Olimpia Carrillo

Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates.

Studies on enzymatic hydrolysis of cell proteins in green microalgae Chlorella vulgaris 87/1 are described. Different proteases can be used for production of hydrolysates from ethanol extracted algae. The influence of reaction parameters on hydrolysis of extracted biomass with pancreatin was considered, and the composition of hydrolysates (Cv-PH) was investigated in relation to the starting materials. Significant changes in the degree of hydrolysis were observed only during the first 2h and it remained constant throughout the process. An enzyme-substrate ratio of 30-45 units/g algae, an algae concentration of 10-15% and pH values of 7.5-8.0 could be recommended. Differences in the chromatographic patterns of Cv-PH and a hot-extract from Chlorella biomass were observed. Adequate amounts of essential amino acids (44.7%) in relation to the reference pattern of FAO for human nutrition were found, except for sulfur amino acids. Cv-PH could be considered as a potential ingredient in the food industry.

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

In vitro digestibility of dietary protein sources for white shrimp (Penaeus vannamei)

In vitro methods to evaluate protein digestibility are rapid and allow close observation of the peptide bonds cleaved while using only small amounts of raw materials. The pH-stat assay using an enzyme preparation from shrimp hepatopancreas, recently developed by us was used to evaluate the degree of hydrolysis (DH) and estimated protein quality in menhaden, anchovy, white fish, tuna waste, soybean protein, and langostilla meals at 23, 25, and 27°C. Data were obtained by the pH-stat method and were compared with those obtained by the pH-drop method and in vivo assays. The arginine content of the meals was compared with shrimp growth and DH by the pH-stat method. The test showed no significant differences between DH obtained at 23, 25, and 27°C. The pH-stat method predicted protein digestibility better than the pH-drop method. A significant correlation between DH and apparent protein digestibility (APD) was observed. Although a significant correlation between the pH-drop method and APD was observed, the value was low. Arginine showed a significant correlation with shrimp growth but not with APD. The relationship between DH by pH-stat at 25 and 27°C and growth, although significant, had a low correlation. The equations obtained from DH had a very high predictive ability. The standard error in percentage was low. The pH-stat assay has a potential for the estimation of growth and digestibility of shrimp feed.

Isolation and biological characterization of a 6-kDa protein from hepatopancreas of lobster Panulirus argus with insulin-like effects

A protein with insulin-like effects was isolated from the hepatopancreas of the lobster Panulirus argus following a classic method for mammalian insulin purification from the pancreas. After acid-alcoholic extraction and ethanol-ether precipitation followed by molecular filtration chromatography, a protein with an apparent molecular weight of 6 kDa was isolated. This protein is characterized by its ability to interact with anti-insulin antibodies and by mimicking insulin actions as the stimulation of glucose oxidation to CO(2) and lipogenesis in isolated rat adipocytes. In addition, this insulin immunoreactive protein (IIP) was able to stimulate the autophosphorylation of the insulin receptor present in rat adipocyte plasma membranes, in a dose-dependent manner. The immunological and biochemical results obtained are consistent with the hypothesis that protein(s) with insulin-like effects occur in the digestive gland of the lobster P. argus and may be of significance to control metabolic and growth related processes in crustaceans.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways

SUMMARY The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency.

Oral Administration of an Enzymatic Protein Hydrolysate from the Green Microalga Chlorella vulgaris Enhances the Nutritional Recovery of Malnourished Mice

This study examined the effects of oral administration of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris (Cv-PH) on the nutritional recovery of malnourished Balb/c mice after a 3-day fasting period. Mice were refed with commercial diet supplemented or not supplemented with Cv-PH (500 mg/kg) for 8 days. Regardless of the diet used during refeeding, animal body weights and serum protein concentrations did not differ between groups. Mice given Cv-PH had a significant increase in hemoglobin concentrations. Most serum amino acid levels were similar in the control and Cv-PH animals. Starved mice refed with Cv-PH showed normal liver functions, as judged by liver weight, protein concentration, and the enzymatic activities of cholinesterase and arginase. Cv-PH increased DNA, protein content, and gut-mucosal weight. In addition, brush-border oligosaccharidase activities were also higher in the Cv-PH group. These findings suggest that Chlorella protein hydrolysate can be used to develop specific formulations suitable for pharmacologic nutrition.

Effect of starvation and refeeding on biochemical and immunological status of Balb/c mice: an experimental model of malnutrition.

Context: Although new methods for the induction of malnutrition disorders in laboratory animals have been developed, the bulk of the models described in the literature are essentially based on dietary restriction/starvation principle. In this context, little data are available about the metabolic and the immune system parameters of Balb/c mice under starvation/refeeding. Objective: This study examined the effects of starvation and refeeding on the biochemical and immunological status of undernourished Balb/c mice. Methods: Female Balb/c mice, weighing 20 g, were starved for 3 days and then refed with commercial pelleted diet for 8 days. The variables considered were as follows: body weight; serum protein and amino acid concentrations; liver protein content, and cholinesterase and arginase activities; jejunal protein and DNA contents as well as oligosaccharidase levels; hematological parameters (bone marrow and peripheral blood cellularity); peritoneal macrophage activation; and humoral and cell-mediated immune functions. Results: Profound alterations in both biochemical and immunological conditions appeared after the starvation period. Refeeding resulted in the normalization of serum albumin levels, the intestinal DNA content and the gut-mucosal associated enzymatic activities, the blood lymphocyte counts, and the number of peritoneal macrophages. The markers of liver metabolic function (cholinesterase and arginase activities), and those of bone marrow hemopoiesis and the adaptive immune response (T-dependent antibody titres and delayed-type hypersensitivity response) remained altered after refeeding compared with control mice. Conclusion: These findings suggest that fasted mice can be used as an animal model of acute starvation that might prove useful in evaluating the effectiveness of nutritional and immunopharmacological interventions.

A Holistic View of Dietary Carbohydrate Utilization in Lobster: Digestion, Postprandial Nutrient Flux, and Metabolism

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2–6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6–12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

Ileal and total digestibility studies in pigs fed molasses type A or starch diets supplemented with torula yeast or soybean meal

Abstract A total of 48 pigs of −40 kg liveweight were used for digestibility studies with molasses Type A, molasses plus torula yeast or plus soybean meal, and cassava starch plus torula yeast. Half of the pigs were prepared with re-entrant cannulas in the terminal ileum and the other half were maintained intact for conventional digestibility studies. Nitrogen-free extract was found to be the main undigested fraction: 39% for molasses Type A and 52–67% for the remaining diets. The ileal digestibility of soluble carbohydrates in molasses diets was 96% which indicated that the undigested nitrogen-free extract fraction was mainly compounds of non-identified organic substances contained in molasses Type A. Energy disappearance in the large intestine accounted for 12% of that digested in the entire gastrointestinal tract in the case of molasses Type A and 18–20% in the other diets. Energy excretion in urine was significantly greater in molasses diets than in those containing starch, indicating a lower metabolizable to gross energy ratio in molasses diets than in starch diets (0.85 vs. 0.90). Digestible and metabolizable energy values for molasses Type A were 14.3 and 13.5 MJ kg −1 dry matter (DM) respectively, and for cassava starch diet 16.8 and 16.6 MJ kg − DM, respectively. According to the high ileal digestibility found in carbohydrates of molasses Type A, the low ileal and total digestibility of non-identified organic substances considerably reduced the dietary energy density, thus acting as an index for the nutritional value of molasses.