Optimization of Cost-Efficient and Effective Culture Media For Lactic Acid Bacteria (LAB)

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 wouldnt be able to experience the rigorous UP training and I wouldnt 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 do. To my bestfriends Rita and Dids, thanks for keeping me sane every time I get stressed. To my teammates from the UPB Womens Basketball Team and to my sisters from the Sigma Alpha Nu Sorority, thank you for all the moral support. To my friend and mentor, maam 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 analysispasteurized Carabaos 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 1 Background of the Study 1 Research Objectives 2 Null Hypothesis 3 Significance of the study 3 Scope and Delimitation 3 Review of Related Literature 4 Lactic Acid Bacteria 4 Probiotics 5 Rice 6 Milk 7 Sugar Sources 8 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 1
Table showing the quantitative and qualitative observations of FRW, LAB serum and pure stock set-ups
22
Table 2
The three kinds of milk used in the experiment, their processing and their content
32
Table 3
The progression of layers in the FRW set-up
43
Table 4
The number of LAB present in fermented rice wash
43
Table 5
The number of LAB present in the serum produced from UHT, Skim and Pasteurized Milk
43
Table 6
The Number of LAB present in the pure stock produced from brown sugar, white sugar and molasses
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Table 7
Cost of Materials
44
Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria________viii LIST OF FIGURES
Figure
Title
Page
Figure 1 Figure 2 Figure 3
Incubated PetriFilm showing the presence of Lactic Acid Bacteria The process of Lactic Acid Fermentation Bacterial count in LAB serum produced from UHT, Skim and Pasteurized milk Diluents used in the experiments Contaminated FRW with grayish-green mold formations Three replicates of the rice wash set up at day 0 at the ITCPH health laboratory The rice wash set up FRW-milk set ups from day 0 to day 5, pasteurized (left), UHT (middle), skim (right) The LAB serum mixed with each type of food source; molasses (left), brown sugar (middle), white sugar (right). Clean PetriFilm from the package Colony count from FRW set ups at different dilutions. a. 100, b. 10-1, c. 10-2 Colony count from each FRW-milk set ups Colony count of each milk mixed with molasses at 6 dilutions, 10-1 to 10-6. a. Pasteurized, b. UHT, c. Skim milk Colony count of each milk mixed with brown sugar at 6 dilutions, 10 -1 to 10-6. a. Pasteurized, b. UHT, c. Skim milk Colony count of each milk mixed with white sugar at 6 dilutions, 10-1 to 10-6. a. Pasteurized, b. UHT, c. Skim milk Microscopic view of the cells, showing violet-blue colored cocci to rod shaped cells that occur in singles and chains
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Figure 4 Figure 5 Figure 6
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Figure 7 Figure 8
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Figure 12 Figure 13
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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 Samples from incubated Petri Plates (from Skim Milk LAB Serum) for Colony Morphology Observation MRS Broth Content Pasteurized Milk; A. 1 Liter of Rosario Pasteurized Carabaos Milk, B. Nutritional Value of Rosario Pasteurized Carabaos Milk, C. Brand Label of Rosario Pasteurized Carabaos Milk Skim Milk; A. 1 Liter of Country Goodness Skim Milk, B. Nutritional Value of Country Goodness Skim Milk, C. Processing information of Country Goodness Skim Milk. UHT Milk; A. 1 Liter of Alaska UHT Milk, B. Nutritional Value Alaska UHT Milk, C. Processing information of Country Goodness Skim Milk
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Figure 19
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Figure 20 Figure 21
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Optimization of Cost-Efficient and Effective Culture Media for Lactic Acid Bacteria________1
INTRODUCTION Background of the Study Organic farming is slowly gaining attention from the scientific world because of the many advantages associated with it. Organic farming may be defined in various ways but all converge to state that it is a system that relies on ecosystem management rather than on external agricultural inputs. It provides alternatives to the use of synthetic inputs, such as synthetic fertilizers and pesticides, veterinary drugs, genetically modified seeds and breeds, preservatives, additives and irradiation (Food and Agriculture Organization of the United Nations, 2012). A group of bacteria, the LAB (Lactic Acid Bacteria) are used in organic farming. LAB is known to improve digestion, stimulate gastrointestinal immunity, and to increase natural resistance from infectious enteric diseases among livestock and fishery animals. LAB is attributed to promote faster and uniform growth rate, prevention of disease development and epidemic decrease in mortality rate and animal stress, improvement of produce quality, easy conversion of animal waste into organic fertilizers and the elimination of foul odour from pens and ponds. The use of LAB in some farms is already gaining popularity because of its benefits as attested by some users like pig farmers. However, there are still misconceptions with regards to the preparation of LAB such as sugar sources, hence, this study. This study aimed to compare the lactic acid bacterial 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 Carabaos 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 sourcemolasses, 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 lactic acid bacteria. In the Philippines, local institutions are already popularizing the use LAB. One institution that does this is the Agricultural Training Institute - International Training Center on Pig Husbandry (ATI-ITCPH). ATI-ITCPH is one of the 17 training centers of the Agricultural Training Institute whose mother agency is the Department of Agriculture. It was established in June 1985 through the joint efforts of the governments of the Philippines and the Netherlands. This study aimed to contribute to the efforts of the ATI-ITCPH as the lead agency in training and extension particularly in pig husbandry and other related fields like animal waste management, artificial insemination, meat processing and organic agriculture. Findings from this research will be useful to both the researchers and ITCPH as first-hand data which can be shared and taught during trainings and short courses offered to the public. Such information dissemination will improve livelihood and business for both small- to medium-scale pig, Sus scrofa domesticus farmers.
Research Objectives The main objective of this research is to characterize and assess the use of three different types of milk and sugar that will produce the LAB culture with the highest population count and to verify the microorganisms present by using confirmatory tests. In addition, this study has the following specific objectives: i. To optimize the conditions of LAB cultures;
ii. To determine and compare the population count of LAB(from the Fermented Rice Wash) when cultured in three different kinds of milk media; iii. To determine and compare the population count of LAB Serum (from the most productive milk media) when cultured in three different kinds of sugars; iv. To identify and characterize LAB using qualitative tests.
Null Hypothesis (Ho): LAB cultures using pasteurized Carabaos milk, UHT fresh milk and skim milk combined with molasses, brown sugar and white sugar will produce LAB that exhibit the same population count.
Significance of the Study This study aims to come up with basic methods using cheap materials for the production of the most efficient lactic acid bacteria serum that can be used for the most cost-effective and efficient means that can be used in better pig-raising in the Philippines.
Scope and Delimitation This study is delimited to utilizing only one kind of riceC4 Dinurado rice; three kinds of milkpasteurized Carabaos milk, UHT fresh milk and skim milk; and three kinds of sugar molasses, white sugar and brown sugar. A quantitative test using the PetriFilm count was used in order to compare the population count of LAB in each experimental set-up. Qualitative tests like simple staining, colony morphology, gram-staining and gas production from glucose were conducted to confirm the presence of the LAB. Application of the produced LAB Pure Stock on actual test organisms was not part of the study.
REVIEW OF RELATED LITERATURE Lactic Acid Bacteria Bacteria are defined as minute, unicellular, plant-like, microscopic organisms that undergo reproduction thru binary fission (Bryan, 1962). They are said to be plant-like because they do not contain chlorophyll but they find ways in which they can produce their own food, usually by the process of fermentation. Lactic acid bacteria are acidic in that they produce acid, but the true Lactic Acid Bacteria (LAB) under which Lactobacillus is classified, is the most famous and largest genus. Lactobacillus is characterized as gram-positive, non-motile, nonspore-forming rods/cocci and their distinctive feature; fermenting carbohydrates and higher alcohols into lactic acid (Kraatz, et al, 2010). Gram positive bacteria are characterized with a cell wall composed of a very thick layer of peptidoglycan, which when tested with crystal violet gram dye, retains the color of the dye and is said to be positive to the gram. Also, Lactobacillus are said to be anaerobic, organisms that can grow low or even in the absence of oxygen. According to Priest and Austin in 1993, Lactobacillus is classified under the Kingdom Procaryotae, Division Firmicules, Order Eubacteriales and Sub-order Eubacteriineae (Bryan, 1962), Family Lactobacillae, and the Genus Lactobacillus. This genus is composed of 180 species. In addition to that, Eubacteriales play an important role in our ecosystem as decomposers. They are responsible for the global cycling of nitrogen, sulfur, iron, phosphate, and other nutrients vital for life to proceed (Weed, 2012). Also, the genus Lactobacillus is included in a special group named LAB sensu stricto, under the Phylum Firmicutes which have low content levels of Guanine and Cytosine that is the main reason for its milk -souring attributes (Kraatz, et al, 2010).
Like all living organisms, microbes, specifically the Lactobacillus demands a certain diet or a specific nutritional requirement. Aside from carbohydrates, amino acids and vitamins are also fermented by the Lactobacillus (Kraatz, 2010). When LABS (Lactic Acid Bacteria Serum) are cultured, it is mixed with molasses (or any sugar source) that can act as its main food source, providing the nutritional requirements it sets. After this, the solution of LABS and molasses are added to the commercial feeds that organically-raised animals eat. These feeds provide the Lactobacillus major nutrient supply in the form of easily fermentable carbohydrates (Kraatz, 2010).
Probiotics Probiotics refer to a live culture of microorganisms that elicit a positive effect on its host, in the action of improved digestion, stimulation of gastrointestinal immunity, and to increase natural resistance from infectious enteric diseases (Turner, et al, 2005). With this, probiotics are used to enhance the growth of livestock, most specifically in organic farming. Lactic Acid Bacteria are used as a probiotic for pig-raising or swine growth. The major opponent in animal farming is the smell coming from the feces of the animals. Portions of ammonia are excreted through the feces of animals and this gives off the foul smell. In a study by Hamilton, et al, in 1998, Sus scrofa domesticus were exposed to ammonia which caused the degradation of their nasal excrement and a progressive atrophic rhinitis. With this problem, the use of LAB was discovered. When LAB is introduced to the feces of the animals, it breaks down the ammonia and eliminates the odor that it gives off (Kraatz, 2010). In general, Lactic Acid Bacteria with respect to Swine Growth are attributed to faster and uniform growth rate, prevention of disease development and epidemic, decrease in mortality rate
and animal stress, improvement of produce quality, easy conversion of animal waste into organic fertilizers and the elimination of foul odors from pens and ponds (ATI-ITCPH Primer, 2012). Lactobacilli are naturally found in the human body; in the stomach, in the large intestine, and in the genitourinary tract. The largest population of Lactobacillus in the human body is found in the large intestine (Prescott, 1996). This is where most digestion takes place and in these processes, the microbes help for proper digestion. On the other hand, other species of Lactobacillus can be found in fermented or cultured food like distilled spirits, meat and fish, bread, vegetables, milk and in grains. In most researches and for household uses, rice is mainly pointed as the most accessible source of Lactobacillus.
