Opening a new gateway towards the applications of chitosan nanoparticles stabilized Pickering emulsion in the realm of aquaculture

In the current study, we synthesized eugenol (EU) based Pickering emulsion (PE) stabilized by food grade ingredients such as chitosan (CS) and tripolyphosphate (TPP) not only to enhance water miscibility of EU but also to decrease stress and damage to the immune system of fish due to anesthetic procedures.

Available online 24 April 2021

( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Opening a new gateway towards the applications of chitosan nanoparticles

stabilized Pickering emulsion in the realm of aquaculture

Bakht Ramin Shah*, Petr Dvoˇr´ak, Josef Velíˇsek, Jan Mr´az

University of South Bohemia in ˇCesk´e Budˇejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of

Hydrocenoses, Czech Republic

A R T I C L E I N F O

Keywords:

Pickering emulsions

Eugenol

Characterization

Fish anesthesia

Common carp

A B S T R A C T

In the current study, we synthesized eugenol (EU) based Pickering emulsion (PE) stabilized by food grade in-gredients such as chitosan (CS) and tripolyphosphate (TPP) not only to enhance water miscibility of EU but also

to decrease stress and damage to the immune system of fish due to anesthetic procedures The formulated EUPEs were characterized in terms of droplet size, size distribution and the effects of environmental conditions e.g pH and temperature on the behavior of the EUPEs The results showed that EU PEs with 5% EU had smaller size with uniform distribution and were stable in the range of pH 5–7.5 and temperature 30–80 ◦C The anesthetic effect of the EUPE was investigated by taking Common carp as a sample species Interestingly, it was found that the induction time to anesthesia and recovery for the fish that received the PE was significantly shorter than that received EU at the same eugenol concentration (50 ppm) Most importantly, the improved hematological and bio-chemical parameters in the PE group further confirmed the immuno-protective and stress control efficacy of the PE The results of this study propose a novel useful and potential application of PE in fishery where sedation

is needed

1 Introduction

Emulsions are regarded as thermodynamically unstable systems of

two immiscible or partly miscible phases e.g oil and water (Berton-

Carabin & Schro¨en, 2015) The interfaces between these phases are

required to be stabilized by some type of amphiphilic or surface active

compounds (Matos et al., 2018) The surfactants used to stabilize

conventual emulsions such as nano or micro (McClements, 2012), can

exert adverse effects on health as well as environment (Perrin et al.,

2020) On the other hand, Pickering emulsion (PE) is an emulsion which

is stabilized by solid particles instead of surfactants (Pickering, 1907;

Ramsden, 1904) The particles are partly wetted by both the phases and

are irreversibly adsorbed at their interfaces thereby conferring these

emulsions with stability for months or even years In contrast with

conventional emulsions (stabilized by small molecular surfactants), PEs

have garnered tremendous attention in scientific community due to its

non-toxic safe and long-term stable nature and therefore, are known as

much more advantageous and efficient option for the encapsulation and

delivery of a wide range of bioactive compounds (Shah & Mraz, 2020)

So far a wide range of particles have been implied to stabilize PEs

including inorganic or synthetic silica, latex, clay and so on (Yang et al., 2017) or natural food grade particles derived from protein such as lac-toferrin, soy protein isolate (SPI), pea protein isolate (PPI) etc (Shao & Tang, 2016); from lipids in the form of fat crystal which have been documented as potential stabilizers for water in oil (W/O) emulsions (Rousseau, 2013) However, carbohydrates-based particles in the form

of modified starches comprise a large group of emulsifiers for PEs This predominantly includes cellulose, starch and chitosan (CS) etc (Chen

et al., 2020; Soltani & Madadlou, 2016) CS is generally obtained by the alkaline deacetylation of chitin (the second most abundant polymer in nature after cellulose) present in the exoskeletons of crustaceans as well

as the cell walls of some algae and fungi (Mohammed et al., 2013) Being inexpensive, commercially available, biocompatible, biodegradable due

to the free amino and hydroxyl groups along its backbone, CS has a wide range of useful applications in biomedicine, pharmaceutics and food industries (Mwangi, Ho, Ooi, et al., 2016) During the last few decades there has been a considerable research interest in using CS nanoparticles (NPs) as drug delivery carriers for sustained and controlled release of the encapsulated drugs and as stabilizers for stabilizing the oil water in-terfaces of PEs To synthesize these NPs, different methods have been

* Corresponding author at: University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, Na S´adk´ach 1780, 370 05 ˇCesk´e Budˇejovice, Czech Republic

E-mail address: bshah@frov.jcu.cz (B.R Shah)

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2021.118096

Received 4 November 2020; Received in revised form 8 March 2021; Accepted 22 April 2021

implied so far including pH induce CSNPs (Liu et al., 2012) or by cross

linking through ionotropic gelation method using different compounds

such as glutaraldehyde (GLA), glyoxal (GO), epichlorohydrin (ECH),

ethylene glycol diglycidyl ether (EGDE) and tripolyphosphate (TPP)

