CA2890965C - Probiotic strains isolated from dogs for use in dog food, treats and/or supplements - Google Patents

Abstract

Isolated probiotic strains from dogs are provided for use in dog food, treats and/or as supplements. Species-specific probiotic bacterial strains for use in products for canines were isolated and characterized. Gram-positive bacteria were recovered from freshly-voided canine feces and duplicate isolates were eliminated using Random Amplified Polymorphic DNA (RAPD) polymerase chain reaction (PCR). Probiotic candidates were confirmed through a series of tests including tolerance to low pH and bile, production of inhibitory substances, ability to alter host immune function, and strain stability. DNA sequencing determined these isolates are a range of lactic acid bacteria.

Description

TITLE:
PROBIOTIC STRAINS ISOLATED FROM DOGS FOR USE IN
DOG FOOD, TREATS AND/OR SUPPLEMENTS
CROSS REFERENCE TO RELATED APPLICATIONS:
This application claims priority of United States Provisional Patent Application Serial No. 61/990,534, entitled "Probiotic Strains Isolated from Dogs for Use in Dog Food, Treats and/or Supplements", filed May 8, 2014.
TECHNICAL FIELD:
The present application relates to probiotics, and more particularly, isolated probiotic strains from dogs for use in dog food, treats and/or as supplements.
BACKGROUND:
By way of background, human health practices (including probiotic consumption) are transitioning into the animal market, for example but not limited to, the companion animal market.
Intestinal dysbiosis, or an imbalance in the immunological state of the Gastrointestinal (GI) tract, is implicated in the development of both acute and chronic GI disorders. The general consensus of what determines a healthy gut from a non-healthy, or diseased, gut is the immunological balance. In a healthy {E6849592 DOCX;

2 animal, there is a balance achieved between tolerance to pathogens and suppression induced by commensal bacteria.
Dysbiosis in microbiota of the intestine causes a shift in the balance of immunostimulatory cytokines. This can throw off the balance of the inflammatory signals in the gut and cause dysbiosis and diarrhea. Probiotics have been shown to ameliorate these issues and restore balance of good bacteria in the Cl tract.
Currently, commercially-available canine probiotics are generally of human origin and are not optimal for animals since probiotic bacteria possess host-specific traits (Oh et al., 2010, Frese et al., 2011).
As such, there remains a need to provide an effective isolated probiotic for use in dog food, treats and/or as supplements that can overcome the shortcomings of the prior art.
SUMMARY:
Isolated probiotic strains from dogs are provided for use in dog food, treats and/or as supplements. Species-specific probiotic bacterial strains for use in products for canines were isolated and characterized. Gram-positive bacteria were recovered from freshly-voided canine feces and duplicate isolates were eliminated using Random Amplified Polymorphic DNA (RAPD) polymerase chain reaction (PCR). Probiotic candidates were confirmed through a series of tests including tolerance to low pH and bile, production of inhibitory substances, ability {E6849592.DOCX; 5}

3 to alter host immune function, and strain stability. DNA sequencing determined these isolates are a range of lactic acid bacteria.
Broadly stated, in some embodiments, isolated strains of canine probiotic bacteria for use in canine products are provided. In some embodiments, the strain of canine probiotic bacteria is of the Lactobacillus genus. In some embodiments, the strain of canine probiotic bacteria is selected from the group consisting of Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus saliva rius. In some embodiments, the strain of canine probiotic bacteria is selected from the group consisting of Willow 2-1-1, Lactobacillus rhamnosus K9-8; Nikita 2-1-1, Lactobacillus paracasei K9-4; Georgia 2-1-1, Lactobacillus fermentum K9-2; Gracie 2-1, Lactobacillus casei K9-1; Coal 2-2-1, Lactobacillus reuteri K9-6; Sullivan 2-1-1, Lactobacillus rhamnosus K9-9; Tucker 2-2-3, Lactobacillus reuteri K9-7, Abby 2-1-1, Lactobacillus reuteri K9-5; Ambar 2-1-1, Lactobacillus rhamnosus K9-10; Mika 2-1-3, Lactobacillus salivarius K9-11; and Mika 2-2-7, Lactobacillus fermentum K9-3.
In some embodiments, a biologically pure culture of the strain is deposited under the Budapest Treaty at an international depositary authority (IDA). In some embodiments, the strain is assigned a specific accession number by the IDA.
In some embodiments, a stock of each isolate, for example, Lactobacillus casei strain K9-1, was deposited in The International Depositary Authority of {E6849592 DOCX,

4 Canada (IDAC) (1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2) on April 21, 2015 and received IDAC Accession No. 210415-01.
In some embodiments, a stock of each isolate, for example, Lactobacillus fermentum strain K9-2, was deposited in The International Depositary Authority of Canada (IDAC) (1015 Arlington Street, Winnipeg, Manitoba, Canada R3E
3R2) on April 21, 2015 and received IDAC Accession No. 210415-02.
In some embodiments, the canine products are an ingestible food product, for example, an ingestible food product selected from the group consisting of food, treats, and supplements.
Broadly stated, in some embodiments, a composition is provided for use in canine products, the composition comprising a first isolated strain of canine probiotic bacteria and a second isolated strain of canine probiotic bacteria.
In some embodiments, the composition can further comprise a third isolated strain of canine probiotic bacteria Broadly stated, in some embodiments, a use of an isolated strain of canine probiotic bacteria in canine products is provided. In some embodiments, a use of an isolated and characterized strain of canine probiotic bacteria in canine products is provided. In some embodiments, the canine products are selected from the group consisting of food, treats, and supplements. In some embodiments, an isolated strain of canine probiotic bacteria is provided for use in canine products for treatment of intestinal dysbiosis.
{E6849592 DOCX, 5) Broadly stated, in some embodiments, a method is provided for isolating a strain of canine probiotic bacteria for use in canine products, the method comprising: recovering bacteria from freshly-voided canine feces; placing the feces into nutrient broth; incubating the feces and nutrient broth under aerobic

