Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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Viable bacteria counts demonstrated that there was no significant difference of total aerobe and total anaerobe counts between the Control group, the Deficiency group, and the Bacteria group at each time interval (Table 19, Table 20). But the total faecal anaerobes of the Bacteria group at Day 81 were significantly lower than that at Day 0 (Table 20). This suggests the lower faecal p-glucuronidase in the Bacteria group may also due to the decrease of intestinal anaerobes or their p-glucuronidase activity, which may result from the inclusion of P. jensenii 702 in the intestinal flora. Further, as the dairy propionibacteria counts reached 108 cfu/g faeces after only 36 days of feeding, without any subsequent significant decrease in anaerobic counts, but with relative increase in faecal (3-glucuronidase levels, it could be speculated that the influence of the P. jensenii 702 on the gut anaerobes does not occur until there is sufficient time for it to adhere and colonise the small intestine. The colonisation of the P. jensenii 702 may be competitive to the other anaerobic bacteria, or the P. jensenii 702 may secrete inhibitory compounds during its growth phase. This would explain why just the transit of the P. jensenii 702 through the gastrointestinal tract exerts no obvious effect on the rats of the Bacteria group during the first month of the feeding period. As the presence of p- glucuronidase in the gastrointestinal tract increases the risk of colon cancer, it is possible that P. jensenii 702 may also act as an anti-cancer agent. To confirm this, a more detailed study would be necessary.
Effective probiotic bacteria must be capable of maintaining their viability in the gastrointestinal tract, despite the numerous adverse factors that may affect them, including acids and enzymes in the stomach, bile salts and enzymes in the intestine and antagonistic bacterial interactions in the gut.

In Example 2, strain P. jensenii 702 has been shown to be resistant to acid and enzyme conditions in the stomach and small intestine in vitro. In this study, P. jensenii 702 proved to be resistant to the in vivo conditions of rat gastrointestinal tract. Dairy propionibacteria were not detected in the faeces of the Bacteria group before the rats were fed with P. jensenii 702 and were not isolated from the Deficiency group or the Control group.


The constant presence of dairy propionibacteria in the rat faeces of the Bacteria group confirms the in vitro results demonstrating that P. jensenii 702 is tolerant to the conditions of the gastrointestinal tract in vivo.
Dairy propionibacteria were also isolated from the small intestine of the male Wistar rats, albeit in relatively small numbers. This probably reflects the small section of intestine sampled rather than giving a true indication of bacteria number.
The in vivo adhesion of P. jensenii 702 to the small intestine was determined by sectioning the small intestine tissue and examining the tissue samples using scanning electron microscopy. Without monoclonal antibodies it is not possible to be certain that the bacteria- shaped structures observed by scanning electron microscopy adhering to the small intestine samples of the Bacteria group were in fact P. jenseltii 702 (Figure 14). However, these structures resembled those observed in in vitro experiments (Figure 10 C, D) and were not seem in either the Deficiency group or the Control groups. It is likely, these were P. jensenii 702, and demonstrate in vivo adherence to gut epithelium of rats.
A bacterial strain that is safe, and survives the gastrointestinal tract still must exert a positive effect on the host to be considered as a probiotic strain. In this study the probiotic property that was selected was vitamin B12 production.
It has been found that vitamin B12 deficient rats show growth retardation, which is caused by a decrease in food intake (Kawata et al. , 1995). In this study, we also found that daily food intake of the Deficiency group was significantly lower than that of the Bacteria group (Table 16). The Control group, however, also had a significantly lower feed intake compared to the Bacteria group (Table 16). There was no significant difference between the food intake of the Control group and that of the Deficiency group (Table 16), which suggests that food intake may not reflect vitamin B I2 status indicated by the Kawata et al. study (Kawata et al. , 1995). The reason the Bacteria group had a higher daily food intake can not be explained by this experiment, and further more detailed studies would be required to determine what the actual mechanism of this appetite stimulation was.
The daily body weight gain of the Deficiency group was lower than that of the Bacteria group, however, not significant (p > 0.05) (Table 16). There are two possible reasons for this. Although the feed intake for both the Control group (p > 0.05) and the Bacteria groups (p < 0.05) were higher than that of the Deficient group (Table 16), these two groups also demonstrated a higher physical activity. Further as the rats used in this study were at an

age of rapid growth, it is likely that this rapid growth period clouded the weight gain, particularly when considering deficiency was not instantaneous, and occurred over a period of one month. It is anticipated that a difference in body weight gain would have been observed had the experiment been extended.


