Expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of temporomandibular joint inflammation

Orofacial pain is a prevalent symptom in modern society. Some musculoskeletal orofacial pain is caused by temporomandibular disorders (TMDs). This condition has a multi-factorial etiology, including emotional factors and alteration of the masticator muscle and temporomandibular joints (TMJs). TMJ inflammation is considered to be a cause of pain in patients with TMD. Extracellular proteolytic enzymes, specifically the matrix metalloproteinases (MMPs), have been shown to modulate inflammation and pain. The purpose of this investigation was to determine whether the expression and level of gelatinolytic activity of MMP-2 and MMP-9 in the trigeminal ganglion are altered during different stages of temporomandibular inflammation, as determined by gelatin zymography. This study also evaluated whether mechanical allodynia and orofacial hyperalgesia, induced by the injection of complete Freund's adjuvant into the TMJ capsule, were altered by an MMP inhibitor (doxycycline, DOX). TMJ inflammation was measured by plasma extravasation in the periarticular tissue (Evans blue test) and infiltration of polymorphonuclear neutrophils into the synovial fluid (myeloperoxidase enzyme quantification). MMP expression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of this process. Furthermore, increases in plasma extravasation in periarticular tissue and myeloperoxidase activity in the joint tissue, which occurred throughout the inflammation process, were diminished by treatment with DOX, a nonspecific MMP inhibitor. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the same treatment.

Free and total GMP (glycomacropeptide) contents of milk during bovine lactation

Individual milk samples taken every two weeks from parturition to the end of lactation from 34 animals of three different herds and breeds were analyzed for free-GMP. A milk pool of each herd was analyzed for free and total GMP (released from k-casein by the action of rennin) and the data were correlated with sanitary conditions of animal and udder, phase of lactation and milk production. Most udder problems were concentrated near parturition, with few and spaced occurrences of clinical mastitis. The Californian Mastitis Test (CMT) results showed oscillations compatible with the phases of lactation period and environmental conditions. The widest variations in free-GMP occurred as a function of lactation period and as a consequence of clinical or subclinical mastitis. Higher levels were observed at the beginning of lactation (5.87mg L-1 of sialic acid), becoming normal with mean values of about 3.30mg L-1 at the end of the second month, and increasing again during the final third of lactation. On average, the same trends were observed for total GMP released by commercial rennet, beginning with slightly high values (35.59mg L-1), becoming normal by the sixth month with values close to 27.15mg L-1, and rising gradually up to the end of lactation, with 58.35mg L-1 of sialic acid. These results prove to be useful for the correct interpretation of tests applied to milk selection with respect to proteolytic status or even to restrain frauds by the addition of whey to milk.

Effect of chickpea (Cicer arietinum L.) germination on the major globulin content and in vitro digestibility

Chickpea seed germination was carried out over a period of 6 days. Little variation in the nitrogen and total globulin content was observed. The major globulin (11 S type) showed higher variation after the 4th day of germination. The elution behaviour and distribution of the isolated major globulin fraction on Sepharose CL-6B chromatography showed little modification at the end of germination. On SDS-PAGE the peak eluted from Sepharose CL-6B showed changes in protein bands between 20 and 30 kDa and above 60 kDa, indicating protein degradation during the period. Proteolytic activity was detected in the albumin fraction of the seeds, which increased up to the fourth and then decreased up to the sixth day, when isolated chickpea total globulin and casein were used as substrates. Chickpea flour, isolated albumin and total globulin fractions did not show an increase for in vitro digestibility; however, the isolated major globulin was more susceptible to hydrolysis after germination.

β-carotene biotransformation to obtain aroma compounds

Carotenoids are important constituents of food due to their color and because their degradation products generate important volatile compounds in foods. Aroma compounds derived from carotenoids are widely distributed in nature, and they are precursors of many important aromas in foods such as fruits and in flowers as well. They present high aromatic potential and are therefore of great interest to the industries of aromas and fragrances. In this study, more than 300 previously isolated microorganisms with potential for biotransformation of β-carotene present in the culture medium were selected using the plate method; about 80 strains presented capacity to produce aroma compounds and 7 strains were selected by an untrained panel of tasters to generate aroma compounds. The β-ionone was the main compound produced by CS1 (34.0 mg.L-1) and CF9 (42.4 mg.L-1) microorganisms at 72 and 24 hours of fermentation, cultured with and without pre-inoculation, respectively. The β-damascone and pseudoionone were found in low concentrations, 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalen (TTN) was tentatively identified and other compounds such as apocarotenoids, apparently obtained from the cleavage of the central part of the carotenoid, were detected.

