Description: DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of aquatic feeds, and in specific relates to an effective bioactive feed that prevent and cure vibrio infections of crustaceans thereby increases survival percentage of crustaceans.
Background of the invention:
[0002] In general, shrimp aquaculture business accounts for 15% of all seafood items exported worldwide. One of the most critical factors for a good shrimp production is to keep the animals healthy. One of the biggest issues faced by aqua culturists has been disease outbreaks in this industry. The shrimp business in the Asia-Pacific area, which is the world's top producer of aquaculture goods, has been afflicted by new diseases, resulting in significant mortalities and economic losses.

[0003] Over the last decade, several diseases, such as luminous Vibriosis, white spot disease (WSD), yellow head disease (YHD), and Taura syndrome (TS), have wreaked havoc on the region's shrimp aquaculture that resulted in the extinction of Penaeus monodon culture. Shrimps are constantly exposed to a range of infections, primarily bacteria and viruses, as they are aquatic animals. While the majority of bacteria are beneficial, some of them can be dangerous. When it comes to bacterial infections, bacteria from the Vibrionaceae family are one of the most common causes of death in cultivated shrimp across the world.

[0004] During 2009–2012, a new emerging disease called acute hepatopancreatic necrosis disease (AHPND) also known as “early mortality syndrome (EMS)” started causing significant production losses in many shrimp consuming countries. The causative agents of AHPND are determined to be Vibrio bacteria such as Vibrio parahaemolyticus, V. campbellii and V. harveyi that contain a binary toxin, PirABvp, known to be responsible for the virulence of the disease.

[0005] Antibiotics are prohibited in shrimp production as a preventive step to stop bacterial resistance and bioaccumulation. As a result, a new chemical defence system is developed using mollusks in order to keep the stock alive. Antimicrobial activity is tested on a large number of marine creatures from a variety of phyla. Mollusks have a chemical defence system for safeguarding their eggs and create secondary metabolites with antibacterial properties, despite their physical protection.

[0006] Molluscs such as Aplysia sp., Phyllialae sp., bivalves, and gastropods are studied extensively for their bioactivity. Recently, bivalves are discovered to have antibacterial properties. Various Anadora granosa organic extracts have found to have modest antibacterial activity. Further, Hemifusus pugilinus and Donax faba acetone extracts have only mediocre effectiveness against Vibrio harveyi. However, the using of Anadora granosa organic extracts for the protection of shrimp is not studied extensively for their bioactivity.

[0007] Therefore, there is a need for an effective bioactive feed to prevent and cure vibrio infections of crustaceans. There is a need for a bioactive extract that is used as an alternative to treat the Vibrio infections in cultured white pacific shrimp for the production of organic culture of shrimp. There is a need for a bioactive extract that exhibits highest antibacterial activity against the pathogenic bacteria infections. There is a need for a bioactive extract that can be used as feed additive to mitigate the lethal effect of the Vibrio infections in the cultural Penaeus vannamei. A bioactive extract is needed that increases the survival percentage and decrease mortality rate of white shrimps.
Objectives of the invention:
[0008] The primary objective of the invention is to provide an effective bioactive feed to prevent and cure vibrio infections of crustaceans such as white shrimp, crabs, lobsters, crayfish, krill, prawns, woodlice, and barnacles thereof.

[0009] The other objective of the invention is to provide a bioactive extract as a feed additive that increases the survival percentage and decrease mortality rate of white shrimps.

[0010] The other objective of the invention is to provide a bioactive extract that is used as an alternative to treat the Vibrio infections in cultured white pacific shrimp for the production of organic culture of shrimp.

[0011] Another objective of the invention is to provide a bioactive extract that exhibits highest antibacterial activity against the pathogenic bacteria infections V. harveyi and S. aureus.

[0012] The other objective of the invention is to provide a bioactive extract that is used as feed additive to mitigate the lethal effect of the Vibrio infections in the cultural Penaeus vannamei

[0013] Yet another objective of the invention is to provide a bioactive extract that shows high Minimum inhibitory concentration against Vibrio infections.
Summary of the invention:
[0014] The present disclosure proposes a bioactive feed additive for vibrio infections in crustaceans and method of preparation for the same. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0015] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide an effective bioactive feed that prevent and cure vibrio infections of crustaceans thereby increases survival percentage of crustaceans.