In this study, serum vitamin B12 was measured as one of the indication of vitamin B, 2 deficiency. The time zero measurement of vitamin B12 levels in all groups was statistically the same, and probably reflects the average normal vitamin B12 level in a healthy rat. After the 1st month of feeding with a vitamin B12 deficient diet, the Deficiency and the Bacteria groups both had a significantly lower serum vitamin B12 level than that of the Control group (Table 22). At Month 2, the vitamin B12 level of the Bacteria group was significantly higher than that of the Deficiency group (p < 0.05), and remained statistically higher until the end of the experiment (p < 0.05). This suggests that feeding with P. jensenii 702 cells has supplemented the diet of the Bacteria group with Vitamin B12. The reason for the initial drop in vitamin B12 in the Bacteria group at Month 1 is explained by the fact that the probiotic bacteria require time to colonise the intestine before exerting any beneficial effects to the host. Once P. jensenii 702 established in the gut and then became an active contributor to the dietary vitamin B 12 source, the serum vitamin B12 levels of the Bacteria group begin to increase (Table 22). This was discussed previously in respect to the total anaerobic count and - glucuronidase activity, and the results of the vitamin B12 levels correlates with this discussion point. It is impossible. to speculate how much higher the vitamin B12 levels of the Bacteria group would go if the study had of been continued. Clearly a maximum population of P. jensenii 702 would eventually be established in the small intestine, which would reflect the number of binding sites, available nutrients, and other ecological criteria. It would be when colonisation is at this level that a constant vitamin B12 level could be reached. It is evident however, that after two months, the vitamin B12 level in the Bacteria group had returned to the same level as its original level (Table 22).
At Month 3, a drop in the serum vitamin B12 levels was observed in the Bacteria group. Although the serum vitamin B12 levels at Month 3 was not significantly different to that at Month 2, it was significantly different to the initial level at Month 0. This does not detract from P. jensenii 702 as a source of vitamin B12. The Bacteria group still had twice the level of serum vitamin B12 (216.0 pmol/L than that of the Deficiency group (105.3 pmol/L). It is likely that the serum vitamin B12 level of the Bacteria group at Month 3 was still within the normal range of serum vitamin B12 levels for male Wistar rats, reflected by the normal homocysteine levels of the Bacteria group at Month 3 (Table 23). The variations among serum vitamin B12 level within each experimental group (Table 22) suggests that a larger population (n > 7) group may be required for a more accurate study.

The large difference between the vitamin B12 levels in the Control group and the Bacteria group was not unexpected. An excess amount of vitamin B12 was provided in the drinking water of the Control group, and the level reached is probably the upper adsorption limit for this vitamin. This is reflected by the fact that the vitamin B12 level increased rapidly after one month and remained relatively constant for the remainder of the experiment (Table 22).


