Chemistry

    The hypothesis of my guiding experiment is that the diversity of LABs will be very large coming from the isolates of fresh milk products. Lactobacillus, Lactococcus and Leuconostoc variants will be observed and level of bacteriocin activity against Pseudomonas flourecens expected. Among the three brands, it is hypothesize that there will not be any significant differences on the amounts of LABs that will be observed as well as bacteriocin activities. The proposed experiment fits the definition of chemical biology or biochemistry because it deals with microorganisms which affect the human health. Furthermore, this experiment utilizes intermediate to advance chemistry methodologies and processes that will entail the researcher to obtain the results as well as test the validity of the hypothesis. Additionally, the product focus of this experiment also dictates that this will be a valuable addition of he knowledge base of consumer and producers in advancing the fortification and production processes of fresh milk.

Background
    Lactic Acid Bacteria (LAB) are extensively used in fermenting a wide variety of food products and are known for their preservative and therapeutic effects. When consumed, LAB such as Lactobacillus acidophilus and Lactobacillus casei can aid lactose digestion, control cholesterol levels and intestinal. LAB can also produce bacteriocins, inhibitory substances other than organic acids which are antagonistic toward other microorganisms. Bacteriocins, are lethal complexes of poly peptides produced by bacteria, are not active against the producer bacterium.  Lactobacillus acidophilus and Lactobacillus casei are commonly known as probiotics for human health, Probiotics are viable microorganisms that exhibit a beneficial effect on health of the host upon digestion by improving the properties of its indigestion microflora. The experiment will be conducted to isolate and identify the LABs from three different commercial brands of fresh milk.

Experimental Plan
    Sampling LAB isolates will be done by extracting sufficient amounts of milk per sample. Consumer brands that will be used are Anchor, Yakult, and Magnolia. Basic chemical extraction techniques will be done to obtain samples that will cover 30 treatment plating per brand.

    Plating Serial dilutions of milk will be prepared and then pour-plated win Man, Rogosa and Sharpe (MRS) agar and incubated at 30 degrees Celsius for 48 hours aerobically and algebraically. Anearobic system enveloped will be used. Individual colonies will be counted and then picked and streaked on MRS agar then this will be incubated aerobically for  30 degrees Celsius for 24 hours. Stock cultures of the isolates will be stored either in the stabs overlaid with mineral oil or in freeze-dried form in skim milk medium

    Culture Preservation The inoculated milk will be incubated for the genera Leuconostoc and Lactococcus. Viable counts in MRS agar will be counted in 6, 12, 18 and 24hours incubation period.

    Characteristics and Identification Morphological characterization will be done by examining the colony growth. Biochemical characterization will follow starting with Bacteriocin activity test. The flip streak method will be used in determining bacteriocin activity against Pseudomonas flourescens.  After incubation, a sterile spatula will be used to dislodge the agar from the bottom of the petridish and with the dish closed, the agar will be flipped onto the cover of the dish After wards, a scoopfull of 24 hr old indicator organism, Pseudomonas flourecens will be streaked perpendicularly to the producer streak on to the surface of the inverted agar, and this will then be aerobically incubated for 18-20 hrs at 20 C. Presence of a clear zone parallel to the producer streak was used as an indicator of inhibitory action or bacteriocin production. Activity test will follow by heating the milk samples in boiling water and cooled to 30 C. After mixing, 9ml of the inoculated milk will be added.

    Analysis Data on total LAB counts will be statistically analyzed using ANOVA. In a completely randomized design. The General Linear model procedure of the SAS software will be used. Data on chemical analysis (pH, moisture and salt) will be analyzed using regression and correlation.

Significance of the Research
    Determination of the levels of specific probiotic and harmful bacteria in fresh milk is detrimental to the whole consumer market. Furthermore, Antimicrobial activity or bacteriocin production against genus Pseudomonas and other psychotropic bacteria is important for the application of LAB as natural preservative of refrigerated milk and various milk products.

Hypothesis
    The hypothesis is that the diversity of LABs will be very large coming from the isolates of fresh milk products. Lactobacillus, Lactococcus and Leuconostoc variants will be observed and level of bacteriocin activity against Pseudomonas flourecens expected. Among the three brands, it is hypothesize that there will not be any significant differences on the amounts of LABs that will be observed as well as bacteriocin activities.

    The experiment will focus on the measuring of bacterial levels on certain milk products. These bacteria levels will be either probiotic (beneficial) or harmful one. Determination of the critical signs of pseudomonas activities will greatly help in creating a new procedure of processing milk or storing mil. Furthermore, the identification and characterization of LAB in three different brands will result in a clear output of bacteria indexing. There results of this experiment heavily relies on the proper execution of the methodologies and the correct evaluation of each treatment.