ZYACTINASE STIMULATES THE PROBIOTIC GUT MICROFLORA WHILST INHIBITING PATHOGENIC MICROFLORA

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1 International Journal of Probiotics and Prebiotics Vol. 3, No. 4, pp , 2008 ISSN print, Copyright 2008 by New Century Health Publishers, LLC All rights of reproduction in any form reserved ZYACTINASE STIMULATES THE PROBIOTIC GUT MICROFLORA WHILST INHIBITING PATHOGENIC MICROFLORA 1 Iona. E. Weir, 2 Robert Peng, 3 Michael L. Bian, 4 Kawal Matharu and 5 Quan Shu 1 Vital Foods Processors Ltd, 70 Ascot Road, Airport Oake, Auckland, New Zealand; 2 Bioactives Research New Zealand Ltd, Mt Albert Research Centre, 120 Mt Albert Rd, Mt Albert, Auckland, New Zealand; 3 Bioactives Research New Zealand Ltd, Mt Albert Research Centre, 120 Mt Albert Rd, Mt Albert, Auckland, New Zealand; 4 Vital Foods Processors Ltd, 67 Airport Oaks Rd, Mangere, Auckland, New Zealand; and 5 Bioactives Research New Zealand Ltd, Mt Albert Research Centre, 120 Mt Albert Rd, Mt Albert, Auckland, New Zealand [Received September 1, 2008; Accepted October 30, 2008] ABSTRACT: Zyactinase a freeze-dried extract of Kiwifruit (Actinidia deliciosa) has been developed as a constipation relief product as well as for long-term gut health. Zyactinase contains a protease complex, fiber, pectin and fructo-oligosaccharides. Clinical Studies have proven that Zyactinase stimulates increased bowel movements and relieves the symptoms of irritable bowel syndrome. This was proposed to be partially due to the stimulation of the gut microflora. To investigate this, the effect of Zyactinase on the growth of the probiotic bacteria Lactobacillus reuteri, Lactobacillus acidophilus, Pediococcus acidilactici, and Lactobacillus planetarium was tested in vitro. Zyactinase was found to significantly increase the growth of these probiotic bacteria, in comparison to isomalt. Zyactinase was also tested in vitro against the pathogenic bacteria E. coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Zyactinase was found to significantly inhibit the growth of E.coli and Salmonella typhimurium with almost significant inhibition of Staphylococcus aureus. However, Zyactinase stimulated the growth of Listeria monocytogenes. Heating of the extract to deactivate the protease complex had no effect on the efficacy of Zyactinase to stimulate probiotic bacteria growth whilst inhibiting pathogenic bacterial growth. Therefore, it is likely that the probiotic-stimulating efficacy of Zyactinase originates from the fructo-oligosaccharides and pectin within Zyactinase. KEY WORDS: Constipation, Kiwifruit, Prebiotic, Zyactinase Corresponding Author: Dr. Iona. E. Weir, Vital Foods Processors Ltd, 70 Ascot Road, Airport Oake, Auckland, New Zealand; Tel.: ; Fax: ; ionaweir@clear.net.nz INTRODUCTION Zyactinase is a patented freeze-dried kiwifruit (Actinidia deliciosa) extract that has been developed for the relief of constipation, irritable bowel syndrome (IBS) and general longterm gut health. Zyactinase contains microbial growth stimulating fructo-oligosaccharides, insoluble fibre and a protease complex that has been shown in in vivo studies to stimulate gut motility (unpublished observation). As Zyactinase has been significantly shown to clinically relieve constipation (unpublished observation) we wanted to examine whether part of this efficacy related to a prebiotic or microbial growth stimulating component of Zyactinase that we surmised was due to the fructo-oligosaccharides. In particular, we needed to determine whether Zyactinase stimulated the growth of the probiotic bacteria with specific health properties such as stimulating gut motility and preventing pathogenic bacterial growth. Furthermore, it was considered necessary to identify whether this microbial growth stimulating bioactivity was specific to the probiotic bacteria, or would Zyactinase also promote the growth of the pathogenic bacteria in the gut and thus be of a detrimental effect or would it inhibit growth of the pathogenic bacteria. Probiotic bacteria are well known as beneficial bacteria that promote gastrointestinal health