5 conditions to allow for the growth of Gram-positive fecal bacteria;
separating the Gram-positive fecal bacteria from the nutrient broth; plating the Gram-positive fecal bacteria onto plates; incubating the plates under aerobic conditions to form resulting colonies; and choosing resulting colonies with different morphologies for further analysis; whereby a biologically pure strain of canine probiotic bacteria is provided for use in canine products.
Broadly stated, in some embodiments, a method is provided for preparing food, the method comprising: providing a canine food product; adding to the canine food product an isolated strain of canine probiotic bacteria for use in canine products.
Broadly stated, in some embodiments, a method is provided for treatment of canine intestinal dysbiosis to derive an economic benefit, the method comprising: providing an isolated strain of probiotic bacteria; adding the isolated strain of probiotic bacteria to a food; and having a canine ingest the food;
whereby an economic benefit is derived by the treatment of canine intestinal dysbiosis.
BRIEF DESCRIPTION OF THE DRAWINGS:
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6 Figure 1 depicts a listing of 16S rDNA, pheS, and rpoA nucleotide sequences of certain isolate embodiments.
Figure 2 depicts the results of Random Amplified Polymorphic DNA
(RAPD) polymerase chain reaction (PCR) of certain isolate embodiments.
Figure 3 depicts the Gram-stain results and bacterial morphology of certain isolate embodiments.
Figure 4a depicts the Low pH Challenge of certain isolate embodiments.
Figure 4b depicts the Simulated Gastric Fluid Challenge (pH1.5) results of certain isolate embodiments.
Figure 4c depicts the Simulated Gastric Fluid Challenge (pH2) results of certain isolate embodiments.
Figure 4d depicts the Bile Tolerance of certain isolate embodiments.
Figure 4e depicts the Simulated Intestinal Fluid (SIF) Challenge of certain isolate embodiments.
Figure 5 depicts the in vitro aggregation assessment results of certain isolate embodiments.
Figure 6a depicts immune modulation ability on DH82 cells results as evaluated by ELISA by certain isolate embodiments.
Figure 6b depicts immune modulation ability on DH82 cells results as evaluated by RT-PCR by certain embodiments.
Figure 6c depicts immune modulation ability on MDCK cells results as evaluated by RT-PCR by certain embodiments.
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7 Figure 7a depicts shelf-life/stability experiment results at 4 C of certain isolate embodiments.
Figure 7b depicts shelf-life/stability experiment results at 25 C of certain isolate embodiments.
Figure 7c depicts shelf-life/stability experiment results at 37 C of certain isolate embodiments.
Figure 7d depicts shelf-life/stability experiment results at 55 C of certain isolate embodiments.
Figure 8a depicts stability in liquid yogurt experiment results at 4 C of certain isolate embodiments.
Figure 8b depicts stability in freeze-dried yogurt experiment results at 4 C
of certain isolate embodiments.
Figure 8c depicts stability in liquid yogurt mixed with pumpkin experiment results at 4 C of certain isolate embodiments.
Figure 8d depicts stability in freeze-dried yogurt mixed with pumpkin experiment results at 4 C of certain isolate embodiments.
Figure 8e depicts stability in freeze-dried yogurt mixed with pumpkin experiment results at 25 C of certain isolate embodiments.
DETAILED DESCRIPTION OF EMBODIMENTS:
Isolated probiotic strains from dogs are provided for use in dog food, treats and/or as supplements. Species-specific probiotic bacterial strains for use in {E6849592 DOCX, 5)

8 products for canines were isolated and characterized. Gram-positive bacteria were recovered from freshly-voided canine feces and duplicate isolates were eliminated using Random Amplified Polymorphic DNA (RAPD) polymerase chain reaction (PCR). Probiotic candidates were confirmed through a series of tests including tolerance to low pH and bile, production of inhibitory substances, ability to alter host immune function, and strain stability. DNA sequencing (16S rDNA, pheA and rpoA, and whole genome) determined these isolates are a range of lactic acid bacteria.
In some embodiments, eleven bacterial strains isolated from canine feces are provided. The bacteria can be isolated using a nutrient medium (LAMVAB, Hartemink et al., 1997) that specifically selects for Lactobacilli.
Resulting bacterial colonies can be differentiated based on their morphology and subsequently subjected to colony purification, for example, two rounds of colony purification. RAPD PCR can be used to eliminate organisms isolated in duplicate from the same starting animal, for example a specific dog. In some embodiments, Gram-staining can reveal the isolates to be Gram-positive rod-shaped bacteria. PCR-amplification and Sanger sequencing of the 16S rDNA
gene or rpoA and pheS genes indicated the eleven isolates belong to the following genus and species: Willow 2-1-1, Lactobacillus rhamnosus K9-8, Nikita 2-1-1, Lactobacillus paracasei K9-4, Georgia 2-1-1, Lactobacillus fermentum K9-2, Gracie 2-1, Lactobacillus casei K9-1, Coal 2-2-1, Lactobacillus reuteri K9-6, Sullivan 2-1-1, Lactobacillus rhamnosus K9-9, Tucker 2-2-3, Lactobacillus reuteri {E6849592 DOCX, 5}

9 K9-7, Abby 2-1-1, Lactobacillus reuteri K9-5, Ambar 2-1-1, Lactobacillus rhamnosus K9-10, Mika 2-1-3, Lactobacillus salivarius K9-11, and Mika 2-2-7, Lactobacillus fermentum K9-3.
The 165 rDNA sequences were identified as SEQ ID NO: 7 for Gracie 2-1, Lactobacillus casei K9-1; SEQ ID NO: 8 for Georgia 2-1-1, Lactobacillus fermentum K9-2; SEQ ID NO: 9 for Mika 2-2-7, Lactobacillus fermentum K9-3;
SEQ ID NO: 12 for Abby 2-1-1, Lactobacillus reuteri K9-5; SEQ ID NO: 13 for Coal 2-2-1, Lactobacillus reuteri K9-6; SEQ ID NO: 14 for Tucker 2-2-3, Lactobacillus reuteri K9-7; SEQ ID NO: 17 for Sullivan 2-1-1, Lactobacillus rhamnosus K9-9; SEQ ID NO: 18 for Ambar 2-1-1, Lactobacillus rhamnosus K9-

