Combination Therapy of Probiotics and Chinese Herbs

Nov 20th, 2008 // Miscellaneous

Contents

1. Overview
2. Introduction
3. Safety Procedures
4. Materials and Methods
5. Results
6. Discussions
7. Conclusions
8. References
10. Annex

Overview

This was a science research project I did with a few of my friends back in 2003, when we were just 13-year-olds. In the following year, we completed the project and participated in the Biotechnology Fair 2004. Against over 70-plus upper-secondary teams from various top schools, we managed to clinched a Merit Award – Top 16! The content here is entirely taken from our research report.

Now, I know this isn’t so much of a laboratory project done AT HOME, nevertheless I thought it was worth sharing since it took a considerable amount of effort.

Team Members: James Hii, Derek Low, Shaun Ng, Joshua Chong
Sponsor Teacher: Ms Chow Wai Hoong
School: Bukit Panjang Government High School

Our team would like to express much gratitude to our mentor, Ms Quek Sern Fern, and Mrs Lee, Education Managers of JAF Enrichment Services, for her support, guidance and advice throughout the entire project. It was largely due to her help and advice that we were able to manage this project. Also, we would like to Ms Chow Wai Hoong, and JAF Enrichment Services and anyone else involved in some way or another. Thank you for all the support you gave us!

Our group’s aims were to breach the gap between western and eastern medical therapy. To do this, we studied how the different types of Chinese herbs would react with Lactobacillus, whether it would promote or retard the growth of Lactobacillus. To do this, 4X concentration of Yoghurt obtained Lactobacillus was added to four different herbs solution, Agastache Rugosa solution, Herba Oldenlandia Diffusa solution, Herba Scutellaria Babata solution and Cordyceps Sinensis solution. The four herbs solutions were labelled A, B, C and D respectively in this experiment. Also added in the four herb solution was 8ml of MRS agar to support the growth of Lactobacillus. Three different dilution of herbal solution were used, 10X, 20X and 1X. We analyze how the nature of the different herbs affects the growth of the Lactobacillus. We also investigated whether the varying concentration would make a difference. Furthermore we wanted to investigate whether Chinese and Western medicine can be mixed together for better results.

Difficulty: 1 2 3 4 5 6 7 8 9 10

Cost: Unknown

Time: 1 2 3 4 5 6 7 8 9 10

Introduction

Lactobacillus is the bacteria found in most yoghurt, especially the well-known Yakult drink. It is an essential element in the production of many diary products. A “probiotic” is a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Although referring to the supplementation of animal feeds for farm animals, the definition is easily applied to the human situation. The major consumption of probiotics by humans is in the form of dairy-based foods containing intestinal species of lactobacilli and bifidobacteria. It known that consumption of the probiotics affects the composition of the intestinal microflora. In this way, the probiotic benefits the consumer. A number of potential benefits have been proposed, including: increased resistance to infectious diseases, particularly of the intestine, decreased duration of diarrhoea, reduction in blood pressure, reduction in allergy, regression of tumours, etc.

Lactobacilli are Gram-positive, non-spore forming rods or coccobacilli (refer to diagrams 1.1 – 1.3). They have complex nutritional requirements and are strictly fermentative, anaerobic and acidophilic. Lactobacilli are found in a variety of habitats where rich, where carbohydrate-containing substrates are available, such as human and animal mucosal membranes, on plants or material of plant origin, sewage and fermenting or spoiling food.

We selected various herbs, taking into consideration their effects on the human body; or rather what kind of sickness they can cure. The following are the herbs used: ‘Herb A‘, Agastache Rugosa, which helps cure stomach aches, diarrhea and can even be used to prevent heat stroke. ‘Herb B‘, Herba Oldenlandia Diffusa, which can be used for clearing heat, fire toxicity, abscesses, toxic sores, ulcerations, swellings, snake bites, sore throat, bronchitis, tonsillitis, and hemorrhoids. ‘Herb C‘, Herba Scutellaria Babata, which can be used to treat Nasal Furuncle, Pustules, Snakebite, Hepatitis and even Cancer. Lastly, ‘Herb D‘ Cordyceps Sinensis, can be used to cure impotence, chronic lower back pain, risk of cold, over abundance of mucus and tears, chronic cough and wheezing. These Herbs were chosen because they shared some properties of lactobacillus, that is, they are used to treat the same symptoms and sicknesses.

