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



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Results General health status

Throughout the experiment rats appeared healthy, inquisitive and active. No illness or death occurred. In the last month of the experiment the activity of the Deficiency group appeared reduced, and they became more irritable on handling. No observable difference in the animal's hair lustre was noticed between the groups.


Feed intake and live weight gain

There was no significant difference (p > 0.05) in the body weight gain among three tested groups (Table 16, Figure 11). The daily feed intake of the Bacteria group was significantly higher than that of the Control group and the Deficiency group. This indicates that feeding with P. jensenii 702 may have stimulated the appetite of rats of the Bacteria group.


Table 16 Compare body weight, body gain (g) and feed intake (g) of different group of male Wistar rats during 81 days treatment Rat groups Initial body Last body weight (g) Daily Body weight Daily feed intake weight (g) gain (g) (g) Control 162. 9 ~ 15.1a 471. 9 + 30. 8 4. 4 0.3 22. 2 0. 1 Deficiency 158. 4 ~ 13.8a 446. 9+334 b 4l+03c 21. 6+0. 6d Bacteria 170.7 ~ 9.4a 482.0 + 30 4 b 4 4 + 0 3 c 22.9 0. 1 e Values are means SD (n=7) ; values within a given column with the same superscripts are not significantly different (p > 0.05, Student's t-test, two tailed) Water intake and bacteria dose for the Bacteria group

The water intake by each cage of rats of the Bacteria group was measured daily, and the average water intake per rat was calculated. Figure 12 presents the average daily water intake (mL/rat) for each cage of the Bacteria group, which was given P. jensenii 702 suspension.


The concentration of P. jensenii 702 given in the water was measured by the pour plate method as previously described, and the average log value of P. jensenii 702 provided in the rat drinking water per day was calculated as 8.79 0.36 (n=12) (raw data not shown).
From this data the average bacteria intake per day was calculated (Table 17). No allowance has been given to the proportional increase in water intake as the rats grew.
Table 17 Average daily water and P. jensenii 702 intakes by male Wistar rats of the Bacteria group, n=7

Intake Average Maximum Minimum Water (mL/rat/day) 49. 8 115 11 P. jensenii 702 (log cfu/rat/day) 10. 61 10. 97 9. 95


Translocation of P. jensenii 702 cells

There was no bacterial growth from swabs taken from the visceral surfaces of the rats of the three experimental groups, which indicates that the visceral surface was not contaminated with bacteria.


No bacteraemia was detected in any of the three experimental groups. There was no growth of P. jensenii 702 in any cultures of blood, spleen, mesenteric lymph nodes and liver samples. This indicates that there is no translocation of P. jensenii 702 to blood and tissues, which suggests that the intake of P. jensenii 702 will not result in any invasion to the tissue cells of the host.
Examination of visceral organs

Macroscopic examination did not reveal any obvious differences in the size and appearance of visceral organs between each group. No hepatomegaly or splenomegaly occurred.


The spleen weight and spleen weight index of the rats are shown in Table 18. There is no significant difference in the spleen weight between the Control group, the Deficiency

Group and the Bacteria group (p > 0.05). The spleen weight index of the Bacteria group is significantly lower than that of the Deficiency group (p 0.05). The difference of the spleen weight index between the

Bacteria group and the Deficiency group may be due to the difference of the body weight, although not significant.
Table 18 Spleen weight (g) Spleen weight index (mg/g) of different groups of male

Wistar rats fed with vitamin B12 deficient diet for 3 month Rat groups Spleen weight (g) Spleen weight index (mg/g) Control 1. 28 0. 07 a 2. 73 + 0. 22 bf c Deficiency 1.22 0. 05 a 2.74 0. 12 b Bacteria 1.21 0. 08 a 2.52 + 0. 23 e

Values are means SD (n=7) ; values within a given column with the same letter superscripts are not significantly different (p > 0.05, Student's t-test, two tailed) Faecal bacteria viable counts

Total faecal aerobe counts

The levels of total aerobes in the faeces of the three experimental groups of male

Wistar rat faeces during the 81 days treatment period are shown in Table 19.


Within each experimental group, the total faecal aerobe counts of Day 36, Day 64, and Day 81 were compared to that of Day 0 (Table 19). The total faecal aerobes of the

Bacteria group at Day 36 and Day 64 were significantly higher than Day 0 (p < 0.05),


however, by Day 81 the total faecal aerobe counts of the Bacteria group reduced to the same level of Day 0 (p > 0.05) (Table 19). The same pattern was also observed in the Control group and the Deficiency group (Table 19). The result indicates that intake of P. jetzsenii 702 did not affect the faecal aerobic population.


