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The purpose of this study was to evaluate the caseinolytic and milk-clotting activities of aqueous crude extracts from leaves and latex of the Pergularia tomentosa, to determine their suitability as a rennet substitute. These extracts were subjected to a series of biochemical tests before being used in the production of cheese. The results showed that the enzymatic latex extract had a higher coagulant activity than the leaf extract. However, under different clotting conditions (pH, temperature, and CaCl2 concentration), both coagulants behaved similarly in the coagulation of Berridge substrate. The SDS-PAGE and zymographic analysis revealed identical protein bands with a single active zone in both extracts, corresponding to a molecular weight of 26.98 kDa and 26.03 kDa in the extract of leaf and latex, respectively. Both extracts were stable to different effectors but strongly inhibited by iodoacetamide and Hg, suggesting it to be a cysteine protease. Both extracts were able to hydrolyze casein and generate peptides of 14 kDa, with excessive hydrolysis of the other casein fractions. The physicochemical parameters of cheese made from latex and leaf extract evolved similarly to control cheese. According to the sensory evaluation, cheese made with latex had a mildly bitter flavor but showed a high acceptance rate (>80%).

Three Phase Partitioning (TPP) system as an elegant non-chromatographic and bulk separation method was successfully applied for the extraction and recovery of papain from the latex of Carica papaya. The optimized parameters of TPP allowed achieving a purification fold of 11.45 and activity recovery of 134% with 40% (NH4)2SO4, 1.0:0.75 ratio of crude extract: t-BuOH at pH and temperature of 6.0 and 25 °C, respectively. The recovered papain had a molecular weight of 23.2 kDa and revealed maximum activity at pH 6.0 and temperature of 50 °C. The maximum values of Km and Vmax parameters were 10.83 mg mL−1 and 33.33 U mL−1, respectively. The protease with 4 isoforms was stable at 40–80 °C and a pH range of 6.0–7.5 against numerous metal ions and none of them inactivated the recovered protease. Moreover, 10 mM Ca2+ improved 2-folds the activity and half-life of the protease at temperatures from 30 to 50 °C. The milk-clotting activity tests revealed high stability of latex papain at storage, namely at −20 °C compared to 4 °C and 25 °C for up than 5 weeks. As a meat tenderizing agent, it showed promising role under different treatments by improving the texture of tough meat. The findings indicated that one-step TPP system is a simple, quick, economical and very attractive process for fast recovery of latex papain compared to other proposed protocols.

This study was designed within the scope of industrial exploitation of antioxidant extracts from Hibiscus sabdariffa. Efficiency in the extraction of antioxidants (total phenolics, flavonoids and anthocyanins) from calyces was assessed through different processes involving solvents (water, 80% acetone and 80% methanol), microwaves and ultrasounds. Results indicated that microwave extraction with 80% acetone gave the highest yield for total phenolics, expressed as gallic acid equivalents (GAE) per g dry weight (DW) (3.73 GAE/100 g DW), while the highest results for total flavonoids and anthocyanins were found in the microwaved aqueous extracts. Antioxidant activity of the extracts reported the highest reducing power for aqueous extracts with microwave extraction, while 80% acetone and methanol samples exhibited the highest antioxidant activity by high-performance thin-layer chromatography (HPTLC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. HPTLC performed for screening of phenolic compounds revealed the occurrence of anthocyanins in dimer-like bands. Laboratory tests showed high protection against lipid oxidation of the whey spread emulsion (22.32 h) incorporated with the sonicated aqueous extract. Results of peptide sequencing data by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, revealed protein–polyphenols interactions by the formation of dimers between polyphenols and protein/peptides. These interactions were found without effect on the oxidative stability of the emulsions. These study suggest that H. sabdariffa’s calyces have the potential to be used like food antioxidants.

A new Rhizopus oryzae strain FSIS4 isolated from wheat seed cultivated in Algerian Sahara was evaluated for the production of a thermostable α-amylase enzyme. Medium components were optimized by a statistical approach for the optimal production of α-amylase using an agro-industrial residue (decommissioned dates). The Plackett–Burman design was performed to screen the fermentation variables and response surface methodology for optimizing the significant factors affecting the production. The optimum levels for maximum production of α-amylase (approx 10,151 IU) were determined. A 1.8-fold increase of production was obtained in comparison to the non optimized medium. Partial characterization of the enzyme showed optimal temperature and pH at 60 °C and 5.0, respectively. The enzyme was found to be stable at 80 and 90 °C, with a half-life of 165 and 105 min, respectively. According to the large literature, this is the first report on the production of α-amylase by the thermophilic R. oryzae strain FSIS4. The thermo and pH stability of the α-amylase suggest its wide application in the food and pharmaceutical industries.

