M. Aspri, D. Tsaltas, P. Papademas. Department of Agricultural Sciences, Biotechnology & Food Science

Transcription

1 M. Aspri, D. Tsaltas, P. Papademas Department of Agricultural Sciences, Biotechnology & Food Science

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

3 Milk Stable part of our diet Critical role in nutrition and health 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.

4 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

5 Benefits for human health and special therapeutic properties: Unique nutrient profile Close to human milk - 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 factoralpha (Mao et al., 2009; Tafaro et al., 2007). - Prevents cardiovascular and autoimmune disorders (Vincenzetti et al., 2007)

6 12,9% 2,3% 1,3% 0,3% Equine-donkey milk million L are produced per annum in Europe but in countries such as Mongolia is higher, probably ~ 9 million L. Cow milk Buffalo milk Goat milk Sheep milk Camel milk Large-scale operations in Italy, France, Belgium, Spain, Germany, Austria, Netherlands, China, Ethiopia and Pakistan. 83,1% Donkey Population Worldwide Year

7 Donkey Milk Human Milk Bovine Milk Total Solids (g/100g) Fat(g/100g) Lactose(g/100g) Ash (g/100g) Protein (g/100g) Casein (g/100g) Whey Proteins (g/100g) ph Nutritional composition of different milks (Guo et al., 2007, Malacarne et al., 2002)

8 Florou-Paneri et al.,2013

9 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 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

10 Collect samples Analyze and isolate LAB Characterization of the strains Design and study of potential of isolated LAB to produce a functional donkey milk Select promising strains

11 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 Molecular Identification 16s rrna Gram staining, physiological and biochemical tests Technological properties (acidification, lipolysis, proteolysis)

12 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)

13 Bacteriocin Production LAB isolates tested for antimicrobial activity using spot and well assays Bacteriocin characterization- Effect of enzymes, ph, temperature and solvent on bacteriocin activity PCR amplification of known bacteriocin genes 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

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15 Parameters Sample 1 13/03/201 3 Sample 3/4/201 3 Sample 3 17/04/201 3 Sample 4 13/05/201 3 Sample 5 06/06/201 3 Sample 6 27/06/201 3 Sample 7 22/07/201 3 Sample 8 27/08/201 3 Sample 9 17/09/201 3 Sample 10 02/10/201 3 Sample 11 14/10/201 3 Enterobacteriacae (ISO :2004) Total Microbial Count (ISO 4833:2003) S. aureus (ISO :1999) Yeast and Moulds (ISO 6611:2004) LAB (ISO 15214:1198) Listeria monocytogenes (ISO :1198) Salmonella spp. (ISO 6579:2002) 1.2*10 3 9* *10 3 <10 <10 <10 <10 TNTC 4.5*10 3 1* * * * * * * * *10 3 TNTC TNTC 8.5* *10 4 <10 9*10 2 9* * * *10 3 5*10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 7.4* *10 3 <10 <10 <10 6*10 2 3*10 3 <10 1.5* * *10 3 TNTC 4.7* * *10 3 Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent ph 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%

16 257 strains belong to LAB isolated from 11 samples of donkey milk according to their Gram staining (positive) and catalase (negative) profile All strains showed good growth at 15, 30 and 37 and 45ºC Growth at different ph(3-6) and salt (2-6.5%) concentration Enterococcus duran/hirae/ thailanticus/ lactis/gallina rum/villorum

17 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

18 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

19 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) OD620nm Growth of L. monocytogenes in the presence of Bacteriocin of sample 33 Bacteriocin of sample 270 Bacteriocin of sample 224 without the presence of bacteriocin (L. monocytogenes 33423) Time (Hours)

20 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)

21 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

22 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

23 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

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