Intense Proteolysis Research Articles

Sourdough fermentation for the valorization of sorghum flour: Microbiota characterization and metabolome profiling

Due to a large adaptability to different cultivation conditions and limited input compared to other cereals, sorghum is considered an emerging crop. Its antioxidant properties, high fiber content and low glycemic index also make it a valuable addition to a healthy diet, nevertheless, the presence of antinutritional factors and the lack of gluten, hamper its use as food ingredient. This study investigated the impact of sourdough fermentation on sorghum nutritional quality. Lactic acid bacteria dominating sorghum flour and sourdough were identified by culture-dependent analysis revealing Lactiplantibacillus plantarum as the dominant species found in the mature sourdough, whereas Weissella cibaria and Weissella paramesenteroides were the species isolated the most after the first refreshment. Among yeasts, Saccharomyces cerevisiae was the most prevalent. Lactic acid bacteria pro-technological and functional performances as starter were evaluated in sorghum type-II sourdoughs through an integrated characterization combining chromatographic and NMR spectroscopic techniques. The metabolic profile of the strains mainly grouped together W. cibaria strains and W. paramesenteroides AI7 which distinguished for the intense proteolysis but also for the presence of compounds particularly interesting from a physiological perspective (allantoin, glutathione, γ-aminobutyric acid and 2-hydroxy-3-methylbutyric acid), whose concentration increased during fermentation in a species or strain specific matter.

Bioactive Peptides in Meat and Meat Products

A number of bioactive peptides with health benefits have been reported to be generated in meat through the proteolysis phenomena taking place during postmortem aging or further processing. Bioactive peptides consist of short sequences, less than 20 amino acid residues, that are inactive while in the parent protein. These sequences may be activated when they are released from the parent protein by peptidases. Such peptides are released through the hydrolysis of major muscle proteins by muscle endopeptidases, like calpains and cathepsins, and resulting protein fragments and polypeptides are further hydrolyzed by muscle exopeptidases, mainly, tri- and di-peptidylpeptidases, aminopeptidases, and carboxypeptidases. As a result, a variety of peptides with different sequences and lengths and large amounts of free amino acids are generated. The highest amounts of bioactive peptides, and their major health benefits, have been reported in dry-cured ham as a result of the intense proteolysis experienced during the long processing time under mild conditions that favor the action of muscle peptidases. The generated peptides must be bioaccesible and bioavailable to exert their physiological benefit.This means that they must be resistant to gastrointestinal digestion and be absorbed through the intestinal barrier. Some bioactive peptides may be generated through hydrolysis during gastrointestinal digestion. This review is focused on the generation of bioactive peptides in meat and processed meats, on the most relevant bioactivities exerted by such peptides(such as inhibition of angiotensin I–converting enzyme and dipeptidyl peptidase IV as well as antioxidant and anti-inflammatory activity), and on their reported benefits to consumers’health.

Proteomics in bovine semitendinosus muscle to assess emerging strategies based on papain injection and ultrasounds on meat tenderization process

The impact of papain and/or ultrasound treatments on tenderization of semitendinosus muscle through a proteomic approach was studied. Sixteen bovine muscles were submitted to the following treatments: aging at 3 °C (Control), papain injection (PI), ultrasound (US), PI followed by US (PIUS) and US followed by PI (USPI). pH, myofibrillar fragmentation indices (MFI), soluble collagen, texture profile and changes of myofibrillar proteins were investigated after 2, 24, 48 and 96 h of storage. The highest MFI and soluble collagen content were found in PI, PIUS and USPI samples while control samples showed the lowest values. PI samples showed the lowest WBSF and hardness values until 48 h of storage while at 96 h meat from USPI treatment showed WBSF value comparable to PI treatment. The lowest values of cohesiveness, gumminess and chewiness were found in PI samples during all storage times.Proteomic analysis revealed a different quantity and expression of proteins among tenderization treatments. US treatment did not exhibit a significant ability to degrade muscle proteins, while, all treatments containing papain, showed a greater ability to hydrolyse and degrade myofibrillar proteins. PI promoted intense proteolysis leading to an early tenderization process; on the contrary, in PIUS and USPI treatments the sequence of treatments was relevant on meat tenderization. Particularly, USPI treatment, after 96 h, reached the same improvement in tenderness of enzymatic treatment but with slower hydrolysing rate; this could be determinant to preserve textural structure.

