Probiotics: what is Africa doing? Ivan Muzira Mukisa, PhD
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Probiotics: what is Africa doing? Ivan Muzira Mukisa, PhD RUFORUM Seminar Presentation at Sokoine University of Agriculture Wednesday 11 Th May 2016 1. Brief biography 2. The gut microflora 3. The concept of probiotics – Definition – Guidelines 4. The probiotics market 5. Diversity of probiotics 6. Health benefits of probiotics Outline of Presentation 7. Mechanisms of action 8. African based studies on probiotics 9. Challenges and opportunities for Africa 10. L. rhamnosus Yoba – Origins – Use in products – Yoba in Uganda • Born 28 November 1977 • B.Sc Food Sci & Tech, MAK 2003 • M.Sc Food Tech, Katholiek University of Leuven and University of Ghent (Belgium), 2007 • PhD, Microbiology, Norwegian University of Life Sciences (Norway), 2012 • Taught at Makerere from 2002 to date • Technologist in the Fish Industry, Uganda, 2003 Brief Biography Areas of interest • Fermentation • Food safety and quality • Functional foods – Antioxidant enriched products – Probiotics Brief Biography The gut microflora NB: Only major groups shown here and mainly faecal flora. Up to 1,000 species reported! Sterile GIT Breastfed infants dominated by bifidobacteria While formula fed have complex adult flora Flora stabilizes at abt 2 years and is essential for optimal gut functioning
Less bifidobacteria More anaerobic and putrefactive flora Gut malfunction
The gut microflora O’Hara and Shanaha. 2006. The gut flora as a forgotten organ. EMB, Vol 7 (7), 688 – 693; Mizutani,(1992) • Changing lifestyles make it difficult to maintain a ‘balance’ – Stress
– Changing dietary patterns – Eating habits – Consumption of antibiotics • Possible shift of ‘balance’! – Reduction of potentially health‐promoting bacteria (e.g lactobacilli and bifidobacteria) – Increase in more harmful flora (e.g clostridia, sulphate‐reducers and proteolytic bacteroides) The gut microflora A cocktail of the good guys and the bad guys The gut microflora The good guys The bad guys Produce vitamins Produce toxins Ferment food Produce carcinogens: Bowel cancer, Modulate the immune system Constipation Enhance digestion and absorptionn Diarrhea
Inhibit harmful species Inflamatory bowel diseases Remove carcinogens (reduce enzymes e.g Β‐glucuronidase, azoreductase, nitroreductase) and toxins Putrefaction Produce short chain fatty acids (SCFAs) Increased susceptibility to transient enteropathogens e.g Salmonela,
Elie Metchnikoff • Earliest mention of the probiotic concept by Metchnikoff in 1908 – Complex microflora in the colon affected ‘autointoxication’ – Longevity of Bulgarian peasants due to consumption of large quantities of yoghurt – The live microbes were believed to promote good health – Origins of the Bacillus bulgaricus (L. delbrueckii subsp. bulgaricus), which together with Streptococcus salivarius subsp. thermophilus form the yoghurt starter
The concept of probiotics: origins ”Inflammation as understood in man and higher anmials is a penomenon that almost always results from the intervention of some pathogenic microbe”, E. Metchnikoff.
