High Proteolytic Activity Research Articles

The absence of PNPase activity in Enterococcus faecalis results in alterations of the bacterial cell-wall but induces high proteolytic and adhesion activities

Enterococci are lactic acid bacteria (LAB) used as starters and probiotics, delineating their positive attributes. Nevertheless, enterococci can be culprit for thousands of infectious diseases, including urinary tract infections, bacteremia and endocarditis. Here, we aim to determine the impact of polynucleotide phosphorylase (PNPase) in the biology of Enterococcus faecalis 14; a human isolate from meconium. Thus, a mutant strain deficient in PNPase synthesis, named ΔpnpA mutant, was genetically obtained. After that, a transcriptomic study revealed a set of 244 genes differentially expressed in the ΔpnpA mutant compared with the wild-type strain, when exploiting RNAs extracted from these strains after 3 and 6 h of growth. Differentially expressed genes include those involved in cell wall synthesis, adhesion, biofilm formation, bacterial competence and conjugation, stress response, transport, DNA repair and many other functions related to the primary and secondary metabolism of the bacteria. Moreover, the ΔpnpA mutant showed an altered cell envelope ultrastructure compared with the WT strain, and is also distinguished by a strong adhesion capacity on eukaryotic cell as well as a high proteolytic activity. This study, which combines genetics, physiology and transcriptomics enabled us to show further biological functions that could be directly or indirectly controlled by the PNPase in E. faecalis 14.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30s.The Lys C gene from M14-150s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Melanoma cells with diverse invasive potential differentially induce the activation of normal human fibroblasts

BackgroundThe tumor microenvironment consists of stromal cells, extracellular matrix, and physicochemical properties (e.g., oxygenation, acidification). An important element of the tumor niche are cancer-associated fibroblasts (CAFs). They may constitute up to 80% of the tumor mass and share some features with myofibroblasts involved in the process of wound healing. CAFs can facilitate cancer progression. However, their interaction with melanoma cells is still poorly understood.MethodsWe obtained CAFs using conditioned media derived from primary and metastatic melanoma cells, and via co-culture with melanoma cells on Transwell inserts. Using 2D and 3D wound healing assays and Transwell invasion method we evaluated CAFs’ motile activities, while coverslips with FITC-labeled gelatin, gelatin zymography, and fluorescence-based activity assay were employed to determine the proteolytic activity of the examined cells. Western Blotting method was used for the identification of CAFs’ markers as well as estimation of the mediators of MMPs’ (matrix metalloproteinases) expression levels. Lastly, CAFs’ secretome was evaluated with cytokine and angiogenesis proteomic arrays, and lactate chemiluminescence-based assay.ResultsAcquired FAP-α/IL6-positive CAFs exhibited elevated motility expressed as increased migration and invasion ratio, as well as higher proteolytic activity (area of digestion, MMP2, MMP14). Furthermore, fibroblasts activated by melanoma cells showed upregulation of the MMPs’ expression mediators’ levels (pERK, p-p38, CD44, RUNX), enhanced secretion of lactate, several cytokines (IL8, IL6, CXCL1, CCL2, ICAM1), and proteins related to angiogenesis (GM-CSF, DPPIV, VEGFA, PIGF).ConclusionsObserved changes in CAFs’ biology were mainly driven by highly aggressive melanoma cells (A375, WM9, Hs294T) compared to the less aggressive WM1341D cells and could promote melanoma invasion, as well as impact inflammation, angiogenesis, and acidification of the tumor niche. Interestingly, different approaches to CAFs acquisition seem to complement each other showing interactions between studied cells.Br5MKGXJnZuusnx4cjbfRkVideo

Gut microbial β-glucuronidases regulate host luminal proteases and are depleted in irritable bowel syndrome.