At each testing time, the faecal aerobe counts of different experimental groups were compared (Table 19). At Day 36, the total faecal aerobic counts of the Deficiency group and the Bacteria group were significantly lower than that of the Control group (p < 0.05).
However, by Day 64 and Day 81, the faecal aerobic counts of the Control group, the Deficiency group and the Bacteria group were not significantly different (P > 0.05).
Table 19 Total faecal aerobe counts (log cfu/g) in male Wistar rats during treatment Groups Day 0 Day 36 Day 64 Day 81 Control 7. 63 + 0. 30 *8. 90 + 0. 22 *8. 39 + 0. 51 8. 19 + 0. 62 Deficiency 7. 80 0. 29 a *8. 48 0. 43 c *8. 48 0. 37 8. 40 0. 67 e Bacteria7. 610. 13"*8. 500. 13'*8. 410. 24" 7. 850. 22" Values are means SD (n=7).
Values of Day 36, Day 64, and Day 81 were compared with that of Day 0 within each group, * p 0.05, Student's t-test, two tailed).
Total faecal anaerobe counts

The levels of total anaerobe counts in male Wistar rat faeces during the 81 days feeding period are shown in Table 20.


Within each group, the faecal anaerobe counts at Day 36, Day 64, and Day 81 were compared to that of Day 0 (Table 20). The total faecal anaerobes in the Control group and the Deficiency group at Day 36, Day 64 and Day 81 were not significantly different from that of Day 0 (p > 0.05) (Table 20). In contrast, the total faecal anaerobes of the Bacteria group at Day 36 and Day 64 were not significantly different from that of Day 0 (p > 0.05) (Table 20); but the total faecal anaerobes of the Bacteria group at Day 81 was significantly lower than that of Day 0 (p < 0.05). The reduction in the total faecal anaerobe counts of the Bacteria groups suggests that feeding with P. jensenii 702 may have an effect on the faecal anaerobic microflora.

* At each testing time, the faecal anaerobes counts of different groups were compared (Table 20). There was no significant difference of faecal anaerobic counts between the Control group, the Deficiency group and the Bacteria group during the 81 days feeding period (p > 0. 05).

Table 20 Total faecal anaerobe counts (log cfu/g) in male Wistar rats during treatment Groups Day 0 Day 36 Day 64 Day 81 Control 9. 22 0. 18 a 9. 46 0. 41 9. 43 0. 36 9. 25 0. 39

Deficiency9. 360. 29'9. 040. 39"9. 300. 26'8. 94 0. 30 Bacteria 9. 66 0. 19 a 9. 30 0. 25 9. 31 0. 33 *9. 15 0. 24 Values are means SD (n=7).


Values of Day 36, Day 64, and Day 81 were compared with that of Day 0 within each group, * p 0.05, Student's t-test, two tailed).
Faecal dairy propionibacteria counts of the Bacteria group

No dairy propionibacteria were detected in the rats of the Bacteria group prior to providing P. jensenii 702 in their drinking water (Figure 13). At the second test time (after 36 days feeding with P. jensenii 702), the dairy propionibacteria level in the faeces of the Bacteria group increased to 8-log value and remained at the level of 8-log to 9-log value until the end of the experiment (Figure 13). No dairy propionibacteria were detected in the faeces of the Control group and the Deficiency group during the 81 days treatment period.


Faecal t-Glucuronidase activities

The P-glucuronidase acivity of P. jensenii 702 suspension (101 cfu/mL) was below the testing limit (IU).


The average faecal 3-glucuronidase activities of different experimental groups of male Wistar rats during 81 days of treatment period are shown in Table 21. There was variation of p- glucuronidase activities among the rats within each group at each testing time.
Within each group, the average faecal p-glucuronidase activity of Day 36, Day 64, and Day 81 were compared to that of Day 0 (Table 21). The average faecal (3-glucuronidase activity of the Control group at Day 36 and Day 64 remained the same level as that of Day 0, but increased significantly at Day 81 (p < 0.05). The average faecal (3-glucuronidase activity of the Deficiency group remained at the same level during the whole 81 days feeding period (p > 0.05). In contrast, the average faecal -glucuronidase activity of the Bacteria group at Day 36 was significantly higher than that at Day 0, then decreased at Day 64 and remained at the same level as that at Day 0 until the end of 81 days of treatment period (p > 0.05).
At each testing time, the average faecal -glucuronidase activities of different groups were compared (Table 21). There was no significant difference of average faecal - glucuronidase activities between the Control group, the Deficiency group and the Bacteria group at Day 0, Day 36 and Day 64. In contrast, at Day 81, the average faecal - glucuronidase activities of the Bacteria group and the Deficiency group were significantly lower than that of the Control group (p < 0.05) (Table 21).