• Several have been proposed over time: – Substances secreted by one microorganism that stimulate the growth of another (Lilly and Stillwell, 1965) – Organisms and substances that influence intestinal microbial balance (Parker, 1974) – A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989) – A live microbial feed supplement that is beneficial to health (Salminen et al., 1998). • Currently acceptable definition: – Live microorganisms which when administered in adequate
(FAO/WHO, 2002) The concept of probiotics: Definition • Probiotic ingestion can be recommended as a preventative approach
– To help main the balance of the intestinal microflora thus enhancing ‘well‐being’. – Prevent or alleviate some conditions • Some studies have shown that effects can be due to: – dead bacteria i.e paraprobiotics (Taverniti and Guglielmetti, 2011) – bacterial products (postbiotics; Tsilingiri and Rescigno, 2013) • However, the definition still emphasizes live microrganisms The concept of probiotics: Definition Guidelines for Probiotics* • FAO/WHO in 2001: need for clear guidelines for systematic evaluation of probiotics and their related health claims • Functional foods
Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002
Strain identification Phenotypic and genotypic methods. Genus, species and strain Deposit strain in International Culture Collection
human trial or other appropriate design with sample size and primary outcome appropriate to determine if strain/product is efficacious
to
comfirm results Phase 3. E ffectiveness trial is appropriate to compare probiotics with standard treatment of a specific condition
Contents: genus, species, strain Minimum number of viable bacteria at end of the shelf life Proper storage conditions Health claims Corporate contact details for consumer information Probiotic Guidelines for evaluation of probiotics for food use. (FAO/WHO, 2002) Probiotic effects are generally strain specific. Save for example S.thermophillus and L.delbruecki ssp. Bulgaricus which enhance lactose digestion Main in vitro tests for study of probiotic strains • Resistance to gastric acidity • Bile acid resistance • Adherence to mucus and/or human epithelial cells and cell lines • Antimicrobial activity against potentially pathogenic bacteria • Ability to reduce pathogen adhesion to surfaces • Bile salt hydrolase activity • Resistance to spermicides (applicable to probiotics for vaginal use) * Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002
Safety considerations of probiotic strains • Lactobacilli and Bifidobacteria associated with food are historically considered to be safe. GRAS status • Probiotics may however, be theoretically responsible for four kinds of side effects: – Systemic infections: few reported in patients with underlying medical conditions – Deleterious metabolic activities – Excessive immune stimulation in susceptible individuals – Gene transfer * Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002
Safety considerations of probiotic strains • Determination of antibiotic resistance patterns • Assessment of certain metabolic activities (e.g D‐lactate production, bile salts deconjugation...) • Assessment of side effects during human studies • Testing for toxin production if strain belongs to a species that is a known mammalian toxin producer • Determining hemolytic actvity if strain belongs to a species with known hemolytic potential • Lack of infectivity in immunocompromized animals * Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002
The pyramid of evidence The concept of probiotics: evidence Meta‐analysis RCTs Randomised control trials Open studies in humans Animal studies In vitro studies Incr easing evidence Marteau, 2003 Health claims related probiotic strains • General health claims allowed in most countries for probiotic containg foods. For example, ’Improves gut health’ • Specific health claims, e.g ’reduces the incidence and severity of rotavirus diarrhea in infants’ can be used where sufficient scientific evidence exists, (FAO/WHO, 2002) • Manufacturer responsible for getting independent third party review by experts to establish if the claim is truthful and not misleading The concept of probiotics: claims Market • Major product categories The probiotics market Foods and beverages: dairy, fermented meat, bakery, breakfast cereals, beverages, meat, fish & eggs and soy products Dietary supplements: tablets, capsules, and powders Animal feed • Global probiotics market valued at USD 32.06 billion in 2013 (Grand View Research, 2016). • Estimated to be valued at USD 33.19 billion in 2015 • Projected to reach USD 46.55 billion by 2020. – For comparison: The global value of the Coca‐Cola brand rose from USD 41.41 billion in 2006 to USD 83.84 in 2015 • Industry growth likely due to: – Increasing global health awareness among consumers – Growth of global functional food industry – Technological advancements – Rising disposable incomes
• Food and beverage applications to increase by 50% in N. America
Projected growth in probiotics in North America probiotics market revenue, by application, 2012 – 2020 (USD Million). (Grand View Research, 2016). The probiotics market • As of 2014 more than 100 companies were involved in marketing probiotic products in the US! • Some of the key players in the international market 1. American Biologics, USA 2. Arla Foods, USA 3. BioGaia Biologics AB, Sweden 4. Chr. Hansen A/S, Denmark 5. ConAgra, USA 6. DuPont Danisco, Denmark 7. General Mills Inc., USA 8. Groupe Dannon, France 9. Institute Rosell, Canada 10. Nestle SA, Switzerland 11. Valio Ltd, Finland 12. Yakult Honsha Co. Ltd., Japan 13. BioGaia AB, Sweden – ...
