Efficiency of Commercial and

Natural Honey on Growth

Inhibition of Escherichia coli and Lactobacillus casei

Alexander Ken Libranza Florijean Aguelo

Shem Rufus Sentones Group 1 Section A-1L

LITERATURE REVIEW

• From an unpublished reaction paper of Bilbao, Roniel, et.al. for Biology I, SY 2011-2012, 1st semester.

• Escherichia coli is a bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls.

• Lactobacillus casei found in dairy products like Yakult, is a beneficial bacterium that is found naturally in both the mouth and intestines of human beings. L. casei is a type of bacteria that helps protect the human body from disease and illness. It produces lactic acid which helps lower pH levels in the digestive system and impedes the growth of harmful bacteria.

• Honey is a sweet food made by bees using nectar from flowers. The variety produced by honey bees is the one most commonly referred to and is primary food source in wax honeycombs inside the beehive.

LITERATURE REVIEW

• The main difference between regular and natural honey is that regular honey is pasteurized and filtered. Commercial honey is smooth and uniform in color. Natural honey is milky in color and may have granules that can be melted in warm water, if desired.

• The zone of inhibition is simply the area on the agar plate that remains free from microbial growth. The size of the zone of inhibition is usually related to the level of antimicrobial activity present in the sample or product - a larger zone of inhibition usually means that the antimicrobial is more potent.

• According to Peter C. Molan (2001), honey may be the “natural cure” for most bacterial infections that could replace man-made antibiotics.

• Researchers say that an enzyme in the honey turns into a tiny amount hydrogen peroxide when combined with bodily fluids killing nearby bacteria. Honey also causes an increase in lymphocyte and phagocyte activity (Cooper et al.,2011).

SIGNIFICANCE OF THE STUDY

• Provide comparison between the antibacterial effect of Natural honey and Commercial honey.

• Pathogenic bacteria such as E. Coli are common food safety threat in the country. Unfortunately, most of these bacteria are resistant to antibiotics. With the specific protein found in honey, called defensin-1, which could combat antibiotic-resistant infections – studies could be further conducted.

• Bee farming can be a good source of income. Visayas and Mindanao are suitable for bees, which are used for honey production. With our perfectly weather condition, there is an immense potential of honey production in the world market (as an agricultural product and as an antibacterial agent)

• Probiotics hold great promise for research on its benefit for digestion and other body processes. Honey as a prebiotic helps stimulate the growth of this probiotic bacteria in the body.

• Promote Natural and Healthy lifestyle through consumption of Natural Honey over commercialized and processed honey.

OBJECTIVES

This comparative study aimed to reinforce and further breed information on microbial growth inhibition and prebiotic microbial growth between commercial and natural honey in E. coli bacteria. The specific objectives were: 1. to determine what type of honey is more effective in

inhibiting the growth of E. coli; • Type of nectar 2. to determine if honey with its antimicrobial effect

inhibits the growth of L. casei or stimulates microbial growth and ;

3. to identify factors that affected the antimicrobial and prebiotic effect of honey.

• Temperature

Figure 3. Placing the E. coli unto the nutrient agar plate using a pipettor.

Fig 1. Materials utilized

Fig 2. Pasteurizing the Natural honey

MATERIALS AND METHODS • Twenty-one nutrient agar plates were prepared, six of which

corresponds to two control set-ups, each with three replicates. The remaining twelve served as experimental set-ups ---- six for the natural honey – two set-ups with three replicates each, and another three for the commercialized honey – two set-ups with three replicates each.

• A 20mL natural honey combined with 20mL 30% sucrose solution was pasteurized using an alcohol lamp as shown in Fig 2. This was done to constitute a difference for comparison for the natural honey. The pasteurized honey will be the representative of a commercial/ regular honey.

• Six controlled set-ups were prepared, three for the E. coli and another 3 for the L. casei. The cultured bacteria were streaked on the nutrient agar gel plates; a piece of filter paper that was dipped in distilled water was placed on the center of the plates.

MATERIALS AND METHODS • One mL of cultured Escherichia coli and a bottle of

Lactobacillus casei from Yakult was carefully placed into each plate (half-way open) by the use of a pipettor as shown in Fig 3.

• The microorganisms was streaked in all areas of the nutrient water agar gel by the use of a sterilized inoculating loop. The same procedure was done to all nutrient water agar gel. This was performed near an alcohol lamp to avoid contamination of the cultured microorganisms by other microscopic organisms and impurities, including other strains of airborne bacteria.

• A small circular filter paper was dipped in natural honey and was placed at the center of the nutrient agar plate by the use of forceps.

MATERIALS AND METHODS • Three wild honey replicates were prepared for E. coli

and L. casei. Another small circular filter paper was dipped in commercialized honey and was placed at the center of the remaining nutrient agar plates.

• Three replicates were also prepared for each bacterium. The eighteen nutrient water agar gels were then stored in a cabinet at room temperature.

• After 3 days, the diameter of the diffused commercialized and natural honey of each nutrient water agar gel was measured by the use of a ruler in centimeters. Results were then recorded and tabulated.

RESULTS AND DIISCUSION

Table 1. Diameter of the zone of inhibition (in cm.) of the E. coli culture agar plates with corresponding type of treatment after 3 days.

