of an excellent starter. Rump meat from Yanbian cattle was dry-aged and then screened for protease-producing fungi. Three protease-producing fungi (Yarrowia hollandica (D4 and D11), Penicillium oxalicum (D5), and Meesziomyces ophidis (D20)) were isolated from 40 d dry-aged beef samples, and their ability to hydrolyze proteins was determined using bovine sarcoplasmic protein extract. SDS-PAGE showed that the ability of Penicillium oxalicum (D5) to degrade proteins was stronger than the other two fungi. In addition, the volatile component content of sarcoplasmic proteins in the D5 group was the highest (45.47%) and comprised the most species (26 types). Metabolic pathway analysis of the fermentation broth showed that phenylalanine, tyrosine, and tryptophan biosynthesis was the most closely related metabolic pathway in sarcoplasmic protein fermentation by Penicillium oxalicum (D5). Dry-aged beef-isolated Penicillium oxalicum serves as a potential starter culture for the fermentation of meat products.

Differentiation of protein types extracted from tilapia byproducts by FTIR spectroscopy combined with chemometric analysis and their antioxidant protein hydrolysates

This research aimed to characterize protein types including sarcoplasmic protein (SP), myofibrillar protein (MP), and alkali-aided protein extract (AP) prepared from tilapia byproducts using water, 0.6 M NaCl, and alkaline solution (pH 11), respectively compared to freeze-dried minced tilapia muscle (CONTROL). Principal component analysis was performed from second derivative FTIR spectra to differentiate protein type. The AP mostly contained β-sheet structure and had low total sulfhydryl content and surface hydrophobicity. SP can be distinguished from MP by the loading plots of the FTIR bands representing the α-helical structure. While the bands for lipids and β-sheet of protein were noted for differentiating AP from CONTROL. After being hydrolyzed by Protease G6, the AP hydrolysate disclosed the highest ABTS radical scavenging activity, while the SP hydrolysate revealed the strongest metal chelating ability. Thus, an understanding of how fish processing waste can be utilized in the production of antioxidant protein hydrolysates has been achieved.

Hydrolysis of pork sarcoplasmic protein extracts by unique staphylococci isolated from low-salt dry-cured ham

Dry-cured hams were produced using two curing agents with low-salt group and full-salt group. The salt content and the free amino acid (FAA) content were measured during the production process. The low-salt group was found to have a lower salt content (P < 0.05) and a higher FAA content (P < 0.05) in the final product than the other group. Three protease-producing strains (Staphylococcus cohni, Staphylococcus saprophyticus and Staphylococcus equorum) were further isolated from the low-salt hams, and their ability to hydrolyze proteins were assessed in porcine sarcoplasmic protein extracts. Gel electrophoresis showed that all strains were similar in their ability to degrade sarcoplasmic proteins. Samples inoculated with Staphylococcus saprophyticus had higher FAA content than those inoculated with the other two strains (P < 0.05). Glycine and alanine were the most increased FAAs compared to the control group. The results suggest that the novel Staphylococcus saprophyticus isolated from dry-cured ham has potential as a starter for fermented meat products.

Метод электронной спектроскопии диффузного отражения в исследовании мышечной ткани диких и домашних животных

The aim of the work was to study the application of diffuse reflection electron spectroscopy (DRES) method for obtaining the optical surface characteristics of slices and chopped muscle tissue, muscle fibre and sarcoplasmic protein extracts of wild (wild boar, elk, deer, freshwater and marine fish) and domestic (pork, beef, lamb) animal tissue depending on the following processing conditions: freezing, water extraction, lyophilisation and enzyme-microbial exposure. Electronic spectra of transverse sections of whole-piece material, ground muscle tissue, muscle fibre and aqueous extracts were recorded using a Specord M-200 spectrophotometer (AIZ Engineering GmbH, Germany) having a vertical pathway relative to the Spectralon standard in the wavelength range of 200-700 nm. During testing of biological tissues of animal origin in solid and liquid state, the high sensitivity, resolution and analytical features of the express DRES method was shown to allow the acquisition of factual information concerning changes in the spectral characteristics of their main components under experimental conditions. Regardless of the species and type of animal organism, proteins, mucopolysaccharides, lipids, pigment protein and various oxidised forms demonstrate absorption bands in four clearly differentiated regions of the electromagnetic spectrum: 200-300, 400-425, 300-380, 540-580 and 635 nm, respectively. A comparative study of the pork and beef muscle tissue demonstrated significant differences in the nature of changes in the spectral characteristics of the main components and components of muscle tissue and muscle fibre, depending on the degree of oxidation, mechanical destruction, strength of myofibrils and pigment protein myoglobin associated with them.

