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Raw microbial enzyme versus human enzyme extract

This experiment was performed to investigate the significance of the enzymatic activity of microbial amylases in hydrolysis of starch. The selected microbial enzyme for this investigation is alpha-amylase, derived from Bacillus subtilis, a species of bacteria. The enzymatic activity of Bacillus subtilis was compared with the activity of human salivary alpha-amylase enzyme. The activity of both sources of enzymes in digestion of starch was represented by the clear zones (brown areas) around each paper disc soaked in both enzymatic agents respectively. The statistical t-test showed that the area of clear zone around the paper disc soaked in human saliva is significantly larger than the one dipped in the culture of Bacillus subtilis’s. The results support the hypothesis. There is a significant difference between the enzymatic activity of human saliva and Bacillus subtilis due to the origin and amount of enzyme present for hydrolysis. The production and activity of bacterial alpha-amylase is significant and this offers a wide range of applications in starch processing industries.

Key words: Alpha amylase; Bacillus subtilis, Starch hydrolysis; Statistical t-test; Starch industries

Research and Rationale

Starch is a polymer of glucose linked to one another by glycosidic bonds. There are two types of polymers in starch: amylase (15-20%) and amylopectin (80-85%). [1]Amylose is a linear polymer consisting of glucose units with alpha-1, 4 glycosidic bonds. Amylopectin consists of short alpha-1, 4 glycosidic bonds linked to linear chains of 10-60 glucose units and alpha-1,6 glycosidic bonds linked to side chains with 15-45 glucose units.[4]

In the past, acid hydrolysis of starch has had a widespread use in starch processing industries. However in the past few decades, this technique was gradually replaced by the use of starch-converting enzymes in the production of modified starches, glucose and fructose syrups as acid hydrolysis requires the use of corrosion-resistant materials, large amounts of energy for heating and the processes were relatively difficult to control.[15]

Conventionally, there are three stages in the conversion of starch, which are:

Gelatinisation, involving the dissolution of the starch granules to form a viscous suspension.

Liquefaction, involving the partial hydrolysis of starch.

Saccharification, involving the production of glucose and maltose by further hydrolysis.

These processes are energy intensive and thus increasing the production cost of starch-based products. To reduce energy consumption, economical raw starch digesting enzymes are increasingly used in starch processing industries.

Raw starch digesting enzymes (RSDE) are enzymes that can act directly on raw starch granules. In spite of the wide distribution of RSDE, microbial sources of these enzymes have many advantages in industries. The benefits include cost effectiveness, consistency, less time, space and labour required for production and ease of process modification and optimisation. [5] Therefore, microbial amylases are currently the most preferred RSDE to meet industrial demands.

C:\Users\User\Downloads\use of enzymes in processing starch.gif

Figure 1 The use of enzymes in processing starch. [15]

Generally, amylases comprise about 30% of the world’s industrial enzyme production. [6]There are four types of starch converting enzymes, which are endoamylases, exoamylases, debranching enzymes, and transferases. Edoamylases composed mainly of alpha-amylases which release oligosaccharides of various lengths by randomly hydrolysing the internal alpha-1, 4 glycosidic bonds. Exoamylases, composed mainly of glucoamylases which hydrolyses the terminal alpha-1, 4 bonds, releasing glucose as the main product. The debranching enzymes act predomainantly on alpha-1, 6 linkages of amylopectin while transferases operate on alpha-1, 4 glycosidic bonds. [7]

Figure 2 Different enzymes involved in the degradation of starch. The open ring structure symbolises the reducing end of a polyglucose molecule. [8]

In this investigation, both human salivary amylase and amylase secreted by Bacillus subtilis are alpha-amylases. This enzyme is found in a wide variety of microorganisms, belonging to the Archaea and the Bacteria domains. The properties of this enzyme vary from one organism to the other due to different origins. The most suitable alpha-amylase for industrial applications is mainly originated from the genus Bacillus. Alpha-amylases produced by this genus have numerous applications in a number of industrial processes such as in food, detergent, and in the manufacture of bioethanol. [9, 10] In addition, the enzymes produced by the bacteria of this genus are thermostable and this is beneficial since the gelatinisation and liquefaction of starch take place at high temperatures (above 90°C). [11, 12] More importantly, Bacillus subtilis appears to have a low degree of virulence to humans. [19]

The results from this study can be used to show that Bacillus subtilis, is able to produce alpha-amylase enzyme to hydrolyse starch. The synthesis and activity of this enzyme could be observed via the clear zones (brown area) present in the starch-agar plates due to absence of starch as a result of hydrolysis, when the surface of the agar is flooded by iodine solution. The regions which turned blue-black after staining by iodine solution indicate the presence of non-hydrolysed starch.The evidence of the production of bacterial alpha-amylase is significant so that potential applications and benefits of this enzyme could be further explored especially in industries.

