This is to report that according to the check % of the content of the above thesis is declared to be plagiarized and covered/not covered according to the plagiarism policy and institutions issued by CASR, Chattogram Veterinary and Animal Sciences University. I am ever grateful and much obliged to the Almighty, without whose grace it would never have been possible for me to pursue this study in this field and complete this thesis written for the degree of Master of Science in Applied Food Science and Nutrition. Goutam Buddha Das, Vice Chancellor, Chittagong Veterinary and Animal Sciences University (CVASU), and Dean, Faculty of Food Science and Technology, CVASU, for their outstanding efforts to arrange industrial placement in collaboration with reputable food industry, oven etc.
It is my pleasure to express my sincere to Kazi Nazira Sharmin (Associate Professor.. of Applied Food Science and Nutrition) for providing me an opportunity to do my MS. Some of the proteins in pyropia orbicularis can serve as starting points for creating bioactive peptides. Peptides as a structural component of bioactive proteins In this virtual experiment, 12 proteins were selected from the pyropia orbicularis.
For this reason, 63 different tripeptides or new peptides were tested to see if any of them could be considered new bioactive peptides. These findings provide support for the use of pyropia orbicularis as a source of bioactive peptides and lay the groundwork for further in-vitro and in-vivo research on these compounds.
Introduction
Significance of study
No in-silico study of pyropia orbicularis proteins as potential building blocks for bioactive peptides has been published. Therefore, the goals of this study were to identify the proteases responsible for the increased release of a dominant bioactive peptide from previously identified pyropia orbicularis proteins, and to determine the probability of the biological activity of the peptides. In addition, the study explored the feasibility of predicting novel peptides and their potential characteristics using in-silico bioinformatics tools.
Objectives
Review of literature
- Sea weed
- Characteristics of sea weed
- Nutritional composition of sea weed
- Pyropia Orbicularis
- Red seaweed protein sequences (pyropia orbicularis)
- Evaluation of pyropia orbicularis as potential precursor of bioactive peptides
- In silico proteolysis
- Virtual screening and characterization of novel tri-peptide
- MALDI-TOF mass spectrometry
- Protein hydrolysis
- Protein extraction method
- Phenolysis buffer Extraction
- Enzyme assisted extraction (EAE)
- Extraction with polysaccharidases …
- Phenol extraction
- Extraction in a deionized water and ultrasonic bath
- Bioactive peptides
- Angiotensin-converting enzyme (ACE) inhibitors
- DPP IV inhibitors
- Alpha-glucosidase inhibitors
- The role of the in silico approach
5 is now given to dietary algae. These adaptable marine plants and algae have also helped expand the economy. Through the use of the BIOPEP-UWM database, we evaluate the protein from Pyropia orbicularis as a potential precursor of bioactive peptides. 6 where a is the fraction of protein sequence that exhibits the desired activity, and N is the total number of amino acids in the protein.
7 where d is the fraction of the protein sequence that is converted to active peptides when treated with the enzyme of choice and N is the total number of amino acid residues. The majority of DPP-IV inhibitory activity sequences reported in BIOPEP-UWN were di- or tri-peptides. The phenolic layer on top of the mixture was separated by centrifugation at 10,000 g for 20 minutes.
The extraction of the lower phase was repeated with the same amount of phenol as before. Numerous studies have demonstrated the effectiveness of the in silico method by demonstrating consistency between their findings and laboratory hydrolysis results.
Materials and Methods
- Study area
- Materials
- Raw material
- Protein extraction
- In silico analysis
- Portparam tools
- Peptide ranker
- Peptide calculator
- Bioinformatics tool for allergenicity prediction
- Peptide cutter
- AHTpin
The NCBI database (https://www.ncbi.nlm.nih.gov/) was used to verify the existence of the 12 proteins. The UniPort database (https://www.uniprot.org/) was consulted to obtain the FASTA sequences and general properties of the 12 proteins. One can provide either a Swiss-Prot/TrEMBL accession number or ID, or a raw sequence to identify the protein of interest. Essential amino acid composition is identified by portparam tools (https://web.expasy.org/protparam).
Peptide Ranker is a web service that uses an innovative N-to-1 neural network to predict which peptides will have biological activity. Peptide Ranker allows users to enter a list of peptides and receive a ranking of the bioactivity potential of the peptides. The technique uses Auto cross covariance (ACC) transformation to convert protein sequences into vectors of the same length.
Protein sequences can be analyzed with PeptideCutter [references / documentation] to identify potential cleavage sites cleaved by proteases or chemicals. Using a single amino acid at a time, this module produces each mutant and makes predictions about the peptides' antihypertensive properties.
Results
Extracted protein identified by MALDI-TOFF mass spectrometry
Essential amino acid percentage
In silico analysis
20 Table 4.5.1: The predicted efficiency of release of bioactive fragments from selected pyropia protein by in silico proteolysis for ACE inhibitor. 21 Table 4.5.2: The predicted efficiency of release of bioactive fragments from selected pyropia orbicularis protein by in silico proteolysis for. 22 Table 4.5.3: The predicted efficiency of release of bioactive fragments from selected Pyropia orbicularis protein by in silico proteolysis for.
Bioactive peptides predicted to be released from pyropia orbicularis protein based on in silico enzymolysis.
Discussions
Protein identified by MALDI-TOFF mass spectrometry
In silico analysis
35 can be done as no in-silico study of pyropia orbicularis protein to produce bioactive peptides has been reported. Among the predicted bioactivity present in the protein were ACE inhibition, antioxidant, DPP-IV inhibition, DPP-III inhibition and stimulatory fragments (table 4.3). ACE inhibitor and DPP IV inhibitor were found to be the maximal biologically active fragments released.
It was also found that different species of pyropia family have proteins with various bioactive peptides mainly ACE inhibitor, antioxidative, DPP IV inhibitor etc. Proteins of the identical family are expected to possess similar bioactive peptides (Sharmin et al., 2022). In the BIOPEP-UWM database there are 33 types of enzymes, but in this study six proteases (Chymotrypsin, Papain, pepsin, thermolysin, trypsin and stem bromelain) are chosen for the in silico proteolysis as they are commercially available.
Compared with our result, it was found that three proteases papain, ficin, stem bromelain were used, and stem bromelain showed the highest value (Sharmin et al., 2022). For the ACE inhibitor, Chymotrypsin enzyme has a degree of hydrolysis ( ) value higher in stem bromelain and papain enzyme. Like that, in DPP-IV inhibitor and Alpha-glucose contain lower Degree of Hydrolysis value in the company of Pepsin and Trypsin enzyme.
Novel peptides
In silico analyzes suggest that these new peptides have high acceptability in pharmaceutical and functional food products. Thus, further in vitro and in vivo analysis is required to understand the current status of these novel peptides.
Conclusion
Recommendations & Future Perspectives
