Aims Whole experiment can be separated into two parts (l) The characterization of the isolate(allocate species) given by * Using simple biochemical tests * Using genetic tests by extraction of total genomic DNA * Amplifying the gene encoding for the ass RNA by PC * Understanding the fermentation growth kinetics in controlled liquid media to know the capability of organisms * The investigation of the digestion of the 1 as radar implicit with Hindi on ass radar strains (II) The amplification and cloning of a specific stress gene given by * Designing primers for cloning * cloning stress gene into cloning vector (page-T) cloning stress gene into expression plasmid(petite) – not done Literature Review Lactic acid bacteria (LAB) LAB are defined as bacteria that produce lactic acid as their major fermentation production.
The examples of LAB include Streptococcus, Intercourse, Leucosis and Lactose’s. They are gram-positive rods or Cisco bacteria with high AT content. (Prescott et. Al, 2008). There are few applications of LAB in food, medical and biotechnological industries. The major use of LAB is on the food industries. The non-pathogenic species, which helps the process of fermentation of different food such as dairy products, meat and vegetables. They mainly convert the hoses sugar exist in food to lactic acid( Macaroon et al. , 2007). Recent research shows that they can produce antimicrobial compounds that have proportion properties, they also helps different medical aspects.
It includes “intramural activity, reduction of serum cholesterol, alleviation of lactose intolerance, stimulation of the immune system, stabilization of gut et. Al, 2009). Another application in medical field is the production of obstetricians by LAB. A more well known one is Nisei, which is a illumination produced by Lactose lactic. (Anastasia et. Al, 2009) Different examples are further illustrated in following paragraphs indicating the industrial importance. Food Production Feta Cheese, which is a Greek cheese traditionally made from raw milk without starter cultures. The contemporary production is in well-structured dairies. Ewe milk is theorized(treated at 63 to ICC for 16 GRIFFITH, 1998) with yogurt as starter.
From Feta Cheese, different species of LAB can be identified, they are responsible for the unique flavor of Feta cheese. (Urbanites et. Al, 2009). LAB can also be used to improve the preservation of ready-to-eat(RET) seafood such as cooked and peeled tropical shrimp and cold-smoked salmon. France is the second largest shrimp exporter in Europe and largest salmon exporter in the world. Technology for preservation is significant for them. Chemicals such as nitrates, sulfates and sorbets can be added to preserve shrimp in France. However, these chemicals are forbidden for smoked salmon. Under these circumstance, preservation plays a significant role for smoke salmon industries. (Cardinal et al. 2009) According to a review from Rodgers, “Preservation is a preservative method that uses the growth of selected microorganisms on a food product to control the undesirable (spoiling and pathogenic) et al. 003) There are few pathogens such as Brio SSP. , Salmonella SSP. And Staphylococcus SSP. Can be found in RET food. Although some of the LAB, such as Lasciviously sakes or Brothering thermostatic , can lead to spoilage in pure culture, some other LAB species still plays a magnificent role in food preservation. Four strains of LAB show the best result(two Leucosis gullied and two Lactose’s picnics strain) in an experiment which can reduce the amount of Listener monocotyledons and S. Erasures for up to four weeks. (Cardinal, 2009) Obstetricians production Obstetricians are synthesized by ribosome.
There are four classes of obstetricians produced by LAB: Illuminations(Class l) which are modified, nonlinguistic(Class II) which are heat-stable and unmodified (Class Ill) which are large heat-liable obstetricians and (Class IV) which are complex molecules with lipid and carbohydrates moieties, As mentioned, chemical preservatives were criticized to be unsafe and the public prefers a more natural way for preservation. Different classes of obstetricians have different activity against different species. For example, nonlinguistic are active against Listener and Colostomies SSP. They ark by parallelize the cytoplasm membrane by inserting the C-terminal regions into the membrane through pore formation. Bacteriologic producing LAB can perform a milder and less expensive method to overcome the whole preservation process, while some enzymes can make the pathogens loses , yet they are more expensive and harder to be controlled. .(Anastasia et. L, 2009) Medical Use LAB can be used as proboscis as well, one of the examples is the compensation of lactate insufficiency. Lactose intolerance means that the concentration of lactate in the small intestine is not enough to digest lactose properly. LAB have acetates activities, when body consumes it, some of the LAB proboscis can resist the bile salt and the pH of the small intestine helping to reduce the lactose intolerance symptoms such as hydrogen exhalation. (De Verse, et. Al, 2001 Overview LAB has significant commercial value in market mainly on food industries. Its main effects are adding health effect on dairy products, assisting the preservation process. It also helps to make cheese have varies flavor acting as the starting culture.
For many countries, instead of using chemical preservatives, LAB is used as preservative that gives consumer a more natural image. Throughout the process of product innovation, stress gene plays an important role. For example, for the production of cheese, it can pass many lab tests. When it is transferred to the real world, the stress gene within the bacteria might affect the cheese taste by the impact of the transportation, storage, fluctuation of the temperature, humidity. Knowing stress gene can help for the controlling of product quality. Identification of a stress related gene from Lactic acid bacteria In the whole experiments, stress related gene was chosen and identified from lactic acid bacteria (LAB).
Stress, in microorganisms, basically means that some change of the environment that might lead to the death or inhibition of that organism. There are different kinds of stress such as temperature, pH, and oxygen level. (Overbear, 2003) The study of stress gene of LAB helps to understand what industrial process can be advocated or avoided to optimize the production and minimize the cost. The oxidative stress for Lactose’s lactic was planned to be studied. It is widely used for food production including the production of Cheddar cheese. During the normal production process, it is not strictly anaerobic. It still involves he contact with oxygen.
LAB use oxygen to regenerate AND+ . In the whole process of converting, there will be an increase of hydrogen peroxide. Hydrogen peroxide inhibits the growth of LAB. LAB can deal with these hydrogen peroxide molecules by using superposed diastase (Sod). During most of the LAB production, there will be an increase in the amount of Lactic acid and thus lowering the PH. In this experiment, the soda gene would be cloned and studied. It would be tested to justify the effect of concentration of hydrogen peroxide on the production of Sod. (Humankind et al. , 1995). This gene will be tested with he difference under aerobic and anaerobic conditions.
The gene will be identified first by giving some specific conditions, in this case, the oxygen, to check if the Sod is present, The “Stress Response Testing” in lab book given should be used. Then use a restrictive enzyme to extract the piece that is responsible for the characteristics. Then use the same restriction enzyme to open the plasmid at the same place in a Vector such as from E. Coli. The gene Sod can then be transferred and have further investigation. (Remark:While in actual experiment, Race was cloned and extracted instead of SodA gene) Flow diagram Whole experiment can be separated into two parts (1) Characterization Fig 1 . Basic biochemical tests such as catalane test and gram stain are done to identify the strain of bacteria being investigated (2) Extraction of Stress gene Fig 2.
Primers are legated into page-T(cloning vector) in order to be replicated inside a bacterial cello 09), the Blue/white selection is to indicate if the legation is successful. Materials and Methods Gram stain: details refer to A. 7 Peg 58 of the lab manual (Call et. Al, 2009) Gram stain is based on the physical and biochemical properties of the cell wall. It can e divided into gram positive and negative. Catalane and Oxides test: refer to A. 8 of the lab report (Call et. Al,2009) Catalane test Hydrogen peroxide is used to test if there is an oxygen production Oxides test An intense deep-purple hue can be seen within 5-10 seconds if it is a positive reaction. PC It is used for amplification of primers in the experiment.