Summary of Biotechnology doctoral thesis: Research and receiving protein from brackish water algae (Chaetomorpha sp.) for application in food industry

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Study on process of extracting and receiving protein concentrate from Chaetomorpha sp. Investigate the biological properties, functional properties and nutritional value of protein concentrate from algae for application in food.

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Nội dung Text: Summary of Biotechnology doctoral thesis: Research and receiving protein from brackish water algae (Chaetomorpha sp.) for application in food industry

MINISTRY OF VIETNAM ACADEMY EDUCATION AND OF SCIENCE AND TRAINING TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- Bach Ngoc Minh RESEARCH AND RECEIVING PROTEIN FROM BRACKISH WATER ALGAE (Chaetomorpha sp.) FOR APPLICATION IN FOOD INDUSTRY Major: Biotechnology Code: 9 42 02 01 SUMMARY OF BIOTECHNOLOGY DOCTORAL THESIS Ho Chi Minh City – 2020
The thesis is completed at: Ministry of Education and Training – Vietnam Academy of Science and Technology. Supervisors 1: Assoc. Prof., Dr. Hoang Kim Anh Supervisors 2: Assoc. Prof., DSc. Ngo Ke Suong Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis shall be defended in front of the Thesis Committee at Ministry of Education and Training – Vietnam Academy of Science and Technology. At...... hour....... date...... month...... year 20… The thesis can be found at: - Library of Ministry of Education and Training - Vietnam National Library
INTRODUCTION 1. The necessity of the thesis Currently, some species of algae (such as Chaetomorpha sp.,) are growing naturally throughout the brackish water shrimp ponds in the Mekong Delta. After harvesting shrimps, algae were removed from the pond, wasting and polluting the environment. Many studies show that the ingredients in algae have high nutritional value. In particular, the protein in algae has a balanced amino acid composition and a high content of essential amino acids. Plant proteins also have many good functional properties that make them widely used in foods industry. In addition, some proteins from algae also exhibit biological properties such as antibacterial and antioxidant properties. Research on protein intake from seaweed opens up opportunities to use plant protein sources to replace protein sources from animals in food industry. 2. Research purpose Study on process of extracting and receiving protein concentrate from Chaetomorpha sp. Investigate the biological properties, functional properties and nutritional value of protein concentrate from algae for application in food. 3. New scientific contributions of the thesis The research focuses on collecting protein concentrate from Chaetomorpha sp., a new source materials that has not been exploited. In this study, we use ultrasound technique combined with a new type of cellulase to hydrolyze into algae cells to enhance protein extraction. Unlike conventional cellulase (with optimal pH in the acid region), the new cellulase works well in the pH range from 6.5 to 7.5, so it is suitable for protein 1
extraction from algae biomass. The protein group dissolves well in a neutral or slightly alkaline medium. In addition, the topic has scientific significance in providing completely new and systematic information about the protein groups in Chaetomorpha sp. such as the amino acid composition, protein fractions and the molecular size of the water-soluble and alkaline protein groups; morphological, antibacterial, antioxidant, functional properties, and digestibility under in vitro and in vivo conditions of the protein concentrate from Chaetomorpha sp. 4. The main research content of the thesis - Investigate the process of extracting and purifying proteins from Chaetomorpha sp. - Determination of amino acid composition, morphology and structure, functional properties, biological activity and nutritional value of protein concentrate from seaweed. Chapter 1: OVERVIEW 1.1. Green algae Chaetomorpha sp. Green algae of the genus Chaetomorpha are multicellular bodies made up of cylindrical cells, characterized by non- branching fibers. Phylum: Chlorophyta Class: Ulvophyceae Order: Cladophorales Family: Cladophoraceae Genus: Chaetomorpha There are about 50 species of Chaetomorpha currently recognized based on several morphological characteristics, such 2
