Acid Protease Activity Research Articles

Changes on digestive enzymes during initial ontogeny in the three-spot cichlid Cichlasoma trimaculatum.

A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.

Effects of koji-making with mixed strains on physicochemical and sensory properties of Chinese-type soy sauce.

Two kinds of soy sauces were prepared with Aspergillus oryzae koji (SSAO) and mixed koji (SSAOM, A. oryzae mouldstarter:Monascus purpureus mouldstarter = 1:2, w/w) respectively. The effects of mixed koji on the essential indices, oxygen radical absorption capacity, color indices, free amino acids and volatile compounds of soy sauce have been studied, followed by a sensory evaluation between SSAO and SSAOM. The contents of non-salt soluble solid, reducing sugar, total acid, total nitrogen and amino nitrogen in SSAOM increased by 21.50%, 9.88%, 15.35%, 5.98% and 41.43%, respectively, compared with the control SSAO, owing to the higher activities of acid protease and glucoamylase in the mixed koji. Moreover, SSAOM showed higher antioxidant activity, higher levels of free amino acids and much more attractive color. Meanwhile, flavor groups such as esters, aldehydes, pyrazines and sulfur-containing compounds in SSAOM were also improved. The contents of free amino acids and aroma compounds were consistent with the sensory evaluation. According to descriptive sensory analysis, SSAOM showed higher intensity for sweet and umami attributes; in addition, higher flowery, burnt, fruity and caramel-like attributes were perceived in SSAOM, while SSAO showed higher ethanolic and sour attributes. The investigated soy sauce prepared with mixed koji can be considered as an effective method to accelerate the fermentation process and improve the flavor of soy sauce.

Unexpectedly strong effect of caffeine on the vitality of western honeybees (Apis mellifera).

We examined the influence of caffeine on honeybee lifespan, Nosema resistance, key enzyme activities, metabolic compound concentrations, and total DNA methylation levels. Caffeine slowed age-related metabolic tendencies. Bees that consumed caffeine lived longer and were not infested with Nosema spp. Caffeine-treated workers had higher protein concentrations. The levels increased with aging but they then decreased in older bees. Caffeine increased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), AST, ALT, ALP, neutral proteases, and protease inhibitors, and the concentrations of uric acid, triglycerides, cholesterol, glucose, and Ca2+. Acidic and alkaline protease activities were lower in the bees treated with caffeine. Creatinine and Mg2+ concentrations were higher in the caffeine-treated workers but only up to 14 days of age. Caffeine significantly decreased DNA methylation levels in older bees. The compound could be considered as a natural diet supplement increasing apian resistance to stress factors. Our studies will enhance possibilities of using Apis mellifera as a model organism in gerontological studies.

Investigation of some virulence factors in Trichosporon spp. strains

The frequency of fungal infections have increased recently in parallel to prolonged survival of patients with chronical infections, common use of the broad-spectrum antibiotics and cytotoxic drugs and surgical interventions. Fungi such as Trichosporon, Fusarium and Geotrichum that were previously evaluated as contaminant/colonization, become important causes of morbidity and mortality especially in neutropenic patients. The aim of this study was to investigate the presence of virulence factors such as acid proteinase, phospholipase, esterase, coagulase and hemolytic activity among Trichosporon species. A total of 40 Trichosporon strains, of them 24 (60%) were T.asahii, 6 (15%) were T.inkin and 10 (25%) were the other species (one of each of T.aquatile, T.asteroides, T.coremiiforme, T.cutaneum, T.dermatis, T.faecale, T.japonicum, T.montevideense, T.mucoides, T.ovoides) were included in the study. Identification of the isolates was performed according to microscopic morphology (blastospores, arthrospores, pseudohyphae and true hyphae) on corn meal agar media, and carbohydrate assimilation patterns (API ID32C; bioMérieux, France). Secretory acid proteinase, phospholipase and esterase activities of the strains were evaluated by 1% bovine serum albumin containing agar, by egg yolk containing solid medium, and by Tween 80 containing solid medium, respectively. Hemolytic activity of the isolates were evaluated by 5-10% sheep blood Sabouraud dextrose agar. Coagulase enzyme activity was determined by using human and rabbit plasma. In our study, all of the 40 Trichosporon spp. strains were found negative in terms of acid proteinase and phospholipase enzyme activity, however all were positive for esterase enzyme activity. Hemolytic enzyme activity were identified in a total of 15 (37.5%) strains, being "+++" in three strains (2 T.asahii, 1 T.japonicum), and "++" in 12 isolates (9 T.asahii, 1 T.inkin, 1 T.asteroides, 1 T.mentevideense). Although 11 of those 15 positive strains were T.asahii, there was no statistical difference between the species in terms of hemolytic enzyme activity (p> 0.05). Coagulase enzyme activity was detected in 5% (2/40; 1 T.asahii, 1 T.inkin) of the strains with human plasma and in 27.5% (11/40; 9 T.asahii, 1 T.inkin, 1 T.montevideense) with rabbit plasma. In conclusion, our data indicated that esterase, coagulase and hemolytic activities detected in Trichosporon spp. might play role in the pathogenesis of Trichosporon infections, however, further large-scaled clinical and mycological studies are needed to prove this relation.

