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



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Aliquots of ImL of cell suspension were dispensed into in 2mL Coming Cryogenic vials, and stored at-70 C.
Simulated upper gastrointestinal transit tolerance tests

The gastric transit tolerance and small intestinal transit tolerance in vitro tests were based on the methods described by Charteris (1998).


Preparation of bacteria suspension

All 13 dairy propionibacteria strains were grown anaerobically in SLB broth at 30 C for 48 hours. Aliquots (1 mL) were transferred to 1. 5mL eppendorf tubes, and bacterial cells were collected by centrifugation (2500 x g, 5 min) and washed three times with lrnL PBS buffer (pH7.0), centrifuging (2500 x g, 5 min) between each wash. Bacterial cells were then resuspended in lrnL PBS buffer (pH7.0). Dilutions of bacterial suspensions were made to acquire a concentration of approximately 108 cfu/mL, and were stored at 37 C prior to use.


Simulated gastric transit tolerance test

The simulated gastric transit tolerance test was performed in a reaction volume of 1. 5mL containing 300AL of 0.5% NaCl or food in liquid form, 200, uL of bacterial suspension, and lrnL of simulated gastric juice. The test mixture was vortexed at a maximum setting for 10sec and then incubated at 37 C in an air forced incubator for 180min. At lmin, 60min, 90min, 180min incubation time, an aliquot (O. lmL) was taken from each test mixture and viable cell counts were determined by the pour plate method.


Small intestinal transit tolerance test

The simulated small intestinal transit tolerance test was performed in a reaction volume of 1. 5mL containing 300, uL of 0.5% NaCl or food in liquid form, 200ttL of bacterial suspension, and lrnL of simulated small intestinal juices with or without 0.3% bile salt. Test mixtures were vortexed at a maximum setting for 10sec, and then incubated at 37 C in an air forced incubator for 240min. At lmin, and 240min incubation time, an aliquot (0. lmL) was taken from each test mixture and viable cell counts were determined by the pour plate method.


Determination of viable cell coufzts using the pour plate method

Viable cell counts were estimated using the pour plate method. Briefly, serial dilutions of 0. lmL aliquots from the test mixture were made with MRD broth. Aliquots (ImL) of selected dilutions were transferred to each of two sterile Petri dishes. SLA agar (15mL), which was stored in a 50 C water bath, was then poured into each plate. The medium and the inoculum were mixed by five to-and-fro movements, followed by five circular clockwise movements, followed by five to-and-fro movements at right angles to the first set, followed by five circular anti-clockwise movements. The Petri dishes were left to stand at room temperature until the medium had set. As soon as the medium had set, the Petri dishes


were inverted, and incubated anaerobically at 30 C. After 6 days incubation, colonies were counted using a colony counter (Stuart Scientific).


Growth of dairy propionibacteria strains in the presence of bile salts

The method was extrapolated from the published methods developed for selecting bile tolerant Lactobacillus and Bifidobacterium strains (Gilliland et al, 1984 ; Chou and Weimer, 1999; Ibrahim and Bezkorovainy, 1993). Bile salts (Oxoid) were chosen in this study because it is a standardised bile extract and is extensively used as a selective inhibitory agent in bacteriological culture media to allow only bile-tolerant organisms to grow.


Propionibacterium strains were screened for their ability to grow in SLB broth containing 0.3% bile salts (Oxoid) at 37 C. Aliquots (100 yL) of 48 hr cultures of Propionibacterium strains were inoculated in lOmL of SLB with or without 0.3% bile salt in triplicate, and incubated anaerobically at 37 C for 5 days. The extent of growth was determined by measuring the optical density at 650nm at 10 intervals during the five day incubation period. Growth curves (Also versus time) of each strain were generated using Microsoft Excel software.
Adhesion to human gut epithelial cell line C2BBel Preparation of bacteria suspension

Strains were grown anaerobically in liquid broth, SLB for Propionibacterium, MRS for L. acidophilus MJLA1, and RCM for B. lactis BDBB2, at 30 C for 48 hours. Aliquots (1 mL) were transferred to 1. 5mL eppendorf tubes, and bacterial cells were collected by centrifugation (2500 x g, 5 min) and washed three times with ImL PBS buffer (pH7.0), centrifuging (2500 x g, 5 min) between each wash. Bacterial cells were then resuspended in PBS buffer (pH7.0) to acquire a concentration of approximately 109 cfu/mL, stored at 37 C for maximum 1 hr prior to the adhesion test.


