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



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201al ; Lane 8: strain 201b; Lane 9: strain 702; Lane 10; strain 801; Lane 11: strain 901; Lane

12 : strain 1001

Figure 9 shows growth curves of dairy propionibacteria strains in SLB with 0. 3% bile salt at 37 C anaerobically. (A) P. freudenreichii CSCC2201; (B) P. freudenreichii CSCC2206; (C) P. freudenreichii CSCC2207; (D) P. freudenreichii CSCC2216; (E) P. freudeyzreichii 201al ; (F) P. freudenreichii 201b; (G) P. jensenii 702 (H) P. freudenreichii 801, (1) P. freudenreichii 901; (J) P. freudenreichii 1001; (K) P. acidopropionici ATCC25562; (L) P. acidopropionici 341 ; (M) P. freudenreichii CSCC2200.


Figure 10 shows scanning electron micrographs of adhesive bacterial strains to human epithelial cell line C2BBel. (A) L. acidophilus MJLA1 ; (B) B. lactis BDBB2; (C, D) P. jensenii 702;

Figure 11 shows the growth curve of the rat groups fed ad libitum (n=7)

Figure 12 shows the water intake by rats of P. jensenii 702 group.
Figure 13 shows the enumeration of dairy propionibacteria in faeces of rats fed with P. jensenii 702, n=7.
Figure 14 shows a scanning electron micrograph of the inner surface of a male Wistar rat small intestine fed P. jensenii 702 for a period. of 81 days.
Figure 15 shows the inverse relationship between serum vitamin B 12 levels and homocysteine levels of male Wistar rats during three-month feeding treatment. A: Deficiency group; B: Bacteria group, each group contained 7 rats.
Figure 16 shows the comparison of the average total cholesterol and triglycerides in the three Wistar rat groups at the completion of the three month feeding trial, each group containing 7 rats.
Figure 17 shows interleukin 2 levels in serum samples from mice

Figure 18 shows a comparison of IFN gamma and IL-4 levels between vaccine groups. Values are mean. Error bars depict standard deviation.


Best Method of Carrying Out the Invention Analysis of vitamin B12

Several analytic methods, including spectrophotometry, microbiological assay, protein-binding assay, radioassay and high performance liquid chromatography (HPLC), are available to determine the levels of vitamin B12 in different samples. No TU for vitamin B 12 activity has been defined, and the assay results are expressed in milligrams, micrograms or nanograms of pure crystalline cyanocobalamin (Ball, 1998).


Extraction procedures are generally required for the determination of total vitamin Bl2 content (Ball, 1998). Vitamin B12 occurs intracellularly in the living tissues of animals and

most biosynthetic microorganisms or is bound to some proteins in food. The extraction procedure liberates protein-bound cobalamins and converts the different forms to a single and stable form, cyanocobalamin or sulphitocobalamin, by reaction with cyanide or metabisulphite respectively (Ball, 1998).


Several extraction methods have been used to extract vitamin B12 from food or microorganism cell samples. In the AOAC [Association of Official Analytical Chemists prior to 1990 but now AOAC International] official method, samples in the extracting solution, which contains 1.3% sodium hydrogen phosphate, 1.2% citric acid. H2O, and 1. 0 g sodium metabisulfite, are autoclaved 10min at 121-123 C (AOAC, 1995). Vitamin B] 2 in P. acidopropionici cells has been extracted by autoclaving the samples at 121 C 10min in a 0. 1M phosphate buffer solution (pH6), containing 0.1% KCN or 0.01% KCN (QuesadaChanto et al. , 1994b, Quesada-Chanto et al. , 1994a, Quesada-Chanto et al. , 1998). Another procedure, which has been described for extracting vitamin B12 from food samples, includes homogenising the food sample with 0.1 M sodium acetate-acetic acid buffer (pH4.5), containing 1 mM potassium cyanide, by sonication; and then autoclaving the mixture for 10min at 121 C (Muhammad et al. , 1993c). Boiling at 100 C for 20 min has also been used for the extraction of vitamin B12 from Propioyaibacteriurra freudenreichii cells in a 0. 1M phosphate buffer solution (pH5.5), containing 0. 01% potassium cyanide (KCN) (Ye et al., 1996). Since autoclaving may result in better extraction than heating by boiling water as the boiling water may not completely extract all of the bound vitamin (Casey et al. , 1982), the method of autoclaving the samples at 121 C for 10 min in 0. 05M phosphate buffer solution (pH5.5), containing 0.01% KCN has been used to extract vitamin B12 from Propionibacterium cells in this study.
Radioassay