Glyphosate on digestive enzymes activity in piava (Leporinus obtusidens)

The effects of glyphosate, a nonselective herbicide (1.0 or 5.0mg L-1) on digestive enzymes activity (stomach and intestine) were evaluated in juveniles of piava (Leporinus obtusidens) after 90 days of exposure. The activity of acid protease, trypsin, chymotrypsin and amylase increased with the increase of glyphosate concentration. These results indicate that glyphosate affects digestive enzyme activities in this species, and may be an indicator of poor nutrient availability when fish survive in herbicide-contaminated water.

Meal timing affects protein-sparing effect by carbohydrates in sea bream: Effects on digestive and absorptive processes

Abstract A commercial diet (C) (48% protein and 20% lipids) and a high-digestible carbohydrate diet (CH) (37% protein, 12.5% lipids and 40% high-digestible carbohydrates) were used to feed sea bream juveniles for an 8-week period. In the commercial diet, more than 60% of ingredients were of plant origin from various sources, whereas the only component of plant origin in the CH diet was wheat. To determine the best time to administer carbohydrates and the possible protein-sparing effect, three different dietary regimes were established: C, CH-M and CH-A, and the corresponding diet was fed to sea bream in the morning (1.6% of bw) and in the afternoon (1% of bw), calculating quantities according to the amount of feed that fish ate during the acclimatization period. After the growth trial, specific growth rate (SGR), relative intestinal length, intestinal pH content, gastric and pancreatic digestive enzyme activities and nutrient absorption capacities were studied 5 h post-feeding after each meal (morning and afternoon). The acid protease activity measured was anticipatory and was higher when the next meal would have more protein. No differences in relative intestinal length or feed buffering capacity were found. The smaller ration given to sea bream in the afternoon led to a lower pancreatic release of alkaline protease and α-amylase and an up-regulation of D-Glc and L-Ala absorption capacity. A higher transit rate was measured when sea bream were fed the CH diet. When high-digestible carbohydrates were administered in the morning and the commercial diet in the afternoon, we observed a better assimilation of both diets due to compensatory mechanisms such as an increase in L-Lys, D-Glc and L-Ala absorption capacity after the morning feed, and a higher pancreatic release of alkaline protease and amylase after the afternoon feed. In contrast, when high-digestible carbohydrates were given in the afternoon, only a significant up-regulation of the capacity to absorb L-Lys was detected. Thus, the inclusion of high-digestible carbohydrates in the diet improved digestion and absorption processes when administered in the morning, leading to a protein-sparing effect that yielded growth comparable to that of fish fed an exclusively commercial diet.

Klinik Aspergillus spp. İzolatlarında Virülans Faktörü Olarak Asit Proteinaz ve Fosfolipaz Aktivitelerinin Araştırılması

