Characterization of potential probiotic lactic acid bacteria isolated from camel milk

Accepted Manuscript Characterization of potential probiotic lactic acid bacteria isolated from camel milk Aisha Abushelaibi, Suheir AlMahdin, Khaled El-Tarabily, Nagendra P. Shah, Mutamed Ayyash PII:

S0023-6438(17)30041-5

DOI:

10.1016/j.lwt.2017.01.041

Reference:

YFSTL 5993

To appear in:

LWT - Food Science and Technology

Received Date: 18 October 2016 Revised Date:

12 December 2016

Accepted Date: 15 January 2017

Please cite this article as: Abushelaibi, A., AlMahdin, S., El-Tarabily, K., Shah, N.P., Ayyash, M., Characterization of potential probiotic lactic acid bacteria isolated from camel milk, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.01.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization of Potential Probiotic Lactic Acid Bacteria Isolated from Camel Milk

3 Aisha Abushelaibia, Suheir AlMahdina, Khaled El-Tarabilyb, Nagendra P. Shah, 2, c, d

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Mutamed Ayyasha1

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a

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(UAEU), PO Box 1555, Al Ain, UAE

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b

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Food Science Department, College of Food and Agriculture, United Arab Emirates University

Department of Biology, College of Science, United Arab Emirates University (UAEU), PO Box

1555, Al Ain, UAE

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c

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Pokfulam Road, Hong Kong

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Adjunct Professor, Victoria University, Melbourne, Australia

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Corresponding authors:

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Dr. Mutamed Ayyash Food Science Department College of Food & Agriculture United Arab Emirates University (UAEU) T: +971 3 713 4552 F: +971 3 767 5336 E: [email protected]

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Food and Nutritional Science, School of Biological Sciences, the University of Hong Kong,

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Prof. Nagendra P. Shah Food and Nutritional Science, School of Biological Sciences, the University of Hong Kong, Pokfulam Road, Hong Kong

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32 Abstract

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The aim of this study was to investigate probiotic characteristics and fermentation profile of

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selected lactic acid bacteria isolated from raw camel milk. Physiological properties, cell surface

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properties (hydrophobicity, autoaggregation, co-aggregation), acid and bile tolerance, bile salt

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hydrolysis, cholesterol removing, exopolysaccharide (EPS) production, hemolytic and antimicrobial

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activities, resistance toward lysozyme and six antibiotics, and fermentation profile (growth, pH, and

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proteolysis) were examined. 16S rRNA sequencing was carried out to identify six presumptive

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LAB isolates. In general, all identified LAB (Lactococcus lactis KX881768, Lactobacillus

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plantarum KX881772, Lactococcus lactis KX881782 and Lactobacillus plantarum KX881779)

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showed auto-aggregation ability, high cholesterol removal ability, high co-aggregation, strong

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antimicrobial activity and EPS production. Among the isolates, Lactococcus lactis KX881768,

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Lactobacillus plantarum KX881772, Lactococcus lactis KX881782 and Lactobacillus plantarum

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KX881779 exhibited remarkable cholesterol removal abilities. Similarly, Lactobacillus plantarum

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KX881779, and Lactococcus lactis KX881782 showed very promising fermentation profiles.

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Keywords: Lactic acid bacteria, probiotic, camel milk, cholesterol removal, Lactobacili

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Chemical compounds studies in this article

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Cholic acid (PubChem CID: 221493); Taurocholic acid (PubChem CID: 6675); Bile salts

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(PubChem CID: 439520); n-Hexadecane (PubChem CID: 11006); Cholesterol (PubChem CID:

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5997); Lysozyme (PubChem CID: 16129749); o-Phthalaldehyde (PubChem CID: 4807)

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1. Introduction

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Probiotics are microorganisms that provide health benefits to humans. FAO/WHO (2002) defines

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probiotics as “living microorganisms which, when administrated in adequate numbers, confer

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benefits to the host”. Numerous studies have reported that probiotics can prevent or alleviate

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symptoms of inflammatory bowel disease, irritable bowel syndrome, constipation, antibiotic-

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associated and acute diarrhea, hypertension, and diabetes (Weichselbaum, 2009). Food products

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containing probiotics, also called functional foods, have several therapeutic benefits including

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antihypertension, anticancer, hypoglycemic properties, antioxidant, and immunomodulatory effects

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(Clare & Swaisgood, 2000; Khan, 2014). Therefore, there has been a lot of medical and industrial

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interests in isolation of new probiotic strains with health-promoting benefits (Khan, 2014).

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Lactic acid bacteria (LAB) together with bifidobacteria are most investigated probiotics since

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decades. The uniqueness of LAB is that they provide several health benefits as well as they are

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involved in food fermentations. Several criteria have been used to consider new LAB isolates as

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probiotic including tolerance to acid and bile conditions, cholesterol lowering potential, ability to

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hydrolyze bile salt, being non-haemolytic, ability to possess antimicrobial properties, and able to

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survive during the fermentation process (Vijaya Kumar, Vijayendra, & Reddy, 2015). Generally,

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potential probiotics are isolated from food matrices in which those microorganisms are used.

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Camel milk is an excellent source where LAB can be isolated with high probiotic potential. Camel

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milk contains greater amount of natural antimicrobial compounds than bovine milk (Elagamy,

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Ruppanner, Ismail, Champagne, & Assaf, 1996). Al haj and Al Kanhal (2010) has reported

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Somalia, Saudi Arabia and United Arab Emirates (UAE) as being the highest camel milk producing

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countries. To the best of our knowledge, few attempts have been made to isolate LAB from camel

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milk and its products (Fguiri et al., 2016; Mahmoudi et al., 2016; Soleymanzadeh, Mirdamadi, &

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Kianirad, 2016; Yateem, Balba, Al-Surrayai, Al-Mutairi, & Al-Daher, 2008). These reports have

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major drawbacks in the procedure section. For example, the study by Fguiri et al. (2016) lacks

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probiotic characterization including their acid and bile tolerance abilities, cholesterol removal

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ability, hemolytic pattern, and antimicrobial activity and use of old non-DNA based methods for

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identification of isolates. The studies of Yateem et al. (2008) and Soleymanzadeh et al. (2016) lack

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many probiotic parameters to provide an evidence that the isolated LABs have probiotics

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characteristics. Yateem et al. (2008), Mahmoudi et al. (2016) and Soleymanzadeh et al. (2016) have

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attempted to identified the isolated LAB but there is no evidence that these isolates were identified

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using DNA based method. Therefore, the objectives of this study were to isolate LAB from raw

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camel milk and investigate probiotic characteristics such as physiological properties, cell surface

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properties (hydrophobicity, autoaggregation, co-aggregation), acid and bile tolerance abilities, bile

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salt hydrolysis, cholesterol removing property, exopolysaccharide (EPS) production ability,

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hemolytic and antimicrobial activities, resistance toward lysozyme and six antibiotics, and

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fermentation profile (growth, pH, and proteolysis) and 16S rRNA sequencing to identify selected

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LAB isolates.