Preparation of C2BBel monolayers C2BBel stock culture (lmL) was thawed in a 37 C water bath and mixed with Supplemented RPMI 1640 medium (7.5 mL) in a 25cm3 tissue culture flask. C2BBel cells were incubated at 37 C in 5% C02/95% air atmosphere until confluent. Fresh Supplemented RPMI 1640 medium was fed to cells when the medium turned yellowish red during incubation. After the cells were confluent, Supplemented RPMI 1640 medium was removed, and 0.25% trypsin, 0.2% EDTA solution (1. 5mL) was added to the flask, and incubated at 37 C in 5% C02/95% air atmosphere for 10 min or until the cells detached. The detached cells were resuspended in 30 mL Supplemented RPMI 1640 medium using sterile Pasteur pipettes. Aliquots (lmL) of cell suspension were dispensed into each well of a tissue culture plate (24 well, Becton Dickinson) containing sterile coverslips (13mm, Sarstedt). The plates were then incubated at 37 C, in 5% C02/95% air atmosphere until there was confluent growth

on the coverslips (around 7-8 days). Fresh Supplemented RPMI 1640 medium was fed to the cells when the medium turned yellowish red during incubation.


In vitro adhesion assay

The in vitro adhesion assay for bacterial strains to C2BBel cells was based on the in vitro assays for adhesion to Caco-2 cells (Tuomola and Salminen, 1998; Sarem et al, 1996).


The monolayers of C2BBel on coverslips in the 24 wells of the microtitre plate were washed with ImL of RPMI 1640 (Trace Biosciences), and supplemented with 950, uL of fresh RPMI 1640. The plate was incubated at 37 C, in 5% CO2/95% air. Aliquots (50, ut) of each bacterial suspension were added in duplicate to each well of the microtitre plate. The cultures were incubated at 37 C, in 5% CO2/95% air atmosphere for 2 hours. The monolayers were then washed four times with 1mL prewarmed (37 C) PBS buffer (pH 7.0) using Pasteur pipettes, and fixed with 500 AL of fixation buffer at room temperature overnight, ready for scanning electron microscopy.
Scanning electron microscopy

The tissue monolayers in the wells of the microtitre plates were washed three times with ImL of 0.1 M Cacodylate buffer. The monolayers were then dehydrated in 1 mL of graded series of ethanol (30%, 50%, 70%, 90%, and 100%), each for 10minutes. The monolayers were stored in ImL of 100% ethanol during transportation to the Electron Microscopy Unit, University of Newcastle, NSW, Australia. The monolayers were then dried by a critical point drier and stored in a desiccator in the Electron Microscopy Unit.


The dried monolayers were observed by scanning electron microscopy by Mr. David Phelan of the Electron Microscopy Unit, University of Newcastle.
Results Production of Vitamin B12

The vitamin B 12 production is shown in Table 10. All strains of Propionibacterium produced vitamin B 12 ranging from 0. 61ng/mL to 20.29ng/mL.


Table 10 Vitamin B12 production of dairy propionibacteria strains in Fermentation Medium Strain Species Source Vitamin B12 (ng/ml) CSCC2200 Pfreudeiireichii Australian Starter Culture Research Centre 10. 920. 58* CSCC2201 P.freudenreichii CSIRO Melbourne, Australia 4. 040. 70 CSCC2206 P. freudenreichii CSIRO Melbourne, Australia 0. 730. 28 CSCC2207 P. freudenreichii CSIRO Melbourne, Australia 5. 990. 34 CSCC2216 P. freudenreichii CSIRO Melbourne, Australia 11. 590. 83 201al P.freudenreichii This study 1. 600. 09 201b P. freudenreichii This study 2. 970. 25

801 P. freudenreichii This study 2. 090. 43 901 Pfreudettreichii This study 15. 691. 02

1001 Pfreudenreichii This study 20. 290. 78 702 P. jensenii This study 12. 840. 65 341 P. acidopropioriici University of Melbourne, Australia 0. 610. 19 ATCC25562 P. acidopropioraici American Type Culture Collection 0. 910. 23

*Results shown are mean (S. D.), n=2.