Aspergillus spp. are widespread in nature and cause severe infections especially in immunocompromised patients. Aspergillus fumigatus complex is the most common species that causes infections in humans; however Aspergillus niger complex and Aspergillus flavus complex are the emerging agents that are isolated frequently from clinical specimens more recently. Besides the host factors such as immunosuppression, hematologic malignancy and corticosteroid use, fungal virulence factors such as elastase, acid protease and phospholipase enzymes are considered among the factors that affect the development of invasive aspergillosis. The aim of this study was to detect the acid proteinase and phospholipase enzyme activities in 30 A.fumigatus complex, nine A.flavus complex and four A.niger complex strains isolated from clinical specimens. Acid proteinase and phospholipase activities of the isolates were investigated by using bovine serum albumin agar (BSA), and egg yolk agar plates, respectively. Acid proteinase and phospholipase activity was detected in 76.7% (23/30) and 93.3% (28/30) of A.fumigatus complex isolates, respectively. None of the nine A.flavus complex isolates exhibited acid proteinase or phospholipase activity. Acid proteinase activity was not detected in any of the A.niger complex isolates (n= 4), however phospholipase activity was detected in one (25%) isolate. All of the acid proteinase positive A.fumigatus complex strains (n= 23) were also positive for phospholipase activity. In conclusion, further larger scale multicenter studies supported by clinical data, are needed to enlighten the roles of acid proteinase and phospholipase in the pathogenesis of infections due to Aspergillus spp.

Efeitos de dietas contendo concentrados proteicos vegetais no desempenho e atividade de enzimas digestivas de jundiá (Rhamdia quelen)

The purpose of study was to evaluate the effect of using protein concentrates crambe and sunflower meal in the diet of silver catfish juveniles, as substitute for animal protein source. A total of 300 silver catfish had been separate in 15 experimental units of 280 L, totaling five treatments with three replications. We evaluated two levels (25% and 50%) replacement of the meat and bone meal by protein concentrates of crambe and sunflower meals. Evaluated growth parameters, biological index and digestive enzymes in fish. There was no statistical difference for mass (g) and standard length (cm), but the fish diet CPFCr-25% had greater total length (cm). No difference in dry matter, crude protein and total protein deposited (calculated). However, there was a higher concentration of ash in the carcass of the animals fed the control diet and CPFCr-50% in relation to diet CPFG- 50%, in addition, higher levels of lipids in fish fed diet CPFG-50%. No significant differences for hepatosomatic index, digestive somatic index and intestinal quotient of animals subjected to different treatments. The activity of digestive enzymes trypsin and chymotrypsin did not change. There was increased activity of acid protease. The quantitative and qualitative increase in protein concentration from this fraction allows the use of bran protein concentrates crambe and sunflower as substitutes for animal protein source.

Farelo de crambe nas formas in natura ou reduzida em antinutrientes na dieta do jundiá

Este estudo avaliou os efeitos da substituição parcial de farinha de peixe e farinha de carne e ossos por farelo de crambe, sobre o crescimento, enzimas digestivas e parâmetros bioquímicos no jundiá (Rhamdia quelen). Este coproduto agroindustrial foi incluído na alimentação de jundiás na forma in natura ou reduzido em antinutrientes, obtido após a aplicação de tratamento químico ao farelo. A inclusão desses ingredientes foi comparada ao tratamento controle (proteína baseada em farinhas de origem animal) durante nove semanas. Crescimento semelhante ao controle foi observado em peixes alimentados com farelo de crambe in natura ou tratado quimicamente na dieta. A inclusão do farelo in natura reduziu a atividade de protease ácida e colesterol plasmático, mas não alterou os parâmetros bioquímicos hepáticos. Menor concentração de glicogênio e glicose no fígado e aumento na aspartato aminotransferase foi encontrada nos peixes alimentados com farelo de crambe tratado, sinalizando uma condição de carência energética. A similar resposta no crescimento, aliada às leves alterações digestivas e metabólicas com o farelo de crambe in natura em relação ao farelo tratado, pode indicar que, no nível de inclusão utilizado, este ingrediente não necessita de um processo prévio para redução de antinutrientes.