10; and SEQ ID NO: 19 for Mika 2-1-3, Lactobacillus salivarius K9-11. The rpoA

sequences were identified as SEQ ID NO: 11 for Nikita 2-1-1, Lactobacillus paracasei K9-4; and SEQ ID NO: 16 for Willow 2-1-1, Lactobacillus rhamnosus K9-8. The pheS sequences were identified as SEQ ID NO: 10 for Nikita 2-1-1, Lactobacillus paracasei K9-4; and SEQ ID NO: 15 Willow 2-1-1, Lactobacillus rhamnosus K9-8. Please refer to Figure 1.
These isolates can be subjected to a series of experiments to characterize the strains and determine which are best suited for use in a dog probiotic product. The characterization experiments can include characterizing tolerance to simulated gastric and intestinal conditions, auto-aggregation ability, carbohydrate fermentation profiles, antibiotic resistance, genome sequencing, host cell binding, immunomodulation ability, and stability.
{E6849592 DOCX, 5) Without any limitation to the foregoing, the present strains, compositions, uses, and methods are further described by way of the following examples.
EXAMPLE 1 - Isolation 5 Bacteria were recovered from freshly-voided canine feces. Using a sterile plastic spatula, a pea-sized piece of feces was collected and placed into a conical tube containing de Man Rogosa and Sharpe (MRS) nutrient broth containing 20 pg/mL nalidixic acid (to prevent growth of Gram-negative bacteria from the solution). The conical tube was either transported to the laboratory the 10 same day, or left at room temperature overnight and then transported to the laboratory. The tube was incubated at 37 C for 24 hours under aerobic conditions to allow for the growth of Gram-positive fecal bacteria. The conical tube was subjected to centrifugation at 5,000 rpm for 10 minutes to pellet the fecal matter. The resulting liquid upper phase (i.e. supernatant) was used to recover bacteria. The supernatant was collected divided into three fractions:
5mL, 1mL, and 0.5mL. The 5mL fraction was centrifuged at 11,000 rpm for 10 minutes and the 1mL portion was centrifuged for were centrifuged at 13,000 rpm for 5 minutes to concentrate/pellet the bacteria.
Following centrifugation, approximately 90% of the supernatant was removed from the tube and the bacterial pellet was resuspended in the remaining supernatant. The resuspended bacteria were then pipetted onto a LAMVAB agar plate (Hartemink et al., 1997) and spread across the plate using an ethanol flame-sterilized glass rod. From the 500pL fraction, 200pL of bacteria were pipetted onto a LAMVAB
{E6849592 DOCX,

11 agar plate and spread using a sterile glass rod. LAMVAB medium was chosen to specifically select for bacteria belonging to the genus Lactobacillus (Hartemink et al., 1997). All agar plates were subsequently incubated at 37 C for 48-72 hours under aerobic conditions.
Resulting colonies were described based on their morphology and those with different morphologies were chosen for further analysis. Colonies were picked using a P-1000 pipette tip, re-streaked onto MRS agar, and incubated for 28 hours at 37 C under aerobic conditions. This re-streaking process was performed a second time to ensure purity of individual isolates. Bacterial colonies resulting from the second purification step were picked using a P-pipette tip, inoculated into 5mL MRS broth, and incubated at 37 C for 24 hours under aerobic conditions.
One freezer stock of each isolate was prepared using 1.5mL of liquid bacterial culture mixed with 0.5mL of sterile 80% glycerol in a 2mL cryovial.
Stocks were placed into the -80 C freezer for long-term storage.
In some embodiments, a stock of each isolate, for example, in a biologically pure culture of bacterial strain, can be been deposited under the Budapest Treaty at an international depositary authority (IDA), for example, the American Type Culture Collection (ATCC) or The International Depositary Authority of Canada (IDAC), and can be assigned a specific accession number.
In some embodiments, the stock of each isolate, for example Lactobacillus casei strain K9-1, was deposited in The International Depositary Authority of {E6849592 DOCX, 5}

12 Canada (IDAC) (1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2) on April 21, 2015 and received IDAC Accession No. 210415-01.
In some embodiments, the stock of each isolate, for example, Lactobacillus fermentum strain K9-2, was deposited in The International Depositary Authority of Canada (IDAC) (1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2) on April 21, 2015 and received IDAC Accession No. 210415-02.
EXAMPLE 2 - Strain Typing 16SrDNA
To determine the genus and species of the isolates, the gene encoding the 16S ribosomal RNA (rDNA) was sequenced. See Figure 1 for sequencing results. Genomic DNA (gDNA) from each canine isolate was extracted using the Presto TM gDNA Bacteria Kit (purchased from FroggaBio, Toronto, ON) according to the manufacturer's recommendations for Gram-positive bacteria. The resulting concentration of gDNA from each preparation was determined using a Nanodrop spectrophotometer and recorded.
The polymerase chain reaction (PCR) was used to amplify the 16S rDNA
gene from prepared gDNA of each isolate. Each PCR reaction contained 10-30pg gDNA and primers 8F, SEQ ID NO: 1 (genetic sequence: AGA GTT TGA
TCC TGG CTC AG) and 805R, SEQ ID NO: 2 (genetic sequence: GAC TAC
CAG GGT ATC TAA TC) which bind to conserved regions of the 16S rDNA gene.
{E6849592 DOCX,

13 Another typing method used was sequencing of the rpoA and pheS genes from K9-8 and K9-4 (Naser et al., 2007). The primers used for the PCR reaction were as follows: PheS Fl, SEQ ID NO: 3 ¨ CAYCCNGCHGYGAYATGC; PheS R1, SEQ ID NO: 4 ¨ CCWARVCCRAARGCAAARCC; RpoA Fl, SEQ ID NO: 5 ¨
ATGATYGARTTTGAAAAACC; RpoA R1, SEQ ID NO: 6 ¨
ACHGTRTTRATDCCDGCRCG.
To confirm successful PCR-amplification of the 16S rDNA gene, 1/5 of the PCR sample was mixed with 6x gel loading dye and subjected to electrophoresis on a 1% agarose gel for 30 minutes at 100 volts. The PCR product was visualized using a UV table lamp. Once the correct sized band was confirmed via agarose gel electrophoresis, the remaining PCR sample was purified using the PCR cleanup kit (QiagenTm). The concentration of the PCR product was determined using a NanodropTM spectrophotometer.
Each sample was submitted for Sanger sequencing using either primer 8F or 805R. Sequencing was performed at The Applied Genomics CentreTM at the University of Alberta.
Resulting sequences and chromatograms were viewed using ContigExpressTM
software and genus and species were determined using the Basic Local Alignment Search Tool (BLAST) nucleotide search algorithm.
RAPD profile To eliminate duplicate strains from the canine isolates, random amplified polymorphic DNA (RAPD) PCR was used. This experiment was performed to compare isolates from the same dog, not isolates from different sources. Here, a {E6849592.DOCX; 5)