Rice The exteriors of freshly harvested grains contain some of the natural flora and contamination from soil and other sources, resulting to a few thousands to millions of bacteria per gram and from none to several hundred thousand mold-spores, and perhaps, spores of smuts and rusts (Frazier, 1967). Thus, washing the rice with water will gather most of the microorganisms from the exteriors of the grains. Scouring and washing of the grains removes part of the microorganisms, but most of the microorganisms are removed with the outer portions of the grain during milling (Frazier, 1967). Microorganisms from grains are mostly from the families Pseumonadaceae, Micrococcaceae, Lactobacilaceae and Bacilaceae (Frazier, 1967). Rice wash can be considered as rice waste in which lactic acid can be gathered. In Greenes (2009) research project, isolated rice bran and rice hulls were treated with acid then heated in an autoclave and treated again with enzymes for increased conversion of waste rice to glucose and production of lactic acid by
bacterium. Yu and Hang (1989) experimented with Rhizopus oryzae NRRL 395 in surface culture to convert D-glucose to a large amount of L(+)-lactic acid in the presence of calcium carbonate.
Milk Generally, milk is rich in microbial foods such as lactose, butterfat, citrate, nitrogenous compounds (proteins, amino acids, ammonia and urea), and other accessory foods and minerals. Aside from its rich food content, it is also high in moisture and nearly neutral in pH which makes it an excellent culture medium for many kinds of microorganisms. Naturally, milk contains few bacteria after being expelled from a healthy cow and accumulates through the stages of handling and productions, unless strict sanitary precautions are employed all throughout (Frazier, 1967). Raw milk is most likely to undergo lactic acid fermentation at room temperature with homofermentative Streptococcus lactis, coliform bacteria, enterococci, lactobacilli and micrococci, being responsible for most acid formation (Frazier, 1967). Pasteurization or the process of treating milk at high temperatures and then cooling immediately to 45 F or lower produces what we call pasteurized milk. This process is employed in order to kill pathogens and improve the storage quality of the milk. Through the process, yeasts, molds and majority of vegetative cells are expected to be killed; however, a number of bacteria survive the high temperature thus, termed thermodurics. Among those that survive are the high-temperature lactobacilli, such as Lactobacilli bulgaricus and Lactobacilli lactis which constitute 90-90% of the thermoduric bacteria present (Frazier, 1967). Commercial ultra-heat treated milk is processed at higher temperature than pasteurized milk. Skim milk, being prepared in dry form, is categorized under dry dairy products. Its microbial content depends on the liquid
product to be dried, temperature, time of preheating, evaporation process and method of drying. Skim milk has low moisture content that prevents the growth of microorganisms by slowing down the oxidation of fats. However, high acid concentration is brought about by accumulation of molds when exposed to air, which also prevents the growth of bacteria and yeasts (Frazier, 1967)
Sugar Sources Commercially acquired sugar comes from natural sources like beets and more commonly, sugar cane. Sugar cane or beets are squeezed so that their juice may be collected. This juice is then boiled that causes the juice to thicken. The product of this heating is what we call molasses. Boiling the mixture for a little longer, sugar crystals settle out. The sugar crystals can be collected by methods of centrifugation and drying. If you mix part of the molasses with part of the crystals, you come up with brown sugar. But, if you totally separate the sugar and process it further by bleaching and purification, you get white sugar. Sugar cane juice may contain the following groups of microorganisms: species from Leuconostoc and bacillus; under the genera Micrococcus, Flavobacterium, Achromobacter, and Aerobacter; a variety of yeasts, chiefly in the genera Saccharomyces, Candida and Pichia; and a few molds (Frazier, 1967). Molasses Sugarcane and sugar beets are used as sources of sugar, most commonly in countries where the two are highly cultivated. Sugar production involves boiling of the extracted juice from the source until the sugar crystallize and precipitate; the left over syrup is then referred to as molasses. Molasses may be available in four varieties, depending on its stage of boiling and
sugar content. Sugarcane juice undergoes first, second and third cycle of boiling and each level produces the light, dark and blackstrap molasses, respectively. Light molasses has the highest sugar content while blackstrap molasses contains the least sugar with the highest concentration of vitamins and minerals. Color and viscosity intensifies by the succeeding boiling cycle; thus light molasses is lightest and least viscous, followed by dark molasses and blackstrap molasses as the darkest and most viscous texture. The fourth variety of molasses is referred to as sorghum molasses and is made from the name itself for animal feeds and ethanol production, among other things (Moncel, n.d.). Over the years, molasses has been used as a substitute for sugar since the production and acquisition of the latter has always been highly costly. Palatability is one of the factors considered in determining the quality of diets used for early weaned Sus scrofa domesticus (Hovell et al, 1975). Cane molasses is used as an additive to enhance the palatability of the feed. Liquid molasses is used particularly in swine feed. It is a black, syrupy solution containing 15-25 percent water and 46 percent sugars (Cheeke, 1991). Molasses has been applied in the field of microbiology, specifically for the growth of Lactobacilli and other bacteria. It is generally used as a source of energy and trace elements (Pond et al., 1995). Because of this property, lactic acid bacteria serum could be kept for 6 months as it provides enough energy source for the microorganism. Ortiz, et al. (2012) used sugarcane molasses as low-cost energy source for the production of mannitol by Lactobacillus reuteri CRL 1101. This study has been successful in producing mannitol from sugarcane
molasses, a low-cost substrate for microbial mannitol synthesis (Ortiz, et al., 2012). Calabia and Tokiwa (2007) grew Lactobacillus delbrueckii on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40C for 72 hours.
Sugarcane molasses, sugarcane juice and sugar beet juice were used as substitutes for the costly production if refined carbohydrates such as glucose and starch is utilized (Calabia and Tokiwa, 2007). On Monteagudo, et al (1997) study on the kinetics of fermentation to convert beet molasses to lactic acid by the homofermentative organism Lactobacillus
delbrueckii C.E.C.T. 286 at controlled pH and temperature under anaerobic batch conditions. It was found out in this study that lactic acid has an inhibitory effect on fermentation of beet molasses (Monteagudo, et al., 1997). Krzywonos and Eberhard, (2011), in an attempt to obtain a low-cost medium for a large scale industry production of Lactobacillus plantarum biomass utilized wheat stillage and sugar beet molasses as carbohydrate and/or nitrogen sources since it contains approximately 50% carbohydrates, where sucrose, glucose and other sugars account for 96%, 3% and 1.4%, respectively. The works of Reyed and El-Diwani (2008) showed that highly active growth promoters for Bifido bacterium bifidum is present in blackstrap molasses due to its high nutrient content such as copper, calcium, iron, potassium, magnesium, chromium, manganese, molybdenum, zinc, phosphorous, pantothenic acid, vitamin E and inositol, as well as biotin which enhance the rate of fermentation. Enhanced fermentation rate resulted in increased. Bifidobacteria and lactic acid bacteria numbers increase with the subsequent increase in acetic and lactic acid concentration (Reyed and El-Diwani, 2008). Furthermore, Reyed and El-Diwani, (2008) mentioned that molasses did not inhibit lactic acid and Bifidobacteria production when added to skim milk at a level of 5 %. As there are positive results for the utilization of molasses for lactic acid production and Lactobacilli growth, there also studies with a different conclusion. Kotzamanidis, et al., (2002) made use of beet molasses for the production of lactic acid by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation. According to
the study, maximum lactic acid concentration was achieved without treatment of molasses, rather sucrose, yeast extract and CaCO3 yielded the maximum concentration of lactic acid (Kotzamandis, et al., 2002).
Brown Sugar There are four more commonly known kinds of brown sugar; the light and brown which are used for baking, the Muscovado Sugar and the Turbinado sugar. According to the Sugar Association, the light and dark brown sugar, are not completely refined as white sugar is, retaining some molasses syrup flavor. The four types of brown sugar differ in the molasses sugar crystals proportion they contain which is comparable to the proportion of processing they undergo. The more crystals a sugar type contains, it is then more processed. Dark brown sugar as its name suggests is darker with a larger proportion of molasses which is used in heavier flavored foods while light brown sugar is for making glazes, condiments and candies, turbinado sugar is characterized as raw sugar with little but large brown crystals and is thicker (because of a larger proportion of molasses, and lastly, muscovado sugar is similar to turbinado but has a stronger taste (Frazier, 1967).
White Sugar The usual processing of sugar starts from sugar cane or beet which is then processed for increased shelf life and to eliminate harmful microorganisms contained hereunto. After harvesting raw sugar from sugar cane juice and beet juice, it is then processed into molasses then into brown sugar and lastly into white sugar. Thus, white sugar is the most processed form of sugar available in the market today. During the manufacture of sugar cane juice to sugar, it gets
more purified and as this happens, the poorer it gets as a culture medium for microorganisms since it contains less vitamins, calcium and iron; and the more processed it becomes, the fewer kinds of microorganisms can grow in it (Frazier, 1967).
Related Studies A study conducted by Karna and Barraquio in 2007 at the University of the Philippines Los Banos, Laguna, Philippines focused on using different kinds of yogurt as media for Lactic Acid Bacteria and comparing the bacterial growth rate in each. They used locally available fermented dairy products, namely: Nestle Yoghurt Natural (YN), Fruit Yoghurt (FY), Non Fat High Calcium Yoghurt (NC), Yoghurt Drink (YD), and Duo Yoghurt (DY). It showed that the most growth of Lactobacillus was found in the Duo Yoghurt and lowest in the Yoghurt Drink (Karna, Emata and Barraquio, 2007). In growing the Lactobacillus, they used MRS Agar. They concluded that the following genera and species of LAB and probiotic bacteria were present in the dairy products examined: Pediococcus acidilactici (YN), Pediococcus pentosaceus (FY), Lactobacillus delbrueckii delbrueckii and Lactobacillus brevis (NC), Lactobacillus acidophilus and Lactobacillus delbrueckii delbrueckii (DY), Lactobacillus delbrueckii delbrueckii and Lactobacillus acidophilus (YD), Pediococcus damnosus and Pediococcus pentosaceus (CO), Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus and Lactobacillus delbrueckii delbrueckii (CP), Lactobacillusparacasei (YK), and Bifidobacterium sp. (Nes and Nan). Another study conducted at the University of the Philippines Los Banos, Laguna by Kisworo, Elegado and again, Barraquio focused on the genetic characterization of Probiotics. Here they spoke in detail of the methodology of the Polymerase Chain Reaction. Probiotic samples were isolated form locally available products like Chamyto and Yakult, serial dilutions
in Ringers solution was done, pour -plated in MRS agar, incubated anaerobically and then microbial count was recorded in CFU ml-1 (Kisworo, Elegado and Barraquio, 2008). Genetic Classification via Phenotypic and Genotypic Identification were done. For the Phenotypic Identification, morphological characteristics of the bacteria were examined by observing the colony growth, cell morphology and Gram reaction (Kisworo, Elegado and Barraquio, 2008). On the other hand, Genotypic Classification was conducted via the Polymerase Chain Reaction Method, PCR-Based Fingerprinting, Analysis of Amplified Products, amplification of the 16S rRNA genes and DNA Sequencing through BLAST. To sum it up, the biochemical reaction profiles of 6 selected isolates showed that they were all Lactobacillus paracasei (Kisworo, Elegado and Barraquio, 2008).
MATERIALS AND METHODOLOGY