El Gueddid is a traditional salted and dried meat with high popularity in Algeria. It is used as an ingredient in various dishes. In this study, different samples of El Gueddid were analyzed at different processing times to follow up their microbiological and physicochemical properties. Changes in the protein profile were also demonstrated by electrophoretic study of myofibrillar proteins. Microbiological determinations included the total viable count, coliforms, Staphylococci, lactic acid bacteria, yeasts, and molds, whereas physicochemical properties were characterized by pH, moisture, salt content and water activity. The results showed that microbial profiles were elevated for all the studied micro-organisms. Staphylococci and lactic acid bacteria were the most abundant micro-organisms in the product. Total coliforms were found in low numbers in fresh meat, being eliminated at the post salting stage of process. The physicochemical characteristics showed that the moisture content decreased in the product during the drying period. The pH also decreased during the drying period, then remained almost unchanged during the rest of the ripening period. Moreover, El Gueddid showed low water activity and high salt content. One of the most important changes in the profile of myofibrillar proteins was a reduction in the myosin heavy chain content., Project AGL2012-32146 from the Spanish Ministry of Economy and Competitiveness is fully acknowledged

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C.

Three phase partitioning or TPP consists of proteins’ precipitation due to simultaneous presence of ammonium sulfate and t-butanol. The technique has been successfully used for the partial purification of ficin (EC 3.4.22.3) from Ficus carica latex. In this study, ficin of Mediterranean F. carica was for the first time partially purified and recovered in a single step using TPP. Optimal purification parameters of the system were 40% ammonium sulfate saturation (w/v) with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 7.0, which gave 6.04 purification fold with 167% recovery of ficin. The enzyme was found to be exclusively partitioned in the interfacial precipitate phase. The Tricine SDS–polyacrylamide gel electrophoresis analysis of the enzyme showed comparatively purification and protein molecular weight of the enzyme was nearly found to be 23.4 kDa. Characterization of enzyme showed that optimal pH of purified enzyme was 6.5 and optimal temperature was 60 °C. The enzyme was found to be stable at 40–70 °C for 1 h. This study showed that TPP is a simple, economical and quick method for ficin recovering from F. carica latex trees and can be performed in a purification process to be used successfully in food industries.

Khliaa Ezir is a traditional and popular meat product, which is produced from whole beef, camel, goat or lamb meat. It is an Algerian and a ready to eat meat product that is marinated, cooked and ripened. The product is for numerous months preserved in an earthenware jar at room temperature. Microbiological investigation on Khliaa Ezir during preparation is a prerequisite and to our knowledge, this is the first study, which provide its preliminary microbiological characterization. Thus, the aim of the present work is to study the evolution of the microflora and microbiological safety of Khliaa Ezir during the main traditional preparation steps. The microbiological counts indicated that Lactic Acid Bacteria (LAB) are the most abundant in the product, namely during the repining and storage period. However, total Coliforms were very low in fresh beef, being eliminated after 3 days of curing. Yeasts and Molds were the highest in fresh beef, and then disappeared after cooking and during the ripening and storage step. None of the pathogenic flora during the whole preparation steps were detected. We think that the cooking temperature applied during thermal treatment (80°C) contributed to the high hygienic quality of Khliaa Ezir. On another hand, a significant increase in pH was observed during the storage period to achieve a final pH of 6.19 ± 0.01 at 30 days of storage.

Actinomycetes are an uncommon agent of human infections and its pathogenic factors are not known. The present study reports a rare case isolation of an actinomycete from a woman pleural fluid; the strain was identified by 16S rRNA gene sequence analysis. This strain was tested to produce an extracellular protease that hydrolysis gelatin, casein and hemoglobin on agar mediums. The purification of the enzyme was carried by ammonium sulfate precipitation, gel filtration and ion exchange chromatographies. The activity of protease was studied at different pH values and temperatures and in the presence of metallic ions and inhibitors. The molecular weight of the enzyme was determined by 12% Tricine SDS-polyacrylamide gel electrophoresis. The strain was identified as Streptomyces cinereoruber ssp. cinereoruber. Extracellular proteolytic enzyme was purified at 19.67 fold and a 3.0% recovery. The enzyme was characterized as having optimal activities at pH 11.0 and 50°C, it keeps more than 50% of activity at pH between 4.0 to 12.0 and it is thermostable at 30 and 40°C. Enzymatic activity is enhanced in the presence of metal ions and inhibited by EDTA and 1,10-phenanthroline. The molecular weight was 53 kDa. This study reports the first case isolation of Streptomyces cinereoruber ssp. cinereoruber from pleural fluid, the extracellular zinc-metalloprotease was proposed as candidate virulence factor.