as growth form, cell size and shape of attached cells (Leliaert, Frederik et al. 2011). 1.2. Algae protein The protein content of algae varies from species to species. Survey results of Kumar (2010) on 18 different species of seaweeds of 3 branches Chlorophyta, Phaeophyta, Rhodophyta in India showed that protein content varies from 10 - 35%. The protein content of seaweed is also affected by the seasonal cycle of the year. Fleurence (1999) pointed to the seasonal change in protein content of 9 - 25% on P.palmate with the highest protein intake during the spring and winter months. In Ulva lactuca, the highest protein intake was August with 32.7% protein on dry weight (Fattah and Sarry, 1987). Protein from seaweed is full of essential amino acid components with a much more proportional ratio than proteins from plants. Algae protein is also rich in aspartic acid and glutamic acid, notably lysine is known for its relatively low concentrations in plant protein sources. Therefore, protein from algae can be a source of nutrition in the human diet. The valuable proteins obtained from algae have been studied many are lectins and phycobiliprotein. In addition, protein concentrate and protein isolate from algae also have the potential to be used in supplements because of their ability to reduce the risk of cardiovascular disease. 1.3. Receiving algae protein The main factor used to extract algae protein is an alkaline solvent. However, protein extraction is restricted by the polysaccharides in the cell wall. The usual option is to use an enzyme system that breaks down the cell wall. Fleurence et al. 3
(1995) studied the use of different enzymes on red seaweed species, resulting in the activity of carrageenases and cellulases that increased protein collection efficiency by 10 times. In addition, using ultrasound to extract protein, carbohydrate components from algae has also been studied a lot. Among the new extraction methods, we can mention SFE method using supercritical CO2 and PLE method using liquid solvent under high temperature and pressure conditions to separate antibacterial, anticancer and antioxidant from S.pallidum and H.elongata (Fitzgeral 2011). After receiving the protein containing supernatant, different methods are used for protein purification. There are many traditional methods of precipitation with different agents such as organic solvents, neutral salts, precipitation at isoelectric point, ... These methods has features of easy implementation, precipitation agents easy to find and popular. However, the efficiency is not high compared to the modern methods. The more common methods is dialysis, gel filtration chromatography, affinity chromatography, ion exchange chromatography, membrane filtration. 1.4. Protein concentrate analysis Algae protein concentrate was assessed through the following criteria: composition and amino acid content, the morphology and structure, functional properties and nutritional value for using in food industry. In addition, protein concentrate was also assessed for a number of special biological properties to evaluate the potential of new protein sources. 4
Chapter 2: MATERIAL AND METHODS 2.1. Material Chaetomorpha sp. collected after 15-20 days of development, collected in extensive shrimp ponds in Gia Rai, Bac Lieu. The algae were transported in a foam tank during the day to the laboratory. Algae was washed to remove impurities, then dried, grinded and dried to 5% humidity, stored in a desiccator at room temperature. Enzyme Cellulase of Genecor firm (USA), trade name is Crestone Conc. able to work well in neutral pH range from 6.0 to 7.5, temperature from 450C - 550C; activity 1,100 UI / ml. 2.2. Research diagrams Research diagram is shown in figure 2.2. 2.3. Experimental arrangement 2.3.1. Identify of protein groups in algae The method of Hu and Esen (1981) is used to identify groups of proteins in algae based on their solubility in various solvents. 2.3.2. Investigate of material pretreatment process Factors studied for material pretreatment process include the influence of the drying temperature and the size of the mill. 2.3.3. Investigate the process of extracting water-soluble proteins with cellulase Survey factors are buffer solution type, pH, substrate and solvent ratio, enzyme concentration, time and temperature of extraction. The objective function is water soluble protein content and phycocyanin content. Optimize enzyme treatment process: optimize extracted condition by using response surface methodology. 5
Algae powder Investigation of material pretreatment process Enzyme Drying, grinding Investigation and optimization of Extraction 1 extraction process of Protein water-soluble protein containing group supernatant Biomass residue Investigation and optimization of Alkaline extraction process of solvent alkaline soluble protein Extraction 2 group Protein containing supernatant Investigation of protein precipitation conditions Tủa protein Dialysis remove salt Freeze-dried Morphology, structure Functional properties Biological properties Nutritional value Water-soluble PC Alkaline soluble PC Figure 2.2. Diagram showing 6the process of obtaining protein concentrate from Chaetomorpha sp.
