Optimization of Enzymatic Extraction of ACE Inhibitory Peptide from Rohu (Labeo rohita) Fish Waste using RSM

Recently there is an increase in cardiovascular disease (CVD) possibly because of reduced physical activities, stressful lifestyle, obesity etc. One of the major CVDs is hypertension. Globally 1.13 billion people are suffering from hypertension, a condition when systolic blood pressure (SBP) reaches at least 140 mm Hg or/and diastolic blood pressure (DBP) to 90 mm Hg or both while the normal SBP/DBP is <120/<80 (Poulter et al., 2015). BP is regulated by the renin-angiotensin system (RAS) with the critical role of angiotensin-I converting enzyme (ACE) (Ngo et al., 2016).
ACE (EC 3.4.15.1) is a membrane bound enzyme found at pulmonary and renal endothelium surfaces. ACE converts Angiotensin I into Angiotensin II removing a dipeptide (His-Leu) from C-terminus (Gao et al., 2018) and there by resulting in high BP. Therefore, any ACE inhibitory biomolecule will reduce BP and there by minimizing hypertension. Pharmaceutical drugs (e.g. enalapril, captopril, lisnopril, ramipril, etc.) target ACE inhibition for lowering BP but these drugs have associated side-effects like cough, angiodema, chest pain etc. (Coleman and Cox, 2012). Therefore, scientists are trying to identify the peptides having potential therapeutic benefits like ACE inhibitory activity from various foods including fish (Yathisha et al., 2018).
       
India is the 2^nd largest producer of freshwater fish in the world. This higher production provides more fish for processing. However, global fish processing dispose >60% of fish biomass as skin, head viscera, trimmings, liver, frames, bones, etc. (Halim et al., 2016) imposing a cost burden on the industry for its disposal. Though it is used traditionally to produce low value products like fish silage, fish meal and sauce (Halim et al., 2016), it can be utilized for high value products with health benefits. The FPH, bioactive peptides including antihypertensive (or ACE-inhibitory) peptides can be extracted from considerable amount of protein in fish waste and novel bioactive peptides can be identified to provide with next generation functional products (He et al., 2013).
       
Indian major carps (IMC) viz. catla, rohu and mrigal contribute 70-75% of total freshwater fish production in India, followed by silver carp, grass carp, common carp and catfish (Kudre et al., 2017). Among IMC, rohu is one of commonly cultured freshwater fish. High growth potential, nutrition, delicious attributes and high market value make rohu an economically important fish. It is sold extensively in the Indian fish market generating approx. 0.9 MT/annum of rohu fish waste (RFW) in India (Kudre et al., 2017). Thus, the utilization of RFW for production of ACE inhibitory peptide would greatly benefit in reducing fish waste disposal. Extracted peptides can also be a suitable ingredient in functional food reducing hypertension without any side effects. Therefore, the objective of this study were to calculate anatomical yield of generated fish waste, to estimate proximate composition and to optimize the independent variables of extraction of ACE inhibitory peptide.

Rohu fish was procured in chilled condition from local fish market to ICAR-CIPHET, Ludhiana. Non-edible tissues (fins, scales, swim bladder and head) separated from edible flesh were mixed in natural proportion to get a homogenized RFW. Analytical grades chemicals, organic solvents and enzymes procured from Sigma, St. Louis, USA; Himedia, Mumbai, India and MP Biomedicals Mumbai, India were used. Importantly, Alcalase® (Protease from Bacillus licheniformis), Angiotensin-I Converting Enzyme (ACE), 2,4,6-Trinitrobenzene Sulphonic Acid (TNBS) and N[3-(2-Furyl)acryloyl]-Phe-Gly-Gly (FAPGG) were obtained from Sigma. Sodium Phosphate Dibasic Heptahydrate, Sodium Phosphate Monobasic Monohydrate, Bovine Serum Albumin, Bradford Reagent, Tricholoroacetic Acid and L-Leucine were procured from Hi-media and Captopril and Hydrochloric acid were received from MP Biomedicals. Experiments were performed during 2018-2020.
       