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2. Materials and Methods

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2.1. Sample Collection

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Fifty raw camel milk samples were collected in sterilized bottles from different camel farms in Abu

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Dhabi, UAE from healthy camels. Due to long distances between farms, samples were kept in ice-

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boxes and were transported to the food microbiology laboratory at the UAE University for isolation

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and characterization studies. All chemicals in this study were purchased from Sigma-Aldrich

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(St. Louis, MO, USA) unless otherwise mentioned.

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2.2. Isolation of lactic acid bacteria

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LAB were isolated using the spread-plate method on MRS agar (de Mann Rogosa Sharpe; Oxoid,

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Hampshire, UK). Plates were incubated at 30°C for 48 h anaerobically. One hundred colonies with

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different morphologies were subjected to Gram stain and catalase test. Twenty-three colonies that

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were Gram-positive and catalase-negative were subjected to further test in section 2.4.1. Colonies

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were subcultured in MRS broth to maintain their purity. Each colony was stored in glycerol stock

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(50%) at -80°C.

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2.3. Reference probiotic and pathogens

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Lactobacillus plantarum DSM 2648 was purchased from Leibniz-Institut DSMZ - Deutsche

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Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and

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was used as a probiotic reference for this study (Anderson, Cookson, McNabb, Kelly, & Roy,

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2010). Listeria monocytogenes ATCC 7644, Salmonella typhimurium 02-8423, Escherichia coli

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O157:H7 1934, and Staphylococcus aureus ATCC 15923 were obtained from Prof. Richard Holley

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Laboratory, University of Manitoba, Canada.

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2.4. Evaluation of probiotic characteristics

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2.4.1. Tolerances to acid and bile

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Acid and bile tolerances of pure isolates were carried out according to Liong and Shah (2005a).

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Acid tolerance was tested in MRS medium adjusted pH to 2.0 during incubation for 2 h at 37°C.

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Bile tolerances of pure isolates were tested against oxgall (0.3% and 1.0%), cholic acid (0.3%),

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taurocholic acid (0.3% and 1.0%) during 6 h of incubation at 37°C.

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2.4.2. Autoaggregation

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Autoaggregation ability of the isolates was carried out according to the method of Angmo, Kumari,

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Savitri, and Bhalla (2016). Autoaggregation percentage was calculated based on: 1 −

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where At represents absorbance at time t and A0 represents absorbance at t=0

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2.4.3. Hydrophobicity

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Cell hydrophobicity of the isolates was according to Mishra and Prasad (2005).

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2.4.4. Co-aggregation

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Co-aggregation of LAB isolates against the four pathogens at 20°C and 37°C during incubation for

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4 h was according to Zuo et al. (2016). Co-aggregation percentage was expressed as: % =

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2.4.5. Antibacterial activity

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Antibacterial activity of cell-free supernatant of LAB isolates was examined according Mishra and

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Prasad (2005).

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2.4.6. Antibiotic susceptibility

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Antibiotic resistance of the isolates was performed according to Das, Khowala, and Biswas (2016).

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Antibiotic discs and cartridge dispenser were from Oxoid (Hampshire, UK).

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2.4.7. Haemolytic activity

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Hemolytic activity of LAB isolates was examined on Colombia blood agar (Himedia, Mumbai,

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India) according to Angmo et al. (2016). LAB isolates exhibiting no clear halos (γ-hemolytic or

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non-hemolytic) were selected as potential probiotics, while those having a clear hemolysis zone (β-

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hemolytic or complete hemolytic) or a greenish halo (α-hemolytic or partial hemolytic) were

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discarded.

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2.4.8. Exopolysaccharide

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Test for exopolysaccharide production for LAB isolates was carried out according to the method of

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Angmo et al. (2016).

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2.4.9. Bile salt hydrolysis (BSH)

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BSH activities of pure isolates were measured by determining the amount of amino acids liberated

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from conjugated bile salts by LAB strains according to Liong and Shah (2005b). BSH activities

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were assayed against sodium glycocholate (6mM), sodium taurocholate (6mM) or conjugated bile

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salt mixture (6mM; glycocholic acid, glycochenodeoxycholic acid, taurocholic acid,

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taurochenodeoxycholic acid, taurodeoxycholic acid).

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2.4.10. Cholesterol removal

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Cholesterol removal of LAB isolates was determined according to Miremadi, Ayyash, Sherkat, and

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Stojanovska (2014).

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2.4.11. Lysozyme tolerance

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Tolerance of LAB isolates toward lysozyme during 90 min of incubation at 30°C was determined

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according to the method of Vizoso Pinto, Franz, Schillinger, and Holzapfel (2006).

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2.4.12. Heat resistance

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Heat resistance of selected bacterial isolates was carried out according to method detailed in Teles

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Santos et al. (2016).

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2.5. Identification of selected isolates by 16S rDNA sequencing

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16S rDNA of selected strains was amplified by PCR procedure described in Angmo et al. (2016).

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PCR primers 27F (5’- AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-

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TACGGYTACCTTGTTACGACTT-3’) were employed during amplification. The DNA sequence

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of PCR product was carried out by Macrogen Sequencing Facilities (Macrogen-Korea, Seoul,

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Korea). Sequence results were aligned with NCBI database using BLAST algorithm. Accession

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numbers were received for selected LAB isolates by GenBank®. Neighber-joining method was

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applied to determine the closest bacterial species (Saitou & Nei, 1987) using MEGA software 7.0.

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2.6. Fermentation profile

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Fermentation capabilities of the selected LAB isolates were assessed according to Angmo et al.

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(2016). Reconstituted bovine skim milk (5% w/v) was sterilized at 105°C for 5 min followed by

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tempering at 37°C and inoculation by 1.5% of actively selected LAB isolates, individually,

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followed by incubation at 37°C for 24 h. Fermented milk samples were stored at 4°C for 21 days.

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Samples were taken at 7d intervals during the storage period for enumerating bacterial count, pH

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measurement and proteolytic activity using OPA.

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2.6.1. Bacterial enumeration and pH

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Appropriate serial dilutions were prepared in 0.1% peptone and water solution, and LAB

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populations were counted using MRS agar. Inoculated plates in duplicate were incubated

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anaerobically at 37°C for 48 h using anaerobic jars. pH values of each sample were measured using

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a calibrated digital pH meter (Starter-3100, Ohaus, NJ, USA).

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2.6.2. Proteolytic activity by OPA

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Proteolytic activities of fermented samples were assayed according to Elfahri, Vasiljevic, Yeager,

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and Donkor (2016). Proteolysis results are presented as absorbance at 340 nm.