14 PCR mixture was prepared that included 1Ong of gDNA from each isolate mixed (independently) with M13 primer (genetic sequence GAGGGTGGCGGTTCT, Rossetti and Giraffa 2005). This primer was chosen because it has low specificity and binds at numerous loci within bacterial gDNA. Agarose gel (1%) electrophoresis of the PCR products provided a banding pattern (visible using a UV table) whereby duplicates of the same strain had the same banding pattern resulting in elimination of the duplicate from further study. Isolates that had unique banding patterns were included for further study (See Figure 2).
EXAMPLE 3 - Strain Characterization Gram-stain A Gram-staining kit (Sigma AldrichTM) containing solutions of crystal violet, Gram's iodine, decolorizing fluid, and safranin was used to confirm isolates were Gram-positive, rod-shaped bacteria. The canine isolates were streaked from the -80 C freezer stock onto MRS agar and grown for 18 hours at 37 C (aerobic). A
5pL drop of saline was placed onto a microscope slide, and one bacterial colony from the MRS agar plate was picked using a P-1000 pipette tip and mixed into the saline on the slide. The bacterial resuspension was allowed to dry on the slide and the slide was then heat-fixed by 3 passes over a flame. A 10pL drop of crystal violet was placed on top of the heat-fixed bacterial spot and incubated for 1 minute. The crystal violet was washed from the slide using deionized water and 10pL of Gram's iodine was placed on top of the bacterial spot and incubated for 1 minute. The iodine was washed from the slide using deionized water.
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Decolorizing solution was washed over the slide until the run-off was clear.
The slide was then washed with deionized water again. A 10pL drop of Safranin was placed on top of the bacterial spot and incubated for 1 minute. The Safranin was washed from the slide using deionized water. The Gram-stained bacteria were 5 visualized using the 100X lens on a OMAX LED 40X-2000X Digital Binocular Biological Compound Microscope and photos were obtained using the 3.0MP
USB camera included in the microscope. The Gram-stain result and bacterial morphology were recorded (See Figure 3).
Acid/Bile sensitivity 10 In order for a probiotic bacterium to be effective, it needs to survive transit through the gastrointestinal (GI) tract. To determine whether the isolates would survive passage through the acidic stomach, or exposure to bile in the intestine, two different experimental in vitro approaches were used.
ACID SENSITIVITY: In a first experiment, nutrient medium (APT broth

15 purchased from BD BiosciencesTm) was prepared and used concentrated hydrochloric acid to lower the pH to 3. As a positive control, nutrient medium at a pH 6.5 was used. A pH 3 was chosen for the simulated stomach acidic condition based on a publication by Parrott and co-workers (2009) who claim that the pH
of the fasted canine stomach is pH 3, thus making this a biologically-relevant experimental setup. All experiments were performed in technical and experimental duplicates.
{E6849592 DOCX; 5)

16 Canine isolates were grown in APT broth for 18 hours at 37 C. Liquid bacterial cultures were mixed well and added to the pH 3 and pH 6.5 media at a concentration of 5% (i.e. 250pL bacterial culture into a tube containing 5mL
medium). The freshly-inoculated tubes were mixed well and a 200pL sample was removed from each tube to enumerate the starting bacterial concentration in each tube. Each 200pL bacterial sample was subjected to 7 serial 1/10 dilutions using a multichannel pipette and 10pL of each dilution was spotted onto an agar plate made of Tryptic soy broth (BD BiosciencesTM) with added yeast extract (BD
BiosciencesTM) (TSBYE) agar plate. Experimental tubes were incubated at 37 C
for 2 hours (the estimated time it takes for food to pass through the fasted canine stomach according to Parrott et al., 2009). After the 2 hour incubation, a 200pL
sample was removed from each tube. Each sample was subjected to seven serial 1/10 dilutions and 10pL of each dilution was spotted onto an TSBYEagar plate. All plates were incubated at 37 C for 48 hours under aerobic conditions.
Colonies were counted in the spot with the highest number of colonies that could be reliably enumerated. Calculations were performed to determine the colony-forming units per milliliter (CFU/mL) for each isolate at time=0 and time=2 hours.
In a second experiment, simulated gastric fluid (or "SGF", purchased from BioRelevantTM) was used to determine which of the isolates are most tolerant to simulated gastric conditions. Because all tested canine isolates tolerated challenge at pH 3, it was decided to test lower pH conditions using SGF to determine which canine isolates were most acid-resistant. SGF was adjusted to {E6849592 DOCX; 5)

17 pH 1.5 and 2.0 using concentrated hydrochloric acid and SGF at pH 6.5 was used as a control. All experiments were performed in technical and experimental duplicates.
Canine isolates were grown in MRS broth (OxoidTM) for 18 hours at 37 C.
Liquid bacterial cultures were mixed well and added to the SGF at pH 1.5, pH
2.0 and pH 6 at a concentration of 5% (i.e. 150pL bacterial culture into a tube containing 3mL SGF). The freshly-inoculated tubes were mixed well and a 200pL sample was removed from each tube to enumerate the starting bacterial concentration in each tube. Each 200pL bacterial sample was subjected to 7 serial 1/10 dilutions using a multichannel pipette and 10pL of each dilution was spotted onto a TSBYE agar plate. Experimental tubes were incubated at 37 C
for 2 hours (the estimated time it takes for food to pass through the fasted canine stomach according to Parrott et al., 2009). After the 2 hour incubation, a 200pL
sample was removed from each tube. Each sample was subjected to seven serial 1/10 dilutions and 10pL of each dilution was spotted onto a TSBYE agar plate. All plates were incubated at 37 C for 48 hours under aerobic conditions.
Colonies were counted in the spot with the highest number of colonies that could be reliably enumerated. Calculations were performed to determine the CFU/mL
for each isolate at time=0 and time=2 hours.
BILE/INTESTINAL TOLERANCE: In a first experiment, nutrient medium (APT broth) was prepared containing 5% purified ox bile (purchased from OxoidTm). As a control, nutrient medium lacking ox bile was used which was {E6849592 DOCX; 5}

