Investigation of the Probiotic Properties of Bacterial Strains from Two Probiotic Drinks and Their Survivability in Artificial Gastric Juice

Investigation of the probiotic properties of bacterial strains from two probiotic drinks and their survivability in artificial gastric juice ABSTRACT: Two probiotic drinks were investigated in vitro to test their ability to survive acidic conditions and their probiotic factors. Both the products: Actimel and Yakult contain gram-positive bacteria, but Actimel also has a gram-negative bacteria. The ability to survive was investigated by adding artificial gastric juice to the products and incubating at different times.Actimel and Yakult were both able to survive the gastric juice. Actimel produced more colonies than Yakult but they both lost the same percentage of viability. The longer the time incubated the more the loss of viability. Introduction: In recent years health promoting functional foods has entered the global market as a result of increased prevalence of lifestyle related diseases (A. A. Aramide et al, 2009). People use functional foods and diet to maintain optimal health. C onsumption of probiotics is one of the ways someone could reach and maintain their optimal health.A probiotic is â€Å"living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition† (Todd R. Klaenhammer, 2000). According to the WHO/FAO report 2001 these probiotics can help prevent disorders associated with the gastrointestinal tract, diarrhoea caused by certain pathogenic bacteria and viruses, inflammatory diseases, allergies and a lot more. Actimel and Yakult is a couple of the said probiotic drinks. They claim to increase your body’s natural defences by fighting off the â€Å"bad† bacteria. Actimel is a yogurt-type drink produced by a company called Danoneâ„ ¢.It has three strains of bacteria, two traditional yoghurt cultures: Lactobacillus bulgaricus  and  Streptococcus thermophiles and a third one called L. casei Imunitass ® (http://www. actimel. co. uk/About/-WhatIsActimel. aspx, Accessed Feb, 28, 2010). Lactobacillus is a genus of bacteria that aid in the conversion of lactose to lactic acid hence increasing acidity in the stomach making it hard for harmful bacteria to survive (http://en. wikipedia. org/wiki/Lactobacillus, Accessed Feb 28, 2010). Actimel contains 10 billion L. casei Imunitass ® bacteria per 100ml bottle.This bacterial strain works under a wide range of pH and temperature hence able to survive the acidic conditions in the stomach. This ensures that the bacteria reach the gut alive and active. It helps by topping up the good bacteria in the stomach and making it hard for the germs to survive. The bacteria also aids in strengthening the gut wall so that only certain nutrients can pass. In 2004 a trial carried out to find the effect of Actimel on the immune response of subjects under academic examination stress showed that Actimel was able to control the number of lymphocytes and CD56 cells in subjects under academic examination stress.Other studies also show that the Actimel bacterial strains can be used in treating allergic rhinitis, prevention of diarrhoea and induce immune responses. On the other hand Yakult is milk based probiotic and contains only one strain of bacteria: Lactobacillus  casei  Shirota. It is produced and distributed by Yakult Honsha Co. Ltd. It contains 6. 5 billion L. casei Shirota per 65ml bottle. A variety of scientific studies have shown that Yakult has an effect on the human NK-cell activity, intestinal micro flora and immune parameters in humans.As a guideline a probiotic microorganisms should be resistant to gastric juices and be able to grow in the presence of bile under conditions in the intestines. The aim of this experiment is to measure the survivability of the strains in artificial gastric juice and to identify the bacterial strains said to be in the product. MATERIALS AND METHODS: Gram Stain: Firstly the bacteria were heat fixed according to the instruction in the lab manual. After heat fixing , crystal violet stain was added to the bacteria for 2 minutes, then washed in water and Lugol’s iodine for 30 seconds.The bacteria were decolorised by adding 95% alcohol for 15 seconds followed by a water wash and counter stain with safranin for 1 minute. This was then washed with water and examined under high power (x100) using oil immersion. A picture of these strains each from Actimel and Yakult directly and pure culture was taken. DNA Extraction: To extract the DNA, 1 ml of culture