PRODUCTION OF LACTIC ACID BACTERIA SERUM (LABS)
Collection of rice wash Five-day fermentation Filtration at day 5
Fermented Rice Wash (FRW)
Mix with Pasteurized Carabaos Milk
Mix with Skim milk
Mix with UHT milk
Confirmatory Tests
Simple staining
Population Count
Colony count using Petrifilm
Five-day fermentation Gram staining Filtration at day 5 Colony observation
LACTIC ACID BACTERIA SERUM (LABS)
Gas production from glucose
Confirmatory Tests
Simple staining Gram staining
Population Count
PASTEURIZED CARABAO MILK

Colony observation Gas production from glucose
Mix with Molasses
Mix with White Sugar
Mix with Brown Sugar
Confirmatory Tests
Simple staining Gram staining Colony observation Gas production from glucose
Population Count
Five-day fermentation Filtration at day 5
PURE STOCK
Optimization of conditions for the culture and collection of LAB From the locally available C4 Dinurado rice, good grains were chosen and washed in distilled water (with a ratio of 1 cup rice to 1 and a half cup water). C4 Dinurado rice was used as the source of lactic acid bacteria to be used in the succeeding parts of the experiment. It was chosen because it is readily available and is relatively cheap in the country. Oxygen level, temperature, ventilation and exposure to light of all setups were constantly monitored to maintain optimal conditions for the growth of LAB. One litre of rice washings was collected and kept in an air-tight container to avoid contamination and to lower oxygen level. The sample was then kept in a cool and dry place with temperature of approximately 23-25 C at ITCPH Health Laboratory. The set-ups were not directly exposed to the sun and the windows were open to allow air to pass through the room. After 5 days, the fermentation process of the samples was checked if layers formed. The sample produced three layers: thin film of rice bran on the very top, the clear liquid in the middle and lastly, the residues on the bottom. The clear liquid in the middle was identified as the fermented rice wash or FRW that was then used in the next step of the research. At day 5 of fermentation, the liquid with its distinctly separated layers was filtered, using a clean cheese cloth, into a clean container. A portion of the FRW was tested with the PetriFilm to determine the bacterial count and another portion was used for the confirmatory tests. Use of Milk as media for the growth of LAB The FRW obtained from the filtration of the rice wash was then mixed with three kinds of milk; (1) Alaska Ultra-Heat Treated (UHT) Milk, (2) Country Goodness Skim Milk, and (3)
pasteurized Carabaos milk. These were used as media for the LAB in order to compare the extent of their population. 100 ml of FRW was mixed with 1L of milk (1:10 ratio). Addition of milk was done to enhance the growth of lactic acid bacteria while eliminating other species of bacteria. The container was again covered tightly to avoid contamination and was kept in an area with 23-25C for 5 days. After day 5 of fermentation; starch, protein, and fat, also characterized as cheese, floated to the surface of the solution. The light yellow or clear liquid below the cheese is the lactic acid bacteria serum (LABS). The cheese was removed immediately to eliminate impurities while the serum was transferred to another air-tight container kept under 1-15C (ATI-ITCPH, 2012). LABS with the highest population count, was further mixed with three types of sugar so as to provide the bacteria with nutrients and to extend the shelf life of the material. On the other hand, a portion of the LABS was tested with the PetriFilm to determine the bacterial count and another portion was used for the confirmatory tests. Preparation of LAB Pure Stock The lactic acid bacteria serum which had the highest population count according to the results from the Petrifilm was mixed with three kinds of sugar: (1) molasses, (2) dark brown sugar and (3) white sugar. After five days of fermentation, the resulting liquid, called the pure stock, can be stored for up to 6 months. A portion of the pure stock was tested with the PetriFilm to determine the bacterial count and another portion was used for the morphological tests.
Identification of population extent obtained from FRW, LAB Serum and Pure Stock Population count using Petrifilm 3M Petrifilm was used in this research with triplicates of 3 dilutions of 10-4, 10-5, and 10-6 from each sample. But for the Pure Stock, six dilutions in triplicates were done: 10 -1, 10-2, 10-3, 10-4, 10-5, and 10-6. Each dilution was mixed with equal amounts of MRS (de Man, Rogosa, Sharpe) broth (http://solutions.3mphilippines.com.ph, 2012). Petrifilms that produced 30-300 were selected to be observed. The Petrifilms were placed on a flat surface and the top film was lifted. With a pipette angled perpendicularly to the Petrifilm, 1ml of sample (dilution mixed with MRS broth) was placed onto the center of bottom film (from which the top film was lifted off from). Then, the top film was carefully rolled down in order to avoid entrapping of air bubbles. A plastic spreader, included with the Petrifilm package, was placed on top of the top film with the spreaders curved side down on the films. Pressure was gently applied on the spreader to distribute the inoculums over a circular area (shaped by the spreader) before the inoculums hardens into a gel. The spreader was then lifted and a minimum of one minute was then allowed for the gel to solidify. The Petrifilm with hardened gels were then stacked up to as much as 20 films and placed inside a sealed glass jar and were incubated using the candle oats method. The research aimed to identify lactic acid bacteria which are anaerobic microorganisms and in order to create an environment which is of very little or absolutely no oxygen; the candle oats method was used wherein the flame of the candle will eventually be extinguished signaling that the environment inside the jar is deprived of oxygen since there is no more oxygen sustaining the life of the candles flame. The Petrifilm were incubated at 30 -35 C in an oven for 24 hours (Dalmacio, 2009). After the incubation period, a red indicator dye in the film reacted with the growing lactic acid bacteria allowing colony visualization. Acid producing bacteria produce a
yellow zone (halo) around the colony (http://solutions.3mphilippines.com.ph). A random box on the PetriFilm was chosen, the lactic acid bacteria present were counted. The CFU/ml and growth rate were the computed in order to measure the rate of microbial growth. Statistical Analysis Results of the study were analysed via one-way ANOVA which include Post-Hoc Tests as in Multiple Comparison and Duncans Test. Significance level was determined at p values equal to 0.05.
Confirmatory Tests Simple Staining In simple staining, cell morphology and purity of the sample was examined. In this staining technique, a loopful of culture broth was transferred onto a microscope slide. The sample was then dried, fixed by exposure to flame 2-3 times for 1-2 seconds and stained with methylene blue for 1-2 minutes. Excess stain was washed under tap water and dried by blotting onto cotton towels and drying. After simple staining, cell shape and arrangements were examined under the light microscope. Samples showing heterogeneous cell morphology were eliminated.
Colony Morphology Colony characteristics of the different samples were observed by using the streak plate method. MRS broth was mixed with agar which was dissolved with water. This mixture was then boiled and cooled until it was ready to be poured into petri plates. When the mixture solidified into a gel, a loopful of inoculum was then streaked unto the plates. This was then sealed in a
glass jar using the candle oats method since LAB are anaerobes. The petri plates were incubated for 3 days, after which morphological characterization of the colonies that grew in the agar were described. With the characteristics portrayed, possible strains of Lactic Acid Bacteria were then identified.
Gram Staining LAB are known to be Gram positive. To confirm the presence of LAB in the sample, this method was performed. A loopful of sample was transferred onto a glass slide, dried in open air and fixed by exposure to flame 2-3 times for 1-2 seconds. It was primary stained with crystal violet for 1 minute. Excess stain was then washed under tap water. Grams iodine stain was applied for another 1 minute and washed with tap water. 95% alcohol was applied for 6 seconds to decolorize the agent. Safranin, which is a counter-stain, was applied for 30 seconds. The slide was dried and observed under the light microscope.
Gas Production from Glucose Lactobacillus is known to be homofermentative. In order to define homofermentative isolates, CO2 production from glucose test was performed. Isolates from the 3M petrifilm were inoculated into MRS broth with durham tubes. These were incubated for 48 hours at 35C, after which, it was observed if gas was present in the tubes or not.
