M. Aspri, D. Tsaltas, P. Papademas

Department of Agricultural Sciences, Biotechnology & Food Science

Introduction-Background Objectives Donkey Milk Methodology Results Conclusions and Future Prospects

Milks from non-traditional animal species are gaining interest

Why donkey milk? ◦ Unique composition, nutritional and bio-functional properties ◦ Could be used as a valuable alternative, especially for sensitive population groups

i.e. infants, the elderly and also for people that suffer from allergic symptoms of bovine milk consumption.

◦ Increased scientific interest: Since 2005, the number of published research papers regarding donkey milk has increased to almost 30 per year. Most of the papers cover the potential of donkey milk use as a substitute of cow's milk for allergic individuals.

◦ Information regarding donkey microbial biodiversity is limited.

Stable part of our diet

Critical role in nutrition and health Milk

General: Provide information about donkey milk produced in Cyprus that can be potentially used for the isolation of beneficial microbial strains and the production of a functional fermented milk Specific aims: - Sample collection during spring and summer period in

order to determine the predominant microbial groups - Isolation and characterization of lactic acid population of

donkey milk - Examine the antimicrobial activity and bacteriocin

production of LAB isolates - Safety evaluation and screening of LAB isolates for

probiotic characteristics and technological properties

Unique nutrient profile Close to human milk

Benefits for human health and special therapeutic properties: - As a breast milk substitute or for children that

suffer from cow milk protein allergy (CMPA) (Polidori et al., 2013; Allesandri et al., 2007; Monti et al., 2007)

- Treat pertussis (whooping cough) (Westermarck,

2013)

- Treatment of cancer patients because its ability to induce release of interleukins (IL) (IL-12, IL-1 beta and IL-10) and tumour necrosis factor-alpha (Mao et al., 2009; Tafaro et al., 2007).

- Prevents cardiovascular and autoimmune

disorders (Vincenzetti et al., 2007)

83,1%

12,9%

2,3% 1,3% 0,3%

Cow milk

Buffalo milk

Goat milk

Sheep milk

Camel milk

Equine-donkey milk ̴1-1.3 million L are produced per annum in Europe but in countries such as Mongolia is higher, probably ~ 9 million L.

Large-scale operations in Italy,

France, Belgium, Spain, Germany, Austria, Netherlands, China, Ethiopia and Pakistan.

41000000

41500000

42000000

42500000

43000000

43500000

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Donk

ey P

opul

atio

n W

orld

wid

e

Year

Donkey Milk Human Milk Bovine Milk

Total Solids (g/100g) 8.8-11.7 11.7-12.9 12.5-13.0

Fat(g/100g) 0.3-1.8 3.5-4.0 3.5-3.9

Lactose(g/100g) 5.8-7.4 6.3-7.0 4.4-4.9

Ash (g/100g) 0.3-0.5 0.2-0.3 0.7-0.8

Protein (g/100g) 1.5-1.8 0.9-1.7 3.1-3.8

Casein (g/100g) 0.64-1.03 0.32-0.42 2.46-2.80

Whey Proteins (g/100g) 0.49-0.80 0.68-0.83 0.55-0.70

pH 7.0-7.2 7.0-7.5 6.6-6.8

Nutritional composition of different milks (Guo et al., 2007, Malacarne et al., 2002)

Florou-Paneri et al.,2013

Chemical composition and nutritional properties of donkey’s milk have been widely investigated

Information regarding the microbiota, especially LAB of this milk is

limited: ◦ Most studies on donkey’s milk microbiota are focused on the detection

of pathogenic bacteria (Salimei et al., 2004; Pilla et al., 2010; Conte et al. 2012; Sarno et al., 2012; Cavallarin et al., 2014)

◦ LAB content of donkey milk range between 1.0-4.2 log cfu/ml, but only

few LAB isolates have been identified (Coppola et al., 2002; Chiavari et al., 2005; Zhang et al., 2008; Saric et al., 2012; Carminati et al., 2014)

◦ According to Conti et al., (2010) fresh donkey milk is a natural source

of probiotic bacteria that are capable to colonize the colon, acting against outcome pathogen bacteria and stimulating the immune system

Collect samples

√

Analyze and isolate LAB

√

Characterization of the strains

√

Select promising strains

Design and study of potential of isolated

LAB to produce a functional donkey milk

Isolation and characterization of LAB present in donkey milk

Sample Collection

Strains isolated on MRS, MRS pH 5.7 and M17 Agars, purification of colonies

Gram staining, physiological and biochemical tests

Molecular Identification 16s rRNA

Technological properties (acidification, lipolysis, proteolysis)

Evaluation of safety and probiotic potential

Virulence activity using specific primers

And physiological tests (DNase, Gelatinase, Lipase and Haemolytic activity)

Antibiotic Resistance (Disc Diffusion assay)

Biogenic Amine Production

pH and bile salt tolerance

Adhesion assay

Antimicrobial activity (bacteriocin production)

Bacteriocin Production Bacteriocin

characterization- Effect of enzymes, pH, temperature and solvent on bacteriocin activity