and are developed by the selection of efficacious strains suitable for human consumption (Brown and Valiere, 2004; Gill et al., 2002). These probiotic bacteria are then encapsulated to survive the gastro-intestinal tract, and sold commercially as a powder, in capsules or within food such as yoghurt. In contrast, foods which promote the growth of probiotic bacteria within the gut are known as prebiotics and have been shown to improve gastro-intestinal function and immune-modulation as well as preventing gastrointestinal infections (Drakes et al., 2004; Ghosh et al., 2004; Hart et al., 2004; Mohamadzadeh et al., 2005; Molis et al., 1996; Shu and Gill, 2002). To validate the efficacy of Zyactinase as a prebiotic we examined whether Zyactinase promoted the

2 232 Zyactinase promotes probiotic growth growth of the critical probiotic bacteria whilst inhibiting the growth of potential pathogenic bacteria. Four commonly used probiotics were selected based on their role in gastrointestinal health as set out below. The first probiotic bacterium selected was Lactobacillus reuteri that is indigenous to the gut, and helps to strengthen the body s natural defenses against harmful bacteria and maintain equilibrium in the gastrointestinal tract by repopulating the non-pathogenic microflora in the gut. L. reuteri secretes reuterin, a substance with antimicrobial properties that helps suppress the growth of pathogenic microorganisms in the gut (Cleusix et al., 2007; Talarico et al., 1988). The second probiotic bacterium of interest was Pediococcus acidilactici a well-known probiotic strain that improves gastrointestinal health by accelerating the rates of lactic acid production and decreasing the gastro-intestinal ph (Brown and Valiere, 2004; Pena et al., 2005). Lactobacillus acidophilus was chosen as it readily adapts to the human gut and has many health benefits including immuno-modulation, symptomatic relief in mucous colitis, irritable colon, idiopathic ulcerative colitis, and various disorders complicated with constipation and biliary symptoms (Brown and Valiere, 2004; Drakes et al., 2004). Lactobacillus plantarum, the fourth probiotic tested colonizes from the saliva of the mouth through to the gut mucosa, and is commonly found in fermented plant material. Research has demonstrated that L. plantarum by its fermentation activity reduces abdominal bloating associated with IBS (Bekkali et al., 2007). L. plantarum is also important in immune-modulation of the gut mucosa and in particular in reducing mucosal inflammation. Thus, L. plantarum plays a major role in managing IBS (Brown et al., 2005; Molis et al., 1996). To investigate the potential inhibition of pathogenic microbial growth by Zyactinase, four common food-borne pathogenic strains were selected which were: 1) E.coli O157:H7; 2) Salmonella typhimurium; 3) Staphylococcus aureus and 4) Listeria monocytogenes. These four bacterial strains are all enteric pathogenic bacteria, in which E.coli O157:H7, Salmonella typhimurium are gram-negative, and Staphylococcus aureus and Listeria monocytogenes are grampositive. Our findings show that Zyactinase had a significant stimulating effect on the growth of the probiotic bacteria, whilst inhibiting the growth of the pathogenic bacteria. MATERIALS AND METHODS Probiotic bacterial strains Lactobacillus reuteri, Lactobacillus acidophilus, Pediococcus acidilactici, and Lactobacillus plantarum from Bioactives Research New Zealand (BRNZ) culture collection were used in this study as representative strains Reagents MRS broth, yeast extract, bacteriological peptone, lactose, and trypticase (Oxiod, England). L-cysteine hydrochloride, K 2 HPO 4, KH 2 PO 4, NaHCO 3, NaCl, CaCl 2, and MgSO 4 (Sigma, USA). Zyactinase and Phloe extract Zyactinase powder supplied by the manufacturer Vital Foods Processors Ltd was mixed with ethanol or water at a concentration of 1g per 20 ml. Phloe is the commercially available form of Zyactinase, and contains Zyactinase (81.82%) plus excipients including isomalt (15.45%), silica dioxide (2%) and magnesium stearate (0.73%). This solution was gently