• Limited information available on market share of probiotics in Africa
– Africa’s market share likely very small in global terms • South Africa has a relatively well established market: – Supplements (capsules) – Fortified food items (especially baby cereals) – Fermented dairy products • Likely need or interest partly based on traditional fermented foods – Fermented foods considered to have health benefits – Some believed to aid in the control of some diseases (intestinal disorders) The probiotics market Both bacteria and yeasts •
– breve – lactis Bb12 – longum BB536 •
•
•
subtilis •
boulardii •
– johnsonii LA1 – acidophilus LA5 – acidophilus NCFB 1748 – rhamnosus “GG” – casei “Shirota” – casei “Imunitass”® – casei Defensis – gasseri – reuteri – salivarius UCC118 – plantarum 299v
Mainly isolated from – Food
– Intestinal contents/stools of humans or animals – The human urinogenital tract – Oral cavity – Human milk Diversity of probiotics Probiotic vehicles • Several foods considered as vehicles for probiotic delivery: – Dairy products – Fermented or brined meat – Fermented vegetables, cereals – Fruit juice – Nutritional supplements (liquid, powder or tablets) • The products are considered as functional foods
Some probiotic products on the African market Product name ProbiFlora Adult Intensive Rescue Company
Danisco Market
South Africa Probiotic 9 strains Claimed
Benefits against
Lactose intolerance Product name ProbiFlora ™ Infant Company
Danisco Market
South Africa Probiotic Not specified Claimed
Benefits against
Colic Product name ProbiFlora ™ Adult Classic Bowel Support Company
Danisco Market
South Africa Probiotic 4 strains Claimed
Benefits against
Bloating Diversity of probiotics Some probiotic products on the African market Product name ProbiFlora ™ Junior Company
Danisco Market
South Africa Probiotic 3 strains Claimed
Benefits against
Lactose intolerance Low immunity Food allergies Product name ProbiFlora ™ Adult Colon Ease Company
Danisco Market
South Africa Probiotic Not specified Claimed
Benefits Enhances digestion And regularity Product name Forever Active Probiotic Company
Forever Living Products Market
Kenya Probiotic 6 strains (B. lactis, L.
Claimed
Benefits Healthy digestive system, enhanced nutrient absorption and immune function Diversity of probiotics Some probiotic products on the African market Product name BioPro Company
Wellington Pharmaceuticals, USA Market South Africa Probiotic L. reuteri Claimed
Benefits against
Colic Product name Combiforte Company
Combiforte BV, Netherlands Market South Africa Probiotic L. acidophilus B. bifidus, B. longum Claimed
Benefits against Antibiotic induced diarrhoea and other intestinal disorders Product name Culturelle Company
Culturelle, UK Market
South Africa Probiotic B. longum, L. acidophilus, L. rhamnosus GG S. thermophilus Claimed
Benefits against
Diarrhoea, bloating, Immune boosting Diversity of probiotics Some probiotic products on the African market Product name BioPro Company
Bioflora CC Market
South Africa Probiotic B. infantis Claimed
Benefits against
Diarrhoea, disturbances which are associated with antibiotic therapy, oral thrush, food allergies, lactose intolerance and nappy rash Product name Vagiforte Plus Company
Bioflora CC Market
South Africa Probiotic lactobacillus Bifidobacteria Claimed
Benefits against Vaginal thrush Product name Lacteol Fort Company
Rameda, Egypt Market
South Africa Probiotic L. fermentum L. delbrueckii Claimed
Benefits against
Bacterial or viral diarrhoea Diversity of probiotics Some probiotic products on the African market Product name Trilac Company
Allergon AB, Sweden Market
Kenya Probiotic L. acidophilus, L. bulgarius and B.animalis Claimed
Benefits against
Diarrhea, constipation Product name Seeking Health Company
Seeking Health, USA Market
Kenya Probiotic S. boulardii Claimed
Benefits against Diarrhea, gut inflamation Product name Kalsis Company
Catalysis Lab., Spain Market
Kenya Probiotic L. acidophilus Claimed
Benefits Fixing of calcium Diversity of probiotics Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Hypercholesterolemia B. lactis Bb12 B. Longum BL1, L. acidophilus L1, L. acidophilus La5 L. reuteri NCIMB 30242 Lower
total cholesterol and LDL cholesterol in probiotic vs. Lowers total cholesterol, LDLcholesterol, and non‐ HDLcholesterol Constipation
Higher defecation frequency, less hard stool. Decreased severity of constipation and improved stool consistency Sources: Taibi and Comelli (2014).
Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Infectious diarrhea (prevention)
Atopic eczema associated with cow’s milk allergy
B. lactis NCC2818 A L. rhamnosus GG Immune response B. lactis DN‐173 010 L. acidophilus LAFT1 L. rhamnosus GG Antibody response to vaccination
Increased antibody response to influenza vaccination in the probiotic group Sources: Taibi and Comelli (2014).
Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Respiratory infection L. casei DN‐114 001 L. rhamnosus GG L. plantarum DSM 15312 (HEAL 9) and L. paracasei DSM 13434 (8700:2)
Decreased duration of Infection Reduced risk of upper RTIs and duration of Symptom Reduced incidence and duration of common cold Infantile colic L. reuteri DSM 17938 Bacterial vaginosis L. rhamnosus GR‐1 L. fermentum RC‐14 Reduced colonization of vagina by potential pathogens Urinary tract infections
Reduction in recurrent UTIs in women Oral health Streptococcus salivarius K12 Reduction of malodour, prevention of pharyngeal infections Sources: Taibi and Comelli (2014).
Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Antibiotic‐associated diarrhea
O/C, NR, SIN, and T B. animalis Bb12 and Sreptococcus Thermophilus Enterococcus faecium LAB SF68
L. acidophilus CL1285 and L. casei LBC80 R L. casei DN‐114 001 L. rhamnosus E/N L. rhamnosus GG S. boulardii lyo Infectious diarrhea (treatment)
Reduced
duration Sources: Taibi and Comelli (2014). Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Clostridium difficile associated diarrhea
and L. casei LBC80 R L. casei DN‐114 001 L rhamnosus GG S. boulardii lyo Clostridium difficile with no diarrhea L. rhamnosus HN001 L. acidophilus NCFM Bacterial vaginosis and vulvovaginal candidiasis L. casei rhamnosus Lcr35 L. rhamnosus GR‐1 L. fermentum RC‐14 Enhanced restoration of the vaginal microbiota after antibiotic treatment of bacterial vaginosis Sources: Taibi and Comelli (2014). Several benefits associated with probiotics Health Benefits of probiotics Condition Probiotic Results Helicobacter pylori Bacillus clausii strains O/C, NR, SIN, and T 1b(t)
Irritable bowel syndromme
Sources: Taibi and Comelli (2014). Adequate probiotic intake? • Still controversial/varrying opinions • Can be expressed as cells per gram product or cells per serving • Studies have used doses of 10 6 – 10
9 cells per day for 1 – 13 weeks • A daily dose of 1–5 × 10 9 cfu for a minimum of 5 days is currently recommended* – About 10 ml of product with 10 8 cells per ml – About 100 ml of of product with 10 7 cells per ml – About 1 L of product with 10 6 cells per ml – About 10 L of product with 10 5 cells per ml Health Benefits of probiotics * Kaur et al. (2002); Health Canada (2009) Cut off for probiotics shelf life
Adequate probiotic intake? Health Benefits of probiotics Log 8 100,000 to 10 million cells per gram at start fermentation
at end fermentation Log 5 ‐ 7 Log 10 per serving 20 billion cells per serving Probiotic Starter culture Assuming a serving of 200 ml of yoghurt
The fate of ingested probiotics Health Benefits of probiotics Administration needs to be continued Mechanisms of action of probiotics Suppression of endogenous pathogens Control of Irritable Bowel Syndromme (IBS) Control of Inflamatory Bowel Disease Alleviate food allergy symptoms in infants Suppression of exogenous pathogens e.g travellors’ diarrhea
Normalised intestinal microbial composition Strenthened innate immunity
mucus, bacteriocins, defensins, enhanced cell binding
effects on lymphocytes e.g cytokine production, stoping proinflamatory responses, preventing apoptosis, icreased Ig release...