(Babakhani & Sartaj, 2020; Shah, Li, et al., 2016) However, TPP is

regarded as low cost and environmentally safe in comparison to the rest

of these cross-linking agents which are known to be toxic and exert

adverse effects on the environment (Vakili et al., 2014) In our previous

studies we successfully synthesized CS cross linked TPP NPs which

proved to be efficient stabilizers for a long-term stable PEs (Shah, Li,

et al., 2016; Shah, Zhang, et al., 2016) Available literature supports the

claim that till date PEs have a broad spectrum of useful applications in

food, cosmetic and pharmaceutical industries including delivery of

bioactive compounds and in the synthesis of porous scaffold,

environment-responsive material and thermo-responsive material so on

(Jiang et al., 2020; Yang et al., 2017) In addition, most recently, a

research group fabricated zein particle stabilized clove oil (CO) PE and

found that the addition of the synthesized COPE boosted up the

anti-microbial efficacy of the chitosan-based edible films (Xu et al., 2020)

Eugenol (EU) an essential oil present in CO (70–90% by weight) is

obtained from the buds, leaves, flowers and stem of the clove tree

(Syzygium aromaticum) (Soltanian et al., 2018) It contains active

com-pounds isoeugenol and methyleugenol and is considered a potential

anesthetic in fish (Hur et al., 2019) However, due to the issue of its poor

dissolution in water, researchers employ alcohols or detergents as

dis-solving agents which can exert unfavorable effects on the fish and other

living organisms (Gholipourkanani et al., 2015) Fish anesthesia is often

required during different experimental procedures or during their

transportation, grading or vaccination to make them sedated for

extended period of time And applying higher doses of anesthetics can

put a huge amount of stress on fish, that can result in anomalous

metabolic rates, oxygen utilization, blood pressure, and blood

physio-logical responses, which can even last for several hours after recovery

from anesthesia and can ultimately affect the nutritional quality of fish

for human consumption (Herrera et al., 2019; Park et al., 2009) Now in

order to avoid these unfavorable conditions, it is desired to reduce the

stress to the fish up to the maximum level during anesthetic procedures

And hence ecofriendly, non-toxic and cost-effective formulations such as

PEs stabilized by food grade ingredients will be the best choice to serve

the role So far no such study has ever been conducted to use EUPEs

based formulations as anesthetizing agents in fishery Therefore, the

main aim of the current study was to fabricate and characterize EUPEs

stabilized by CSTPP NPs and evaluate their anesthetizing role in

com-mon carp as a sample specie

2 Experimental section

2.1 Materials and methods

CS (3 × 105–7 × 105 Da), ≥99.5% acetic acid (AA), TPP, NaOH were

purchased from Sigma-Aldrich, Sokolovska, Prague, Czech Republic EU

(100%) was purchased from Mach chemical spol s.r.o Ostrava, Czech

Republic NaCl and 36–38% HCl were purchased from PENTA

phar-maceuticals Radiova, Prague, Czech Republic All of the materials

guaranteed analytical grade and were used without further purification

Water used in the preparations of all solutions was purified by

deion-ization and filtration with a IWA 30 iol, WATEK apparatus (Czech

Re-public) to a resistivity higher than 18.0 MΩ⋅cm

2.2 Preparation of chitosan tripolyphosphate nanoparticles (CSTPP NPs)

CSTPP NPs were synthesized using the same method as described in

our previous work (Shah, Li, et al., 2016) Briefly, 0.5 wt% CS solution

was prepared by dissolving CS powder in AA solution maintaining CS to

AA ratio of 2:3 and was diluted with deionized water to obtain the

required volume The resultant mixture was then stirred for overnight at

room temperature (≈25 ◦C) After overnight stirring the solution was filtered to remove the large dust or undissolved particles A solution of TPP at the concentration of 1.0 mg/ml (0.1 wt%) was prepared by dis-solving TPP powder in ultrapure water and stirred to ensure its complete dissolution The NPs were then prepared by dropwise addition of TTP solution onto the CS solution at a ratio of 1:1 (w/w) under continuous stirring

2.3 Preparation of the required EUPEs

Using the synthesized CSTPP NPs as stabilizer, the desired PEs were prepared with different EU contents (5, 10, 20, 30, 50 and 70 wt%) To

do so, aqueous phase containing NPs (95, 90, 80, 70, 50 and 30 v% respectively) was taken in a glass vial and was mixed with the oil phase (EU) at the specified amounts This mixture was then vigorously ho-mogenized using UltraTurrax® T25 device equipped with a S25N-18G shaft (IKA, Germany) rotating at a speed of 10,000 rpm for 3 min The emulsions so prepared were immediately transferred to tightly covered glass bottles and stored at room temperature (≈25 ◦C) for further analysis

2.4 Emulsions characterizations 2.4.1 Mean droplet size & droplet size distribution

Particle size (mean diameter) and size distributions of the PEs sam-ples were determined using a Malvern Mastersizer 3000 instrument (Malvern Instruments Ltd., Worcestershire, UK) The samples were dispersed in deionized water (≈1000 ml) and stirred at a rate of 2200 rpm to avoid multiple scattering effects The refractive indices of 1.53 and 1.33 were used for clove oil droplets and water (the dispersant), respectively

2.4.2 Effect of pH

The effect of pH was assessed by adjusting pH of the samples to different values i.e 5, 6, 7 and 8 using either 0.1 M NaOH or 0.1 M HCl solution Thereafter, mean particle diameter and size distributions of these samples were measured in the same way as described above in Section 2.4.1

2.4.3 Effect of temperature

In order to evaluate the effect of temperature on the behavior of the emulsions, emulsion samples were incubated in a water bath at 30, 50,

