1. KEFIR MICROBIOLOGY AND NUTRACEUTICAL BIOTECHNOLOGY (OVERVIEW)
Dr. Cheba Ben Amar
Associate Professor of Biotechnology (MCA)
Dept .of Biotechnology, Faculty of Nature and Life Sciences, (USTOMB), Oran, Algeria

2. KEFIR :MICROBIOLOGIE ET BIOTECHNOLOGIE NUTRACEUTIQUE (VUE GLOBALE)
Dr. Cheba Ben Amar
Maitre de Conferences (MCA) on Biotechnologie
Dept .de Biotechnologie, Faculté SNV -(USTOMB), Oran, Algerie

3. الكفير : الميكروبيولوجيا والبيوتكنولوجيا التغذودوائية (نظرةعامة)
مقدمة من قبل
د . شبة بن عمر
أستاذ محاضر أ في البيوتكنولوجيا
كلية علوم الطبيعة والحياة -جامعة العلوم والتكنولوجيا محمد بوضياف

5. Les 20ème Journées Nationales de Microbiologie
ACKNOWLEDGEMENTS
Les 20ème Journées Nationales de Microbiologie

6. Nutraceutical biotechnology
PRESENTATION PLAN
kefir
Generalities
Microbiology
Nutraceutical biotechnology

7. kefir ????

8. Kefir definition
Kefir is viscous ,slightly sour, carbonated , and sparkling dairy beverage that contains small quantities of alcohol, and posses high nutritional and functional properties produced by the action of the symbiotic association of bacteria and yeasts in kefir grains

9. “what means the word kefir ?

10. kefir Etymology
Kefir (pronounced kuh-FEER) comes from the turkish word ‘keif ’ which can be translated as “good feeling” (joy/pleasure) after its ingestion يتكيَف ان الرثيئة تطفئ الغضب

11. Alternative names
alternative names : kefīrs, keefir, kephir, kewra, talai, mudu kekiya, milkkefir,bulgaros), purportedly from either the Turkish "kopur" ((milk) froth, foam), Commercial names: kephir, kiaphur, kefer, knapon, kepi and kippi (Koroleva 1988a)

12. Kefir history
Kefir is originated by - North Caucasian shepherds - produced in mountain villages of North Caucasus for thousands of years

13. Satellite image of the Caucasus, from NASA’s Visible Earth

14. Kefir history
spread from the North Caucasus mountains to Northeastern steps of Mongolia and Tibet countries
- It spread through the centuries in the Anatolian Region, the Balkans and Eastern Europe

15. Kefir diffusion kies -Ottoman Empire
- Irina Sakharova Moscow (Russia )
-Mike Smolyansky Russian immigrant - Lifeway Foods manufacture 1986 (USA)

16. KEFIR CONSUMPTION COUNTRIES Ref. Caucasus
Mountains in the former Soviet Union
Hungary
and Poland
(Komai and Nanno, 1992).
Sweden, Norway, Finland and
Germany
(Kroger, 1993)
Greece, Austria, and Brazil
(Hallé et al., 1994)
Chile, Latin America NM
United States and Japan
Worldwide

17. KEFIR PREPARATION

18. MAMMALIAN MILKS
Animal milks
cow
Buffalos
yaks
ewes
Goat
Camel
Deer

19. MILK SUBSTITUTES
Plant milks
soy milk
coconut milk
rice milk

20. sugar and molasses solutions
SUGARY LIQUIDS
sugary liquids
fruit juice
sugar and molasses solutions
coconut water
beer wort
ginger beer

22. kefir-related products
Omaere
South-West Africa
"yogurt de pajaritos"
Chile,
Rob or Roba
some Arab countries
Zabady
Egypt
KjaKlder MjoKlk
Norway
Osobyi
modified Kefir in Russia
Kellermilch
Germany
tibicos
Water kefir
Tarag
Mongolia
khymus
Kefir
Turkey
kombucha
china

24. Kefir Grains
These grains are composed of microorganisms immobilized on polysaccharide - protein matrix, where several species of bacteria and yeast coexist in symbiotic association (Farnworth and Mainville, 2008; Garrote et al., 2010)
SCOBY [Symbiotic Culture Of Bacteria and Yeast]

26. Kefir Grains
Kefir grains small cauliflower-florets shaped, characterized by an irregular, multi-lobular surface, united by a single central section
their color varies from white to yellowish white,
vary in size, from rice grain to walnut
The grains are elastic and have a viscous and firm texture (Farnworth and Mainville, 2008; Magalhães et al., 2011; Rea et al., 1996).

