Ultrasound‐assisted extraction of guava and papaya leaves for the development of functional shrimp patties

Abstract The current study was aimed to evaluate the effects of guava and papaya leaves extract on the antioxidant profile and their outcomes in the storage stability of shrimp patties. Total of seven treatments were prepared by employing guava leaf extract (GLE) and papaya leaf extract (PLE) including control. Both the extracts were employed in the concentration of 1% and 2% each and in combination as 1:1% and 2:2%, respectively. The shrimp patties were kept in ziplock bags at refrigeration temperature, and further analysis was done after 21 days of storage period with intermittent evaluation interval of 7 days. The antioxidant capability of functional shrimp patties was determined by total phenolic content (TPC), 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP). Higher significant values of TPC, DPPH, and FRAP were observed in the functional shrimp patties enriched with GLE2%:PLE2% at start of the experiment. The physicochemical parameters were observed by hunter color, TVBN, TBARS, and peroxide value (POV). Higher significant values of TVBN, TBARS, and peroxide were observed in the control samples as compared to treatment group GLE2%:PLE2%. The bacterial counts were also higher in control samples as compared to the treatment group GLE2%:PLE2%. The sensorial attributes were observed regarding appearance, taste, texture, odor, and overall acceptability. The maximum scores related all parameters were gathered by control group but significantly lower scores were for the group GLE2%:PLE2%. In conclusion, functional shrimp patties enriched with GLE2%:PLE2% showed better antioxidant capacity, stability, and sensory characteristics during storage.

tides, and PUFAs in special along with other useful nutrients. They are being referred as one of the vastly developing food products in the global market, possibly because of their apparent nutritional advantages, culinary traits and being a significant source of protein (Ajifolokun, Basson, Osunsanmi, & Zharare, 2018). Their shelf life is limited due to the presence of high moisture content, various degradable compounds, and neutral pH (Viji, Venkateshwarlu, Ravishankar, & Gopal, 2017).
The shelf life of shrimps can be increased by using synthetic as well as natural antioxidants. Presence of synthetic antioxidants play vital role in reducing free radical damage. It also helps to confer antiaging effects. However, at the end level consumers may suffer from their harmful effects, that is, propyl gallate, BHA (butylated hydroxyanisole) and BHT (butylated hydroxyltoluene) (Mona, Isis, Muhammad, & Samir, 2012). On the other hand, natural preservatives such as plant extracts and essential oils are being widely used to replace synthetic preservatives for having admirable antimicrobial and antioxidant potential (Smaldone et al., 2011). They account for lowering the losses by microbial contamination and lipid peroxidation in the food (Erkan, Gunlü, & Genç, 2014).
Guava (Psidium gujava) is considered beneficial fruit tree inherent from Mexico. Guava tree is rich in terpenoids, phenolic acids, flavonoids, and tannins (Barbalho et al., 2012;Pérez Gutiénez, Mitchell, & Vargas Solis, 2008); however, the leaves contain mainly tannins, flavonoids, and less amount of terpenoids (Metwally, Omar, Ghazy, & Harraz, 2011). Particularly, the focused area of research is terpenoid content of the leaves and the fruit as well as phenolic contents. Until now, 204 volatile components have been identified in the fruit (Joseph & Priya, 2011). While in the leaves, 86 compounds have been found (Garcia, Quijano, & Pino, 2009). White guava fruit was found to be rich in β-caryophyllene and β-bisabolene. Among white and pink fruit forms, variations in the composition have been noticed in the essential oil of fruit (Thuaytong & Anprung, 2011) and of the leaves (Chalannavar, Venugopala, Baijnath, & Odhav, 2014).
Papaya (Carica papaya) belongs to family Caricace. It has likewise been accounted for having cancer-preventing agents and free radical scavenging potential in its leaves separately (Okoko & Ere, 2012). The leaves of papaya contain numerous dynamic parts that can induce antioxidant activity in blood and diminish lipid peroxidation level like paper chymopapain, cyanogenic glycosides, glucosinolates, tocopherol, ascorbic corrosive, and flavonoids along with cystatin (Otsuki et al., 2010). These mixes are identified with mitigating action. C. Papaya leaf extracts additionally found to have antibacterial potential (Romasi, Karina, & Parhusip, 2012), antiproliferative to tumor and immunomodulation experiences. It is declared as nonpoisonous in light of the fact that it's LD > 15 g per kg body weight. The leaves additionally contain cardiovascular glycosides, anthraquinones, carpaine, pseudocarpaine, and phenolic mixes (Owoyele, Adebukola, Funmilayo, & Soladoye, 2008). Keeping in view the above facts, the main objective was to minimize the quality loss of shrimp patties during storage as well as enhancing their antioxidant profile with the incorporation of natural extracts of guava and papaya leaves.

