Coconut milk beverage fermented by Lactobacillus reuteri: optimization process and stability during refrigerated storage

  • Carolina Saori Ishii MauroEmail author
  • Sandra Garcia
Original Article


This study aimed to establish the optimal conditions of temperature (31–43 °C) and coconut pulp concentration in water 1:3–1:9 (w/v) for the growth of Lactobacillus reuteri LR 92 or DSM 17938 in coconut milk beverage, using a central composite face centered design. The optimized conditions were used for analysis of the viability during the fermentation process, pH, production of sugars and organic acids by High Performance Liquid Chromatography (HPLC) and reuterin production. Coconut milk provided adequate substrate for L. reuteri growth without supplementation. The optimal parameters for L. reuteri viability were: concentration 1:3 (w/v) and 37 °C for LR 92 and concentration 1:3 (w/v) and 34 °C for DSM 17938. Chemical analysis showed that the naturally occurring sucrose in the matrix (ca. 4.4 g/L) was used for cell multiplication and the strains differed in the production and content of organic acids. After fermentation until pH 4.5 ± 0.1, the samples were stored at 4 °C for 30 days and the final cell viability in coconut milk was 7.55 ± 0.07 log CFU/mL for L. reuteri LR 92 and 8.57 ± 0.09 log CFU/mL for DSM 17938. It was detected 0.15 ± 0.03 mM and 0.14 ± 0.04 mM of reuterin produced by DSM 17938 and LR 92, respectively. L. reuteri DSM 17938 presented a great decrease of pH and post acidification after storage. The LR 92 strain showed low post acidification. These results showed that coconut milk provides adequate matrix for the development of new fermented functional beverages.


Plant-based milk Probiotic Response surface Sugars profile Organic acids profile Reuterin 



This research was developed with the support of the State University of Londrina, which provided the infrastructure and facilities. This study was supported by a scholarship from the Coordination for the Improvement of Higher Education Personnel (CAPES).


