Presentation on theme: "INVESTIGATING THE DIGESTIVE ENZYMES INHIBITORS OF BEAN EXTRACTS"— Presentation transcript:
1 INVESTIGATING THE DIGESTIVE ENZYMES INHIBITORS OF BEAN EXTRACTS Kiu Yan YuFoo Toon Min
2 INTRODUCTIONBeans are known to be a rich source of defensins, small, cysteine rich proteins found in most plants.Some of these defensins are known to have enzyme-inhibiting properties
3 INTRODUCTIONFor example, defensins from soybeans are known to inhibit human and bovine trypsin.Defensins from the common bean has also been found to inhibit the alpha-amylase activities of adzuki bean weevil (C.chinensis L.) and cowpea weevil (C. maculatus)
4 Rationale The large scale utilization of chemical pesticides has caused detrimental effectsto human health and environmentEnzyme inhibitors could provide a key role inplant defence against pest by inhibiting majorinsect digestive enzymes thus hampering thegrowth of these insects.Need to find new and natural alternative pesticides which has minimal impact on human health and environment
5 Rationale Enzyme inhibitors from beans, being a type of antinutrient, havebeen known to cause diarrhea andflatulence when consumed.RationaleThere is also a need to characterize thenature of these enzyme inhibitors tofind out how they can be deactivated.This is caused by an increase in resistant (undigested) starch passing into the colon, where it is broken down by flatulence-causing bacteria.
6 OBJECTIVESTo determine the alpha-amylase inhibitory activity of various bean extracts.To separate and identify the fragment or fragments responsible for the inhibition of these digestive enzymes.To find out the effect of different treatment on these enzyme inhibitors in bean extracts
7 HYPOTHESISDifferent bean extracts will have varying levels of amylase inhibitory activity.The bean extracts contains one or more fragments responsible for digestive enzyme inhibition.Certain treatments, such as boiling and soaking the beans before consumption would decrease its enzyme-inhibitory activity.
9 DEPENDENT VARIABLES Enzyme Activity Amylase Inhibition Assay Decrease in the amount of maltose liberated in percentage
10 CONSTANT VARIABLES Extraction method Volume and Concentration of starch solutionTemperature of incubationDuration of IncubationVolume and concentration of the enzymes, substrates and bean extract used
12 MATERIALS 20mM sodium phosphate buffer, pH 6.9, containing 300mM NaCl 2mg/ml standard maltose solutionAmylase Inhibition Assay3,5-Dinitrosalicylic acid solution (reacts with reducing sugars to form a colored compound which absorbs light strongly at 540 nm.50 mM Sodium Phosphate, 50 mM Sodium Chloride, 0.5 mM Calcium Chloride,0.1% Bovine Serum Albumin Buffer, pH 6.9 (or BSA buffer to stabilise the enzyme and prevent adhesion of enzymes to the reaction tubes and tip surfaces)1% starch solutionHuman salivary α-Amylase solutionDeionized Water
14 METHODOLOGY 1. Maltose 4. Treatment Standard curve Test 2. Preparing crudeExtracts of bean3. AmylaseInhibition Assay5. Purification andreverse HPLC
15 METHODOLOGY Maltose standard curve To establish the relationship between maltose concentration and its absorbance value.Maltose was used as standard solution in the concentration as 2mg/ml.Pipette 1ml, 0.8ml, 0.6ml, 0.4ml and 0.2ml of maltose standard solution into respective containersMake up the solution of each container to 2ml by adding different volumes of deionized water. (Blank tube contains only 2ml deionized water)
16 METHODOLOGY Maltose standard curve Add 1ml of 1% DNS solution to each containerPlace the containers in a boiling water bath for 5 minutesCool the containers on ice to room temperatureAdd 8ml to each container to dilute the solutionMix the solution by inversion and record the absorbance value for the blank and test using the spectrometer at 540nm
17 METHODOLOGY Preparing Crude Extracts of Beans 25g of beans were blended and homogenized in 50ml of 20mM sodium phosphate buffer , pH 6.9, containing 300mM NaClThe crude extract was then centrifuged at 8000rpm for 10 minutes.Collect the supernatant in eppendorf tubes, and centrifuge again at 12,000 rpm for 20minutes.
