FIELD OF THE INVENTION
The purpose of this invention is to develop a synergistic formulation comprising commercially available natural antimicrobial compounds against Early Mortality Syndrome (EMS) causing bacterial pathogen Vibrio parahaemolyticus. Although quite a few phytochemical compounds have already been reported for their antimicrobial activity against several bacterial pathogens, their efficacy at the commercial level is yet to be standardized. Hence, the present invention is indented to evaluate the synergistic activity of certain phytochemicals and their effective formulations with the viable cost to control the virulence of EMS-specific V. parahaemolyticus infections in shrimp aquaculture.
BACKGROUND FOR THE INVENTION
Cultivation of Litopenaeus monodon and L. vannamei is becoming the preferable choice of aquaculture worldwide. Despite the apparent success, in terms of production and economy, currently, this sector is experiencing a steep decrease in the rate of production and heavy economic loss worldwide due to the outbreaks of microbial diseases. In the year 2012, Ruwandeepika et al. reported that the bacterial infectious diseases commonly ensue at all stages of the shrimp life cycle and confer results in severe economic losses. For instance, in the year 2015, Zorriehzahra and Banaederakhshan stated that the shrimp farming is most commonly affected by bacterial diseases especially EMS which was also known as “Acute Hepatopancreatic Necrosis Disease” (AHPND). It is an emerging and most devastating disease caused by a bacterial pathogen, which most commonly causes infection on farmed penaeid shrimp. EMS typically targets the shrimp between the age group of 20 to 40 days old post-larvae and results in 100% mortality within a week. The syndrome is more often fatal to shrimp rather than other aquatic animals. Though various technologies were well standardized for control of bacterial diseases, the control of EMS in early stages of shrimp larvae still remains unexplored.
In this fact, many researchers described that a wide range of antibiotics is being used to treat established bacterial infections. Although control of bacterial diseases in aquaculture has been partially achieved through the administration of antibiotics, the indiscriminate use of antibiotics has resulted in the widespread occurrence of antibiotic-resistant bacteria in the

aquatic environment and posing severe threats to both aquatic animals and human health (Cabello, 2006). Therefore, the recent research interest in disease management has been focused towards finding alternatives to antibiotics, which are more effective and environment-friendly. Thus, a novel alternative control strategy is highly essential to combat EMS.
The use of commercially available natural compounds in combination is a robust emerging strategy to reduce microbial infections and to potentiate the biological activity of each other. Al-Yaqout et al. (2014) have patented an anesthetic and therapeutic composition of thyme oil against aquatic bacterial pathogens (Patent No: US 2014/0044812 A1).
The current study is aimed to formulate an effective synergistic formulation with an enhanced antimicrobial activity against EMS specific V. parahaemolyticus using non-antibiotic commercially available phytochemical compounds.
SUMMARY OF THE INVENTION
The present invention relates to finding suitable formulations to control the devastating disease viz., EMS, caused by V. parahaemolyticus in shrimp especially in the cultures of L. vannamei, which ruins the shrimp aquaculture industry worldwide.
In the present invention, the antimicrobial potential of various commercially available phytotherapeutic compounds was evaluated against EMS disease. Further, in order to enhance their antimicrobial activity, a combinatorial approach was followed to formulate effective formulations with environmentally friendly and economically viable qualities. Further a formulated feed comprising the present synnergistic formulation was developed.
Thus, 20 different commercially available compounds such as catechin, eugenol, furfural, hexadecanoic acid, curcumin, geraniol, quercetin, pyrogallol, vanillin, tannic acid, citral, mangiferin, morin, naringin, rosmarinic acid, 2,6-ditert-butyl-4-methylphenol, linalool, phytol, cinnamaldehyde, and menthol were purchased from Sigma-Aldrich, St. Louis, MO, USA and were screened against the growth of V. parahaemolyticus. Among these, eight