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2.7. Statistical analysis

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One-way ANOVA was carried out to examine the significant effect of differences in LAB isolates

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on quantitative parameters (p < 0.05). Tukey’s test was employed to examine differences between

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means at p < 0.05. All tests were repeated at least three times to calculate the means and standard

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error. All statistical analyses were performed using Minitab version 17.0 (Minitab, Ltd., Coventry,

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UK).

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3. Results and Discussion

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3.1. General characterization of isolates

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Twenty-three isolates out of 100 isolated colonies were Gram-positive, rod-shape, and catalase-

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negative. All 23 isolates showed better growth capabilities at incubation temperature 37°C

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compared to 30°C (data not shown). These 23 strains were subjected to acid and bile tolerance test.

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3.2. Tolerances to acid and bile

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Table 1 presents results of acid tolerance of 23 isolates at pH 2.0 and bile tolerances against oxgall,

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colic acid, and taurocholic acid at different concentrations. The growth of the isolates decreased (p

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< 0.05) during 2 h of incubation at 37°C under acidic condition pH 2.0. The growth reduction

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ranged from 0.29 to 6.78 log10 CFU/mL. Isolates 15, 70, 34, 66, 76, 22, 51, 73, and 47, showed

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highest acid resistance, hence were used for further characterization. The growth decreased with

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incubation time. Isolates showed remarkable resistance toward cholic and taurocholic acids

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compared with Oxgall. Overall, isolates 15, 70, 34, 66, 76, 22, 51, 73, and 47, among other, showed

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higher bile resistance. Based on acid and bile tolerances, isolates 15, 70, 34, 66, 76, 22, 51, 73, and

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47 were selected for subsequent characterization.

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Tolerances to acid and bile stresses are important properties for any potential probiotic bacteria. The

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ability of probiotic to survive, in adequate numbers, after subjected to gastric acidity (low pH) and

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intestine condition (bile salts) is important to be used in the food industry (Chalas et al., 2016). Acid

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and bile tolerances of all isolates varied significantly due to strain or species specificity. The

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resistant mechanism toward low pH or bile concentration is strain and species dependent (Montville

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& Matthews, 2013). Similar results to our study have been reported by Angmo et al. (2016) and Das

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et al. (2016) for LAB isolated from Ladakh beverages and marine samples. Acid resistances of the

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selected isolates in this study were higher as compared with those reported by Angmo et al. (2016)

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at pH 2.0.

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3.3. Autoaggregation and hydrophobicity

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Percentages of autoaggregation during 24 h of incubation at 37°C and hydrophobicity against three

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hydrocarbons namely hexadecane, xylene and octane are presented in Table 2. The nine isolates

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(isolates 15, 70, 34, 66, 76, 22, 51, 73, and 47) exhibited a good percentage of autoaggregation

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ranging from 0.1 - 10% and 0.6 - 38% during 3 h and 24 h of incubation. ANOVA showed that

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autoaggregation increased (p < 0.05) during the incubation period. After 24 h, isolates 51, 70, 22,

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47 and 66 exhibited highest autoaggregation than other isolates. Results in Table 2 revealed that

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hydrophobicities of the 9 isolates toward xylene and octane were higher (p <0.05) than that in

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hexadecane. The percentages of hydrophobicity ranged from 0.6 – 16.2%, 1.6 – 57.9%, 2.7 – 67.0%

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for hexadecane, xylene and octane, respectively (Table 2). In general, isolates 51, 76, 47, 22, and 66

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showed higher hydrophobicity than other investigated isolates.

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Cell surface properties tested by hydrophobicity and autoaggregation are indicative parameters for

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probiotic cells adhesion to epithelial cells in human intestine. Several researchers have reported that

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aggregation (Botes, Loos, van Reenen, & Dicks, 2008) and hydrophobicity (Duary, Rajput, Batish,

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& Grover, 2011) tests correlated with adhesion properties of probiotic bacteria. However, Zuo et al.

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(2016) have reported the opposite trend. In the current study, LAB isolates showed remarkable

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percentages of autoaggregation and hydrophobicity toward hydrocarbons. However, significant

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differences existed among the investigated strains which may be attributed to the variation in

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hydrophilic/hydrophobic extensions in the cell wall of LAB isolates. Angmo et al. (2016) have

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reported hydrophobicity against hexadecane and results ranged from <5% - 47% for LAB strains.

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The drawback of the previous study (Angmo et al., 2016) was that hydrophobicity of LAB was

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tested against one hydrocarbon (hexadecane). In our study, LAB isolates, however, exhibited

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comparatively higher hydrophobicity as compared with the previous study, in particular against

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xylene and octane. Das et al. (2016) have also reported hydrophobicity and autoaggregation for 3

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LAB isolates from Indian marine sources. These authors have reported the hydrophobicity range

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from 22.2% to 25.0%, which is lower compared with our results, whereas autoaggregation

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percentages are in accordance with our results. Tareb, Bernardeau, Gueguen, and Vernoux (2013)

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have studied autoaggregation percentages for Bifidobacterium strains. These authors have reported

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similar autoaggregation results to our study.

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3.4. Co-aggregation

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The results of co-aggregation of 9 LAB isolates in the presence of E. coli O157:H7, S.

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typhimurium, L. monocytogenes, and S. aureus separately at 2 h and 4 h of incubation at 20°C and

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37°C are shown in Table 3. ANOVA revealed that co-aggregation increased (p < 0.05) with

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incubation at each incubation temperatures. In general, the co-aggregations percentages of LAB

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isolates with all pathogens at incubation temperature 37°C were higher (P > 0.05) compared with

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20°C, particularly in the presence of L. monocytogenes, and S. aureus. As shown in Table 3, LAB

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isolates exhibited higher co-aggregation (p > 0.05) toward Gram-negative pathogens (E. coli

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O157:H7 and S. typhimurium) during the first 2 h compared with Gram-positive (L.

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monocytogenes, and S. aureus). This trend had changed when incubation time prolonged to 4 h.

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Overall, isolates 22, 47, 34, and 70 demonstrated higher co-aggregation ability compared with other

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investigated isolates.

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Several workers have reported that co-aggregation ability of LAB in the presence of gut pathogens

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will enhance probiotic properties of the LAB. Different authors have revealed that cellular

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aggregation will be positive in promoting cell colonization of probiotic bacteria (Cesena et al.,

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2001; Collabo, Gueimonde, Hernández, Sanz, & Salminen, 2005). The co-aggregation results of our

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study agree with the reported by others (Angmo et al., 2016; Ben Taheur et al., 2016; Collado,

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Meriluoto, & Salminen, 2008). The ability of current LAB isolates to co-aggregate with pathogens

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may be attributed to cell surface components, but the mechanisms still need more investigation.

