As we begin to write these lines after completion of my thesis, my heart is filled with deepest sense of gratitude. We shall ever remain thankfully indebted to all those who have encouraged us to achieve our goal and enlightened us with the tough of their encouragement.
First and foremost we would like to thanks to our beloved Mother, Father, Sister and brother for their affection, blessing, emotional support and love which has actually inspired us throughout our dissertation work. Without them we would have never come to this proliferative stage and engaged ourselves in career building. Having such a wonderful family who supported us whole heartily no matter what the situation was. They have listened our every problem and suggested us the possible solutions. We have shared the every moment of excitation after getting positive results and disappointment after not getting success in some work during the dissertation. We have no words to pay regards to this moral support.
We consider, it as a blessing to pursue my dissertation under the guidance of Dr. Atulkumarupadhaya, Assistant Professor, L.P.U, Phagwara. We are grateful to our mentor for being a great support and true guide throughout our dissertation work. No words are enough to express our gratitude for his whole hearted encouragement, supervision and support. He would take a great concern in troubleshooting problems and was always available to discuss the problems even in his busy schedules. We heartily thanks to him for being aa patient listener to our problems that we came across during our work and for providing us the useful guidance.
We are obliged and deeply indebted to Dr. Neeta Raj, Head of School of Biotechnology and Biosciences for her splendid guidance and Dr. Himanshu Singh, Head of Department of Biotechnology and Biosciences for providing us a wonderful opportunity that has brought a revolutionary change in our life. We would like to thank the whole department of Biotechnology and Biosciences, Lovely Professional University, Phagwara for accepting and allowing us to conduct the experiment regarding the dissertation.
At last we would like to thank almighty GOD for supporting us spiritually throughout our life and providing us everything that we needed. With all these people it would have possible for us to successfully complete our project.
JasleenBadyal (Regn No-11508149)
ParwinderKaur (Regn No-11510627)
IqbalKaur (Regn No-11507432)
This report includes sequence structure and functional analysis of 2 specific proteins present in mushroom and milk namely polyphenol oxidase 3 (mushroom) and Beta 2 microglobulin (milk) respectively. Multiple sequence alignment of different related proteins were performed using CLUSTAL OMEGA and domain analysis of selected two proteins were done on PFAM separately. Motif detection was carried out using PROSITE database whereas to calculate the predicted molecular weight and isoelectric properties of given proteins PEPTIDE PROPERTY CALCULATOR (version 3.1) was used. Furthermore, structural analysis of these proteins were done using three databases namely PDB,SCOP and CATH. On the basis of resolution(<3?) specific crystal structure of each protein were selected from PDB and structural analysis were undertaken . Based on the results of scope, the number of macromolecules structures, protein sequences and bioactive molecules can be detected and studied further. In addition to it, CATH results reflected matching superfamilies of our given proteins. Eventually, alignment of PDB structure and query structure was performed and its similarities were studied.
Chapter 1 Introduction
Consumers need nutritious, wholesome, pure and safe food. In recent years, consumers have placed increased emphasis on food safety, which include safety, purity, wholesomeness and value. Because milk contribute a very significant proportion in our daily diet. Milk considered to be closest to the natures perfect food, is an excellent source of calcium, a good source of minerals and high-quality proteins, the only source of lactose and lipids, the most valuable component, which also forms the basis of milk pricing. India is largest milk producing country in the world, with 127 million tonnesof milk produced annually. Milk is more prone to adulteration by addition of vegetable oil, starch, sugar and flour to adjust its composition illegally. Addition of urea, detergents and pond water in sour and spoiled milk is also in practice to make it fit for processing and consumption. This lead to deterioration of quality of milk and a risk to consumers safety.
As milk is very perishable and deteriorate rapidly due to high moisture content, chemical preservatives are added to milk samples so that composition does not changes. Various studies have been conducted to report the effects of preservatives on composition of milk, but the studies describing preservatives and their limitations have not been previously described.
Chemical milk preservative has been practiced for a long time since milk fat testing become a routine exercise. Several substaces and formulations have been used in the past, but the search for ideal milk sample preservative continues. The type of preservatives depends upon the purpose for which it is required. If the sample requires short-term preservative, for example, for carrying sample from the farm to the laboratory for analysis, a preservative providing small increase in shelf life will suffice. On the other hand, in situations where samples are collected from the market by food authorities and sent to a food laboratory for analysis and when disputes require the intervention of referral laboratory, a preservative providing longer shelf life is required.
Coliform bacteria and staphylococci are more sensitive to the effect of sodium azide than mesophillic bacteria, enterococci and lactobacilli, whereas enterococci are more resistant to chloramphenicol than salmonella sp., Esherichia coli, Lysteriasp., and staphylococcus aureus. Research dealing with combinations of antibacterial agents and bacteriostatic such as chloramphenicol and nitrofurazone brought our new mixtures that were good enough to preserve milk for 3 days without affecting the instrumental analysis of milk by Milkoscan, Bactoscan and Fossomatic systems. One of these mixtures Azidol(containing chloramphenicol and sodium azide) was most commonly used in all milk testing laboratories in Spain.