different compounds such as citral, curcumin, geraniol, pyrogallol, quercetin, tannic acid, vanillin, and menthol showed killing effect at their minimal concentrations against V. parahaemolyticus. Further, to evaluate the combinatorial effect of all these compounds, a total of 28 different combinations were designed to investigate their synergistic activity. Among the 28 different combinations, eight combinations showed synergistic effect such as curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral against the growth of V. parahaemolyticus. Out of eight combinations tested, tannic acid + geraniol effectively inhibited the growth of V. parahaemolyticus. Then the third compound vanillin with higher synergistic score was decided to added with tannic acid + geraniol combination in order to further enhance the activity.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein.
Figure 1: Shows synergistic antimicrobial analysis of tannic acid and geraniol against V. parahaemolyticus. Green highlighted box represent the higher synergistic concentration.
Figure 2: Shows effect of test formulation on the growth of V. parahaemolyticus (MIC -Tannic acid + Geraniol + Vanillin- in a ratio of 4: 1: 2 (50+12.5+25 μg/mL).
Figure 3: Shows effect of test formulation on preformed biofilm of V. parahaemolyticus (MIC - Tannic acid+Geraniol+Vanillin in a ratio of 4: 1: 2 - (50+12.5+25) μg/mL).

Figure 4: Shows microscopic analyses of biofilm. a) Light microscopic analysis b) CLSM analysis (MIC - Tannic acid+Geraniol+Vanillin in a ratio of 4: 1: 2 - (50+12.5+25) μg/mL).
Figure 5: Shows effect of test formulation on swarming motility of V. parahaemolyticus (MIC - Tannic acid+Geraniol+Vanillin in a ratio of 4: 1: 2 - (50+12.5+25) μg/mL).
Figure 6: Analysis of CFU count in Shrimp HP region. Effect of test formulation on bacterial colonization at different concentrations (4xMIC - Tannic acid+Geraniol+Vanillin - in a ratio of 4: 1: 2 (200+50+100) μg/mL).
Figure 7: Schematic diagram of formulated feed preparation for 1 kg.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification are to be understood as being modified in all instances by the term "about". It is noted that, unless otherwise stated, all percentages given in this specification and appended claims refer to percentages by weight of the total composition.
Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner.
The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the

invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to a “solvent” may include two or more such solvents. The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
The term “EMS” as used herein refers to a new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS).
The expression “Combinatorial therapy” as used herein refers to the use of combinations of commercially available phytochemical compounds and antibiotics as therapeutic agents to microbial infections.

The term “Vibrios” as used herein refers to the major disease-causing bacterial pathogen and are ubiquitous in the marine environment.
The term “Antibiotics” as used herein refers to the chemical substances which kill or inhibit the growth of bacteria by interfering with the cell wall, nucleic acid, protein synthesis and other enzymatic pathways.
The term “Phytochemicals” as used herein refers to the biologically active chemical substances extracted from natural resources.
The term “Virulence” as used herein refers to the ability of an agent of infection to produce disease. The virulence of a microorganism is a measure of the severity of the disease caused by them.
The term “Pathogens” as used herein refers to either bacteria, fungi or virus which cause the infectious disease to the host cell.
The term “Biofilms” as used herein refers to a self-developed hydrated matrix made up of microbial cells and biomolecules such as polysaccharides, proteins, nucleic acids, and lipids.
The term “MIC” as used herein refers to the Minimum Inhibitory Concentration (MIC) and defined as the lowest concentration of phytochemicals, alone or in combination with other compounds, which visibly inhibits the growth of bacteria.
The term “FIC” as used herein refers to the Fractional Inhibitory Concentration. The degree of synergy between natural phytochemical drugs is often expressed in terms of the FIC.
The FIC index (FICI) was calculated using the following formula: FIC index = FICA + FICB
7