Vitamin B12 deficiency has been found to cause hyperhomocysteinaemia in humans (Bjorkergren and Svardsudd, 2001). Serum/plasma homocysteine levels show an inverse association with serum/plasma vitamin B12 levels (Mann et al. , 1999). In this study, this inverse relationship was clearly observed between serum vitamin B12 and serum homocysteine levels of the rats from the Deficiency group and the Bacteria group (Figure 15).
The decreased serum vitamin B12 levels were correlated with the increase of serum homocysteine levels in the Deficiency group (Figure 15). This confirms that vitamin B12 deficiency result in the increase of homocysteine, and the measurement of serum homocysteine is a complementary diagnosis tool for vitamin B12 deficiency (Bjorkegren and Svardsudd, 2001).
High levels of homocysteine are now recognised as a risk factor for cardiovascular disease (Shaw et al. , 1999; Wilcken and Wilcken, 1998). At Month 0, there were no statistical differences between the homocysteine levels between the three rat groups (Table 23), but thereafter the change in homocysteine levels (Table 23) inversely reflecyed the vitamin B 12 levels (Figure 15). Intake of P. jensenii 702 lowered the serum homocysteine levels of the Bacteria group (Figure 15), and at the end of the two and three months, there was no significant difference in the homocysteine levels of the Bacteria group and the Control group (p > 0.05) (Table 23). This result suggests that the vitamin B12 level in the Bacteria group at the completion of the study was within the normal rat range. The application of P. jensenii 702 as probiotic bacteria can therefore be extended to reduce the risk of cardiovascular disease.
In the study by Kawata et al (1997), one measure of vitamin B12 deficiency was an abnormality of the testes. In this study, no abnormality of testes due to diet was indicated. It was not possible to compare the vitamin B12 levels between the two studies, as Kawata et al (1997) measured testicular tissue not serum levels, and these are not comparable.
Furthermore, the rat model used in this study was a modification of the Kawata et al. (1997) model, in that unlike the Kawata et al (1997) model, the rats in this study were born to healthy mothers and not made deficient until after weaning. The diet used also was different. In a previous experiment, the Kawata et al (1997) diet was tested, and the rats became malnourished, suffered stunted growth and were euthanated prior to the completion of the study (data not shown). The diet lacked more than just vitamin B12, and therefore for this

study a commercial pelleted preparation, Vitamin B12 Deficient Diet Modified (ICN) was used.


The association between cardiovascular disease and cholesterol is well established. A number of studies have identified the possibility of using probiotics to lower cholesterol, and with dietary management being the preferred method of treatment, the potential market for a cholesterol lowering probiotic is substantial. The Wistar rat model used in this study was primarily selected as a vitamin B12 deficient model, and hence not optimum for a cholesterol study. Regardless of this, a shift in cholesterol was observed between the groups, with a significantly lower cholesterol identified for the Bacteria group compared to the Deficiency group (Table 25).
Previous studies suggest that probiotics can only alter cholesterol in subjects with already high serum cholesterol levels (de Rossa and Katan, 2000). In this experiment the rats were not feed with a high cholesterol diet. Furthermore, as one of the mechanisms of cholesterol reduction by probiotics is the deconjugation of bile, any measurable effects of this mechanism could not be determined in this study due to the rats lack of a gall bladder. For these reasons, we anticipate a more significant response when this bacteria is trailed in a more suitable model.
After the three-month feeding period, the Bacteria group had significantly lower triglycerides than both the Vitamin B12 supplementation and the Deficiency groups (Table 25). High triglycerides are identified as a risk factor in cardiovascular disease due to their influence on cholesterol metabolism. The ability of P. jenenii 702 to lower triglycerides is likely to be related to its influence on fatty acid metabolism, and has likewise been demonstrated with other intestinal and probiotic bacteria (Kankaanpaa et al. , 2002).
Conclusion

As a new probiotic P. jensenii 702 appears to have remarkable potential. It clearly exerts a positive effect on vitamin B12 and homocysteine, and its applications in this area extends beyond purely diet supplementation, to an effective treatment in patients with gastric atropy, R-factor disorders and other physiological causes of vitamin Bi2 deficiency. Due to the potential of intrinsic factor independent mass pharmaceutical uptake of vitamin B12 to occur in the small intestine (Herbert 1988), P. jensenii 702 may offer a cheaper alternative to treatment of those patients with pernicious anaemia. In addition, the fact that P. jensenii 702 may have a positive effect on serum cholesterol and triglycerides gives it an added advantage over food fortification as has been used extensively in the vegetarian food industry. Finally as a naturally occurring bacterial strain, P. jensenii 702 is likely to have high consumer acceptability, which is important, in particular for those who maintain a strict dietary program.

EXAMPLE 4 THE USE OF THE PROBIOTIC BACTERIUM P. JENSENII 702 AS AN IMMUNE STIMULANT, IMMUNE MODULATOR AND ADJUVANT

There are many reports on the role of probiotics on immune function, however these studies are primarily on Lactobacilli sp. and Bifidobacteria sp. The type of immune response includes enhancement of phagocytosis (Fooks et al., 1999), stimulation of immunoglobulin (Ig) -A production (Fooks et al. , 1999), stimulation of the cell-mediated immune response (Kaur et al. , 2002), enhancement of immune response to oral vaccines (Salminen et al., 1998b), immunomodulation (Kitazawa et al. , 1992), and mitogenic B lymphocyte stimulation (Takeda et al. , 1997). Inactivated Propionibacterimm acnes is know to act as a non-specific immunostimulant (Julia et al., 1998). There are no reports of dairy propionibacterium being used to enhance immune function.