60, 80 and 90 ◦C for 30 min The visual changes of the emulsions were observed Then the mean particle diameter and size distributions of the samples were determined after the samples cooled down to room tem-perature following the same procedure mentioned in Section 2.4.1

2.4.4 Optical images

Optical micrographs of the ZNCSPs based PEs were taken by an Optical microscope OLYMPUS (U-TVO.63XC, Tokyo, Japan) fitted with

a digital camera (Olympus, DP 50) Emulsions samples were placed directly onto a microscope slide and softly covered with a cover slip The images were recorded at 25 ◦C under the magnification from 10 to 40×

2.5 Fish trials 2.5.1 Experimental fish

A total of 28 common carps with average body weight 206 ± 41, total body length 223 ± 21 and specific length of 182 ± 12 were selected from the already acclimatized common carp in the aquarium of the Institute

of Aquaculture and Protection of Waters, Faculty of Fishery and Pro-tection of Waters, University of South Bohemia in ˇCesk´e Budˇejovice The fish were individually stocked in plastic tanks (10 L) equipped with aeration Parameters such as water dissolved oxygen, pH and tempera-ture of the water were measured to be 90%, 7.3 and 19 ◦C respectively

In order to test the anesthetizing efficacy of the designed EUPEs, the

selected fish were divided into seven groups (six experimental and one

control with no EU) with four fish in each group EU at three different

concentrations i.e 12.5, 25 and 50 ppm either in the form of PE (0.25

ml, 0.5 ml & 1 ml of 5% PE/l respectively) or free (original form) were

applied to the respective tanks with the individual fish

2.5.2 Onset and recovery from anesthesia

The observations of the stages 5 & 6-anesthesia onset and recovery

(according to Table 1) were made after using either EUPE or EU under

completely similar experimental conditions The duration to achieve

stages 5 & 6 anesthesia (A5 & A6) for each replicate were noted using a

stopwatch Once an individual fish would reach the onset of stage 6

anesthesia, a dip net was used to immediately remove it from the

experimental tank The fish was then washed off in clean water tank and

transferred to another 10 L, well-oxygenated tank termed as ‘Recovery’

tank (i.e., no anesthesia present) maintained at 19 ◦C and observed until

it fully recovered During this recovery period, the fish behavior was

observed and times to observe stages 5 & 6 recovery (R5 & R6) were

noted using a stopwatch

2.5.3 Blood sample collection, hematological and biochemical assay

Blood samples were collected from all the anesthetized fish included

in six experimental groups and one control group (without EUPE and EU

administration) The samples were drawn from vena caudalis using a

syringe with heparin as anticoagulant (Heparin inj., Leciva, Czech

Re-public) at a concentration of 5000 IU heparin sodium salt in 1 ml

Plasma samples obtained by centrifugation of blood at 3000g for 15 min

were stored at − 80 ◦C until analysis The hematological parameters

were measured that included red blood cell count (RBC), haemoglobin

concentration (Hb), hematocrit (PCV), white blood cell count (WBC)

mean erythrocyte volume (MCV), mean corpuscular haemoglobin

con-centration (MCHC), mean corpuscular haemoglobin (MCH), differential

% WBCs (lymphocyte, monocyte, neutrophile granulocytes segment,

neutrophile granulocytes bands, developmental phases – myeloid

sequence) The indices used to evaluate hematological profile were

based on unified methods for hematological examination of fish

(Svo-bodova et al., 1991) The bichemical parameters were measured that

included glucose (GLU), total protein (TP), albumin (ALB), globulin

(GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST),

lactate (LACT), inorganic phosphate (PHOS), cholesterol (CHOL)

Globulin content was calculated by subtracting albumin values from

serum total protein Measurement of biochemical plasma parameters

was conducted using the Catalys One analyser (IDEXX Laboratories Inc.,

Maine, USA)

3 Statistics

All experiments were done at least in triplicates The data were presented as mean ± SD Statistical evaluation of the anesthesia in-duction and recovery times as well as hematological and biochemical parameters were carried out by analysis of variance (ANOVA) procedure using the Statistix 8.1 software Least significant difference (LSD) at 5% level was used for multiple comparison tests among the treatments

4 Results and discussion

4.1 Fabrication and characterization of EUPEs 4.1.1 Optimization and droplet size of the EUPEs

In order to prepare stable PE systems, the oil to water ratio should be optimized as it will affect particles concentration which is the vital factor in controlling the droplet size of PEs With this consciousness, initially we prepared the required EUPEs with different oil (EU) frac-tions of 5, 10, 20, 30, 50 and 70 wt% shown in Fig 1a As can be seen, the formulations with higher oil contents of 30, 50 and 70 wt% didn’t turn into well homogenized emulsions systems and the oil fractions were clearly seen separated soon after the homogenization On the other hand, formulations with lower oil contents i.e 5, 10 and 20 wt% gave well homogenized and stable emulsions systems at the same homoge-nizing speed and time The reason for this emulsification failure at higher oil contents could be due to the poor coverage of the oil water interface by the stabilizing CS-TPP NPs Based on these outcomes, we therefore, didn’t further characterize the formulations with higher oil contents but rather proceeded with the ones having lower oil contents Optical images in Fig 1b, c and d for emulsions with oil contents 5, 10 and 20 wt% respectively, exhibited an increase in size with increasing oil contents This trend in increase with increasing oil contents was further confirmed by measuring mean droplet diameter through laser diffraction instrument (Malvern Mastersizer 3000, UK)