RESULTS
After conducting two unsuccessful and mold-contaminated set-ups (Appendix B, Figure 5), physical conditions such as low oxygen level, temperature range of 23-25 C, high ventilation and low light exposure were noted to provide optimal conditions for the growth of LAB. Lactic Acid Bacterial count was considered present in a PetriFilm if there were small pinkish-violet dots observed (Figure 1).
Figure 1. Incubated PetriFilm showing the presence of Lactic Acid Bacteria
From the optimized conditions established, a fermented rice wash with microbial population count of 6.40 x 105 cfu / ml based from Petrifilm set-ups. FRW was noted to have a pH of 5 and a sour odor (Table 1). LAB Serum obtained from UHT Milk as identified to have the least number of microbes (1.493 x 1010) than LAB serum from Skim Milk that contained more microbes (2.091 x 1010),
while LAB Serum from Pasteurized Carabaos Milk had the most number of microbes (3.619 x 1010). Qualitative observations revealed that the LAB Serum and pH from UHT Milk had the lightest color and the pH of 4.5 whereas Skim Milk had a yellowish serum and the highest pH of 5 and Pasteurized Carabaos Milk obtained the darkest serum with the lowest pH of 4. Cheese was formed after a five-day fermentation period. Cheese characteristics obtained from UHT Milk weighed 297.9 g and was observed to be the darkest with very large lumps and medium-sized air holes, while Skim Milk weighed the lightest cheese of 269.9 g which was light yellow with small lumps and numerous small sized air holes and lastly, Pasteurized Carabaos Milk weighed the heaviest cheese of 321.3 g being the lightest and smoothest cheese with large air holes (Appendix B, Figure 8). Upon identifying that Pasteurized Carabaos Milk had the highest population count; it was mixed with three kinds of sugars. All pure stock setups resulted to highly numerous microbial counts that are insignificantly different from one another. Results from direct microscopic observations of all setups showed that cells were cocci to rod-shaped, confirming the presence of lactic acid bacteria. Gram staining revealed that in FRW Gram positive and Gram negative bacteria were present, this indicates that other bacteria aside from LAB exists in the sample. On the other hand, all LAB Serum and pure stock set ups showed only Gram positive bacteria, indicating that lactic acid bacteria persisted in the mixtures. No gas was produced from the Gas production from Glucose test which confirmed that all setups contained homofermentative bacteria. Colony characteristics of all setups were observed to be round, smooth and convex, white, cream or light yellow, and non-transparent (appendix B, Figure 19).
Table 1. Table showing the quantitative and qualitative observations of FRW, LAB serum and pure stock set-ups; LAB Serum Assessment Fermented Rice Wash UHTTreated Milk 1.493 x 1010 Skim Milk Pasteurized Carabaos Milk Pure Stock Molasses Dark Brown Sugar White Sugar
Quantitative Petrifilm Count (cfu/ml) Qualitative LAB Serum Color pH Weight of Cheese 5 N/A 6.40 x 105 2.091 x 1010 3.619 x 1010 TNTC TNTC TNTC
Lightest 4.5 297.9 g Darkest with very large lumps and mediumsized air holes
Yellowish 5 269.9 g Light yellow with small lumps and numerous small sized air holes
Darkest 4 321.3 g N/A N/A N/A N/A N/A N/A
Cheese characteristic
N/A
Lightest and smoothest with large air holes
N/A
N/A
N/A
Confirmatory Tests Cell Morphology Gram Staining Cocci to rod-shaped cells; some cells appeared in chains, some individually Presence of Gram + and bacteria Gram +
Gas Production No gas production from Glucose Colony Round, smooth and convex, white, cream or light yellow, and non-transparent Characteristics
*TNTC (Too Numerous To Count) as per readings from the PetriFilm.
Pasteurized Carabaos Milk produced the most number of lactic acid bacteria, the most acidic LAB Serum and the heaviest cheese. All three sugars produced high population count of lactic acid bacteria that were insignificantly different from each other (Table 1). Gram negative bacteria were only observed at the Fermented Rice Wash level.
DISCUSSION
Results showed that using Dinurado rice washings, pasteurized milk and molasses is the best combination for producing Pure Stock of Lactic Acid Bacteria. The Dinurado rice-washings were the main source of the LAB used in this research, not from pure cultures that are commercially available. The C4 Dinurado rice was chosen because this is readily available and is relatively cheap in the country. The healthy appearance of rice seeds does not directly suggest that it is free from any fungal infection (Paderes, 1987). The rice wash contains various kinds of microorganisms accumulated from the soil, the transportation and the packaging of these grains. A number of isolated fungi from rice were identified in 1982. These are: Alternaria, Curvularia, Drechslera, Fusarium, Epicoccum, Nigrospora, Pyricularia, Trichoconis, Aspergillus and Penicillum. Fungi have long been distinguished as major causes of the decay of organic materials, particularly in the spoilage of storage grains and grain products (Paderes, 1987). For the study to be accurate, more LAB population and lesser of all the other microorganisms are needed. In other words, the environmental conditions of the LAB were experimentally controlled in such a way that it supported their growth optimally. The first two attempts in coming up with a fermented rice wash failed due to contamination with molds because conditions were not conducive to LAB growth. Trial and error guided by the results of previous works eventually led to the identification of the optimal conditions necessary in culturing the LAB, namely; low oxygen level, a temperature range of 2325 C, high ventilation and low light exposure. These conditions are consistent with the researches of Pederson.
Molds and bacteria commonly grow in cereal and seed fermentations which is clearly associated with spoilage (Pederson, 1971).The researchers had to go back to the drawing board and review the optimal growing conditions of the LAB and had to repeat the set-ups by placing the containers in a different environment which will be explained in detail further into the discussion. In order to solve the problem of contamination with molds, the researchers identified why the molds appeared what they could change in order to prevent this from happening in the future set-ups. In trying to solve the two questions, the physical factors like the level of oxygen, the temperature, moisture content and pH that affect the set-up and the microorganisms living and growing in the environment were considered. The types of organisms that will grow and develop in the milk depend on some factors such as sanitary conditions, prevailing temperatures, and other environmental factors (Pederson, 1971). As was mentioned, Lactobacillus which make up the largest chunk of lactic acid bacteria are anaerobic in that they live optimally in very low or even no oxygen. LAB are referred to as microaerophilic as they carry on essential metabolic biological processes without oxygen by means of a complex series of intramolecular oxidations and reductions. Molds, as opposed to Lactobacillus, are oxygen requiring. Upon the exposure of food to air, the oxidation-reduction potential increases and the growth of molds and some microorganisms are permitted and favoured (Pederson, 1971). It may be possible that the containers were not sealed properly and high levels of oxygen were exhibited leading to the favourable growth of the molds which then overshadowed and dominated over the growth of the Lactic Acid Bacteria. In other words, this
failure of fermentation may be accounted for by the dominance of the undesirable organisms (molds in this case) and the delay of growth of the desirable organisms (LAB) (Pederson, 1971). Another physical factor we should consider is the temperature. Most yeasts and molds thrive in the lower mesophilic range which is near 20 to 25C while Lactic acid bacteria grow well at 20 to 40C and optimally between 30 to 32C (Pederson, 1971). It can be said that molds and yeast grow optimally at colder environments and temperature cannot be considered as the main reason as to why the molds predominantly existed in the first two trials. The third physical factor considered is the moisture of the environment. Some factors such as the nutrients, temperature and oxygen affect the moisture requirements of microorganisms. The moisture content required by the microorganism to grow is the quantity needed to supply enough nutrients for growth (Pederson, 1971). The laboratory in which the first two set-ups were conducted was constantly exposed to direct sunlight and thus leading to a hotter environment which supposedly favoured the growth of the lactic acid bacteria. But along with this high level of temperature, a humid environment in the containers was achieved which led to moisture in the form of water droplets forming at the inner walls of the containers. It is a fact that moist conditions favour the growth of molds. This could be accounted for as the second reason for the visible growth of the molds. The fourth and last physical factor that was taken into consideration was the level of pH. Most molds may grow in pH 2.0-8.5 which exceeds the range of pH developed in food fermentations. This is a very wide range of growth for molds. And in order to prevent mold growth, oxygen requirement should be restricted (Pederson, 1971).