rotated at either 36 C or 45 C for 24 h. After 24 h, the solution was then centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.45 µm sterile filter and then stored in the freezer for further use as the Zyactinase extract. The isomalt was provided in encapsulated granule form. 10% (w/v) ethanol extracts were prepared by taking 1.0 g of the powder and dissolving in 10ml of Milli-Q water and gently rotated and incubated at either 36 C or 45 C for 24 hours. Bacterial Growth Medium MRSC broth: 5.2 % (w/v) MRS -water solution containing L-cysteine hydrochloride 0.02 % (w/v) was autoclaved. The PY salts solution was prepared according to Holdeman and Moore (1972), with the PY (Peptone, yeast-extract) broth containing 10 g of bacto-yeast extract; 10 g of bacteriological peptone, and 40 ml of the salts solution in 930 ml pre-boiled distilled H 2 O. The PYL broth was autoclaved and cooled to 50 C. To the cooled media, 20 ml of 40% lactate, 10 ml of 5% cysteine-hcl and 160 μl of resazurin solution (5 mg/ml) were added to the PY broth aseptically. Finally, the ph was adjusted to 6.0 ± 0.1 with 0.1M NaOH. The prepared PYL broth was subsequently stored in 4 C fridge overnight for pre-reduction of oxygen. Bacterial culture The bacteria were incubated with the Zyactinase extract in PYL broth and the optical density (OD) value was measured using a Genova UV/Vis Spectrophotometer SS-002 Jenway, UK. Bacteria were cultured in MRSC broth at 37 C for 20 hours. A 0.1 ml aliquot of the culture was transferred into 5 ml of PYL broth (ph 6.0 ± 0.1) containing Zyactinase extract (final concentration 0.5 % (w/v)), and incubated at 37 C for 20 hours. PYL broth without bacteria was used as a blank for zeroing the OD value. PYL broth without Zyactinase extract was used as the control. Since Zyactinase is slightly turbid, the background turbidity was determined by subtracting the sample OD from the control OD. After incubation, each of the broth were mixed well prior to measuring the OD (610 nm) value that was indicative of the growth of the bacteria. The experiment was carried out in triplicate, twice. Pathogenic Bacteria Common food-borne pathogen strains used included E.coli O157:H7 (strain 2988), Salmonella typhimurium

3 Zyactinase promotes probiotic growth 233 (ATCC1772), Staphylococcus aureus (ATCC 2592) and Listeria monocytogenes (Scott A ATCC49594), which were obtained from Bioactives Research New Zealand. Pathogens were first recovered from -80 C, with Brain Heart Infusion (BHI, Oxoid, England) broth. A loop full of each thawed pathogen was transferred aseptically to 5 ml of BHI in respectively marked 20ml bottles, and then incubated at 37 C for 20 h. After 20 h incubation, pathogen growth was checked using a haemocytometer via light microscopy to confirm log-phase state. A 0.1 ml aliquot of 1/4 diluted pathogen culture was transferred into 4 ml of BHI broth (ph 7.4 ± 0.2) containing 0.1ml of each extract (final concentration 0.25 % (w/v), and incubated at 37 C for 20 h. BHI broth with 0.1 ml 1/4 diluted pathogens only was used as negative control. No positive control was assigned. Pure BHI broth was used as a blank for equilibrating the spectrophotometer (Perkin Elmer, Sydney) to zero optical density (OD) value. After incubation, each extracted sample was mixed well prior to reading on the spectrophotometer; OD values were measured at wavelength 610 nm at room temperature. The OD values collected from the spectrophotometer at 610nm, are indicative of pathogen growth (higher the optical density, greater the number of living bacteria). All controls and samples had to be diluted 2 times with Mili-Q water in order to remove the turbidity and obtain zero for blanks. The test was carried out in triplicate, with the results analyzed using t-test. FIGURE 1. The effect of either water or ethanol Zyactinase extracts 0.5 % (w/ v) on the growth of Lactobacillus planetarium (n = 3). Vertical errors bars represent the standard error of the mean. * P < 0.05, ** P < All treatments were significantly higher than the comparative blank control and compared to placebo (isomalt) control. FIGURE 