FICA = [A] / MICA; FICB = [B] / MICB Here [A] and [B] represents the MIC of compound A and B in combination, respectively.
The FIC index obtained was interpreted as follows: <0.5 denoting synergy; 0.5-0.75 denoting partial synergy; 0.76-1 denoting an additive effect; 1-4 denoting indifference; and >4 denoting antagonism.
Problem to be solved by the Invention
As discussed under the background of the invention, the problem identified in the present invention is the lack of an eco-friendly , economical and natural formulation in effectively treating the devastating disease EMS caused by parahaemolyticus in shrimps.
The objective of the present invention
The present invention is intended to provide an alternate non-antibiotic
Combinatorial/Synergistic antimicrobial phytochemical formulations against V.
parahaemolyticus causing EMS in shrimps.
Solution provided
It was surprisingly observed by the present inventors that the synergetic formulation according to the present invention satisfies the need in the field. Particularly, the present synergetic formulation was found to be highly effective in reducing or eliminating EMS caused by V. parahaemolyticus in shrimps. Thus a synergetic formulation comprising geraniol : tannic acid : vanillin in a concentration of about 50 to about 400µg/mL : about 12.5 to about 100µg/mL: about 25 to about 200µg/mL was achieved. The present formulation is found to be economical, userfriendly, and highly effective with no side effects. In aquaculture, this considered to be a good tool for targeting bacterial challenges and act as a good alternative to antibiotic usage. Especially in critical phases such as disease occurrence, it exhibits its potential in treating the infections and thereby decreases the mortality rates. Under standard conditions, it protects the shrimp health, shortens the time to harvest and therefore increases the productivity. Most interestingly, the application this

formulation will prevent the selectve pressure for the development of antibiotic resistance among aquatic microbial pathogens and bioaccumulation of antibiotic residues in the edible tissues of cultured animals. Hence, it is worth to mention that this formulation not only enhances the survival of the shrimp, also it promotes the productivity and quality of the shrimp.
It was studied by the present inventors that when phytochemicals compounds are combined in the manner disclosed herein the description, provided enhanced efficacy compared to the same compounds used in single. It is a novel approach, which totally ruled out the utilization of antibiotics, it reduces the production costs and enhances the growth and quality of the shrimp. It is interesting to note that it is highly stable over the years, long lasting activity, doesnot require any storage specifications and more ecofriendly in nature.
The embodiment of the present invention comprises a synergistic formulation comprising
tannic acid, geraniol and vanillin.
The formulation is an anti microbial formulation with an anti vibriosis activity .
The anti microbial activity is particularly against V. parahaemolyticus.
The formulation is for the control of shrimp disease.
The formulation comprises Tannic acid, Geraniol and vanillin in a weight ratio of about
1:0.25: 0.5 to about 8: 2: 4.
The formulation comprises of about 50 µg/mL to about 400µg/mL of tannic acid.
The formulation comprises of about 12.5 µg/mL to about 100µg/mL of Geraniol.
The formulation comprises of about 25 µg/mL to about 200 µg/mL of vanillin.
The formulation is for the control of shrimp disease Early Mortality Syndrome [EMS].
The minimum inhibitory concentration of the formualtion has a microbial inhibitory activity
of about 50 % to about 60%.
Another embodiment of the present invnetion is a formualted feed encapsulated by the
present formualtion.
The formualted feed encapsulated by the present formualtio has binder as an excipient.

Accordingly, the first aspect of the present invention relates to the screening of about 20
different commercially available phytochemical compounds against the growth of
V.parahaemolyticus. These compounds include catechin, euginol, furfural, hexadecanoic acid, curcumin, geraniol, quercitin, menthol, pyrogallol, vanillin, tannic acid, citral, mangiferin, morin, naringin, rosmarinic acid, 2,6-ditert-butyl-4-methylphenol, linalool, phytol, cinnamaldehyde, and menthol. From the above list of compounds, 8 compounds were found to have high killing effect of V.parahaemolyticus at its minimal concentration. These 8 compounds identified were citral, curcumin, geraniol, pyrogallol, quercetin, tannic acid, vanillin, and menthol. Table-1.
In the second aspect of the invention, to evaluate the combinatorial effect of the 8 selected compounds, 28 different combinations were formulated and tested as mentioned in Table -2. Among the 28 combinations tested, 8 combinations showed synergistic anti microbial activity such as curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral against the growth of V. parahaemolyticus.
In the third aspect of the invention, the highly effective synergistic anti V. parahaemolyticus combination was selected comprising Tannic acid and geraniol as shown in Figure 1.
In the fourth aspect of the present invention, a test formulation was prepared comprising Tannic acid, geraniol and 25µg/mL vanillin. The third compound vanillin with higher synergistic score was decided to be added with tannic acid + geraniol combination in order to further enhance the activity. Hence, 25 μg/mL concentration of vanillin was added which surprisingly reduced the concentration of tannic acid + geraniol upto 50% (Table 3). Hence, the MIC of the formulation (Tannic acid + Geraniol + Vanillin) is in a ratio of 4: 1: 2 [50 + 12.5 + 25 µg/ml (Figure 2).