Replicates

Treatment

T0 - Control T1 - Natural honey T2 - Commercial Honey

1 NA 3.4 1.7

2 NA 5.9 0.8

3 NA 3.2 1.7

Average NA 4.17 1.4

• As seen in Table 1, the zones of inhibition in each of the E. coli culture agar plates with the corresponding types of honey induced are presented. The control setup and its two other replicates served as the basis for any change or reaction in the setups as a whole. In the other six setups, three setups were prepared for natural honey cultures (T1) and three setups for commercial honey cultures (T2) showed positive results of the formation of distinguishable zones of inhibition in areas surrounding the original site of honey introduction. T1 showed diameters of zones of inhibition ranging from 3.2 to 5.9 cm, while T2 showed diameters of zones of inhibition ranging from 0.8 to 1.7 cm.

• Measuring for the average diameter, results show that the average diameter of T1 was 4.7 cm and 1.4 cm for T2. This significant difference indicates that the T1 (natural honey) was more effective in inhibiting the growth of E. coli compared to T2 (commercial honey).

Fig 4. E. coli culture control set-ups: on the conduct and 3 days later

Fig 5. E. coli culture experimental set-ups: on the conduct and 3 days later

Natural Honey Commercial Honey

• Fig 4. and Fig 5. illustrates growth of E. coli bacterium around the nutrient agar plate.

• In Fig 4. It shows no zone of inhibition around the filter paper dipped in distilled water. The main reason for this is water as a medium does not inhibit the growth of microorganisms like E. coli, instead it promotes microbial growth. Water, as a mineral is an important nutritional requirement along with oxygen and temperature are just some of the factors that influence bacterial growth.

• Fig 5. on the other hand, showed positive result on the formation of distinguished zones of inhibition. The efficacy can be attributed to the composition of natural honey in which the enzymes found in the natural honey were unaffected by purifications and processes that the commercial honey was subjected to. Also bioflavonoids, which exhibit antimicrobial effects, are found in great variety in the natural honey due to its multifocal origin rather than the single floral origin exhibited by the commercial honey (Lindauer, 2001).

Table 2. Diameter of the largest L. casei colony (in cm) and its equivalent number of colonies with

corresponding type of treatment after 3 days.

Replicates

Treatment

S0 - Control S1 - Natural honey S2 - Commercial Honey

Diameter of

largest colony

No. of

colonies

Diameter of

largest colony

No. of

colonies

Diameter of

largest colony

No. of

colonies

E1 0.79 16 2.7 47 1.2 22

2 1.1 8 1.7 42 1.5 26

2 0.9 24 1.3 37 1.5 18

Average 0.93 16 1.9 42 1.4 22

• Table 2 shows the zones of inhibition in each of the L. casei culture agar plates with the corresponding types of honey induced are presented.

• The control set-up (S0) and its two other replicates served as the basis for any change or reaction in the setups as a whole.

• In all the 9 setups, three setups were prepared for natural honey cultures (S1) and three setups for commercial honey cultures (S2) showed NEGATIVE results of the formation of distinguishable zones of inhibition in areas surrounding the original site of honey introduction, instead, honey stimulates the growth and activity of bacteria that after 3 days, numerous colonies were formed and there is no zone of inhibition around the introduced substance.

• Here we can say that honey is a prebiotic. The table showed the number of colonies and the diameter of the corresponding largest colony in each treatment. S1 exhibited the largest average diameter and number of colonies (1.9cm, 42 colonies) than S0 (0.93cm, 16 colonies) and S2 (1.4cm, 22 colonies).

Fig 6. L. casei culture control set-ups: on the conduct and 3 days later

Fig 7. L. casei culture experimental set-ups: on the conduct and 3 days later

Natural Honey Commercial Honey

• Fig 6. and Fig 7. illustrates colonial growth of L. casei bacterium around the nutrient agar plate.

• All set-ups showed positive result on the stimulated growth of the bacteria but NEGATIVE result on the formation of zones on inhibition. The efficacy can be attributed to the Pre-biotic nature of honey.

• According to heathwise.com, honey stimulates the growth of digestive flora – the good bacteria in our intestines. It is assumed that a prebiotic should increase the number and/or activity of bifid bacteria and lactic acid bacteria. The presence of these good bacteria within our bodies helps to maintain a healthy digestive system.

CONCLUSION & REMCOMMENDATION

• it is shown that Natural honey is more effective as an antimicrobial agent and as a prebiotic instrument. It is also shown that honey with its antimicrobial effect does not inhibit the growth but stimulates the growth and activity of L. casei bacterium.

• Many factors have affected the result of the experiment like interval of time for observation and some humanly unpredicted errors. It is recommended that other confirmatory tests be under taken due to the multiple weaknesses of zone of inhibition testing.

CONCLUSION & REMCOMMENDATION

• Bee farming could be a good source of income. Raw honey, as proven to have a number of health benefits could help alleviate the bee farming industry. Whether it be for natural or commercial purposes, honey is still has a great market value in any types of industry.

• According to the department of trade and Industry, Honey production is an existing livelihood activity in the rural areas of Davao City and Davao del Norte but a government study showed that this remains a backyard activity. Despite the high market price which should have been an incentive for more commercialized production, bee farming was put into rejection.

• From health benefits to market and economic growth, bees are seen to be a source of nit just both, but a number of yet to be explored benefits.