Radioassay was first used to determine the vitamin B, 2 content of serum in the clinical environment (Lau et al. , 1965). Now radioassay techniques have been developed to determine vitamin Bi2 levels in food (Casey et al. , 1982, Richardson et al., 1978).


Radioassay is based on the competitive binding capacity of radioactive vitamin B12 and the vitamin B12 in samples for binding protein. Cyanocobalamin is recommended as the calibration standard for the competitive binding radioassay (Muhammad et al. , 1993b, Muhammad et al. , 1993a). A preparation procedure, which converts different vitamin Bi2 forms (except methylcobalamin) into dicyanocobalamin, is suggested to provide accurate results (Muharnmad et al. , 1993a). This is because the binding affinity for hog intrinsic factor by hydroxocobalamin, adenosylcobalamin and sulphitocobalamin has been found to be statistically different from that of cyanocobalamin, but the relative binding affinity of

dicyanocobalamin or methylcobalamin does not differ significantly from that of cyanocobalamin (Muhammad et al. , 1993a).

* There are several commercial radioassay kits available for vitamin B12 determination (Richardson et al. , 1978), including Pharmacia B12 Test 50 Radioassay (Poston and Hemmings, 1979), and Bio-Rad B, 2/Folate (used in this study). The Bio-Rad commercial radioassay kit utilises [57Co] cyanocobalamin as the tracer, hog intrinsic factor as the binding protein and cyanocobalamin as the calibration standard. The testing range of this commercial kit is from 50pg/ml to 2000pg/ml.
EXAMPLE 1 ISOLATION AND IDENTIFICATION OF DAIRY PROPIONIBACTERIA STRAINS FROM RAW MILK, CHEESE AND HUMAN SMALL INTESTINE BIOPSY SAMPLES

The purpose of this study was to isolate strains of dairy propionibacteria species from raw milk, cheese products and human small intestine biopsy samples, and further identify the isolated strains into species. For this purpose, different identification methods, including traditional identification methods, API 50 CH carbohydrate fermentation profiles, PCR identification methods and whole cell water- soluble protein analysis by SDS-PAGE, were used.


Materials and Methods Reference strains

Reference strains used throughout this study are listed in Table 4.


Table 4 Details of reference strains used
STRAIN SPECIES SUPPLIER

CSCC 2200 Propionibacteriuoa Australian Starter Culture Research Centre, Australia

freuderzreichii

CSCC2201 P. freudenreichii CSIRO Melbourne, Australia

CSCC2206 P. freudenreichii CSIRO Melbourne, Australia

CSCC2207 P. freuderareiclzii CSIRO Melbourne, Australia

CSCC2216 P. freudenreichii CSIRO Melbourne, Australia

ACM365 P. tlioetzii Australian Collection of Microorganisms, University

of Queensland, Australia

ATCC25562 P. acidopropiozlici American Type Culture Collection, the United States

