• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1369-1379
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• 2010

This study investigated purification and milk-clotting activity of the enzymes produced by Bacillus subtilis var, natto and Rhizopus oligosporus compared with that of commercial rennet. The clotting time, viscosity, tension and microstructure of the curd and electrophoretic patterns of milk proteins were determined. The milk-clotting activity/proteolytic activity ratios (MCA/PA ratio) of B. subtilis, R. oligosporus and commercial rennet were also compared. The results revealed that the curd formed by the commercial rennet had the highest viscosity and curd tension and the shortest clotting time among the three enzymes. However, curd produced by Rhizopus enzymes was ranked as second. From the MCA/PA ratio and electrophoretogram analyses it could be concluded that the enzyme produced by B. subtilis had the highest proteolytic activity, while the commercial rennet had the highest milk-clotting activity. Observations of microstructures of SEM showed that the three-dimensional network for curd formed by commercial rennet was denser, firmer and more smooth. The milk-clotting activity, specific activity, purification ratio and recovery of the purified enzymes produced by both the tested organisms were also determined with ion exchange chromatography and gel filtration.

• Food Science of Animal Resources
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• v.20 no.1
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• pp.1-7
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• 2000

The influence of the ditary containing boiled eggs on the plasma cholesterol level and antithrombotic activity in rats was studied. Rats were fed basal diet(0% boiled eggs) as a control group or diets containing 25% and 50% boiled eggs or a mixed diet with 95% boiled eggs plus 5% $\alpha$-cellulose powder as a experimental groups for 30 days. The bleeding time and whole blood clotting time were significantly(P<0.05) increased by feeding diet containing 25% boiled eggs compared to groups of basal diet, 50% or 95% boiled eggs diets. The plasma clotting time was high in group of 25% boiled eggs diet. However, there were no difference in plasma clotting time among rats fed the dietary boiled eggs. The levels of plasma total cholesterol(TC) and low density plus very low density lipoprotein cholesterol(LDL, VLDL-C) were significantly(P<0.05) highest in group 95% boiled eggs diet compared to others. There were no differences in high density lipoprotein cholesterol(HDL-C) among rats fed the dietary boiled eggs. The levels of plasma TC, HDL-C, LDL$.$VLDL-C and the ratios of HDL-C/TC were not significant among the basal diet, 25% and 50% boiled eggs diets. These results suggest that the intakes of the dietary boiled eggs have the antithrombotic activity and plasma cholesterol lowering effect.

• Journal of Food Hygiene and Safety
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• v.11 no.2
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• pp.77-82
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• 1996

Green tea catechins(GTC) were studied for its inhibitory effect on human platelet aggregation in vitro, for its antithrombotic effect in mice in viro, and bleeding and clotting time in rats. The catechins were isolated and purified from green tea, which were composed of (-)-epigallocatechin gallate, (-)-epigallocatechin, (-)epicatechin gallate and (-)-epicatechin, GTC produced a potent inhibition of human platelet aggregation in a dose-dependent manner against the stimulants such as ADP, collagen, epinephrine and ristocetin n vitro. GTC also prevented death due to the formation of pulmonary thrombosis by platelet aggregates in mice in a dose-de-pendent manner in viro. GTC increased the bleeding time, whole blood clotting time and plasma clotting time in rats, too. These results suggest that GTC is a promising antithrombotic agent.

• Journal of Pharmacopuncture
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• v.18 no.4
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• pp.7-11
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• 2015

Objectives: Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations $(SDs)={\pm}0.71$; however, clotting was observed in the control group 13.8 s, $SDs={\pm}0.52$. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The $LD_{50}$ of the crude BV was found to be $177.8{\mu}g/mouse$. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase $A_2(PLA_2)$ and melittin, and that can increase the blood clotting times in vitro.

• Archives of Pharmacal Research
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• v.24 no.2
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• pp.109-113
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• 2001

Curdlan is a natural $\beta$-1,3-glucan produced by Agrobacterium biovar 1. In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan. Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time. Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time. However, the chain length of the substituent did not play a role in prolonging the clotting time. The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III. The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.4
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• pp.418-422
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• 1992

Mucor mucedo C-7 selected as a potent fungus for producing milk-clotting enzyme was cultured on wheat bran solid medium and the optimum culture conditions for the production of milk clotting enzyme were ot tamed as follows. Amount of water added to wheat bran was 100% to the weight of wheat bran and culture temperature and time was 3$0^{\circ}C$ and 72hrs, respectively. The production of milk-clotting enzyme was markedly increased by the addition of Macllvaine buffer solution (pH4.5) instead of water added to wheat bran solid medium and milk-clotting activity was stable for culture period.