In this experiment, we intend to find out if the growth, and probiotic functions of lactobacillus are affected by Chinese herbs.

Fig 1.1: Rod-shaped Lactobacilli

Fig 1.2: Gram-positive Lactobaciili

Fig 1.3: Gram-positive Lactobacilli

Safety Procedures

The following procedures were carried out to ensure sterile conditions so as to prevent contamination:

Our hands were washed with anti-bacteria soap and sterilized with 70 % alcohol.
Use of laminar flow.
Sterilization of laminar flow.
All apparatus and materials were autoclaved.
Only sterile equipment and materials were used.

To ensure safety, all bacterial wastes were autoclaved for 20mins at 121’C, 2atm prior to disposal.

Materials and Methods

Preparation of Herbal Solution

Before lactobacillus can be tested with the selected herbs, the herbs had to be prepared in a liquid form, in order to have an even distribution in the plate. To do so, we added 50 grams of dry herbs and 50 ml distilled water into a Duran bottle. The mixture was then autoclaved for 20 mins, 121OC and 2atm. After this was done, we opened the mixture in a laminar flow, in order to prevent contamination, and transferred the herbal extract into sterile falcon tubes for subsequent uses.

Preparation of Different Dilutions of Herbal Solutions

Different dilutions of herbal solutions were used in our project to see if the concentration of the herb affects the growth of lactobacilli. To do so, we used two dilutions, mainly 10X dilution and 100X dilution. To obtain the 10X dilution, we added one part of herbal solution to nine parts of autoclaved water. To obtain the 100 times dilution, we added one part of 10X diluted herbal solution to nine parts of autoclaved water. All the steps taken to dilute the herbal solutions were done in a laminar flow, so as to prevent contamination. We also shook the falcon tubes to ensure a more even and accurate dilution in each step.

Dilution of Yoghurt

The yoghurt was diluted to 4X concentration. This was done to ensure that the yoghurt solid particles do not interfere with the experiment.’ A sterile dropper was used to transfer 2ml of yoghurt solution into a sterile falcon tube. 6ml of autoclaved water was added into the falcon tube and the mixture was shaken well to dilute the yoghurt.

Preparation of MRS Agar

26 grams of MRS was weighted out into a Duran bottle. 5 grams of agar powder and 500ml of distilled water was added. Mixture was autoclaved for 20 mins, 121’C and 2atm.

Experiment to Find Out the Effect of Selected Herbs on Lactobacillus

In every set of experiments we did, triplicates were obtained. To make these triplicates of this experiment, many different materials were required. These materials include 39 petri dishes, 39 strips of parafilm, permanent markers, 12 sterile droppers, 500ml of MRS agar, 6 falcon tubes, 10-100ul micropipette, autoclaved micropipette tips, a packet of marigold yoghurt and 39 sterile inoculation loops.

Firstly, we labeled 39 plates, three times each for each herb and concentration, including a control experiment. Below are the details for each set of experiment:

YG (Yoghurt)
YG + Original Herb A
YG + Herb A (10X)
YG + Herb A (100X)
YG + Original Herb B
YG + Herb B (10X)
YG + Herb B (100X)
YG + Original Herb C
YG + Herb C (10X)
YG + Herb C (100X)
YG + Original Herb D
YG + Herb D (10X)
YG + Herb D (100x)

Next, we poured 1ml of herbal solution into their respective Petri dishes using sterile droppers, one for each concentration of herbal solution, thus preventing the chances of cross-contamination. Thirdly, using sterile falcon tubes, we added 8ml of MRS agar to each Petri dish. After this, the Petri dishes were swirled and left to cool and solidify.