2. The effect of Zyactinase water and ethanol extracts 0.5 % (w/v) on the growth of Pediococcus acidilactici (n = 3). Vertical errors bars represent the standard error of the mean. ** P < All treatments produced significantly higher growth than the control and the placebo isomalt. The water placebo (isomalt) was significantly higher than the blank control. RESULTS Concentration-related effect of Zyactinase A dose response curve of Zyactinase water and ethanol extracts (0, 0.1%, 0.25%, 0.5%, 1.0% and 2.0% w/ v) was undertaken and revealed no concentrationrelated effect of Zyactinase. Therefore, Zyactinase did not inhibit the growth of the probiotic bacteria at any of the concentrations tested. For this study a concentration of 0.5% w/v was used for both the ethanol and water extracts for testing efficacy with the probiotic bacteria, whilst 0.25% was used in testing inhibition of pathogenic bacteria. Efficacy of Zyactinase in Stimulating growth of probiotic bacteria in vitro Effect of Zyactinase on the growth of Lactobacillus plantarum Broth containing either water or ethanol Zyactinase extract had significantly higher OD value than that without the Zyactinase extract as well as the placebo (isomalt) control (Fig 1). Results demonstrate that Zyactinase promoted the growth of Lactobacillus plantarum. Effect of Zyactinase on the growth of Pediococcus acidilactici Broth containing either water or ethanol Zyactinase extracts had a significantly higher OD value than that without the Zyactinase extract or the placebo control (Fig 2). Results show that Zyactinase promoted the growth of Pediococcus acidilactici.

4 234 Zyactinase promotes probiotic growth Effect of Zyactinase on the growth of Lactobacillus acidophilus Broth containing either water or ethanol Zyactinase extract had a significantly higher OD value than that without the Zyactinase extract (Fig 3). These results demonstrate that Zyactinase promoted the growth of Lactobacillus acidophilus. Effects of Zyactinase on the growth of Lactobacillus reuteri Broth containing Zyactinase water and ethanol extracts had a significantly higher OD value than that without Zyactinase extract (Fig 4). Results show that Zyactinase promoted the growth of Lactobacillus reuteri. The ethanol placebo isomalt had a significant inhibitory effect on the growth of Lactobacillus reuteri. FIGURE 3. The effect of Zyactinase water and ethanol extracts 0.5 % (w/v) on the growth of Lactobacillus acidophilus. All treatments resulted in significantly higher growth than the control and the placebo isomalt. The water placebo (isomalt) was significantly higher than the blank control. (n = 3). Vertical errors bars represent the standard error of the mean. * P < 0.05, ** P < FIGURE 4. Effect of Zyactinase water and ethanol extracts 0.5 % (w/v) on the growth of Lactobacillus reuteri (n = 3). All treatments resulted in significantly higher growth than the control and the placebo isomalt. The ethanol placebo (isomalt) was significantly lower than the blank control and thus had inhibited the growth of Lactobacillus reuteri. Vertical errors bars represent the standard error of the mean. * P < 0.05, ** P < Efficacy of Zyactinase in inhibiting growth of Pathogenic bacteria in vitro In order to analyze whether Zyactinase extract would promote or inhibit the growth of pathogenic bacteria, the effects of water extracts of Zyactinase, Phloe and the isomalt placebo were tested on four pathogenic bacteria: E.coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. The effect of Zyactinase on the growth of E.coli O157:H7 Both Zyactinase and Phloe have significantly (P<0.05) lower OD values than their respective controls, while there is no obvious difference between the placebo and blank controls. Therefore, the water extracts of Zyactinase and Phloe had an inhibitory effect on the growth of E.coli O157:H7, with Zyactinase having a greater effect than Phloe (Fig. 5). FIGURE 5. Effect of 0.25% final concentration of Zyactinase water extracts on E.coli O157:H7 growth, n=3. Vertical errors bars represent standard error of mean. * P < 0.05.