Anatomical yield was determined based on total length (TL) and total weight (TW) of fish (Table 1). The percentage of fish head (FH), fins (FN), scales (SC) and swim bladder (SB) were calculated. Moisture, fat, ash and protein of rohu wastes were analyzed individually and after mixing them in natural proportion (RFWP) (AOAC, 2020).
 

       
Homogenized RFW added with a buffer (PBS, 50 mM, pH 7.5) and alcalase enzyme was incubated for 30 min followed by 240 min hydrolysis and subsequent enzyme inactivation at 90°C for 15 min and centrifugation (5000Xg, 20 min). The supernatant filtered was named rohu fish hydrolysate (RFH). Its DH was determined by Adler-Nissen (1979) method. RFH (0.25 ml) added with 2 ml PBS (0.2125 M, pH 8.2) and 2 ml TNBS (0.1%) was incubated in the dark at 50°C (1 h). The reaction was terminated using HCl (0.1 M, 5 ml) and the absorbance was measured at 340 nm. DH expressed Q-amino acids in terms of L-Leucine (Eq. 1).

Where, 
Lt= Amount of a-amino acids released at time ‘t’.
Lo= Amount of a-amino acids released in acid-solubilized substrate.
Lmax= Amount of maximum a-amino acids found.
 
Ultrafiltration
 
Rohu fish hydrolysate (RFH) obtained at the end of enzymatic hydrolysis was filtered twice with filter paper (hi-media, 12.5 cm dia; retention: 10 µm; grade 631 equivalent to grade 4) and then by a syringe filter (hi-media, PES, 0.45 µm) and subsequently again through a syringe filter (hi-media, PES 0.2 µm pore size). The filtrate was then passed through a MWCO (10 kDa and subsequently 3 kDa). The filtrate (<3 kDa) named RFW peptides (RFWP) was used to assess the ACE inhibitory activity.
 
ACE inhibitory assay
 
A modified method of Murray et al., (2004) and Theodore and Kristinsson (2007) using FAPGG (a synthetic substrate) was used for ACE inhibitory assay. RFWP (100 μL, 0.2% protein w/v), ACE enzyme (15 mU, 50 μL) and substrate (1 mL, 0.5 mM FAPGG) were mixed together, incubated at 25°C (60 min) and the absorbance was measured at 340 nm. FAPGG without ACE enzyme served as a control. Captopril was used as standard. Hydrolysis of FAPGG by ACE resulted in a decrease in absorbance. ACE inhibition calculated as (Eq. 2).  Estimation of peptide yield (PY)
 
PY was determined as per Wu et al., (2019). RFWP (5 ml) dissolved in TCA (10%, 1ml) was kept at room temperature (20 min.) and then centrifuged (10000xg, 10 min). Biuret method was used to find peptide content in supernatant taking bovine serum albumin as standard. PY was calculated (Eq. 3) considering conversion coefficient of nitrogen to protein as 6.25.

                                                               
Statistical analysis
 
Single factor experiments were executed at different alcalase concentrations, hydrolysis temperatures, time and S/L ratios to find ACE-inhibitory peptide. Design-Expert ver.12 was used for design of experiments, optimization of independent variables and statistical calculations. Response surface methodology applied with Box Behnken Design (RSM-BBD) (32 runs) optimized 4 independent variables for highest DH, ACE inhibition and PY. All experiments were done in triplicate and expressed as the mean±standard deviation (n=3).

Anatomical yield and proximate composition of RFW
 
The anatomical yield of RFW (FH, FN, SC and SB) varied significantly (p<0.05) among small, medium and large rohu fish (Table 1). Small fish generated more waste (49.45%) than that of medium (34.51%) and large (34.54) fish. Lower yield of head (27.62, 32.43 and 22.11% in common carp, bighead carp and grass carp respectively) (Skałecki et al., 2015) than found in our study might be due to species difference.
       