27. Kefir Grains size vary from 0.3 to 3.0 cm in diameter

28. (from rice grain to walnut)

29. Kefir Grains viscosity

30. KEFIRAN
Kefiran produced by L. kefiranofaciens is a branched, water-soluble polysaccharide, containing equal amounts of D-glucose and D-galactose. The production of this polysaccharide is stimulated when L. kefiranofaciens grows in co-culture with S. cerevisiae (Cheirsilp et al., 2003).

31. kefir microbial diversity

32. Somme Fermented Milks and their specific starter culture(s)
Acidophilus Milk:
Lactobacillus acidophilus.
Yoghurt
Symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
Alternate Culture Yoghurt:
Cultures of Streptococcus thermophilus and any Lactobacillus species.
Kumys:
Lactobacillus delbrueckii subsp. bulgaricus and Kluyveromyces marxianus
Kefir:
Starter culture prepared from kefir grains, Lactobacillus kefiri, species of the genera Leuconostoc, Lactococcus and Acetobacter growing in a strong specific relationship.
Kefir grains constitute both lactose fermenting yeasts (Kluyveromyces marxianus) and non-lactose-fermenting yeasts (Saccharomyces unisporus,Saccharomyces cerevisiae and Saccharomyces exiguus).

33. Effecting factors Effecting factors degree of agitation
fermentation time
temperature
degree of agitation
type of milk
grain/milk inoculum ratio
microorganism distribution

34. kefir microbial composition
The FAO/WHO (2001) have proposed a definition of kefir based on the microbial composition of both kefir
grains (the starter culture used to produce kefir) and the final kefir product
The FAO/WHO (2001) have proposed a definition of kefir based on the microbial composition of both kefir
grains (the starter culture used to produce kefir) and the final kefir product
The FAO/WHO (2001) have proposed a definition of kefir based on the microbial composition of both kefir
grains (the starter culture used to produce kefir) and the final kefir product
kefir microbial composition
The FAO/WHO (2001) have proposed a definition of kefir based on the microbial composition of both
- kefir grains
-Final kefir product

35. Units Count of Microbes in Gram Stained Kefir Grains Bacilli [single cells, pair, chains] Streptococci [pair, chains] Yeast [single cells] The Means Range Bacilli 66, 62-69% Yeast 18, % Streptococci 16, %

36. KEFIR BACTERIA Lactobacilli Lactobacillus kefir
Lactobacillus kefiranofaciens
Lactobacillus kefirgranum
Lactobacillus parakefir
Lactobacillus delbrueckii
Lactobacillus rhamnosus
Lactobacillus casei
Lactobacilli paracasei
Lactobacillus brevis
Lactobacillus fructivorans
Lactobacillus plantarum
Lactobacillus hilgardii
Lactobacillus helveticus
Lactobacillus fermentum
Lactobacillus acidophilus
Lactobacillus viridescens
Lactococci
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. cremoris
Streptococci
Streptococcus thermophilus
Enterococci
Enterococcus durans
Streptococcus durans
Leuconostocs
Leuconostoc sp.
Leuconostoc mesenteroides
(reported as Leuconostoc kefir
Pediococcus damnosus
Acetic acid bacteria
Acetobacter lovaniensis
Acetobacter pasteurianus
Acetobacter aceti,
A fabarum ,A. syzygii ,

37. KEFIR YEASTS Kluyveromyces marxianus
(reported as Saccharomyces lactis or
Kluyveromyces lactis
Saccharomyces sp.
Saccharomyces cerevisiae
Saccharomyces carlbergensis
Saccharomyces unisporus
Saccharomyces exiguus
(reported as Torolopsis holmii
Saccharomyces turicensis
Saccharomyces delbrueckii
Saccharomyces dairensis
Torulaspora delbrueckii
Brettanomyces anomalus
Issatchenkia occidentalis
Kazachstania exigua
Yarrowia lipolytica
Candida friedrichii
Candida pseudotropicalis
Candida tenuis
Candida inconspicua
Candida maris
Candida lambica
Candida tannotelerans
Candida valida
Candida kefyr
Candida holmii
Pichia fermentans
Zygosaccharomyces sp. Wallemia,Eurotium,Microdochium
Cryp-tococcus,Teratosphaeria
,Debaromyces,Cyberlindnera
,Malassezia,Hetero-basidion
Neofabraea,Peziza ,Ganoderma
,Mycena , Naumovozyma,and Dioszegia, Lachancea meyersii,
Zygotorulaspora florentina
Hanseniaospora valbyensis
Dekkera anomalap, Hansenula yalbensis
Kloeckera apiculata

38. Contaminant microflora
Dipodascus capitus and Trichosporon coremiiforme, considered pathogens
Geotrichum candida, Penicillium and Aureobasidium
Bifidobacterium spp ,Solirubrobacter,,Pediococcus, Weissella, Gluconobacter japonicus,
Pseudomonas putida
Escherichia coli, Shewanella,,Pelomonas,
Acinetobacter, Halococcus spp. Micrococcus sp.,
Bacillus subtilis ,Bacillus sp,Clostridium