After studying the physical factors, the researchers washed rice again, transferred it into a clean container and was then sealed very tightly to lower the level of oxygen inside the set-up. Next, in order to control the moisture of the set-up, the containers were kept in a cool place where windows were opened to allow air circulation and set-ups were not directly exposed to sunlight. This maintained a temperature range of 23-25C. LAB produced acid as they ferment. The incubation of 5 days made sure that the LAB produced enough acid to make their environment acidic to eliminate the existence of microorganisms that could not tolerate high levels of acid. The LAB from the FRW were then maintained in a culture medium pasteurized milk. Like any other living organism, Lactobacilli from the rice wash need food in order to survive. This genus of microorganisms ferments carbohydrates and higher alcohols into lactic acid (Kraatz, et al, 2010). A good source of carbohydrates is milk and other sugar products that is why these are used as the next media after having gathered enough population of Lactobacilli from the rice wash. In order to characterize the lactic acid bacteria cultured in the different milk media and to be able to assess the better food source of these microorganisms, we must first understand the processes that they undergo to survive. In the fermentation process, microorganisms such as bacteria, yeasts and molds furnish the energy needed for metabolism and growth by fermenting foods. Microorganisms that bring about desirable changes from the enzymes they produce (that chemically transform the organic substances present in the substance) can be distinguished from those that are responsible for spoilages, illnesses and death (Pederson, 1971). Along with the production of desirable changes, little loss in nutritive values is observed when fermentation
occurs. Specifically, the metabolism process of lactic acid bacteria are so similar to the metabolic processes occurring in the animal body that humans can utilize the fermented foods. It is only logical that the growth of one microorganism, such as the Lactobacillus, will be promoted if it is supplied with its preferred food. In this sense, lactic acid bacteria are said to be fastidious. Lactic acid bacteria, in general, require carbohydrates, amino acids and peptones, lipids, vitamins and minerals for their growth (Pederson, 1971). Carbohydrates are found in foods rich in sugar and starch. These foods are the main source of energy and are then called the GO foods. As for the lactic acid bacteria, carbohydrates are necessary in the anaerobic mechanism of energy utilization. Carbohydrates are fermented into lactic acid with some by-products like volatile acid, alcohol and carbon dioxide. These substances make carbohydrates essential for good development (Pederson, 1971). Lactic acid bacteria need proteins, vitamins and mineral elements certain amounts. Proteins, as explained in the molecular level, are the enzymes that permit or stop processes in the cell to occur. Proteins regulate the processes that occur inside the cell and are also responsible for the proper functioning of the cell. Proteins are the most valuable content of milk and can be split into two major components: caseins and whey products and in connection to this, the ratio of casein to the total protein in milk can determine the amount of cheese that can be made, if the ratio is lower than the usual ratio of 80, it can be difficult to make cheese (Boland, 2003). Just like humans, lactic acid bacteria also need supplements in the form of vitamins. Vitamins are needed in small amounts for growth and proper functioning of the body and are taken in when the organism cannot provide this kind of substance. B vitamin factors, vitamins A, C, D, E and K are needed by lactic acid bacteria (Pederson, 1971).
Lipids are found in plant and animal cells. These are substances that are insoluble to water and needed in maintaining the stability of cells and providing a source of energy for the body. During lactic acid fermentations, fats and complex phospholipids undergo certain chemical reactions exemplified by hydrolysis and increase in fatty acids and in choline that when combined with lactic and acetic acids, they form lacteal and acetyl cholines (Pederson, 1971). After determining the needed substances for fermentation to take place, it is essential to consider the process itself and lastly the products of the process (Figure 1). Glycolysis, a process which aims to gather energy in the form of ATP is involved in the fermentation of lactic acid. 2 ATP molecules; and lactate are the main products seen in the image. But, specifically, during homofermentation, lactic acid is the primary product (Pederson, 1971).
Figure 2. The process of Lactic Acid Fermentation
Milk was identified as the next medium for lactic acid bacteria. The overall chemical composition of cows milk is an ideal growth medium for heterotrophic microorganisms, including the nutritionally fastidious gram-positive lactic acid bacteria (Jay, 2005).
Milk provides enough supply of simple disaccharides and nitrogen source (Law, 1997). The type of growth of microorganisms in milk is controlled by the process through which it was produced. The microorganism should also be able to obtain energy from lactose and utilize milk proteins (Law, 1997). Some microorganisms capable of fermenting milk should be considered since they would be expected to be the ones found in the results of the research. Aside from Streptococcus lactis and Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus lactis are also important milk-fermenting bacteria (Pederson, 1971). In fermented milks, the lactose of the original milk is converted to lactic acid, citric acid is converted in part to diacetyl, and some other minor changes occur but none decrease the original nutritional value, so we can say that acid fermentation always results to fermented milks where the sugar in the milk is changed to lactic acid and other products (Pederson, 1971). Milk also helps eliminate other microorganisms and thus, promote the growth of the LAB. Competition always occurs among many microbial species present in fresh milk. Many cause undesirable effects even to the consumer. When and if lactic acid bacteria grow over the number of the other species present, they produce acid, lowering the oxidation-reduction potential, and causing death of other microorganisms (Pederson, 1971). Milk may be initially fermented by Streptococci and coliforms, continued by acid tolerant high acid-producing Lactobacilli, followed by film yeasts and molds that consume the acid. The presence of proteolytic bacteria may convert the milk to its simple elements (Pederson, 1971). This is parallel to the results obtained from the experiment; the Fermented Rice Wash tested gram positive and negative bacteria, meaning there was a mixture of lactic acid bacteria and other microorganisms. Upon the addition of milk, and after allowing the mixture to ferment for five