18 expected to grow the isolates well. All experiments were performed in technical and experimental duplicates.
Canine isolates were grown in APT broth for 18 hours at 37 C. Liquid bacterial cultures were mixed well and added to the medium with and without ox bile at a concentration of 5% (i.e. 250pL bacterial culture into a tube containing 5mL medium). The freshly-inoculated tubes were mixed well and a 200pL
sample was removed from each tube to enumerate the starting bacterial concentration in each tube. Each 200pL bacterial sample was subjected to 7 serial 1/10 dilutions using a multichannel pipette and 10pL of each dilution was spotted onto a TSBYE agar plate. Experimental tubes were incubated at 37 C
for 7 hours (the estimated time it takes for food to pass through the canine intestine according to Parrott et al., 2009). After the 7 hour incubation, a 200pL
sample was removed from each tube. Each sample was subjected to seven serial 1/10 dilutions and 10pL of each dilution was spotted onto a TSBYE agar plate. All plates were incubated at 37 C for 48 hours under aerobic conditions.
Colonies were counted in the spot with the highest number of colonies that could be reliably enumerated. Calculations were performed to determine the CFU/mL
for each isolate at time=0 and time=7 hours.
In a second experiment, simulated intestinal fluid (or "SIF", purchased from BioRelevantTM) was used to determine which of the isolates are most tolerant to simulated intestinal conditions. Here, a buffer was prepared to which SIF powder was added. The buffer lacking SIF powder was used as a positive {E6849592 DOCX,

19 control for bacterial survival. All experiments were performed in technical and experimental duplicates.
Canine isolates were grown in MRS broth for 18 hours at 37 C. Liquid bacterial cultures were mixed well and added to the SIF and buffer control at a concentration of 5% (i.e. 150pL bacterial culture into a tube containing 3mL
SIF).
The freshly-inoculated tubes were mixed well and a 200pL sample was removed from each tube to enumerate the starting bacterial concentration in each tube.

Each 200pL bacterial sample was subjected to 7 serial 1/10 dilutions using a multichannel pipette and 10pL of each dilution was spotted onto a TSBYE agar plate. Experimental tubes were incubated at 37 C for 7 hours (the estimated time it takes for food to pass through the fasted canine stomach according to Parrott et al., 2009). After the 7 hour incubation, a 200pL sample was removed from each tube. Each sample was subjected to seven serial 1/10 dilutions and 10pL of each dilution was spotted onto a TSBYE agar plate. All plates were incubated at 37 C for 48 hours under aerobic conditions. Colonies were counted in the spot with the highest number of colonies that could be reliably enumerated.
Calculations were performed to determine the CFU/mL for each isolate at time=0 and time=7 hours.
See Figures 4A- 4E for the acid/bile sensitivity testing results.
In vitro aggregation assessment {E6849592.DOCX, 5}

The ability to bind host cells is an important characteristic of probiotic bacteria. Work by Kos and co-workers (2002) suggests there is a positive correlation between probiotic bacteria that autoaggregate (or floc out of solution) and those that bind host cells. To determine whether the canine isolates 5 autoaggregate, liquid cultures (5mL MRS broth) of each strain were inoculated from the -80 C freezer stock and incubated for 18 hours at 37 C. Liquid cultures were mixed by vortexing and a 100pL sample was removed from each tube and used to make a 1/10 dilution in MRS broth. The 0D600 of the 1/10 dilution was read using a spectrophotometer (Fisher ScientificTm). Without agitating the liquid, 10 the tubes were placed into the 37 C incubator. A 100pL sample was taken from the very top of the liquid culture every hour for 5 hours and was used to make a 1/10 dilution in MRS broth. The 0D600 of this dilution was recorded for each time point. Strains that are capable of autoaggregation will exhibit a significant drop in 0D600 over the 5 hour experiment. Based on the work of Kos et al., this 15 data can be extrapolated to predict which strains will also have the ability to bind host cells.
It was observed that strains K9-11, K9-3, K9-9, and K9-6 experienced auto-aggregation when left stationary in liquid culture as shown in the graph depicted in Figure 5.
Production of inhibitory substances {E6849592 DOCX, 5}

Lactic acid bacteria are known to produce metabolites that inhibit the growth of other bacteria. Each of the eleven canine isolates were tested to determine if they could prevent the growth of the common canine intestinal pathogens Clostridium perfringens, Salmonella enterica serovar Typhimurium, and Enteropathogenic Escherichia coli E2348/69. The inhibitory activity against four other strains of pathogenic bacteria was also tested: Listeria monocyto genes, Meth icillin-resistant Staphylococcus aureus, Vancomyci n-resistant Enterococcus species, and Clostridium difficile. Table 1 is a summary of experimental data indicating bacterial inhibition by the canine isolates.
{E6849592 DOCX, Table 1 ¨ Production of inhibitory substances INHIBITORY ACTIVITY SUMMARY
C.difficile 293 C.difficile 54 C. perfringens 8533 C. perfringens 15 MRSA R667 MRSA R776 L. monocytogenes 19112 L. monocytoge nes 5578 Salmonella enterica E. coil 2348/69 Antibiotic susceptibility testing Antibiotic resistant microorganisms are a serious concern in modern society and probiotic bacteria, which are meant to provide a health benefit, should not introduce the possibility for transfer of antibiotic resistance genes to other bacteria. The minimum inhibitory concentration (MIC) for 16 different antibiotics was determined for the K9 isolates using the broth micro-dilution method. The 16 antibiotics tested cover a wide range of inhibitory mechanisms and include clinically-relevant antibiotics such as ampicillin, gentamycin, kanamycin, vancomycin, streptomycin, and erythromycin. The European Food {E6849592.DOCX; 5}

