Optimization of cost efficient and effec

OPTIMIZATION OF COST-EFFICIENT AND EFFECTIVE CULTURE MEDIA FOR LACTIC ACID BACTERIA (LAB) by RODRIGUEZ, KATHERENE ARASULA SISON, JELICA AYE MARI AGCAOILI TAN, TERESA TING A thesis submitted to the Department of Biology College of Science University of the Philippines Baguio Baguio City In partial fulfillment of the requirements for the Degree of BACHELOR OF SCIENCE IN BIOLOGY March 2013 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria i Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria ii CERTIFICATION This is to certify that this undergraduate thesis entitled ―Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria (LAB)‖ and submitted by KATHERENE ARASULA RODRIGUEZ, JELICA AYE MARI AGCAOILI SISON and TERESA TING TAN to fulfil part of the requirements for the Degree of Bachelor of Science in Biology, is hereby endorsed PROF CELIA M AUSTRIA Thesis Adviser The Department of Biology accepts this undergraduate thesis in partial fulfillment of the requirements for the Degree of Bachelor of Science in Biology _ PROF CELIA M AUSTRIA Chair, Department of Biology The undergraduate thesis is hereby accepted as partial fulfillment of the requirements for the Degree of Bachelor of Science in Biology ROSEMARY M GUTIERREZ, Ph.D, Dean, College of Science Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria iii ACKNOWLEDGEMENTS This thesis would not have been possible without the assistance of people who in one way or another have contributed and extended their valuable support in the preparation and completion of this study First, to our thesis adviser, Prof Celia Austria, who became a strict yet very understanding mother and mentor We thank her for attending to our thesis with utmost patience and enthusiasm and for always being available for consultation We are also grateful for her intellectual and constructive criticisms that have inspired us to improve and finally finish our thesis We are very grateful to the officers of the Agricultural Training Institute - International Training Center on Pig Husbandry (ATI-ITCPH) Lipa, Batangas; Center Director Alexander Castillo and Dr Ruth Sonaco, Chief of the Technical Support Services for providing financial and technical support of the study We appreciate the advice and suggestions that Dr Rosemarie Gutierrez and Sir Jaime Canedo have shared for the development of this thesis They have helped us narrow down our objectives into realistic and attainable goals We also thank the staff and researchers of ATI-ITCPH namely; Mariquez Sison, Editha Cabilitazan, Ramilo Ostil, Liberty Onciong, Amy Eguia, Filipina Nava, and Noemie Tesco, who have imparted their knowledge, ideas and suggestions The support they have given us during our stay in their research facility means much to us We thank Manang Alice Naniong and Manang Romelyn Fernandez for helping us contact suppliers of glassware, reagents and other materials that were essential in conducting our experiment To Dr Marichu Lubrica, Professor of Statistics at the Benguet State University (BSU), for the statistical analysis of our data and for the help in interpretation and discussion of our results, we extend gratitude To Jun Barnes of 3M Philippines for his effort in personally teaching us the proper usage of PetriFilm which was the heart of our study and for his enthusiasm towards the success of our study, we thank you Also, to Kuya Sanpa and Kuya Bobby of RTC Laboratory for personally making sure that the materials ordered reached us in good condition To the library staff of UPB, BSU and UPLB Libraries, our study would not have been credible if not for the updated and comprehensive resources which each library provided, we thank you Lastly, to Kuya Gary of MMP Catering who made sure that our tummies were full anytime of the day while we conducted our study at Lipa -Jam, Kathie and Tere Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria iv In our daily work, I have been blessed with a cheerful and supportive mother, Mariquez Sison, who personally accompanied us during our late night experiments and made sure that none of us starved, and with a caring father, George Sison who helped in making our budget proposal I would also like to thank my brother, Jan Irvin Mark, who never failed to fascinate me with his ability in Statistics and had given me some