days, the LAB Serum tested purely of gram positive bacteria, meaning the gram negative bacteria were eliminated by the presence of milk. Lactic acid is a by-product of the fermentation process. Most bacteria thrive in pH near neutrality while a few are favoured by acidity or alkalinity; those favoured by a reaction in acid are lactic acid bacteria, acetic acid bacteria, propionic acid bacteria and a few more. They produce acid in foods which is lower than the pH tolerance of a large majority of other bacteria including pathogens (Pederson, 1971). The production of lactic acid does not entirely inhibit the growth of molds. In several fermentations, this acid permits the growth of molds responsible for the aroma, texture and flavour characteristics of several mold-fermented foods such as in the Roque and Camembert cheese fermentations (Pederson, 1971). But the growth of these molds and other microorganisms must be controlled so that they may not overshadow the growth of the lactic acid bacteria that we want to produce. The researchers maintained the set-up where the container is sealed in order to lower the level of oxygen and thus, milk was added. As a form of self-defence, the LAB produced substances that favour their growth and are unfavourable to other microorganisms. Lactic acid bacteria secrete antagonistic or antimicrobial compounds as natural preservatives that could be used as preparations for increasing the shelf-life and safety of minimally processed foods and thereby, modifying their environment by their metabolism hence excluding other microbes present (Niku, et al, 1999). Raw milk is complex and is made up mostly of water and other components like proteins, fats, lactose, a wide range of vitamins and minerals and many other trace elements in minor components, plus active enzymes including acid and alkaline phosphatases, lactoperoxidase and
lactoferrin (Lewis, 2003). But, raw milk was not considered to be used in the experiment for various reasons; it was hard to acquire fresh and uncontaminated raw milk and this type of milk had a short shelf-life. Originally, raw milk has low microbial count, but, once it is contaminated, pathogenic microorganisms thrive in it (Lewis, 2003). For raw milk to be safe for intake of humans, it must undergo special processes that would eliminate these pathogenic microorganisms. UHT Milk, Skim Milk and Pasteurized Milk were chosen because they are relatively cheap, readily available, and were those used by the ATI-ITCPH. Different parameters would be used in order for a medium for growth of LAB to be better than the other. One parameter would be the number of LAB population that will be produced by the milk media. And secondly, when tested with the confirmatory tests, it would test positively for lactic acid bacteria (cocci shaped, gram positive, does not produce gas). UHT Milk and Skim Milk were obtained from Cows Milk while the Pasteurized Milk is from a Carabao. The source of milk, along with the treatment it has undergone greatly affect the contents of the milk and thus, the contents of each milk varies from one another (Table 2). Milk is heat-treated in order to reduce the microbial population in raw milk and to inactivate enzymes that may cause chemical reactions to take place (Lewis, 2003). Different levels of heat-treatment may be conducted and would lead to the production of different kinds of milk. Raw milk is pasteurized to the major pathogenic and spoilage bacteria that should also minimal chemical, physical and organoleptic changes in the product that when cooled and packed hygienically and stored under refrigerator conditions, it should have a shelf-life of over 10 days (Lewis, 2003). Pasteurized milk is available in the public market but usually, milk is
processed further so that its shelf-life may be extended. After being pasteurized, milk must undergo sterilization for it to be having a longer shelf-life. Sterilization involves heating milk in a sealed container in the temperature range 114-120C for 20-30 minutes where the milk is subjected to considerable changes in its nutritional value (Lewis, 2003). Some pasteurized milk is not sterilized. More recently, a continuous sterilization processes that involve temperatures in excess of 135C for, followed by aseptic packaging which subject milk to a much lesser chemical change compared to sterilized milk, in terms of colour development, thiamine inactivation, lactulose formation and whey protein denaturation is introduced; this is the Ultra Heat Temperature (UHT) treatment (Lewis, 2003). UHT (ultra-high temperature) treatment is another thermal treatment that destroys non-spore-forming pathogens in milk, but in addition some spore-formers are severally reduced in numbers (Jay, 2005).
Table 2. The three kinds of milk used in the experiment, their processing and their content. Pasteurized Carabaos Milk Alaska Fresh Milk Country Goodness Skim Milk Treatment Pasteurized Ultra Heat Treated Ultra-Pasteurized Ultra Heat Treated Protein (grams) 3.20-3.5 g 3.3 g 5.4 g Fat 2.20-4 g 3.5 g 0.15 g Carbohydrates 4-5 g 4.7 g 5.0 g Other content Vitamins: Vitamin B-carotene, Salts: Sodium, Salt: Sodium Vitamin A,Vitamin C, Thiamin Potassium
UHT Milk and Skim Milk were obtained from Cows Mi lk while the Pasteurized Milk was from a Carabao. The source of milk, along with the treatment it has undergone greatly affects the contents of the milk and thus, the content of each kind of milk varies from one another.
Pasteurized Carabaos Milk was the most productive milk medium to be used for the growth of the LAB. A higher number of LAB count can be associated to the acidic pH of the serum which eliminated microorganisms that could not tolerate acidic environments. Also, the great amount of cheese and very low pH produced was expected since more LAB were present. As compared to the LAB Serum from Skim Milk that produced the second largest number of LAB, but the least cheese and the most basic pH. The number of LAB cannot be correlated to the acidity of the lactic acid they produce; the LAB Serum from UHT Milk produced the least number of LAB, the second heaviest cheese and a pH of 4.5 (pH in between that of the LAB Serum of Pasteurized Carabaos Milk and that of Skim Milk). A small number of LAB can produce a more acidic serum.
4E+10 Microbial Count (cfu/ml) 3.5E+10 3E+10 2.5E+10 2E+10 1.5E+10 1E+10 5E+09 0 UHT Skim Pasteurized
Figure 3. Bacterial count in LAB serum produced from UHT, Skim and Pasteurized milk.
Pasteurized Carabaos Milk obtained the highest number of lactic acid bacteria. Pasteurized Milk underwent the least processing; pasteurization. This level of processing led to a larger starter culture of lactic acid bacteria. Also, Pasteurized Carabaos Milk, in comparison to UHT Milk and Skim Milk, contained more protein, fats, carbohydrates and vitamins in the right
proportions. All nutrients were supplied in excess. Whereas, UHT Milk and Skim Milk portrayed an imbalance of content, other contents were very high while others were very low. It was stated that lactic acid bacteria are fastidious microorganisms in that they need excess of all their food requirements because they use up much sources and produce very little lactic acid (Figure 3). For purposes of storage and preservation of LAB Serum, sugar is necessary to be provided. Lactic acid bacteria require carbohydrates as their source of energy. Carbohydrates are commonly found in foods rich in sugar and starch. For the metabolic and functional processes of LAB, it is necessary that they utilize carbohydrates from sugar and ferment it into lactic acid with some by-products like volatile acid, alcohol and carbon dioxide. Some studies have shown that yoghurt culture bacteria ( L. bulgaricus and S. thermophilus) preferentially metabolize and transport a few to many sugars such as lactose, glucose, sucrose and to a lesser extent galactose (Sobowale et al, 2011). This supports the idea that supplementation of sugar increases their metabolic processes. In determining which sugar is best to use for the culture medium, the results showed that all the three kinds of sugar tested provided the requirements for the growth and maintenance of the LAB. This is proven by the population count of the LAB in the medium using any of the three sugars. Results showed that the Petrifilms for any of the sugars equally recorded that the LAB were too numerous to count. Molasses is the least costly (Php15 per kg) in comparison with brown and white sugar, which are Php43.50 and Php45.00, respectively. In spite of these findings, the molasses is still the best sugar to use for two reasons: it costs the least and does not need further handling because it is already in the liquid form.
That the bacteria isolated and cultured were indeed LAB as confirmed by the following tests: simple staining, gram staining, colony morphology and gas production from glucose. Simple staining confirmed that the bacteria were cocci to rod-shaped and they occur individually or in chains. These results helped confirm that the bacteria present in the samples were lactic acid bacteria. Results from Gram staining in the fermented rice wash showed that Gram negative bacteria were also present. Actinomyces, together with Arthrobacter, Mycobacterium, and