Safety Authority (EFSA) provides a list of MIC breakpoint values which indicate acceptable ranges of antibiotic resistance for direct-fed nnicrobials.
Using the broth micro-dilution dilution (according to ED ISO 10932:2012) allowed for high-throughput testing of 10 concentrations per antibiotic in a 96-well plate format. Bacteria were added to the positive control well and each of the wells containing 1:1 dilutions of the antibiotic. No bacteria were added to the final negative control well. After incubation for 48 hours at 37 C, the plates were visually inspected for opacity or presence of a pellet indicating bacterial growth.
This experiment was performed in experimental duplicate. The lowest antibiotic concentration for which there is no growth was deemed the MIC. The MIC
values obtained for each K9 isolate are presented in Table 2. Notably, K9-5 and K9-7 had MICs that exceeded the break point values specified by the EFSA and were eliminated from further study. At this point, K9-6 was also eliminated from further study due to general growth defects.
{E6849592 DOCX, 5) Table 2 - An ti b i oti c Resistance Profiles MIC (ug/mL) Isolate NEOMYCIN VANCOMYCIN NALIDIXIC ACID AMOXI CI
LLI N
K9-1 (Grade 2-1) SENSITIVE RESISTANT RESISTANT 0.125 K9-2 (Georgia 2-1-1) SENSITIVE RESISTANT RESISTANT 0.5 K9-3 (Mika 2-2-7) 4 32 RESISTANT 0.25 K9-4 (Nikita 2-1-1) 2 RESISTANT RESISTANT 1 K9-5 (Abby 2-1-1) SENSITIVE RESISTANT RESISTANT 2 K9-6 (Coal 2-2-1) SENSITIVE RESISTANT RESISTANT 1 K9-7 (Tucker 2-2-3) SENSITIVE RESISTANT RESISTANT 1 K9-8 (Willow 2-1) 8 RESISTANT RESISTANT 1 K9-9 (Sullivan 2-1-1) 2 RESISTANT RESISTANT 1 K9-10 (Amba r 2-1-1) 8 , RESISTANT RESISTANT 1 K9-11 (Mika 2-1-3) 2 RESISTANT RESISTANT 0.5 L. fernnentum 14931 (control) 2 32 RESISTANT
0.5 L. reuteri 23272 (control) 256 RESISTANT RESISTANT

P. a eruginosa (control) 8 RESISTANT 8 64 E. fa eca I i s 29212 (control) 256 4 RESISTANT
0.5 Isolate TRIMETHOPRIM TYLOSIN METRONIDAZOLE RI
FAMPI CI N
K9-1 (Gracie 2-1) SENSITIVE SENSITIVE RESISTANT SENSITIVE
K9-2 (Georgia 2-1-1) 2 SENSITIVE RESISTANT SENSITIVE
K9-3 (Mika 2-2-7) SENSITIVE SENSITIVE RESISTANT SENSITIVE
K9-4 (Nikita 2-1-1) 2 SENSITIVE RESISTANT SENSITIVE
K9-5 (Abby 2-1-1) 16 SENSITIVE RESISTANT SENSITIVE
K9-6 (Coal 2-2-1) 16 SENSITIVE RESISTANT SENSITIVE
K9-7 (Tucker 2-2-3) 16 SENSITIVE RESISTANT SENSITIVE
K9-8 (Willow 2-1) 2 SENSITIVE RESISTANT 0.25 K9-9 (Sullivan 2-1-1) 4 SENSITIVE RESISTANT 0.5 K9-10 (Amba r 2-1-1) 16 SENSITIVE RESISTANT SENSITIVE
K9-11 (Mika 2-1-3) SENSITIVE SENSITIVE RESISTANT SENSITIVE
L. fermentum 14931 (control) SENSITIVE 1 RESISTANT
SENSITIVE
L. reuteri 23272 (control) 16 2 RESISTANT 4 P. aeruginosa (control) 0.032 RESISTANT RESISTANT 8 E. fa eca I i s 29212 (control) SENSITIVE 1 RESISTANT 1 {E6849592.DOCX; 5}

____________________________________________________________________ MIC
(ug/mL) Isolate GENTAMYCI N KANAMYCIN
STREPTOMYCIN TETRACYCLINE
K9-1 (Grade 2-1) SENSITIVE 16 2 2 .
K9-2 (Georgia 2-1-1) SENSITIVE 16 8 8 K9-3 (Mika 2-2-7) SENSITIVE 16 4 8 K9-4 (Nikita 2-1-1) 1 16 8 2 K9-5 (Abby 2-1-1) SENSITIVE 4 4 32 K9-6 (Coal 2-2-1) SENSITIVE 8 1 8 K9-7 (Tucker 2-2-3) SENSITIVE 8 4 32 K9-8 (Willow 2-1) 2 32 8 1 K9-9 (Sullivan 2-1-1) 2 32 8 2 K9-10 (Amba r 2-1-1) 2 32 2 2 K9-11 (Mika 2-1-3) 1 64 16 4 L. fe rmentum 14931 (control) 16 32 4 32 L. re ute ri 23272 (control) 32 256 256 32 P. aeruginosa (control) 1 SENSITIVE RESISTANT

E. fa eca I i s 29212 (control) 64 128 256 32 Isolate ERYTHROMYCIN CLINDAMYCIN
CHLORAMPHENI COL AMPICILLIN
K9-1 (Gracie 2-1) SENSITIVE 0.125 2 0.5 K9-2 (Georgia 2-1-1) 0.063 0.125 4 1 K9-3 (Mika 2-2-7) 0.032 0.063 4 1 K9-4 (Ni kita 2-1-1) 0.032 SENSITIVE 4 1 K9-5 (Abby 2-1-1) 0.032 SENSITIVE 4 4 K9-6 (Coal 2-2-1) 0.032 SENSITIVE 4 2 K9-7 (Tucker 2-2-3) 0.032 SENSITIVE 4 2 K9-8 (Willow 2-1) 0.032 0.125 4 2 K9-9 (Sullivan 2-1-1) 0.063 0.25 4 2 ..
K9-10 (Amba r 2-1-1) SENSITIVE 0.125 4 1 K9-11 (Mika 2-1-3) 0.125 SENSITIVE 2 0.125 L. fermentum 14931 (control) 0.063 0.063 8 0.5 L reuteri 23272 (control) 1 RESISTANT 8 2 P. a eruginosa (control) RESISTANT RESISTANT 32 32 E. fa eca I i s 29212 (control) 2 RESISTANT 16 2 Carbohydrate fermentation profile Microorganisms can be differentiated based on their ability to ferment certain carbohydrates. To determine the carbohydrate fermentation profiles for {E6849592.DOCX; 5}