interpretations in statistical analyses and some good laughs To Regin Ruis Oliveros who never got tired of accompanying me on my UPLB trips for some library research works, thank you To my best friend, Karina Ruth Sonaco, who provided me with stories during our stay in Lipa, thank you and I miss you To my second family, Philippine Association of Food Technologists, Inc, Beta Chapter in UPLB who had provided me with good arguments regarding our study, great thanks to you brothers and sisters And finally, to my friends and the rest of our family members, specially my cute little Angel, thank you for giving me the inspiration to work hard on this research and motivated me to push through my goals I love you all Xie xie - Jam My deepest gratitude goes to my family, for the understanding and the support that they have given me throughout the length of this study To my sorority sisters from the Sigma Beta Sorority, for their confidence in me and their thoughtfulness as to the progress of this study To my college clique Flor, Cla, and Dez who remained patient and available for story-telling during the cyclic moments of fun and hardship To Sheena, Keichi, JM and all the people who believed in us and encouraged us to succeed, along with all the friendships created during the course of this study To Anton, for you have witnessed the development of this study and for the encouragement, constructive criticisms and time you have listened to all my stories and rants To labs Tere and labs Jam, for the perseverance and memorable friendship we have created Finally to the Creator, for without Him I wouldn’t be able to experience the rigorous UP training and I wouldn’t be here to thank each and every one of you - Kathie Thanking my Nanay and my older brothers Jet, Gil and Abing will not be enough for all the financial assistance, emotional support and confidence they have given me, but finishing this thesis will To my bestfriends Rita and Dids, thanks for keeping me sane every time I get stressed To my teammates from the UPB Women’s Basketball Team and to my sisters from the Sigma Alpha Nu Sorority, thank you for all the moral support To my friend and mentor, ma’am Wilen Mina, thank you for your patience each time you listen to me rant and for all the times you helped me figure out a solution To my ever loyal friends, Ella, Nica, Jiggy, Alice, Sha, Meryl, Benson and Deg, thanks for giving me courage and hope when everything was just as hard To AKMA UP, thank you for making me realize that my extension in school was never a waste To Kathie and Jam, my thesis group mates who have extended their patience, understanding and support to the ends of this world just to finish this thesis And lastly, to my Tatay, you may not see me finish me thesis, you may not see me graduate, but this is for you - Tere Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria v ABSTRACT (216 Words) Organic farming makes use of lactic acid bacteria (LAB) which is known to improve digestion, stimulate gastrointestinal immunity, and increase natural resistance from infectious enteric diseases among livestock and fishery animals This study aimed to produce the most costefficient and effective LAB serum for organic farmers by comparing the LAB population count of the fermented rice wash from the locally acquired C4 Dinurado rice, the LAB serum and the pure stock Three kinds of milk were used for analysis—pasteurized Carabao’s milk, UHT (Ultra Heat-Treated) fresh milk and skim milk Upon determining which milk media promoted the highest population count, three kinds of sugars were used as food source—molasses, white sugar and brown sugar After which, confirmatory tests such as simple staining, colony morphology, Gram staining and gas production from glucose were conducted to verify the presence the LAB Petrifilm count of LAB was highest for pasteurized milk (3.619 x 10 10 cfu/ml) compared to UHT and skim milk which were 2.091 x 1010 cfu/ml and 1.493 x 1010cfu/ml, respectively All three sugars produced high population count of LAB that were insignificantly different from each other Therefore, the most cost-efficient and effective LAB stock is produced from the combination of Dinurado rice wash, pasteurized milk and molasses which can then be applied in organic swine farming Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria vi TABLE OF CONTENTS