Experimental hypothesis

Bacillus subtilis is able to produce alpha-amylase, an extracellular enzyme to hydrolyse starch. There is a significant difference between the enzymatic activity of human saliva and Bacillus subtilis due to different origins and concentrations of enzyme. Human saliva hydrolyses more starch than the culture of Bacillus subtilis.

Null hypothesis

There is no significant difference between the enzymatic activity of human saliva and Bacillus subtilis.

Variables

Manipulated enzyme : Type of enzymatic agent

Responding variable : Area of clear zone (brown area) around paper disc soaked in respective agents

Fixed variable : Size of filter paper discs, temperature of incubation (30°C), time of incubation (24 hours), thickness and composition of agar, type of enzyme present in both enzymatic agents, concentration of iodine solution

Apparatus

Petri dishes, sterilised filter paper discs, pairs of sterile forceps, weighing balance, dropper, Bunsen burner, water bath, spatula, beakers, glass rods and measuring cylinders

Materials

Freshly-prepared human saliva, sterilized distilled water, 70% ethanol, Bacillus subtilis nutrient broth culture, starch powder, agar powder (only agar, without nutrients), antiseptic solution (Dettol), 1% Grams iodine solution, ruler, marker pens and labels

Planning

A trial experiment was conducted to determine the suitable concentration of starch in agar medium to induce the production of alpha-amylase by Bacillus subtilis for the most marked effect on enzymatic reaction. Two different agar mediums containing distinctive concentrations of starch were used.

Three Petri dishes were used for each agar medium. Two paper discs were soaked in both enzymatic agents respectively and one paper disc were soaked in sterile distilled water, as a control experiment. After incubation, the surfaces of both types of agar mediums in the Petri dishes were flooded with 1% iodine solution. The diameters of clear zones were determined.

Concentration of starch

(gcm-3)

Mean diameter of clear zone around enzymatic agents (mm)

Human saliva

Bacillus subtilis

1

29.0

18.5

0.1

20.5

12.0

Table 1 The diameters of clear zones with respect to different concentrations of starch in agar medium.

The result shows that 1 gcm-3 of starch had the most marked effect on the enzymatic reaction. The larger the diameter of clear zone, the greater the amount of hydrolysed starch. Thus, the agar medium containing 1 gcm-3 of starch was chosen.

Two different reagents were used to determine the region of hydrolysed starch. The selected reagents were 1% Grams iodine solution and 1% Benedict’s solution.

Iodine test is used to detect the presence of starch. Starch forms a dark blue-black complex after reacting with iodine. Benedict’s test is used to test the presence of reducing sugars (sugars with a free aldehyde or ketone group). The blue copper sulphate present in Benedict’s solution is reduced to form copper oxide, a red-brown precipitate. If iodine was used in this experiment, non-hydrolysed starch would be stained, leaving brown areas around each paper disc due to absence of starch, as a result of hydrolysis by alpha-amylase enzyme. If Benedict’s solution was used instead, products of hydrolysed starch (maltose, reducing sugars) would be stained, leaving blue areas of non-hydrolysed starch. Thus, the hydrolysed starch is represented by the red-brown regions observed around each paper disc.

Reagents

Mean diameter of clear zone around enzymatic agents (mm)

Human saliva

Bacillus subtilis

Grams iodine solution

28.5

19.0

Benedict’s solution

10.5

3.0

Table 2 The diameters of clear zones with respect to different reagents.

Based on the results, the diameter of the clear zones of hydrolysed starch is larger and more significant when iodine solution is used as compared to Benedict’s solution. This is such as iodine can detect presence of starch at room temperature while Benedict’s solution can only detect and oxidise reducing sugars at higher temperatures. Since the Petri dishes were incubated at room temperature, the most suitable reagent for this experiment is iodine solution.

Real Experimental Procedures

Making up starch-agar medium

2g of soluble starch powder was measured using a measuring balance and was transferred into an empty, clean 250mL beaker.

20cm3 of sterilised hot water was measured using a measuring cylinder and was poured into the beaker containing starch powder to make a soluble paste.