Design of automatic control system for the precipitation of bromelain from the extract of pineapple wastes

In this work, bromelain was recovered from ground pineapple stem and rind by means of precipitation with alcohol at low temperature. Bromelain is the name of a group of powerful protein-digesting, or proteolytic, enzymes that are particularly useful for reducing muscle and tissue inflammation and as a digestive aid. Temperature control is crucial to avoid irreversible protein denaturation and consequently to improve the quality of the enzyme recovered. The process was carried out alternatively in two fed-batch pilot tanks: a glass tank and a stainless steel tank. Aliquots containing 100 mL of pineapple aqueous extract were fed into the tank. Inside the jacketed tank, the protein was exposed to unsteady operating conditions during the addition of the precipitating agent (ethanol 99.5%) because the dilution ratio "aqueous extract to ethanol" and heat transfer area changed. The coolant flow rate was manipulated through a variable speed pump. Fine tuned conventional and adaptive PID controllers were on-line implemented using a fieldbus digital control system. The processing performance efficiency was enhanced and so was the quality (enzyme activity) of the product.

Culture alternative medium for the growth of extreme halophilic bacteria in fish products

Halotolerant or halophilic (Archaeabacteria) microorganisms can be found in salted and ripening fish products that are not affected by salt. They can be moderate or extremely halophilic bacteria. The extremely halophilic bacteria require between 15-30% of NaCl for growth. The extremely halophilic archaeobacteria may be selectively isolated in different media. The aim of this work was to determine the effectiveness of the Salt-Agar-Milk medium, a medium modified in our laboratory through the addition of MgSO4 and KCl - named SAMm, and its effect on the bacterial growth by means of comparison with other media, with and without milk, determining time of incubation and counting. Two samples of salted fish from local fish salting factories and two laboratory strains were used. The factory samples were matured anchovy and anchovy fillets in oil, and the laboratory strains were: Haloarcula spp. (proteolytic) and Halococcus spp. (non-proteolytic). The following media were alternatively used for the isolation of extremely halophilic bacteria: IRAM; Formulation of Gibbons and collaborators, Cod Milk agar, and SAMm. IRAM and Gibbons were also used enriched with milk. In the SAMm medium, there were obtained count values similar or higher than the ones of the traditional media; besides the simplicity of its elaboration, the possibility to obtain positive results two or three days earlier also added to its benefit. Consequently, it can be considered an alternative to the media traditionally used for the studied halophilic bacteria.

Isolation and characterization of bacterial strains with a hydrolytic profile with potential use in bioconversion of agroindustial by-products and waste

There is a trend towards the use of novel technologies nowadays, mainly focused on biological processes, for recycling and the efficient utilization of organic residues that can be metabolized by different microorganisms as a source of energy. In the present study the isolation of bacterial strains from six different agro-industrial by-products and waste was performed with the objective of evaluating their hydrolytic capacities and suitability for use in bioconversion of specific substrates. The 34 isolated strains were screened in specific culture media for the production of various hydrolytic enzymes (lipase, protease, cellulase, and amylase). It was found that 28 strains exhibited proteolytic activity, 18 had lipolytic activity, 13 had caseinolytic activity, 15 had amylolytic activity, and 11 strains exhibited cellulolytic activity. The strains that showed the highest hydrolytic capacities with biotechnological potential were selected, characterized genotipically, and identified as Bacillus, Serratia, Enterococcus, Klebsiella, Stenotrophomonas, Lactococcus, and Escherichia genera. It was concluded that the strain isolates have a high potential for use in the bioconversion of agro-industrial waste, both as a pure culture and as a microbial consortium.

Potential of quixaba (Sideroxylon obtusifolium) latex as a milk-clotting agent

There are several obstacles to the use of chymosin in cheese production. Consequently, plant proteases have been studied as possible rennet substitutes, but most of these enzymes are unsuitable for the manufacture of cheese. The aim of this study was to evaluate the potential of latex from Sideroxylon obtusifolium as a source of milk-clotting proteases and to partially characterize the enzyme. The enzyme extract showed high protease and coagulant activities, with an optimal pH of 8.0 and temperature of 55 °C. The enzyme was stable in wide ranges of temperature and pH. Its activity was not affected by any metal ions tested; but was inhibited by phenylmethanesulfonyl fluoride and pepstatin. For the coagulant activity, the optimal concentration of CaCl2 was 10 µmol L- 1. Polyacrylamide gel electrophoresis showed four bands, with molecular weights between 17 and 64 kDa. These results indicate that the enzyme can be applied to the cheese industry.

Peptide separation of commercial fermented milk during refrigerated storage

Milk is an important source of bioactive compounds. Many of these compounds are released during fermentation and refrigerated storage. The aim of this study was to determine the release of peptides by lactic acid bacteria in commercial fermented milk during refrigerated storage. The size and profile of peptides were analyzed by polyacrylamide gel electrophoresis and sizeexclusion HPLC. During electrophoresis, it was observed that the peptides were released from caseins, whereas β-lactoglobulin was the whey protein with the highest degradation. HPLC analysis confirmed the pattern of peptide formation observed in electrophoresis. Two fractions lower than 2 kDa with aromatic amino acids in their structure were separated. These results were consistent with those reported for structures of peptides with antihypertensive activity. Therefore, the presence of aromatic amino acids in the peptide fractions obtained increases the likelihood of finding peptides with such activity in refrigerated commercial fermented milk. In conclusion, during cold storage, peptides with different molecular weights are released and accumulated. This could be due to the action of proteinases and peptidases of the proteolytic system in lactic acid bacteria.