Proteomic based approach for characterizing 4-hydroxy-2-nonenal induced oxidation of buffalo (Bubalus bubalis) and goat (Capra hircus) meat myoglobins.

BackgroundMyoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins.MethodsPresent study characterize the Mb extracted from water buffalo and goat cardiac muscles using two-dimensional gel electrophoresis (2DE), OFFGEL electrophoresis and mass spectrometry (MS). Purified buffalo and goat bright red oxymyoglobin were reacted with HNE in-vitro at physiological pH (7.4) and temperature (37 °C) conditions and the formation of oxidised brown metmyoglobin was measured. The Mb-HNE adducts were detected using MALDI-TOF MS, whereas specific sites of adduction was determined using ESI-QTOF MS/MS.ResultsPurified buffalo and goat Mb samples revealed a molecular mass of 17,043.6 and 16,899.9 Daltons, respectively. The 2DE analysis exhibited 65 (sarcoplasmic protein extract) and 6 (pure Mb) differentially expressed (P < 0.05) protein spots between buffalo and goat samples. OFFGEL electrophoresis revealed an isoelectric point of 6.77 and 7.35 respectively, for buffalo and goat Mb’s. In-vitro incubation of HNE with bright red buffalo and goat oxymyoglobin’s at pH 7.4 and 37 °C resulted in pronounced (P < 0.05) oxidation and formation of brown metmyoglobin. MALDI-TOF MS analysis of Mb-HNE reaction mix revealed covalent binding (via Michael addition) of 3 and 5 molecules of HNE with buffalo and goat Oxy-Mb’s, respectively. ESI-QTOF MS/MS identified seven and nine histidine (HIS) residues of Mb that were readily adducted by HNE in buffalo and goat, respectively.ConclusionThe study demonstrated better redox stability of buffalo Mb than goat Mb. Our findings confirm the hypothesis that relative effect of HNE was greater for Mb’s with 12 ± 1 HIS residues than Mb’s with 9 HIS residues and helps meat processors in developing species-specific processing strategies to reduce the color variability.

Flavour formation from hydrolysis of pork sarcoplasmic protein extract by a unique LAB culture isolated from Harbin dry sausage

The lactic acid bacteria Pediococcus pentosaceus, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum isolated from Harbin dry sausage were assessed for their protein hydrolysis and flavour development in pork muscle sarcoplasmic protein extracts. Gel electrophoresis indicated that sarcoplasmic proteins were degraded by all of the strains, especially by P. pentosaceus and L. curvatus. Trichloroacetic acid-soluble peptides increased in all of the samples (P<0.05), especially samples inoculated with P. pentosaceus. Samples inoculated with P. pentosaceus and L. curvatus had higher free amino acid contents than did the other two strains (P<0.05), and glutamic acid and alanine appeared to be the predominant free amino acids. The volatile compound analysis indicated that the highest aldehydes, alcohols and acid contents were found in the sample with P. pentosaceus followed by L. curvatus. The results revealed that P. pentosaceus could be appropriate for use as a meat starter culture.

Solubilidad proteica, contenido de mioglobina, color y pH de la carne de pollo, gallina y codorniz

The meat proteins are responsible for diverse characteristics of their derived products. The solubility of the meat proteins can be an index of other important functional properties for the meat processing (as the emulsification), while the myoglobin content influences the meat color. In order to evaluate the solubility of the sarcoplasmic and myofibrillar proteins as well as the myoglobin content in poultry meat, there were obtained chicken, hen and quail carcasses from commercial slaughterhouses; it was applied a completely random design with factorial arrangement 3x2: type of poultry (chicken, hen and quail) and body portion (breast and leg meat), with six treatments, three repetitions in each treatment. There was found interaction in the factors: lightness (L), redness (a), pH, extractability of sarcoplasmic and myofibrillar proteins. The myoglobin content was affected by the body portion and the type of poultry, being higher in the leg than in the breast (0.99 and 0.47 mg/g meat, respectively), the quail had the highest value (1.13 mg/g) and the chicken the lowest value (0.37 mg/g). In all the cases the leg pH was higher than the breast pH. There was a tendency to obtain a higher extractability of the sarcoplasmic proteins from the breast meat, while the myofibrillar proteins were obtained in a higher proportion from the leg portion. The information generated about the characteristics of these poultry meat is novel in Venezuela and can contribute to explain the behavior of these raw materials during the processing activities.