1.5g of agar (without growth medium) was measured using a measuring balance and was added to the beaker containing starch paste. The mixture was stirred well using a glass rod.

200cm3 of sterilised distilled water was measured using a measuring cylinder and was gradually added into the mixture of starch and agar.

Then, the mixture was transferred into a sterilised container and was heated in a boiling water bath up to 95°C. The mixture in the water bath was continuously stirred using a spatula to ensure uniform distribution of starch and agar in the mixture.

After 10 minutes of heating, the starch-agar was removed from the water bath and was left in a water bath which was set to a temperature of 50°C. The medium was ready to be used in the experiment after sterilisation.

Preparation of agar plates and observation of results

A Bunsen burner was lighted and was set to a hot, roaring flame.

A sealed flask of sterilised starch-agar was collected from the oven.

20mL of agar medium was measured and poured into a Petri dish.

The dish was gently rotated on the table to ensure that the medium covers the base of the dish evenly. The base of the plate must be covered, agar must not touch the lid and the surface must be smooth with no bubbles.

The agar medium in the Petri dish was left aside to cool. The medium then solidified.

At the base of the Petri dish, three distinct sections (A, B and C) were labelled using a marker pen. Each label represented a paper disc with a type of treatment, such as:

Section

Treatment

A

Bacillus subtilis

B

Natural human saliva

C

Distilled water

Table 3 Types of treatment for paper disc of each section.

A pair of sterilised forceps was passed through the Bunsen burner flame. The tips of the heated forceps were allowed to cool and were used to pick up one of the sterile paper disc.

The lid of the bottle containing bacterial culture of Bacillus subtilis was untightened and removed. The mouth of the bottle was flamed and the disc was dipped into the broth.

The saturated paper disc was placed on the surface of the agar in the Petri dish according to its respective location, for example, disc immersed in Bacillus subtilis was placed at section A.

Steps 7 to 9 were repeated using another discsdipped in freshly prepared natural human saliva and distilled water respectively. The paper disc soaked in distilled water was a control experiment.

The lid of the Petri dish was opened as little as possible.

Steps 3 to 11 were replicated ten times to obtain ten identical Petri dishes with three labelled sections each.

The dishes were placed in inverted positions in the incubator which was set at the temperature of 30°C (room temperature) for 24 hours.

At the end of 24 hours, the dishes were removed from the incubator.

The lids of the Petri dishes were open slightly and a dropper was used to transfer just enough Grams iodine solution to stain the surface of the agar. The lids were then replaced.

The clear zone (brown area) around each disc was observed.

The diameter of the clear zone including the diameter of paper disc, 6mm was measured vertically, horizontally and diagonally using a ruler and the mean value was obtained.

The area of the clear zone was determined by using this formulae:

The data of diameters and areas of clear zones were recorded in Table 5 and 6. A bar chart of mean area of clear zone against enzymatic agents was plotted.

A t-test was used to analyse the data statistically.

Risk Assessment

Aseptic technique was used throughout this experiment to prevent bacterial contamination in the specimen and the place of experiment. The table top was cleaned using an antiseptic solution and 70% ethanol before and after the experiment. Hands were washed with antiseptic solution before handling sterilised apparatus to avoid the materials handled during experiment from being contaminated. Gloves were worn and hands were washed after the experiment. Sterile apparatus such as Petri dishes and forceps were used to terminate any other bacteria that might affect the results. A cloth was used while handling the hot flask containing starch-agar. Petri dish lids were lifted as slight as possible when pouring the agar and placing the paper discs soaked in both enzymatic agents respectively. This is also to prevent entry of other bacteria. While covering the surface of agar with iodine solution to observe results, gloves were worn to prevent stain by iodine. The Petri dishes were incubated at 30°C instead of 37°C (body temperature) to prevent growth of pathogenic bacteria which possess harm to humans. The covers were not open while taking measurements to prevent exposure to Bacillus subtilis and stain by iodine solution. The mouths of the flask containing agar and bottle containing Bacillus subtilis were flamed to ensure there was no contamination by any other microorganisms. After using the sterilised forceps, the apparatus were left in a container containing disinfectant solution to avoid spreading of bacteria. After the experiment, the Petri dishes were sent for autoclaving before disposal.