COENZYME Q10 TREATMENTS INFLUENCE THE LIFESPAN AND KEY BIOCHEMICAL RESISTANCE SYSTEMS IN THE HONEYBEE, Apis mellifera

Natural bioactive preparations that will boost apian resistance, aid body detoxification, or fight crucial bee diseases are in demand. Therefore, we examined the influence of coenzyme Q10 (CoQ10, 2,3-dimethoxy, 5-methyl, 6-decaprenyl benzoquinone) treatment on honeybee lifespan, Nosema resistance, the activity/concentration of antioxidants, proteases and protease inhibitors, and biomarkers. CoQ10 slows age-related metabolic processes. Workers that consumed CoQ10 lived longer than untreated controls and were less infested with Nosema spp. Relative to controls, the CoQ10-treated workers had higher protein concentrations that increased with age but then they decreased in older bees. CoQ10 treatments increased the activities of antioxidant enzymes (superoxide dismutase, GPx, catalase, glutathione S-transferase), protease inhibitors, biomarkers (aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase), the total antioxidant potential level, and concentrations of uric acid and creatinine. The activities of acidic, neutral, and alkaline proteases, and concentrations of albumin and urea were lower in the bees that were administered CoQ10. CoQ10 could be taken into consideration as a natural diet supplement in early spring before pollen sources become available in the temperate Central European climate. A response to CoQ10 administration that is similar to mammals supports our view that Apis mellifera is a model organism for biochemical gerontology.

쌀과 미입국의 배합비율에 따른 쌀 당화액의 품질특성

The effects of Aspergillus oryzae rice koji (AO) and Asp. kawachii rice koji (AK) as enzyme preparations, on the quality characteristics of rice mash were invested in this study. The amount of AORM (Asp. oryzae rice mash) and AKRM (Asp. kawachii rice mash) were 50, 100, 200% (w/w) based on 100 g of rice. Firstly, in the titer measurement result on the α-amylase and glucoamylase activities of AO and AK. On the other hand, the acid protease activity has values of 31.56 unit for AO and 849.17 unit for AK. The sugar solid of the AORM and AKRM groups significantly increased as the rice koji ratio on rice was higher, which were shown with values as high as 17.63∼20.53 and 17.51∼19.28, respectively. Glucose and maltose were detected for free sugar of AORM. Only glucose was found in AKRM. Citric acid, malic acid, and lactic acid were detected as the organic acid of KORM; oxalic acid, citric acid, and succinic acid were detected for AKRM, and the content increased as the rice koji ratio on rice increased (P<0.05). From the above result, rice koji with useful mold is expected to be used broadly in foods by looking at the fact that it has starch degradation ability and organic acid producibility.

Why specific anti-integrase antibodies from HIV-infected patients can efficiently hydrolyze 21-mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus-infected patients possess anti-integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti-myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti-MBP and anti-IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti-IN abzymes efficiently hydrolyze a 21-mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti-MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG-mediated and IgM-mediated proteolysis of OP21 by anti-IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti-IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti-IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non-cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease-like abzymes against different proteins, anti-IN abzymes possess serine, thiol, acidic, and metal-dependent protease activities.

Identification of a fungus able to secrete enzymes that degrade regenerated cellulose films and analyses of its extracellular hydrolases

The LW03 strain was isolated from Chinese farmland soil and found to be able to secrete certain enzymes degrading regenerated cellulose films at low temperature. The LW03 strain was systematically identified as Rhizopus arrhizus var. arrhizus by morphological, physiological, and molecular methods. Incubation of regenerated cellulose films with the extracted crude enzyme of LW03 was done to measure morphological changes by using scanning electron microscopy. Microscopic observations showed that the morphology of the regenerated cellulose films changed drastically due to enzymatic hydrolysis. The extracellular hydrolases of LW03 strain incubated on bran medium were also assessed. The predominant activity in the crude enzyme was glucoamylase activity, followed by acid proteinase, phytase and pectinase activity. Interestingly, activities of β-glucosidase, endoglucanase, exoglucanase, and cellulase were also observed, but at a much lower extent. Based on initial evidence, the crude enzyme is most likely to contain some new constituents capable of degrading regenerated cellulose films.

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea)

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine-alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine-alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.

Bone Marrow Injury Induced via Oxidative Stress in Mice by Inhalation Exposure to Formaldehyde