Table 21 Faecal p-glucuronidase activities (MU/g) of male Wistar rats during treatment Groups Day 0 Day 36 Day 64 Day 81 Consol 334. 04"12. 263. 42'' 11. 003. 69' *13. 251. 73" Deficiency 11.34 ~ 5.30a 18. 16 6. 86' 13. 07 ~ 1.97c 9.33 ~ 1.65e Bacteria7. 512. 98'*14. 624. 13" 13. 41 + 5. 59C 7. 61 + 1. 81 f Values are means SD (n=7).


Values of Day 36, Day 64, and Day 81 were compared with that of Day 0 within each group, * p 0.05, Student's t-test, two tailed).
Dairy propionibacteria in small intestine

Dairy propionibacteria was isolated from the contents and tissues of the ileum sections of the rats from the Bacteria group. The viable counts of dairy propionibacteria from these samples was quite low (101 to 102 cfu/g), however, as only 10 cm of the small intestine was sampled, this does not reflect the total intestinal number. No dairy propionibacteria was isolated from the contents and tissues of ileum sections of the rats from the Deficiency group and the Control group.


Observation of Scanning Electron Microscope micrographs of the small intestine tissue samples were difficult to interpret as there were no monoclonal antibodies available to specifically stain for P. jensenii 702. No structures that resembled bacteria in the samples of small intestine tissue were observed for the Deficiency group and the Control group. In the case of the Bacteria group, short rod shaped structures of the expected shape and size were present (Figure 14). These rod shaped structures were not observed for the other two groups.
Serum vitamin B12 and homocysteine levels Serum Vitamin B 12 levels

The mean serum vitamin B 12 levels (pmol/L) for the three experimental groups of Male Wistar rats during the three-month feeding period are presented in Table 22.


The serum vitamin B1z levels for the Control group increased significantly after one month feeding (p 0.05) (Table 22). The results may indicate that the rats of the Control group absorbed vitamin B12 from the drinking water to build up serum vitamin B12 during the first month, and then reached their upper absorption limit for this vitamin after one-month treatment.
The serum vitamin B12 levels of the Deficiency group decreased significantly from 444.4 pmol/L to 131.3 pmol/L after one month (p < 0.05), and remained at the significant

lower level till the end of 3-month feeding period (p < 0.05) (Table 22). This indicates the Vitamin B12 Deficient Diet resulted in the depletion of serum vitamin B12 in rats.


The serum vitamin B12 levels of the Bacteria group decreased significantly after the first month feeding period (p < 0.05) (Table 22). After two-months feeding period, the serum vitamin B12 levels of the Bacteria group increased significantly compared to that of Month 1 (p 0.05). The serum vitamin Bl2 level of the Bacteria group at Month 3 was significantly lower than that at Month 0 (Table 22), but significantly higher than that at Month 1, and not significantly different from that at Month 2 (p < 0.05).
Table 22 Comparison of serum vitamin B12 levels (pmol/L) of male Wistar rats during feeding period Groups Month 0 Month 1 Month 2 Month 3 Control 464. 9 50. 4"*914. 6 200. 8"*1096. 2 365. 7 *949. 2 375. 0 g Deficiency 444. 3+28. 2 *131. 3+54. 4 *135. 7+50. 8e *105. 5+34. 1 Bacteria 387. 2 62. 3a * 119. 9 67. 9 241. 6 101. 5 *216. 072. 8' Values are means SD (n=7).
Values of Month 1, Month 2, and Month 3 were compared with that of Month 0 within each group, * p 0.05, Student's t-test, two tailed)

The serum vitamin B12 levels of different group were compared at each testing time (Table 22). At the beginning of the feeding period, there was no significant difference in the serum vitamin B12 levels between the Control group, the Deficiency group, and the Bacteria group (p > 0.05). After the Ist month, the serum vitamin B12 levels of the Deficiency group and the Bacteria group were significantly lower than that of the Control group (p < 0.05). This is attributed to the vitamin B12 depletion effect of the Vitamin B12 Deficient Diet Modified.


The fact that the serum vitamin B 12 level increased in the Control group indicates that the pharmaceutical form of vitamin B12 in the drinking water provided adequate dietary source of the vitamin B 12. After two months until the end of the experiment, the serum vitamin B12 level of the Bacteria group was significantly higher than that of the Deficiency group. This confirms that P. jenseiiii 702 cells provided vitamin B, 2 to the rats of the Bacteria group. The fact that the Bacteria group had significantly lower serum vitamin B12 than that of the Control group at the end of the study is not of concern, and will be addressed in the subsequent discussion.