341 P. acidopropionici Microbiology Department, University of Melbourne,

Australia

NCFB571 P. jense) lii Dr. Franca Rossi, University of Verona, Italy

NCFB572 P. jensenii Dr. Franca Rossi, University of Verona, Ital

MJLA1 Lactobacillus Gist Brocades, Australia

acidophilus


Strain recovery

Strains were recovered in liquid medium, Sodium Lactate Broth (SLB) for Propionibacterium, de Man, Rogosa and Sharp medium (MRS, Oxoid) for Lactobacillus at 30 C anaerobically, and then streaked onto agar plates, Sodium Lactate Agar (SLA) for Propionibacterium, and MRS agar (Oxoid) for Lactobacillus to establish purity. Pure colonies were Gram stained, and catalase tested. All strains were examined microscopically for correct cell morphology.
Strain preservation Propionibacteriasm strains were harvested from SLA plates and resuspended in Sodium Lactate Glycerol Broth, which is SLB with 20% glycerol (Sigma). IniL of the bacterial suspension was dispensed into a 2mL Coming Cryogenic vial (Corning Glass Works), frozen in dry ice, and stored at-70 C.
L. acidophilus MJLA1 was harvested from MRS plates and resuspended in MRS broth containing 20% glycerol. lrulL of the bacterial suspension was dispensed into a 2mL Coming Cryogenic vial (Corning Glass Works), frozen in dry ice, and stored at-70 C.
Isolation of dairy propionibacteria strains Ethics approval

Ethics approvals for studies of human biopsy samples, were obtained from both the Ethics Committee of the University of Newcastle of Australia, and the Ethics Committee of the Sydney Adventist Hospital, Sydney, NSW, Australia. The ethics certificate number is H6700499.


Human biopsy sample, milk and cheese sample collection

Human gut biopsy samples were collected at the Sydney Adventist Hospital, Sydney, NSW, Australia, stored in 2mL Coming Cryogenic vials, containing ImL Maximum Recovery Diluent (MRD, Oxoid), and delivered to the laboratory at ambient temperature.


Raw milk samples were obtained from a dairy farm at Cooranbong, NSW, Australia.
Cheese samples, including Parmesan cheese (Bonlac), Swiss cheese (Dairy Good), Best Quality Gouda (New Zealand), Grana Papano cheese (Bengodi) and Jarlsberg cheese (Norway), were purchased from Woolworths supermarket in Gosford, NSW, Australia.
Procedures for isolation of dairy propioraibacteria strains

1 in 5 dilutions of cheese samples were prepared by mixing lOg of each cheese sample with 40mL of MRD in a stomacher bag, and then stomaching for 1 min in a stomacher (Calworth).


Coming Cryogenic vials containing human biopsy samples were vortex mixed at maximum setting for 10s using a vortex mixer (Ratex).
A loop of each cheese suspension, human biopsy suspension, and each raw milk sample was streaked onto duplicate Yeast Extract Lactate Agar (YELA) plates, and incubated

anaerobically at 30 C for 10 days. After 10 days incubation, different single colonies on YELA plates were picked and streaked onto SLA plates, and incubated anaerobically at 30 C for 7 days. Then pure single colonies were streaked onto SLA slopes, and stored at 4 C for up to 3 month for identification.


Traditional identification method Species ide7ztification scheme

A quick traditional identification scheme was designed for the identification of isolated strains: 1 Gram positive, catalase positive, non spore-forming, irregular shaped short rods ......... 2 Propionibacterium genus 2 Gelatin hydrolysis positive....... cutaneous propionibacteria

Gelatin hydrolysis negative....... 3 dairy propionibacteria 3 Fermentation of maltose and sucrose positive......... 4

Fermentation of maltose and sucrose negative.......... P. freSenreich 4 hemolysis positive.......... P. thoezii hemolysis negative.......... 5 5 Reduction of nitrate positive.......... P. acidopropionici

Reduction of nitrate negative......... P. jensenii

This scheme was formulated from the information contained in'A scheme for the identification of Gram-positive bacteria' (Harrigan, 1998b) and the differentiation characteristics of dairy propionibacteria in Bergey's Systematic Bacteriology (Cummins and Johnson, 1986). The identification is based on phenotypic characteristics, including morphological properties and biochemical properties (catalase test, gelatin hydrolysis, maltose and sucrose fermentation, 13-hemolysis, and reduction of nitrate).


Gram-stain

A single colony of each strain was selected from an SLA plate. A smear of bacteria was prepared on a clean, grease-free slide. After the smear was air dried and heat fixed, it was flooded with Crystal violet stain (Micro Diagnostics) for 30 seconds, rinsed with tap water and blot dried; the smear then was flooded with Gram Iodine stain (Micro Diagnostics) for 30 seconds, rinsed with tap water and blot dried; the smear then was decolourised with 95% ethanol (Micro Diagnostics) for 5-15 seconds, and counterstained with safranine stain (Micro Diagnostics) for 30 seconds. The slides were rinsed with tap water and the stain deposits were carefully removed from the back of the slide with filter paper. The slide was blot dried and examined under a light microscope at 1 OOOx magnification.

