Studies on the role of exogenous proteolytic enzymes in digestion processes in fish

Studies were carried out on the proteolytic activity of the fry of common carp, rainbow trout, grass carp, and whitefish, as well as on the activity of digestive organs of adult common carp and rainbow trout. Activity of exogenous enzymes in relation to endogenous ones was assessed on the basis of the proteolytic value of fish food and the activity of digestive organs. It was found that the share of proteolytic enzymes of natural food in the digestion process in fish was high. Beginning from a weight of 50–100 g for common carp and 10 g for rainbow trout, the relation between the daily enzymatic ration and the weight of fish indicates the cooperation of an approximately constant amount of exogenous enzymes.     

Studies on the proteolytic enzymes of invertebrates constituting fish food

Studies were carried out on the proteolytic activity of trypsin and pepsin-like enzymes of invertebrates consituting fish food. Relations between proteolytic activity of enzymes, pH, and temperature were established.     

Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes

Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and flavor enhancers in food, beverage and pharmaceutical industries. Antioxidants from fishery resources have attracted the attention of researchers as they are cheaper in cost, easy to derive, and do not have side effects. Thus the present investigation was designed to produce protein hydrolysate by pepsin and papain digestion from the backbones of Rastrelliger kanagurta (Indian mackerel) and evaluate its antioxidant properties through various in vitro assays. The results reveal that both hydrolysates are potent antioxidants, capable of scavenging 46% and 36% of DPPH (1,1-diphenyl-2 picrylhydrazyl) and 58.5% and 37.54% of superoxide radicals respectively. The hydrolysates exhibit significant (p < 0.05) reducing power and lipid peroxidation inhibition. Among the two hydrolysates produced, pepsin derived fraction is superior than papain derived fraction in terms of yield, DH (Degree of hydrolysis), and antioxidant activity.     

Cloning of fish enzymes and other fish protein genes

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

In vitro culture of bovine egg fertilized either in vivo or in vitro

Three-quarters of in vivo and one-third of in vitro fertilized bovine eggs reached blastocyst stage when cultured on tubal cell monolayers (TCM), but no hatching occurred in B2 medium supplemented with estrous cow serum. When after 3 days of culture on TCM, morulae were transferred on endometrial cell monolayers (UCM), the same proportion of blastocysts was obtained and one-third of them hatched. Histological studies of hatched blastocysts showed that the number of inner cells was significantly lower than in hatched blastocysts recovered in vivo 8-8.5 days after ovulation. Moreover, the number of pycnotic cells was higher than normal, although mitosis were present. On the contrary, there was no difference in either the number or the appearance of trophoblastic cells between blastocysts obtained in vitro and in vivo. The addition of transforming growth factor (TGF-beta) to either TCM or UCM co-cultures at the very beginning of blastocyst formation specifically stimulated growth of the inner cell mass (ICM). The number of cells at hatching was about double (120) and significantly higher than that found in 8-8.5-day blastocysts in vivo. Moreover, hatching percentages were similar to the controls, even when eggs were cultured for 8 days only on TCM. However the proportion of pycnotic cells remained higher than normal, although many mitotic cells were unevenly distributed in ICM) In vivo during hatching, there were always pycnotic cells in ICM, but their number was limited and approximately similar to the number of mitosis. The uterine factors which control both mitosis and pycnosis in ICM remain to be discovered.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

Effective extraction of astaxanthin pigment from shrimp using proteolytic enzymes

The present study was to investigate efficient extraction conditions of astaxanthin from shrimp wastes for utilizing it as a functional food additive. In order to enhance the stability of pigments, proteolytic enzymes were applied to extract astaxanthin as carotenoprotein. Also, various conditions such as acid ensilaging of samples, using EDTA solution and adding various enzymes were examined to optimize extraction processing. After extraction, all of the extracts were partitioned in a separate funnel and each astaxanthin content was analyzed by using UV/VIS spectrophotometer. Carotenoprotein was effectively extracted from non-acid ensilaged shrimp wastes by using EDTA medium and a proteolytic enzyme. In that case, the reddish top layer showed 91.9% of recovery and the blackish bottom layer did 2.3% and its separation ratio was about 0.2 (v/v); therefore concentration and purification of reddish top layer were more desirable than those of whole extract.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

The role of proteolytic enzymes in protein degradation during senescence of rice leaves

The role of proteolytic enzymes in protein degradation of detached and intact leaves of rice seedling ( Oryza sativa L. cv. Taiching Native 1) during senescence and of mature leaves during reproductive development was investigated. The amount of soluble protein decreased by about 50% in 2, 4, and 15 days for detached, intact and mature leaves, respectively. Three proteolytic enzyme activities were monitored with pH optima of 4.5 for hemoglobin-digesting proteinase, 5.5 for carboxypeptidase and 8.0 for aminopeptidase. No azocoll-digesting proteinase activity could be detected in rice leaves. Dialysis did not alter the activities of any of the three proteolytic enzymes. Acid proteinase activity and aminopeptidase activity were highly unstable during storage of the enzyme extracts at 4°C. Proteolysis was stimulated by inclusion of meroaptoethanal either in the extraction medium or the assay medium.
Acid proteinase, carboxypeptidase and aminopeptidase were all present in detached, intact and mature leaves throughout senescence. There seems to be a direct correlation between protein degradation and increases of acid proteinase and carboxypeptidase activity in seedling leaves (detached and intact) during senescence. In senescing (detached and intact) leaves of seedlings the acid proteinase activity developed first, while that of carboxypeptidase developed later. Acid proteinase and carboxypeptidase may play major roles in protein degradation of leaves from seedlings during senscence. During reproductive development, protein degradation was associated with decreases in the activities of acid proteinase, carboxypeptidase and aminopeptidase in mature leaves suggesting that the enzymes were less important for protein degradation in this system. Hence, the role of protelytic enzymes in protein degradation during senescence of rice leaves appears to depend largely on the leaf system used.