Relationship between muscle exudate protein composition and broiler breast meat quality

The objective of this study was to determine the relationship between meat quality and the protein content and composition of muscle exudate from broiler breast fillets. Deboned breast fillets (n = 48) were obtained from a commercial processing facility and segregated into 2 groups based on color (light and dark). Meat pH, color, moisture content, 3 measures of water-holding capacity (drip loss, salt-induced water uptake, cook loss), protein solubility, and the protein content of muscle exudates were determined in breast fillets. The protein composition of the muscle exudate was evaluated using SDS-PAGE analysis. Light breast fillets had lower meat pH (4 and 24 h postmortem) and higher L* (lightness) and b* (yellowness) values than dark fillets. Light breast fillets exhibited greater drip loss after 2 and 7 d of storage, lower salt-induced water uptake, and higher cook loss than dark fillets. Neither sarcoplasmic nor total protein solubility differed between light and dark fillets. Protein concentration of muscle exudates was greater in dark fillets and was negatively correlated to drip loss after 2 d of storage (r = −0.50) and salt-induced water uptake (r = 0.42). Electrophoretic protein banding patterns were similar between muscle exudates and sarcoplasmic protein extracts. Gel electrophoresis data from muscle exudates showed that the relative abundance of 4 bands corresponding to 225, 165, 90, and 71 kDa was higher in dark breast fillets. The relative abundance of 3 bands corresponding to 47, 43, and 39 kDa was higher in light breast fillets. Muscle pH and measurements of water-holding capacity were significantly correlated to the abundance of several individual protein bands within the protein profile of muscle exudates. Data from this study showed that protein differences in breast muscle exudates are related to meat pH, color, and water-holding capacity and suggest that muscle exudate could be a potential source of protein markers for fresh meat quality attributes in broiler fillets.

Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages

The aim of this study was to determine the technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase and enterotoxigenic activities) of 38 strains of Staphylococcaceae (11 of Staphylococcus epidermidis, 15 of Staphylococcus equorum, 5 of Staphylococcus pasteuri and 7 of Staphylococcus saprophyticus) isolated from Androlla and Botillo, two Spanish traditional sausages, in order to evaluate their suitability as potential starter cultures in the manufacture of these sausages. Most strains were able to grow at 10 °C, in the presence of 10% and 15% NaCl and at pH values of 5.5 and 5.0, except for S. equorum strains, growth of which was reduced at these pH values. The proteolytic activity assessed by the agar plate method showed that 89.5% and 52.6% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were not confirmed by electrophoretic assays as only 47.2% of the strains changed the SDS-PAGE profile of actin, myosin and/or sarcoplasmic protein extracts. The assessment of the lipolytic activity by titration showed that only 21.0% of the strains can hydrolyze pork fat to any extent; whereas the profiles of the freed fatty acids were different in the different strains. Most of the strains showed decarboxylase activity against histidine, lysine, ornithine and tyrosine, but the quantities of biogenic amines produced were in most cases <25 ppm and <5 ppm for putrescine and cadaverine, respectively. Only four strains (10.5%), of S. epidermidis, produced enterotoxin C.

Assessment of the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of hydrolysates of bovine brisket sarcoplasmic proteins produced by papain and characterisation of associated bioactive peptidic fractions

The main objective was to investigate the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of sarcoplasmic proteins isolated from the brisket muscle (Pectoralis profundus) of 3 (Bos taurus) cattle and hydrolysed with papain for 24h at 37°C. Sarcoplasmic protein hydrolysates were ultra-filtered using molecular weight cut off (MWCO) membranes and 10-kDa and 3-kDa filtrates were obtained. The total sarcoplasmic protein extracts and the 3-kDa filtrates were tested for angiotensin I-converting enzyme inhibitory (ACE-I) activities. The total hydrolysates, 10-kDa and 3-kDa filtrates were also tested for their associated antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe2+ metal chelating ability assay. The peptidic content of the total hydrolysates, the 10-kDa and the 3-kDa filtrates were analysed using an ORBITRAP mass spectrometer, and mass spectral data obtained were analysed using TurboSEQUEST. Eleven peptides were characterised from the total hydrolysates, fifteen from the 10-kDa filtrate fractions, whilst nine peptides were characterised from the 3-kDa filtrate fractions. Similarities between the amino acid sequences of the peptides identified in this study and previously identified antioxidant and ACE-I inhibitory peptides detailed in the BIOPEP database were outlined.

Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity

To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures.

Monoclonal antibody specific to a major fish allergen: parvalbumin.

The major fish allergen, parvalbumin, is a low-molecular-weight (10 to 13 kDa), heat-stable protein. Monoclonal antibody (MAb) 3E1, developed against heat-treated catfish sarcoplasmic protein extract, recognizes a thermal-stable protein with the molecular-weight range of parvalbumin in fish extracts. We further investigated the antigen-binding characteristics of this antibody by comparing its immunoreactivity against various fish and other animal species, with a commercially available anti-parvalbumin antibody, MAb PARV-19. Soluble proteins were extracted from 67 cooked (100 degrees C for 20 min) finfish, shellfish, meat, and poultry species. Indirect enzyme-linked immunosorbent assay (ELISA) was performed to examine the immunoreactivity of both MAb 3E1 and MAb PARV-19 with sample extracts. Western blot was performed to compare the antigenic protein banding patterns in cooked fish extracts by using these two MAbs. The ELISA results revealed that both MAbs had identical reaction patterns to the fish species tested. Removal of Ca2+ from the fish extracts increased the overall immunoreactivity of both MAbs. Western blot results confirmed that the antigenic protein banding pattern in various fish species blotted by MAb 3E1 corresponded to the molecular weights of parvalbumins recognized by PARV-19. However, screening with non-finfish extracts revealed MAb 3E1 to be strictly finfish specific, while PARV-19 cross-reacted with frog, rat, and rabbit extracts. Based on the heat stability, molecular weight, immunoreactivity, and Ca2+-dependent binding of the antigenic proteins, MAb 3E1 is specific to fish parvalbumin. It would therefore be a useful probe for investigating the major fish allergen in both raw and processed food.

Production and Characterization of Monoclonal Antibodies Specific to Pangasius Catfish, Basa, and Tra

Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

Proteolytic and lipolytic modifications during the manufacture of dry-cured lacón, a Spanish traditional meat product: Effect of some additives

The extractability of sarcoplasmic and myofibrillar proteins, the myofibrillar proteins and their degradation products, classical nitrogen fractions, free amino acids, acidity of the fat, and free fatty acids were determined throughout the manufacturing process of dry-cured lacón, a traditional dry-salted and ripened meat product made in the northwest of Spain from the foreleg of the pig, following a similar technological process to that of dry-cured ham. The effect of the use of additives (glucose, sodium nitrite, sodium nitrate, sodium ascorbate and sodium citrate) on the proteolytic and lipolytic changes was also studied.Throughout the manufacture, approximately 87% of the sarcoplasmic proteins and 91% of the myofibrillar proteins became insoluble. There was a significant (p<0.05) decrease of the myosin heavy chain, actin, and myosin light chains 1, 2 and 3, and also a significant (p<0.05) increase in the components generated as a result of the degradation of these myofibrillar proteins. The content of the different nitrogen fractions and of the free amino acids indicated that protein degradation during the manufacture of dry-cured lacón is only moderate. Data on the acidity of fat and of free fatty acids also indicated that lipolysis in dry-cured lacón is lower than in hams. The use of additives did not significantly influence the protein and lipid degradation, which occur throughout the manufacturing process.

Interactions between strains of Staphylococcus xylosus and Kocuria varians isolated from fermented meats

To evaluate the interactions of Staphylococcus xylosus on Kocuria varians strains isolated from fermented meat products. Interactions were assessed in vitro by agar spot test, agar well diffusion assay and spectrophotometric assay. The growth of K. varians (five strains) alone was compared with that in the presence of growing cells of S. xylosus (50 strains) or in the presence of heat-treated or untreated supernatants of S. xylosus. Sixteen strains stimulated the growth of K. varians K4, while four strains inhibited the K4 strain. Heated cell-free supernatants of S. xylosus did not have any effect on K. varians. The proteolytic activity of single strains or their combinations was assessed in vitro and in vivo by sodiumdodecylsulfate-polyacrylamide gel electrophoresis of sarcoplasmic protein extracts. Combinations of stimulatory strains of S. xylosus and K. varians showed a higher proteolytic activity compared with that of S. xylosus or K. varians alone. The interactions between strains may influence both the growth of the co-cultured strains and proteolysis, technologically relevant characteristics. The study of interactions between coagulase-negative cocci may guide the formulation of mixed strain starters for the production of fermented sausages.