5 Zyactinase promotes probiotic growth 235 Effect of Zyactinase on the growth of Salmonella typhimurium Both Zyactinase and Phloe had significantly (P<0.05) lower OD values and thus bacterial growth than their respective controls. Water extracts of Zyactinase and Phloe inhibited the growth of Salmonella typhimurium, with Zyactinase having a greater effect than Phloe. No obvious effect was found with the placebo (Fig. 6). FIGURE 6.. The effect of 0.25% final concentration of Zyactinase water extracts on Salmonella typhimurium growth, n=3. Both Phloe and Zyactinase significantly inhibited bacterial growth. There was no difference between placebo and blank. Vertical errors bars represent standard errors of means. * P < 0.05, **P < The effect of Zyactinase on the growth of Listeria monocytogenes. Both Zyactinase and Phloe water extracts promoted the growth of Listeria monocytogenes, unlike the previous inhibition shown with the other pathogenic bacteria. The placebo isomalt had no effect on the growth of Listeria monocytogenes (Fig. 8). FIGURE 8. The effect of 0.25% final concentration of Zyactinase water extracts on Listeria monocytogenes growth compared to the controls, n=3. Growth of Listeria monocytogenes was promoted by the Zyactinase extracts. Vertical errors bars represent the standard error of the mean. * P < The effect of Zyactinase on the growth of Staphylococcus aureus Since the standard error of the mean is slightly high, no significant effect was found with either of the tested extracts on the growth of Staphylococcus aureus. However, it is obvious that the trends have shown slight inhibition of Staphylococcus aureus growth by the addition of the Zyactinase extracts, as well as the placebo (Fig. 7). FIGURE 7. The Effects of 0.25% final concentration of Zyactinase water extracts on Staphylococcus aureus growth, show slight inhibition n=3. Vertical errors bars represent the standard error of the mean. DISCUSSION Zyactinase is a freeze-dried patented kiwifruit extract that has been developed to gently treat constipation, to relieve irritable bowel syndrome (IBS) and to promote long-term gut health. Zyactinase has been extensively clinically tested (unpublished observation) and has been found to significantly relieve constipation and to return the number of bowel movements to normal as well as relieving IBS symptoms such as bloating, flatulence and abdominal pain. From these results it was hypothesized that Zyactinase had a three way mode of action in which the protease complex gently stimulated gut motility, the fibre bulked and softened the stool and that there was a prebiotic component that regulated the gut microflora. This prebiotic component or probiotic bacterial growth stimulant we believed was the fructo-oligosaccharides and pectin contained within the Zyactinase complex. To test this hypothesis we tested the efficacy of Zyactinase in vitro in stimulating the growth of four very fundamental probiotic bacteria for the relief of constipation and IBS. In addition, we compared this to the placebo that we had used in the clinical trial; isomalt that generally also has a probiotic bacteria growth stimulating effect (Code, 2006). Our results shown here demonstrate that Zyactinase does significantly stimulate the growth of these four probiotic bacteria and furthermore is statistically significant over isomalt. The protease complex within Zyactinase is heat sensitive and is deactivated at 38 C. Our hypothesis was that there was more than just the protease complex stimulating the probiotic bacteria and so to test this hypothesis we deactivated the enzyme complex by heating to 45 C. For both 36 C and 45 C there was no