Results

Diameter of clear zone (mm)

Petri dish

Human saliva

Bacillus subtilis

1

2

3

Mean

1

2

3

Mean

A

28.0

28.0

28.0

28.0

21.0

25.0

20.0

22.0

B

28.0

28.0

28.0

28.0

21.0

17.0

19.0

19.0

C

29.0

29.0

29.0

29.0

24.0

15.0

17.0

18.7

D

29.0

29.0

29.0

29.0

16.0

15.0

17.0

16.0

E

28.0

28.0

28.0

29.0

30.0

23.0

19.0

24.0

F

27.0

27.0

27.0

27.0

23.0

19.0

18.0

20.0

G

28.0

28.0

28.0

28.0

21.0

22.0

18.0

20.3

H

29.0

29.0

29.0

29.0

19.0

18.0

17.0

18.0

I

27.0

27.0

27.0

27.0

23.0

18.0

17.0

19.3

J

28.0

28.0

28.0

28.0

24.0

17.0

16.0

19.0

Table 4 Diameters of clear zones with respect to enzymatic agents.

Petri dish

Area of clear zone (mm2)

Human saliva

Bacillus subtilis

A

615.7

380.1

B

615.7

283.5

C

660.5

274.6

D

660.5

201.1

E

660.5

452.4

F

572.6

314.2

G

615.7

323.7

H

660.5

254.5

I

572.6

292.6

J

615.7

283.5

Mean

625.0

306.0

Table 5 Areas of clear zones with respect to enzymatic reagents.

Mean Area of Clear Zone against Enzymatic Agents

Mean area of clear zone (mm2)

Enzymatic agents

Figure 3 Bar chart of mean area of clear zones with respect to enzymatic agents.

Statistical Analysis

The clear zone around the paper disc due to human saliva is significantly wider than that due to Bacillus subtilis. The calculated t-value (13.73) shows that it is significant whereby it vastly exceeds the critical t-value, which is 2.10 (level of significance, p<0.05; d.f. =18). The means of clear zones of both enzymatic agents are significantly different at p= 1.34 x 10-10. Therefore, the experimental hypothesis is accepted and the null hypothesis is rejected. This analysis assumes that the variances of both treatments are equal.

Formulae

Human saliva

Bacillus subtilis

Table 6 Calculations for t-test.

Where = mean of sample 1 (Human saliva)

= mean of sample 2 (Bacillus subtilis)

= number of subjects in sample 1

= number of subjects in sample 2

= variance of sample 1

= variance of sample 2

Data analysis

The mean area of the clear zone due to human saliva is 319mm2 larger than Bacillus subtilis’, with a percentage difference of 51.04%. Error bars are displayed on the graph to represent the overall distribution of the data. Upper error bar of Bacillus subtilis does not overlap with the range of values within the error bar of human saliva. Thus, these two mean area values differ significantly.

Both enzymatic agents caused clear zones because both contains enzyme to digest starch. The alpha-amylase enzyme of natural human saliva diffused out of the paper disc into the agar medium and hydrolyses the surrounding starch. As for Bacillus subtilis, the presence of starch stimulates the production of extracellular alpha-amylase enzyme. [13] Hydrolysis of external starch molecules results in maltose, maltotriose, and a mixture of branched alpha-1, 6 oligosaccharides, non-branched oligosaccharides, and some glucose. [1] The microbes then absorb the sugar produced as they are small enough to pass into their cells.

Due to hydrolysis by both enzymatic agents, starch is not present in the clear zones. The presence of starch is detected by Gram’s iodine solution. When starch is present, iodine binds to the coils of amylose molecules. There is some transfer of charge between the iodine and starch in the complex. As a result of the altered energy levels, light is absorbed, giving the complex its intense blue colour.[16]

The enzymatic activity is higher near the disc and reduces slowly as it gets further from the disc. This is such as the concentration of enzyme is more saturated near the disc. Therefore, the size of the clear zone is a measure of the amount of alpha-amylase present. The larger the clear regions around the disc, the higher the concentration of alpha-amylase. Distilled water which acted as a control, showed no signs of starch hydrolysis as no clear zone was produced.

The difference in the sizes of clear zones is due to the difference in the concentration of alpha-amylase enzyme. Generally, amylase is a component found in relatively high concentrations in human saliva. The larger the concentration of alpha-amylase enzyme, the greater the formation enzyme-substrate complexes, as the amount of active sites available for binding of starch molecules is greater. [17]As a result, more substrates are hydrolysed, forming more products (sugars).

On the other hand, the quantity of alpha-amylase enzyme secreted by Bacillus subtilis in the given period of time (24 hours) is lesser than the concentration of enzyme already present in human saliva. This is such as various stages are involved in the formation of alpha-amylase, thus Bacillus subtilis requires a longer time than human saliva to hydrolyse the same amount of starch. As a result, lesser starch molecules are hydrolysed.