Certification Page ii Acknowledgement iii Abstract v Table of Contents vi List of Tables vii List of Figures viii Introduction Background of the Study Research Objectives Null Hypothesis Significance of the study Scope and Delimitation Review of Related Literature Lactic Acid Bacteria Probiotics Rice Milk Sugar Sources Related Studies 12 Materials and Methodology 14 Optimization of conditions for the culture and collection of LAB 15 Use of Milk as media for the growth of Lactic Acid Bacteria 15 Preparation of LAB Pure Stock 16 Identification of population extent obtained from FRW, LAB Serum and Pure Stock 17 Population count using PetriFilm 17 Statistical Analysis 18 Confirmatory Tests 18 Simple Staining 18 Colony Morphology 18 Gram Staining 19 Gas Production from Glucose 19 Results 20 Discussion 23 Conclusion 37 Recommendations 38 Literature Cited 39 Appendix A –TABLES 44 Appendix B –FIGURES 45 Appendix C –PREPARATION OF DILUENTS 53 Appendix D – CULTURE MEDIA 56 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria vii LIST OF TABLES Table Title Page Table Table showing the quantitative and qualitative observations of FRW, LAB serum and pure stock set-ups 22 Table The three kinds of milk used in the experiment, their processing and their content 32 Table The progression of layers in the FRW set-up 43 Table The number of LAB present in fermented rice wash 43 Table The number of LAB present in the serum produced from UHT, Skim and Pasteurized Milk 43 Table The Number of LAB present in the pure stock produced from brown sugar, white sugar and molasses 43 Table Cost of Materials 44 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria viii LIST OF FIGURES Figure Title Page Figure Incubated PetriFilm showing the presence of Lactic Acid Bacteria 20 Figure The process of Lactic Acid Fermentation 28 Figure Bacterial count in LAB serum produced from UHT, Skim and Pasteurized milk 33 Figure Diluents used in the experiments 45 Figure Contaminated FRW with grayish-green mold formations 46 Figure Three replicates of the rice wash set up at day at the ITCPH health laboratory 46 Figure The rice wash set up 47 Figure FRW-milk set ups from day to day 5, pasteurized (left), UHT (middle), skim (right) 47 Figure The LAB serum mixed with each type of food source; molasses (left), brown sugar (middle), white sugar (right) 48 Figure 10 Clean PetriFilm from the package 48 Figure 11 Colony count from FRW set ups at different dilutions a 100, b 10-1, c 10-2 48 Figure 12 Colony count from each FRW-milk set ups 49 Figure 13 Colony count of each milk mixed with molasses at dilutions, 10-1 to 10-6 a Pasteurized, b UHT, c Skim milk 49 Figure 14 Colony count of each milk mixed with brown sugar at dilutions, 10 -1 to 10-6 a Pasteurized, b UHT, c Skim milk 49 Figure 15 Colony count of each milk mixed with white sugar at dilutions, 10-1 to 10-6 a Pasteurized, b UHT, c Skim milk 50 Figure 16 Microscopic view of the cells, showing violet-blue colored cocci to rod shaped cells that occur in singles and chains 50 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria ix Figure Title Page Figure 18 Preparation for Colony Morphology observation from FRW, LAB Serum and LAB Stock Samples 51 Figure 19 Samples from incubated Petri Plates (from Skim Milk LAB Serum) for Colony Morphology Observation 52 Figure 20 MRS Broth Content 54 Figure 21 Pasteurized Milk; A Liter of Rosario Pasteurized Carabao’s Milk, B Nutritional Value of Rosario Pasteurized Carabao’s Milk, C Brand Label of Rosario Pasteurized Carabao’s Milk 55 Figure 22 Skim Milk; A Liter of Country Goodness Skim Milk, B Nutritional Value of Country Goodness Skim Milk, C Processing information of Country Goodness Skim Milk 56 Figure 23 UHT Milk; A Liter of Alaska UHT Milk, B Nutritional Value Alaska UHT Milk, C Processing information of Country Goodness Skim Milk 56 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 44 APPENDIX A TABLES Table The progression of layers in the FRW set-up Day Temperature Presence of Molds or other foreign particles Day 27 °C None Day 27°C None Day 25.5°C None Day 26°C None Day 25°C None Day 25°C None Layers formed No layers yet Thin film at the surface of the liquid Thin film as thick as Day 1’s film Thicker film at the surface Three distinct layers Three distinct layers Table The number of LAB present in fermented rice wash Replicate Average