Metabolic effects Reduced risk of colon cancer Lowered serum
cholesterol Bile salt deconjugation and excretion Lactose hydrolysis Improved lactose tolerance Lower levels of toxigenic/mutagenic reactions in the gut Supply of SCF’s and vitamins (e.g folate) to the colonic epithelium Maitai, C. K., & Kokonya, D. M. (2008). A Brief Review of Probiotic Use. East and Central African Journal of Pharmaceutical Sciences, 11(3). Some studies on probiotics in Africa Source: Franz, C. M., Huch, M., Mathara, J. M., Abriouel, H., Benomar, N., Reid, G., ... & Holzapfel, W. H. (2014). African fermented foods and probiotics. International journal of food microbiology, 190, 84‐96.Global Probiotics Market Outlook (2014‐2022) . • Mainly in vitro studies focussed on functional and technological properties • A few human studies Some studies on probiotics in Africa Source: Franz, C. M., Huch, M., Mathara, J. M., Abriouel, H., Benomar, N., Reid, G., ... & Holzapfel, W. H. (2014). Strains Source Inoculated/consu med product Region were product is consumed E. faecium Raw cow milk for nono production
Nigeria
L. plantarum, L. fermentum Fremented maize dough, cassava, Gari
Ghana
L. plantarum, Pediococcus spp Fufu and Ogi In vitro study Nigeria
Pediococcus spp, Lactobacillus spp, L. fermentum Pearl millet slurry In vitro study Burkina Faso, Ghana L. Fermentum Kimere: pearl millet dough
In vitro study Kenya
L. Johnsonii (BFE 6128, BFE 6154) L. plantarum (BFE 5092, BFE 5759, BFE 5878), L. acidophilus, L. paracasei, L. rhamnosus. L. fermentum Kule naato (Maasai fermented milk) Kwerionik (fermented milk)
In vitro study Kenya
Uganda L. acidophilus, L. pentosus Fermented cow’s milk and cassava Fermented cereal maize gruel West Africa LAB, mainly W. confusa and L.
from millet porridge Koko sour water Ghana
L. rhamnosus GR‐1 Milk yoghurt and milk yoghut with Moringa powder Tanzania
• High costs of randomized control trials – Lack of government investment in R & D – Lack of industrial and philanthropic funding • High costs of developing and storing of cultures • Getting the consumer to accept probiotics – The fear of eating microbes • Legislation – Non existent in a number of countries Major challenges in Africa • Increasing interest in natural, non‐drug related remedies • Limited knowledgeable about probiotics – About 95% of medical practitioners in Nigeria not familiar with probiotics yet 64% if favour of their use (Anakum, 2006) • Populations exposed to: – Poor hygiene conditions – Toxic compounds (aflatoxins) – Malnutrition (e.g vitamin deficiency, iron deficiency) – Chronic enteric infections (e.g diarrhea which accounts for 37% of childhood deaths in Sub‐Saharan Africa) – Increasing NCDs
• Unavailability of locally sourced strains with documented health benefits • A great diversity of traditional fermented foods – Potential sources of probiotics – Potentially acceptable probiotic vehicles • Exisistence of several well document probiotic strains from the developed countries – No need for expensive trials – Could be evaluated for use in local foods
• A generic strain of Lactobacillus rhamnosus GG • LGG isolated in 1983 from the intestinal tract of a healthy human being • Patent filed by Sherwood Gorbach and Barry Gordin on 17
th April 1985 – Strain initially identified as Lb. acidophilus GG (ATCC Accession No. 53103) • Patent of LGG expired in 2006