was centrifuged for 5 minutes. The pellet was re suspended in 480 ? l of 50mM EDTA with 60 ? l of 10mg/ml lysozyme then incubated at 370C for 45 minutes, centrifuged for 2 minutes and re suspended in 600 ? of nuclei lysis solution and incubated at 800C for 5 minutes. After cooling down 3 ? l of RNAase was added and left to incubate at 370C for 30 minutes. The mixture was left to cool and 200 ? l of protein precipitation solution was added, left on ice for 5minutes followed by high speed (13000 rpm) centrifuging for 5 minutes. The supernatant was then added to 600 ? l of isopropanol and mixed until DNA â€Å"threads† were formed and centrifuged for 15 minutes. The DNA pellet was washed with 200 ? l of 70% ethanol and centrifuged for 2 minutes. The ethanol was then removed and the DNA left to air dry and then re suspended in 50 ? of sterile water. PCR of chromosomal DNA: A 2 ? l of the DNA was added to 1 ? l of AmpF primer(GAGAGTTTGATYCTGGCTCAG), 1 ? l of AmpR (AAGGAGGTGATCCARCCGCA) primer, 2 ? l of dNTP’s, 10 ? l of x10 PCR buffer, 83 ? l of water and 1 ? l of Taq polymerase was added. This mixture was placed in the Promega Wizard Chromosomal DNA preparation kitâ„ ¢ and run according to the manufacturer’s guidelines. PCR Purification: The PCR reaction contents were added to a 1. 5 ml Eppendorf tube with 500 ? l of buffer PB1. This was centrifuged at high speed in the spin column for 30 seconds.A 750 ? l of buffer PE was added to the spin column and centrifuged for 1 minute. The spin column was then placed in an Eppendorf tube and 50 ? l of water was added and centrifuged for a further 1 minute. A 15 ? l of this PCR product was added to 5 ? l of Gel loading buffer and was run at 50 V for 2 hours. 20 ? l of the PCR product was then sent to the John Innes sequencing service for sequencing. Media Preparation: To media was prepared by adding 37g of Brain Heart Infusion (BHI) to 1 litre of distilled water and mixed using a magnetic stirrer.This was then added to a conical flask with 3g of agar and autoclaved at 1210C, 15 psi for 10 minutes. The media was then microwaved and poured onto petri dishes with Bunsen burner going, to sterilise the air around. Survival Studies: For carrying out the survival studies, 5 ml of the product was added to 25 ml of artificial gastric juice and left to incubate at 370C for 30, 60 and 90 minutes. The product was taken from different bottles to ensure replicates. After incubation the mixture was then diluted to 10-5 for Yakult and 10-7 for Actimel. This was spread onto a petri dish and was left to incubate.The plates were then counted and the number of CFU/ ml was calculated. RESULTS: Culturing bacteria: Firstly the number of colony forming unit (cfu) per ml was worked out by culturing the bacteria from the probiotic products and counting the number of colonies formed. This was then used to work out cfu/dose by using the volume they are produced in, which are 100 ml and 65 ml of Actimel and Yakult respectively. Table 1: Class data of cfu/ml and cfu/dose of bacteria in the product Yakult(cfu/ml)| Yakult(cfu/dose)| Actimel(cfu/ml)| Actimel(cfu/dose)| 4. 21. x 109| 2. x 1011| 4. 36 x 109| 4. 36 x 1011| 4. 14 x 109| 2. 86 x 1011| 2. 6 x 108| 2. 6 x 1010| 9. 7 x 10 9| 7. 8x 1010| 2. 1 x 109| 2. 1 x 1011| 1 x 109| 6. 3 x 109| 7. 5 x 108| 7. 5 x 1010| 1. 6 x 109| 6. 5 x 1010| 5. 5. 2x 108| 5. 5 x 1010| 9 x 107| 5. 8 x 109| 1 x 1010| 1 x 1012| 7 x 107| 4. 5 x 109| 2. 5 x 109| 2. 5 x 101 1| 4. 6 x 109| 2. 99 x 1011| 1. 21x 109| 1. 21x 1011| 1. 68 x 108| 1. 09 x 1010| 4. 3 x 1010| 4. 3 x 1012| 4. 02 x 108| 2. 61 x 1010| 1. 18 x 109| 1. 18 x 1011| 9. 1 x 107| 5. 9 x 109| 2. 89 x 108| 2. 89 x 1010| 1 x 108| 6. 5 x 109| 2. 7 x 109| 2. 7 x 1011| x 108| 3. 2 x 1010| 3. 6 x 109| 3. 6 x 1011| 3. 4 x 107| 2. 2. x109| 2. 7 x 109| 2. 7 x 1011| 2. 39 x108| 1. 5 x 1010| 3. 78 x 109| 3. 78 x 1011| 9. 7 x 107| 6. 3 x 109| 5. 0 x 1010| 5. 0 x 1012| 1 x 108| 6. 5 x 109| 1. 4 x 109| 1. 4 x 1011| 1 x 108| 6. 5 x 109| 2. 6 x 109| 2. 6 x 1011| To compare the mean differences between these two products an independent t test was carried out assuming equal variance. Table 2: Independent t-test of the class data for cfu/dose on Actimel and Yakult Independent t-test| | | Mean| Standard Deviation| SE Mean| P Value| cfu/dose| Actimel| 7. 9 x 1011| 1. 45 x 1012| 3. 41 x 1011| 0. 056| | Yakult| 6. 29 x 1010| 1. 04 x 1011| 2. 46 x 1010| | The mean shows that Actimel contains 10 times more bacteri a than Yakult on average. But only the mean is not significant to come to a conclusion as this could be because of sample variation. The P value from the t-test is 0. 