Extraction and identification of bioactive peptides from Panxian dry-cured ham with multifunctional activities

Panxian dry-cured ham, a typical representative of traditional Chinese dry-cured ham, has a minimum time of ripening of 12 months, which results in intense proteolysis and the generation of numerous peptides. The purpose of this study was to measure the peptides with the activities of antioxidant, angiotensin-converting enzyme (ACE), and platelet-activating factor-acetulhydrolase (PAF-AH) inhibitory, from the Panxian dry-cured ham. The results showed that Panxian dry-cured ham extracts had antioxidant, ACE, and PAF-AH inhibitory activities, and these activities increased significantly with increasing concentrations of extracts (P < 0.05). The <3 kDa peptide fraction prepared by 3 kDa cutoff membrane had significantly stronger inhibitory activity, compared with 3–10 kDa and >10 kDa fractions (P < 0.05). It was identified for the peptide fractions of below 3 kDa to use Nano-LC-ESI-MS/MS. Totally, 66 peptides were identified containing 2–16 amino acid residues. Among these peptides, 22.7%, 15.2%, and 9.09% were derived from myosin, beta-enolase, and actin, respectively. The bioactive peptides of Panxian dry-cured ham, generated during long ripening processing, are positively contributing to human health.

Treatment With Simvastatin and Rifaximin Restores the Plasma Metabolomic Profile in Patients With Decompensated Cirrhosis.

Patients with decompensated cirrhosis, particularly those with acute‐on‐chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this study was to investigate the effect of treatment with simvastatin and rifaximin on plasma metabolites of patients with decompensated cirrhosis, specifically on compounds characteristic of the ACLF plasma metabolomic profile. Two cohorts of patients were investigated. The first was a descriptive cohort of patients with decompensated cirrhosis (n = 42), with and without ACLF. The second was an intervention cohort from the LIVERHOPE‐SAFETY randomized, double‐blind, placebo‐controlled trial treated with simvastatin 20 mg/day plus rifaximin 1,200 mg/day (n = 12) or matching placebo (n = 13) for 3 months. Plasma samples were analyzed using ultrahigh performance liquid chromatography–tandem mass spectroscopy for plasma metabolomics characterization. ACLF was characterized by intense proteolysis and lipid alterations, specifically in pathways associated with inflammation and mitochondrial dysfunction, such as the tryptophan–kynurenine and carnitine beta‐oxidation pathways. An ACLF‐specific signature was identified. Treatment with simvastatin and rifaximin was associated with changes in 161 of 985 metabolites in comparison to treatment with placebo. A remarkable reduction in levels of metabolites from the tryptophan–kynurenine and carnitine pathways was found. Notably, 18 of the 32 metabolites of the ACLF signature were affected by the treatment. Conclusion: Treatment with simvastatin and rifaximin modulates some of the pathways that appear to be key in ACLF development. This study unveils some of the mechanisms involved in the effects of treatment with simvastatin and rifaximin in decompensated cirrhosis and sets the stage for the use of metabolomics to investigate new targeted therapies in cirrhosis to prevent ACLF development.

Lactic Acid Bacteria Fermentation and Endopeptidase Treatment Improve the Functional and Nutritional Features of Arthrospira platensis.

This study aimed at investigating the effect of fermentation and enzymatic treatment on the degree of proteolysis of wet (WB), dried at low temperature (DB), and freeze-dried Spirulina (LB) proteins that affect the nutritional (e.g., amino acid content and profiles, and protein digestibility) and functional (e.g., antioxidant and antimicrobial activities) properties. The desiccation treatments influenced the unprocessed Spirulina characteristics because, compared with that in WB, peptides and free amino acids content was 73% lower in DB and 34% higher in LB. An integrated approach, including chromatographic and electrophoresis analyses, was used to evaluate the effect of the different bioprocessing options on protein profiles, release of peptides and amino acids, and the overall protein digestibility. Compared with the application of fermentation with the selected Lactiplantibacillus plantarum T0A10, the treatment with the endopeptidase Alcalase®, alone or combined, determined the most intense proteolysis. Moreover, the treatment with Alcalase® of LB allowed the release of potentially bioactive compounds that are able to inhibit Penicillium roqueforti growth, whereas the combination of fermentation with L. plantarum T0A10 and Alcalase® treatment increased Spirulina antioxidant properties, as determined by the scavenging activity toward ABTS radical (up to 60%) and antimicrobial activity against food pathogen Escherichia coli.

