Toxicity evaluation of collagen hydrolysates from chrome shavings and their potential use in the preparation of amino acid fertilizer for crop growth

Resource utilization of chrome shavings (CS) has attracted a lot of attention from scientists and technologists in leather industry. Especially, the collagen hydrolysates extracted from CS are expected to find potential application values in agricultural field. However, there is no biotoxicity analysis of collagen hydrolysates from CS. Herein, the collagen hydrolysates with different molecular weights were produced from CS by three hydrolysis dechroming methods including alkaline hydrolysis, enzymatic hydrolysis and alkaline-enzymatic synergistic hydrolysis, and the optimal hydrolysis process of CS was designed and conducted. To evaluate their toxicity, the three collagen hydrolysates were formulated into a nutrient solution for zebrafish development. The obtained results indicated that the hydrolysates with low concentrations (less than 0.6 mg/mL) were safe and could promote the development for zebrafish embryos. Furthermore, the three collagen hydrolysates were utilized as organic nitrogen sources and formulated into amino acid water-soluble fertilizers (AAWSF) including alkaline type fertilizer (OH), enzymatic type fertilizer (M) and alkaline-enzymatic type fertilizer (OH–M) for the early soilless seeding cultivation of wheat, soybean and rapeseed. It is worth mentioning that the chromium contents in the prepared AAWSF were less than 10 mg/kg, which is far less than the limit value in the standard (China, 50 mg/kg). The growth and development of seedlings (germination rate, plant height, fresh weight of leaves, soluble sugar content and chlorophyll content) were investigated. The corresponding results showed that the growth of seedlings watered with AAWSF was better compared with the other treatments, and the OH–M fertilizer had the best promoting effect on the seedlings growth and development, followed by the M and OH fertilizers. The safe toxicity assessment of the collagen hydrolysates will expand their application scope, and the use of collagen hydrolysates extracted from CS for seedlings growth also provides an effective and reasonable way to deal with the chromium-containing leather solid waste, which is an effective way to realize its resource utilization. Graphical Abstract

Effects of type II collagen hydrolysates on osteoarthritis through NF-ĸB, Wnt/β-catenin and MAPK pathways

Osteoarthritis (OA), a degenerative disease, has attracted extensive attention all over the world. In the study, medial meniscus resection (MMx) and anterior to medial collateral ligament (ACL) operation rat model...

Yield results obtained in maize crop following the foliar fertilization with new biostimulators based on keratin and collagen hydrolysates of animal origin

The aim of the paper is to show the reaction of maize crop to the foliar application of new biofertilizers, obtained from the residues left after the wool tanning process and which are based on protein hydrolysates of keratin and collagen. The tests have been performed in microplots (10 sqm) and macroplots (1000 sqm), in three repetitions, in southern Romania. All data obtained as a result of the 2020 autumn harvest have been centralized in tables and statistically processed, in order to establish the influence of each product, compared to the control. The bifactorial analyses show that, in climatic stress conditions (prolonged drought – Calarasi), very significant increases in yield have been obtained with K4 (2.5 and 5.0 l/ha), K5 (5.0 l/ha) and C (5.0 l/ha), with increases between 700 and 1300 kg/ha, ie up to +24%. In macroplots, subject to a moderate drought (Teleorman), product C (5.0 l/ha) brought a yield increase of 1537 kg/ha (+26%), noting that in this location almost all tested biostimulators offer very significant yield growth (except KC). Based on the 2020 yields, it was possible to identify which the foliar biostimulators that will continue to be tested in 2021.

FUNCTIONAL PROPERTIES OF CATFISH SKIN COLLAGEN HYDROLYSATES

Collagen hydrolysates were obtained from catfish skin collagen hydrolysis using catfish collagenase, pepsin and trypsin individually and mixed for 15-300min. The degree of hydrolysis , antioxidant activity based on, DPPH radical-scavenging activity (RSA), and reducing power (RP) for all obtained hydro lysates were studied , then the collagenase hydrolysate was (CH) was selected to be evaluated for antibacterial activity , functional properties including solubility, emulsification and foaming properties besides the toxicity . The highest values for RSA (72.5%) has been noticed when DH reached (24.30%) after 30 min. of hydrolysis by collagenase (CH). The water and oil holding capacities for this hydro lysate was compared to that for ASC (acid soluble collagen and PSC (pepsin soluble collagen), the results showed that the values recorded by CH were significantly higher than ASC & PSC. The molecular weight of CH peptides ranged from 180 to 11 Da. as analyzed by SDS- PAGE. The toxicity assay result revealed that CH is safe for human consumption. There was no antibacterial function for CH toward E.coli and S. aureus. At concentration of 2mg/ml. This study suggests that catfish collagen hydro lysate could be a good natural alternative for synthetic antioxidants in food industries.

Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS. Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis

High molecular weight (Mw) collagen hydrolysates have been demonstrated to produce a higher synthesis of collagen type I mRNA. Mw determination is a key factor maximizing the effect of collagen hydrolysates on collagen type I synthesis by fibroblasts. This work aimed to achieve a high average Mw in Blue Shark Collagen Hydrolysate, studying different hydrolysis parameters by GPC-LS analysis and testing its effect on mRNA Type I collagen expression. Analysis revealed differences in blue shark collagen hydrolysates Mw depending on hydrolysis conditions. Papain leads to obtaining a significantly higher Mw hydrolysate than Alcalase at different times of hydrolysis and at different enzyme/substrate ratios. Besides, the time of the hydrolysis factor is more determinant than the enzyme/substrate ratio factor for obtaining a higher or lower hydrolysate Mw when using Papain as the enzyme. Contrary, Alcalase hydrolysates resulted in similar Mw with no significant differences between different conditions of hydrolysis assayed. Blue shark collagen hydrolysate showing the highest Mw showed neither cytotoxic nor proliferation effect on fibroblast cell culture. Besides, it exhibited an increasing effect on both mRNA expression and pro-collagen I production.