Effect of sifnulated gastric juices (pH2, pH3, pH4) on tlze viability of dairy propioraibacteria strains

The effect of simulated gastric juices (pH2, pH3, and pH4) on viability of 13 dairy propionibacteria strains is presented in Table 11. The average final pH of the simulated transit mixture was 2.33, 3.80, and 5.97 for pH2, pH3, and pH4 gastric juices.


Each strain showed higher viability in pH3 or pH4 simulated gastric juice than pH2 simulated gastric juice (Table 11).
When the simulated gastric juice was at pH2, all the strains showed progressive reduction in viability during the 180 min simulated gastric transit. After incubation with simulated gastric juice (pH2) for only 1 min, strains P. freudenreichii CSCC2206, P. acidopropionici 341 and P. acidopropionici ATCC25562 had 2 to 3-log reduction in viability (Table 11). After 60 min incubation, viability of most of the strains decreased substantially, except for Pfreudeizreichii 801, and P. freudenreichii 901 with only 1-log decrease in viability. After 90 min to 180 min incubation with simulated gastric juice (pH2), all the strains showed significant reduction (P < 0.05) in viability at different levels (Table 11), especially P. freudenreichii CSCC2200, Pfreudenreichii CSCC2206 and P. freudenreichii CSCC2216, which completely lost viability after 180 min simulated gastric transit (Table 11).
This indicates all the strains are sensitive to simulated gastric juice at pH2.
When the simulated gastric juice was at pH3, ten out of 13 tested strains retained similar viability during simulated gastric tract transit for up to 180 min (Table 11). Three strains, P. freudenreichii CSCC2206, P. acidopropionici 341 and ATCC25562 CSCC2206 showed only 1-log cycle reduction in viability after 180 min simulated gastric tract transit (Table 11). This indicates that the majority of the tested strains were tolerant to simulated gastric juice at pH3 and may survive gastric passage.
When the simulated gastric juice was at pH4, all of the tested strains retained the same level of viability during 180 min simulated gastric tract transit (Table 11). This indicates that the cells of 13 tested dairy propionibacteria strains may survive gastric passage, when gastric juice is at pH4.
Table 11 Effect of simulated gastric juices (pH2, pH3, pH4) on the viability of dairy propionibacteria strains

Strains Simulated Viable Count (log cfu/ml) during simulated gastric transit tolerance gastric juices 0 min 1 min 60 min 90 min 180 min P. freudenreichii pH2a 8. 46 (0.42) 8.19 (0.02) 4.16 (0.08) ** 0. 84 (0.09) *** < 1 CSCC2200 pH3b 8. 46 (0.42) 8.25 (0.24) 8.32 (0.05) 8.28 (0.20) 8.28 (0.28)


pH4c 8. 46 (0.42) 8.26 (0.17) 8.38 (0.18) 8.35 (0.21) 8.38 (0.18)

P. freudetireichii pH2 8. 60 (0.43) 8.80 (0.09) 3.98 (0.13) * 3. 72 (0. 21)* 2. 00 (0.00) **

CSCC2201 pH3 8.60 (0.43) 8.86 (0.09) 8.88 (0.00) 8.90 (0.01) 8.87 (0.03) pH4 8.60 (0.43) 8.70 (0.04) 8. 89 (0.02) 8. 84 (0.03) 8.83 (0.04) P. freudenreichii pH2 7. 65 (0.08) 4.67 (0.00) *** 2.72 (0.00) *** < 1 < 1

CSCC2206 pH3 7.65 (0.08) 7. 75 (0.00) 6.84 (0.34) * 6.84 (0.34) * 6.75 (0.21) * pH4 7.65 (0.08) 7.63 (0.14) 7.80 (0.02) 7.89 (0.03) 7.86 (0.09) P. freudelreicllii pH2 8. 63 (0.09) 8.77 (0.05) 4.85 (0.05) ** 2. 87 (0.21) ** 1.36 (0.03) ***