| Procurement of materials and sample preparation
Mature guava and papaya leaves were procured from Ayub Agricultural Research Institute Faisalabad. Frozen shrimps were purchased from supermarket from Faisalabad. All the glassware and chemicals for further testing were used of analytical grade and purchased from Sigma-Aldrich ® . Guava and Papaya leaves were sundried for several days and then grounded to fine powder using grinder and sieved to remove woody parts and then stored in air tight bags to avoid moisture gain prior to extraction. Shrimps were thawed and minced, and patties were formed.

| Extraction of GLE and PLE
Powdered samples of dried guava and papaya leaves were taken in 500 ml beaker with 70% ethanol at 1:10 solid to solvent ratio. The extraction was carried out employing an ultrasonic probe Sonics & Materials). Frequency was set at 20 KHz with 50% amplitude. The extraction was carried out at room temperature for 10 min (Liu, Wang, Lu, & Chiang, 2014).

| Filtration and evaporation of solvent
The slurries obtained from two extractions were filtered individually by using Whatman No.1 filter paper. The filtrate of all samples was evaporated using a rotary evaporator attached with a vacuum pump (Model VP18R, Lab tech) and a refrigerated cooling system. Total solvent was dried at 79°C temperature of water bath at a vacuum pressure of 0.07 MPa.

| Development of shrimp patties
Minced shrimps were shaped into patties weighing about 5 g each. 6 treatments were given to patties except control as 1% and 2% for GLE, 1% and 2% for PLE, and 1% and 2% in combination for both, respectively. Mincer was used for mincing purpose. 0.5 ml of each solution was added to patties and then stored them in airtight plastic zip lock bags at refrigeration temperature for 21 days. Physiochemical analysis was done at 7-day intervals.

| Total phenolic contents (TPC)
Folin-Ciocalteu method was used to determine the phenolic content of enriched shrimp patties according to Tezcan, Gültekin-Özgüven, Diken, Özçelik, and Erim (2009). 1 ml of 10% Folin-Ciocalteu reagent was applied to 0.5 ml of a known sample concentration. The mixture was blended and left for 6 min; then, 2 ml of 20% sodium carbonate solution was added in the above mixture. The spectrophotometer (Specord 200/plus, Germany) was used to test the phenols at 760 nm after reacting at 30°C for 60 min. By using standard gallic acid solution, a calibration curve was prepared and the total phenols were expressed as 1 g of equivalent gallic acid (GAE) per gram of sample.

| Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP test was performed on the shrimp patties as per the method described by Benzie and Strain (1996) with few modifications. The FRAP mixture was designed by blending 10 mM 2,4,6-tri(2pyridyl)-1,3,5-triazine (TPTZ) in 40 mM HCl; 300 mM acetate buffer (93.6 ml 2 M acetate acid and 6.4 ml 2 M sodium acetate, and pH 3.6,); and 20 mM of ferric chloride in 10:1:1 ratio (v/v/v). Afterward, 200 μl of formulated FRAP chemical compound was added with 50 μl at each test. The blend was placed in the shadow at ambient temperature for 30 min, during which a spectrophotometer was used to measure the absorbance at 593 nm. Different Trolox concentrations (5-50 μg/ml) have been used in the preparation of the standard curve, and the results were measured as (TEAC) mostly in the type of Trolox/gram microgram samples.