The results showed that hydrolysis of starch by microbial amylase is significant and this knowledge could be applied in a wide field of industries. Bacterial amylases, which are stable at high temperatures, are used by the textile industries. Threads used in weaving are pre-strengthened with starch. After weaving has been completed, the cloth is soaked in a dilute solution of amylases to remove the starch. [2]

Sometimes microbial enzymes are immobilised on water-insoluble materials to increase their versatility. With proper supporting materials, immobilised enzymes are more stable and retain their activity longer than unbound enzymes. Furthermore, they can be added and removed from reaction at will, enabling greater control of the reaction. An example is in the production of high-fructose syrup as sweetening agents. [3]

Evaluation

The agar was poured at about 50°C as it was not too hot to be handled and to reduce the amount of condensation in the Petri dishes. The agar was not allowed to cool to much as it will start to solidify at about 42°C. Once disc soaked in sterile distilled water was placed in each Petri dish as a controlled experiment. Three discs were placed far apart from each other in Petri dishes and not too close to the side of Petri dishes, so that hydrolysis of starch which results in clear zones around disc can be seen clearly. The Petri dishes were left for 24 hours so that enough time was provided for the synthesis of extracellular alpha-amylase by Bacillus subtilis.

Based on the measurements obtained, the diameters of clear zones due to human saliva were consistent. The areas of clear zones due to human saliva were perfect circles in shape and were constant throughout the experiment. As compared to treatment by human saliva, diameters and areas of clear zones due to Bacillus subtilis were inconsistent. This may be due to uneven distribution of bacteria on each soaked paper disc. To minimise this inaccuracy, the diameter of the clear zone was measured horizontally, vertically as well as diagonally, and the sum of the three widths was divided by three to determine the mean diameter. A large sample of 10 replicates was taken to decrease the possibility of inconsistent data, such as due to irregular area of clear zones and to increase the reliability of results. Natural human saliva was used instead of synthetic human salivary amylase as the latter is unavailable in the laboratory. Furthermore, starch agar without nutrient medium was used to prevent multiplication of bacteria which results in increased production of enzyme, since human saliva which is non-living is unable to replicate and increase in amount. Thus, starch agar acted as a control variable which maintained the amount of bacteria in each Petri dish, and indirectly regulating the quantity of amylase secreted.

Modifications

This experiment could be modified by replacing natural human saliva with synthetic human salivary amylase. This is such as human saliva contains impurities such as electrolytes, mucus, antimicrobial compounds and various proteins that vary among individuals, especially since the composition of saliva is partially due to the genotype of individuals. The results would be more reliable as the concentration of enzyme present is known and fixed under all circumstances.

Besides that, the investigation could be carried out by replacing bacterial source of enzyme with fungal source of alpha-amylase. Common species of fungi that produce alpha-amylases are Aspergillus sp. and Rhizopus sp. [14] This requires starch malt agar, which is made by adding 100cm3 of 4% starch suspension to each 100 cm3 of malt agar. If Aspergillus sp. was used, a culture grown in malt extract broth for 7 days is required.[18]

Moreover, human saliva and culture of Bacillus subtilis can be mixed and the synergistic effects of both sources of alpha-amylase on hydrolysis of starch can be studied.

Conclusion

Experimental hypothesis is accepted. Bacillus subtilis is able to produce alpha-amylase, an extracellular enzyme to hydrolyse starch. Human saliva hydrolyses more starch than the culture of Bacillus subtilis as the former has a greater concentration of alpha-amylase.

Source Evaluation

Sources 1 to 3 are published text books by experts and are credible sources as their content would have been reviewed by other experts prior to publication. Therefore, the information from these books is reliable and useful.

Sources 4 to 14 are online scientific journals written by scholars and experts of their respective field. These journals must have undergone intensive reviews by professionals before being published, thus can be trusted. For instance, Source 4 is a journal on applications of alpha-amylase enzyme in industries. This study has been cited by many sources and this proves that the research is generally accepted by the scientific community. Source 7 is a website run by the U. S. National Library of Medicine which is a well-established organisation. The scientific literatures presented on this site include facts from the most reputable resources, thusare reliable.

Source 15 is a website of the London South Bank University. It is a research website from the Faculty of Engineering, Science and the Built Environment, hence the information is scientifically valid and trustable.


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