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Interactions between carbohydrate-lectin and proteinaceous components present on the cell surface

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may be involved (Tareb et al., 2013). Nonetheless, co-aggregation percentages are strain (probiotic

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and pathogen) and time of incubation dependent (Collado et al., 2008). The ability of LAB to co-

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aggregate in the presence of pathogen microbes will form a defensive barrier that will not allow for

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pathogens to colonize in the human gut (Vidhyasagar & Jeevaratnam, 2013). Our study showed that

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incubation temperature did not significantly influence the co-aggregation. Our result contradicts

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with that of Collado et al. (2008) who reported that incubation temperature did affect co-

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aggregation of studied probiotics, but data were not shown. Therefore, authors selected temperature

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to carry out coaggregation assay at 20°C (Collado et al., 2008). We believe that co-aggregation

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influenced by environmental conditions need to be investigated further to standardize coaggregation

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assay. The unpredicted co-aggregation trend of LAB isolates in this study toward Gram-positive

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pathogens during the first 2 h of incubation need further studies.

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3.5. Antibiotic resistant and antimicrobial activity

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Table 4 presents the antimicrobial activities against 4 foodborne pathogens and antibiotic resistance

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of 9 LAB isolates against 6 antibiotics. Antimicrobial activities of LAB isolates ranged from small

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(0.1 mm zone) to high (>2.0 mm zone). Isolates 15, 34, 47, 66, 70 and 76 exhibited strong

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antimicrobial activities against all 4 pathogens. Interestingly, these isolates showed greater

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influence against S. aureus compared with other pathogens (Table 4). In general, all LAB strains

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were able to resist the impact of antibiotics. However, isolates 22, 51 and 73 were susceptible to

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antibiotics.

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Antimicrobial activity of LAB strains may be attributed to some compounds produced during LAB

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growth including metabolites, organic acids, and bacteriocins (Zuo et al., 2016). Antimicrobial

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activities of the current study are in agreement with the results reported by other authors (Angmo et

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al., 2016; Das et al., 2016; Zuo et al., 2016). These studies have also revealed that antimicrobial

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activity was species- and strain-dependant. In general, all isolates were moderately susceptible and

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sensitive toward penicillin, ampicillin, clindamycin, and erythromycin. However, isolates were

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resistant to trimethoprim and vancomycin. The resistant against particular antibiotic may be due to

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the absence of target site of that particular antibiotic in LAB cell (DeLisle & Perl, 2003). These

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results are almost in agreement with those of Angmo et al. (2016) and Teles Santos et al. (2016)

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who isolated LAB from fermented Indian product and cocoa fermentation, respectively. However,

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minor differences compared with our study may be attributed to strain and species differences.

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3.6. Bile salt hydrolysis (BSH) and cholesterol removal

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BSH activities and cholesterol removal of 9 LAB isolates are presented in Table 5 and Figure 1,

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respectively. All LAB strains showed the ability to hydrolyze all three investigated bile salts

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producing free cholic acid. Isolates 70, 22, 34, 76 and 15 exhibited high BSH activities compared

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with other LAB isolates (Table 5). In general, the differences in BSH among 3 bile salts at each

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isolate were insignificant (p < 0.05). As shown in Figure 1, LAB isolates were able to remove

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cholesterol from MRS media. Isolates 15, 34, 76 and 70 demonstrated higher (p < 0.05) cholesterol

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removal ability than other LABs.

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Cholesterol removal is one of the desirable characteristics for probiotics. Several mechanisms art

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proposed for cholesterol removal including: cholesterol incorporation in the cell wall, adhesion to

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the cell wall, and enzymatic reduction via cholesterol reductase (Ishimwe, Daliri, Lee, Fang, & Du,

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2015). Moreover, the ability of LAB to hydrolyze bile salts in human intestine would reduce

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cholesterol absorption inside intestine (Jones, Tomaro-Duchesneau, Martoni, & Prakash, 2013). In

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our study, investigated isolates exhibited cholesterol lowering and BSH activities. Cholesterol

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lowering ability results in our study are in agreement with the results reported by Das et al. (2016),

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while BSH activities are in accordance with those reported by Miremadi et al. (2014).

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3.7. Heat and lysozyme tolerances

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Resistant to heat and impact of lysozyme of 9 LAB isolates are presented in Table 6. The growth of

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all isolates decreased (p < 0.05) after they were subjected to heat treatment at 60°C for 5 min. The

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reduction in LAB growths ranged from 0.9 to 2.5 log10 CFU·mL-1. Isolates 34, 76, 51, 66, and 73

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exhibited higher heat resistant compared with other strains. On the other hand, the reduction in the

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growth of LAB occurred during incubation for 90 min in the presence of 100 mg·L-1 lysozyme was

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insignificant (Table 6). The growths’ reductions were < 1.0 log10 CFU·mL-1.

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LAB isolates displayed good resistant toward heat which would be a positive property for these

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probiotics to be applied in food for food industry. The reduction range in the growths of LAB due to

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heat treatment in our study was lower than the reduction range reported by Teles Santos et al.

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(2016) who isolated LAB from cocoa fermentation. This suggests the LAB isolated from camel

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milk were more resistant than those isolated from cocoa fermentation. Turchi et al. (2013) have

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reported that survival rates of 37 investigated LAB strains from Italian food products were > 80%.

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Those results are in agreement with our results (Table 6). The results of lysozyme resistant in our

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study are also in accordance with results reported by Angmo et al. (2016).

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3.8. Haemolytic activity and EPS production

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Table 7 presents haemolytic activity and ability to produce EPS of 9 LAB isolates. Based on EPS

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test, all LAB isolates exhibited the ability to produce EPS, except isolates 51 and 73. With regard to

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haemolytic activity, LAB isolates exhibited no haemolytic activity, except isolates 22, 51 and 73.

330

Isolates 22 and 51 showed β-haemolysis, whilst isolate 73 displayed ⍺-haemolysis.

331

The presence of ropy white colored mucous on skimmed milk-ruthenium red plates producing

332

lactobacilli are the EPS producers. Several workers have reported similar EPS results using same

333

tests of this study (Angmo et al., 2016; Kumari, Angmo, Monika, & Bhalla, 2016). The 3 isolates

334

demonstrated haemolytic activities of either ⍺- or β-haemolysis were not considered for

335

identification.

336

3.9. Identification of selected isolates by 16S rDNA sequencing

337

Six potential probiotics were identified by 16S rRNA gene sequence. PCR amplicons were between

338

1200 – 1400 bp. Alignment were carried out using BLAST. The identified isolates were designated

339

as probiotic lactic acid bacteria. Molecular phylogeny analysis and phylogenic tree were performed

340

to identify LAB to species level based on the 16S rDNA sequences from evolutionary distances by

341

neighbor-joining method. Phylogenic tree of the 6 isolates is shown in Figure 2. Sequence analysis

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exhibited that 50% of the 16S rDNA clustered with the sequence of lactobacilli and the other 50%

343

grouped with the sequence of Lactococci. The accession numbers from Genbank for each isolates

344

are presented in Table 8.