[0016] According to an aspect, the invention provides a bioactive feed additive for prevention of vibrio infections in crustaceans. A bioactive feed comprises a pellet feed, 0.1 to 0.9 grams of a bioactive extract per kilogram of body weight of the pellet feed, and a protein gel binder. In specific, the bioactive extract is extracted from visceral tissue mass of marine bivalve Anadara granosa. The bioactive extract is coated on the pellet feed using the protein gel binder to obtain the bioactive feed. The bioactive feed prevents and cures vibrio infections of crustaceans.

[0017] In specific, one of the preferable quantity of the bioactive extract coated on the pellet feed is 0.2 grams per kilogram of the pellet feed. The bioactive extract is (((2Z,4Z)-dodeca-2,4-dienoyl)oxy)-3-hydroxypropan-2-yl tetradecanoate. The pellet feed includes 32 to 33% of protein, 3.5% of fat, 4% of fiber, and 11% of moisture. The bioactive feed prevents and cures vibrio infections of crustaceans such as white shrimp, crabs, lobsters, crayfish, krill, prawns, woodlice, and barnacles thereof.

[0018] According to another aspect, the invention provides a method for preparation of a bioactive feed for prevention of vibrio infections in crustaceans. The bioactive extract is extracted from visceral tissue mass of marine bivalve Anadara granosa (A. granosa). Next, the bioactive extract is coated on the pellet feed using the protein gel binder to obtain the bioactive feed. In specific, the amount of bioactive extract coated on the pellet feed is based on average weight of at least 100 shrimps and feeding rate of 10% body weight. Later bioactive feed is subjected for air drying and then the dried bioactive feed is stored at 4°C.

[0019] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0020] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0021] FIG. 1 illustrates an exemplary method for preparation of a bioactive feed for prevention of vibrio infections in crustaceans in accordance to an exemplary embodiment of the invention.

[0022] FIG. 2A illustrates inhibitory activity of different fractions on Vibrio harveyi in accordance to an exemplary embodiment of the invention.

[0023] FIG. 2B illustrates inhibitory activity of different fractions on Staphylococcus aureus in accordance to an exemplary embodiment of the invention.

[0024] FIG. 3A depicts an exemplary proton NMR spectra of bioactive extract obtained from fraction 9 in accordance to an exemplary embodiment of the invention.

[0025] FIG. 3B depicts an exemplary C13 NMR Spectra of bioactive extract obtained from fraction 9 in accordance to an exemplary embodiment of the invention.

[0026] FIG. 3C depicts an exemplary 2D COSY NMR Spectra of bioactive extract obtained from fraction 9 in accordance to an exemplary embodiment of the invention.

[0027] FIG. 4A depicts a graphical representation of mean % survival of Post larvae Penaeus vannamei with Vibrio harveyi in accordance to an exemplary embodiment of the invention.

[0028] FIG. 4B depicts a graphical representation of mean % survival of Post larvae Penaeus vannamei fed the bioactive feed (positive control) in accordance to an exemplary embodiment of the invention.

[0029] FIG. 4C depicts a graphical representation of mean % survival of Post larvae Penaeus vannamei tested with Vibrio harveyi and fed with the bioactive feed in accordance to an exemplary embodiment of the invention.

[0030] FIG. 4D depicts a graphical representation of mean % survival of Post larvae Penaeus vannamei with pellet feed (negative control) in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0031] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0032] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide an effective bioactive feed that prevent and cure vibrio infections of crustaceans thereby increases survival percentage of crustaceans.

[0033] According to an exemplary embodiment of the invention, a bioactive feed additive for prevention of vibrio infections in crustaceans. A bioactive feed comprises a pellet feed, 0.1 to 0.9 grams of a bioactive extract per kilogram of body weight of the pellet feed, and a protein gel binder. In specific, the bioactive extract (bioactive compound) of 15 grams is extracted from 1350 grams of Anadara granosa (A. granosa) visceral tissue mass. The bioactive extract is coated on the pellet feed using the protein gel binder to obtain the bioactive feed.

[0034] In specific, the preferable quantity of the bioactive extract coated on the pellet feed is 0.2 grams per kilogram of the pellet feed. The bioactive extract is (((2Z,4Z)-dodeca-2,4-dienoyl)oxy)-3-hydroxypropan-2-yl tetradecanoate. The pellet feed includes 32 to 33% of protein, 3.5% of fat, 4% of fiber, and 11% of moisture. The bioactive feed prevents and cures vibrio infections of crustaceans such as white shrimp, crabs, lobsters, crayfish, krill, prawns, woodlice, and barnacles thereof. The proposed bioactive feed prevents vibrio infections in crustaceans, but in particular shrimps.