2.3.4. Investigate the process of extracting alkaline protein Survey factors are substrate and solvent ratio, solvent concentration, time and temperature of extraction. The objective function is alkaline protein content. Optimize alkaline treatment process: optimize extracted condition by using response surface methodology. 2.3.5. Study on protein purification process Investigation the process of protein precipitation by different solvents: isoelectric precipitation with HCl, precipitate with ethanol, precipitate with ammonium sulphate and improve the purity by dialysis method. 2.3.6. Study on morphological and structural characteristics of protein concentrate Protein purification by gel filtration chromatography, determination of amino acid composition by HPLC, morphology observation by SEM scan, structure determination by electrophoresis and LC-MS / MS. 2.3.7. Identify biological properties of protein concentrate − Antibiotic activity was tested on 3 strains of bacteria causing human disease are S.aureus, E.coli, P.aeruginosa. − Antioxidant activity was tested by the following mechanisms: DPPH free radical, ABTS•+ free radical, interaction with Fe2+ ions and in vivo cell lipid peroxidation inhibitory activity. 2.3.8. Identify functional properties of protein concentrate Protein solubility and absorb water, foaming ability and foam stability, emulsion capacity and stability, least gel concentration. 2.3.9. Evaluate nutritional value in in vitro condition − In vitro protein digestibility (IVPD) − Amino acid score (AAS) 7
− Protein digestibility-corrected amino acid score (PDCAAS) 2.3.10. Evaluate nutritional value in in vivo condition In vivo modeling was performed on white mice with 4 groups with separate diets. Group 1: Diets without protein Group 2: Normal diet (food provided by the Pasteur Institute) Group 3: The AIN 93 diet contains APC protein Group 4: The AIN 93 diet contained soy protein Evaluation criteria: protein efficiency ratio (PER), net protein ratio (NPR) and biological value (BV). 2.4. Analytical method − The biochemical components were determined according to the LAPS method (Dien et al., 2010). − Soluble protein content was determined by Lowry and Bradford method. − Phycocyanin content was determined by spectroscopy (Bennett and Bogorad, 1973). − Observe the structure of algae and protein concentrate using a scanning electron microscope (SEM). − The amino acid composition was analyzed by HPLC method. − MW of protein was determined by SDS PAGE electrophoresis method and by LC-MS / MS method − The indicators of glucose, triglyceride, HDL-C and LDL-C in rat blood were analyzed at Hospital 115. 2.5. Data processing methods Experiments were performed 3 times. The results were statistically processed by Statghraphics software, the level of significance α = 0.05. 8
Optimized data of the extraction process using response surface methodology using Moodle 5.0 software. Chapter 3: RESULTS AND DISCUSSIONN 3.1. Chaetomorpha protein content 3.1.1. Biochemical composition The carbohydrate and protein content in Chaetomorpha biomass are 47.19% and 12.68%, respectively. The protein content in Chaetomorpha is not high (12,68%-20%) when compared to the protein content in soybeans, which is the plant source commonly used to obtain protein concentrate and protein isolate. However, the extraction of protein from algae, followed by the use of algae residue after protein extraction to produce bioethanol and microbiological fertilizers will create a closed value chain of materials with many diversified products, creating jobs for farmers, opening up a sustainable development direction for this wasted material source. 3.1.2. Determination of protein groups in Chaetomorpha Algae Chaetomorpha will be treated in turn with distilled water; NaCl 0.5M; 70% ethanol and 0.1M NaOH to identify protein groups. The results showed that the percentage of protein from Chaetomorpha with the highest alkaline protein was 55.27%, the water soluble protein accounted for 34.33% compared to the total protein. The two groups of proteins dissolved in salt and alcohol have a low ratio of 6.07% and 4.32%. The analysis results is the basis for the selection of suitable solvents for protein extraction. From the above results, we decided to choose to investigate the extraction process of two 9