Aerobic stability of alfalfa silage and methods of its improvement

Alfalfa is a non-silage crop, the silage of which became possible after the effect of drying was detected. A special feature of alfalfa is the intensive proteolysis that occurs during silage, and the prolonged development of undesirable microflora due to the slow acidification of feed. The objective of the research was to determine the effectiveness of the use of Biotrof, Biotrof 111, Biotrof 2+ and Biotal Axfast NS Gold in the silage of dried (37.07% SV) alfalfa mass. It was found that the use of these drugs contributed to the rapid acidification of feed to a pH of 3.97-4.08, against a pH of 4.96 in silage without additives, which led to the suppression of the vital activity of undesirable bacteria, a decrease in the breakdown of nutrients to gaseous products by 1.7-2.3 times and the accumulation of ammonia by 1.5-4.0 times. Due to the high resistance of alfalfa silage to aerobic spoilage, the use of bacterial preparations did not lead to an improvement in the aerobic stability of the silage, but by restraining the development of some yeasts, including pathogenic ones, and fungi of the Aspergillus sp. species, it helped to improve the sanitary status of the feed. Yeasts of the genera C. gattii and D. hansenii serve as marker organisms, an increase in the number of which indicates the occurrence of aerobic spoilage in the feed.

Preliminary Study of Extended Ripening Effects on Peptides Evolution and DPPH Radical Scavenging Activity in Mexican Goat Cheese

Cheese ripening causes intense proteolysis, particularly when the cheese contains starter cultures. Several studies have shown the presence of bioactive peptides in goat’s milk cheeses with antioxidant activity. Mexican goat cheeses’ peptide fractions were evaluated at different ripening stages. Additionally, they were correlated with their antioxidant activity. Proteolysis was measured in the acid-soluble nitrogen and non-protein nitrogen fractions using reverse-phase high-performance liquid chromatography. While the antioxidant activity in both nitrogenous fractions was determined using a 2,2-diphenyl-1-picrylhydrazyl solution. Analyzed cheeses showed peptides fraction in the retention time of 2.05, 18.36, and 50.11 min for acid-soluble fraction and non-protein protein nitrogen, and showed antioxidant activity from the first day of ripening to 73% discoloration in the DPPH solution at 55 ripening days. Obtained results suggested that ripened Mexican goat cheese had a DPPH radical scavenging activity related to peptides present originally in the milk or released by starter culture action during cheese ripening.

Features of alfalfa fermentation depending on the dry matter content and silage method

Quite intensive proteolysis, accompanied by deamination of amino acids to ammonia, is observed during silage of alfalfa even when it is dried to a dry matter content of 31-36%. As a result, the feed can accumulate up to 0.5% of ammonia in the dry matter of the feed. Our research has shown that the ammonia content depends more on the degree of drying than on the level of acidification of alfalfa silage. And reaches a minimum at 50-55% dry matter. Drying increases both the concentration of water-soluble carbohydrates (1.3 times) and the amount of malic acid (up to 4% per day), which can later be used by lactic acid bacteria acidifying the feed to a pH of 4.3-4.4. Preparations based on homofermentative lactic acid bacteria have shown themselves more effectively in silage of dried alfalfa to a dry matter content of 40-45%.

Impact of Enzymatic and Microbial Bioprocessing on Antioxidant Properties of Hemp (Cannabis sativa L.).