A few attempts have been made to improve and maintain the shelf life of milk by using chitosan, which is a natural modified biopolymer derived by deacetylation of chitin a major component of shells of crustacean. Lee(2000b) studied the effects of water soluble chitosanswith 3 different molecular weights (0.2 to 3, 3 to 10 and 10 to 30 KDa) on the physiochemical and sensory properties of milk. Consistency of chitosan added milk increased with increasing molecular weight and concentrations. Milk containing 0.5% and 1.0% chitosan could be sterilized at 730 for 15 minutes without protein coagulation. Addition of 0.5% water soluble chitosan to milk negatively affected the sensory quality of color, taste and flavor, browning in color and chemical off-flavor. However there is no differences in sensory quality between coffee flavored milk containing chitosan, this may be due to masking effects of coffee.
Ha and lee(2001) studied the effectiveness of water soluble chitosan(0.03%) to minimize the microbial(bacterial and yeast) spoilage of processed milk. Complete inhibition of microbial growth was observed in banana-flavored milk in contrast to that observed in control milk(without chitosan), during chitosan also control relatively high pH than that of control milk during storage.
Till now there is no way to increase the shelf life without having effects on color, appearance, taste, aroma of milk. So we take initiative through the knowledge of bioinformatics tools to increase the shelf life of milk, for that we select the Beta-2-microglubulin (B2M) gene of bostaurus cattle commonly called Zebu, which is found in hot climates and tropical parts of the world such as sub- saharan Africa, India, china and Southeast Asia.B2M gene help in presentation of peptide antigen to the immune system. It encode for serum protein found in MHC class 1heavy chain on surface of all nucleated cells. As it encode for antimicrobial protein displays antimicrobial activity in amniotic fluid. The Beta -2-microglubulin also called lactollin.
In mushrooms Polyphenol oxidase enzyme responsible for enzymatic browning is a group of copper proteins that catalyses the oxidation of phenolic compounds to quinones, which produce brown pigments, commonly found in fruits and vegetables. It plays crucial part in melanin synthesis and help in defence mechanism. At the same time it protect from UV radiations,free radicals and prevents dehydration of fungal bodies. It is basically belongs to tyrosinase family and has length about 576 and mass about 66,267 dalton. The isoelectric point of ppo3 is 4.95 and charge -7.
Chapter 2 Review of literature
2.1 Need to extend the shelf life
Consumers demands are increasing for high food quality and have expectations that theproduct quality will be maintained at high level during the time of purchase and utilization. The expectations are not only regarding the food quality should be safe, but also need to minimize unwanted changes in sensory quality. In the UK, the date coding to be used is checked by the total life of the product: for microbiologically highly perishable foods, a use by date is needed, while for other foods, including foods with more than 18 months a shelf-life, a best before or a best before end date is needed.
McGinn et al., (1982) reported that microbiological changes are of primary importance for short-life products and chemical and sensory changes for medium- to long life products; all three types of changes can be necessary for perishable products.
The more recent IFST Guidelines (1993) provide a more workable explanation of shelf-life:
Shelf-life is explained as the period during which the food product will:
(i) remain safe;
(ii) retain desired sensory, phsyiochemical and microbiological characteristics;
(iii) comply with any label declaration of nutritional data,
when stored under the recommended conditions.
2.2 Factors influencing shelf life
Shelf life can be affected by various factors and categorized into intrinsic and extrinsic factors (IFST, 1993). The final properties of product are called intrinsic factors and include the following:-
Water activity (aw) (available water).
pH value and total acidity; type of acid.
Natural microflora and surviving microbiological counts.
Natural biochemistry of the product formulation (enzymes, chemical reactants).
Use of preservatives in product formulation (e.g. salt).
Extrinsic factors are those factors the final product encounters as it moves through the food chain. They include the following:
Timetemperature profile during processing; pressure in the headspace.
Temperature control during storage and distribution.
Relative humidity (RH) during processing, storage and distribution.
Exposure to light (UV and IR) during processing, storage and distribution.
Environmental microbial counts during processing, storage and distribution.
Composition of atmosphere within packaging.
Subsequent heat treatment (e.g. reheating or cooking before consumption).
The interaction of such intrinsic and extrinsic factors can inhibits or stimulates a number of processes which limit shelf-life. These processes can be conveniently classified as:
2.2.1 Microbial changes:- During storage period the growth microbes occur due to various reasons, i.e, microbes present starting of storage; the physicochemical properties of the food, like moisture content, pH, presence of preservatives; the method used in the production of the food; and the outerenvironment of the food; the surrounding gas composition and storage temperature. The growth of food-poisoning organisms such as Salmonella species will not be accompanied by changes in appearance, odor, flavor or texture that could be detected by the human senses and it cause serious health concerns.