Propionibacterium were originally reported as Gram-positive bacteria. In 1989, these bacteria were observed to be Gram-negative as it approaches exponential phase. As cultures of these bacteria aged to stationary phase, it was observed that there was a relatively slight increase toward Gram negativity due to the increased lysis of non-dividing cells by means of lesions in the side walls (Beveridge, 1990). In conducting the test for colony morphology, the presence of LAB were confirmed by inoculating the samples in MRS broth. The colonies were round, smooth and convex, white, cream or light yellow, and non-transparent. These characteristics were identified to be that of lactic acid bacteria, hence verifying their presence in the samples. One important characteristic used in differentiation of LAB is the mode of glucose fermentation under standard conditions which are non-limited supply of glucose, growth factors such as amino acids, vitamins, and nucleic acid precursors, and limited oxygen availability. Homofermentative LAB convert sugars almost quantitatively to lactic acid while
heterofermentative bacteria produce not only lactic acid but also, ethanol/acetic acid.
Obtaining homofermentative LAB from all samples suggests that the samples are free from ethanol and are safe for consumption by monogastrics. Given these results, a cost-efficient and effective culture medium for lactic acid bacteria can be produced from C4 Dinurado rice wash, pasteurized milk and molasses under optimal conditions. It is recommended that these new information be shared with pig raisers.
CONCLUSION
Physical conditions such as low oxygen level, a temperature range of 23-25 C, high ventilation and low light exposure were noted to provide optimal conditions for the growth of LAB. Dinurado rice wash contained enough LAB to provide as a starter culture. Differences in the treatment of milk result to differences in their content. Pasteurized milk is the most efficient media for LAB growth because of its contents. Regardless of the kind of sugar used as a preserving media, LAB stock will still produce high LAB count. Thus, 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.
RECOMMENDATIONS
The researchers of this study would like to recommend the following for further studies; i. To compare LAB population count between different kinds of fermented rice grains; ii. Quantitative analysis of more diluted samples of the Pure Stock to come up with a definite number of population count; iii. Species identification of all microorganisms found in the FRW, LAB Serum and LAB Stock via genomic analysis; iv. The procedures of making LAB Serum from C4 Dinurado Rice Wash, Pasteurized Milk and Molasses, be re-done by local farmers and to assess if the procedures could be replicated without errors; v. Application of produced LAB Pure Stock made from Pasteurized Milk and Molasses on actual test organisms(swine) vi. Information dissemination to local pig raisers.
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Ortiz, M. E.,Fornaguera, M. J., Raya, R. L., and Mozzi, F. (2012).Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source. Applied Microbiology and Biotechnology.95, 4. pp. 991-999, DOI: 10.1007/s00253-012-3945-z. Pederson, C. 1971. Microbiology of Food Fermentations. Connecticut: The Avi Publishing Company. Pond WG, Church DC and Pond KR. (1995). Basic Animal Nutrition and Feeding. 4 th Edition. USA: John Wiley and Sons. Pages: 295, 331 Priest, F. and Austin, Brian. (1993). Modern Bacterial Taxonomy, Second Edition. Chapman and Hall; London, UK. Prescott, Lansing M.; Harley, John P.; Klein, Donald A. (1996). Microbiology, Third Edition. WM. C. Brown Publishers; IA, USA. Reyed, R. M., El-Diwany, A. (2008). Molasses as Bifidus Promoter on Bifidobacteria and Lactic acid Bacteria growing in skim milk. The Internet Journal of Microbiology.5, 1. DOI: 10.5580/1f0 Robbins, G. and Lewis, K. 1939. Fermentation of sugar acids by bacteria. Nebraska. Ronzio, R. (2003). The Encyclopedia of Nutrition and Good Health, Second Edition. Library of Congress Catalouging-in-Publication Data. USA. p. 499 Seeley HW and VanDenmark PJ. (1967). Microbes in Action: A Laboratory Manual of Microbiology. 2nd Edition. San Francisco: W. H. Freeman and Company. Pages: 51-55, 274-275 Sobowale, A. A., Efuntoye M. O. and O. O. Adesetan. 2011. Energy sources of yoghurt bacteria and enhancement of their galactose uptake. African Journal of Biotechnology Vol. 10(21), pp. 4457-4463 Todar, K. (2008). Todar's Online Textbook of Bacteriolody. Accessed http://textbookofbacteriology.net/lactics_5.html. Retrieved on: August3, 2012. from:
Turner, J., Pas, S., Dritz, S., Minton, J.E. (2005). Review: Alternatives to Conventional Antimicrobials in Swine Diets. The Professional Animal Scientist. 17:217-226. Weeks, B. and Alcamo, E. (2012). Microbes in the Society, Second Edition . Jones and Bartlett Publishers; Boston, USA. Yang, S., Ji, K., Baik, Y., Kwak, W. and McCaskey, T. (2005). Lactic acid fermentation of food waste for swine food. Bioresource Technology, Elsevier Publishing.
Yu, R. and Hang, Y. D. (1989).Kinetics of direct fermentation of agricultural commodities to L(+) lactic acid byRhizopusoryzae. Biotechnology of Letters.11, 8. pp. 597600, DOI: 10.1007/BF01040043. [URL 1]http://www.horizonpress.com/pcr/real-time-pcr-detection-foodborne-pathogens.html [URL 2] http://tribes.tribe.net/effectivemicro/thread/d6b8fd03-e2c7-4650-a658-51fdf4f013ad [URL3]http://www.becomenatural.com/blog/2007/03/natural-brown-raw-sugar-vs-brown-sugarvs-white-sugar/
APPENDIX A TABLES
Table 3. The progression of layers in the FRW set-up Day Temperature Presence of Molds or other foreign particles Day 0 27 C None Day 1 27C None Day 2 25.5C None Day 3 26C None Day 4 25C None Day 5 25C None Table 4. The number of LAB present in fermented rice wash Layers formed No layers yet Thin film at the surface of the liquid Thin film as thick as Day 1s film Thicker film at the surface Three distinct layers Three distinct layers
Replicate 1 2 3 Average
Number of LAB (cfu/ml) 6.48 x 105 6.40 x 105 6.32 x 105 6.40 x 105
Table 5. The number of LAB present in the serum produced from UHT, Skim and Pasteurized Milk
Number of LAB Replicate UHT Skim 10 1 1.280 x 10 2.048 x 1010 2 1.664 x 1010 2.304 x 1010 3 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 6. 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
+ Molasses TNTC TNTC TNTC
Table 7. Cost of materials Material C4 Dinurado Rice Rosario Pasteurized Carabaos Milk Alaska UHT Milk Country Goodness Skim Milk Molasses Dark Brown Sugar White Sugar
Quantity 1 Kilo 1 Liter 1 Liter 1 Liter 1 Kilo 1 Kilo 1 Kilo
Price (Php) 40.00 100.00 67.75 73.50 15.00 43.50 44.50
APPENDIX B FIGURES
Figure 4. 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.
Figure 5. Contaminated FRW with grayish-green mold formations; A. and B. contaminated set-ups 1 and 2, C. Contaminated set-up 1, D. Contaminated set-up 2
Figure 6.Three replicates of the rice wash set up at day 0 at the ITCPH health laboratory.
Figure 7. 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 8. FRW-milk set ups from day 0 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 5 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.
Figure 9. 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 0. 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
Figure 12. Colony count from each FRW-milk set ups
Figure 13. Colony count of each milk mixed with molasses at 6 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 6 dilutions, 10-1 to 10-6. a. Pasteurized, b. UHT, c. Skim milk.
Figure 15. Colony count of each milk mixed with white sugar at 6 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
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
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
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): 0 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 78.4C (173.1F) (Ethanol). Melting Point: May start to solidify at -114.1C (-173.4F) 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 (@ 20C) (Ethanol).p. 4 Vapor Density: The highest known value is 1.6 (Air = 1) (Ethanol). Volatility: Not available.
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 @ 20C. Odor Threshold: N/A. Vapor Density: (air=1) 0.7 (water). Solubility: Soluble in water. Melting Point: 0C. Evaporation rate: < 1 (Butyl acetate = 1). Boiling Point/Range: 100C. Partition Coefficient: N/A (log POW). Vapor Pressure: (20C) 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
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): 7 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 Potassium iodide 0.5-2.0 Physical State: Liquid Color: Dark amber Odor: No information found pH: 4.4-4.8 Vapor Pressure: No information found Vapor Density: >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
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.
Figure 21. Pasteurized Milk; A. 1 Liter of Rosario Pasteurized Carabaos Milk, B. Nutritional Value of Rosario Pasteurized Carabaos Milk, C. Brand Label of Rosario Pasteurized Carabaos Milk
Figure 22. Skim Milk; A. 1 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. 1 Liter of Alaska UHT Milk, B. Nutritional Value Alaska UHT Milk, C. Processing information of Country Goodness Skim Milk.