It is evident from Fig 2 that although the three emulsions had uni-form particle distributions, the droplet sizes in terms of the Sauter mean diameter (d32) and volumetric diameter (d43) increased significantly with increasing the oil contents from 5 to 20 wt% These results are in agreement with the previous study where increase in sizes with increase

in oil contents of olive oil based PE was observed at all tested pH values (i.e 2, 4 and 7) (Jutakridsada et al., 2020) As a matter of fact, this can

be attributed to the alteration in the oil to particles ratio caused by increasing the oil contents in the system This phenomenon has been well explained in previous work by Arditty and colleagues who described the phenomenon of droplet size variation with the amount of particles by a so-called limited coalescence The emulsion droplets tend

to coalesce as a result of the decrease in the total particles concentration required to completely cover the oil-water interfaces (Arditty et al., 2003) Similar results of the link between particles concentration and droplet size were also presented by Tzoumaki et al., who obtained emulsions with smaller droplets, enhanced stability and distinct elastic responses by increasing the concentrations of chitin nanocrystals (ChNs) used to stabilize oil-in-water emulsion (Tzoumaki et al., 2011)

4.1.2 Effects of temperature and pH on the behavior of the EUPEs

Droplet size of an emulsion has a crucial role in its stability The smaller the emulsion droplets, the more stable the emulsions are and vice versa This higher stability of an emulsion with smaller droplets can

be due to the strong resistance of smaller droplets against different de- stabilization phenomena like aggregation, coalescence, and floccula-tion because of the Brownian mofloccula-tion that overcomes gravitafloccula-tional forces (Hidajat et al., 2020) Thus a stable emulsion is the one capable of resistance to the instability mechanisms such as Ostwald ripening (development of the larger droplets by the expense of the smaller ones)

or very much similar to that coalescence as well as the flocculation, where the droplets collide and bunch together to make flocs (larger

Table 1

Detailed description of different stages of anesthesia induction and recovery in

EU based tests performed in common carp (Park et al., 2009; Yousefi et al.,

2018)

Stage Characteristic behavior

Anesthesia

A1 Normal swimming; opercular movement and normal general movement

A2 Swimming speed slowed; rolling from side to side

A3 Partial loss of equilibrium; swimming erratic

A4 Complete loss of equilibrium; swimming perfectly inside out; pectoral fin,

pelvic fin and dorsal fin movement stop

A5 Little sedation; anal fin and tail fin movement stop

A6 Perfect sedation; only opercular movement

Recovery

R1 Resume opercular movement

R2 Preferential movement of pectoral fin and tail fin

R3 Dorsal fin, pelvic fin and anal fin movement

R4 Swimming perfectly inside out

R5 Swimming erratic; redress the balance

R6 Normal swimming; responsiveness to visual stimuli

droplets) which then finally lead to sedimentation or creaming of the

emulsion due to gravitational separation (Low et al., 2020)

Based on these facts, the emulsion prepared with 5 wt% EU was

selected to evaluate the effects of temperature and pH on its stability in

terms of visual observations and droplet sizes, as it had smaller sizes

compared to the ones prepared with 10 and 20 wt% (Fig 2)

In the first instance, the effect of temperature was evaluated by

incubating emulsion samples (original temperature 20 ◦C) in a water

bath at 30, 50, 60, 80 and 90 ◦C for 30 min Visual observations of the

samples showed that there was no cream formation or even

sedimen-tation of the particles on the bottom of the sample tubes till 80 ◦C And

hence, although the mean droplet diameter slightly increased at

tem-perature 30 ◦C, no significant changes were observed in the sizes

thereafter till 80 ◦C (Fig 3a) Also, all the samples showed uniform size

distributions (Fig 3b) which further confirmed the capability of the PE

to withstand the influence of temperature However, when the tem-perature was raised to 90 ◦C, the emulsion apparently turned into cream and therefore at this temperature no mean droplet size of the emulsion was measured This observation can be attributed to the enhanced en-counters among the particles due to the rise in temperature and ulti-mately their average kinetic energy, thereby leading to enhancing their motion and rate of collision finally making them to agglomerate (Mwangi, Ho, Tey, & Chan, 2016) Similarly, the effect of pH on the emulsion stability was monitored by adjusting pH of the samples (original pH 4.3) to 5, 6, 7, 7.5 and 8 As no destabilization occurred during changing pH from 5 to 7.5, it can be stated that emulsion was highly stable against droplet coalescence and creaming till pH 7.5 The droplet sized increased linearly with increasing pH values (Fig 3c) but

Fig 1 Digital photographs of the PEs prepared with different EU contents (wt%) a) and optical micrographs of the ones prepared with 5 b) 10 c) and 20 d) wt%

of EU

Fig 2 Mean droplet sizes and size distributions of the EUPEs prepared with 5, 10 and 20 wt% EU

exhibited uniform distribution (Fig 3d)