Although the hemp seed boasts high nutritional and functional potential, its use in food preparations is still underestimated due to scarce technological properties and the presence of several anti-nutritional factors. Here, an optimization of a biotechnological protocol aimed at improving the antioxidant properties and the protein digestibility of the whole hemp seed has been proposed. Processing based on the use of commercial food grade enzymes and ad hoc selected lactic acid bacteria was tested and the phenolic and protein profiles were investigated through an integrated approach including selective extraction, purification, and identification of the potentially active compounds. The influence of the bioprocessing on the antioxidant activity of the hemp was evaluated both in vitro and on human keratinocytes. The lactic acid bacteria fermentation was the best method to significantly improve the antioxidant potential of the hemp through intense proteolysis which led to both the release of bioactive peptides and the increase in the protein digestibility. Moreover, changes in the phenolic profile allowed a significant protective effect against oxidative stress measured on the human keratinocyte cell line.

The effects of curing agents on the proteolysis and lipid oxidation of pastırma produced by the traditional method.

In the study, the effects of nitrate and/or nitrite (150mg/kg KNO3, 300mg/kg KNO3, 150mg/kg NaNO2, and 150mg/kg KNO3 + 150mg/kg NaNO2) on proteolytic changes (free amino acid composition, SDS-PAGE) in pastırma were investigated. Pastırma samples were also analyzed in terms of some qualitative (pH, aw, TBARS, residual nitrite, salt) properties. The lowest total free amino acid content (1818.3mg/ 100g DM) was observed in the combination of 150mg/kg KNO3 + 150mg/kg NaNO2, while the highest content (2847.49mg/ 100g DM) was observed in 150mg/kg KNO3. Although the pastırma groups generally exhibited similar SDS-PAGE profiles, differences were detected at some band intensities. The lowest TBARS value (22.24μmol MDA/kg) was observed in 150mg/kg KNO3 + 150mg/kg NaNO2. As a result, the use of 150mg/kg KNO3 in the pastırma curing process causes more intense proteolysis.

Gastrointestinal Interaction between Dietary Amino Acids and Gut Microbiota: With Special Emphasis on Host Nutrition.

The gastrointestinal tract (GIT) of humans and animals is host to a complex community of different microorganisms whose activities significantly influence host nutrition and health through enhanced metabolic capabilities, protection against pathogens, and regulation of the gastrointestinal development and immune system. New molecular technologies and concepts have revealed distinct interactions between the gut microbiota and dietary amino acids (AAs) especially in relation to AA metabolism and utilization in resident bacteria in the digestive tract, and these interactions may play significant roles in host nutrition and health as well as the efficiency of dietary AA supplementation. After the protein is digested and AAs and peptides are absorbed in the small intestine, significant levels of endogenous and exogenous nitrogenous compounds enter the large intestine through the ileocaecal junction. Once they move in the colonic lumen, these compounds are not markedly absorbed by the large intestinal mucosa, but undergo intense proteolysis by colonic microbiota leading to the release of peptides and AAs and result in the production of numerous bacterial metabolites such as ammonia, amines, short-chain fatty acids (SCFAs), branched-chain fatty acids (BCFAs), hydrogen sulfide, organic acids, and phenols. These metabolites influence various signaling pathways in epithelial cells, regulate the mucosal immune system in the host, and modulate gene expression of bacteria which results in the synthesis of enzymes associated with AA metabolism. This review aims to summarize the current literature relating to how the interactions between dietary amino acids and gut microbiota may promote host nutrition and health.

Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF

Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. Therefore, we aimed to analyze the blood metabolome in patients with cirrhosis, with and without ACLF. We performed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 650 patients with AD (acute decompensation of cirrhosis, without ACLF), 181 with ACLF, 43 with compensated cirrhosis, and 29 healthy individuals. Of the 137 annotated metabolites identified, 100 were increased in patients with ACLF of any grade, relative to those with AD, and 38 comprised a distinctive blood metabolite fingerprint for ACLF. Among patients with ACLF, the intensity of the fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that the fingerprint reflected not only decreased kidney excretion but also altered cell metabolism. The higher the ACLF-associated fingerprint intensity, the higher the plasma levels of inflammatory markers, tumor necrosis factor α, soluble CD206, and soluble CD163. ACLF was characterized by intense proteolysis and lipolysis; amino acid catabolism; extra-mitochondrial glucose metabolism through glycolysis, pentose phosphate, and D-glucuronate pathways; depressed mitochondrial ATP-producing fatty acid β-oxidation; and extra-mitochondrial amino acid metabolism giving rise to metabotoxins. In ACLF, intense systemic inflammation is associated with blood metabolite accumulation and profound alterations in major metabolic pathways, in particular inhibition of mitochondrial energy production, which may contribute to the development of organ failures. Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. We identified a 38-metabolite blood fingerprint specific for ACLF that revealed mitochondrial dysfunction in peripheral organs. This may contribute to organ failures.