LAB isolates tested for antimicrobial activity using spot and well assays

Bacteriocin purification using RP- HPLC, Determination of molecular mass using MALDI TOF-MS

Antimicrobial activity of crude bacteriocin using well assays and inhibition of growth of foodborne pathogens using crude bacteriocin

PCR amplification of known bacteriocin genes

Parameters Sample 1 13/03/2013

Sample 3/4/2013

Sample 3 17/04/2013

Sample 4 13/05/2013

Sample 5 06/06/2013

Sample 6 27/06/2013

Sample 7 22/07/2013

Sample 8 27/08/2013

Sample 9 17/09/2013

Sample 10 02/10/2013

Sample 11 14/10/2013

Enterobacteriacae (ISO 21528-2:2004)

1.2*103 9*102 1.7*103 <10 <10 <10 <10 TNTC 4.5*103 1*103 1.3*103

Total Microbial Count (ISO 4833:2003)

3.4*103 3.1*103 4.5*103 3.6*103 1.6*104 1.1*104 5.6*103 TNTC TNTC 8.5*103 4.6*104

S. aureus (ISO 6888-1:1999)

<10 9*102 9*102 1.1*103 3.6*103 3.5*103 5*10 <10 <10 <10 <10

Yeast and Moulds (ISO 6611:2004)

<10 <10 <10 <10 <10 <10 <10 7.4*103 7.2*103 <10 <10

LAB (ISO 15214:1198)

<10 6*102 3*103 <10 1.5*103 1.0*103 2.3*103 TNTC 4.7*103 3.7*103 2.9*103

Listeria monocytogenes (ISO 11290-1:1198)

Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent

Salmonella spp. (ISO 6579:2002)

Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent

pH 7.32 7.39 7.41 7.6 7.35 7.22 7.03 7.17 7.39 7.23 7.28 Titratable Acidity 0.05% 0.09% 0.07% 0.08% 0.15% 0.14% 0.05% 0.05% 0.07% 0.09% 0.07%

Antibiotics - (Neg) - (Neg) -Neg -Neg -Neg -Neg -Neg - Neg - Neg -Neg -Neg Fat(Gerber) 0.15% 0.4% 0.5% 0.9% 0.6% 0.5% 0.5% 1.2% 0.6% 0.6% 0.8%

All strains showed good

growth at 15, 30 and 37 and 45ºC

Growth at different pH(3-6) and salt (2-6.5%) concentration

257 strains belong to LAB isolated from 11 samples of donkey milk according to their Gram staining (positive) and catalase (negative) profile

Enterococcus duran/hirae/ thailanticus/ lactis/gallinarum/villorum

• 79 isolates acidified RSM (pH<5) in 24 hours and showed proteolytic activity on agar medium composed of 10% RSM and 2% agar

• None of the strains showed any lipolytic activity

Tolerance to low pH- all of the 79 isolates were able to survive at pH 3.0 during the three hours of incubation

Tolerance against bile salt- able to survive at bile

concentration 0.3% during 4 hours of incubation Antimicrobial activity –Bacteriocin production ◦ 15 isolates show antimicrobial activity against indicator

strains

After Molecular ID 3 strains were tested for bacteriocin production ◦ Enterococcus faecium ◦ Streptococcus gallolyticus ◦ Enterococcus villorum

Stable over a wide range of pH (2-8), temperature (30◦C-100◦C, autoclave temperature-121◦C), sensitive to protein enzymes ( a-chymotrypsin, trypsin and pronase)

Antimicrobial activity: very active against L. monocytogenes (bactericidal). E. villorum show antimicrobial activity also against S. aureus, B. cereus and some other LAB

PCR amplification with known enterocins shows that produce three different types of bacteriocins (A, B and P)

Growth of L. monocytogenes 33423 inthe presence of

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 240.0

0.5

1.0

1.5Bacteriocin of sample 33Bacteriocin of sample 270Bacteriocin of sample 224without the presence ofbacteriocin (L.monocytogenes 33423)

Time (Hours)

OD

620n

m

None of the strains showed any Dnase, gelatinase and lipase production 2 of the 79 strains showed β-haemolysis and

1 strain α-haemolysis Antibiotic susceptibility profile

Preliminary study: Bacteriocin producing strains were susceptible to vancomycin (5μg/ml) and ampicilin (2μg/ml) (EUCAST,2015)

Low total microbial flora: healthy base

ingredient for feeding in case of low immune defense system, elderly, children with CMPA and when breast-feeding is not possible

Serve as a medium for the isolation of technologically important, bacteriogenic and probiotic LAB

Vehicle for delivering probiotic bacteria

Need to complete the safety-status analysis of the enterococci population in order to show non pathogenic characteristics

Application of these potential bacteria for the production of a functional fermented donkey milk product

Asst Prof Photis Papademas, CUT, Cyprus Asst Prof Dimitris Tsaltas, CUT, Cyprus Prof Colin Hill, UCC, Ireland Dr Des Field, UCC, Ireland

Golden Donkeys Farm for financially

supporting our research