6 236 Zyactinase promotes probiotic growth difference in growth of probiotic or pathogenic bacteria (results not shown) and thus the protease complex does not appear to play a role in the prebiotic activity of Zyactinase. Two forms of Zyactinase were tested; the capsule form of Zyactinase known as Phloe, which contains the excipients isomalt, magnesium stearate and silica dioxide that was used in the clinical trials, and XRF45 that is the pure Zyactinase powder without the excipients. As can be seen, there is some variation in response between the pure Zyactinase and the capsule form across all four probiotic bacteria that directly correlate to the concentration difference between the two. The placebo is that which was used for the clinical trials and contains isomalt that is a potent food source for bacteria and thus was expected that there would be some growth of the bacteria in the presence of the placebo (Code, 2006). For the clinical trials we had wanted to confirm that the relief of constipation was more than just a stimulus of probiotic bacteria effect and isomalt is currently an excipient of Phloe. The Zyactinase extracts were found to be statistically significant in comparison to the placebo demonstrating that the promotion of probiotic bacterial growth was more than just as a food source but a true stimulant effect. The results show that Zyactinase is a potent stimulant of probiotic bacteria growth, but that Zyactinase also inhibited the growth of the gram-negative pathogenic bacteria E. coli and Salmonella typhimurium. This suggests that within Zyactinase there is more than just a food source, but rather some bioactive component that stimulates probiotic bacterial growth whilst inhibiting pathogenic bacteria. Across all four probiotic bacteria zyactinase had a statistically significant promotion of growth, which was not dosedependent. Zyactinase (with and without the enzyme complex) was found to significantly inhibit the growth of the gram-negative bacteria E.coli O157:H7, and Salmonella typhimurium. However, this inhibition effect was not obvious for the gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes. In fact the water extracts of Zyactinase have even promoted the growth of Listeria monocytogenes. These results suggest that there is a food source effect stimulating the growth of the bacteria, in particular when considered in relation to the deactivation of the protease complex had no effect. However, the fact that there is a difference between the water and ethanol extracts and that there is inhibition of the pathogenic bacteria indicates a possible other component within Zyactinase that is stimulating probiotic bacterial growth whilst inhibiting gram negative pathogenic growth. Prebiotic oligosaccharides, and in particular galactooligosaccharides have been found to prevent the adherence of pathogenic bacteria to the gut epithelium and thus prevent infection (Shoaf et al., 2006). Anecdotal evidence from human use of Zyactinase has indicated a reduction in gut pathogenic infections and we surmise it maybe due to the oligo-saccharides preventing this adherence of the pathogen to the gut. However, both fructooligosaccharides and inulin have previously been shown to lower resistance to Salmonella in the gastrointestinal tract as demonstrated by increased colonization and translocation of Salmonella in Wistar rats (Ten Bruggencate et al., 2004). This study was of concern and thus we wished to explore this potential issue in regards to Zyactinase. Zyactinase was found to significantly inhibit the growth of Salmonella, but did increase the growth of Listeria. Previous studies have used calcium phosphate to inhibit the increased susceptibility to Salmonella (Ten Bruggencate et al., 2004) and alternatives are now being trialed for Zyactinase to see if it is possible to increase resistance to Listeria whilst continuing to promote the growth of probiotic bacteria in in vivo studies. CONFLICT OF INTEREST Dr Iona Weir is affiliated with Vital Foods Processors Ltd, the manufacturer of Zyactinase. REFERENCES Bekkali, N. L. H., Bongers, M. E. G., Van den Berg, M.M., Liem, O. and Benninga, M. A. (2007) The role of a probiotics mixture in the treatment of childhood constipation: a pilot study. Nutrition Journal 6, 17. Brown, A. C., Shovic, A., Ibrahim, S., Holck, P. and Huang, A. (2005) A non-dairy probiotic s (POI) influence on changing the gastrointestinal tract s microflora environment. Alternative Therapy Health Medicine 11, Brown, A. C. and Valiere, A. 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8 238 Zyactinase promotes probiotic growth