056 which is greater than 0. 05 (P>0. 05) hence the difference between the mean of the two products are not significantly different from zero at the 5% confidence level. Gram Stain: Figure 1 shows the gram stain images from Actimel (i) and Yakult (ii).Figure 1 shows the gram stain images from Actimel (i) and Yakult (ii). Gram stained slides of both Actimel and Yakult were captured onto a computer at x1000 magnification. From the images you can see that Yakult is stained all in one colour but the Actimel contains two different coloured stains. Survival studies: To test the survivability of the bacteria they were incubated with artificial gastric juice for 30 60 and 90 minutes. The colonies were then counted Table 3: Viable counts of survival studies at different time and different replicates | Actimel|Time/min| 1| 2| 3| Mean| CFU/ml| CFU/dose| 0| 329| 69| 1088| 371. 5| 3. 72 x 1010| 3. 72 x 1012| 30| 321| 39| 880| 322. 5| 3. 23 x 1010| 3. 23 x 1012| 60| 309| 28| 740| 286. 8| 2. 87 x 1010| 2. 87 x 1012| 90| 204| 24| 642| 238. 8| 2. 39 x 1010| 2. 39 x 1012| | Yakult| | 1| 2| 3| Mean| CFU/ml| CFU/dose| 0| 312| 135| 53| 125. 0| 1. 25 x 108| 8. 13 x 109| 30| 190| 134| 11| 96. 3| 9. 63 x 107| 6. 26 x 109| 60| 159| 130| 11| 92. 5| 9. 25 x 107| 6. 01 x 109| 90| 149| 84| 8| 81. 5| 8. 15 x 107| 5. 3 x 109| The table shows that colonies on both Actimel and Yakult decrease over time in all the replicates.Both the products decreased to about 65% of its original count. A graph (Figure 2) was plotted with the CFU/dose against time on a log scale and it showed a linear decline over time in both the products. DNA Extraction: Figure 3 shows the Chromosomal DNA gel image. Figure 3 shows the Chromosomal DNA gel image. The DNA from the bacteria was extracted and gel electrophoresis was carried out to ensure that a DNA was obtained from the extraction procedure. Lanes 3 and 4 have migrated towards the positive side showing that chromosomal DNA was obtained.PCR Purification: After the DNA underwent the PCR process, the PCR product was purified and run on a gel electrophoresis to check if PCR product has been obtained. Figure 4 shows the image of PCR product run under electrophoresis. Figure 4 shows the image of PCR product run under electrophoresis. As the image shows there is a PCR product obtained as there is a distinct band in lanes 2 and 3. DNA Sequencing: The PCR product was then sent to the John Innes centre for sequencing and the following sequence was obtained.Actimel: GGGTCGGGGCGGGTGCTATACATGCAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCC AAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA Yakult: TAGGAGTGGGCGCGTGCCTATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC CACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGCCCGCACAAGCGGTGGGA Figure 5 shows the graphical summary of â€Å"strong† hits in the database of Yakult (i) and Actimel (i). Figure 5 shows the graphical summary of â€Å"strong† hits in the database of Yakult (i) and Actimel (i).This sequence was then run through the BLAST analysis to identify the probiotic isolate. Discussion: A Probiotic must be able to survive the conditions of the stomach and pass through to the gu t without significant loss. The bacteria found in the probiotics are cultured on petri dishes to test the amount of colonies present in the product. As mentioned above Actimel contains 10 billion per 100 ml and Yakult contains 6. 5 billion per 65 ml. From the t-test there was no significant difference in the content of the two products (Table 1). This was due to the fact that they both contain 100 million bacteria per ml of product. From the gram stain images both Actimel and Yakult was stained with the same conditions.But Yakult had only one stain whereas Actimel had two different stains. This is due to the fact that there is more than one species of bacteria in Actimel. The colour of the staining represents two different types of bacteria: gram-negative and gram-positive. All species of the lactobacillus genus are gram-positive. Gram-positive organisms retain the stain when they are stained with crystal violet but gram negative organisms lose their purple/violet stain when washed with alcohol but when retain safranin stain. Therefore the Yakult contains only gram positive bacteria (L. casei Shirota ®) while Actimel contains both gram positive and gram negative bacterium (Figure 1). From the survival studies we can