each of the canine isolates, API 50 CH strips were purchased from BioMerieuxTm.
The carbohydrate fermentation profiles for applicable K9 isolates are presented in Table 3.
Table 3 ¨ Carbohydrate Fermentation Profiles Characteristic K9-1 K9-2 K9-3 K9-4 K9-8 K9-9 K9-10 Acid Produced from L-Arabinose - + + _ - - -D-Ribose - + + + + + +
D-Xylose - + - - - - -D-Adonitol - - - + - -D-Galactose + + - + + + +
D-Glucose + + + + + + +
0-Fructose + + + + + + +
D-Mannose + - - + + + +
L-Sorbose - - - + + + +
L-Rhamnose - - - - + + +
D-Mannitol + - _ + + + +
D-Sorbitol - - - + + + +
Methyl-aD-Glucopyranosid e - - - - + + -N-AcetylGlucosamine + - - + + + +
Amygdalin + - - - + + +
Arbutin + - - . + + +
Esculin Ferric Citrate + + + + + + +
Saliin + - - + + + +
D-Cellobiose + - - + + + +
D-Maltose - + + + + + -0-Lactose (bovine origin) + + - + + -r +
D-Melibiose - + - - - _ -D-Saccharose (sucrose) - + + - - - +
D-Trehalose + - _ + + + +
lnulin - - - - - - _ D-Melezitose + - - - + + +
D-Raffinose - + + - - - _ Gentiobiose + - - + - + +
D-Turanose - - - + + + +
D-Lyxose - - - - - - _ D-Tagatose + - - + + + +
potassium gluconate + + + + + + +
potassium 5-ketogluconate - + + - - - _ {E6849592.DOCX; 5}

The resulting data from the API 50 CH strips was submitted to the apiwebTM identification database which predicts the genus and species of the organism based on which carbohydrates it fermented. These results can be used to corroborate the ribotyping of strains using 16s rDNA Sanger sequencing The predicted genus and species for each applicable K9 isolate based on their carbohydrate fermentation profile is depicted in Table 4.
Table 4 - Predicted Genus and Species based on Carbohydrate Fermentation Profiles K9 Isolate Predicted genus and species % Identity K9-1 Lactobacillus paracasei subspecies paracasei 92.3 K9-2 Lactobacillus fermentum 62.6 K9-3 Lactobacillus fermentum 95.5 K9-4 Lactobacillus paracasei subspecies paracasei 99.7 K9-8 Lactobacillus rhamnosus 99.7 K9-9 Lactobacillus rhamnosus 99.8 K9-10 Lactobacillus rhamnosus 95.5 Genome sequencing Genome sequencing was performed for further strain typing and characterization. GenomicDNA from K9-1 and K9-2 was isolated using the PrestoTM gDNA Bacteria Kit (purchased from FroggaBioTM, Toronto, ON) and shipped to Fusion GenomicsTM (Vancouver, BC) for lIIuminaTM sequencing.
Following contig assembly, bioinformatics software (GENEi0usTM) was used to identify the presence of specific genes (e.g. those encoding bacteriocins or host adhesion factors) and confirm the absence of certain harmful genes (e.g.
antibiotic resistance genes, or those encoding virulence factors).
This {E6849592.DOCX; 5}

information can further characterize the strains and indicate that the strain(s) are safe to use as direct-fed microbials.
Immune modulation assessment Intestinal dysbiosis, or an imbalance in the immunological state of the GI
tract, is implicated in the development of both acute and chronic gastrointestinal disorders. The general consensus of what determines a healthy gut from a non-healthy, or diseased gut, is the immunological balance. In a healthy animal, there is a balance achieved between tolerance to pathogens and suppression induced by commensal bacteria. Microbiomes can be specific/adapted to particular hosts.
Dysbiosis in microbiota of the intestine causes a shift in the balance of immunostimulatory cytokines. This can throw off the balance of the inflammatory signals in the gut and cause dysbiosis and diarrhea. Probiotics can ameliorate these issues and restore balance of good bacteria in the GI tract after antibiotic associated diarrhea. One proposed theory by which probiotic bacteria exert their health-promoting properties is by stimulating production of anti-inflammatory cytokines (such as IL-10 and TGF-f3) by host immune cells (Smits et al., 2005).
Furthermore, the cytokine IL-6 has been shown to have a role in repair of the intestinal epithelium (Kuhn et al. 2014). Key to maintaining this balance of tolerance versus stimulation of the GALT, are pro- (IL-12 and IFN-13) and anti-(IL-10 and TGF-E3) inflammatory cytokines. Increased expression of these {E6849592 DOCX, 5) cytokines can alter the balance and disease state within the gut. Specific cell types are known to produce different cytokine profiles which are responsible for maintaining this homeostatic immunological balancing act.
The immune modulation ability of the K9 isolates can be determined using two different cell lines: (1) Madin-Darby Canine Kidney (MDCK) cells, which are a canine kidney epithelial cell line and (2) a canine-derived macrophage-like cell line called DH82 (from ATCC) which has been previously shown to produce IL-10 (Barnes et al. 2000). The levels of anti-inflammatory cytokines (IL-10, IL-6, and TGF-6) and inflammatory cytokines (IL-12, and IFN-6) produced in the presence or absence of the K9 probiotic isolates were determined using semi-quantitative real-time PCR. The levels of IL-10 and IL-12 produced by DH82 cells in response to incubation with the K9 isolates were also quantified using enzyme-linked immunosorbent assays (ELISAs).
For all immune modulation experiments, E. coil lipopolysaccharide (LPS) (Sigma AldrichTM) was used as a cell stimulant. MDCK and DH82 adherent cell lines were incubated for 24 hours in the presence or absence of K9 isolates. Supernatants were collected and analyzed by ELISA (R&D Systems). Canine IL-10 and IL-12 ELISA kits were purchased from R&D SystemsTM and were used as per the manufacturer's recommendations. Cell monolayers were harvested using TRIzolTm reagent (Life TechnologiesTm) and RNA was collected by chloroform extraction. Total RNA
was quantified and 50Ong was used as a template for cDNA synthesis using MMLV reverse transcriptase (PromegaTM) and oligo dT primers. Real-time PCR
{E6849592 DOCX;

probes were designed according to Peters et al. (2005). RT-PCR samples were prepared in technical triplicate and the AACT method was used for relative quantification of gene expression compared to the canine G3PDH
(Glyceraldehyde 3-phosphate dehydrogenase) endogenous control.
Data 5 pertaining to immune modulation studies (ELISA and RT-PCR) are presented in Figure 6.
Strain stability assessment A major concern regarding probiotic products currently on the market 10 (both for humans and animals) is they are unstable and often the claims on the label regarding the number of probiotic bacteria present are inaccurate. The viability, quality, and stability of a probiotic product are important to ensure the human or animal is ingesting a suitable number of living microorganisms.
Stationary phase cultures of K9-1 and K9-2 grown in appropriate media, for 15 example All-purpose Tween (APT) from BD (Becton Dickinson and CompanyTm), were mixed with 10% maltodextrin (w/v) and freeze-dried in a LyoStar II
lyophilizer (FTS Systems). Samples were frozen for 24 hours at -20 C followed by drying at +40 C with a vacuum of 500 mTorr applied for 24-48 hours.
The stability/survivability of K9 isolates was assessed over an 8-week

