THE SAME

FIE L D OF T H E INVE NTION

The present invention relates to a shrimp feed and a method for producing the same. More particularly, the present invention relates to a shrimp feed additive that increases survival rates of shrimp fry at the post larval stage, and a method for producing the same.

BAC KG ROU ND ART

The following discussion of the background of the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the date of the application.

S hrimp farming is a multimillion dollar industry. P lagued with emerging diseases, land-based farmers face mass mortality of shrimp fry from the zoeae stage at the post larval stage right up till 30-60 culture days (DOC 30-60). Others are overwhelmed by disease such as E nterocytozoon hepatopenaei (E H P) that do not kill the shrimp outright but instead severely hinders or retards the growth of the shrimp and raising the F eed C onversion Ratio (FC R). E H P- infected shrimps therefore take a much longer time to reach a minimum size of 10-12g at 100 DOC where farmers should be getting an average of 15-18g of shrimp at DOC 100.

To improve survival and growth rates of cultured shrimp fry at the post larval stage, farmers currently cultivate shrimp by feeding shrimp fry at the post larval stage with several varieties of antibiotic drugs, chemicals, or pharmaceutical agents to overcome mass mortality of the shrimp fry which are often toxic and cancer-causing to the human body when the shrimp is consumed. Bacillus subtilis (hay bacillus) is a spore-forming aerobe with no known toxicity. It occurs in nature, including dry grass, soil, sewage and in the air and is capable of enzymatically curding milk, saccharifying starch, and degrading lipids. Bacillus subtilis is also extensively utilized in industrial fields. It optimally grows at a pH of 7 - 8.5 with a temperature set to be 37 - 40eC .

The present invention attempts to overcome at least in part some of the aforementioned disadvantages and to provide for an alternative solution that improves survival and growth rates of cultured shrimp and decreases the incidence of diseases emanating from the hatchery.

S U MMARY OF T H E INVE NTION

Throughoutthis document, unless otherwise indicated to the contrary, the terms ^' comprising _, ^' consisting of., and the like, are to be construed as non- exhaustive, or in other words, as meaning ^' including, but not limited to_.

In accordance with a first aspect of the invention, there is a method for producing a shrimp feed additive, comprising

i. a first process of preparing a pre-condition mixture of germinated soy beans;

ii. a second process of preparing an activated bacillus subtilis;

iii. a third process of adding the activated bacillus subtilis to the pre condition mixture of germinated soy beans to produce a first mixture; iv. a fourth process of removing an activated soy bean stock from the first mixture;

v. a fifth process of combining the activated soy bean stock and an acidified fructose solution to sterilized shrimp shells to obtain a second mixture, fermenting the second mixture to produce a fermented mixture; and

vi. a sixth process of removing a top layer of the fermented mixture.

P referably, the pre-condition mixture of germinated soy beans is prepared by: i. repeatedly washing soy beans with distilled water and draining the distilled water in a container;

ii. adding pre-heated distilled water to the soy beans to produce a soybean mixture and incubating the soybean mixture to stimulate production of enzymes and heat shock protein in the soy beans; and iii. draining the distilled water from the soybean mixture to obtain expanded soy beans, and adding citric acid to the expanded soy beans.

P referably, the activated bacillus subtilis is prepared by modifying a bacillus subtilis culture to induce overstimulation of tricarboxylic acid enzymes.

P referably, overstimulation of the tricarboxylic acid enzymes is induced through an acidified sugar molecule.

P referably, the acidified sugar molecule is an acidified glucose molecule or an acidified fructose molecule.

P referably, the acidified fructose solution is prepared by adding acetic acid, and a proprietary extract comprising fermented elderberries, to fructose.

P referably, the bacillus subtilis culture is prepared using Bacillus S Z-2010 with Luria Bertani or Lysogeny Broth (LB) media.

In accordance with a second aspect of the invention, there is a shrimp feed additive, produced in accordance with the method of the first aspect of the invention.

P R E F E R R E D E MB ODIME NT OF T H E INV E NTION

Particular embodiments of the present invention will now be described in detail. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Other embodiments may be utilized, and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. Additionally, unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the specification, the term :comprising ^“ shall be understood to have a broad meaning similar to the term line luding ^“ and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term 'comprising ^“ such as :comprise ^“ and :comprises ^“

In accordance with a first embodiment of the invention, there is a method for producing a shrimp feed additive comprising of the following steps.

S tep One

The method comprises a first step of preparing a primed pre-condition mixture of germinated soy beans as follows:-

(a) 3 kilograms (kg) of soy beans are washed with 5 litres (L) of 15 degree

Celsius (- C) distilled water. This process of washing is repeated at least 3 times. The soy beans are then placed in a 10 L container and 15 - C distilled water added and abrasively hand washed. The washed soy beans are then drained in a metal sieve and transferred to another 10 L container where the soy beans are soaked in 15 - C distilled water for between 12 (+/-1 ) hours before the distilled water is drained off completely. The parameters mentioned above are preferably increased or decreased in multiple ratios. Thus, if 6 kg of soy beans are used, 10 L of distilled water should be used correspondingly to ensure adequate distilled water is imbibed by the soy beans. It was observed that a 10L container will retain near constant temperature of the soy beans and distilled water mixture for between 12 (+/-1 ) hours.