2.2.2 Chemical deteriorative changes:-most of the food spoilage occur due to reaction within food or with outer component, for example oxygen. Rancidity is mainly occur in fat-containing foods and can occur through different mechanisms, for example oxidative reactions and flavor reversion reactions. Enzymatic reactions limit the shelf-life of fruits and vegetables, and oxidation reactions limit the shelf-life of meat. Chemical hydrolysis can occur in food containing intense sweeteners, reducing sweetness.
2.2.3 Physical deteriorative changes:-Moisture can be removed and added during processing of food or through surrounding migration of moisture, which is reason for physical deterioration. This is easily observed in dry products such as breakfast cereals and biscuits can lose their crispness through moisture uptake. Freezer burn is also a result of moisture migration from the surface of frozen foods. Other migration phenomena can limit shelf-life, such as migration of fat from one component to another and the bleeding of colors in composite products such as chilled desserts. Physical changes in packaging materials by chemical reaction can aslo limit the shelf life. Migration of chemical components from the packaging material due to change in permeability can produce taints and this can be particularly serious in products with a long shelf-life.
2.2.4 Temperature-related deteriorative changes:- Spoilage can occur at both high and low temperatures. The minimum required temperature for growth of pathogens and spoilage organisms define the importance of temperature control in preventing microbial spoilage. The rise in temperature can lead to chemical reaction, which could be a cause of spoilage. In foods containing fats, solid fat turn into liquid and act as a solvent for reactions to occur, e.g forming bloom in chocolate. High temperature can also change crystalline characterstics of food. Varying of temperature can be reason of ice crystal formation in frozen foods such as ice-cream. In disparity, high temperatures can reduce the of staling in bread, although the condition with other baked products can be complex and unpredictable.
2.3 causes of spoilage
The class bacillus and clostridium are only bacteria that can produce heat resistant spores, which are major concern in food microbiology. New researches have shown that many organisms are classified to the genus Bacillus actually represent a number of class within the class Bacilli. The twelve new classes of aerobic spore forming bacteria have been defined,
Shida and et.al (1996) reported Brevibacillus, which is gram positive bacteria and associated with infectious diseases. It is commonly located in soil, water and decaying matter.
Nazina and et.al( 2001) reported geobacillus. It is rod shaped gram positive bacteria and found in soil, hot springs, ocean sediments and cause of food spoilage.
Ash and et.al(1993) reported paenibacillus. It is particularly associated with food spoilage.
Heyndrickx and et.al(1998) reported virgibacillus. It do not cause major spoilage but can be problem for some specific foods.
Scheldeman and et.al( 2004) reported that the paenibacillus can be isolated from both raw and heat treated milk.
Carey and et.al(2005) reported that these microbes increase interest in pasteurized milk products, but now the microbiological quality of these products is not sufficient to achieve the goals.
2.4 Attempts for the transfer of B2M
Smith and Little et.al (1922), Mikulska et.al. (2000) reported that the placental transfer of immunoglobulin does not occur in cattles.
Butler et.al (1974), Stott et.al (1976, 1979a) reported that the antibodies from cattle can be transfer to calves through colostrum (which is IgG and protein rich milk produce near parturition) only.
Besser and Gay et.al(1985) reported that in bovine neonates the colostrum is carry across intestine by nonselective pinocytosis.
Yadav et.al (2012) reported the quantitative PCR is very crucial for gene expression study because of its high specificity and sensitivity.
Mansur et.al (1993) reported that the sometime the transcription level of reference gene vary with tissue type and developmental stage.
Chapter 3 Materials and Methods
A laptop or computer with easily and fast access to internet, so that bioinformatics tools can be explored easily.
3.2.1 Tools to know basic about B2M
1. To take the protein sequence of B2M gene in Bos Taurus we visit the website www.uniprot.org. After selecting the beta-2-microglobulin gene we go for download option and download the sequence in fasta format.
2. After downloading the sequence we visit the website www.ebi.ac.uk/Tools/msa/clustalo/ (clustal omega which is multiple sequence alignment tool) and paste the sequence of bos Taurus, rat, human, sheep and chick to know the evolutionary change and if they having common ancestary.
3. Then we visit the website which is protein family database tools. By adding the accession id of B2M gene we came to know about the domain, start and end of domain in B2M sequence and its role in the organisms. It also give data about that how many amino acids making the sequence.
4. By knowing all this information about B2M, we came to know about the motifs, location of disulphide bond though PROSITE a protein database, for this we visit the website //prosite.expasy.org.
5. To know the properties of protein i.e ionic strength, chemical formula, molecular weight, net charge and sequence composition we use peptide property calculator by visiting the website
3.2.2 Tools for structural analysis of B2M