Catalase test

3% Hydrogen peroxide (Chem-Supply) was freshly prepared daily and stored at 4 C until use. A single colony of each isolated strain was picked from the SLA plate and emulsified with the 3% hydrogen peroxide on a clean grease-free slide using a wooden tooth pick. Production of gas bubbles indicated the presence of catalase in the tested culture.


Gelatin hydrolysis

Nutrient broth (NB) (Oxoid) containing 12% gelatin (Oxoid) was prepared in 10 x

130 mm test tubes with screw caps. A single colony of each isolated strain on an SLA plate was inoculated into the medium with a straight stabbing wire, and incubated anaerobically at 30 C for 30 days. The tubes containing cultures were removed from the incubator and placed in a 4 C fridge for 4h or until the medium in the negative control tube set. Liquefaction of medium indicated a positive gelatin hydrolysis reaction.
J3-Haemolysis reaction

A single colony of each isolated strain on an SLA plate was streaked onto a sheep blood agar plate (HAPS), and incubated anaerobically at 30 C for 7 days. The plates were then checked for signs of hemolysis. An uninoculated sheep blood agar plate was used as negative control to prevent false positive results, since sheep blood agar can be naturally hemolyzed during storage or incubation.


Carbohydrate fermentation

The carbohydrate fermentation medium consists of Sodium Lactate Base (SL) containing 1% each of maltose or sucrose. Aliquots of lOmL of each carbohydrate medium were prepared in 20mL bottles (Oxoid) with screw caps, and inoculated with one single colony of each isolated strain grown on sheep blood agar anaerobically at 30 C for 5 days.


Each strain was tested in duplicate. The inoculated bottles were incubated anaerobically at 30 C for 7 days. Turbidity or precipitation of the broth indicated a positive growth. Yellow colorization of the broth or pH of the broth below 5.7 indicated positive fermentation.
Dairy propiozibacteria genus and species specific PCR Preparation of genomic DNA

Purification of genomic DNA of 6 isolated strains and reference Propioflibacteriurra strains was based on the method described by Rossi (1998), and as outlined briefly below:

Strains were grown anaerobically in SLB broth for 48-72 hr. 1mL of each strain was transferred to a 1. 5mL eppendorf tube (Sarstedt), and centrifuged at 2 500 x g (Beckman) for 5 min. The supernatant was decanted. The cell pellet was then resuspended with 900 tL of TE buffer. Lysozyme (100 L) (Sigma, 100mg/mL) was added to the cell suspension and mixed by inverting the tubes 30 times. The mixture then was incubated in a 37 C water bath

for 2 hours. After incubation, the treated cells were collected by centrifugation (2500 x g, 5min). The supernatant was removed and each of the pellets was resuspended in TE buffer (370 L), and 20% (w/v) sodium dodecyl sulfate (10 uL, SDS), then mixed by inverting the tube 10 times. Nase-free RNAase (20 yL) (Sigma, 2 mg/mL) was added to the suspension and mixed by inverting the tube 20 times and incubated in a 37 C water bath for 30 min.


Proteinase K (8 gel) (Sigma, 1 mg/mL) was added and mixed by inverting the tubes 20 times, and incubated in a 37 C water bath for 1 hour. Sodium perchloride (3M, 215 uL) (Sigma) was added and mixed by inverting the tubes 30 times. The aqueous phase was extracted with an equal volume of phenol : chloroform: iso-amylalcohol (Sigma), followed by chloroform (Ajax): iso-amylalcohol (Ajax) (24: 1), centrifuging (3500 x g, 5min) in between, and transferring to a new Eppendorf tube after each extraction. The DNA was precipitated with an equal amount of isopropanol (Unilab) by gently mixing the tubes 50 times. The DNA was pelleted by centrifugation (3500 x g, 1 min). The DNA pellet was then washed with cold 70% (vol/vol) ethanol (Rhone-Poulenc), air dried for 30 min, and resuspended in 100, uL of PCR- grade water (Biotech) at 4 C overnight. The DNA solution was mixed gently for 10 s by a vortex mixer (Ratex), and stored at 20 C.
Determination of DNA concentration

DNA concentration was determined as described by Towner (1999).