Effect of ionic strength of different salts on the binding of volatile compounds to porcine soluble protein extracts in model systems

Abstract The effect of different salts on the binding ability of porcine soluble protein extracts was studied using solid-phase microextraction (SPME) and GC/MS analysis. Previously, the effect of the different salts (NaCl, KCl, MgCl2 and CaCl2) on the volatile compound headspace concentration in water solution was determined. KCl produced a similar salting out effect as NaCl increasing the volatile compound headspace concentration up to 5–10 times. However, MgCl2 and CaCl2 did not produce a salting out effect on the volatile compounds studied. Finally, NaCl and KCl produced a significant reduction on the binding ability of sarcoplasmic protein extracts to branched aldehydes, hexanal and methional while no effect was produced on octanal and 2-pentanone. On the other hand, the effect of MgCl2 and CaCl2 was much lower even at high ionic strength, except for the branched aldehydes where the presence of MgCl2 at 1.0 ionic strength produced the complete liberation of the volatile compound bound. Consequently, the partial NaCl substitution in meat products by other salts must be considered not only due to their salting out effect but also due to their effect on protein binding ability.

Iron‐Binding Properties, Amino Acid Composition, and Structure of Muscle Tissue Peptides from in vitro Digestion of Different Meat Sources

The enhancing effect of meat on nonheme iron bioavailability in humans is thought to be due to the release of low-molecular-weight (LMW) iron-binding peptides during digestion. To better characterize the LMW iron-binding peptides from meat digests. Cooked beef, chicken, cod, lamb, and pork myofibrillar or sarcoplasmic protein extracts, casein, and egg albumin were digested in vitro with pepsin or pepsin/pancreatin. Ultrafiltrates were analyzed for N and iron and further characterized by gel filtration with added 59Fe, amino acid analysis, and LC-MS. 84% to 98% of total iron in enzymic digests was associated with soluble LMW peptides (< 10 kDa) of the myofibrillar proteins compared to only 2% to 20% in the corresponding sarcoplasmic protein digests. Pepsin digestion alone of the myobrillar proteins generated > 80% soluble LMW iron, compared to < 5% with casein and egg albumin. Iron-binding peptides from myofibrillar protein with an estimated 2 kDa molecular mass were separated by gel filtration. Peptides in this fraction were enriched in aspartic and glutamic acid residues and included potential peptide fragments of myosin. LMW (< 10 kDa) peptides in enzyme digests of myofibrillar proteins were the major facilitators of iron solubility. Unlike with casein, egg albumin, and most sarcoplasmic proteins, these LMW peptides were generated on pepsin digestion. One group of iron-binding peptides had a mass of approximately 2 kDa and was enriched in glutamic and aspartic acids. Such early generation of a multitude of LMW iron-binding peptides could explain the enhancing effect of muscle tissue on iron absorption.

Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE > 10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE > 10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.

Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates

Sarcoplasmic protein extracts from beef rump ( biceps femoris) were hydrolyzed (for 0, 4, 8, 12, and 24 h) with three enzymes or their paired combinations. Ultrafiltration, gel-filtration, and RP-HPLC were used to separate angiotensin converting enzyme (ACE) inhibitory peptides from the hydrolysates. The highest ACE inhibitory activity of enzyme hydrolysates resulted from 4 h incubation with enzymes or their paired combinations. The activities of gel filtrated fractions from these hydrolysates were assayed in vitro, demonstrating that the 3rd peak of enzyme thermolysin + proteinase A hydrolysate had the highest ACE inhibition activity (52.8%). The 3rd peak of this hydrolysate was separated by RP-HPLC into five peaks, of which peak 3 showed 30.1% ACE inhibition activity. Its peptide sequence was determined to be Val-Leu-Ala-Gln-Tyr-Lys. The results suggested that this peptide may be a potent ACE inhibitor which might perhaps be used to develop beef with a bioactive peptide to lower blood pressure.

Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii

Strains of Debaryomyces hansenii originally isolated from sausages were screened for proteinase and aminopeptidase activity towards synthetic substrates. On the basis of these results, D. hansenii CT12487 was selected for further assays. The activities of the whole cells (WC), cell-free extracts (CFE) and a combination of both from the selected strain on pork muscle sarcoplasmic protein extracts were determined by protein, peptide and free amino acid analyses. There was a pronounced hydrolysis of protein bands of 110 kDa and 27–64 kDa regardless the incorporation of WC, CFE or a combination of both. The proteolytic activity also resulted in the generation of polar and non-polar peptides showing noticeable differences depending on the addition of WC or CFE. Whole cells generated greater amounts of free amino acids than the cell-free extracts.