Number of LAB (cfu/ml) 6.48 x 105 6.40 x 105 6.32 x 105 6.40 x 105 Table The number of LAB present in the serum produced from UHT, Skim and Pasteurized Milk Number of LAB Replicate UHT Skim 10 1.280 x 10 2.048 x 1010 1.664 x 1010 2.304 x 1010 1.536 x 1010 1.920 x 1010 Average 1.4933 x 1010 2.091 x 1010 Pasteurized 3.560 x 1010 3.584 x 1010 3.712 x 1010 3.619 x 1010 Table The Number of LAB present in the pure stock produced from brown sugar, white sugar and molasses Milk + Brown Sugar TNTC UHT TNTC Skim Pasteurized TNTC *TNTC – too numerous to count + White Sugar TNTC TNTC TNTC Table Cost of materials Material C4 Dinurado Rice Rosario Pasteurized Carabao’s Milk Alaska UHT Milk Country Goodness Skim Milk Molasses Dark Brown Sugar White Sugar Quantity Kilo Liter Liter Liter Kilo Kilo Kilo + Molasses TNTC TNTC TNTC Price (Php) 40.00 100.00 67.75 73.50 15.00 43.50 44.50 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 45 APPENDIX B FIGURES Figure Diluents used in the experiments; A-C Ethanol, guidelines to usage of ethanol and ethanol contents, respectively, D-F Pure Methylene Blue in powder from and guidelines to usage of Methylene Blue, G Safranin, Gram Iodine and Crystal Violet, H Xylol, I Chemicals properly diluted and transferred to sterilized and air-tight containers Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 46 Figure Contaminated FRW with grayish-green mold formations; A and B contaminated set-ups and 2, C Contaminated set-up 1, D Contaminated set-up Figure 6.Three replicates of the rice wash set up at day at the ITCPH health laboratory Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 47 Figure The rice wash set up a All three replicates have started to form a thin layer at the surface of the liquid b A closer look at the formed layers c The act of filtering the fermented rice wash Figure FRW-milk set ups from day to day 5, pasteurized (left), UHT (middle), skim (right) a.-c Fermented rice wash mixed with the three variations of milk at day 0, d.-e After days, the set up formed two layers, an upper cheese and a lower liquid form, g.-i The liquid filtered which becomes the lactic acid bacteria serum, j.-l The cheese formed from each FRW-milk set up Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 48 Figure The LAB serum mixed with each type of food source, molasses (left), brown sugar (middle), white sugar (right) a.c LAB serum-food source set up at day d.-f After some days, the same set up produced a layered appearance Figure 10 Clean PetriFilm from the package Figure 11 Colony count from FRW set ups at different dilutions a 10 0, b 10-1, c 10-2 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 49 Figure 12 Colony count from each FRW-milk set ups Figure 13 Colony count of each milk mixed with molasses at dilutions, 10-1 to 10-6 a Pasteurized, b UHT, c Skim milk Figure 14 Colony count of each milk mixed with brown sugar at dilutions, 10-1 to 10-6 a Pasteurized, b UHT, c Skim milk Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 50 Figure 15 Colony count of each milk mixed with white sugar at dilutions, 10 -1 to 10-6 a Pasteurized, b UHT, c Skim milk Figure 16 Microscopic view of the cells, showing violet-blue colored cocci to rod shaped cells that occur in singles and chains Figure 17 Gas production tests done for FRW, LAB serum and pure stock rendered negative, confirming that LAB present were homofermentative Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 51 Figure 18 Preparation for Colony Morphology observation from FRW, LAB Serum and LAB Stock Samples A Diluting samples, B Pouring MRS Agar into Petri Plates, C Candle Oats Set-Up with APPENDIX C lighted candle, D Candle Oats Set-Up after it was sealed and the flame of the candle has died PREPARATION OF DILUENTS Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 52 Figure 18 Samples from incubated Petri Plates (from Skim Milk LAB Serum) for Colony Morphology Observation; A and B From dilution of 10-6 ,C and D From dilution of 10-5,E and F From dilution of 10-4 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 53 APPENDIX C PREPARATION OF DILUENTS Reagents were acquired from RTC Laboratory, Quezon City Methylene Blue (Basic Blue 9,trihydrate; Methelyne blue trihydrate; 3,7-Bis(dimethylamino) phenazathionium chloride trihydrate ) Molecular Weight: 373.91 Chemical Formula: C16H18ClN3S 3H2O Physical and