345

3.10. Fermentation profile

346

Bacterial count, proteolysis assessment and pH values of fermented milk during 21 days of storage

347

are provided in Table 9. All 6 isolates maintained high bacterial count during 21 days of storage at

348

4°C. A slight drop (p > 0.05) in bacterial counts of L. lactis KX881768, L. plantarum KX881772, L.

349

garvieae KX881774, and L. reuteri KX881777 occurred during storage except L. lactis KX881782

350

which dropped significantly. On the other hand, L. plantarum KX881779 populations increased (p

351

> 0.05) during storage (Table 9). Table 9 shows that proteolysis index (OPA) increased (p < 0.05)

352

during 21 days of storage. L. plantarum KX881779 (isolate 70) exhibited the highest proteolysis

353

compared with other investigated isolates. pH values of fermented milk dropped significantly after

354

24 h of fermentation. In general, pH values of fermented milk with all isolates as potential

355

probiotics kept constant during storage except milk fermented by L. plantarum KX881779 (Table

356

9).

357

Capability of probiotic to ferment milk and survive in adequate numbers in fermented milk during

358

storage is essential to support probiotic claims and health benefits. In this study, bacterial viability

359

in all fermented milk was at the adequate level to provide claimed health benefits. Our results are in

360

agreement with those reported by Angmo et al. (2016). Also, the increase in proteolysis in

361

fermented milk is another critical parameter that would support health benefit claims including

362

antioxidant, antihypertension and anticancer properties (Clare & Swaisgood, 2000). Our OPA

363

results indicated that our isolates had a robust proteolytic activity which in turn would produce

364

sufficient level of bioactive peptides.

365

4. Conclusions

366

Selected isolates from camel milk exhibited probiotic characteristics. Results of this study showed

367

that camel milk isolates, especially L. plantarum KX881779 and Lactococcus lactis KX881782 had

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368

great potential to be used in foods. Further studies are required to explore the health benefit of these

369

isolates of fermented foods made by these isolates.

370 371

RI PT

372 373 Acknowledgement

375

Authors would like to thank Dr. Mohamed Enan from the Biology Department for providing

376

technical support for PCR runs. Also, authors would like to acknowledge UAE University for the

377

financial support.

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Table 1: Acid and bile tolerances for 23 LAB isolates from camel milk

Isolates

Acid Tolerance (Log10 CFU)

Bile Tolerances (%)