[0035] According to an exemplary embodiment of the invention, FIG. 1 refers to an exemplary method 100 for preparation of a bioactive feed for prevention of vibrio infections in crustaceans. At step 102, the bioactive extract is extracted from visceral tissue mass of A. granosa. At step 104, the bioactive extract is coated on the pellet feed using the protein gel binder to obtain the bioactive feed. In specific, the amount of bioactive extract coated on the pellet feed is based on average weight of at least 100 shrimps and feeding rate of 10% body weight. At step 106, the bioactive feed is subjected for air drying and then the dried bioactive feed is stored at 4°C.

[0036] Further, the pellet feed is either in solid particle form or in liquid emulsion form or in dissolved form. Ingredients in the pellet feed are any ingredients that are needed to culture crustaceans. The choice depends on the nutritional value or on certain theological characteristics which is obtained by the activity of the ingredients.

[0037] According to another aspect of the invention, FIG. 2A and FIG. 2B refer to inhibitory activity of different fractions on Vibrio harveyi (V.harveyi) and Staphylococcus aureus (S.aureus) respectively. An intertidal bivalve mollusc is tested for inhibitory activity against pathogenic bacteria of the shrimp, and fish thereof. The bioactive extract exhibits an inhibition zone of 11 mm and 10 mm against Vibrio harveyi and Staphylococcus aureus, respectively, in antibacterial activity studies. Fractional separation process is performed on the bioactive extract to separate the fraction. For Vibrio harveyi, the purified fraction shows a minimum inhibitory concentration (MIC) of 32 µg/ml. For Staphylococcus aureus, the purified fraction shows MIC of 64 µg/ml. Further, Nuclear Magnetic Resonance (NMR) analysis is used to identify the purified compound's structural elucidation.

[0038] In the bioautography experiment a major spot is obtained at Rf 0.4 with a clear zone. Further, for separation and identification of antibacterial bioactive extract (bioactive compound), a total of 12 fractions are separated by column chromatography. Out of 12 fractions, 5 fractions (7, 8, 9, 10 & 11) exhibited inhibitory activity against V. harveyi as shown in FIG. 2A and 2 fractions (8 & 9) exhibited inhibitory activity against Staphylococcus aureus as shown in FIG. 2B. Fraction no. 9 exhibits inhibitory zones of 12 mm and 11 mm against V. harveyi and S. aureus respectively. The fraction no. 9 is further processed for structural elucidation.

[0039] For example, table 1 depicts inhibitory zones (mm) of various fractions on V harveyi and S aureus.

[0040] Table 1:

Fraction Number Concentration of fractions
(µg/µl) Zone of inhibition
(Diameter in mm.)
V harveyi S aureus
1 100 NA NA
2 100 NA NA
3 100 NA NA
4 100 NA NA
5 100 NA NA
6 100 NA NA
7 100 10.3±0.6 NA
8 100 10.7±0.6 10.3±0.6
9 100 12.0±1.0 10.7±0.6
10 100 8.7±0.6 NA
11 100 10.7±0.6 NA
12 100 NA NA
NA= No activity

[0041] According to another exemplary embodiment of the invention, FIG. 3A, FIG. 3B and FIG. 3C refer to proton NMR spectra, C13 NMR Spectra, and 2D COSY NMR Spectra of the compound obtained from fraction 9 respectively. On recrystallization of the fraction 9, a compound is obtained in the form of off-white crystals. The molecular formula of the compound is C29H52O5. The 1H NMR spectrum shows the presence of 5 protons on oxygenated carbons at δ 3.95 (m, 2H), a set of geminal protons at δ 4.09 (m, 1H), δ 4.12 (m, 4.12) and a methine at δ 4.85, which indicates the presence of glycerol in the structure.

[0042] FIG. 3B shows the presence of a D2O exchangeable proton at δ 5.52 and appearance of two carbon signals in the carbonyl region at δ 160, confirms di-glyceride chemistry in the compound. Appearance of a triplet at δ 2.01 indicates that one of the fatty acid with functionality on α-carbon. FIG. 3C shows, the proton NMR shows four mutually coupled protons present on sp2 carbons in the region δ 5.35–6.65. The 1 H–1 H correlations confirmed the connectivity in the four protons and ascertained presence of Δ2,4 unsaturated fatty acid. The mass fragmentation confirms the chain length and placement of unsaturated fatty acid on C-1 and saturated fatty acid on C-2 of glycerol.