After the MRS agar had solidified, 50?l of 4X diluted yoghurt was added into all the 39 petri dishes. After adding the yoghurt, a sterile inoculation loop was used to spread the yoghurt evenly across the plate and the petri dishes were sealed with parafilm. It was then left to incubate at 37’C for 48 hours.

Results

Date of experiment: 15/07/2004 400 microlitres

Date of experiment: 22/7/2005 (4X Dilution of Yoghurt ) 20 microlitres

Date of experiment: 24/7/2004 (4X Dilution of Yoghurt) 40 microlitres

Discussion

During the process of experimenting, we have collected many results and encountered many shortcomings. These results and shortcomings will be discussed below, in the discussion section.

At the beginning of the project, we hypothesized that the effect of the herbs chosen would either retard or promote the growth of lactobacillus. We also hypothesized that the dilution of these herbs would affect how much the herb retards or promotes its growth. From the results as shown above, it shows clearly that both the concentration and the nature of the herbs themselves affect the growth of lactobacillus greatly. In the cases of herbs A, B and C, it is seen that the herbs actually retard the growth of lactobacillus. This retardation causes very little or no growth in the plates. In the results collected, there was more growth seen on the plates as the dilution of the herb increased. Herb D, however, is an exception and seems to have properties which promote the growth of lactobacillus. In this case, the higher the concentration of herb D in the plates, the more colonies of lactobacillus were seen.

Also, during the course of experimentation, we noticed ho the count of the lactobacillus colonies were rather low. We suspected that this had to do with the viscosity of the yoghurt used. The milk solids present could also interfere with our results. To resolve this problem, we used a simple dilution method, diluting the yoghurt to a 4 times dilution, and as it turned out, we were right and the amount of colonies seen increased immediately.

During the course of any experiment, setbacks and shortcomings are inevitably present. Needless to say, we have encountered many of these shortcomings and have come up with solutions for our setbacks.

Our first and most serious setback was contamination in our experiment. Contamination could occur in any experiment, due to even the slightest carelessness or failure to comply with precautions. In our project, we noticed a large amount of contamination in the first few phases of experimentation. After a month or so, the amount of plates being contaminated had a sharp drop. This was probably due to the fact that we were more familiar with the safety precautions after carrying out a few experiments. Once, there was a mix up in the equipment we used and this led to much contamination in the plates, not to mention results which did not make much sense. After this incident, we learned to take more precautionary measures before starting any experiment.

Secondly, we experienced time constrains in many cases. Classes sometimes ended late for us, so some experiments had to be postponed. Personal matters also interfered with the experimentation period. Despite these constraints, we took into consideration the needs of each other, and used our free time to account for any time lost, so as to be able to proceed with the project. We learnt how to be interdependent through this setback, learning that no man is an island on its own.

Also, we collected many inconclusive and self-contradicting results from our experiments. These results were unexpected and we did not know how to counter this problem. In some of these experiments, we took into consideration of many external factors, and tried to reduce the way these factors could affect how the lactobacillus grew. Some of these factors include the temperature at which it is incubated, the anaerobic conditions required for it to grow and the type of medium used. After troubleshooting and finding more solutions to this problem for some time, we managed to find out how to grow lactobacillus better.

From these setbacks, we have learnt how to deal with problems and hiccups more effectively now, and troubleshooting a glitch is not a problem to us. These problems and setbacks, although require much effort and strength to overcome, are indeed very good experience for us.

Conclusion

From the results of our experiment, we can conclude that different herbs have different effects on the growth of lactobacillus. These effects include the retardation and promotion of lactobacillus growth. Therefore, eastern and western medicine should not be combined.

From this project, we have learnt many new things. We have learnt how external factors can affect the growth of bacteria. We have also grasped some basic skills in the culturing, experimenting and working with bacteria. These skills will be very useful in the near future, when Singapore expands its horizons, moving towards bio-technology. Most importantly, we have learnt that teamwork is very important. Without it, we would not have been able to complete this project on time, with many sets of conclusive results.

Further experiments could be carried out to test if there are other herbs that can be combined with lactobacillus, to make the probiotic effect even more effective.

References