| Hunter color (Lab)
The surface color estimations of the GLE-and PLE-enriched shrimp patties were assessed by Hunter colorimeter, with the assistance of measurements that were established according to reference plate (L = 89.2, a = 0.921, and b = 0.783). The CIE L (lightness), CIE a (redness), and CIE b (yellowness) color parameters were estimated by utilizing a mean from 9 arbitrary readings to investigate statistically.

| Total Volatile Basic Nitrogen (TVBN)
The TVBN experiment was performed by the method of Malle and Tao (1987) with minor modifications. Samples were blended in a ratio of 1:2 (w/v) with trichloroacetic acid (TCA) and homogenized with blender at speed 2. The solution was then centrifuged at 1008 g for 5 min and passed through filter paper Whatman No.1. In the Kjeldahl refining tank, 25 ml of samples were pipetted and 5 ml of 10% sodium hydroxide was applied to them. Steam processing was carried out, and the distillate was titrated against 0.05 M sulfuric acid until the shade turns pink.

| Thiobarbituric Reactive Substances (TBARS)
The TBARs value was determined by the method described by Schmedes and Hølmer (1989). 5 g of sample was mixed in a homogenizer with 25 ml of 20 percent trichloroacetic acid (200 g/L) in a solution of 135 ml/L of phosphoric acid for 30 s. The homogenized sample was gone through Whatman filter paper number 4 to dispose of solid particles from the filtrate. 2 ml of 0.02 M TBA solution (3 g/L) was applied in a test tube at that stage with 2 ml filtrate.
Samples containing test tubes were incubated at 100°C for 30 min from that point forward and then cooled in running tap water. Using UV-VIS spectrophotometer, the absorption of supernatant formulations was measured at 532 nm. The TBA values were determined from a standard curve (1,1,3,3-tetraethoxipropane [TEP]) and expressed as mg malondialdehyde per kilogram (MDA/kg) of sample.

| Peroxide Value (POV)
The peroxide value of shrimp enriched with GLE and PLE was measured by using the method outlined by Sallam, Ishioroshi, and Samejima (2004). 3 g sample was collected in the Erlenmeyer flask.
To melt the fat in sample, it was heated at 60°C for 3 min in the water bath. After that, to digest the fat, the container was stirred for 3 min with 30 ml of acetic acid-chloroform solution (3:2 v/v).
Whatman channel paper number 1 was used to remove the solid particles from the filtrate. The process was accompanied by adding the starch solution as a marker, after the addition of potassium iodide solution (0.5 ml) to filtrate. The titration was carried against the standard sodium thiosulfate solution. POV was calculated by following equation and expressed as milliequivalent peroxide per kilogram of sample.

| Total Bacterial Count (TBC)
Total bacterial counts were performed on the outlines given by Linton, Mc Clements, and Patterson (2003) and Karim, Rahman, Muhammad, Zainol, and Ikhwanuddin (2018). A minimum of 10 g sample solution was homogenized with 90 ml of maximum recovery diluent (MRD). Serial dilution has been made before getting desired dilution. Using a sterile glass spreader, 0.1 ml of the dilution was applied to the agar plate. The total count of bacteria was represented as log colony forming units per gram of test samples (log10 CFU g −1 ).

| Sensory Evaluation of Shrimp Patties
The shrimp patties were cooked and evaluated by 10 trained panelists using a 9-point hedonic scale ranging from 9 = like extremely to 1 = dislike extremely (Meilgaard, Civille, & Carr, 2007). All panelists were asked to evaluate appearance, texture, taste, odor, and overall acceptability. The rational assessment was done by giving the mineral water, unsalted crackers, and cups to neutralize their receptors during the sensory evaluation process. The data was recorded during the storage period of 21 days with the interval of 7 days.