39. Kefir Mineral Composition
Mineral content (g)
content
Trace elements
Calcium
0.12
Iron (mg)
0.05
Phosphore
0.10
Zinc (mg)
0.36
Magnesium 12
Copper (μg)
Potassium
0.15
Manganese (μg) 5
Sodium
Molybdenum (μg)
5.5
Chloride

40. kefir chemical composition and nutritional values
Components
100 g
Energy
65 kcal
Tryptophan
0.05
Vitamins (mg) A
0.06
Fat (%)
3.5
Phenylalanin+tyrosine
0.35
Carotene
0.02
Protein (%)
3.3
Leucine
0.34 B1
0.04
Lactose (%) 4
Isoleucine
0.21 B2
0.017
Water (%)
87.5
Threonine
0.17 B6
Milk acid (g)
0.8
Methionine+cystine
0.12
B12
0.5
Ethyl alcohol (g)
0.9
Lysine
0.27
Niacin
0.09
Lactic acid (g) 1
Valine
0.22 C
Cholesterol (mg) 13
Aromatic compounds D
0.08
Phosphatateds (mg) 40
Acetaldehyde ND E
0.11
Essential amino acids (g)
Diacetyl
Acetoin
acide linoléique conjugué (CLA)

41. Regularly kefir consumption can be help to
easily digestion
provides beneficial bacteria and yeast (probiotics)
vitamins and minerals and complete proteins and are nourishing food that contribute in the healthy immune system
Historically, kefir has been recommended for the treatment of several clinical conditions such as gastrointestinal problems, hypertension, allergies, and ischemic heart disease(CVD) tuberculosis, AIDS, chronic fatigue syndrome, herpes and cancer

42. Impact on the Gastrointestinal Tract (GIT)
relieve all intestinal disorders,
promote bowel movement,
reduce flatulence and create a healthier digestive system.
It cleans effectively the whole body that helps to establish a balanced inner ecosystem for optimum health and longevity

43. Impact on the Gastrointestinal Tract (GIT)
-direct pathogen inhibition by acids and bacteriocin production, besides competitive pathogen exclusion in the intestinal mucosa (Rattray and O'Connel, 2011).
- reduced enterobacteria and Clostridia populations. Furthermore, kefir consumption also
- prevented C. jejuni colonization in chick ceca (Zacconi et al., 2003),
-treatment of peptic ulcers in the stomach and duodenum of human patients (Farnworth and Mainville, 2008).
-oral treatment with a mixture of kefir-isolated bacteria and yeasts was able to prevent diarrhoea and enterocolitis triggered by C. difficile.-According to Marquina et al. (2002) kefir consumption significantly increased LAB counts in the intestinal mucosa
-Kefir fermented milk and kefiran promote growth of Bifidobacterium bifidum PRL2010 and modulate its gene expression

44. Kefir and Lactose Intolerance
-due to the presence of β-galactosidase activity in grains and fermented milk products and make them suitable for consumption by people classified as lactose intolerant
(Farnworth and Mainville, 2008; Sarkar, 2007).(Hertzler and Clancy, 2003).

45. Anti-diabetic Effect Kefir reduces the level of glucose in the blood and maintains the normal blood sugar level.

46. Hypocholesterolemic Effect
the hypocholesterolemic activity in LAB may
involve the inhibition of the exogenous cholesterol absorption in the small intestine, by the
-binding and incorporation of cholesterol to bacterial cells and cholesterol uptake,
-as well as suppression of bile acid reabsorption by enzymatic deconjugation of bile salts, promoted by the bile salt hydrolase (BSH) enzyme (Wang et al., 2009).

47. Hypocholesterolemic Effect
- Wang et al. (2009) observed a significant reduction in serum levels of total cholesterol, low density lipoproteins (LDL) and triglycerides, while there was no change in high density lipoprotein (HDL-C) levels in mice fed cholesterol-rich a diet supplemented with Lactobacillus plantarum MA2.
-The soyamilk-kefir diet also elicited a significant decrease in the serum ratio of non-HDL-cholesterol to HDL-cholesterol, compared with the control,

48. Anti-inflammatory and Healing Activity
-treating a number of disorders like arthritis, gout, rheumatism,periodontitis,pancreatitis, gastritis, irritable bowel syndrome (IBS) and ulcers
Healing and anti-inflammatory activities of kefir and kefiran in mice were observed after a seven-day treatment with kefir gel (Rodrigues et al., 2005), as well as antinflammatory activitiy (Diniz et al., 2003) on a model in which granulomatous tissue and writhing is induced by acetic acid in mice.
-Husseini et al. (2012) corroborated the healing activity in burns infected with Pseudomonas aeruginosa in mice.