This finding can be well explained by the fact of relatively low net

droplet charge at high pH with lowered electrostatic repulsion between

the droplets (Shah, Li, et al., 2016) which could even be reason for the

complete destabilization of the emulsion at pH 8 (shown in the inset of

Fig 3c) These results can be regarded to present an ideal scenario of

releasing the encapsulated EU as main anesthetizing agent into the

water (with pH ≈ 7.3) in which the experimental fish are stocked

Furthermore, to assess the influence of storage time on its stability,

the emulsion was stored at room temperature for three months and was

observed visually with time It was found that no visual change appeared

in the behavior of the emulsion during this time which clearly indicated

its long-term stable nature

4.2 Evaluating anesthetizing efficacy of the synthesized EUPE in fish

model (common carp)

With the aim to introduce EU based PE an unprecedented non-toxic

formulation composed of biocompatible and biodegradable materials as

anesthetizing agent in fish, we evaluated the anesthetizing role of the

prepared EUPE by choosing common carp as a sample species The

selected fish were divided into different groups, and three different

concentrations of EU i.e 12.5, 25 and 50 ppm in the form of EUPE or free

EU were tested to evaluate their anesthetizing efficacy It was found that

the EUPE ensured complete dissolution of the loaded EU as no oil

droplets were visualized on the water surface after the administration

Conversely, the oil droplets were seen on the water surface even after

powerful agitation when free EU was applied And therefore, it could be

the reason that even at lower concentration of EU i.e 12.5 and 25 ppm,

anesthesia up-to stage 4 (A3 & A4 shown in Table 1) was achieved in the

PE groups, whereas no change in the behavior of fish was observed even after 30 min of applying EU in their respective water tanks This dif-ference was also reflected in the analysis of different blood parameters of both the groups For example, it can be seen in Table 2, that hematocrit (PCV) of 0.3925 in the PE group with 25 ppm of EU was significantly

higher (P < 0.05) than EU group (0.3425) These results comply with the

previous studies where significant increase in PCV levels were observed

after administrating EU as an anesthetic in O tshawytscha (Cho & Heath,

2000) and olive flounder (Paralichthys olivaceus) (Hur et al., 2019) Interestingly, when the EU at a concentration of 50 ppm in the form

of EUPE or EU was administrated, the induction and recovery times from

anesthesia were significantly lower (P < 0.05) for the EUPE group than

EU As shown in Fig 4a, the induction time of stage 5 and 6 anesthesia

(A5 and A6) with ≈3.3 min, was significantly lower (P < 0.05) than the

EU group with A5 and A6 of 12.4 and 14.2 min respectively Similarly, the recovery time for stage 5 and 6 recovery (R5 and R6) of 11.9 and 15

min respectively for EUPE group was significantly lower (P < 0.05) than

R5 and R6 of 15 and 19.9 min respectively for the EU group (Fig 4b) These findings of EUPE are closely coincided with the claims of previous studies mentioning optimum anesthetic concentrations to induce anes-thesia around 3 min and recovery around 10 min (Park et al., 2009) This promising capability of EUPE was further hallmarked by different hematological and biochemical plasma parameters as given in Tables 2 and 3 respectively It is evident from Table 2 that neutrophil granulocytes segment (%) of 1.8000 ± 1.1106 for EUPE group was

significantly higher (P < 0.05) than EU group with 0.5000 ± 0.7572, but

was non-significant with the control group (1.4750 ± 0.4717) where no

EU (neither EUPE nor EU) was administered Similarly, another important indicator developmental phases – myeloid sequence (%) with

1.8000 ± 1.2410 for EUPE group was significantly higher (P < 0.05)

Fig 3 Influence of temperature on the droplet size a) size distribution b) and influence of pH on the droplet size c) and size distribution d) of the EUPE prepared

with EU contents of 5 wt%

than 0.7000 ± 0.5944 for the EU group

Furthermore, as given in Table 3, we also analyzed different

biochemical plasma parameters It is important to mention that being

one of the most common stress indicators in fish, glucose cannot be

ignored while speaking about stress Because under stress conditions,

the release of stress hormones is altered which cause changes in the

blood as well as tissue chemistry including increasing blood glucose

levels, in an attempt to prepare the fish for emergency situations

(Makaras et al., 2020) Referring to the fascinating results of EUPE group

for the glucose levels in Table 3, it can be seen that the glucose values of

4.7950 ± 0.7735 mmol/l for EUPE was significantly lower (P < 0.05)

than for the EU with 7.1800 ± 0.7963 mmol/l What’s more, the glucose

levels of the EUPE group (4.7950 ± 0.7735 mmol/l) was almost similar

with the control group (4.0775 ± 0.9859 mmol/l) having no significant

difference (P < 0.05) Looking at these findings it can postulated that the

EUPE reduced the stress level of fish by ≈50% than free EU These re-sults clearly favor the immuno-protective as well as stress reducing potentials of the synthesized EUPE The fast induction of anesthesia and recovery of fish in the EUPE group due to short time of exposure would favor less stress in these fish compare to the ones in the EU group exposed for longer time This can be the key immuno-protective factor as there is a well-established link between stress and immune system modulation of fish (Tort, 2011)

Table 2

Hematological parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU

0.0150 bc 0.3238 ± 0.0075 c 0.3250 ±0.0220 c 0.3925 ± 0.0419 a 0.3425 ±0.0328 bc 0.3725 ±0.0260 ab 0.3650 ± 0.0297 ab