Serum homocysteine levels

The mean serum homocysteine levels (, umol/L) for the three experimental groups of male Wistar rats during the three-month feeding period are presented in Table 23.
The mean serum homocysteine levels of the Control group remained at the same level during the 3 months feeding period (p > 0.05) (Table 23). The serum homocysteine level of the Deficiency group increased significantly after the 1st month (p < 0.05) and remained at a significantly higher level than that of Month 0 until the end of 3 months feeding period (p < 0. 05). The serum homocysteine level of the Bacteria group initially increased significantly after the 1st month (p < 0.05) ; but then decreased significantly at Month 2 and Month 3 (p 0.05).
The serum homocysteine levels of the different groups, at each testing time, were compared (Table 23). At the beginning of the feeding period, there was no significant difference in the serum homocysteine levels between the Control group, the Deficiency group, and the Bacteria group (p > 0.05). At Month 1, the serum homocysteine level of the Deficiency group and the Bacteria group were significantly higher than that of the Control group. At Month 2 and Month 3, the serum homocysteine level of the Deficiency group still significantly higher than that of the Control group (p < 0.05). In contrast, the serum homocysteine level of the Bacteria group decreased at Month 2, and was not significantly different from that of the Deficiency group or the Control group; at Month 3, the serum homocysteine level of the Bacteria group decreased further to a significantly lower level than that of the Deficiency group (p 0.05). The gradual decrease in serum homocysteine level of the Bacteria group indicates that feeding with P. jensenii 702 reduces the serum homocysteine. As serum homocysteine levels reflect the store of vitamin B 12 in an animal, it is likely that the low levels of homocysteine reached by the Control group are the low ends of homocysteine values found in a rat.
Table 23 Comparison of serum homocysteine levels (llmol/L) of different groups of male Wistar rats during 3 months feeding period Groups Month 0 Month 1 Month 2 Month 3 Control 18.1 ~ 2.4a 15.4 ~ 3.4b 16.8 ~ 2.6d 16.4 ~ 2.0 Deficiency 15. 4 1. 5'*24. 8 4. 7 c *23. 1 + 5 3 e *26. 4 + 7-4 g Bacteria 13.3 ~ 1.9a *24.1 ~ 5.9c *18.9 ~ 2.2d,e 16.7 ~ 2.6 Values are means SD (n=7).
Values of Month 1, Month 2, and Month 3 were compared with that of Month 0 within each group, * p < 0.05 (student's t-test, two tailed).

Values of different groups at each testing time were compared with each other; values within a given column with the same superscripts are not significantly different (p > 0.05, Student's t-test, two tailed) Testes weight and morphology

There was no significant difference in the wet weight of testes between the Control group, the Deficiency group, and the Bacteria group (Table 24). However, the relative testes weight (g/lOOg of body weight) of the Control group was significantly lower than that of the Deficiency group and the Bacteria group (Table 24). One possible reason for this result is that one rat in the Control group was noted to have smaller testes (data not shown), which apparently sometimes occurs in healthy Wistar rats (Dr. Mary Bate, Animal Ethics Officer, University of Newcastle, personal communication). This probably skewed the results.
According to the report from the IDEXX-Veterinary Pathology Services, Brisbane, Australia (not shown), the testicular morphology of all tested three groups was also normal.

Table 24 Testes weight (g) and relative testes weight (g/100 g body weight) of male

Wistar rats fed with vitamin B12 deficient diets

Rat groups Testes weight (g) Relative testes weight (g/100 g body weight)

Control 3. 19 0. 21a 0. 67 0. 03b

Deficiency 3.50 0. 35a 0. 79 0. 08C

Bacteria 3.47 0. 25a 0.72 0. 052

Values are means SD (n=7);

Values within a given column with the same superscripts are not significantly different (p > 0.05, Student's t-test, two tailed)

Cholesterol and triglycerides

The mean serum lipid concentrations for the Control, Deficiency and Bacteria Group are shown in Table 25. The mean serum cholesterol concentration of the experimental groups were compared at month 3. The Bacteria group had lower cholesterol levels than the Control group, but not significantly (p > 0.05). The Bacteria group, however, were found to have significantly lower total cholesterol concentration than the Deficiency group (p < 0.05). No significant difference was observed for lipoproteins between the three experimental groups.
The serum triglyceride concentrations of the experimental groups were compared at the end of the experiment (Table 25). The Bacteria group had significantly lower triglycerides than both the Control and Deficient groups (p < 0.05). The rats in the Deficient group had significantly lower concentration of triglycerides than the Control group (p < 0.05), but significantly higher than the rats in the Bacteria group (p < 0.05).
Table 25 : Serum lipid parameters (mmol/l) of the three experimental groups of male Wistar rats at the end of three-month feeding treatment