The aim of this Example is to demonstrate that Propionibacterium jensensii 702 acts to: (1) enhance humoral immune response; (2) enhances and modulates cell-mediated response ; and (3) enhances response to vaccines (acts as an adjuvant). In this example live P. jetisenii 702 will be used, however one can extrapolate that inactivated, killed or selected part or parts of P. jensenii 702 will also produce an equivalent response.
Furthermore this example provides evidence of the ability to produce an oral vaccine for tuberculosis. No previous studies have been able to do this due to the fact that no oral adjuvant currently approved for human use can stimulate an immune response as demonstrated in this example.
Materials and Methods Stimulation and modulation of humoral immune response

In Example 3 above, P. jensenii 702 was fed to Wistar male rats for a period of three months. At the conclusion of this study blood was collected. In this example some of the blood collected will be used to measure total IgA, IgG and IgE, using a standard Enzyme Linked Immunosorbent Assay (ELISA). The expected outcome is that the group fed the bacteria will have a higher IgA and IgG and a lower IgE than the other two groups.


Stimulation, modulation and enhancement of immune response to vaccine (humoral and cell-mediated) Vaccine

In this example a non-living bacterial vaccine will be given orally. It can be extrapolated that the results would be equivalent if the vaccine was (a) living or attenuated, (b) the whole organism or part or parts of the organism, (c) viral, bacterial or fungal and (d) given orally, parenterally or subcutaneously. The vaccine in this example will be given as two types: (i) soluble protein extracted from the whole bacteria combined with soluble protein excreted from the bacteria during growth and (ii) soluble protein excreted from the bacteria during growth.

Further, this example uses M. tuberculosis as the antigen demonstrating the first evidence of a successful oral vaccine for tuberculosis. Application of the principle demonstrated can be extrapolated for animal tuberculosis and other oral vaccines.
Finally from the immune response produced in this example, it is possible to extrapolate that P. jensenii 702 can act as an immune modulator, that is it would have efficacy in either (a) living or attenuated form, or (b) as the whole organism or part or parts of the organism, to modify the immune response correcting an allergy-type immune system.
General equipment

All work involving live M. tuberculosis organisms was carried out in a Class II Biological Safety cabinet (Gelman Sciences). Reagents used in the following experiments were obtained either ready-made from the respective suppliers or formulated as listed above.


Unless otherwise indicated all pipetting was performed using Gilson automatic pipettes and Bonnet Equipment pipette tips.
Growth of live M. tuberculosis Lowenstein Jensen Slopes

Only one Mycobacteriufn strain was used in this research, Mycobacterimn tuberculosis H37Rv strain (ATCC 27294), and it was grown and maintained on Lowenstein Jensen (LJ) slopes (Micro Diagnostics). LJ slopes were maintained throughout the duration of this research for stock culture purposes. The inoculated LJ slopes were incubated at 37 C in air for up to three weeks, and then were subcultured onto fresh slopes to ensure continued growth and survival of the bacteria.


Modified Sauton's Medium

Modified Sauton's Medium (MSM) was used for supporting the rapid growth of M. tuberculosis in this experiment. A culture was set up on the Modified Sauton's Medium by firstly transferring, via a loop, M. tuberculosis from the LJ slope to a 0. 45um cellulose ester membrane (Advantec MFS). Using sterile forceps the membrane disc was floated on the surface of the liquid Modified Sauton's Medium. The culture vessel was then incubated (Thermoline) at 37 C in air for 2 weeks. After this time had expired, the bacteria was separated from the membrane using a teaspoon ladle and transferred to a fresh Modified Sauton's Medium.


Subcultures were made every 4-7 days depending on the extent of bacterial growth.
From the growth on the MSM a two-fold collection of protein occurred. Firstly there was the harvesting of whole M tuberculosis cells from the surface of the medium and secondly there was the sterile filtration of the excreted proteins, known as Short-Term Culture Filtrate (STCF). These two sources were further processed to produce the protein used for the vaccine.