groups of proteins with high content in seaweed, namely the water-soluble group and the alkali-soluble group. 3.2. Investigation of material pretreatment process The algae material was investigated for the pretreatment condition which is drying temperature and material size. The results show that the drying temperature at 600C and material size less than 0.1mm is the best for protein extraction. 3.3. Investigate the process of extracting water-soluble proteins with cellulase The use of cellulase will facilitate cell wall hydrolysis and the combination of physical methods such as ultrasound helps to accelerate the extraction process and achieve higher yields. In this study, the factors examined are the type of buffer; buffer pH ranges from 5.0 to 8.0; ratio of substrate and buffer solution from (1: 5) to (1:30). The cellulase concentration used varied from 50UI / g substrate to 150UI / g substrate. Extraction time from 1 to 8 hours, temperature from 300C to 700C. The results showed that phosphate buffer pH 7 was suitable for enzyme activity. The cellulase concentration used is 100 UI/g. The optimal extraction time and temperature are 60 minutes and 500C respectively. These conditions are also suitable conditions for phycocyanin absorption. Phycocyanin content obtained the highest is 0.39 mg / g dried algae, accounting for about 1% of the total content of the water-soluble protein group (38.36 mg / g dried algae). Optimize enzyme treatment process Enzymatic hydrolysis is a reaction in the heterogeneous system, so the potential for enzyme exposure to substrates is important. The ratio of raw materials (substrates): high buffers will 10
increase the environmental viscosity, limit the ability to transfer mass. In contrast, using a low substrate concentration will dilute the extraction solution and make further extraction difficult with alkaline solvents. In this experiment pH 7 and the ratio of ingredient / substrate: solvent 1:20 are kept at optimal levels. The optimization process is set up with 3 variable parameters namely enzyme concentration (X1), temperature (X2) and extraction time (X3), using a level 2 orthogonal optimization model. has a rotating center with 3 experiments in the center. The regression equation describes the soluble protein content obtained in the extract as follows: Y1 = 37,024 + 3,452 X1 + 2,322 X3 – 3,137 X12 – 1,417 X32 Enzyme concentration and extraction time significantly affect protein extraction efficiency, but the interaction of these factors has no effect on the protein content obtained in the extract (p> 0.05). The optimal conditions of the extraction process are the enzyme concentration of 121 UI / g substrate, the temperature and the extraction time is 400C; 90 minutes. Soluble protein content was predicted and experimental 38.921 and 37.651 mg / g dried , 3.27% difference. 3.4. Investigate the process of extracting alkaline protein with NaOH solvent The single factor survey results show that, optimal parameters for the extraction of alkaline-soluble protein groups include the ratio of substrates and solvents (1:20), NaOH concentration 1%, extraction time and temperature are 60 minutes and 500C respectively. 11
Based on these results, optimization was performed according to orthogonal model 2 with vortex center with 3 experiments at center. The factors that have a great influence on the extraction process selected as the variable parameters are extraction time (X4) and NaOH concentration (X5). The regression equation describes the soluble protein content obtained in the extract as follows: Y2 = 65,180 + 6,321 X4 + 10,224 X5 – 5,509 X4 X5 – 8,259 X52 The results showed that NaOH concentration and extraction time showed the mutual effect of protein content. When using a low concentration of NaOH, it will take a long time for the extraction to achieve optimal efficiency. In contrast, when using NaOH at a high concentration, the extraction process will quickly reach equilibrium and the time extension will not significantly increase the extraction efficiency. Dissolved protein content was predicted to reach the highest of 68,651 mg / g of dry seaweed at the NaOH concentration of 1.2% and the extraction time was 72 minutes, 0.8% difference compared to the experimental results. Evaluate the efficiency of protein extraction by methods In this experiment, Elma ultrasonic bath at 35kHz frequency was used in combination with cellulase to increase the efficiency of cell wall hydrolysis. The complex crystal structure Figure 3.5. Efficient extraction of water- of cellulose makes it soluble and alkaline protein groups by different methods resistant to hydrolytic 12
