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May 05, 2014

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  1. by: Aishwarya Singh Gangwar M.Tech-4th sem.

  2. Introduction  Fermentation.  Functional foods  Probiotics  Lactic

    acid bacteria  Spore forming probiotics
  3. Bacillus coagulans  Bacillus coagulans is a lactic acid-forming bacterial

    species within the genus Bacillus.  The organism was first isolated and described as Bacillus coagulans in 1915 by B.W. Hammer.  Separately isolated in 1935 and described as Lactobacillus sporogenes in the Fifth edition of Bergey's Manual.  It exhibits characteristics typical of both genera Lactobacillus and Bacillus; However, in the seventh edition of Bergey’s, it was finally transferred to the genus Bacillus.
  4.  Scientific classification:  Kingdom: Bacteria  Phylum: Fermicutes 

    Class : Bacilli  Order: Bacillales  Family: Bacillaceae  Genus: Bacillus  Species: Coagulans  Binomial name: Bacillus coagulans, (Hammer, 1915)
  5.  Bacillus coagulans as a probiotic bacterium:  Bacillus coagulans

    has been added by the EFSA to their Qualified Presumption of Safety (QPS) list.  Approved as GRAS by the U.S. Food and Drug Administration's Center as well as by the European Union.  It is listed by AAFCO for use as a direct fed microbial in livestock production.
  6. Tender coconut water  Tender Coconut water is the clear

    liquid inside young green coconuts.  Chief of FAO’s : "It’s a natural isotonic beverage with the same level of electrolytic balance as we have in our blood. It’s the fluid of life.“  The major chemical constituents of coconut water are sugars , minerals, vitamins and minor ones are fat and nitrogenous substances.
  7. Aim  The aim of this study was to assess

    the growth, survival and fermentation performance of probiotic bacteria Bacillus coagulans in coconut water.  The main goal of this project are to study the fitness of Tender coconut water to sustain probiotic bacteria Bacillus coagulans to made a novel non dairy probiotic beverage.
  8. Objectives  Morphological, Physiological and Biochemical characteristics of Bacillus coagulans.

     Probiotic properties of Bacillus coagulans.  Physio-chemical analysis of Tender coconut water and Fermented coconut water.  Evaluation of sensory characterstics of the fermented coconut water.
  9. Methods  The Probiotic bacteria: Capsules under the trade name

    ‘Vizylac’, which contain probiotic bacteria Bacillus Coagulans in powdered form were purchased from a Pharmacy shop in Delhi. A capsule contains 120×108 million viable spores of Bacillus Coagulans. Propagation: The spores of Bacillus Coagulans were propagated separately in sterile de Man Rogosa Sharpe (MRS) broth for up to 48h at 37 °C aerobically
  10. Propagation of bacterial spores

  11. Preparation of Bacterial suspension

  12. Characterization of Species  The sub-cultured species were subjected to

    various Morphological, Physiological and Biochemical Identification tests. Various tests like:  Gram’s staining  Methyl-Red test  Voges-Proskauer test
  13. Gram’s staining

  14. MR test & VP Test

  15. Analyzing Probiotic attributes:  Qualitative test for lactic acid production:

  16. Acid tolerance test

  17. Bile tolerance test

  18. Morphology, physiology and biochemistry of Bacillus coagulans

  19. Preparation of Tender Coconut Water

  20. Preparation of Inoculum

  21. Plating and Counting

  22.  The viable spore count = Number of colonies per

    plate × Final dilution factor. Dilution factor No. of colonies/ml 10-7 102 10-8 73 10-9 58
  23. Fermented Coconut Water

  24. Analytical Determination  pH  Total soluble solids  Rheological

    measurement  Titrable acidity  Biomass/cell density  Viable cell counts  Total sugars  Reducing sugars  Sensory evaluation
  25. Biomass/cell density  The cell density of fermented coconut water

    determined at 540nm.  According to P. H. A. The absorbance of fermented product to 0.600 that correspond to 9.00 Log CFU/mL, using Mac Farland scale.  This is ideal for probiotic beverage functionality
  26. Titrable acidity  Formula:  % Titrable acidity= (ml of

    NaOH used) (Normality of NaOH) (Eq. Wt. of lactic acid/Citric acid) (Total vol. Made) (100) (Volume of diluted sample taken for titration) (Weight of the sample taken) (1000)
  27. Total sugars by Phenol sulphuric acid method  Absorbance from

    tender coconut water at 490nm: 0.52  Absorbance from fermented coconut water at 490nm: 0.31 y = 11.33x + 0.0708 R² = 0.9943 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.02 0.04 0.06 0.08 0.1 0.12 Absorbance @ 490nm. Glucose concentration (mg/ml).
  28.  Tender coconut water: g/mL in tube using equation from

    Standard Curve:  y = 11.33x + 0.0708  Absorbance corresponds to 0.1 mL of the test = x mg of glucose  10mL of the sample solution contains = x / 0.1 × 10 ml of glucose =3.96 % of total sugars present in sample solution.  Fermented coconut water: =2.37 % of total sugars present in sample solution.
  29.  Total sugars estimation by Phenol-Sulphuric acid method: showing increasing

    amount of sugar concentration.
  30. Calculation of reducing sugars by DNSA method  Absorbance from

    tender coconut water at 540nm: 0.264  Absorbance from fermented coconut water at 540nm: 0.190 y = 7.95x + 0.009 R² = 0.998 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 0.02 0.04 0.06 0.08 0.1 0.12 Absorbance @ 540nm. Glucose concentration (mg/ml).
  31.  Tender coconut water: g/mL in tube using equation from

    Standard Curve:  y = 7.95x + 0.009  Absorbance corresponds to 0. mL of the test = x mg of glucose  10mL of the sample solution contains = x / 0.1 × 10 ml of glucose  =3.15 % of reducing sugars in tender coconut water.  Fermented coconut water: =2.27 % of reducing sugars in tender coconut water.
  32.  Reducing sugars estimation by DNSA Method - showing increasing

    amount of sugar concentration.
  33. Physico-chemical characteristics of TCW & FCW.

  34. Sensory evaluation of Fermented Coconut Water.

  35. Conclusion  The good adaptation of B. coagulans in the

    tender coconut water advocate the fact that if a potentially probiotic strain is used as a starter culture then it might produce a fermented product with defined and consistent characteristics and possibly health- promoting properties.  These results suggest the feasibility of fermenting coconut water in to a probiotic beverage, especially for sports nutrition, with the dual benefits of electrolytes and probiotics.
  36. None