pH 2.0

0.3 Oxgall

0h

2h cdef

Iso15

9.9±0.05

Iso18

9.5±0.10defgh efgh

9.5±0.00

3h a

17.6±0.40

6h ij

3.7±0.00efg

25.2±0.75gh

efg

de

73.1±1.95

38.4±0.95i

1.3±0.05

48.8±1.45bc

68.9±1.80

58.1±1.40fg

1.7±0.05

60.1±1.75a

33.9±0.95def

77.3±1.70ab

24.5±0.70hi

73.5±1.65abcd

0.3±0.00e

Iso27

9.2±0.05h

4.4±0.10def

33.9±1.00def

75.0±1.65abc

18.9±0.55jk

71.5±1.55abcd

Iso28

9.5±0.00defgh

4.4±0.10def

37.8±1.10cde

75.2±0.05abc

23.3±0.70hij

75.5±1.85a 21.8±0.45

bcd

3h

17.3±0.45

14.8±0.45d

5.9±0.15ij

11.8±0.25 6.4±0.15

ij

14.9±0.35

bc

5.9±0.15

6h i

43.5±1.15bc

24.9±0.70bc cd

18.0±0.55

d

12.3±0.30kl 33.3±0.70def

18.7±0.55bc

13.7±0.30de

29.5±0.85a

34.6±0.75de

0.3±0.00e

20.5±0.45cde

12.4±0.35e

12.4±0.25def

22.9±0.65c

36.5±0.80d

72.6±1.05abcd

0.3±0.00e

22.6±0.40bc

20.1±0.60b

13.2±2.00def

30.4±0.90a

32.2±1.85def

bcde

e

17.3±0.55efgh

12.1±0.35e

14.6±0.45cde

25.1±0.75bc

29.1±0.90fg

Iso39

9.3±0.15gh

3.2±0.05efg

29.3±0.55fg

38.3±1.25i

30.6±0.60g

51.4±1.60g

0.1±0.00e

0.2±0.05j

4.3±0.05k

8.2±0.25hi

11.2±0.25gh

11.2±0.35lm

Iso41

9.2±0.10gh

3.1±0.05fg

36.5±0.70de

67.4±2.15cdef

30.4±0.60g

67.6±2.15bcdef

0.3±0.00e

15.0±0.45ghi

5.8±0.15ijk

12.1±0.40def

12.9±0.25fg

21.2±0.65hi

Iso45

9.2±0.10

gh

de

12.0±0.40kl

Iso47

9.6±0.05cdefgh

5.4±0.05cd

34.5±0.65def

80.8±2.55a

17.8±0.35k

72.6±2.25abcd

3.4±0.10bc

23.0±0.75bc

17.1±0.35c

17.4±0.55bc

25.9±0.50b

46.4±1.45ab

Iso49

9.8±0.15cdefg

3.2±0.05efg

35.8±0.70de

54.3±1.70gh

44.4±0.85cd

75.9±2.40ab

2.0±0.05cde

0.2±0.00j

5.4±0.10ijk

8.7±0.25ghi

15.1±0.30ef

11.2±0.35lm

Iso51

9.7±0.05cdefgh

6.4±0.05bc

38.2±0.75cd

73.0±2.30abcd

31.1±0.60fg

72.2±2.25abcd

0.3±0.00e

17.8±0.55efg

10.7±0.20ef

11.5±0.35efg

18.4±0.35d

31.3±1.00ef

Iso57

9.6±0.05

cdefgh

cd

abcd

e

Iso66

10.9±0.05a

9.1±0.10a

16.3±0.55j

67.6±1.20cdef

1.0±0.05m

61.1±1.05efg

1.4±0.05cde

13.8±0.25i

9.3±0.30fg

24.3±0.40a

5.1±0.20i

47.5±0.80ab

Iso70

10.6±0.20ab

9.6±0.15a

49.5±1.65a

62.5±1.05efgh

70.7±2.40a

81.0±1.40a

0.2±0.05e

1.0±0.00j

14.5±0.50d

0.9±0.00k

15.1±0.50ef

5.2±0.10n

Iso73

9.7±0.05cdefgh

6.2±0.05bc

33.3±1.15def

62.7±1.05efgh

40.5±1.35de

74.1±1.25abcd

0.6±0.00de

3.2±0.05j

1.4±0.00l

2.2±0.05k

1.8±0.10j

6.7±0.10mn

Iso76

9.7±0.15

cdefgh

gh

cdef

de

Iso78

9.7±0.10cdefgh

4.5±0.05de

45.0±1.50ab

70.3±1.85bcde

36.4±1.25ef

67.6±1.85bcdef

2.6±0.10bcd

Iso82

10.1±0.00bc

3.3±0.00efg

43.2±0.95bc

53.7±1.45h

49.5±1.10bc

64.5±1.75def

Iso84

10.0±0.05cde

3.5±0.00efg

49.2±1.10a

63.5±1.70defg

51.0±1.15b

Iso85

bcd

efg

DSM26482

502 503 504

1

9.1±0.08

3.5±0.10 5.9±0.10

38.1±1.25

23.0±0.50 32.1±0.90

cd

hi

71.8±1.25

abcde

74.6±2.00

63.0±1.60

abc

68.6±2.15

TE D

58.2±1.00

fgh

36.3±0.70

45.0±0.90

EP

7.2±0.00

b

35.6±1.20

de

56.5±1.80

28.6±1.00

AC C

3.4±0.00

efg

32.5±0.65

M AN U 1.0±0.05de

74.0±1.25

0.3±0.00

18

0.1±0.00

4.7±0.10

j

1.9±0.05

6.1±0.10

ij

15.8±0.30

0.2±0.00

18.5±0.35ij

8.4±0.25gh

6.3±0.15ij

9.2±0.30h

16.4±0.40ijk

1.0±0.00de

18.6±0.50def

14.7±0.30d

14.1±0.40de

23.2±0.50bc

24.4±0.70gh

71.7±1.95abcd

0.0±0.00e

16.5±0.40fghi

24.3±0.55a

26.9±0.75a

31.1±0.65a

26.0±0.70g

abcd

b

e

b

71.8±1.95

23.9±0.80

67.6±1.70

4.3±0.10 3.0±0.12

16.5±0.30

22.2±0.60 14.9±0.40

bc

7.0±0.20

12.4±0.25 8.7±0.31

19.8±0.55 13.1±0.40

fgh

j

14.3±0.40hi

0.9±0.00

fghi

l

3.9±0.10

10.5±0.20

65.5±1.10

Values are mean ± standard error of triplicates DSM2648 is indicative probiotic and not part of means differences. a-n Means in same column with different lowercase letters differed significantly (p < 0.05) 2

0.6±0.05

jk

hi

m

3.4±0.10

0.3±0.00

jk

15.7±0.45

41.3±0.90c

74.8±2.35abc

j

14.1±0.30

de

23.5±0.70hij

e

8.6±0.25

de

77.5±2.40ab

bcde

24.4±0.55

gh

35.8±0.70de

3.1±0.00

0.4±0.05

b

9.1±1.10a

ef

69.5±1.55

SC

22.2±0.45bc

10.8±0.05a

gh

10.0±0.25

22.0±0.60

6h e

Iso34

ef

72.7±1.60

3h

bc

9.4±0.25

efg

25.6±0.75

1.0 TA

Iso33

10.1±0.05

2.9±0.05

0.3±0.00

e

7.1±0.25b

l

72.1±1.55

abcd

0.3 TA

6h

cde

9.8±0.05cdefg

abcd

21.9±0.65

ijk

3h bcde

Iso22

gh

73.5±1.60

abc

6h m

9.5±0.05

g

35.7±1.05

3h abcd

0.3 Colic acid

Iso21

fgh

3.4±0.00

1.0 Oxgall

RI PT

501

10.7±0.35

5.6±0.15

i

14.2±0.40

gh

14.1±0.25jkl

49.1±1.30a 23.2±0.70

ACCEPTED MANUSCRIPT

505 506

Auto-aggregation (%) 3h Iso15 1.6±0.05c Iso22 9.9±0.55a Iso34 2.4±1.80bc Iso47 8.4±0.05a Iso51 10.2±0.25a Iso66 8.2±1.05a Iso70 5.6±0.50abc Iso73 6.8±0.65ab Iso76 1.5±1.50c DSM26482 4.0±0.71 1 Values are mean ± standard error of triplicates Bacteria

Hydrophobicity (%) Hexadecane 0.7±0.10d 11.8±0.45b 0.7±0.00d 15.3±0.90a 15.1±1.00a 1.5±0.90d 1.4±0.10d 3.0±0.20cd 5.9±0.15c 5.8±0.40

M AN U

TE D

DSM2648 is indicative probiotic and not part of means differences.

a-f

EP

2

Means in same column with different lowercase letters differed significantly (p < 0.05)

AC C

508 509 510 511 512 513

24h 2.7±0.40e 26.9±0.00b 2.4±1.80e 25.7±0.55b 38.8±0.05a 16.8±1.30c 29.7±0.10b 8.6±0.05d 9.0±1.45d 14.8±0.60

RI PT

Table 2: Autoaggregation (%) during 24 h and hydrophobicity (%) against hexadecane, xylene and octane during 18 h at 37°C

SC

507

514 515 516 517

19

Xylene 23.2±0.60cd 18.1±0.95d 2.2±0.60e 27.3±1.15c 46.6±1.05b 22.8±3.25cd 3.1±0.20e 3.9±0.15e 57.8±0.15a 23.0±0.91

Octane 3.2±0.45f 28.0±0.40c 15.6±0.50de 46.8±0.10b 66.7±0.35a 19.7±2.45d 13.1±2.20e 15.7±0.15de 45.4±0.01b 25.0±0.69