Lactobacillus rhamnosus GG improves the sensorial profile of Camembert-type cheese: An approach through flash-profile and CATA

Camembert is a white-mould ripened cheese in which intense proteolysis, caused mainly by the fungus Penicillium candidum, among other factors, leads to a substantial production of aromatic and flavor compounds. The aim of this study was to evaluate the sensory profile of the Camembert-type cheese made from raw milk and from pasteurized milk with or without the addition of Lactobacillus rhamnosus GG. The cheeses were analyzed after 50 days of ripening through a combination of Flash-Profile (n = 15) and Check-All-That-Apply (CATA) (n = 80) sensory methods with global acceptance. The 51 sensory attributes obtained allowed the separation of the samples in three clusters. Through the CATA, a significant difference (p < 0.05) was observed for 21 of the 51 descriptors evaluated. Camembert-type cheese obtained from pasteurized milk with the addition of Lactobacillus rhamnosus GG showed greater global acceptance when compared to the other cheeses, which did not significantly differ between them. The addition of the adjunct culture was a good technological strategy to improve the sensory characteristics of industrial Camembert-type cheese obtained from pasteurized milk, bringing its sensory attributes closer to the ideal product and the sensorial characteristics of a cheese produced with raw milk, but in a standardized and microbiologically safe way.

Bioactive peptides and free amino acids profiles in different types of European dry-fermented sausages

A wide variety of dry-fermented sausages are produced in European countries, where are considered valued traditional products. An intense proteolysis takes place during the processing of dry-fermented sausages due to the combined action of muscle and microbial peptidases, generating large amounts of peptides and free amino acids. These compounds participate in the development of the characteristic flavor of dry-fermented products, but some peptides can also exert certain bioactivities such as antioxidant and ACE inhibitory activities. This study has evaluated the changes in peptide profile and amino acid contents of three European dry-fermented sausages produced in Spain, Italy and Belgium, proving the intense degradation of proteins, mainly myofibrillar, and the generation of high amounts of different size peptides and free amino acids. The changes observed between the profiles of European sausages could be due to differences in product formulation, processing conditions and starter cultures used, which influence the activity of enzymes, both from muscle and bacterial origin. On the other hand, the bioactivity profile of each type of dry-fermented sausage was evaluated through the measurement of the ACE inhibitory and antioxidant activities in water-soluble peptide extracts fractionated by size-exclusion chromatography. Spanish and Belgian dry-fermented sausages showed values of ACE inhibition around 85%, whereas Belgian samples presented the highest DPPH radical-scavenging activity and ferric reducing power capacity. These results evidence the potential of Spanish, Italian and Belgian dry-fermented sausages as natural sources of bioactive peptides, giving an added-value to these traditional products.

Vacuolar Targeting and Characterization of Recombinant Antibodies.

Plant-based platforms are extensively use for the expression of recombinant proteins, including monoclonal antibodies (mAbs). Generally, immunoglobulins (Igs) are sorted to the apoplast, which is often afflicted with intense proteolysis. Here, we describe methods to transiently express mAbs sorted to central vacuole in Nicotiana benthamiana leaves and to characterize the obtained IgG. Central vacuole is an appropriate compartment for the efficient production of Abs, consequently vacuolar sorting should be considered as an alternative strategy to obtain high protein yields.