In the fifth aspect of the present invention a formulated feed was prepared by encapsulating the shrimp feed with the present formulation and further comprising excipiemt. The excipient comprises a binder.
Hereinafter, the present invention will be described in detail with reference to the following examples. The following examples are set forth to assist in understanding the invention and should not, of course, be construed as specifically limiting the invention described and claimed herein.
EXPERIMENTAL DETAILS
General Information and Materials: All the ingredients of the present composition were procured from the commercial suppliers as shown below:
Tannic acid - S. V. Enterprises, Mumbai
Vanillin - Rankem & Kemphasol, Mumbai
Geraniol - Sigma-Aldrich, St. Louis, USA
EXAMPLE 1
Screening of phytochemicals for its antimicrobial efficacy against V. parahaemolyticus
(Determination of MIC)
The antimicrobial activity of commercially available phytochemicals against V. parahaemolyticus was tested by broth dilution method as described by the Clinical and Laboratory Standards Institute (CLSI, 2006). The assays were performed in a 24-well microtitre plate (MTP) with 1 ml of LB broth in each well. The LB broth was supplemented with phytochemicals in a two-fold increased concentration ranging between 25 - 1280 μg/ml. Then 1% V. parahaemolyticus cell suspension (1 × 106 cells/ml) was inoculated and incubated at 30°C for 24 h. After incubation, the optical density (OD) of the grown culture was measured at 600 nm. The lowest concentration of compounds which inhibited the

visible growth of the bacteria was considered as MIC value(s). The results are shown in Table -1.
Table-1 List of commercially available phytochemicals screened for antimicrobial potential against V. parahaemolyticus

Results: Among these, eight different compounds such as citral, curcumin, geraniol, menthol, pyrogallol, quercetin, tannic acid, and vanillin showed efficient killing activity at their minimal concentrations against V. parahaemolyticus (Table 1). Hence, the compounds as it alone and in combinations were taken for further experimental analysis.
EXAMPLE: 2
Evaluation of synergistic antimicrobial potential of phytochemicals against V.
parahaemolyticus by checkerboard method.
The degree of synergy between phytochemicals is often expressed in terms of the fractional inhibitory concentration (FIC). The FIC is the MIC of the drug in combination divided by the MIC of drugs acting alone. A checkerboard technique was used to examine the synergism between two active compounds against V. parahaemolyticus. In this method, MIC was determined, for the test compounds in their paired combinations (Isenberg 2007) in two fold increased concentrations up to the MIC of alone. Then 1% V. parahaemolyticus cell suspension (106 cells/mL) was inoculated and incubated at 30°C for 24 h. After incubation, the optical density (OD) of the grown culture was measured at 600 nm. The lowest concentration at which no visible growth occurred was recorded to be the MIC value of the individual and combined test compounds. FIC was calculated from the MIC of test compound A and the MIC of test compound A in combination with test compound B. Therefore, FIC of A = MIC of test compound A in combination/MIC of test compound A alone. The FIC of test compound B was calculated in the same manner and the sum of the two FIC compounds combined to give the ΣFIC index.
ΣFIC index = FIC of test compound A + FIC of test compound B The calculated FIC index was used to detect the nature of the interaction between the two test compounds and the interaction either synergism or indifference or antagonism type. The values published by the American Society of Microbiology were used to decide the nature of the interaction FICI < 0.5 synergy, 0.5 ≤ FICI < 1 partial synergy, FICI = 1 additive, 2 ≤ FICI < 4 indifferent, and 4 < FICI antagonism (Botelho 2000). Table -2; and Figure 1 and 2.
Table-2 List of phytochemicals selected for synergistic effect against V. parahaemolyticus