CSCC2207 pH3 8. 63 (0.09) 8. 94 (0.10) 8.92 (0.07) 8. 89 (0.05) 8. 88 (0.00) pH4 8.63 (0.09) 8.87 (0.02) 8.83 (0.00) 8.91 (0.09) 8.84 (0.05)

P. freudenreichii pH2 8. 61 (0.03) 8.49 (0.02) 2.00 (0.00) *** 0.69 (0.12) *** < 1

CSCC2216 pH3 8.61 (0.03) 8.72 (0.11) 8.65 (0.13) 8.67 (0.05) 8.66 (0.13) pH4 8.61 (0.03) 8.64 (0.03) 8.63 (0.05) 8.72 (0.03) 8.65 (0.04)

P. freuderreicllEi pH2 8. 43 (0.03) 8.36 (0.04) 5.04 (0.12) *** 3.93 (0.04) *** 3.47 (0. 18) *** 201al pH3 8.43 (0.03) 8.29 (0.01) 8.30 (0. 05) 7.82 (0.64) 8.27 (0.01) pH4 8.43 (0.03) 8.30 (004) 8.25 (0.03) 8.43 (0.15) 8.46 (0.12) P. pH2a 8. 46 (0.07) 8.26 (0.12) 5.58 (0.17) *** 5.18 (0.07) *** 4.89 (0.00) *** freudenreichii 201b pH3b 8. 46 (0.07) 8.29 (0.08) 8.34 (0.03) 8.36 (0.01) 8.43 (0.22) pH4c 8. 46 (0.07) 8.17 (0.13) 8.44 (0.05) 8.36 (0.00) 8.38 (0.00) P. jensenii702 pH2 8. 21 (0. 12) 8.29 (0.20) 5.96 (0.00) *** 4.03 (0.05) *** 2.61 (0.01) *** pH3 8.56 (0.05) 8.60 (0.04) 8.50 (0.02) 8.44 (0.03) 8.50 (0.03) pH4 8.56 (0.05) 8.62 (0.04) 8. 83 (0. 12) 8.26 (0.29) 8.50 (0.22) P. pH2 8. 78 (0.11) 8.54 (0.00) 7.83 (0.00) *** 5.89 (0.15) *** 3.49 (0.16) *** freudenreichii

801 pH3 8.78 (0.11) 8.83 (0.02) 8.83 (0.04) 8. 82 (0.06) 8.94 (0.03) pH4 8.78 (0.11) 8.77 (0.04) 8.87 (0.11) 8.91 (0.03) 8.91 (0.05) P. pH2 8. 71 (0.06) 8.78 (0.14) 7. 58 (0.15) ** 6. 75 (0.00) *** 3.89 (0.22) *** . freudeizreiclaii


freudenreichii pH3 8.76 (0.01) 8.83 (0.03) 8.78 (0.03) 8.85 (0.01) 8.94 (0.03) 901 pH4 8.76 (0.01) 8.77 (0.04) 8.96 (0.01) 8. 87 (0.02) 8.94 (0.01) P. pH2 8. 60 (0.00) 8.46 (0.00) 7.25 (0.09) *** 4.67 (0.09) *** 3.13 (0.25) *** freudenreichii

1001 pH3 8.60 (0.00) 8. 64 (0.02) 8.61 (0.00) 8.49 (0.00) 8.69 (0.00) pH4 8.60 (0.00) 8.61 (0.00) 8.76 (0.00) 8.72 (0.00) 8.76 (0.00) P. pH2 8. 25 (0.03) 6.00 (0.00) ** 4.90 (0.17) *** 4.64 (0.23) *** 2.95 (0.00) *** acidopropionici 341 pH3 8.25 (0.03) 8.20 (0.00) 7.96 (0.08) * 8.01 (0.01) ** 7.54 (0.34) *** pH4 8.25 (0.03) 8.13 (0.02) 8.23 (0.07) 8.29 (0.10) 8.18 (0.10) P. pH2 9. 09 (0.00) 6.50 (0.28) ** 5.54 (0.08) *** 5.40 (0. 16) *** 4.90 (0.00) *** acidopropiozlici ATCC25562 pH3 9.09 (0.00) 9.05 (0.00) 8.96 (0.00) * 8. 45 (0.02) *** 8.03 (0.01) *** pH4 9.09 (0.00) 9.08 (0.00) 9.24 (0.00) 9.16 (0. 00) 9.20 (0.00) Results are shown as mean (S. D.), n=2.
Compare the values of different incubation time with 0 min, use Anova, single factor statistics *: P value < 0.05, **: P value < 0.01, ***: P value < 0.001, 0, b, c, mixture pH was 2.34, 3.80, 5.97 respectively Effects offood addition on viability of dairy propionibacteria strains during simulated gastric tract transit tolerance (pH2 gastric juice)