Comparison of effectiveness of various bacterial proteases preparations in collagen hydrolysis

Гидролизаты коллагена широко используются в пищевой, фармацевтической и косметической промышленности. Для обработки коллагена используют различные протеолитические ферменты растительного, животного и микробного происхождения, из которых самыми перспективными являются бактериальные протеазы. Наиболее известные и часто используемые в пищевой отрасли коммерческие препараты бактериальных протеаз различаются по компонентному составу и могут содержать сериновые, металлопротеазы либо их смесь. С целью определения эффективности препаратов бактериальных протеаз с различным компонентным составом при получении гидролизатов коллагена мы провели ферментативную обработку говяжьего коллагена с использованием коммерческих препаратов Alcalase, Neutrase, Protamex и двух образцов ферментных препаратов, полученных в лабораторных условиях на основе новых отечественных штаммов Bacillus. Эффективность гидролиза оценивали по накоплению аминного азота, содержанию низкомолекулярного белка и по интенсивности белковых полос на электрофореграммах полученных продуктов. Все исследуемые препараты обеспечивали эффективный гидролиз коллагена. Наиболее интенсивно коллаген расщепляли препараты, содержащие только сериновые протеазы: Alcalase и ФП-145, полученный из культуральной жидкости штамма B. licheniformis-145. Высокую эффективность показали коммерческий препарат Protamex и ФП-96, полученный на основе мутантного варианта штамма B. subtilis-359 - продуцента субтилизина BPN’ и бациллолизина. Наименьший выход аминного азота и низкомолекулярных белков наблюдался в варианте с препаратом Neutrase, протеолитическая активность которого полностью представлена действием нейтральной протеазы бациллолизина. Лабораторные образцы ферментных препаратов, полученные на основе новых отечественных продуцентов, не уступали зарубежным коммерческим аналогам, а ФП-96 обеспечил более высокий выход аминного азота и низкомолекулярных белков в сравнении с широко используемым в пищевой промышленности препаратом Protamex. Collagen hydrolysates are widely used in the food, pharmaceutical and cosmetic industries. For collagen hydrolysis, various proteolytic enzymes of plant, animal and microbial origin are used, of which the most promising are bacterial proteases. The most well-known and commonly used in the food industry commercial preparations of bacterial proteases differ in their component composition and may contain serine protease, metalloproteases, or their mixture. In order to determine the effectiveness of bacterial protease preparations with different component composition in obtaining collagen hydrolysates, we carried out hydrolysis of beef collagen using commercial preparations Alcalase, Neutrase, Protamex and two laboratory samples of enzyme preparations obtained from new domestic Bacillus strains. The efficiency of hydrolysis was assessed by amine nitrogen accumulation, low-molecular-weight protein content, and by the intensity of the protein bands on the electrophoregrams of the hydrolysates. All investigated preparations provided effective collagen hydrolysis. Collagen was hydrolyzed most intensively by preparations containing only serine proteases: Alcalase and FP-145, obtained from B. licheniformis-145 culture liquid. The commercial preparation Protamex and FP-96, obtained from a mutant variant of the B. subtilis-359 strain, a producer of subtilisin BPN' and bacillolysin, showed high efficiency. The lowest yield of amine nitrogen and low molecular weight proteins was observed when using Neutrase, which contains only the neutral protease bacillolysin. Laboratory samples of enzyme preparations obtained from the new domestic producers were not inferior to foreign commercial counterparts, and FP-96 provided a higher yield of amine nitrogen and low molecular weight proteins in comparison with Protamex, which is widely used in the food industry.

Assessment of Bioavailability after In Vitro Digestion and First Pass Metabolism of Bioactive Peptides from Collagen Hydrolysates

Collagen hydrolysates (CHs) are composed of bioactive peptides (BAPs), which possess health enhancing properties. There is a knowledge gap regarding the bioavailability of these BAPs that involves intestinal transport and hepatic first pass effects. A simulated gastrointestinal model was used to generate digesta from two CHs (CH-GL and CH-OPT), which were applied to a novel transwell co-culture of human intestinal epithelium cell line-6 (HIEC-6) and hepatic (HepG2) cells to simulate in vivo conditions of absorption and first pass metabolism. Peptide transport, hepatic first pass effects, and bioavailability were determined by measuring BAPs (Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, Gly-Pro-Hyp) using an innovative capillary electrophoresis method. All peptides were transported across the intestinal cell layer to varying degrees with both CHs; however, Gly-Pro-Hyp was transported only with CH-GL, but not CH-OPT. Notable hepatic production was observed for Ala-Hyp with both CH treatments, and for Pro-Hyp and Gly-Pro with CH-GL only. All peptides were bioavailable (>10%), except for Gly-Pro-Hyp after CH-OPT. Overall, a high degree of transport and hepatic first pass effects on CH-derived BAPs were observed. Further research is needed to explore the hepatic mechanisms related to the production of BAPs and the bifunctional effects of the bioavailable BAPs noted in this study.

Efficacy of Chondroprotective Food Supplements Based on Collagen Hydrolysate and Compounds Isolated from Marine Organisms

Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.