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OPTIMIZATION OF COST-EFFICIENT AND EFFECTIVE CULTURE MEDIA FOR LACTIC ACID BACTERIA (LAB)

____________________________ PROF. CELIA M. AUSTRIA Thesis Adviser

___________________________ PROF. CELIA M. AUSTRIA Chair, Department of Biology

__________________________________ ROSEMARY M. GUTIERREZ, Ph.D, Dean, College of Science

-Jam, Kathie and Tere

The progression of layers in the FRW set-up

The number of LAB present in fermented rice wash

Figure 1 Figure 2 Figure 3

Figure 4 Figure 5 Figure 6

MATERIALS AND METHODOLOGY

Fermented Rice Wash (FRW)

Mix with Pasteurized Carabaos Milk

Mix with Skim milk

Mix with UHT milk

Five-day fermentation Gram staining Filtration at day 5 Colony observation

LACTIC ACID BACTERIA SERUM (LABS)

Gas production from glucose

PASTEURIZED CARABAO MILK

Mix with Molasses

Mix with White Sugar

Mix with Brown Sugar

Five-day fermentation Filtration at day 5

Figure 1. Incubated PetriFilm showing the presence of Lactic Acid Bacteria

Darkest 4 321.3 g N/A N/A N/A N/A N/A N/A

Lightest and smoothest with large air holes

Figure 2. The process of Lactic Acid Fermentation

Pasteurized 3.560 x 1010 3.584 x 1010 3.712 x 1010 3.619 x 1010

+ White Sugar TNTC TNTC TNTC

+ Molasses TNTC TNTC TNTC

Quantity 1 Kilo 1 Liter 1 Liter 1 Liter 1 Kilo 1 Kilo 1 Kilo

Price (Php) 40.00 100.00 67.75 73.50 15.00 43.50 44.50

Figure 10. Clean PetriFilm from the package

Figure 12. Colony count from each FRW-milk set ups

APPENDIX D CULTURE MEDIA MRS Broth

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