Since tested dairy propionibacteria strains were sensitive to pH2 simulated gastric juice (Table 11) the effect of two kinds of vegetarian food, So-good soymilk (Sanitarium) and Up & Go cereal breakfast (Sanitarium), were tested for their effects on the viability of dairy propionibacteria strains during 180 min simulated gastric transit tolerance with pH2 gastric juice. The average final pH of the simulated transit mixture was 2.33, 5.23, and 5.34 for control, So-Good soymilk, and Up & Go cereal breakfast groups.


In general, addition of So-good soymilk and Up & Go cereal breakfast improved the viability of each strain significantly (P < 0.05) when simulated gastric juice was at pH2 (Table

12).
All the 13 tested dairy propionibacteria strains showed 3.5 to 8-log reduction in viability during the 180 min simulated gastric tract transit with pH2 simulated gastric juice without any food addition (Table 12).


In the presence of So-good soymilk, twelve out of 13 tested dairy propionibacteria strains exhibited complete tolerance to simulated gastric transit with pH2 gastric juice with no loss of viability after the 180 min simulated gastric transit (Table 12). Only strain P. freudenreichii CSCC2200 showed 0.3-log reduction in viability (Table 12). Interestingly, strain P. freudeiireichii CSCC2206 showed a slight reduction in its viability after 1 min of

simulated gastric transit but recovered to the same level of viability as that at 0 min at the end of 180 min gastric transit.


In the presence of Up & Go cereal breakfast, eleven out of 13 tested dairy propionibacteria strains maintained the same viability through the 180 min simulated gastric transit with pH2 gastric juice (Table 12). The other two strains, P. freudenreichii CSCC2200 and strain P. jensenii 702 showed only 0.3-log reduction in viability. However, they still had viability of about 8-log value compared to 2. 6-log and < 1-log for strain P. jensenii 702 and P. fre7ude7treichii CSCC2200 respectively without the addition of Up & Go after 180 min simulated gastric tract transit with pH2 gastric juice. These results indicate that all 13 tested dairy propionibacteria strains may survive passage through the human stomach, even when the gastric juice is at pH2, if tested strains are ingested with So-Good soymilk or Up & Go cereal beverages.
Table 12 Effect of food addition on the viability of Propionibacterium strains during gastric transit (pH2)

Viable Count (log cfu/ml) during simulated gastric transit tolerance Strains Conditions 0 min 1 min 180 min P.freudenreichii controla 8.46 (0.42) 8.19 (0.02) < 1 CSCC2200 So-goodb 8. 83 (0. 00) 8.48 (0.12) * 8.51 (0.00) *

Up & Goc 8.83 (0.00) 8.46 (0.09) * 8.53 (0.01)

P. freudenreichii control 8. 60 (0.43) 8.80 (0.09) 2.00 (0.00) **

CSCC2201

So-good 8.70 (0.00) 8. 76 (0.01) 8.91 (0.05)

Up & Go 8.70 (0.00) 8.76 (0.00) 8.91 (0.02)

P. freudenreichii control 7. 65 (0.08) 4.67 (0.00) *** < 1

CSCC2206

So-good 7.69 (0.00) 7.45 (0.00) *** 7.81 (0.04)

Up & Go 7.69 (0.00) 7.60 (0.07) 7.84 (0.04) P. freudenreichii control 8. 63 (0.09) 8.77 (0.05) 1. 36 (0.03) ***

CSCC2207

So-good 8.70 (0.00) 8.87 (0.02) 8.81 (0.09) Up & Go 8.70 (0.00) 8.80 (0.10) 8.89 (0.00)