2.3276 a 80.481 ± 5.4021 a 78.153 ±11.806 a 86.658 ± 15.477 a 88.627 ±6.5280a 88.806 ±6.7754 a 85.315 ± 4.6171 a

0.0981 a 1.7975 ± 0.3715 a 1.7525 ±0.2323 a 1.8900 ± 0.3153 a 1.9725 ±0.2478 a 2.0050 ±0.2478 a 1.8325 ± 0.1047 a

1.3672 a 13.275 ± 4.9466 a 7.8750 ±2.4281 a 6.7500 ± 3.3789 a 11.525 ±4.5353 a 9.7500 ±2.5331 a 9.5000 ± 3.9370 a

2.5597 a 45.896 ± 7.8457 a 45.272 ±10.132 a 47.721 ± 16.919 a 45.780 ±9.1281 a 44.837 ±5.7419 a 46.753 ± 4.9958 a

13.556 a 185.37 ± 33.917 a 187.05 ±19.772 a 210.04 ± 22.324 a 176.46 ±32.849 a 187.97 ±23.121 a 200.01 ± 23.743 a

4.6701 a 248.68 ± 17.346 a 240.10 ±28.368 a 223.40 ± 53.929 a 260.24 ±27.369 a 239.60 ±28.651 a 235.19 ± 25.914 a Differential WBC count

1.2083 bc 97.950 ± 0.8583 ab 98.975 ±0.6076 a 95.350 ± 2.2368 c 98.100 ±1.3928 ab 94.425 ±2.2232 c 96.625 ± 2.2750 abc

0.3403 ab 0.6000 ± 0.6928 b 0.3250 ±0.3948 b 2.4500 ± 1.7823 a 0.9500 ±1.2557 ab 1.7250 ±1.0468 ab 1.8500 ± 1.8448 ab Neutrophil granulocytes segment

(%) 1.4750 ±0.4717 ab 1.0250 ± 0.2754 abc 0.5750 ±0.4272 c 0.8250 ± 0.2217 bc 0.5000 ±0.4082 c 1.8000 ±1.1106 a 0.5000 ± 0.7572 c Neutrophil granulocytes bands (%) 0.1000 ±

0.2000 a 0.0000 ± 0.0000 a 0.1250 ±0.2500 a 0.2375 ± 0.2750 a 0.0000 ±0.0000 a 0.2500 ±0.5000 a 0.3250 ± 0.2217 a Developmental phases – myeloid

sequence (%) 1.6000 ±0.8446 ab 0.4250 ± 0.6131 cd 0.0000 ±0.0000 d 1.1375 ± 0.5764 abc 0.4500 ±0.5196 cd 1.8000 ±1.2410 a 0.7000 ± 0.5944 bcd

Similarly, significant difference (P < 0.05) in lymphocyte (%) i.e 95.350 ± 2.2368 and 98.100 ± 1.3928 for EUPE and EU group respectively was found between the

two groups As lymphocytes have fundamental importance in body’s immune system, these findings highlight significant influence of anesthesia on the immunity of the fish

Fig 4 Comparison between the times elapsed during the stages of anesthesia induction and recovery in the EUPE and EU groups of the experimental common carp

5 Conclusion

In the current study we for the first time introduced EU based PE as

an anesthetizing formulation in fish As drawn schematically in Fig 5,

the EUPEs were successfully fabricated using CS-TPP NPs for stabilizing

the oil water interfaces The preparation of the required EUPE was

initially optimized by its preparation with different oil contents Droplet

size and size distribution of all the samples were measured and the one

with the smallest size was chosen for further analysis Effects of

influ-encing environmental conditions such as temperature and pH on the

behavior of the EUPE were evaluated The EUPE was found to be stable

against different tested temperature ranges up-to 80 ◦C as no significant

change in droplet size was observed On the other hand, the droplet size

was found to increase with increasing pH until pH 7.5 as at pH 8 the

emulsion was completely destabilized Thereafter, the anesthetizing

efficacy of the formulated EUPE was evaluated by testing three different

concentrations of EUPE based on EU in common carp Although no full sedation was achieved at lower concentrations (i.e 12.5 and 25 ppm EU), however with higher concentration (50 ppm), the times for anes-thesia induction and recovery was significantly lower for EUPE group than EU groups We also analyzed different hematological and biochemical plasma parameters which showed that compared to EU, EUPE significantly control the stress with no attenuating effects on the immunity of the fish Overall outcomes of the current study categorically demonstrated EUPE to be a potential anesthetizing formulation in fish-ery with immuno-protective and stress control capabilities

CRediT authorship contribution statement

Dr Bakht Ramin Shah: Conceptualization, Methodology, Formal

analysis, Data curation, Writing - original draft, Visualization, Ac-tivities administration