  1. Årsköld E, Lohmeier-Vogel E, Cao R, Roos S, Rådstrom P, Van Niel EWJ (2008) Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis. J Bacteriol 190:206–212. CrossRefPubMedGoogle Scholar
  2. Barros Neto BB, Scarminio IS, Bruns RE (2010) Como Fazer Experimentos: Pesquisa e desenvolvimento na ciência e na indústria, 4ª edn. Bookman, Campinas, Porto AlegreGoogle Scholar
  3. Bernat N, Cháfer M, González-Martínez C, Rodríguez-Garcia J, Chiralt A (2015a) Optimisation of oat milk formulation to obtain fermented derivatives by using probiotic Lactobacillus reuteri microorganisms. Food Sci Technol Int 21:145–157. CrossRefPubMedGoogle Scholar
  4. Bernat N, Cháfer M, Chiralt A, González-Martínez C (2015b) Probiotic fermented almond “milk ” as an alternative to cow-milk yoghurt. Int J Food Stud 4:201–211. CrossRefGoogle Scholar
  5. Dinleyici EC, Dalgic N, Guven S, Metin O, Yasa O, Kurugol Z, Turel O, Tanir G, Yazar AS, Arica V, Sancar M, Karbuz A, Eren M, Ozen M, Kara A, Vandenplas Y (2015) Lactobacillus reuteri DSM 17938 shortens acute infectious diarrhea in a pediatric outpatient setting. J Pediatr (Rio J) 91:392–396. CrossRefGoogle Scholar
  6. Fatheree NY, Liu Y, Taylor CM, Hoang TK, Cai C, Rahbar MH, HessabI M, Ferris M, McMurthy V, Wong C, Vu T, Dancsak T, Wang T, Gleason W, Bandla V, Navarro F, Tran DQ, Rhoads JM (2017) Lactobacillus reuteri for infants with colic: a double-blind, placebo-controlled, randomized clinical trial. J Pediatr 191:170–178.e2. CrossRefPubMedGoogle Scholar
  7. Food and Agriculture Organization of the United Nations, World Health Organization (FAO/WHO) (2001) Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba, ArgentinaGoogle Scholar
  8. Food and Drug Administration (FDA) (1982) Cultured and acidified milks, cultured and acidified buttermilks, yogurts, and eggnog; confirmation of effective date and further amendments; and stay of effective date of certain provisions. Fed Regist 74(183):41522Google Scholar
  9. Garcia MJ, Zuniga M, Kobayashi H (1992) Energy production from L-malic acid degradation and protection against acidic external pH in Lactobacillus plantarum CECT 220. J Gen Microbiol 138:2519–2524. CrossRefGoogle Scholar
  10. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME (2014) Expert consensus document: the International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11:506–514. CrossRefPubMedGoogle Scholar
  11. IFT (2018) U.S. non-dairy milk sales surpass $2 billion in 2017.$2-billion-in-2017.aspx. Accessed 11 Feb 2018
  12. Jeske S, Zannini E, Arendt EK (2018) Past, present and future: the strength of plant-based dairy substitutes based on gluten-free raw materials. Food Res Int 110:42–51. CrossRefPubMedGoogle Scholar
  13. Kechagia M, Basoulis D, Konstantopoulou S, Dimitriadi D, Gyftopoulou K, Skarmoutsou N, Fakiri EM (2013) Health benefits of probiotics: a review. ISRN Nutr 2013:1–7. CrossRefGoogle Scholar
  14. Kraft-Bodi E, Jørgensen MR, Keller MK, Kragelund C, Twetman S (2015) Effect of probiotic bacteria on oral candida in frail elderly. J Dent Res 94:181S–186S. CrossRefPubMedGoogle Scholar
  15. Langa S, Landete JM, Martín-Cabrejas I, Rodríguez E, Arqués JL, Medina M (2013) In situ reuterin production by Lactobacillus reuteri in dairy products. Food Control 33:200–206. CrossRefGoogle Scholar
  16. Liu XT, Hou CL, Zhang J et al (2014) Fermentation conditions influence the fatty acid composition of the membranes of Lactobacillus reuteri I5007 and its survival following freeze-drying. Lett Appl Microbiol 59:398–403. CrossRefPubMedGoogle Scholar
  17. Mäkinen OE, Uniacke-Lowe T, O’Mahony JA, Arendt EK (2015) Physicochemical and acid gelation properties of commercial UHT-treated plant-based milk substitutes and lactose free bovine milk. Food Chem 168:630–638. CrossRefPubMedGoogle Scholar
  18. Mauro CSI, Guergoletto KB, Garcia S (2016) Development of blueberry and carrot juice blend fermented by Lactobacillus reuteri LR92. Beverages 2:37. CrossRefGoogle Scholar
  19. McCarthy KS, Parker M, Ameerally A, Drake SL, Drake MA (2017) Drivers of choice for fluid milk versus plant-based alternatives: what are consumer perceptions of fluid milk? J Dairy Sci 100:6125–6138. CrossRefPubMedGoogle Scholar
  20. Moraes Filho ML, Busanello M, Garcia S (2016) Optimization of the fermentation parameters for the growth of Lactobacillus in soymilk with okara flour. LWT: Food Sci Technol 74:456–464. CrossRefGoogle Scholar
  21. Ortiz-Rivera Y, Sánchez-Vega R, Gutiérrez-Méndez N, León-Félix J, Acosta-Muñiz C, Sepulveda DR (2017) Production of reuterin in a fermented milk product by Lactobacillus reuteri: inhibition of pathogens, spoilage microorganisms, and lactic acid bacteria. J Dairy Sci 100:4258–4268. CrossRefPubMedGoogle Scholar
  22. Østlie HM, Helland MH, Narvhus JA (2003) Growth and metabolism of selected strains of probiotic bacteria in milk. Int J Food Microbiol 87(1–2):17–27CrossRefGoogle Scholar
  23. Pallin A, Agback P, Jonsson H, Roos S (2016) Evaluation of growth, metabolism and production of potentially bioactive components during fermentation of barley with Lactobacillus reuteri. Food Microbiol 57:159–171. CrossRefPubMedGoogle Scholar
  24. Pauli ED, Cristiano V, Nixdorf SL (2011) Método para determinação de carboidratos empregado na triagem de adulterações do café. Quím Nova 34(4):689–694CrossRefGoogle Scholar
  25. Rosander A, Connolly E, Roos S (2008) Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938. Appl Environ Microbiol 74:6032–6040. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Santos CCA do A, Libeck B da S, Schwan RF (2014) Co-culture fermentation of peanut-soy milk for the development of a novel functional beverage. Int J Food Microbiol 186:32–41.
  27. Santoso U, Kubo K, Ota T, Tadokoro T, Maekawa A (1996) Nutrient composition of kopyor coconuts (Cocos nucifera L.). Food Chem 57:299–304. CrossRefGoogle Scholar
  28. Seow CC, Gwee CN (1997) Review coconut milk: chemistry and technology. Int J Food Sci Technol 32:189–201. CrossRefGoogle Scholar
  29. Sethi S, Tyagi SK, Anurag RK (2016) Plant-based milk alternatives an emerging segment of functional beverages: a review. J Food Sci Technol 53:3408–3423. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Siriphanich J, Saradhuldhat P, Romphophak T, Krisanapook K, Pathaveerat S, Tongchitpakdee S (2011) Coconut (Cocos nucifera L). Woodhead Publishing Limited, CambridgeCrossRefGoogle Scholar
  31. Spinler JK, Sontakke A, Hollister EB, Venable SF, Oh PL, Balderas MA, Saulnier DMA, Mistretta T, Deveraj S, Walter J, Versalovic J (2014) From prediction to function using evolutionary genomics: human-specific ecotypes of Lactobacillus reuteri have diverse probiotic functions. Genome Biol Evol 6:1772–1789. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Talarico TL, Axelsson LT, Novotny J, Fiuzat M, Dobrogosz WJ (1990) Utilization of glycerol as a hydrogen acceptor by Lactobacillus reuteri: purification of 1,3-propanediol:NAD + oxidoreductase. Appl Environ Microbiol 56:943–948PubMedPubMedCentralGoogle Scholar
  33. Tobajas M, Mohedano AF, Casas JA, Rodríguez JJ (2007) A kinetic study of reuterin production by Lactobacillus reuteri PRO 137 in resting cells. Biochem Eng J 35:218–225. CrossRefGoogle Scholar
  34. Yildiz F (2009) Development and manufacture of yogurt and other functional dairy products, 1st edn. CRC Press, Boca RatonCrossRefGoogle Scholar
  35. Zannini E, Jeske S, Lynch K, Arendt EK (2018) Development of novel quinoa-based yoghurt fermented with dextran producer Weissella cibaria MG1. Int J Food Microbiol 268:19–26. CrossRefPubMedGoogle Scholar
  36. Zhao X, Gänzle MG (2018) Genetic and phenotypic analysis of carbohydrate metabolism and transport in Lactobacillus reuteri. Int J Food Microbiol 272:12–21. CrossRefPubMedGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2019

Authors and Affiliations

  1. 1.Department of Food Science and TechnologyState University of LondrinaLondrinaBrazil

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