P. freudenreichii control 8. 61 (0.03) 8. 49 (0.02) < 1

CSCC2216

So-good 8. 64 (0.00) 8. 74 (0.11) 8.75 (0.10)

Up & Go 8.64 (0.00) 8.54 (0.11) 8.73 (0.18)

P. freudenreichii201al control 8. 43 (0.03) 8.36 (0.04) 3.47 (0.18) ***


So-good 8.43 (0.03) 8.34 (0.06) 8.40 (0.05)

Up & Go 8.43 (0.03) 8.31 (0.05) 8.39 (0.01) P. freudenreichii 201b control'8. 46 (0.07) 8.26 (0.12) 4.89 (0. 00) *** So-goodb 8. 46 (0.07) 8.43 (0.16) 8.32 (0.00) Up & Go'8. 46 (0.07) 8.32 (0.15) 8. 37 (0.01) P. jensenii 702 control 8. 21 (0.12) 8.29 (0.20) 2.61 (0. 01) ***

So-good 8.21 (0.12) 8.22 (0.14) 8.30 (0.18)

Up & Go 8.21 (0.12) 8.23 (0.07) 7.95 (0. 07) * P. freiideizreichii 801 control 8. 78 (0.11) 8.54 (0. 00) 3.49 (0.16) ***

So-good 8.78 (0.11) 8.56 (0.00) 8.64 (0.00)

Up & Go 8.78 (0.11) 8.52 (0.00) 8.35 (0.14) P. freudenreichii 901 control 8. 71 (0.06) 8.78 (0.14) 3.89 (0.22) ***

So-good 8.71 (0.06) 8.73 (0.00) 8.81 (0.00)

Up & Go 8.71 (0.06) 8.68 (0.00) 8.79 (0.00) P. freudenreichii1001 control 8. 60 (0.00) 8.46 (0.00) 3.13 (0. 25) ***

So-good 8.60 (0.00) 8.60 (0.00) 8.66 (0.00)

Up & Go 8.60 (0.00) 8.60 (0.00) 8.72 (0.00)

P. acidopropionici control 8. 25 (0.03) 6.00 (0.00) *** 2. 95 (0. 00) ***

341


So-good 8. 25 (0.03) 8.08 (0.06) 8.33 (0.01)

Up & Go 8.25 (0. 03) 8.09 (0.06) 8.38 (0.10)

P. acidopropionici control 9. 09 (0.00) 6.50 (0.28) *** 4. 90 (0. 00) ***

ATCC25562

So-good 9.09 (0.00) 9.06 (0.00) 9.28 (0.00)

Up & Go 9.09 (0.00) 9.17 (0.00) 9.24 (0.00)

Results are shown as mean (S. D. ), n=2.
Compare the values of different incubation time with 0 min, use Anova, single factor statistics *: P value < 0.05, **: P value < 0.01, ***: P value < 0.001, a,b,c, mixture pH was 2.34, 5.23, 5.35 respectively

Simulated small intestinal transit tolerance of dairy propionibacteria strains

The effect of simulated small intestinal transit on the viability of 13 dairy propionibacteria strains is presented in Table 13.

All tested strains retained the same viability during 240 min simulated small intestinal transit in the absence of bile salt (Table 13). In contrast, in the presence of 0.3% bile salts, eleven of 13 tested dairy propionibacteria strains maintained the same viability during 240 min simulated small intestinal transit; the other two strains, P. freudenreichii CSCC2207 and P. acidopropionci 341, showed slight reduction in viable counts, of 0. 2-log and 0. 97-log respectively (Table 13).


Table 13 Effect of simulated small intestinal transit on viability of dairy propionibacteria strains

Viable count (log cfu/mL) during simulated small intestinal transit tolerance

Strains Absence of bile salts In the presence of 0.3% bile salts

0 min 1 min 240 min 0 min 1 min 240 min

CSCC2200 8. 17 (0.04) 8.24 (0.01) 8.29 (0. 03) 8.42 (0.02) 8.54 (0.04) 8.46 (0.03)

CSCC2201 8.80 (0.01) 8.82 (0.01) 8.86 (0. 10) 8.75 (0.01) 8.75 (0.12) 8.76 (0.01)