Chemical Properties Appearance: Dark green crystals with bronze luster or crystalline powder Odor: Odorless Solubility: Soluble in water Specific Gravity: No information found pH: No information found % Volatiles by volume @ 21C (70F): Boiling Point: Decomposes Melting Point: 100 - 110C (212 - 230F) Vapor Density (Air=1): 13 Vapor Pressure (mm Hg): Not applicable Evaporation Rate (BuAc=1): No information found *Methylene Blue Stain was prepared by adding a small amount of methylene powder in distilled water until desired color was obtained Safranin Composition Safrnin: 1% Ethanol: 99% Chemical Name: Safranin 1%, Alcoholic Solution Chemical Formula: Not applicable Physical and Chemical Properties Physical state and appearance: Liquid Odor: Not available Taste: Not available Molecular Weight: Not applicable Color: Not available pH (1% soln/water): Neutral Boiling Point: The lowest known value is 78.4°C (173.1°F) (Ethanol) Melting Point: May start to solidify at -114.1°C (-173.4°F) based on data for: Ethanol Critical Temperature: Not available Specific Gravity: The only known value is 0.789 (Water = 1) (Ethanol) Vapor Pressure: The highest known value is 40 mm of Hg (@ 20°C) (Ethanol).p Vapor Density: The highest known value is 1.6 (Air = 1) (Ethanol) Volatility: Not available Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 54 Odor Threshold: The highest known value is 180 ppm (Ethanol) Water/Oil Dist Coeff.: Not available Ionicity (in Water): Not available Dispersion Properties: See solubility in water, methanol, diethyl ether Solubility: Easily soluble in cold water, hot water, methanol, diethyl ether Crystal Violet (Gentian Violet, Water Solution ) Composition / Information on Ingredients Crystal Violet (548-62-9), 1% Water (7732-18-5), 99% Molecular formula C25H30CIN3 Appearance: Green crystalline powder Molecular weight: 407.99 Odor: No odor Specific Gravity: 1.00 g/mL @ 20°C Odor Threshold: N/A Vapor Density: (air=1) 0.7 (water) Solubility: Soluble in water Melting Point: 0°C Evaporation rate: < (Butyl acetate = 1) Boiling Point/Range: 100°C Partition Coefficient: N/A (log POW) Vapor Pressure: (20°C) 14 (water) pH: N/A Ethanol Composition: Water: 5% Ethanol: 95% Chemical formula: 2C2H5OH Melting Point -1150C Boiling Point 780C Specific Gravity 0.79 Xylol Composition: M-XYLENE: 46% P-XYLENE: 20% ETHYL BENZENE: 19% O-XYLENE: 15% TOLUENE: 0.5% BENZENE: 0.01% Melting Point: -48 C - -25 C Boiling Point: 280 F - 290 F Autoignition Pt: 430 C Flash Pt: 81 F Method Used: Closed Cup Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 55 Explosive Limits: LEL: AP 1% UEL: AP 7% Specific Gravity (Water = 1): 0.87 Density: 7.18 LB/GL at 77 F Vapor Pressure (vs Air or mm Hg): MM HG at 20 C Vapor Density (vs Air = 1): No data Evaporation Rate (vs Butyl No data Acetate=1): Solubility in Water: No data Licensed to W.M Barr and Company ANSI Z400.1 formatRevision: 06/16/2011 Printed: 06/27/2011 Percent Volatile: 100 % by weight VOC / Volume: 870 G/L HAP / Volume: 100 % WT Appearance and Odor: Sweet, pungent aromatic hydrocarbon Gram Iodine Composition: Deionized Water 98-99 Iodine 1.0 Evaporation Rate: No information found Viscosity: No information found Boiling Point: No information found Freezing/Melting Point: No information found Decomposition Temperature: No information found Solubility in water: Soluble Specific Gravity/Density: No information found Molecular Formula: Solution Molecular Weight: No information found Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 56 APPENDIX D CULTURE MEDIA MRS Broth Figure 20 MRS Broth Content Dissolve ingredients in distilled water Adjust medium to pH 6.2 to 6.6 before sterilization for 15 minutes at 121 °C The pH after sterilization should be between 6.0 and 6.5 Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 57 Figure 21 Pasteurized Milk; A Liter of Rosario Pasteurized Carabao’s Milk, B Nutritional Value of Rosario Pasteurized Carabao’s Milk, C Brand Label of Rosario Pasteurized Carabao’s Milk Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria 58 Figure 22 Skim Milk; A Liter of Country Goodness Skim Milk, B Nutritional Value of Country Goodness Skim Milk, C Processing information of Country Goodness Skim Milk Figure 23 UHT Milk; A Liter of Alaska UHT Milk, B Nutritional Value Alaska UHT Milk, C Processing information of Country Goodness Skim Milk