ACCEPTED MANUSCRIPT

518 Table 3: Co-aggregation (%) of LAB with 4 pathogens during 4 h at two different incubation temperature.

520 521 522 523

1

S. typhimurium

L. monocytogenes

S. aureus

Iso22

7.3±0.43a

7.4±0.43ab

3.3±0.45a

5.8±0.44ab

Iso34

1.0±0.92

b

bc

a

bc

Iso47

2.9±0.91ab

2.1±0.92c

1.3±0.92a

7.5±0.87a

Iso51

1.9±0.91b

10.7±0.83a

3.5±0.90a

1.1±0.92c

Iso66

2.9±0.90ab

4.4±0.89bc

1.2±0.91a

3.5±0.90abc

1.1±0.46

c

1.8±0.91

1.6±0.91

3.0±1.92

1.6±1.38d

10.0±1.26

a

1.6±0.46

bc

524 20

S. typhimurium 2.9±0.76

bc

6.1±1.31abc

ab

7.9±1.28

ab

L. monocytogenes

S. aureus

bcd

2.4±0.11bc

3.3±1.35bcd

7.2±1.29ab

3.0±2.01

10.4±1.25

a

9.7±1.26a

4.3±1.34bcd

2.3±1.36c

1.8±1.37d

5.0±1.33abc

9.0±1.27abc

5.7±1.32ac

10.7±1.24a

6.4±1.30abc

4.2±0.45bcd

6.0±0.44abc

3.0±0.45bcd

6.2±0.44abc

a

abc

abc

Iso73

1.9±1.37b

4.2±1.34bc

2.7±1.36a

Iso76 DSM26482

2.4±0.46b 2.3±0.80

9.2±0.42a 5.0±0.67

1.8±0.46a 1.67±0.76

Iso15

8.6±1.51a

7.2±1.53b

Iso22

8.3±1.29

a

13.0±1.23

Iso34

4.1±1.79a

8.5±1.71b

10.8±1.66b

5.7±1.76a

12.7±1.63abc

17.2±1.54ab

17.7±1.54a

14.6±1.60ab

Iso47

9.1±1.71a

5.5±1.78b

7.4±1.75b

10.4±1.69a

3.0±1.81d

5.2±1.77c

9.4±1.69ab

7.9±1.72abc

Iso51

5.5±1.77a

12.2±1.64ab

6.2±1.75b

8.3±1.71a

14.1±1.61ab

9.5±1.69bc

17.0±1.55a

9.3±1.70abc

Iso66

4.3±1.79

a

b

a

bcd

bc

ab

Iso70

7.2±1.75a

4.1±1.81b

Iso73

4.0±2.25a

6.5±2.19b

Iso76

8.8±1.28a

DSM2648

6.1±1.55

4.3±1.58b ab

b

a

20.9±1.11

6.2±1.75

11.2±0.42

5.9±0.44

8.4±0.43

7.6±0.43a

1.0±0.46

2.0±1.37bc

14.8±0.40a

10.2±0.42a

8.6±0.43ab

9.3±0.42a

1.5±0.46bc 2.6±0.56

3.1±0.45cd 6.5±0.98

2.2±0.46c 5.1±0.87

2.0±0.46cd 5.2±0.45

1.7±0.46c 6.0±0.70

4.0±1.58a

3.4±1.80cd

5.0±1.78c

3.4±1.80b

4.1±1.79c

10.7±1.26

4.7±1.78

a

11.9±1.64

5.5±1.78

abc

12.6±1.63

8.6±1.72

abc

12.1±1.65

9.0±1.71

ab

10.2±1.68abc

7.1±1.76bc

6.8±1.76b

8.0±1.74a

11.5±1.67abc

13.7±1.63abc

16.2±1.58a

15.3±1.60ab

8.6±2.14b

5.5±2.21a

16.2±1.58a

19.0±1.53a

17.3±1.56a

16.9±1.57a

21.0±1.11a

8.3±1.29b

4.2±1.35a

5.9±1.78bcd

5.9±1.78c

5.3±1.78b

4.0±1.81c

9.1±1.34

8.0±1.44

6.1±1.90

8.4±1.55

9.5±1.01

10.5±1.22

8.6±1.71

Values are mean ± standard error of triplicates DSM2648 is indicative probiotic and not part of means differences. a-d Means in same column with different lowercase letters differed significantly (p < 0.05) 2

cd

Iso70

5.7±1.76

0.7±0.47

1.3±0.69

E. coli O157:H7

bc

5.1±0.67

3.4±0.90

2.1±0.69

a

Iso15

b

4.0±0.67

bc

RI PT

ab

SC

E. coli O157:H7

Incubation 37°C

M AN U

4h

Incubation at 20°C

TE D

2h

Bacteria

EP

Incubation time

AC C

519

ACCEPTED MANUSCRIPT

525 526 Table 4: Antimicrobial activity against 4 pathogens and antibiotic resistant toward 6 different antibiotics.

RI PT

527

Antimicrobial activity1 Antibiotics resistant2 E. coli O157:H7 S. typhimurium L. monocytogenes S. aureus PEN TRI AMP CLI VAN Iso15 + + + ++ R MS R R R Iso22 + S R S S MS Iso34 + + ++ ++ MS R MS R R Iso47 + + + +++ R R S MS MS Iso51 + + S S MS S S Iso66 ++ + + ++ MS R S MS R Iso70 ++ ++ +++ +++ MS R MS MS R Iso73 + S MS S S S Iso76 ++ + + +++ S R MS S R 3 DSM2648 + ++ ++ + S R S S R 1 528 (-) no inhibition, (+) inhibition zone 0.1 to 1.0 mm; (++) inhibition zone 1.1 to 2.0 mm; (+++) inhibition zone > 2.1 mm 529 530 2 PEN: penicillin (10 µg); TRI: trimethoprim (25 µg); AMP: ampicillin (10 µg); CLI: clindamycin (2 µg); VAN: vancomycin (30 µg); ERY: 531 erythromycin (15 µg), R: resistant, MS: moderately susceptable, S: sensitive. 532 533 3 DSM2648 is indicative probiotic and not part of means differences. 534

AC C

EP

TE D

M AN U

SC

Bacteria

535 536 537 538 21

ERY MS MS MS S MS MS MS S MS MS

ACCEPTED MANUSCRIPT

539 540

a-c

2

M AN U

SC

Sodium taurocholate 0.84±0.05bc 1.26±0.05abc 2.89±0.21a 0.56±0.11c 0.33±0.02c 0.48±0.09c 2.35±0.70ab 0.77±0.43bc 1.46±0.32abc 1.00±0.18

TE D

Bacteria Sodium glycocholate Iso15 0.79±0.20bc Iso22 1.29±0.32bc Iso34 3.06±0.52a Iso47 0.61±0.10bc Iso51 0.45±0.08c Iso66 0.65±0.22bc Iso70 2.26±0.26ab Iso73 0.59±0.10bc Iso76 1.42±0.51abc 2 DSM2648 0.90±0.22 1 Values are mean ± standard error of triplicates

Means in same column with different lowercase letters differed significantly (p < 0.05)

EP

543 544 545 546 547 548

Table 5: Bile salt hydrolysis activity (specific activity; U/mg)

DSM2648 is indicative probiotic and not part of means differences.