20 period (sampling occurred once per week) with cultures in the lyophilized (freeze-dried) state stored at 4 C, 25 C, and 37 C. An accelerated shelf life test was {E6849592.DOCX, 5) performed at 55 C where lyophilized cultures were assessed for viability at 0, 4, 8, 24, 28, 32, and 48 hours of incubation.
The number of recovered bacteria for each temperature was recorded and graphed and the resulting slope of the line can be used to calculate the long-term shelf-life of the product at different temperatures. Data from the stability assessment is presented in Figure 7.
Product formulation Probiotics can be incorporated into food for consumption, for example, probiotics can be incorporated into liquid yogurt for human (or animal) consumption. To test how K9-1 and K9-2 fare in a yogurt-based matrix, the stability of K9-1 and K9-2 was determined in both a liquid yogurt and freeze-dried yogurt formulations.
Yogurt can be prepared in-house using a CuisinartTM yogurt maker, skim milk, and commercially-available plain yogurt as a starter culture. Liquid cultures of K9-1 and K9-2 were used to inoculate the fresh yogurt. A sample of fresh yogurt without added K9 isolate was set aside as a "background flora control"
which contained the bacteria used to produce the yogurt. Half of the fresh yogurt was refrigerated, and the other half was lyophilized in -1mL portions according to the parameters described above. Liquid and freeze-dried yogurt (stored at 4 C
or 25 C) were sampled once weekly for 8 weeks (plated onto MRS with (E6849592 DOCX, 5) vancomycin) to determine K9 strain stability. Data pertaining to stability in liquid and lyophilized yogurt is presented in Figure 8.
The scope of the claims should not be limited by the embodiments as set forth in the examples herein, but should be given the broadest interpretation consistent with the description as a whole.
Although a few embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications can be made to the embodiments described herein. The terms and expressions used in the above description have been used herein as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that the invention is defined and limited only by the claims that follow.
While the above description details certain embodiments of the invention and describes certain embodiments, no matter how detailed the above appears in text, the invention can be practiced in many ways. Details of the apparatuses and methods may vary considerably in their implementation details, while still being encompassed by the invention disclosed herein. These and other changes can be made to the invention in light of the above description.
Particular terminology used when describing certain features or aspects of the invention should not be taken to imply that the terminology is being redefined {E6849592 DOCX;

herein to be restricted to any specific characteristics, features, or aspects of the invention with which that terminology is associated. In general, the terms used in the following claims should not be construed to limit the invention to the specific embodiments disclosed in the specification. Accordingly, the actual scope of the invention encompasses not only the disclosed embodiments, but also all equivalent ways of practicing or implementing the invention.
The above description of the embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed above or to the particular field of usage mentioned in this disclosure.
While specific embodiments of, and examples for, the invention are described above for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize.
The elements and acts of the various embodiments described above can be combined to provide further embodiments.
While certain aspects of the invention are presented below in certain claim forms, the inventors contemplate the various aspects of the invention in any number of claim forms. Accordingly, the inventors reserve the right to add additional claims after filing the application to pursue such additional claim forms for other aspects of the invention.
(E6849592 DOCX, 5}

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Claims (14)

WE CLAIM:

1. An isolated strain of canine probiotic bacteria for use in canine products, wherein the isolated strain is selected from the group consisting of Lactobacillus casei strain K9-1 IDAC Accession number 210415-01 and Lactobacillus fermentum strain K9-2 IDAC Accession number 210415-02.

2. The isolated strain of claim 1 wherein the canine products are ingestible canine products.

3. The isolated strain of either one of claims 1 or 2 wherein the canine products are selected from the group consisting of food, treats, and supplements.

4. A composition for use in canine products, the composition comprising a first isolated strain of canine probiotic bacteria and a second isolated strain of canine probiotic bacteria, wherein the first isolated strain is Lactobacillus casei strain K9-1 IQAC Accession number 210415-01.

5. A composition for use in canine products, the composition comprising a first isolated strain of canine probiotic bacteria and a second isolated strain of canine probiotic bacteria, wherein the first isolated strain is Lactobacillus fermentum strain K9-2 IDAC Accession number 210415-02.

6. The composition of either one of claims 4 or 5 further comprising a third isolated strain of canine probiotic bacteria.

7. A use of an isolated strain of canine probiotic bacteria in canine products, wherein the isolated strain of canine probiotic bacteria is selected from the group consisting of Georgia 2-1-1, Lactobacillus fermentum K9-2 IDAC
Accession number 210415-02; and Gracie 2-1, Lactobacillus casei K9-1 IDAC Accession number 210415-01.

8. The use of claim 7, wherein the canine products are ingestible canine products.

9. The use of either one of claims 7 or 8, wherein the canine products are selected from the group consisting of food, treats, and supplements.

10.An isolated strain of canine probiotic bacteria selected from the group consisting of Lactobacillus casei strain K9-1 IDAC Accession number 210415-01 and Lactobacillus fermentum strain K9-2 IDAC Accession number 210415-02 for use in canine products for treatment of intestinal dysbiosis.

11.A method of preparing food, the method comprising:
providing a canine food product;
adding to the canine food product an isolated strain of canine probiotic bacteria for use in canine products, wherein the isolated strain is selected from the group Lactobacillus casei strain K9-1 IDAC Accession number 210415-01 and Lactobacillus fermentum strain K9-2 IDAC
Accession number 210415-02.

12.The method of claim 11 wherein the canine food product is selected from the group consisting of food, treats, and supplements.

13. Lactobacillus casei strain K9-1 IDAC Accession number 210415-01.

14. Lactobacillus fermentum strain K9-2 IDAC Accession number 210415-02.