Moisture, fat, crude protein and ash differed significantly (p<0.05) among all wastes (Table 2). Though SB constitutes a small fraction (1.42-1.91% by wet weight) of total generated waste, it can be mostly preferred for peptide extraction because of its highest protein content (>34%) among fish wastes. Protein content in scales of farmed grass carp (Ctenopharyngodon idella) (21.74%) (Naqvi et al., 2014) was closer to that found in rohu scales (22.9%). More protein (4% in FH), fat (11% in FH) and ash (17% in SC) were found in rohu in our study than grass carp (Naqvi et al., 2014). In contrast comparatively less moisture (3% in FH and 1% in SC), ash (11% in FH and 2.5% in SC) and protein (1% in SC) were also observed in rohu (in our study) than grass carp (Naqvi et al., 2014).
 

 
Alcalase concentration
 
With increase in alcalase concentration from 0.5 to 2% (v/w) there was a significant (p<0.05) increase in DH, ACE inhibition and PY (Fig 1A). However, ACE inhibition and PY decreased beyond 2.5% (v/w) alcalase concentration but DH continued to increase. In salmon (Salmon salar) skin hydrolyzed by alcalase, See et al (2011) found 2.5% enzyme as optimum for highest DH (77.03%). In eel (Monopterus sp.) protein hydrolysate with alcalase (1.5-2%) showed DH ranging 36-69% (Baharuddin et al., 2016). In grass carp (Ctenopharyngodon idella) skin hydrolysate obtained with increasing alcalase enzyme to substrate ratio (0.12 to 1.08) showed increasing DH (5.02 to 14.9%) (Wasswa et al., 2007). In our study ACE inhibition reduced from 61.1 to 56.6% as alcalase concentration increased from 2 to 2.5% (v/w). Similar result is reported in lizard fish (Saurida elongata) muscle protein hydrolysate wherein ACE inhibitory activity decreased from 78 to 70% with increase in E/S from 1:1 to 1.5:1 (Wu et al., 2012). Optimum PY (amount of peptide/s (g) produced per 1 g crude protein) in pig bone collagen peptide was 36% with alcalase (6000/ u/g powder) at 6% substrate concentration (Wu et al., 2019).
 

 
Hydrolysis temperature
 
DH, ACE inhibition and PY increased significantly (p<0.05) with increase in hydrolysis temperature (Fig 1B). Higher percentage of all these reduced dependent variables were noticed between 50-60°C which further reduced beyond 60°C. This may be due to degradation of enzyme at higher temperature. Similar increase in DH (36 to 69%) with increasing temperature (40 to 60°C) observed in eel (Monopterus sp.) protein hydrolysate (Baharuddin et al., 2016). In our study a DH reduced from 19.9 to 15.9% beyond 65°C. Similar decrease (71 to 61%) in DH reported in alcalase hydrolysate of salmon (Salmon salar) skin (See et al., (2011) when temperature increased from 55.3°C to 70°C. The result was consistent with the optimal temperature (55°C) of alcalase enzyme. Highest ACE inhibition (62.2% at 60°C) did not increase at >60°C might be due to inactivation of enzyme at higher temperature. Alcalase hydrolysate of Basa fish skin showed 33.3% ACE inhibition at 52°C (Zhang et al., 2016). Minced striped snakehead hydrolyzed with alcalase (3% g/mL) showed 70% ACE inhibition at 55°C (Ma, et al., 2021). Our study showed PY 51.8% at 60°C.
 
Hydrolysis time
 
There was a significant (p<0.05) increase in DH, ACE inhibition and PY with increase in hydrolysis time (Fig 1C). A rapid hydrolysis of RFWP at 60 min of hydrolysis indicated that large number of peptide bonds were cleaved. Later on, DH decreased significantly (p<0.05) until 180 min. and further reduced beyond 180 min. Similar to our study, DH increased from 5.02 to 14.9% in grass carp (Ctenopharyngodon idella) skin hydrolysate when hydrolysis time increased from 75 to 120 min. (Wasswa et al., 2007) and 36, 48 and 69% in eel (Monopterus sp.) protein hydrolysate at 120, 180 and 300 min. of hydrolysis, respectively (Baharuddin et al., 2016). PY (48.2%) found maximum at 240 min when DH and ACE inhibition showed reducing trend. This may be due to fact that more hydrolysis period allowed higher cleavage of protein leading to higher PY. However, optimum PY (36%) of pig bone collagen peptide found at 5 h of hydrolysis (Wu et al., 2019).
Solid/liquid ratio
 