CSCC2206 7.53 (0.21) 7.84 (0.04) 7.78 (0.12) 7.87 (0.02) 8.00 (0.06) 7.85 (0.06)

CSCC2207 8.87 (0.04) 8.81 (0.03) 8.81 (0.04) 8.70 (0.01) 8.71 (0.08) 8.49 (0. 01) **

CSCC2216 8.62 (0.04) 8.67 (0.12) 8.54 (0.03) 8.58 (0.01) 8.74 (0.02) 8.61 (0.01)

201al 8.59 (0.04) 8.43 (0.01) 8.46 (0.05) 7.56 (0.03) 7.54 (0.10) 7. 61 (0.05)

201b 8.20 (0. 10) 8.23 (0.07) 8.45 (0.06) 7.55 (0.01) 7.61 (0.13) 7.70 (0. 05)

702 8.56 (0.05) 8.64 (0.02) 8.50 (0.06) 8.64 (0.06) 8.98 (0.03) 8.66 (0.26)

801 8.78 (0.11) 8.63 (0.04) 8.86 (0.02) 7.75 (0.14) 7.70 (0.08) 7.72 (0.06)

901 8.76 (0.01) 8.83 (0.02) 8.85 (0.03) 7.58 (0.05) 7.66 (0.01) 7.73 (0. 10)

1001 8.68 (0.03) 8.82 (0.03) 8.83 (0.02) 7.72 (0.03) 7.65 (0.04) 7.68 (0.07)

341 8.08 (0.07) 8.15 (0.04) 8.31 (0.16) 7.75 (0.09) 6.54 (0.09) ** 6.78 (0.25) *

ATCC25562 7.97 (0.09) 8. 14 (0. 13) 8.61 (0. 14) 7.71 (0. 01) 7.7 (0.14) 7.83 (0. 05)

Results are shown as mean (S. D. ), n=2.


Compare the values of different incubation time with 0 min, use Anova, single factor statistics *: P value < 0.05, **: P value < 0.01, *** : P value < 0.001.
Growth of dairy propionibacteria strains in SLB with 0. 3% bile salt

The growth of the 13 dairy propionibacteria strains in this experiment was evidenced by the increase in optical density (A6so) of the growth broth. The higher the optical density of the broth, the more bacterial cells in the broth. The total viable cell counts of dairy propionibacteria strains were found to be around 108 cfu/mL when the value of A6so was 0.5 (Table 14). Probiotic bacteria cannot affect their environment unless the population reaches a certain minimum level around 106 to 108 cfu/g of intestinal contents (Charteris et al, 1998). Therefore, A6so = 0. 5 was considered as the threshold for effective growth of dairy propionibacteria strains in this experiment.

Table 14 Total viable cell counts of dairy propionibacteria strains at Also of 0. 5.
Strains Total viable cell counts (cfu/mL)

P. freudenreichii CSCC2200 5.11 *108

P. freudenreichii CSCC2201 8.31 * 108

P. freudenreichii CSCC2206 3.41 * 108

P. freudenreichii CSCC2207 5.91 * 108

P. freudenreichii CSCC2216 7.51 * 108

P. freudenreichii 201al 7.11 * 108

P.freudenreichii 201b 7.78 * 108

P. jensenii 702 3.32 * 108

P. freudenreichii 801 9.94 * 108

P. freudenreichii 901 9.92 * 108

P. freudenreichii 1001 5.96 * 108

P. acidopropionici 341 2.67 * 108

P. acidopropionici ATCC25562 3.39 * 108

Figure 9 and Table 15 show the growth characteristics of 13 dairy propionibacteria strains, including six isolated strains, 5 strains of P. freudenreichii and 2 strains of P. acidopropionici, in SLB with 0. 3% bile salts incubated anaerobically at 37 C.
Nine out of 13 tested dairy propionibacteria strains reached their maximum growth (A6so) during a 5-day incubation period (Figure 9 A, C, D, E, F, G, I, K, M). The other four strains, including P. freudenreichii CSCC2206, P.freudenreichii 801, P.freudenreichii 1001, and P. acidopropionici 341 (Figure 9 B, H, J, L), did not reach their maximum growth (A6so) during the 5-day incubation period. This was particularly evident for P. freudenreichii

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