| Statistical analysis
The data obtained from different parameters were statistically analyzed by using the statistical package, Statistic 8.1, by following the guidelines of Steel and Torrie (2012). The level of significance was observed by using the factorial design under CRD. Three replications were done for each parameter, whereas 10 measurements were recorded for hunter color and 10 panelists were used for sensory evaluation. The means were compared by using least significant differences.

| Total phenolic contents
Phenolic is one of the main compounds that serve as key antioxidants or free radical terminators. One of the essential criteria for estimating the amount of antioxidants is the calculation of maximum phenolic compounds. Polyphenols demonstrate high antioxidant behavior when plant polyphenols contribute hydrogen atoms to stop fat rancidity by stopping the radical chain reaction through transferring free radicals into more stable atoms. Regarding this manner, they are established as strong antioxidants. The total phenolic content of GLE-and PLE-incorporated shrimp patties is shown in Figure 1. Higher value of total phenolic contents found in functional patties enriched with 2%GLE: 2% PLE at the start of the experiment, whereas minimum value of total phenolic contents was observed in control at day 0. With the passage of time, significant reduction was found in all treatments up to 21 days. The results depicted that higher total phenolic contents was observed in treatment having mixture of 2% GLE and PLE both at day 0 and day 21, whereas minimum contents were found in control. The results are in agreement with the findings of Verma, Rajkumar, Banerjee, Biswas, and Das (2013) who reported that guava leaf powder supplemented in sheep nuggets showed higher antioxidant potential. Similarly, another study reported by Ekaluo, Ikpeme, Ekerette, and Chukwu (2015) showed higher total phenolic contents in treatment with guava leaves than the bitter leaf (Vernonia amygdalina). Our results are also supported by the findings of Arshad et al. (2019), who reported that turmeric powder-treated chicken patties showed higher antioxidant potential.
Furthermore, Asghar et al., 2016 depicted that higher phenolic contents were found in papaya leaves than other parts of plant.

| 2,2-Diphenyl-1picrylhydrazyl (DPPH)
2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging approach is a technique to test the antioxidant ability of a drug, sample, or other biological sources. This is the modest approach in which F I G U R E 1 Total phenolic contents (TPC) of PLE-and GLE-enriched functional shrimp patties during storage the prospective compound or extract is combined with DPPH solution and absorbance is reported after a specified period of time.
DPPH offers the first tactic for assessing the antioxidant potential of a compound, an extract, or other biological sources. This is proved to be the modest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded Citrus limon.  reported the same synergistic effect of antioxidants from wheat germ oil and alpha-lipoic acid, induced in the chicken and found the maximum DPPH inhibition in the formulated nuggets.

| Ferric reducing antioxidant power
One of the most widely used methods for determining the overall antioxidant power of a specimen is the ferric decreasing/antioxidant power (FRAP), which is fairly simple, quick, delicate, and rational to conduct.
There are many decentralized studies that have used the FRAP method, and they have produced an extremely huge list of overall antioxidant assessment of a food product that can help in picking diet for greater intakes of antioxidants. Therefore, the FRAP experiment was used to evaluate the bioavailability of antioxidants in food and to investigate the impact of changing environments, processing, handling, and cooking techniques on the overall antioxidant potential of food (Prior, Wu, & Schaich, 2005)

| Hunter color
The values regarding color parameters of GLE-and PLE-enriched shrimp patties are shown in the  Furthermore, Kim, Jin, et al. (2013) depicted that with the addition of antioxidant source in patties increased the yellowness (b*) which is in line with the outcomes of present study.

| Total volatile basic nitrogen
Microbiological spoilage causes the formation of volatile bases (mainly ammonia, dimethylamine, and trimethylamine) during the TA B L E 2 Total volatile basic nitrogen (TVBN) and total bacterial count of PLE-and GLE-enriched functional shrimp patties during storage postmortem storage of a wide variety of seafood, which have often been determined to indirectly measure the freshness quality of such seafood. Total volatile nitrogen is almost exclusively composed of ammonia. When ammonia production increases during spoilage due to protein deamination, its assessment is an easy way to assess seafood value (Howgate, 2010). The results regarding the TVBN content of GLE-and PLE-enriched shrimp patties were shown in the Table 2.
The maximum value of TVBN was observed in the control samples at

| Total bacterial counts
The total number of bacteria is one of the key indicators of hygiene control in food processing. The total bacterial counts of GLE-and PLE-supplemented shrimp patties are shown in who observed that the antimicrobial activities of GLE were considerably due to it is rich in tannins.