Intestinal proteases mediate digestion and immune signaling, while increased gut proteolytic activity disrupts the intestinal barrier and generates visceral hypersensitivity, which in common in irritable bowel syndrome (IBS). However, the mechanisms controlling protease function are unclear. Here we show that members of the gut microbiota suppress intestinal proteolytic activity through production of unconjugated bilirubin. This occurs via microbial β-glucuronidase-mediated conversion of bilirubin conjugates. Metagenomic analysis of fecal samples from patients with post-infection IBS (n=52) revealed an altered gut microbiota composition, in particular a reduction in Alistipes taxa, and high gut proteolytic activity driven by specific host serine proteases compared to controls. Germ-free mice showed 10-fold higher proteolytic activity compared with conventional mice. Colonization with microbiota from high proteolytic activity IBS patients failed to suppress proteolytic activity in germ-free mice, but suppression of proteolytic activity was achieved with colonization using microbiota from healthy donors. High proteolytic activity mice had higher intestinal permeability, a higher relative abundance of Bacteroides and a reduction in Alistipes taxa compared with low proteolytic activity mice. High proteolytic activity IBS patients had lower fecal β-glucuronidase activity and end-products of bilirubin deconjugation. Mice treated with unconjugated bilirubin and β-glucuronidase overexpressing E. coli, which significantly reduced proteolytic activity, while inhibitors of microbial β-glucuronidases increased proteolytic activity. Together, these data define a disease-relevant mechanism of host-microbial interaction that maintains protease homeostasis in the gut.

The Texture of Camel Milk Cheese: Effects of Milk Composition, Coagulants, and Processing Conditions.

Numerous people in African, Middle Asian, Middle Eastern, and Gulf Cooperation Council (GCC) countries highly value camel milk (CM) as it plays a vital role in their diet. The protein composition of CM as well as the structure of its casein micelles differs significantly from bovine milk (BM). Cheeses made from CM have a weak curd and soft texture compared to those made from BM. This review article presents and discusses the effect of milk protein composition, processing conditions (pasteurization and high-pressure treatment), and coagulants (camel chymosin, organic acids, plant proteases) on the quality of CM cheeses. CM cheese's weak texture is due to compositional characteristics of the milk, including low κ-casein-to-β-casein ratio (≈0.05 in CM vs. ≈0.33 in BM), large micelle size, different whey protein components, and higher proteolytic activity than BM. CM cheese texture can be improved by preheating the milk at low temperatures or by high pressure. Supplementing CM with calcium has shown inconsistent results on cheese texture, which may be due to interactions with other processing conditions. Despite their structure, CM cheeses are generally well liked in sensory studies.

Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation.

Evaluation of the proteolytic activity of new cryoresistant lactobacillus strains

The search for new functionally active strains of lactic acid bacteria, together with the development of domestic competitive starter cultures on their basis, are important directions of contemporary food biotechnology. Proteolytic activity represents one of the criteria for selecting lactic acid bacteria strains for their subsequent use in food production and largely determines the quality characteristics of the finished product. The present study aims to evaluate the proteolytic activity of 15 new cryoresistant strains of Lactobacillus genus lactic acid bacteria having a number of functional and technological properties. According to the results obtained, all strains demonstrated higher proteolytic activity in alkaline media and those close to neutral (pH = 6.5). In slightly acidic media, the strains under study showed minimal values of proteolytic activity, except for L. fermentum 12 and L. plantarum 21 strains. Strains L. casei 32, L. casei 36, L. fermentum 10, and L. acidophilum 9 (48.9–52.3 µg tyrosine/mL·min) showed the maximum proteolytic activity. The minimal proteolytic activity was characteristic of L. fermentum 12, L. fermentum 24, and L. plantarum 1 (27.7–28.9 µg tyrosine/mL·min). The studied paramenter depends on the conditions of proteolysis (substrate, medium pH) and represents an individual strain characteristic independent of the lactobacillus species membership. According to the results obtained, L. casei 32, L. casei 36, and L. fermentum 10 cryoresistant strains, manifesting high proteolytic activity and effectively affecting various protein substrates (casein, albumin, haemoglobin) in a wide range of medium pH values, can be recommended for inclusion in the composition of starter cultures for the production of fermented food products.

Investigating the biotechnological potential of lactic acid bacteria strains isolated from different Algerian dairy and farm sources.

Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

Anti-Inflammatory, ACE Inhibitory, Antioxidative Activities and Release of Novel Antihypertensive and Antioxidative Peptides from Whey Protein Hydrolysate with Molecular Interactions

Objective The aim of the study was to evaluate the whey protein hydrolysate with bio-functional attributes viz. antioxidative, anti-inflammatory and ACE inhibition efficacy and release of bioactive peptides with antioxidative and ACE-inhibitory activity by employing Pepsin. Method The antioxidant, Anti-inflammatory, ACE inhibitory and proteolytic activities of the whey protein hydrolysates were studied followed by SDS-PAGE analysis and IEF. Anti-inflammatory activity of whey protein hydrolysate was also studied on RAW 264.7 cell line. The separation of the bioactive peptides from whey protein hydrolysate was achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS. Results WPC (Whey protein concentrate) hydrolysate with pepsin showed proteolytic activity ranging between 14.46 and 18.87 mg/ml. Using the ABTS assay, the highest antioxidative activity was observed in 10 kDa retentate (84.50%) and 3 kDa retentate (85.96%), followed by the highest proteolytic activity (13.83 mg/ml) and ACE inhibitory activity (58.37%) in a 5% WPC solution at 65 °C after 8 h of pepsin hydrolysis. When the protein hydrolysate concentration was low, the production of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages (RAW 264.7) was reduced. SDS-PAGE results exhibited very little protein bands when comparing with WPC hydrolysates to insoluble WPC. There were no protein spots on 2 D gel electrophoresis and “in-solution trypsin digestion” technique have been utilized to digest protein samples directly from WPC hydrolysates. Novel antioxidative peptides and ACE inhibitory peptides were also observed by comparing two databases, i.e., BIOPEP and AHTPDB respectively. The peptide sequences used in this study were found to have excellent potential to be used as inhibitors of hACE as all of them were able to show substantial interactions against the enzyme’s active site. Conclusions The antihypertensive and antioxidative peptides from whey protein hydrolysates may be beneficial for the future development of physiologically active functional foods. Further, in vivo investigations are required to establish the health claim for each individual bioactive peptide from whey protein hydrolysate. Supplemental data for this article is available online at

Isolation and identification of proteolytic lactic-acid bacteria of the common carp (Cyprinus carpio) by spontaneous fermentation to obtain functional peptides.

High proteolytic activity and several biological functions (antimicrobial, antioxidant, antihypertensive, among others) have been attributed to lactic-acid bacteria (LAB) isolated from fish and peptides obtained from proteolysis. Therefore, the objective of this research was isolating, characterizing, and identifying LAB with proteolytic activity by spontaneous fermentation from common carp (Cyprinus carpio) reared in ponds and wild ones obtained from Lago de Chapala, Jalisco, Mexico. Spontaneous fermentation from complete carp specimens was observed, considering two sampling points (skin and intestines) at 15°C at 5 and 10days. Isolated LAB-from both reared and wild specimens-were identified and morphologically characterized; identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Proteolytic activity was assessed by the presence of the proteolytic halo. A total of five genera and eight different LAB proteolytic species were isolated from all the carp samples. At 10days, greater proteolytic LAB diversity was obtained from the intestine (Tukey's, p < 0.05); the proteolytic halo with the greatest diameter was recorded in wild carp skin with Lactiplantibacillus plantarum S5P2 (2.8cm) at 5days of fermentation, followed by Leuconostoc mesenteroides S5I1 (2.73cm) and Leuconostoc pseudomesenteroides S5P2 (2.66cm) (p < 0.05). In conclusion, proteolytic capability of LAB isolated from carp (Cyprimus carpio)-both wild and reared-is influenced by the ecosystem where they develop. These proteolytic LAB may be used in biotechnological industries to obtain bioactive peptides by fermenting substrates rich in proteins.

Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the “Tanche” olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had β-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

Smart proteolysis samplers for pre‐lab bottom‐up protein analysis – Performance of on‐paper digestion compared to conventional digestion

AbstractHere the relation between digestion of proteins by trypsin covalently bound to paper and trypsin in‐solution is investigated. The trypsin acting on paper is covalently bound. A trypsin concentration of 0.5% (w/v) results in the highest digestion activity of all concentrations tested. Additionally, it can be seen that trypsin on‐paper has retained approx. 50% of its activity. Unlike trypsin in‐solution, the stability of the smart proteolysis samplers was regarded to be stable for at least four months when kept refrigerated. Autolysis was very small for covalently bound trypsin: less than 2% compared to in‐solution trypsin. Proteomic analysis of diluted human serum showed more protein identifications (214) in‐solution digestions than on‐paper digestions (76). Also, higher coverage for the in‐solution digestion was obtained. Those proteins identified after on‐paper digestion with no or few disulfide bonds seem to have more similar sequence coverages compared to those identified after in‐solution digestion. Smart samplers allow the determination of at least 70–75 proteins without performing the overnight digestion. All in all, trypsin covalently bound to paper shows to retain high proteolytic activity and is a stable alternative for conventional digestions. In this way, smart proteolytic samplers show their feasibility in pre‐lab sample preparation.

Wash-Free Amperometric Escherichia coli Detection via Rapid and Specific Proteolytic Cleavage by Its Outer Membrane OmpT.

Various methods have been developed for the detection of Escherichia coli (E. coli); however, they are complex and time-consuming. OmpT─a cell membrane endopeptidase of E. coli─strongly embedded in the outer membrane of only E. coli, exposed to external solutions, with high proteolytic activity, could be a suitable target molecule for the rapid and straightforward detection of E. coli. Herein, a wash-free, sensitive, and selective amperometric method for E. coli detection, based on rapid and specific proteolytic cleavage by OmpT, has been reported. The method involved (i) rapid proteolytic cleavage of consecutive amino acids, after cleavage by OmpT, linked to an electrochemical species (4-aminophenol, AP), by leucine aminopeptidase (LAP, an exopeptidase), (ii) affinity binding of E. coli on an electrode, and (iii) electrochemical-enzymatic (EN) redox cycling. OmpT cleaved the intermediate peptide bond of a peptide substrate containing alanine-arginine-arginine-leucine-AP (-A-R-R-L-AP), forming R-L-AP, followed by the cleavage of two peptide bonds of R-L-AP sequentially by LAP, to liberate an electroactive AP. Affinity binding and EN redox cycling, in addition to rapid proteolytic cleavage by OmpT and LAP, enabled high electrochemical signal amplification. Two-sequential-cleavage was employed for the first time in protease-based detection. The calculated detection limit for E. coli cells in tap water (approximately 103 CFU/mL after 1 h incubation) was lower than those obtained without affinity binding and EN redox cycling. The detection method was highly selective to E. coli as OmpT is present in only E. coli. High sensitivity, selectivity, and the absence of wash steps make the developed detection method practically promising.

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.

Atypical response to bacterial coinfection and persistent neutrophilic bronchoalveolar inflammation distinguish critical COVID-19 from influenza.

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non–COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1β, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766).

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Proteomics and metagenomics reveal the relationship between microbial metabolism and protein hydrolysis in dried fermented grass carp using a lactic acid bacteria starter culture

A microbial starter culture is expected to improve the quality of traditional fermented fish products. Lactobacillus plantarum was selected for grass carp fermentation due to its high proteolytic activity. To investigate its effects on muscle proteolysis of dried fermented fish, the protein profile and microbial community were analysed by using proteomics and metabolomics. The myofibrillar protein and collagen profiles showed remarkable variation after processing, changes that were related to the development of flavour and texture in fish samples. The starter culture had a marked effect on the microbial composition. Macrococcus and Staphylococcus were the dominant genera, with a relative abundance of 24.79% and 12.53%, respectively. There were significant correlations (P < 0.05) between the dominant genera and the major peptidase genes and quality-related proteins. These findings suggest that microbial activity is involved in proteolysis and affects the flavour and texture of dried fermented fish.

