Preparation,Isolation And Characterization Of Protein Hydrolysate From A Hybrid Sturgeon(Acipenser Ruthenus Linnaeus X Huso Dauricus(Georgi))

Nowadays,there is an increasing interest in how to utilize enzymatic hydrolysis processes to produce valuable nutrients from fish proteins.Fish protein hydrolysates?FPH?are products made from fish by the enzymatic hydrolysis.The peptides produced from FPH have smaller molecular mass and used for the treatment of various diseases such as atherosclerosis,carcinogenesis,coronary heart,and cardiovascular.The FPH prepared by exploiting combination of the enzymatic hydrolysis process and modern techniques including ultrasonic,microwave irradiations and combined ultrasonic-microwave to improve the enzymatic hydrolysis process and product properties.Firstly,protein hydrolysate was prepared from the muscles of a hybrid sturgeon?Acipenser ruthenus Linnaeus x Huso dauricus?Georgi??,and the effects of different conditions on the degree of hydrolysis?DH?by using papain were investigated.The DH of 24.89%was conducted under the optimum conditions including a solid-to-liquid mixing ratio of 1:1,an enzyme-substrate ratio of 3%,pH 6,a temperature of 70°C,and an incubation time of 6 h.The yield of protein hydrolysate was 17.47%,in which the protein content accounted for 79.67%,and the amino acid content represented 96.35 g/g protein.The molecular weight of peptides decreased with the progress in hydrolysis time.Protein hydrolysate solubility ranged between 86.57 and98.74%;emulsifying activity index was 11.0–13.27 m²/g,emulsion stability index was more than94%at different pH levels,water holding capacity was 1.93 g water/g protein,oil holding capacity was 2.59 g oil/g protein,and foam capacity was 76.67%.In the second part,protein hydrolysates from a hybrid sturgeon were prepared using Alcalase 2.4L enzyme.Under the optimum conditions?enzyme-substrate ratio of 3.5%,pH of8.5,and temperature of 55°C?,the DH was 13.8%,16.7%,and 19.1%after 1,3,and 6 hrs,respectively.At the DH of 19.1%,the contents of protein content and amino acid increased to86.97%and 78.29 g/100g protein hydrolysate,respectively.There was a noticeable increase in the low-molecular-weight peptides,which could enhance the hydrolysate's functional properties such as solubility,representing more than 90%at different pH levels.The obtained protein hydrolysates revealed excellent emulsification properties and high oil absorption.Furthermore,suitable antioxidant activities,such as 1,1-diphenyl-2-picrylhydrazyl?DPPH?inhibition,ferric reducing power,and ferrous ion(Fe²⁺)chelating ability were attained depending on the solution concentration.In the third part,the effects of the enzymatic hydrolysis and enzyme type on the structural and physicochemical characteristics of protein hydrolysates from a hybrid sturgeon were evaluated.Two different enzymes,including papain and alcalase 2.4L were used in the hydrolysis process.In this part,the papain enzyme significantly increased the DH?20.62%?and decreased the zeta potential?12.4±1.31 mV?as compared to the alcalase enzyme,which were15.55%and 15.53±0.77 mV,respectively.Alcalase 2.4L hydrolysate exhibited a smaller particle size?822.047±61.26 nm?than papain hydrolysate?1,425.39±44.82 nm?.Hydrolysis by papain and alcalase 2.4L enzymes decreased the molecular weights?MW?1000 Da?to 98.27 and86.84%,respectively.The surface hydrophobicity and turbidity of the hydrolysates significantly affected by enzyme type and protein concentrations.By using the X-ray diffraction analysis,the papain hydrolysate showed a higher relative crystallinity degree?30.33%?than alcalase 2.4L hydrolysate?29.40%?.The Fourier transform infrared spectroscopy showed more clearly peaks for the amide bands of alcalase hydrolysate.The thermal properties also affected by hydrolysis,since the melting temperatures were 159.17°C and 149.58°C,while the rate of mass loss was67.04%and 62.8%,for papain and alcalase hydrolysates,respectively.In the fourth part,ultrasonic and/or microwave approaches have been employed to evaluate the efficiency of protein hydrolysate production from a hybrid sturgeon and its antioxidant properties.Initially,the samples were subjected to thermal pretreatments in order to reach optimal temperature of 55°C.The Degree of hydrolysis?DH?recorded 7.63,5.55,and6.02%for ultrasonic?US?,microwave?MW?and combined ultrasonic-microwave pretreatment?US+MW?,respectively at the optimal time?8 min?.The enzymatic hydrolysis?EN?of pretreated samples increased the DH to 19.41,14.18 and 16.91%for US+EN,MW+EN and US+MW+EN,respectively.The US+EN treatment was more effective for obtaining higher DH and yield,which were 19.41%and 18.62%,respectively.The use of US+EN also resulted in an increase in the percentage of molecular weights??1000 Da?,amino acid content and protein solubility,which reached 89.24,80.08 and 98.58%,respectively.While,US+MW+EN pretreatment has achieved the highest antioxidant activities by IC₅₀ of DPPH and ABTS,which were 3.01 and 1.85 mg/mL respectively,in addition to the reducing power assay,which was0.528 at a protein concentration of 5 mg/m L.In the fifth part,the fractionation of peptides obtained from the enzymatic hydrolysis process using two different enzymes according to their molecular weights was carried out to obtain four fractions?<1000,1000–2000,2000–3000,and>3000 Da?.The antioxidant activities of the different fractions were evaluated by DPPH,ABTS and reducing power assays.Peptide fractions with molecular weights<1000 Da of both protein hydrolysate achieved higher antioxidant activities for the assays used.While the alcalase peptide fraction?MW<1000 Da?was more active in all assays which were 84.81%?DPPH?,93.96 mg trolox/g protein?ABTS?and 1.51 Absorbance unit for reducing power.The results of the current work demonstrated that the use of the enzymatic hydrolysis process led to improved properties of the resulting peptides that could be useful in many applications of the food industry.The application of ultrasound technology enhanced the DH while the combined use of ultrasound and microwave led to improved protein hydrolysate antioxidant properties.