[0043] According to another aspect of the invention, challenge studies are conducted on Penaeus vannamei (white shrimp) with and without Vibrio harveyi (V. harveyi). First, healthy post larvae of Penaeus vannamei (PL’s) are collected and stored in 12 troughs. Each trough contains 100 post larvae of Penaeus vannamei. The Penaeus vannamei are feed at the rate of 10 % of body weight for the acclamation period of 3 days. A temperature of 28°C and pH between 8.2-8.5 are maintained to culture the Penaeus vannamei. The collected Penaeus vannamei are tested for V. harveyi by PCR using species specific primer. The acclimatized Penaeus vannamei, which are tested negative to V. harveyi used for the experiments.

[0044] Then, lethal doses (LD 50) of V.ordalii, V.harveyi, V. proteolyticus, V. natriegen, V.parahaemolyticus, V.ganogenus, V. tubiashii, V.vulnificus, V. funissii against to the post larvae of Penaeus vannamei are found to be 108, 106, 109, 108, 107, 108, 107, 108 and 109 CFU/ml respectively at 48 hours post challenge study.

[0045] Each of the 12 fibre troughs, labelled V1, V2, V3, M1, M2, M3, T1, T2, T3, C1, C2, C3, is filled with 100 acclimatized Penaeus vannamei. The acclimatized Penaeus vannamei from V1, V2, and V3 are bathed and tested with Vibrio harveyi at a concentration of 107 CFU/ml before feeding the pellet feed at 10% of body weight. The acclimatized Penaeus vannamei in the M1, M2, and M3 troughs (positive controls) are fed with the bioactive feed (experimental diet) at a 10% body weight that contains 0.2 g of bioactive extract/kg of pellet feed. The acclimatized Penaeus vannamei in the T1, T2, and T3 are tested with Vibrio harveyi and fed with the bioactive feed at 0.2 g of bioactive extract/kg of pellet feed.

[0046] The acclimatized Penaeus vannamei in C1, C2, and C3 troughs are fed the pellet feed at 10% of body weight each day (negative control). The acclimatized Penaeus vannamei in all treatment 12 fibre troughs are checked every 12 hours for up to 48 hours. Under the preparation of the bioactive feed, a loss of 50% antibiotic due to leaching and a loss of 25% due to reduced feed intake in Penaeus vannamei infected conditions are assumed.

[0047] According to another exemplary embodiment of the invention, FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D depict percent (%) survival of post larvae Penaeus vannamei in different conditions. FIG. 4A depicts mean % survival of post larvae Penaeus vannamei with Vibrio harveyi. The post larvae Penaeus vannamei from V1, V2, and V3 are bathed and tested with Vibrio harveyi at a concentration of 107 CFU/ml before feeding the pellet feed at 10% of body weight. FIG. 4B depicts mean % survival of post larvae Penaeus vannamei that are fed the bioactive feed (positive control) at 0.2g of bioactive extract/kg body weight. The post larvae Penaeus vannamei in the M1, M2, and M3 troughs (positive controls) are fed with the bioactive feed at a 10% body weight that contains 0.2 g of bioactive extract/kg of pellet feed.

[0048] FIG. 4C depicts mean % survival of post larvae Penaeus vannamei tested with Vibrio harveyi and are fed the experimental feed. The acclimatized Penaeus vannamei in the T1, T2, and T3 are tested with Vibrio harveyi and fed with the bioactive feed at 0.2 g of bioactive extract/kg of pellet feed. FIG. 4D depicts mean % survival of post larvae Penaeus vannamei with normal feed (negative control). The post larvae Penaeus vannamei in C1, C2, and C3 troughs are fed the pellet feed at a rate of 10% of body weight each day (negative control).

[0049] For instance, table 2 depicts effect of the bioactive extract on the survival of post larvae Penaeus vannamei. Mean (%) survival of post larvae Penaeus vannamei in challenge experiments under different treatments. Mortality rate of 82% is observed in post larvae Penaeus vannamei after 48 hours post challenge with V.harveyi when fed with normal diet. The post larvae Penaeus vannamei tested with the same bacteria and fed with the bioactive feed showed 82.66% survival. Survival % in both positive and negative controls is 99.3%.