AC C

542

RI PT

541

549 550 551 552 22

Bile salts mixture 0.79±0.20b 1.25±0.06b 2.82±0.27a 0.92±0.33b 0.45±0.08b 0.38±0.05b 3.58±0.15a 0.37±0.05b 1.18±0.42b 1.20±0.22

ACCEPTED MANUSCRIPT

553 554

RI PT

555 556

Table 6: Heat (60°C/5 min) and lysozyme resistant (log10 CFU/mL)

557 558 559 560

Heat resistant (Log10 CFU/mL) Lysozyme resistant (Log10 CFU/mL) 0 min 5 min 0 min 30 min Iso15 9.4±0.04a* 8.3±0.11ab* 9.3±0.05a 9.4±0.05a Iso22 9.8±0.12a* 7.2±0.08c* 9.4±0.15a* 9.1±0.45a* Iso34 9.8±0.01a* 8.8±0.16a* 9.4±0.00a 9.8±0.45a a* a* a Iso47 9.7±0.03 8.6±0.12 9.4±0.20 9.2±0.40a Iso51 9.8±0.01a* 8.7±0.03a* 9.3±0.20a 9.3±0.35a Iso66 9.8±0.01a* 8.6±0.01a* 9.6±0.15a 9.3±0.20a Iso70 9.7±0.01a* 8.7±0.13a* 9.4±0.10a 9.3±0.05a a* b* a Iso73 9.6±0.01 8.0±0.05 8.7±0.20 8.8±0.20a Iso76 9.7±0.01a* 8.6±0.03a* 9.6±0.25a 9.4±0.30a DSM26482 9.5±0.02 8.2±0.10 9.2±0.37 9.1±0.29 1 Values are mean ± standard error of triplicates 2 DSM2648 is indicative probiotic and not part of means differences a-b Means in same column with different lowercase letters differed significantly (p < 0.05) * Means in same row significantly differed (p < 0.05)

AC C

EP

TE D

M AN U

SC

Bacteria

561 562 563 564

23

90 min 9.1±0.15a 9.4±0.05a* 9.2±0.10a 9.3±0.05a 9.3±0.10a 9.3±0.00a 9.1±0.10a 8.7±0.50a 9.2±0.20a 9.0±0.10

ACCEPTED MANUSCRIPT

565 566 567

Exopolysaccharides2 + + + + + + + +

RI PT

569

Bacteria Haemolysis1 Iso15 Iso22 β Iso34 Iso47 Iso51 β Iso66 Iso70 Iso73 ⍺ Iso76 3 DSM2648 1 (-) no haemolysis; (⍺) haemolysis; (β) hemolysis

SC

Table 7: Haemolytic activity and EPS production

M AN U

568

570

2

(-) EPS negative; (+) EPS positive

571 572

3

DSM2648 is indicative probiotic and not part of means differences.

TE D

573 574

Table 8: Identified LAB isolates by 16S rDNA gene sequencing and their Genbank accession

575

numbers.

576 577 578 579 580

24

EP

Species Lactococcus lactis Lactobacillus plantarum Lactococcus garvieae Lactobacillus reuteri Lactobacillus plantarum Lactococcus lactis

AC C

Isolate Iso15 Iso34 Iso47 Iso66 Iso70 Iso76

NCBI accession No. KX881768 KX881772 KX881774 KX881777 KX881779 KX881782

ACCEPTED MANUSCRIPT

Table 9: Fermentation profile (bacterial count, OPA (340 nm), pH) of 6 identified LAB during 21 days of storage at 4°C.

9.4±0.02a 9.0±0.11bcd 8.9±0.07bc 8.8±0.05bc 8.8±0.05c 9.1±0.07b 9.2±0.07

Lactococcus lactis KX881768 Lactobacillus plantarum KX881772 Lactococcus garvieae KX881774 Lactobacillus reuteri KX881777 Lactobacillus plantarum KX881779 Lactococcus lactis KX881782 DSM2648

0.08±0.003e 0.15±0.007c 0.15±0.003c 0.13±0.004d 0.45±0.003a 0.20±0.004b 0.16±0.001

SC

Lactococcus lactis KX881768 Lactobacillus plantarum KX881772 Lactococcus garvieae KX881774 Lactobacillus reuteri KX881777 Lactobacillus plantarum KX881779 Lactococcus lactis KX881782 DSM26482

RI PT

0

EP

Lactococcus lactis KX881768 4.2±0.00c Lactobacillus plantarum KX881772 4.6±0.05b Lactococcus garvieae KX881774 4.4±0.03bc Lactobacillus reuteri KX881777 5.1±0.01a Lactobacillus plantarum KX881779 4.9±0.08a Lactococcus lactis KX881782 4.4±0.08bc DSM2648 4.5±0.03 1 Values are mean ± standard error of triplicates 2 DSM2648 is indicative probiotic and not part of means differences a-f Means in same column at each parameter with different lowercase letters differed significantly (p < 0.05)

AC C

582 583 584 585

Storage at 4°C (days) 7 14 Log10 CFU/mL ab 9.2±0.08 8.5±0.12b 9.4±0.02a 9.1±0.04a 9.0±0.07ab 8.8±0.04ab ab 9.1±0.05 9.0±0.06a 9.3±0.04ab 8.9±0.06ab 8.3±0.14c 7.3±0.17c 8.7±0.23 8.6±0.03 OPA at 340 nm b 0.12±0.004 0.11±0.004d 0.17±0.005b 0.14±0.016cd 0.17±0.009b 0.20±0.003bc 0.16±0.018b 0.15±0.026cd a 0.40±0.078 0.56±0.010a 0.25±0.006b 0.25±0.007b 0.19±0.009 0.20±0.005 pH values d 4.3±0.01 4.3±0.01e c 4.7±0.05 4.9±0.01c 4.6±0.02c 4.6±0.01d 5.1±0.02b 5.1±0.01b 5.5±0.09a 5.8±0.01a d 4.2±0.04 4.0±0.00f 4.5±0.02 4.4±0.29

M AN U

Bacteria

TE D

581

25

21 9.0±0.06bc 9.3±0.03a 8.9±0.01c 9.0±0.02bc 9.1±0.03ab 7.4±0.06d 8.0±0.11 0.10±0.002d 0.15±0.005c 0.17±0.005c 0.16±0.005c 0.51±0.013a 0.23±0.004b 0.18±0.011 4.3±0.01e 5.0±0.01c 4.6±0.01d 5.1±0.00b 5.9±0.03a 4.1±0.00f 4.2±0.05

ACCEPTED MANUSCRIPT

Figure Captions

3

Figure 1: Cholesterol removal (%) of LAB after 24 h of incubation at 37°C (values are mean ± standard error)

4

Figure 2: Neighbor-joining phylogenetic tree based on 16S rDNA sequences. Numbers in parentheses are accession numbers of identified sequences.

5

Filled circles are the reference strains from NCBI.

RI PT

1 2

SC

6

M AN U

7 8 9

13 14 15 16 17 18

EP

12

AC C

11

TE D

10

ACCEPTED MANUSCRIPT

19 20

RI PT SC

50.0 40.0

M AN U

Cholesterol Removal %

60.0

30.0 20.0

21 22 23 24 25

Figure 1.

AC C

EP

0.0

TE D

10.0

ACCEPTED MANUSCRIPT

26 27

29 30 31 32

Figure 2.

AC C

EP

TE D

M AN U

SC

RI PT

28

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Highlights • Isolated LABs are excellent candidates to produce functional foods • Probiotic characteristics of isolated LAB from camel are remarkable • Isolates survived well under low pH 2.0 and heat at 60°C • Isolates exhibited outstanding cholesterol removal and BSH activity • Isolates were non-haemolytic and sensitive to several antibiotics