Results: In 28 different combinations tested, eight combinations showed a synergistic effect against the growth of V. parahaemolyticus (Table 2). This includes curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral. Among these eight combinations, the tannic acid + geraniol combination exhibited effective inhibitions in the growth of V. parahaemolyticus at a ratio of 4: 1 [100 + 25 µg/mL concentration] respectively (Figure 1).
Tannic acid (A) + Geraniol (B) = (100 + 25) μg/mL
MICA = 400 μg/mL
MICB= 200 μg/mL
FIC index = FICA + FICB
FICA = 100 / 400 = 0.25
FICB = 25 / 200 = 0.125
FIC index = 0.25 + 0.125 = 0.375
0.375 < 0.5 - Synergism
Further, a third compound vanillin with higher synergistic score was decided to be added with tannic acid + geraniol combination in order to further enhance the activity. Hence, 25

μg/mL concentration of vanillin was added which surprisingly reduced the concentration of tannic acid + geraniol upto 50% (Table 3). Hence, the MIC of the formulation (Tannic acid + Geraniol + Vanillin) is 50 + 12.5 + 25 µg/ml (Figure 2).
Furthermore, the active formulation tannic acid + geraniol + vanillin at MIC and 2xMIC has effectively eradicated the preformed biofilm of V. parahaemolyticus to the level of 55% and 88%, respectively (Figure 3).
Table-3 Final concentration of test formulation (Tannic acid + Geraniol + Vanillin)

EXAMPLE: 3
Preformed biofilm eradication assay
Eradication efficiency of the test formulation tannic acid + geraniol + vanillin against preformed biofilm formation of V. parahaemolyticus was studied. Initially, V. parahaemolyticus was allowed to form biofilm for 24h, in a sterile 24well MTP in which one mL of fresh media supplement with 1% inoculum. Then planktonic cells were removed and replaced with fresh media supplement with MIC and 2xMIC of tannic acid + geraniol + vanillin and extended incubation up to 24h. After incubations, the planktonic cells were discarded and the wells were washed with sterile distilled water and allowed to dry. The

biofilm cells were stained using 0.4% crystal violet (CV) dye for 10 min. After staining, the unbound dye was washed with distilled water and the wells were destained using 20% glacial acetic acid (GAA) for 30 min and OD570 nm was measured using a UV spectrometer (Hitachi U- 2800, Tokyo, Japan). The amount of dye present after washing is directly proportional to the amount of cells in the biofilm. The control was maintained without the addition of test formulation. The percentage of biofilm inhibition was calculated using the formula:
% of inhibition = [(Control OD570 nm –Treated OD570 nm) / Control OD570 nm]× 100.
Results: The active formulation tannic acid + geraniol + vanillin at MIC and 2xMIC has effectively eradicated the preformed biofilm of V. parahaemolyticus to the level of 55% and 88%, respectively .The results are shown in Figure 3.
EXAMPLE:4
Microscopic analyses of preformed biofilm of V. parahaemolyticus
The knowledge about the growth and architecture of biofilms is much important to understand the biofilm eradication potential of phytocompounds. Therefore, microscopic analysis was performed to study the effect of active formulation tannic acid + geraniol + vanillin induced alterations on the surface and the three-dimensional architecture of vibrios biofilm through a light microscope and Confocal Laser Scanning Microscope (CLSM).
In the microscopic analysis, V. parahaemolyticus was allowed to form a biofilm on glass slides (1 × 1 cm) for 24 h at 30ºC. Then the slides were treated with MIC and 2xMIC of tannic acid + geraniol + vanillin and incubated as previously metioned conditions. Then the slides were washed with sterile PBS and processed as below (Santhakumari et al. 2016).
In light microscopic analysis, the control and treated glass slides were stained with 0.4% CV for 5 mins and washed with sterile distilled water. Then the stained slides were imaged under a light microscope (Nikon Eclipse Ti 100, Japan) at 400x magnification.