Experimental Total HDL (mmol/1) LDL (mmol/1) Triglycerides

Groups Cholesterol (mmol/1) (mmol/l) Control 2. 70 0. 49 a'0. 76 0.15c 1.08 ~ 0.14d 1. 92 0. 42 e Deficiency 2. 56 0. 06 b 0. 69 0. 02 c 1. 33 0.14d 1.19 ~ 0.14 Bacteria 2. 06 0. 14 a 0. 51 ~ 0.04c 1. 18 0. 08 0. 82 0.08 g HDL = High-density lipoprotein; LDL = low density lipoprotein. The LDL was calculated by the equation of Friedewalds et al. (1972).
Values are means standard deviation (n=7)

Values within a given column with the same superscripts are not significantly different (p >

0.05, one way-ANOVA)

Discussion

Safety is the most important criterion for selection of new probiotic strains, and it is unfortunate that there are no general guidelines or specific policies for safety assessment. In this study, recommended safety testing was undertaken, including measurement of acute oral toxicity, bacterial translocation to blood and organ tissues, and the production of harmful enzymes.
Acute oral toxicity has been advocated as a fundamental test for assessing safety (Donohue et al. , 1998; Stine and Brown, 1996). Acute oral toxicity has been applied previously in the safety assessment of lactic acid bacteria (Zhou et al. , 2000). In these assessments, appetite, activity, and live weight gain have been regarded as general and sensitive indicators for the health status of animals.
On average, the rats of the Bacteria group consumed a very high number of viable bacteria per day (101 cfu/rat/day) (Table 17). All rats in this group were healthy, as indicated by their activity, feed intake, daily weight gain, growth curves, and general appearance, and no adverse effect of P. jeriserii 702 was observed. The average dose of P. jefasefaii 702 (1010 cfu/day) used in this study for rat weight ranging from 150 g to 500 g would correspond to an average dose of P. jensenii 702 (1012 cfu/day) for a 70 kg human. The general suggested level of probiotic bacteria in food is 106 cfu/mL (or cfu/g). Therefore, this study suggests that consumption of P. jensenii 702 at general probiotic food level would unlikely result in adverse effect to humans.
In addition to acute oral toxicity, bacterial translocation is another highly recommended indicator for probiotic safety assessment (Marteau et al. , 1997; Zhou et al., 2000). Bacterial translocation is a measure of infectivity. In this study, no translocation of P. jensenii 702 from the gut to tissues including spleen, liver, mesenteric lymph nodes and blood was observed.
From the evidence above it is apparent that P. jensenii 702 is not pathogenic in rats.
This result was expected as dairy propionibacteria strains have been consumed by people over long periods of time without any adverse consequences. The limitation of this study is that it was performed on healthy rats, and therefore does not provide data on the effect of consumption of large quantities of dairy propionibacteria by an immunosuppressed population. Further a single animal model always has limitations as the physiology between species is different.
Another safety concern for probiotic bacteria is the production of potentially harmful enzymes, one of which is p-glucuronidase. Bacterial p-glucuronidase can hydrolyze glucuronide in the diet and release steroids and certain carcinogenic compounds. The glucuronidase activity for strain P. Jy2S1211 702 in vitro was below the testing limit (1 U)

using the method described previously. In this study, the faecal 8-glucuronidase activities of the three experimental groups of male Wistar rats were difficult to interpret due to the large variation between rats within the same group. The faecal p-glucuronidase activity of the Bacteria group, however, at Day 81 (Table 21) was significantly lower than that of the Control group. It is important to note that the level of faecal p-glucuronidase in the Deficiency group at Day 81 was also significantly lower than that of the Control group. This may indicate that vitamin Bl2 in the diet alters the type of microbiota in the gut.


The reduction of the faecal p-glucuronidase has also been found in a dairy propionibacteria strain, P. acidipropionici CRL 1198 (Perez-Chaia et al. , 1999). It has been suggested that the anaerobes and coliform bacteria are the major contributor to the faecal glucuronidase activity; and the lower activity of faecal p-glucuronidase results from the change of faecal population and their metabolic activities after the inclusion of P. acidipropionici CRL 1198 (Perez-Chaia et al. , 1999).



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