Briefly, 5 HL of DNA solution was added to an eppendorf tube containing 995 StL distilled water, and mixed gently by a vortex mixer (Ratex). The absorbance of the DNA solution was measured at 260 nm and 280 nm. The ratio of OD26o/OD280 indicated the purity of the DNA sample. Samples with a ratio above 1.7 were accepted. The concentration of DNA (ng/L) is equivalent to 104 * OD260.
Dairy propionibacteria genus specific and species specific primers

Primers were constructed by Genes Works, Perth, WA, Australia, according to the description by Rossi (1999), as in Table 5. Primer pairs are PB 1-PB2, PF-PB2, PJ-PB2, PA- PB2, and PT3-PB2.


Table 5 Primer sequences

Primer name Primer sequence PB 1 (SEQ ID No. 14) 5'-AGTGGCGAAGGCGGTTCTCTGGA-3' PB2 (SEQ ID No. 15) 5'-TGGGGTCGAGTTGCAGACCCCAAT-3' PF (SEQ ID No. 16) 5'-CTTTCATCCATGACGAAGCGCAAG-3' PJ (SEQ D No. 17) 5'-GACGAAGTGCCTATCGGGGTG-3' PA (SEQ ID No. 18) 5'-GACGAAGGCATTCTTTTAGGGTGT-3' PT3 (SEQ m No. 19) 5'-GGACAAAAGGCCTTTCGGGGTTT-3'


PCR amplification procedure

PCR was performed in a reaction volume of 20 iL containing lx reaction buffer (Biotech), 1.5 mM MgCl2 (Biotech), 100 M of each dATP, dCTP, dTTP, and dGTP (Biotech), 0.5 ! 1M each primer, 1 UI of Taq DNA polymerase, and l, uL of diluted target DNA solution (around 20 ng DNA). PCR was performed in a thermal cycler (Corbett Research). After an initial denaturation at 94 C for 4 min, the PCR conditions used consisted of 40 cycles of denaturation at 94 C for 30 s, annealing for 15s at different temperatures for each different upstream primer (at 68 C for PA and PJ, at 69 C for PF, and at 70 C for PT3 and PB1), and extension at 72 C for 1 min. After the final cycle, the temperature was maintained at 72 C for 5 min to complete extension.


Analysis of dairy propionibacteria genus specific PCR products

The PCR mixtures were analysed for amplification products by mixing 5 L of the mixture with 1 L of 6 x sample buffer (Biotech) and observing after agarose gel electrophoresis at 100 volts for 20 min using a 1.5% agarose gel containing 0.5 ! lg/rnL ethidium bromide (Bio-Rad) in 0. 5x TAE buffer (Sigma). Following electrophoresis the agarose gel was examined and photographed using Gel-Doc Computer software (Bio-Rad).


RAPD-PCR using prifner OPL-OS for identification of dairy propionibacteria

Genomic DNA preparations and DNA concentration determination were performed as previously described.


Primer OPL-05

Primer OPL-05 (5'-ACGCAGGCAC-3') was constructed by Genes Works, Perth, WA, Australia.


RAPD-PCR amplification procedure

PCR was performed in a reaction volume of 25 L containing lx reaction buffer (Biotech), 1. 5 mM MgCl2 (Biotech), 100)-) M of each dATP, dCTP, dTTP, and dGTP (Biotech), l, uM primer OPL-05, 2.5 UI of Taq DNA polymerase, and 5 RL of diluted target DNA solution (around 25 ng DNA). RAPD-PCR reaction was performed in a thermal cycler (Corbett Research). After an initial denaturation at 94 C for 4 min, the PCR conditions used consisted of 45 cycles of denaturation at 94 C for 1 min, annealing at 35 C for lmin, and extension at 72 C for 2min. After the final cycle, the temperature was maintained at 72 C for 5min to complete extension.