18 METHODOLOGY Amylase Inhibition Assay The following reagents were added:The reaction mixtures were incubated for 15 minutes at 25°C.BlankControl solutionBean-only solutionBean + amylase solutionBAS Buffer (ml)1Alpha-amylase (µl)-10Bean extract (µl)50Deionised water (µl)60
19 METHODOLOGY Amylase Inhibition Assay 250µl of reaction mixture was added to 500µl of starch solution and 250 µl of deionized water before further incubating at 37°C for 10 minutesThe reaction was stopped by adding 500µl DNS solution heating the contents in a boiling water bath for 15 minutes
20 METHODOLOGY Amylase Inhibition Assay 5. The solution was allowed to cool before the volume in each tube was made up to 5 ml by adding de-ionized water and mixing the contents by inversion6. The absorbance values of the blank, control, bean-only and test solutions were measured at 540 nm.7. Triplicates are done for each bean extract used.
21 METHODOLOGY Treatment Test Three types of treatment are carried out Pre-soakingBoilingDehulling (future work)
22 METHODOLOGY Treatment Test Pre-soaking Beans were soaked in water (1:5 (weight/volume) bean to water ratio) overnightBoilingBeans were heated in boiling water bath for 30 minutesDehullingBeans were manually dehulled with sharp knife.
23 RESULTS AND DISCUSSION Maltose Standard CurveFrom our experiment, we have obtained the following results and plotted the maltose standard curve:
24 RESULTS AND DISCUSSION Maltose Standard CurveThis shows that amount of maltose liberated is directly proportional to absorbance value.Amylase inhibitory activity can be expressed as the decrease in the amount of maltose liberated in percentage.Using this standard curve, we can determine the presence of amylase inhibitors denoted by a lower absorbance value for the solution containing the bean extract and amylase.
26 RESULTS AND DISCUSSION Amylase Inhibition AssayInhibition (%) can be calculated by the formula:((a-(c-b) )/a)*100%Decrease in absorbance value (due to decrease in amount of maltose liberated)Inhibited amylolytic activity without the influence of the endogenous amylolytic activity of the bean
27 RESULTS AND DISCUSSION Amylase Inhibition Assay
28 RESULTS AND DISCUSSION Amylase Inhibition AssayFrom the graph, kidney bean is the only extract that has consistently inhibited human alpha-amylase.Human alpha-amylase is also highly inhibited by it (84.3%)Conversely, the other bean extracts recorded an average of near zero or even negative inhibition.However in general, this shows that all but kidney bean extract did not show any significant inhibition of human alpha-amylase.
29 RESULTS AND DISCUSSION Treatment test63.6%95.1%
30 RESULTS AND DISCUSSION Only the kidney bean was treated as it has the highest alpha-amylase inhibitory activity.From the graph, soaking the bean reduced its inhibitory activity by more than 60%Boiling the bean almost completely stopped its inhibitory activityThis shows that the inhibitor is probably a heat-labile protein
31 RESULTS AND DISCUSSION This also suggests that presoaking and cooking the beans would have already removed most or all of the inhibitorsTherefore improving the digestibility of the beans and reducing flatulence.
32 LIMITATIONSAbsorbance values may also vary due to errors in pipetting.Different varieties of the same species of bean may contain varying contents of enzymes inhibitors
33 APPLICATIONSBean extracts with effective digestive enzyme inhibitors can be further studied to make insecticides, increasing production yield.Bean extracts with amylase inhibitory activity can also be used as a novel medicine to combat diabetes
34 FUTURE WORKEffect of dehulling on the alpha-amylase inhibitory activity of bean extractIdentification and purification of fragment or fragments responsible for the inhibition of these digestive enzymes using HPLC
35 BIBLIOGRAPHYSivakumar, S. , Mohan, Franco , O.L. , Thayumanavan, B. (2006) Inhibition of insect pest-amylases by little and Wnger millet inhibitors. Pesticide Biochemistry and Physiology ,85, 155–160.Kokiladevi, E., Manickam, M. , Thayumanavan, B. (2005) Characterization of Alpha-amylase inhibitor in Vigna sublobata. Botanical Bulletin of Academia Sinica, 46,Broekaert, W. F., Cammue, B. P., De Bolle, M. F., Thevissen, K., De Samblanx, G. W., & Osborn, R. W. (1997). Antimicrobial Peptides from Plants. Critical reviews in plant sciences (16),Broekaert, W. F., Terras, F. R., Cammue, B. P., & Osborn, R. W. (1995). Plant Defensins: Novel Antimicrobial Peptides as Components of the Host Defense System. Plant Physiol (108),Hartmut, E. S., Stephanie ,G., Andrew, M., Linda, M.T., Stuart, C., & Darryl,C. H.,(1995)Bean a-Amylase lnhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas(Pisum sativum ). Plant Physiol (107),Osborn, R. W., Samblanx, G. W., Thevissen, K., Goderis, I., Torrekens, S., & Leuven, F. V. (1995). Isolation and characterisation of plant defensins from seeds of Asteraceae,. FEBS Letters (368),
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