In Confocal Laser Scanning Microscopic (CLSM) analysis, the control and treated slides were stained with 0.1% acridine orange in dark for 5 mins and subsequently washed with sterile distilled water. Then the stained slides were imaged under 488 nm argon laser in CLSM (Zeiss LSM 710, Carl Zeiss, Germany) and processed with Zeiss LSM Image Examiner (Version 4.2.0.121).
Results: The light microscope, as well as CLSM images, clearly revealed that the visual reduction of surface coverage and thickness of biofilm on the slides treated with the as-designed formulation consisting tannic acid + geraniol + vanillin was observed, than that of untreated controls. The results are shown in Figure 4(a)&(b).
EXAMPLE: 5 Motility assessment
The flagellar-mediated swarming motility plays an important role in biofilm formation in a bacterial pathogen. It is believed that any interruption in such motility could possibly inhibit the biofilm development. In the present study, the efficacy of active phytochemical formulation was further assessed for its ability to interfere with swarming motility of V. parahaemolyticus.
The effect of test formulation on swarming motility of V. parahaemolyticus was determined by placing 5 μL of V. parahaemolyticus cell suspension in the center of the swarm agar plates (1% peptone, 0.5% NaCl, 0.5% agar and 0.5% of filter-sterilized d-glucose) with and without active formulation. Then the plates were then incubated at 30°C in an upright position for 24 h and observed for reduction in swimming and swarming migration zones (Srinivasan et al. 2016).

Results: The results clearly showed that the formulation tannic acid + geraniol + vanillin in a ratio of 4: 1: 2 (50+12.5+25 μg/mL) effectively reduced the swarming motility of V. parahaemolyticus. Results are shown in Figure 5 (a) & (b).
EXAMPLE: 6
Preparation of formulated feeds
The effective test formulation was encapsulated at the specific concentrations with the commercial feed using XL BindTM supplement (TIL Biosciences), as a binder. Initially, the test formulation (tannic acid + geraniol + vanillin) at various concentration of MIC, 2xMIC and 4xMIC was completely dissolved in 100ml of distilled water separately and the binder was diluted with equal volume of distilled water to prepared 50% diluted binder. Then, 150 µl of the diluted binder was added to 100 µl of test formulation mixture. Then, 1 g of commercial feed was gently mixed with obtained mixture solution and allowed to air dry at room temperature for 1 h. The schematic diagram showing the feed preparation is shown in figure 7. Three different ratio of test formulation for feed encapsualtion: (MIC= µg/ml = µg/g)
> 1xMIC - Tannic acid + Geraniol + Vanillin 4: 1: 2 (50µg+12.5µg+25µg)/g
> 2xMIC - Tannic acid + Geraniol + Vanillin 4: 1: 2 (100µg+25µg+50µg)/g
> 4xMIC - Tannic acid + Geraniol + Vanillin 4: 1: 2 (200µg+50µg+100µg)/g
EXAMPLE: 7
In vivo challenge study
The efficacy of encapsulated feed was assessed their antipathogenic activity against V. parahaemolyticus challenged Penaeus semisculcatus shrimps in a feeding manner. Healthy, pathogen-free active shrimps were selected for this study. The immersion challenge was carried out following the protocol is given below. In the experiment, the shrimps were immersed or infected in the suspensions of V. parahaemolyticus (107 CFU/ml) for 6 h. Subsequently, a group of six immersed shrimp was transferred into glass aquaria containing