effects. When combined with cellulase, the ultrasonic waves help break down the crystalline structure of the cellulose, allowing the enzyme to have better access to the substrate for efficient hydrolysis of algae cells. Therefore, using a combination of both ultrasound and enzyme helps to increase the efficiency of protein extraction from algae biomass significantly compared to the control (67.9 vs. 43.34 mg / g of dried algae). The structure morphology of algae and algae residue was studied by scanning electron microscope (SEM). a b Figure 3.7. Image on scanning electron microscope (magnification 250x, scale 50µm) of algae residue Chaetomorpha sp. after protein extraction (a) Do not use ultrasound and enzymes (b) Use ultrasound and enzymes In the absence of supportive measures, algae cell walls are ineffectively broken down, so many cells still have intracellular components that have not been extracted (Figure 3.7a). In contrast, with the help of ultrasound and enzymes, almost all algae cells atrophy due to the complete release of intracellular components (Figure 3.7b). 3.5. Study on protein purification process The isoelectric precipitation and ethanol precipitation process for protein collection efficiency is not high, the purity of protein precipitate
group reached 62.86% and 58.77%, respectively, of the alkaline soluble protein group 65.29% and 62.29%, respectively. The sample was dialysated with a 14kDa semi-permeable cellophane membrane to remove salt, the recovery efficiency of the protein group dissolved in water and alkaline was 70.81%, 71.08%; The respective purity is 72.80% and 75.50%. 3.6. Determination of the properties of protein concentrate 3.6.1. The biochemical composition of protein concentrate To obtain the protein concentrate, algae are extracted two protein segments: water-soluble and alkaline-soluble, then precipitated with ammonium salt and subjected to the salt dialysis under optimal conditions. Protein concentrates were freeze-drying to moisture below 10% w/w. Protein content in the two preparations APC-N and APC-K are 72.8% and 76.3%, respectively. 3.6.2. Amino acid composition in protein concentrate Table 3.17. Amino acid composition in protein concentrate Amino acid content in protein Acid amin concentrate (g/100g protein) APC-N APC-K Aspatic acid 11.7 ± 0.20 11.5 ± 0.15 Glutamic acid 16.1 ± 0.09 15.7 ± 0.14 Serine 5.3 ± 0.03 5.1 ± 0.1 Histidine 1.4 ± 0.07 1.7 ± 0.25 Arginine 5.3 ± 0.15 6.2 ± 0.1 Glycine 4.9 ± 0.18 4.9 ± 0.12 Threonine 4.1 ± 0.16 5.1 ± 0.23 Alanine 7.3 ± 0.11 7.8 ± 0.09 14
Tyrosine 4.5 ± 0.09 4.1 ± 0.06 Methionine 3.6 ± 0.15 3.5 ± 0.15 Valine 5.4 ± 0.18 6.5 ± 0.09 Phenylanine 5.9 ± 0.34 5.9 ± 0.18 Isoleucine 3.5 ± 0.15 4.6 ± 0.01 Leucine 8.3 ± 0.14 8.6 ± 0.09 Lysine 5.1 ± 0.18 5.6 ± 0.07 Cystine 3.0 ± 0.05 1.8 ± 0.09 Proline 4.6 ± 0.09 1.4 ± 0.05 Content of essential amino acids in protein of Chaetomorpha sp. accounting for a high percentage, the total content of 8 non- substituting amino acids in the protein APC-N and APC-K accounts for 37.3% and 41.5% respectively of the total protein content, respectively. 3.6.3. Morphological of protein concentrate a b c d Figure 3.9. Image on a scanning electron microscope of protein concentrate at 500x magnification, 50µm scale and at 1000x magnification, 10µm scale (a), (b) APC.N; (c), (d) APC.K Figure 3.9 shows the structure of the protein concentrate inoculant form. water-soluble protein APC-N has a flat and large plate structure, while alkaline soluble protein APC-K has a 15
