Research and receiving protein from brackish water algae (chaetomorpha sp.) for application in food industry

industry. In addition, protein concentrate was also assessed for a number of special biological properties to evaluate the potential of new protein sources. 5 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. 6 Investigation of material pretreatment process Drying, grinding Enzyme Investigation and optimization of extraction process of water-soluble protein group Protein containing supernatant Extraction 1 Extraction 2 Alkaline solvent Investigation and optimization of extraction process of alkaline soluble protein group Tủa protein Investigation of protein precipitation conditions Dialysis remove salt Freeze-dried Morphology, structure Functional properties Biological properties Nutritional value Biomass residue Protein containing supernatant Water-soluble PC Alkaline soluble PC Algae powder Figure 2.2. Diagram showing the process of obtaining protein concentrate from Chaetomorpha sp. 7 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) 8 − 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. 9 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 10 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 11 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. 12 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 of cellulose makes it resistant to hydrolytic Figure 3.5. Efficient extraction of water- soluble and alkaline protein groups by different methods 13 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). 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 <50%. When using 70% saturated (NH4)2SO4, the precipitation efficiency and purity of the water-soluble protein 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 14 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 Acid amin Amino acid content in protein 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 15 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 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 a b c d 16 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 / 17 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). 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 Figure 3.16. Analysis results of MS APC-K Figure 3.15. Analysis results of MS APC-N 18 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 19 resistant to oxidation by many different mechanisms. APC-N is protein concentrate with a protein concentration of 72.8% w/w. Besides, water-soluble proteins and phycocyanins, APC-N may also contain other components that may contribute to antioxidant activity such as soluble polysaccharides, plant polyphenols, chlorophyll ... Due to impurities, the antioxidant resistance of APC-N compared to the control of pure chemicals such as vitamin C, Na2EDTA of Sigma is still low (about 5-20 times lower). However, in the mouse meningeal cell experiment, APC-N exhibited similar inhibition of membrane lipid peroxidation similar to pure Trolox (Calbiochem Ltd. Co.). In general, the antioxidant activity of APC-N is from strong to weak: DPPH scavenging ability, membrane lipid peroxidation inhibition, Fe2+ ion binding ability, and neutralization ABTS•+ with IC50 values of 19.11 µg / ml, 21.52 µg / ml, 31.46 µg / ml and 52.24 µg / ml, respectively. Good oxidation resistance by many different mechanisms at the concentration of µg/ml shows the prospect of APC-N for antioxidant protection in functional foods. 3.8. Functional properties of protein concentrate Solubility is an important indicator of protein used in beverage technology. Protein from algae is soluble in both neutral and alkaline media, while soy protein is good for both acid, neutral and alkaline media. APC-N have higher solubility than APC-K (12.6 and 10.3% at pH7), possibly due to the ratio of hydrophilic groups on the surface of protein molecules in APC-N is higher. 20 The water absorption capacity of APC-K was quite low (18.5%), lower than that of APC-N (57.8%) and soy protein concentrate (49.33%). The protein molecule in the APC-N may have extended hydrophilic regions or a coiled helix structure to conceal the internal hydrophobic regions. Hydrophilic regions are where hydrogen bonds will form with surrounding water molecules, helping to absorb water onto the protein macromolecule. Gel-forming abilities play a role in giving the texture properties of many foods. The gel also increases viscosity, adhesion, and improves water and oil / fat bonding. The results showed that both APC-N and APC-K from Chaetomorpha sp. have a minimum gel forming concentration (LGC) of 10%, demonstrating good gel-forming ability compared to soy protein and many other plant proteins. APC-K has very good foaming and foam stability. The soy protein concentrate and APC-N have the same foaming capacity as APC-K (40-43%) due to its good solubility at pH 7. However, it is possible that due to the lack of hydrophobic amino acid groups that can interact well with the phase division surface (which helps to create a protein film around the tough, durable bubbles), the resulting foam system has less durability (75 -78% vs 90%). APC-K are also highly capable of emulsifying and stabilizing emulsions. The hydrophobic groups occupy a high proportion of the alkaline soluble proteins that help them interact well with the oil phase of the fat droplets. At the same time, the ratio of amino acids such as aspartic acid, glutamic acid is quite high in protein of 21 Chaetomorpha sp. can help create an electrostatic repulsion between fat droplets and improve the ability to endure the emulsion system. 3.9. Evaluate nutritional value in in vitro condition This study explores the nutritional value under in vitro conditions of protein concentrate from the green algae Chaetomorpha sp. to evaluate their potential to use them as a new source of plant protein in foods. With a protein content of 76.3% and the IVDP digestibility more than 83.8%, the APC-K has a PDCAAS value of essential amino acids greater than 1 except for histidine. The content of histidine in the APC-K meets 95% of the adult requirement as recommended by WHO (2007). The APC-N has a protein content of 72.8%, the digestibility of IVDP is 79. This preparation is deficient in two essential amino acids including lysine and histidine with PDCAAS index of 0.95 and 0.73 respectively. Thus, APC-K inoculant has higher nutritional value than APC-N. 3.10. Evaluate nutritional value of APC-K in in vivo condition After 4 weeks, there was a difference in the increase in body weight of 3 groups of mices; the mice that ate foods containing soy protein and APC-K increased 60-63%, and the group that ate normal foods increased 54%. Protein efficiency ratio (PER) is an index used to measure the quality of protein. PER value when using APC-K protein from Chaetomorpha reached 2.12, lower than the PER of animal proteins such as egg (3.9), beef (2.6) and fish ( 3.5). However, this value is higher than some vegetable proteins such as flour (0.6), beans (1.5). 22 Net protein ratio (NPR) shows that protein is not only needed for body development, but also for sustaining life.