(b) 6L of sterilized distilled water is further heated up to 30· C and added to the washed and drained soy beans as described in (a) above. The mixture is then allowed to incubate for between 4-6 hours inside a plastic container which is preferably made of styrofoam to retain near constant temperature of the soy beans and distilled water mixture. It was observed that a temperature of 30· C stimulates the production of enzymes in the soy beans which causes the soy beans to produce superoxide di-methane.

(c) The water from the soy beans and distilled water mixture is next drained and another 6L of 42· C sterilized distilled water is added to the washed and drained soy beans and the resulting mixture is allowed to incubate for 24 hours, after which the distilled water is drained off completely leaving the expanded soy beans. Low molecular weight heat shock protein (HS P) is activated and produced by the soy beans.

(d) The expanded soy beans are next transferred to a 5L container, preferably a non-metallic container such as a glass jar. A 2L solution of 0.1 % citric acid is prepared and added to the expanded soy beans. The entire mixture is kept at 37 (+/- 2)· C for 24 hours or till the radicles or roots of the expanded soy beans are observed to be between 2-3 millimetres (mm) in length.

Step Two:

A bacillus subtilis culture is next modified to induce overstimulation of tricarboxylic acid enzymes, such overstimulation of tricarboxylic acid enzymes producing activated bacillus subtilis. The overstimulation of the tricarboxylic acid enzymes is induced through an acidified sugar molecule. In the present embodiment of the invention, preferably an acidified fructose molecule is used. An acidified fructose solution is prepared by adding 5 mL of 1 % wA/ acetic acid to 10g of fructose in a closed enclosure such as a container to prevent crystallization of the acidified fructose solution. The acidified fructose solution is allowed to react for 1 -6 hours at 60· C before use, preferably for 1 hour. The acidified fructose solution must not be allowed to react for more than 6 hours to prevent extraneous metabolites from forming in the solution. After 1 hour, 1 -5 millilitres (mL) of a proprietary extract (P roprietary E xtract) is added to the acidified fructose solution. The P roprietary E xtract comprises fermented elderberries using Bacillus S Z-0097. P referably, 1 mL of the P roprietary E xtract is added to the acidified fructose solution. A volume in excess of 100 mL of the acidified fructose solution is prepared.

The bacillus subtilis culture is prepared by using a proprietary single colony of Bacillus S Z-2010 or variations of this together with Luria Bertani or Lysogeny Broth (LB) media for 1 8h at room temperature. 10 mL of Bacillus S Z-201 0 is aliquoted to 900 mL of 1 /1 0 LB media concentration and incubated at 37- C for 3 hours.

90 mL of acidified fructose solution is next added to the proprietary Bacillus S Z- 2010 to make 1 L and incubated at43- C for at least 8 hours to produce activated Bacillus S Z-2010.

S tep 3:

500mL of the activated Bacillus S Z-2010 is then added to 500g of primed pre condition mixture of germinated soy beans produced in step one to form a mixture and allowed to ferment at room temperature for 21 -28 days.

S tep 4:

After 21 -28 days, the liquid top layer of the resulting mixture (activated soy bean stock) produced in step 3 is removed. P referably, 60-1 50mL of the activated soy bean stock is removed as it is observed that a volume of more than 150 mL will contain sediments produced through the fermentation of the primed soy beans and the presence of such sediments will decrease the efficacy of the activated soy bean stock. More preferably, 100mL of the activated soy bean stock is removed. The activated soy bean stock that is removed is stored at 4- C for no more than 365 days. The activated soy bean stock must not be stored at less than 4- C to prevent the liquid from freezing which would affect its efficacy.

S tep 5:

100mL of the activated soy bean stock and 100 mL of acidified fructose solution prepared as described in step 2 above are added to 1 Kg of sterilized shrimp shells. The mixture is allowed to ferment for 365 days.

After around 6 months of fermentation, another 100mL of the acidified fructose solution is added. This mixture is now allowed to ferment for another 6 months.

S tep 6:

After 1 year of fermentation, the top liquefied layer of the resulting fermented mixture, also known as the P rime U p Toll-Like R eceptor (T LR) is removed and is ready to be used as a shrimp feed additive to increase the survival rate of post larval shrimp fry at the hatchery.

The present invention has the following advantages:-

1 . The present invention reduces diseases and mortality rate among cultured shrimp at the hatchery state before transfer to grow-outs.

2. S hrimp fed with the feed produced in accordance with the invention have been shown to be resistant to protozoan attachment at the Zoeae stage where there is previously no known cure for this protozoan attachment. Studies have shown that once protozoan attachment takes place, 50-90% of the shrimp fry will face mortality in 2 days. 3.The shrimp feed is produced using natural ingredients which is not toxic or harmful to the human body.

It should be appreciated by the person skilled in the art that the above invention is not limited to the embodiment described. In particular, the following modifications and improvements may be made without departing from the scope of the present invention:

6 Different strains of naturally occurring bacteria or fungus could be used in place of a pre-condition mixture of germinated soy beans.

It is to be understood that the above embodiments have been provided only by way of exemplification of this invention, and that further modifications and improvements thereto, as would be apparent to persons skilled in the relevant art, are deemed to fall within the broad scope and ambit of the present invention described herein. It is further to be understood that features from one or more of the described embodiments may be combined to form further embodiments.