Table 3

Biochemical plasma parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU

Biochemical plasma

parameters Control Group I (12.5 ppm EU) PE EU Group II (25 ppm EU) PE EU Group III (50 ppm EU) PE EU

a 10.750 ± 0.5000 b 11.750 ± 0.5000 ab 10.750 ± 0.9574 b 10.750 ± 0.9574 b 10.500 ± 1.2910 b 12.250 ± 0.5000 b

a 23.250 ± 1.5000 ab 21.750 ± 1.5000 bc 19.250 ± 0.9574 d 20.000 ± 1.4142 cd 21.250 ± 0.5000 bcd 25.000 ± 1.1547 a

a 141.25 ± 23.796 a 176.25 ± 44.192 a 102.50 ± 16.862 a 122.00 ± 33.872 a 165.50 ± 42.587 a 149.00 ± 49.214 a PHOS (mmol/l) 1.3125 ± 0.1204

a 1.3650 ± 0.0311 a 1.2800 ± 0.0455 a 1.1400 ± 0.0906 b 1.2950 ± 0.1480 a 1.3250 ± 0.1439 a 1.4250 ± 0.0954 a

ab 33.500 ± 1.7321 bc 33.750 ± 0.9574 bc 30.000 ± 0.8165 d 30.750 ± 0.9574 d 31.750 ± 1.7078 cd 37.750 ± 2.3629 a

c 5.4650 ± 1.3986 bc 6.9825 ± 0.6547 a 6.1050 ± 0.9425 ab 4.6825 ± 1.1598 bc 4.7950 ± 0.7735 bc 7.1800 ± 0.7963 a

ab 2.2325 ± 0.1056 ab 1.9500 ± 0.1013 cd 2.0250 ± 0.1318 bcd 1.9200 ± 0.1454 d 2.2900 ± 0.1525 a 2.1900 ± 0.2652 abc

c 4.5675 ± 2.8222 a 5.4675 ± 0.9652 a 2.3625 ± 0.8982 bc 4.0575 ± 0.9251 ab 5.4725 ± 0.5861 a 4.7850 ± 1.4121 a CHOL (mmol/l) 3.0875 ± 0.2708

abc 2.8025 ± 0.2702 c 2.8300 ± 0.0648 bc 2.4700 ± 0.1564 d 2.3050 ± 0.1666 d 3.1250 ± 0.1028 ab 3.1550 ± 0.2908 a Glucose (GLU), total protein (TP), albumin (ALB), globulin (GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST), lactate (LACT), inorganic phosphate (PHOS), cholesterol (CHOL) The values are expressed as mean ± SD Measurements were performed in triplicate (n = 4/group) Means in the rows with different letter

are significantly different (LSD test, P < 0.05)

Fig 5 Schematic representation of the NPs and EUPEs synthesis and the potential application of the EUPE as an anesthetizing formulation in comparison with free

EU in common carp

Dr Peter Dvoˇr´ak: Methodology, Investigation,

Professor Josef Velíˇsek: Visualization, Investigation

Associate Professor Jan Mraz: Funding acquisition, Writing -

re-view & editing

Declaration of competing interest

There is no conflict of interest in this manuscript

Acknowledgement

The authors are grateful to the Ministry of Education, Youth and

Sports of the Czech Republic the CENAKVA project (LM2018099),

CENAKVA Center Development (CZ.1.05/2.1.00/19.0380) and

Biodi-versity (CZ.02.1.01/0.0/0.0/16_025/0007370) and National Agency for

Agricultural Research (QK1810296), for financial support The authors

would like to express their sincere gratitude to the colleagues of

labo-ratory of Nutrition, Institute of Aquaculture and Protection of Waters

(IAPW), Faculty of Fisheries and Protection of Waters (FFPW),

Univer-sity of South Bohemia in ˇCesk´e Budˇejovice, Czech Republic for their

huge support in conducting fish trials

References

Arditty, S., Whitby, C., Binks, B., Schmitt, V., & Leal-Calderon, F (2003) Some general

features of limited coalescence in solid-stabilized emulsions The European Physical

Journal E, 11(3), 273–281

Babakhani, A., & Sartaj, M (2020) Removal of cadmium (II) from aqueous solution

using tripolyphosphate cross-linked chitosan Journal of Environmental Chemical

Engineering, 8(4), Article 103842

Berton-Carabin, C C., & Schro¨en, K (2015) Pickering emulsions for food applications:

Background, trends, and challenges Annual Review of Food Science and Technology, 6,

Chen, L., Ao, F., Ge, X., & Shen, W (2020) Food-grade pickering emulsions: Preparation,

Cho, G., & Heath, D (2000) Comparison of tricaine methanesulphonate (MS222) and

clove oil anaesthesia effects on the physiology of juvenile chinook salmon

Gholipourkanani, H., Gholinasab-Omran, I., Ebrahimi, P., & Jafaryan, H (2015)

Anesthetic effect of clove oil loaded on lecithin based nano emulsions in gold fish,

Herrera, M., Mancera, J., & Costas, B (2019) The use of dietary additives in fish stress

mitigation: comparative endocrine and physiological responses Frontiers in

Endocrinology, 10

Hidajat, M J., Jo, W., Kim, H., & Noh, J (2020) Effective droplet size reduction and

excellent stability of limonene nanoemulsion formed by high-pressure homogenizer

Colloids and Interfaces, 4(1), 5

Hur, J W., Gil, H W., Choi, S H., Jung, H J., & Kang, Y J (2019) Anesthetic efficacy of

clove oil and the associated physiological responses in olive flounder (Paralichthys

Jiang, H., Sheng, Y., & Ngai, T (2020) Pickering emulsions: Versatility of colloidal

particles and recent applications Current Opinion in Colloid & Interface Science, 49,

Jutakridsada, P., Pimsawat, N., Sillanp¨a¨a, M., & Kamwilaisak, K (2020) Olive oil

stability in Pickering emulsion preparation from eucalyptus pulp and its rheology