Effect of milk origin on proteolysis and accumulation of biogenic amine during ripening of Dutch-type cheese

Dairy products from goat's milk are characterized by their distinctive aroma and their specific taste. However, the strong aroma can discourage some consumers. Properties of cheese can be modified by the combination of goat's and cow's milk. On the other hand, chemical diversity from different milk origin may affect the changes during ripening. The aim of the study was to compare the intensity of changes during ripening of model cheese samples produced with various ratios of goat's and cow's milk. The combinations 100:0 (100% goat's milk), 75:25, 50:50, 25:75 and 0:100 (100% cow's milk) were used for the manufacture of Dutch-type cheeses, which were ripened during a period of 84 days. Protein profile, free amino acid content and biogenic amine content were used for the description of cheese properties during storage. Cluster analysis showed different changes in the protein matrix of the examined samples. The results indicated that even low addition of cow's milk significantly affected the protein profile. However, the homology of protein profiles rose with the increasing ripening time. More intensive proteolysis occured in the samples with predominance of goat's milk. Moreover, cheese samples produced only from goat's milk presented a significant increase inthe amount of free amino acids after 14 days of ripening. The effect of milk origin on the production of biogenic amines was also examined. However, higher concentrations of biogenic amines were detected in samples manufactured from goat's milk. Tyramine, putrescine, histamine and phenylethylamine were detected during the storage of the samples. The total biogenic amine content exceeded 100 mg/kg in samples with predominance of goat's milk.

Proteolysis during fermentation of Suanyu as a traditional fermented fish product of China

ABSTRACTProteolytic changes in Suanyu, a traditional fermented fish product of China, were determined during 6 weeks of fermentation. The gradual decrease of pH value and aw was detected, accompanied with an increasing trend detected in trichloroacetic acid–soluble C-1: peptides concentration. Protein composition changed throughout the fermentation. A decrease in sarcoplasmic and myofibrillar protein fractions was accompanied by an increase in the insoluble fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed most bands of sarcoplasmic, myofibrillar, and insoluble proteins lightened or disappeared due to proteolysis or denaturation. New bands emerged especially in insoluble proteins. Fluctuation of molecular weight distribution of the water-soluble nitrogen in Suanyu was found during fermentation. The highest percentage in Suanyu end-products was observed in the fraction of <0.18 kDa, followed by 0.18–0.5 kDa, 66.02 ± 0.38% and 10.79 ± 0.27%, respectively. Free amino acids analysis showed that nearly all amino acids increased, especially for Asp, Glu, Met, Phe, Leu, Val, and Ile. Overall, intense proteolysis in Suanyu brought numerous of small peptides and free amino acids, which contribute to the taste and flavor of Suanyu.

Volatile compounds associated with desirable flavour and off-flavour generation in ewe´s raw milk commercial cheeses

This study investigated relationships between the volatile profile and balanced flavour of high sensory quality ewe’s raw milk commercial cheeses, and those relations between certain volatile compounds and off-flavours in defective cheeses. Commercial cheeses were selected in the routine sensory quality control from 27 farmhouses. High sensory quality cheeses showed a particular desirable balanced flavour and volatile profile. However, an imbalanced composition of volatile compounds was found in off-flavour cheeses. Rancid off-flavour was mainly related to an excessive concentration of n-butanoic and n-hexanoic acids, and their corresponding ethyl esters, generated by strong lipolysis during ripening. Animal off-flavour was associated with the generation of 3-methylbutan-1-ol, 2-phenylethanol and 4-methylphenol originated by amino acid catabolism after intense proteolysis. Finally, acid off-flavour was related to an excessive abundance of acetic acid generated mainly by strong glycolysis at early stages of ripening.

White cheeses as a potential source of bioactive peptides

In addition to the nutritional value, cheese and other fermented milk-based products are a good source of biologically active proteins and peptides. Bioactive peptides in cheese are mainly formed during cheese production, especially during ripening, which is one of the most important stages in cheese production. Since the ripening process differ significantly depending on the cheese type, it can be assumed that their bioactivity is also substantially different. White brined cheeses are specific for the region of South-Eastern Europe. They are characterized by relatively intensive proteolysis and as such may be of interest as products with favorable functional characteristics. Yet, their functionality is still insufficiently characterized. This paper presents an overview of the current knowledge on white brined cheese’s bioactive peptides.