ObjectiveFormaldehyde, a ubiquitous environmental pollutant has been classified as a human leukemogen. However, toxicity of formaldehyde in bone marrow, the target site of leukemia induction, is still poorly understood.Methodology/Principal FindingsTo investigate bone marrow toxicity (bone marrow pathology, hematotoxicity) and underlying mechanisms (oxidative stress, inflammation, apoptosis) in formaldehyde-exposed mice. Male Balb/c mice were exposed to formaldehyde (0, 0.5, and 3.0 mg/m3) by nose-only inhalation for 8 hours/day, over a two week period designed to simulate a factory work schedule, with an exposure-free “weekend” on days 6 and 7, and were sacrificed on the morning of day 13. Counts of white blood cells, red blood cells and lymphocytes were significantly (p<0.05) decreased at 0.5 mg/m3 (43%, 7%, and 39%, respectively) and 3.0 mg/m3 (52%, 27%, and 43%, respectively) formaldehyde exposure, while platelet counts were significantly increased by 109% (0.5 mg/m3) and 67% (3.0 mg/m3). Biomarkers of oxidative stress (reactive oxygen species, glutathione depletion, cytochrome P450 1A1 and glutathione s-transferase theta 1 expression), inflammation (nuclear factor kappa-B, tomour necrosis factor alpha, interleukin-1 beta), and apoptosis (activity of cysteine-aspartic acid protease 3) in bone marrow tissues were induced at one or both formaldehyde doses mentioned above.Conclusions/SignificanceExposure of mice to formaldehyde by inhalation induced bone marrow toxicity, and that oxidative stress, inflammation and the consequential apoptosis jointly constitute potential mechanisms of such induced toxicity.

Long‐term oropharyngeal colonization by C. albicans in children with cystic fibrosis

This longitudinal prospective study aimed to determine the prevalence of oropharyngeal colonization by C. albicans in children with cystic fibrosis (CF), and observe the continuity of candidal colonization and the changes in production of virulence factors, susceptibility to antifungal agents and RAPD patterns of the isolates. Thirty-seven children with CF were followed-up for oropharyngeal C. albicans colonization for 18 months. The colonization rate was detected in 54%. All isolates were susceptible to amphotericin B, but those isolated from one patient were resistant to fluconazole. Biofilm production, secretory acid proteinase, phospholipase and esterase activity rates were 30%, 60%, 75% and 80%, respectively. RAPD analysis with the primers OPE-03 and OPE-18 was performed for genotyping. RAPD patterns of the strains isolated from the same patient were related to each other, whereas they were not related with other strains isolated from different patients. Two C. albicans strains isolated from the same patient were found to be unrelated to one another. As a result, long-lasting colonization of the oropharyngeal mucosa of children with CF by endogenous C. albicans isolates having the same RAPD pattern was demonstrated. Colonization prevalance and development of resistance to antifungal agents and the increased production of virulence factors were not correlated.

Ca 2+ release-activated Ca 2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca(2+) concentration inside pancreatic acinar cells ([Ca(2+)]i), due to excessive release of Ca(2+) stored inside the cells followed by Ca(2+) entry from the interstitial fluid. The sustained [Ca(2+)]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca(2+) release-activated Ca(2+) channels (CRAC) would prevent sustained elevation of [Ca(2+)]i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca(2+) ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca(2+). Ca(2+) entry then occurred upon admission of Ca(2+) to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca(2+) entry in a concentration-dependent manner within the range of 1 to 50 μM (IC50 = 3.4 μM), but had little or no effect on the physiological Ca(2+) spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca(2+)]i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca(2+)]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.

Effect of organic and inorganic carbon on extracellular enzyme activity of acid phosphatase and proteases in three kinds of fungal hyphae.

Most researches have manifested that mycorrhizal fungi can promote plant nutrient absorption and improve plant growth,but the effects of the hyphae on soil nutrient exploitation were less reported.Through laboratory control test of 3 kinds of fungi,Lactarius deliciosus,Russula integra and Gomphidius viscidus in this paper,N,P related extracellular enzymes exuding from variable fungal hyphaes(acid phosphatase and proteases) was found.In most cases,each enzymatic activity of these 2 enzymes was the highest in 2 weeks culture in MMN medium,while short(one week) or long(3 weeks) may reduce their activities.However, different fungus significantly differed in the enzyme activity,and G.viscidus had the lowest activity of acid phosphatase,while the highest in protease activity.No significant difference was found in the other two fungus; Secondly,addition of organic carbon(humic acid,HA) and inorganic carbon(calcium carbonate) could significantly affect the activities of acid phosphatase and protease.When HA was added,the activity of acid phosphatase steadily increased in 3 fungus,while fungus-specific patterns were found in protease activity,i.e., continuous increasing activity of protease in L.deliciosus protease was observed,while less HA-sensitivity was found in R.integr and G.viscidus;Thirdly,small amount of calcium carbonate addition could enhance the exudation of these two enzymes of acid phosphatase and protease,while adding more may reduce the enzymatic activities(e.g.0.1 g).To sum up,fungal hyphaes can secrete acidic phosphatase and acid protease,and these enzymes may promote the activation of ineffective organic P and N and enhance plant growth.Soil organic carbon accumulation and depletion of inorganic carbon could directly increase the extracellular enzyme secretions of all tested fungal hyphae,showing the function of hyphae in soil carbon cycle.