DH, ACE inhibition and PY increased significantly (p<0.05) with increase in S/L ratio (Fig 1D). Initially (at <0.4) there was a gradual increase followed by a sharp increase (from 0.4 to 0.8) and finally very minimal increase (beyond >0.8). The increase in solid (homogenized RFWP) per unit of liquid (reaction buffer) increases the substrate availability and thereby provide more active sites for the enzymatic activity. In alcalase hydrolysate of Basa fish skin highest ACE inhibition was reported at S/L ratio 1:8.05 g/mL (Zhang et al., 2016) while in the minced striped snakehead (62% ACE inhibition) at 1:15 S/L (Ma et al., 2021). Highest PY found in our study is 49.9%.
 
Optimization of enzymatic extraction process variables
 
RSM-BBD (32 runs) was performed to study the combined effect of 4 independent factors (X₁: Alcalase Concentration, X₂:  Temperature, X₃: Time and X₄:  S/L Ratio) on 3 dependent responses (DH, Y₁; ACE inhibition, Y₂ and PY, Y₃). A multiple regression analysis was performed using RSM to determine all the coefficients of linear (X₁, X₂, X₃, X₄), quadratic (X₁², X₂², X3², X₄²) and interaction (X₁ X₂, X₁ X₃, X₁ X₄, X₂ X₃, X₂ X₄, X₃ X₄) terms to fit a full response surface model for the responses. In our study, a linear model term X₁ (alcalase concentration) found significant (p<0.05) for all the responses while X₂ (temperature) was significant (p<0.05) for PY only. X₃ and X₄ did not affect any response significantly (p>0.05). Additionally, a quadratic model term, X₂² showed significant (p>0.05) effect on all responses while other quadratic model term X₃² showed significant (p>0.05) effect on ACE inhibition and PY only.
       
Fig 2 illustrates contour (2D) and response surface (3D). Alcalase concentration significantly (p<0.05) influenced all the dependent variables. Temperature affected PY significantly (p<0.05) but not DH and ACE inhibition, however, temperature demonstrated significant effect on all the dependent variables of 2^nd order reaction but with negative effect on DH and PY. Time and S/L ratio were insignificant (p>0.05) for all the dependent variables. A final response surface regression equation obtained by RSM is as below (Eq. 4).
 

 
In our study the desirability profile indicated optimum DH (19.27%), ACE inhibition (54.98%) and PY (51.37%) with 1.08 % (v/w) alcalase concentration, 52.10°C temperature, 129.18 min hydrolysis time and 0.8:1 solid-liquid ratio. Taking the actual operations these may be adjusted to 1.1% (v/w), 52°C and 129 min. respectively. Similar results were reported in salmon skin hydrolysate (2.5% v/w alcalase, 55.3°C temperature resulted in 77% DH) (See et al., 2011) and minced striped snakehead (5% w/v alcalase, 55°C temperature,  3 h hydrolysis and 1:25 g/mL S/L ratio exhibited 54.35% ACE inhibition) (Ma et al., 2021).

First author thanks the Director, ICAR-CIPHET, Ludhiana for providing necessary facilities to conduct this study and acknowledge informal supports of Dr. K. Narsaiah, Dr. D.N. Yadav and Dr. Sandeep Mann. Authors acknowledge the Dean, College of Fisheries, Ludhiana for providing lyophilizer.

Authors declare no potential conflict of interest.

ICAR-CIPHET, Ludhiana, Punjab [Grant No. 1 (254)/ICAR-CIPHET/TOT/IRC/2018].