[0050] Table 2:
Post larvae Penaeus vannamei Troughs

Time Mean % of survival
0 Hrs. 12Hrs. 24Hrs. 36Hrs. 48Hrs.
challenge with V.harveyi V1 100/100 78/100 50/100 38/100 15/100 18
V2 100/100 77/100 62/100 40/100 22/100
V3 100/100 75/100 59/100 40/100 17/100
challenge with V.harveyi and fed with medicated feed T1 100/100 98/100 92/100 89/100 85/100 82.6
T2 100/100 95/100 93/100 90/100 80/100
T3 100/100 95/100 92/100 89/100 83/100
Fed with experimental feed
(positive control ) M1 100/100 100/100 100/100 100/100 100/100 99.3
M2 100/100 100/100 100/100 99/100 99/100
M3 100/100 100/100 100/100 100/100 99/100
Fed with normal diet
(Negative
Control) C1 100/100 100/100 100/100 99/100 99/100 99.3
C2 100/100 100/100 100/100 100/100 99/100
C3 100/100 100/100 100/100 100/100 100/100

[0051] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, an effective bioactive feed that prevent and cure vibrio infections of crustaceans is disclosed here.

[0052] The proposed effective bioactive feed to prevent and cure vibrio infections of white shrimps. The proposed bioactive extract as a feed additive increases the survival percentage and decrease mortality rate of white shrimps. The proposed bioactive extract is used as an alternative to treat the Vibrio infections in cultured white pacific shrimp for the production of organic culture of shrimp. The proposed bioactive extract exhibits highest antibacterial activity against the pathogenic bacteria infections V. harveyi and S. aureus. The proposed bioactive extract is used as feed additive to mitigate lethal effect of the Vibrio infections in the cultural Penaeus vannamei. The proposed bioactive extract shows high Minimum inhibitory concentration against Vibrio infections.

[0053] The proposed bioactive extract can be applied to other types of feed pellets as well, including pellets for monogastric animals and polygastric animals, such as pigs, horses, calves, young sheep, cows, sheep, goats, fowl and poultry, such as chickens, turkeys, and ducks, deer, reindeer, cats and dogs.

[0054] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application. , Claims: CLAIMS:
We Claim:
1. A bioactive feed additive for prevention of vibrio infections in crustaceans, comprising:
a pellet feed;
0.1 to 0.9 grams of a bioactive extract per kilogram of body weight of said pellet feed, where said bioactive extract is extracted from visceral tissue mass of marine bivalve Anadara granosa; and
a protein gel binder, where said bioactive extract is coated on said pellet feed using said protein gel binder to obtain a bioactive feed,
whereby said bioactive feed prevent and cure vibrio infections of crustaceans.
2. The bioactive feed additive for prevention of vibrio infections in crustaceans as claimed in claim 1, wherein preferable quantity of said bioactive extract coated on said pellet feed is 0.2 grams per kilogram of body weight of said pellet feed.
3. The bioactive feed additive for prevention of vibrio infections in crustaceans as claimed in claim 1, wherein said bioactive extract is (((2Z,4Z)-dodeca-2,4-dienoyl)oxy)-3-hydroxypropan-2-yl tetradecanoate.
4. The bioactive feed additive for prevention of vibrio infections in crustaceans as claimed in claim 1, wherein composition of said pellet feed includes 32 to 33% of protein, 3.5% of fat, 4% of fibre, and 11% of moisture.
5. The bioactive feed additive for prevention of vibrio infections in crustaceans as claimed in claim 1, wherein said crustaceans includes white shrimp, crabs, lobsters, crayfish, krill, prawns, woodlice, and barnacles thereof.
6. A method for preparation of a bioactive feed for prevention of vibrio infections in crustaceans, comprising:
extracting a bioactive extract from visceral tissue mass of marine bivalve Anadara granosa;
coating said bioactive extract on a pellet feed using a protein gel binder to obtain said bioactive feed, and
subjecting said bioactive feed for air drying and then storing said dried bioactive feed at 4°C.
7. The method for preparation of a bioactive feed for prevention of vibrio infections in crustaceans as claimed in claim 3, wherein amount of said bioactive extract is based on average weight of at least 100 shrimps and feeding rate of 10% body weight.