International audience; Khliaa Ezir is a typical Algerian meat product, prepared according to a traditional process based on empirical observations and traditional recipes, consisting of steps of trimming, marinating, cooking, ripening and ageing in earthenware jar (Ezir). Understanding the biological mechanisms behind the development of the sensory properties of this cultural product is of great interest for eventual product marketing. The aim of the present study was first a preliminary proteomic characterization of myofibrillar and sarcoplasmic protein extracts of the product during preparation, ripening and ageing in earthenware jar. A comparison of the proteome of the two extracts at different days and steps was carried out with 1-DE electrophoresis, showing quantitative difference in several bands. Some bands might be potential markers of the proteolysis that takes place during the preparation steps, especially during marinating and ripening. This first and preliminary proteomic data from both myofibrillar and sarcoplasmic protein extracts of Khliaa Ezir are consistent with a differential release of specific proteins as a function of preparation step.

A new thermostable α-amylase from Rhizopus oryzae FSIS4 was purified for first time and recovered in a single step using a three-phase partitioning (TPP) system. The fungal α-amylase, at a concentration of 1.936 U per kg of flour, was used in bread-making and compared to the commercial enzyme. The results showed a significant effect of the recovered α-amylase in the prepared bread and allowed us to improve the quality of the bread. The study indicated clearly that the recovered α-amylase is a potential candidate for future applications in the bread-making industry and in other food biotechnology applications.

A rapid overview of the Three Phase Partitioning (TPP) system as an efficient non-chromatographic tool is given. This elegant non-chromatographic tool is able to purify numerous proteins and especially proteases in a one step protocol. TPP is able to do not only purify proteins, but also concentrate them into one of the phases and enhance their proteolytic activity. The application of TPP for the extraction and purification of plant milk-clotting enzymes and meat tenderizers agents are given. In addition, some proteases from other materials are summarized. This short communication stress showed that TPP is a simple, economical and quick method for proteases recovery from plant proteases. This elegant non-chromatographic tool may be performed in a purification process to be used successfully in food industries, namely to provide enzymes for cheese-making and meat tenderizing.

International audience; Objective: A total of 16 spent hens (ISA F15) were slaughtered at about 220 and 337 days of age and studied for their muscle characteristics and sensory attributes. Methodology: Tenderness was estimated from breast muscle using penetrometer PNR 10 and sensory trained panel. Water Holding Capacity (WHC), percentage of released water, cooking loss, pH and Myofibril Fragmentation Index (MFI) were studied. Results: Penetration values were found negatively correlated with tenderness and MFI. Tenderness scores were negatively correlated with animal age and positively with pH. Overall acceptability of the meat of spent hens was judged higher irrespective of age. Multiple regressions analysis revealed that penetration depth was mainly explained by juiciness and MFI. Conclusion: In addition, the results revealed that sensory parameters representing textural properties of meat (tenderness, cohesiveness, chewiness and residues) were mainly explained by cooking losses.

Postmortem (PM) meat tenderization is a complex biochemical process that involves multiple endogenous proteolytic enzymes and that is not completely understood. Cell death by apoptosis is recently proposed as a novel mechanism in this process. The signification of the caspases implication in muscle proteins degradation is that the cells are dying through apoptosis, a major cell death programme. Our purpose was therefore to verify this statement using different well known hallmarks of the apoptotic process including cell shrinkage, phosphatidylserine (PS) externalization. Cells start to shrink few minutes after animal death and reach a maximum shrinkage about 24 h postmortem while they showed the typical rounding appearance of apoptotic cells. Externalization of PS is detectable within 1 h postmortem and increased gradually with time. The present work is therefore the first to provide direct evidence supporting the onset of apoptosis in post-mortem muscle. The present findings should lead us to reconsider as a whole, the mechanisms contributing of the development of meat qualities (tenderness, flavor, juiciness …) by a detailed analysis of the apoptosis associated biochemical and physiological modifications taking place within the cells.

International audience; In living cells, after activation, protein inhibitors constitute the last step of proteases activity regulation. This review intends to provide original information about a group of bovine muscle serine proteases inhibitors belonging to the Serpin superfamily and characterized at the gene and protein level. This report is the only one and the first to provide much information on this group of proteases inhibitors of the serpin type and their potential biological functions. Amongst the eight genes identified in bovine, three serpins were purified from the muscle tissue and characterized. These are two members of the bovSERPINA3 family, i.e., bovSERPINA3-1 and A3-3, and the last one is antithrombin III (AT-III or BovSERPINC1). BovSERPINA3 family comprises at least eight protein members encoded by different genes mapped on chromosome 7q23–q26 cluster. BovSERPINA3-1 and A3-3 were shown to locate within muscle cells and are cross-class inhibitors strongly active against trypsin as well as against human initiator and effector caspases 8 and 3. They constitute a key apoptosis control in mammals. They were thus expressed in proliferating and confluent myoblasts phases where cells must be alive but not in myotubes. Antithrombin III inhibits trypsin and, in a heparin dependent manner, thrombin. AT-III and its mRNA were expressed in muscle cells and in differentiating primary myoblasts in culture.