• Archives of Pharmacal Research
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• v.16 no.3
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• pp.209-212
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• 1993

The anti-coagulant activity of ilexoside D isolated from the roots of ilex pubescens Hook. et Am. was investigated in in vivo models of blood coagulation in rats. On oral administration, ilexoside D prolonged the bleeding time and the whole blood recalcified clotting time, but not the plasma recalcified clotting time. In vitor, ilexoside D did not affect the recalciffed clotting times of whole blood, platelet-rich plasma(PRP), and platelet-poor plasma(PPP), while in the presence of tissue factor the compound prologed the reduced proth-rombin times of whole blood, PRP and PPP in the dose-dependent manner. These results indicate that ilexoside D has the anit-tissue factor activity as well as the antithromobotic activity.

• The Korea Journal of Herbology
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• v.26 no.4
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• pp.125-132
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• 2011

Objectives : An aim of study is to investigate effects of curculiginis rhizoma in vitro (factor Xa (FXa) inhibitor assay, prothrombinase assay, prothrombin time (PT) assay, activated partial thromboplastin time (aPTT) assay) and in vivo experiment (blood clotting time, thromboxane B2 content assay in serum and weight of thrombus by AV-shunt rat model). Methods : We gained a human serum and used serum in vitro study such as factor X activity (FXa) inhibition, prothrombinase inhibition, prothrombin time (PT) and activated partial thromboplastin time. Fifteen SD rats were divided into three groups (intact control group and two experimental group treated with extract of Curculiginis Rhizoma(ECR)). Rats were orally administrated DW (intact control group), 600 mg/kg concertration of ECR and 200 mg/kg concertration of ECR. After one hour, we anesthetized rats and made arteriovenous (AV) shunt rat models to study weights of thrombus, took a hole blood to study content of thromboxane B2 and blood clotting time. Results : In vitro, ECR increased a inhibitory activity of FXa, prothrombinase and aPTT compared than intact control group. Especially ECR made significant increase of FXa and prothrombinase inhibitory activity (p<0.05, p<0.01). And PT were increased in ECR control group compared with intact control group. In vivo, a blood clotting time of experiment group treated with ECR 600 mg/kg were significantly increased compared with that of intact control group (p<0.05) and content of thromboxane B2 was significantly decreased in group treated with ECR 600 mg/kg in seum. The weight of thrombus were significantly reduced in group treated with ECR 600 mg/kg compared with intact control group (p<0.05). But in vivo experiment study, those of group treated with ECR 200 mg/kg were reduced compared with those of intact control group without statistical significance. Conclusions : ECR has a antithromboic activity in internal course with inhibitory activity of FXa and prothrombinase in vitro, it required to research more study for effective compounds.

• Microbiology and Biotechnology Letters
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• v.6 no.3
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• pp.109-113
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• 1978

The process of milk clotting by enzymes was confirmed to be consisted of two distinct stages: an enzymic coagulation stage followed by a nonenzymic clotting stage. The endpoint of the enzymic stage was determined simply by measuring flow distance of milk-enzyme system in a regular test tube being laid down to five degree slope after the reaction. By measuring the time elapsed during the enzymic stage a new method of evaluating the power of milk clotting enzymes was proposed.

• Applied Biological Chemistry
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• v.12
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• pp.7-11
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• 1969

Mucor-rennin, the crystalline milk-clotting enzyme, isolated from Mucor pusillus var. Lindt, has an acid protease activity. The optimum pH for the digestion of k-casein is 4.5, while that for hemoglobin digestion is 4.0. The skim milk solution was easily clotted acidic solution than alkalin solution, and the milk clotting activated by Ca ion. The enzyme was heat stable against heat from pH 4.0 to 6.0 but was more stable at pH 5.0. The activity of the enzyme at pH 5.0 did not decrease at 30 C for 15 days and the activity was not effected by sodium propionate and salicilic acid. Therefore, the enzyme of liguid type could store for a long time and could be transported from Erzyme production Co. to Manufacture of cheese Co. by adding the antiesptic and by adjusting pH to 5.0.