49. ANTIOXIDANT / ANTI-AGEING EFFECT
Guven and Gulmez (2003) reported that mice treated with kefir had a higher protective effect against damage induced by carbon tetrachloride, indicating that kefir can also act as an antioxidant
Kefir is rich in antioxidants which help the aging process to slow down by neutralizing the free radicals by oxidizing them and reducing the impact of the damage
Kefir administration reduced progression of renal injury in STZ-diabetic rats by lowering oxidative stress

50. Immunomodulatory effect
The formation of bioactive peptides during fermentation or digestion processes has shown a variety of physiological activities,
- including stimulation of the immune system in animal models (Farnworth, 2005).
-Thoreux and Schmucker (2001), after feeding mice with kefir, observed an increase in the specific mucosal immune response (IgA) against the cholera toxin.
-Stimulation of the immune system may also occur due to the action of exopolysaccharides found in kefir grains (Farnworth, 2005; Furukawa et al., 1992).
- Medrano et al. (2011) observed that kefiran was able to modify the balance of the immune cells in the intestinal mucosa-
- The administration of kefir also induced a response in the intestinal mucosa, suggesting that components of kefir can stimulate cells of the innate immune system,
-oral feeding of heat-inactivated Lactobacillus (L b.) kefiranofaciens M1 from kefir grains effectively inhibited immunoglobulin (Ig) E production in response to ovalbumin (OVA) in vivo).
- Recently, Hong et al. (2009) demonstrated, in vitro, the immunomodulating capacity of LAB isolated from kefir grains, suggesting their influence on the secretion of the proinflammatory cytokines IL-6 and TNF-α by TLR-2.

51. antimicrobial activity
Salmonella Typhimurium,S. enteritidis,S.kedougou,Shigella flexneri ,S. sonnei,EHEC,Y. enterocolitica,Pseudomonas aeruginosa
Listeria monocytogenes ,Bacillus cereus,B. subtilis, Enterococcus faecalis ,Staphylococcus aureus
Candida albicans ,candidiasis,psoriasis,
(yeast infection) and eczema
HIV / AIDS,kefir antimicrobial activity is associated with the production of organic acids, peptides (bacteriocins), carbon dioxide, hydrogen peroxide, ethanol and diacetyl

52. Antitumor /anticarcinogenic Activity
The anticarcinogenic role of fermented dairy products can be attributed, in general, to cancer prevention and the suppression of early-stage tumors,
-by the delaying of enzyme activities that convert pro-carcinogenic compounds to carcinogens,
-or by immune system activation(Sarkar, 2007).
-Kubo et al. (1992) reported the kefir inhibition of the proliferation of subcutaneously transplanted Ehrlich ascites tumors in mice.
-Liu et al. (2002) observed the inhibition of tumor growth, the induction of apoptotic cell lysis in tumors and significant increases in IgA levels in mice, suggesting that kefir potentially has anti-tumoral properties and promotes resistance of the mucosa to intestinal infections..
-.prevent certain type of cancers like colon
cancer, breast cancer etc. and reduce the size of tumors

53. exopolysaccharides (kefiran)
Bioactive Compounds (Garrote et al., 2010; Rattray and O'Connel, 2011).
bioactive compounds
organic acids
CO2
H2O2
Ethanol
bioactive peptides
exopolysaccharides (kefiran)
Bacteriocins

54. Conclusion
kefir is an amazing dairy beverage with unique nutraceutical benefits which need more investigations for full exploitation

55. SOMME REFERENCES
Dobson A, O'Sullivan O, Cotter PD, Ross P, Hill C (2011) High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. FEMS Microbiol Lett 320:56-62.
Guzel-Seydim, Z. B., Kok-Tas, T., Greene, A. K. and Seydim, A. C Review: Functional properties of kefir. Critical Reviews in Food Science and Nutrition 51:
  Farnworth ER (2005) Kefir -a complex probiotic. Food Sci Technol Bull: Functional Foods 2:1-17.         [ Links ]
1Sarkar, S Potential of kefir as a dietetic beverage – a review. British Food Journal 109(4):
2Sarkar, S Biotechnological innovations in kefir production: A review. British Food Journal 110(3):
Angulo L., Lopez E. & Lema C. (1993). Microflora present in kefir grains of the Galician region (North-West of Spain). J. Dairy Res. 60,
Farnworth ER, Mainville I (2008) Kefir -A Fermented Milk Product. In: Farnworth, E. R. (2th ed.), Handbook of Fermented Functional Foods (2 ed). CRC Press Taylor & Francis Group, Boca Raton, London, New York, p        

56. Thank you for your kind interest