50 ml of fresh UV sterilized sea water. This experimental setup contains control and a treatment group which was maintained in triplicate. In the experimental control group, the post challenged shrimp was fed with unformulated commercial feed, while in the treated group, the formulated feed was added to post challenged shrimp containing aquaria. The survival rate and the impact of test formulation (tannic acid + vanillin + geraniol) at MIC, 2xMIC and 4xMIC on bacterial colonization in shrimp’s hepatopancreatic(HP) region were analyzed after 72 hrs. Then the treated and untreated shrimps were sacrificed and their HP part was crushed and immersed in sterile distilled water. Then, 100 µl of each control and treated samples were spread on TCBS agar plates separately and incubated for 24 h at room temperature for count the bacterial load of V. parahaemolyticus.
Results: The results showed the CFU count of V. parahaemolyticus on HP region of the formulated (4x MIC - Tannic acid + Geraniol + Vanillin: 200 µg + 50 µg + 100 µg) feed treated post challenged shrimp was significantly reduced compared to the untreated control. (Figure 6).
CONCLUSION
In the present study, a screening of commercially available phytochemicals belonging to different compound categories were randomly selected and tested for its antibacterial efficacy against V. parahaemolyticus. Furthercombinatorial usage of phytochemicals with potent antimicrobial effect comprising , tannic acid + geraniol exhibited effective inhibitions in the growth of V. parahaemolyticus at 100 + 25 µg/mL concentration respectively. Further, a third compound vanillin when added at a concentration of , 25 μg/mL surprisingly reduced the concentration of tannic acid + geraniol upto 50% (Table 3).
The invivo efficacy of this active formulation (Tannic acid + Geraniol + Vanillin), when fed to shrimps in a concentration of (4xMIC - Tannic acid + Geraniol + Vanillin: 200 µg + 50 µg + 100 µg) showed significant reducedion of bacterial count compared to the untreated

control (Figure 6). It clearly proves the action of in vivo bactericidal effect of formulated feed was enhanced the immune response of shrimp model against V. parahaemolyticus infection.
Thus the present invention delineates novel synergistic formulations comprising commercially available phytochemicals against EMS specific V. parahaemolyticus. Further, it accentuates the high pharmaceutical and economical values of individual phytochemicals and an effective formulation comprising of tannic acid + geraniol + vanillin to treat EMS associated V.parahaemolyticus infection in shrimp aquaculture. The formulation of the present invention is ecologically friendly without any side effects to the cultured organisms. It is user-friendly and very economical and highly effective in controlling EMS.

WE CLAIM
1. A synergistic formulation comprising:
i. Tannic acid,
ii. Geraniol and iii.Vanillin.
2. The formulation as claimd in claim 1 wherein the said formulation is an anti microbial formulation.
3. The formulation as claimed in claims 1 and 2 wherein the antimicrobial activity is anti vibriosis activity.

4. The formulation as claimed in claims 1 to 3 wherein the microorganism is V. parahaemolyticus.
5. The formulation as claimd in claim 1 to 4 wherein the said formulation is for the control of shrimp disease.

6. The formulation as claimd in claims 1 wherein the formulation comprises Tannic acid, Geraniol and vanillin in a weight ratio of about 1:0.25: 0.5 to about 8: 2: 4.
7. The formulation as claimed in cliams 1 wherein the formulation comprises of about 50 µg/mL to about 400µg/mL of tannic acid.
8. The formulation as claimd in claim 1 wherein the formulation comprises of about 12.5 µg/mL to about 100µg/mL of Geraniol.
9. The formulation as claimd in claim 1 wherein the formulation comprises of about 25 µg/mL to about 200 µg/mL of vanillin.

10. The formulation as claimed in claim 1 to 9 wherein the shrimp disease is Early Mortality
Syndrome [EMS].
11. The formulation as claimed in claim 1 to 10 wherein the minimum inhibitory
concentration of the formualtion has a microbial inhibitory activity of about 50 % to about
60%.
12. A formualted feed encapsulated by the formualtion as claimed in claim 1 to 11 and
excipients.
13. The formulated shrimp feed as claimed in claim 12 wherein the excipient is a binder.