The Influence of Mild Pulsed Electric Field (PEF) on Acid Tolerance, Bile Tolerance, Growth and Protease Activity of the Dairy Culture Bacteria Lactobacillus acidophilus LA-K

The objective of this study was to determine the influence of mild PEF conditions on acid tolerance, growth, bile tolerance and protease activity of L. acidophilus LA-K. The treatments were positive square unipolar pulse width of 3 µs, pulse period of 0.5 second, voltage of 1 kV/cm, delay time of 20 µs and flow rate of 60 ml/min at 40.5°C PEF treatment temperature. The control was passed through the PEF system at the same flow rate (60 ml/min) without receiving any pulsed electric field condition. The acid tolerance was determined every 30 minutes for 120 minutes of incubation in acidified MRS broth at pH 2. Growth of the culture was determined hourly for 32 hours of incubation at 37°C in MRS broth. The bile tolerance was determined hourly for 16 hours of incubation in MRS-Thio broth supplemented with 0.3% (w/v) Oxgall (bovine bile) and 0.2% sodium thioglycolate. Samples were plated in duplicates using Lactobacilli MRS agar. The petriplates were incubated anaerobically at 37C for 48 hours. Protease activity was determined by o-phthaldialdehyde (OPA) spectrophotometric assay at 0, 12, 24, 36 and 48 hours of incubation of inoculated skim milk at 40°C three replications were conducted. The experimental design was repeated measurements on complete randomized block, replications were the blocks. Lactobacillus acidophilus subjected to mild PEF conditions as well as the control were acid tolerant until the end of the 120 minutes of incubation but there was a significant (P&lt;0.0001) decrease in the viable bacterial counts after each incubation time of 30 minutes. Mild PEF conditions studied significantly improved acid tolerance of the bacterium. The mild PEF treated culture reached the logarithmic phase of the growth an hour earlier than the control. Lactobacillus acidophilus LA-K exhibited tolerance to the bile conditions and exhibited similar growth patterns in the presence or absence of bile acids but the bacterium reached the stationary phase after 12 hours of incubation in bile conditions compared to 16 hours of incubation in the absence of bile conditions. Mild Pulsed Electric Field conditions had insignificant effect on bile tolerance of L. acidophilus LA-K. Mild Pulsed Electric Field treatment significantly (P&lt;0.0001) enhanced the protease activity of the bacterium compared to the control.

콩 코오지의 최적 제조조건 설정

강릉원주대학교 생명과학대학 식품가공유통학과To establish the optimal manufacturing conditions of soybean koji, soybean Koji prepared with Aspergillus oryzae 6-M-1 and Bacillus subtilis 3-B-1 isolated from traditional Korean meju. During 7 days of making Koji, the amount of amino-type nitrogen was getting more increase. The amount of amino-type nitrogen of Koji prepared with A. oryzae 6-M-1 was 686.16 mg% (w/w), that of Koji with B. subtilis 3-B-1 was 643.46 mg% (w/w) at seventh day of making Koji. The α-amylase activity of Koji prepared with A. oryzae 6-M-1 was 1472.54 unit/g, that of Koji with B. subtilis 3-B-1 was 791.00 units/g on the seventh day of the making. The acidic protease activity of Koji prepared with A. oryzae 6-M-1 was 309.00 unit/g, that of Koji with B. subtilis 3-B-1 was 135.88 unit/g at 7th day of making. The amount of amino-type nitrogen and enzyme activities of soybean Koji prepared with A. oryzae 6-M-1 and B. subtilis 3-B-1 were produced more than those of wheat flour Koji made in factory. Sensory evaluation on a commercial doenjang and doenjangs prepared with A. oryzae 6-M-1 and B. subtilis 3-B-1 was not significantly different at p<0.05.Key words Koji, soybean, doenjang, Aspergillus oryzae, Bacillus subtilis