Isolation and identification of protease‐producing bacteria from sludge and sediment soil around Adama, Ethiopia

Proteases are enzymes used in industries such the production and processing of detergents, food, leather, and silk. The aim of this study was to isolate and identify protease‐producing bacteria from a sludge disposal site and from sediments. Soil samples were collected separately from the selected area. Samples weighing 1 g were serially diluted and spread onto skim milk agar. A total of 16 bacteria species were isolated from the study samples. Four bacterial isolates showed high proteolytic activity and were selected for enzymatic study based on their zone of proteolysis. The isolates were identified based on biochemical tests. The results indicated that the isolated bacteria were E. coli (99.69%), Pseudomonas putrefaciens (Shewanella putrefaciens) (91.61%), Bacillus carboniphilus (92.78%), and Lysinibacillus sphaericus (98.4%). The crude protease enzymes produced by these bacterial isolates showed promising results for application in dehairing and destaining as detergent additives. Bacillus carboniphilus showed the best level of activity and was selected as the most potent protease‐producing bacteria for both dehairing and destaining ability. Soils from sludge disposal sites and sediments from around tannery wastes could be good sources from which to isolate alkaline protease‐producing bacteria.

Скрининг микроорганизмов, способных ускорять процесс микробной трансформации птичьего помета

Abstract. The purpose of the research. Screening of collection strains of microorganisms with enzymatic properties to accelerate the processes of microbial biodegradation of bird droppings. Research methods. The proteolytic activity of the grown cultures was studied according to GOST 20264.2-88, the total microbial number in the chicken droppings (CFU/ml) was analyzed, and the ammonium nitrogen was determined. Research results. As a result of the experiments, it was found that the highest proteolytic activity was demonstrated by the strain Pseudomonas putida 90 biovar A (171), which amounted to 74.6 units/g. When analyzing the effect of the studied collection strains on the decomposition processes of droppings, it was revealed that the largest number of microbial cells in bird droppings was achieved using Pseudomonas putida 90 biovar A (171), which was 104 CFU/ml at the beginning of the researches, and was the maximum and amounted to 1011 CFU/ml by the 15th day. The content of ammonium nitrogen in droppings treated with this culture decreased from 340 mg/l from the beginning of the experiment to 174 (15th day) and 169 mg/l (20th day) and it was the best indicator. When selecting the dose and concentration of the strain-producer Pseudomonas putida 90 biovar A (171) under introduction to bird droppings, it was found that to accelerate the process of biodegradation of bird droppings, the optimal dose for applying the studied culture is 4.0 % of organic waste mass with preliminary dilution by 2 times with water. At the same time, the optimal time of droppings keeping and the studied culture is 15 days. Scientific novelty. It was established for the first time that the treatment of chicken manure with the collection strain Pseudomonas putida 90 biovar A (171) accelerates the process of its microbial transformation.

Characterization of nettle leaves (Urtica dioica) as a novel source of protease for clotting dromedary milk by non-destructive methods

This study investigates the valorization of the nettle leaves (Urtica dioica) as a novel source of a protease for clotting dromedary milk. The aim of this work is to study the effect of extracting pH on the enzymatic activity of nettle leaves extracts. The extraction was achieved in phosphate citrate buffer at different pH values (from 3 to 6.5) and the obtained extracts were used to coagulate dromedary milk. The characterization of the obtained extracts was carried out using non-destructive methods namely FT-MIR, fluorescence spectroscopy and turbiscan instrument. The extract prepared at pH = 4 had the highest proteolytic activity. The fluorescence and turbiscan measurements revealed a substantial effect of the pH value on chlorophyll residues extraction and stability, respectively. At an acidic environment (pH range of 3 – 4), the enzymatic extracts were unstable (with turbiscan stability index (TSI) values ~ 20), while at a nearly neutral pH value (pH range of 5 – 6.5), they were found to be more stable as indicated by the low TSI values ~ 1. The maximum milk-clotting activity (MCA) (0.021 U/mL) was obtained for the extracts prepared at pH = 4.