Analysis of RAPD-PCR products

The PCR mixtures were analysed for amplification products by mixing 5, uL of the mixture with lu, L of 6 x sample buffer (Biotech) and observing after agarose gel electrophoresis at 80 volts for 40 min using a 1.5% agarose gel containing 0.5 sug/mL


ethidium bromide (Bio-Rad) in 0. 5x TBE buffer (Sigma). Following electrophoresis the agarose gel was examined and photographed using a Gel-Doc (Bio-Rad).


SDS-PAGE of whole cell water-soluble protein Preparation of whole cell water-soluble proteins

Whole cell soluble proteins extraction was based on the method described by Fessler (1999). In brief, each strain was grown anaerobically in lOmL of SLB for 48 hours at 30 C.


Cells were centrifuged (3500 x g, 10 min) and the pellets were washed in ImL 0. 9% NaCl twice. The pellets were then resuspended in 200 FL of TES buffer. Approximately 0. 5g of glass beads (0.25 mm-0. 5mm, Australian Scientific) were added to the suspension and the suspension was vortexed at maximum speed five times at 1 min intervals. The suspensions were chilled on ice for at least 1 min between vortexing. The suspensions were centrifuged (600 x g, 10 min) and the supernatants, containing whole cell soluble proteins, were collected for analysis by SDS-PAGE.
Determination of protein concentration

The protein concentration of each sample was determined using the Bio-Rad Protein Assay, Microtiter Plate Protocols. A standard curve was constructed using bovine serum albumen (Bio-Rad) concentrations of 0.073, 0.137, 0.247, and 0.336 mg/mL protein. Each standard and sample (10 gL) was placed in triplicate in a 96 well microtitre plate. Protein samples were diluted if required, so the reading fell within the boundaries of the standard curve. Bio-Rad dye reagent (100 L) was added to each well and mixed gently. Reaction was allowed between 5 min to 1 hour. The absorbance of each well was read and the protein concentration of the tested sample was automatically calculated using a Bio-Rad Microplate Reader (Model 550).


SDS-PAGE electroplioresis

SDS-PAGE electrophoresis conditions were a modification of those described by Baer (1987). Briefly, vertical 12% acrylamide gels of 0.75 mm thickness were prepared using the Bio-Rad Protean II xi Cell cooled vertical slab apparatus. Each gel was prepared by slowly pipetting the 12% acrylamide separation gel mix along the edge of the glass plates, leaving 4 cm at the top for the stacking gel. Sterile water was layered over the top and the acrylamide gel was allowed to set for 40-45 min at room temperature. The water layer was carefully drained and 4-4.5mL of 4% acrylamide stacking gel was added to the top of the separating gel, leaving 0. 5cm at the top. A 15-well comb was then inserted into the stacking gel. The gels were left to solidify for 30-35 minutes at room temperature. Samples were diluted with Sample Buffer to a final protein concentration of 10-15 Fg/llL, and heated to 94 C for 3 min. Samples (30gel) were added to each well. Double gels were run fully immersed in lx Running Buffer at a constant current of 30 mA at room temperature for 5 hr.

Coomassie Blue stainizg SDS-PAGE gel

The gels were carefully removed from the glass plate, immersed in 1 x Coomassie Blue stain (Bio-Rad) and incubated at room temperature for 30 min with constant shaking on a rotating incubator (lOrpm).


The gels were then destained by immersion in Destaining Solution for 90 min at room temperature with constant shaking and replacement of destaining solution a few times until the gel background was clear.
A7zalysis of SDS-PAGE gels

The gels were photographed and band profiles analysed using GelDoc computer software (Bio-Rad).


Storage of SDS-PAGE gels

Gels were stored by drying in cellophane. Briefly, gels were washed with deionised water three times and immersed in gel-dry solution (Bio-Rad) for 30 minutes with constant shaking, then dried in cellophane by the Bio-Rad GelAir Drying System.


API CH 50 carbohydrate profile using SL

The Propionibacterium strains were assessed for carbohydrate profile using API CH 50 strips. The preparation and analysis of isolates were conducted according to the manufacturer's instructions. SL broth was adopted as the growth medium.




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