| Thiobarbituric Acid Reactive Substances (TBARS)
TBARS is most probably the oldest and one of the most commonly employed methods to measure lipid peroxidation end product malondialdehyde, a volatile aldehyde produced by polyunsaturated fatty acid lipid peroxidation. The statistical results regarding TBARS value of GLE and PLE enriched functional patties with different storage intervals are given in Table 3. The results showed that higher TBARS value was observed in the control, whereas minimum was found in shrimp patties enriched with GLE 2%:PLE 2% at day 0. As the time increased, the TBARS values were also increased significantly in control followed by PLE 2% while the minimum value was exhibited by the patties treated with GLE 2%:PLE 2%. The total amount of 2 mg malondialdehyde/kg TBARS is taken in the test sample as an acceptable limit in frozen or dried goods, according to the National Iranian Standards Organization (Gh et al., 2018).

| Peroxide value
The POV is the indicator of lipid peroxidation in the meat samples (Domínguez et al., 2019). The results regarding the POV of enriched shrimp patties are shown in Table 3. Higher POV was showed by control samples, while the lowest values were revealed by the patties enriched with GLE2%:PLE2% at the start of research. As like as TBARS, the POV were also intensified with the storage intervals, the highest POV was seen in the control followed by PLE 1% while the minimum value was shown by shrimp patties treated with 2%GLE:2% PLE at the 21st day of storage. As in our study, the POV in control was higher both at the start and end of the experiment. The POV was observed to be significantly decreased in the patties treated with 2%GLE:2%PLE as compared to all treatments because of the combination of antioxidants provided by both GLE and PLE extracts (Karim et al., 2018;Patil, Patil, Patil, & Patil, 2014). Arshad

| Sensory evaluation of GLE and PLE enriched shrimp patties
Sensory characteristics of a food product are very important in consumer appeal. They play a vital role in the consumer demand toward that product (Sharif, Masoos, Hafiz, & Muhammmad, 2017).
The sensory evaluation of GLE-and PLE-enriched shrimp patties was carried by trained panelists. The mean score for appearance, texture, taste, odor, and overall acceptability of functional shrimp patties enriched with GLE and PLE is presented in Table 4. The results showed that the sensory attributes were decreased with the passage of storage time. The results showed that, with the increase in the concentration of the extracts applied, the sensory attributes decreased slightly but remained in the acceptable stage throughout the storage period which is correlated with the results of lightness which also increased due to increase the amount of extracts applied. The results regarding the odor also directly re-

| CON CLUS IONS
This study revealed the significance of GLE and PLE on the storage stability and antioxidant activity of shrimp patties during storage.
The antioxidant potential of shrimp patties was found to significantly higher in the treatments with combination of GLE (2%) and PLE (2%), whereas lower was observed in control. The stability parameters showed that TBARS and peroxide value were found to be higher in control as compared to the other treatments during 21 days of storage. Total aerobic counts were also found to be higher in control, and minimum was observed in treatments with the combination of GLE (2%) and PLE (2%). The sensory score given by the panelist was found to be higher in the control as compared to the other treatments but found acceptable score in other treatments. It is concluded that the shrimp patties made by the combination of GLE and PLE found significantly better results regarding the physical, microbiological, and antioxidant profile during storage.

ACK N OWLED G M ENTS
The authors are highly thankful to Department of Food Science, Government College University Faisalabad, for providing the facilities in conducting the research work.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study has nothing to do with human and animal testing.

D ECL A R ATI O N
The authors did not use the human subjects.