• Concept of ’generic probiotics’ introduced by Kort and Sybesma (2012). – Patent‐expired probiotics are free to be used by others • Remco Kort and Wilbert Sybesma (Yoba For Life Foundation) isolated LGG from a probiotic product • Clone of LGG named L.rhamnosus Yoba – Deposited in the BCCM/LMG labs in Belgium – Identity and genome sequence evaluated • Yoba for Life Foundation introduced L. rhamnosus Yoba in Uganda in 2013 – First introduced in a small dairy plant in Mukono district – Mainly used in yoghurt production Lb. rhamnosus Yoba Other African products in which Lb. rhamnosus Yoba has been tried Lb. rhamnosus Yoba Product Description Country Reference Mutandabota Milk + baobab fruit pulp Zimbabwe
Mpofu et al., (2014) Zoom‐kom
Wheat, milk and water Burkina Faso Kort et al., (2015) Uji
Fermented maize/sorghum beverage Kenya Kort et al., (2015) Obushera Fermented sorghum/millet beverage Uganda
Kort et al (2015) Meeme and Mukisa (unpublished) Soy yoghurt Fermented soy milk Uganda
Lutalo, Nanyonga and Mukisa (unpublished) Fermented vegetable salads
Mixed vegetables Uganda
Kakeeto, Bion and Mukisa (unpublished) Fruit juice cocktail Mixed fruits Uganda Agaba and Mukisa (unpublished) • First prototype of non‐dairy based probiotic in Uganda produced at MAK in May 2016 • Obushera fermented using the
– Acceptable – Probiotic viable for at least 8 weeks during cold storage (about log 8 cfu/ml) Lb. rhamnosus Yoba in Obushera • The Food Technology Business Incubation Center, Makerere
• Yoba for Life Foundation • RUFORUM • iAGRI • SUA
Acknowledgement Thank You • Anukam, K. C., Osazuwa, E.O., and Reid, G. (2006). Knowledge of probiotics by Nigerian clinicians. International Journal of Probiotics and Prebiotics, 1(1), 57‐62. • Elliott, E and Teversham, K. 2004. An evaluation of nine probiotics available in South Africa, August 2003. Vol. 94, No. 2 SAMJ. • Fooks L. J. and Gibson G. R. 2002. Probiotics as modulators of the gut flora. British Journal of Nutrition, 88, Suppl. 1, S39–S49. • Franz, C. M., Huch, M., Mathara, J. M., Abriouel, H., Benomar, N., Reid, G., ... & Holzapfel, W. H. (2014). African fermented foods and probiotics. International journal of food microbiology, 190, 84‐96.Global Probiotics Market Outlook (2014‐2022) . http://www.strategymrc.com/report/global‐probiotics‐market‐outlook‐2014‐ 2022
• Grand View Research. 2016. Probiotics Market Analysis By Application (Probiotic Functional Foods & Beverages, Probiotic Dietary Supplements, Animal Feed Probiotics), By End‐Use (Human Probiotics, Animal Probiotics) And Segment Forecasts To 2020. http://www.grandviewresearch.com/industry‐analysis/probiotics‐ market
• Health Canada. 2009. Guidance Document. The Use of Probiotic Microorganisms in Food. Available from http://www.hc‐sc.gc.ca/fn‐an/legislation/guide‐ld/probiotics_guidance‐orientation_probiotiques‐eng.php. [Accessed 12 February 2014.] • Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002. • Kaur, I.P., Chopra, K., and Saini, A. 2002. Probiotics: potential pharmaceutical applications. Eur. J. Pharm. Sci. 15(1): 1–9. doi:10.1016/S0928‐0987(01)00209‐3. PMID:11803126. • Kort, R. and Sybesma, W. (2012) Probiotics for every body. Trends Biotechnol. 30, 613–615 References • Kort, R., Westerik, N., Serrano, L.M., Douillard, F., Gottstein, W., Ananta, E., Mukisa, I.M., Tuijn, C., Basten, L., Hafkamp, B., Meijer, W., Teusink, B., de Vos, W., & Sybesma, W. (2015). A novel consortium of Lactobacillus