Liu, H., Wang, C., Zou, S., Wei, Z., & Tong, Z (2012) Simple, reversible emulsion system

switched by pH on the basis of chitosan without any hydrophobic modification

Langmuir, 28(30), 11017–11024

Low, L E., Siva, S P., Ho, Y K., Chan, E S., & Tey, B T (2020) Recent advances of

characterization techniques for the formation, physical properties and stability of

Makaras, T., Razumien˙e, J., Gureviˇcien˙e, V., ˇSakinyt˙e, I., Stankeviˇci¯ut˙e, M., &

Kazlauskien˙e, N (2020) A new approach of stress evaluation in fish using β-d-

Matos, M., Laca, A., Rea, F., Iglesias, O., Rayner, M., & Guti´errez, G (2018) O/W emulsions stabilized by OSA-modified starch granules versus non-ionic surfactant:

Stability, rheological behaviour and resveratrol encapsulation Journal of Food Engineering, 222, 207–217

McClements, D J (2012) Nanoemulsions versus microemulsions: Terminology,

Mohammed, M H., Williams, P A., & Tverezovskaya, O (2013) Extraction of chitin

from prawn shells and conversion to low molecular mass chitosan Food Hydrocolloids, 31(2), 166–171

Mwangi, W W., Ho, K.-W., Ooi, C.-W., Tey, B.-T., & Chan, E.-S (2016) Facile method for forming ionically cross-linked chitosan microcapsules from Pickering emulsion

Mwangi, W W., Ho, K.-W., Tey, B.-T., & Chan, E.-S (2016) Effects of environmental factors on the physical stability of pickering-emulsions stabilized by chitosan

Park, M., Im, S.-Y., Seol, D.-W., & Park, I.-S (2009) Efficacy and physiological responses

of rock bream, Oplegnathus fasciatus to anesthetization with clove oil Aquaculture, 287(3–4), 427–430

Perrin, L., Gillet, G., Gressin, L., & Desobry, S (2020) Interest of pickering emulsions for

sustainable micro/nanocellulose in food and cosmetic applications Polymers, 12

Pickering, S U (1907) Cxcvi.—emulsions Journal of the Chemical Society, Transactions,

91, 2001–2021

Ramsden, W (1904) Separation of solids in the surface-layers of solutions and

mechanical coagulation)—Preliminary account Proceedings of the Royal Society of London, 72(477–486), 156–164

Rousseau, D (2013) Trends in structuring edible emulsions with Pickering fat crystals

Current Opinion in Colloid & Interface Science, 18(4), 283–291

Shah, B R., Li, Y., Jin, W., An, Y., He, L., Li, Z., … Li, B (2016) Preparation and optimization of Pickering emulsion stabilized by chitosan-tripolyphosphate

Shah, B R., & Mraz, J (2020) Advances in nanotechnology for sustainable aquaculture

Shah, B R., Zhang, C., Li, Y., & Li, B (2016) Bioaccessibility and antioxidant activity of curcumin after encapsulated by nano and Pickering emulsion based on chitosan-

Shao, Y., & Tang, C.-H (2016) Gel-like pea protein Pickering emulsions at pH 3.0 as a potential intestine-targeted and sustained-release delivery system for β-carotene

Food Research International, 79, 64–72

Soltani, S., & Madadlou, A (2016) Two-step sequential cross-linking of sugar beet pectin for transforming zein nanoparticle-based Pickering emulsions to emulgels

Carbohydrate Polymers, 136, 738–743

Soltanian, S., Hoseinifar, S H., & Gholamhosseini, A (2018) Modulation of rainbow trout (Oncorhynchus mykiss) cutaneous mucosal immune responses following

anesthesia: A comparative study on different anesthetic agents Fish & Shellfish Immunology, 80, 319–324

Svobodova, Z., Pravda, D., & Palackova, J (1991) Unified methods of haematological examination of fish (p 31) Vodˇnany, Czech Republic: Research Institute of Fish

Tort, L (2011) Stress and immune modulation in fish Developmental & Comparative Immunology, 35(12), 1366–1375

Tzoumaki, M V., Moschakis, T., Kiosseoglou, V., & Biliaderis, C G (2011) Oil-in-water

emulsions stabilized by chitin nanocrystal particles Food Hydrocolloids, 25(6),

Vakili, M., Rafatullah, M., Salamatinia, B., Abdullah, A Z., Ibrahim, M H., Tan, K B., … Amouzgar, P (2014) Application of chitosan and its derivatives as adsorbents for

dye removal from water and wastewater: A review Carbohydrate Polymers, 113,

Xu, Y., Chu, Y., Feng, X., Gao, C., Wu, D., Cheng, W., … Tang, X (2020) Effects of zein stabilized clove essential oil Pickering emulsion on the structure and properties of

chitosan-based edible films International Journal of Biological Macromolecules, 156,

Yang, Y., Fang, Z., Chen, X., Zhang, W., Xie, Y., Chen, Y., … Yuan, W (2017) An overview of Pickering emulsions: Solid-particle materials, classification,

Yousefi, M., Hoseini, S M., Vatnikov, Y A., Nikishov, A A., & Kulikov, E V (2018) Thymol as a new anesthetic in common carp (Cyprinus carpio): Efficacy and