smaller plate structure with a rough and porous surface. Usually panels that are small and porous will have higher solubility. The microstructures of the two protein concentrate can be used to explain their solubility (Hu et al., 2013). 3.6.4. Structural characteristics of protein concentrate The protein concentrate from algae will be segmented by gel filtration chromatography and molecular size determination using SDS-PAGE electrophoresis. Protein molecules have a very complex structure. The spatial structure of the protein as well as its ability to be denatured in the medium containing SDS can affect the protein's mobility. In addition, protein can bind with other non- protein components and it is difficult to separate proteins from these components under normal SDS-PAGE electrophoresis conditions. The influence of the shape and size of the non-protein binding components on protein mobility is difficult to predict. According to many studies, the proteins in algae are usually glycoproteins. Glycoproteins are proteins containing covalently linked oligosaccharides / polysaccharides with appropriate amino acids. The glycoproteins have low electrophoresis mobility and high molecular weight, thereby creating banding in the electrophoresis gel. Two samples of water-soluble and alkaline-soluble proteins were sent to the Biochemistry Laboratory, University of Natural Sciences, National University of Ho Chi Minh City to determine molecular size by LC-MS / MS method, using electrospray ionization (ESI) and molecular mass determination by QTOF. Chromatogram of two protein samples when analyzing LC / MS / 16
MS is shown in figure 3.15 and figure 3.16. The results of mass spectrometry analysis of these peaks are as follows. - The water-soluble protein concentrate contains protein fragments of 11.4 kDa, 13.6 kDa, 15.4 kDa, 27.2 kDa, and 38.6 kDa sizes, respectively (Figure 3.15). - The alkaline protein concentrate contains protein fragments of 11.4 kDa, 21.1 kDa, 27.2 kDa, 34.1 kDa 40.5 kDa and 63.8 kDa sizes, respectively (Figure 3.16). Figure 3.15. Analysis results Figure 3.16. Analysis results of MS APC-N of MS APC-K 3.7. Biological properties of protein concentrate 3.7.1. Antimicrobial activity test The bacteria tested to cause disease in humans provided by ATCC: S.aureus, E.coli, P.aeruginosa. When compared to the Kanamycin antibiotic control with the recommended concentration of 10µg/ml, the antimicrobial effect of the algae protein concentrate is quite good against S. aureus and P. aeruginosa (The antibacterial ring diameter for these 2 bacteria is 6 mm and 6.33 mm, respectively). Antibiotics are twice as effective against E. coli than when using protein concentrate from algae. However, the MIC value (the lowest concentration at which the inoculant exhibits antibacterial 17
activity) of the algae protein was much higher than the control, Kanamycin. That shows, the antibacterial activity of algae protein products is quite low, not enough to be able to put into practical application. 3.7.2. Antioxidant activity test The antioxidant activities of the two Chaetomorpha protein concentrate were tested by different mechanisms. At the same concentration, APC.N preparation has much higher DPPH free radical activity than APC.K. At the same test concentration of 78,125 µg / ml, the percentage of active of DPPH of the two protein APC.N and APC.K were 73.97% and 22.22%, respectively. DPPH capture activity of APC-K preparations at the test concentration of 312.55 µg / ml was 55.18%. The higher DPPH capture activity of APC.N is probably due to the Phycocyanin is a water-soluble protein with antioxidant activity. In addition, protein molecules obtained from algae may also contain antioxidant amino acid components such as cysteine (containing thiol group), amino acids containing phenolic groups such as tyrosin. With the purpose of assessing the applicability of APC-N in antioxidant-rich functional foods, APC-N continues to be used to further study the antioxidant capacity by different mechanisms. . The experiments were conducted at the Laboratory of Biochemistry, Center of Ginseng and Medicinal Materials, University of Medicine and Pharmacy, HCMC. Experiments to evaluate the oxidation resistance of water- soluble protein from Chaetomorpha sp. showed that APC-N is 18