• Lei, V. et al. (2006) Spontaneously fermented millet product as a natural probiotic treatment for diarrhoea in young children: an intervention study in Northern Ghana. Int. J. Food Microbiol. 110, 246–253 • Maitai, C. K., & Kokonya, D. M. (2008). A Brief Review of Probiotic Use. East and Central African Journal of Pharmaceutical Sciences, 11(3). • Markets and Markets.com. 2015. Probiotic Ingredients Market by Function (Regular, Preventative, Therapy), Application (Food & Beverage, Dietary Supplements, & Animal Feed), End Use (Human & Animal Probiotics), Ingredient (Bacteria & Yeast), and by Region ‐ Global Trends & Forecast to 2020. http://www.marketsandmarkets.com/Market‐Reports/probiotic‐market‐advanced‐technologies‐and‐global‐ market‐69.html • Mathara, M.J., Schillinger, U., Kutima, P.M., Mbugua, K.S., Guigas, C., Franz, C.M.A.P., Holzapfel, W.H., 2008a. Functional Properties of Lactobacillus plantarum strains isolated from Maasai traditional fermented milk products in Kenya. Curr. Microbiol. 56, 315–321. • Mathara, M.J., Schillinger, U., Guigas, C., Franz, C.M.A.P., Kutima, P.M., Mbugua, S., Shin, H. K., Holzapfel, W.H., 2008b. Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya. Int. J. Food Microbiol. 126, 57–64. • Mpofu A, Linnemann AR, Sybesma W, Kort R, Nout MJ, Smid EJ. (2014). Development of a locally sustainable functional food based on mutandabota, a traditional food in southern Africa. Journal of Dairy Science, 97(5):2591‐2599. References • Ng, S. C., Hart, A. L., Kamm, M. A., Stagg, A. J., & Knight, S. C. (2009). Mechanisms of action of probiotics: recent advances. Inflammatory bowel diseases, 15(2), 300‐310. • O’Hara and Shanaha. 2006. The gut flora as a forgotten organ. EMB, Vol 7 (7), 688 ‐ 693 • Patrignani, F., Lanciotti, R., Mathara, J.M., Guerzoni, M.E., Holzapfel, W.H., 2006. Potential of functional strains isolated from traditional Maasai milk as starters for the production of fermented milks. Int. J. Food Microbiol. 107, 1–11. • Reid, G. 2016. Probiotics: definition, scope and mechanisms of action. Best Practice & Research Clinical Gastroenterology 30: 17‐25 • SCAN. (2000). Report of the Scientific Committee of Animal Nutrition on the Safety of use of Bacillus Species in Animal Nutrition. European Commission of Health and Consumer Protection, Directorate‐General. http://europa.eu.int/comm/food/fs/sc/scan/out41.pdf • Sybesma, W., Kort, R., and Lee, Y. 2015. Locally sourced probiotics, the next opportunity for developing countries? Trends in Biotechnology, Vol. 33 (4): 197 – 200. • Taibi, A and Comelli, E.M. 2014. Practical approaches to probiotics use. Appl. Physiol. Nutr. Metab. 39: 980– 986. • Taverniti, V., and Guglielmetti, S. 2011. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept). Genes Nutr. 6(3): 261–274. doi:10.1007/s12263‐011‐0218‐x. PMID:21499799. Technavio. 2016. Global Probiotics Market 2015‐2019. http://www.technavio.com/report/probiotics‐market • Tsilingiri, K., and Rescigno, M. 2013. Postbiotics: what else? Benef. Microbes, 4(1): 101–107. doi:10.3920/BM2012.0046. PMID:23271068. • Viana, JV, da Cruz, A.g, Zoellner, SS., Silva, R., Batista ALD. 2008. Probiotic foods: consumer perception and attitudes. International Journal of Food Science and Technology, 43, 1577–1580 •
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