Antigen production Collection of Whole Mycobacteriunz tuberculosis Cells

After 4 days of growth the cells were collected from the MSM. The cells were scooped from the surface of the medium and collected into a 10ml sterile centrifuge tube (Sarstedt). The cells were spun at 4000rpm for 15 minutes for a total of 3 times to wash the cells. Between each session the supernatant was pipetted off using a 3ml disposable pipette and sterile distilled water was added up to 10 ml. The water was pipetted off and the cells were frozen at-80 C until required in the next stage of the experiment.
Short-Term Culture Filtrate (STCF) (Based on methods described by Andersen et al. 1991)

Short-term culture filtrate (STCF) consisted of the Modified Sauton's Medium containing excreted proteins from 4 days of logarithmic M. tuberculosis growth. The Modified Sauton's Medium with whole cells removed was sterile filtered using a 60 ml syringe and a Ministart (0. 22 um) sterile filter head into a sealed vessel and frozen at-80 C until required for use.


Concentration of STCF protein Two Step Ultrafiltration of STCF

This process aimed to remove the STCF via filtering straight from the MSM growth medium. Specifically, it involved using a 10 kDa molecular weight cut off regenerated cellulose ultrafiltration membrane (Millipore) to filter the STCF and medium. The liquid from the waste line of the ultrafiltration unit was collected in a sterile 70ml pot (Sarstedt). After the sample had filtered through until there was approximately 2ml left, ultrafiltration was paused and this concentrated solution was removed and placed in a sterile 5ml tube (Sarstedt). This contained all proteins larger than 10 kDa and was refrigerated while the next step of ultrafiltration was performed. The next stage involved replacing the lOkDa filter (Millipore) with a 3kDa molecular weight cut off and refiltering the initial filtrate from the waste line, which contains proteins less than lOkDa. Once this sample had reduced to 2ml, the remaining sample from the refrigerator was added. Fluid from the waste line was discarded during this filtration stage. Once the total volume of the final sample had reduced to less than 2ml it was removed and placed in a cryogenic tube and stored at-80 C. Once a considerable volume of STCF had been collected it was washed to remove any residual MSM. This involved defrosting the sample and ultrafiltering it through a 3kDa filter and running 20 ml of sterile distilled water through. This wash step was repeated 3 times.


Sonication of whole M. tuberculosis cells

Cells that were removed from the top of the Modified Sauton's Medium were washed prior to sonication in sterile PBS at 4000rpm for 15 minutes, and the supernatant was


removed. All sonication performed in this research utilised a Branson Digital Sonifier 250 with a Microtip Tapered 1/8"horn.


For each round of sonication, 6ml of packed cells were placed in a 10ml round bottomed test tube (Crown Scientific), along with 6ml of glass beads and 7. 5ml of PBS. This test tube was fixed in position with the sonicator tip inside it by a clamp attached to a retort stand. The sonicator itself was mounted on a retort stand with two clamps securing it at a 45 angle, to accommodate it within the Class II Biological Safety Cabinet. Also included inside the test tube, alongside the tip of the sonicator was a temperature probe that monitored the temperature of the sample. The temperature limit was set to 20 C, and when this temperature was reached the sonicator would pause until the temperature had lowered. To further control the temperature of the sample a beaker filled with ice and 50ml of 100% ethanol was fixed with another clamp and attached to the first retort stand, so as to envelop the bottom of the test tube containing the sample. This aimed to further minimise the heat damage to vital proteins due to the heat created by the sonication process. The sonication was performed at an amplitude of 35% for 20 minutes with a temperature limit of 20 C as mentioned previously.
Following sonication the sample was transferred from the test tube to an ultracentrifuge tube, via a transfer pipette leaving the glass beads behind. The sample was centrifuged (Beckman, J2-MC centrifuge) 14000rpm for 30 minutes at 4 C to remove any insoluble proteins. The sample was then sterile filtered using a 0. 2211m Sartorius Ministart filter into a sterile container and stored at-80 C until required. The protein concentration of the sample was determined by performing a protein assay.
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