TITLE: Formulation offish feed using fusarium venenatam and its applications on ariopsis seemanni growth
FIELD OF THE INVENTION
The present invention relates to the field of aquaculture, more particularly to fish feed
formulation using fusarium venenatum to promote growth effect in Ariopsis seemanni.
BACKGROUND OF THE INVENTION
Aquaculture is known as growing fish, molluscs, crustaceans and algae etc.,
with controlled conditions. The Food and Agriculture Organization (FAO) considers that about 16% of the consumed animal protein comes from fish proteins, [FAO 2012]. At the World Food Summit organized by the Food and Agriculture Organization (FAO) in Rome in 1996, the participating countries committed to reduce the number of malnourished people in the world by half by the year 2015 (Haylor, 2004). The increasing world population has resulted in a rising demand for protein for human consumption and animal production. The demand for protein is certain to become more serious with over exploitation of the sea and the depletion of most of the available arable land with the ever-rapid population growth. There is, therefore, the need to find novel protein sources that production wuuld require less of the agricultural land, with lower production cost (Israelidis and Conduonis, 1982). It well known fact that fish contains enormous amount of proteins, essential amino acids, calcium, vitamin A and B12 and omega-3 fatty acids (Jayasankar 2018). The current status of aquaculture for over 50% of the global food fish consumption

(Subasinghe et al., 2009). Aquaculture and aqua feed production is providing income generation in rural area population. Aqua feed production also plays an important role in aquaculture by providing rich nutrients source from soya protein, cereals, oil cake and dried whey, incorporated probiotics and single cell protein (SCP). In further 60% of total production cost spends for commercial aquaculture. Feed management is important factor for fish growth, feed should be contains suitable energy sources like proteins, minerals, vitamins. If poor quality feed and poor feeding strategy have severe environmental impacts in aquaculture farming system. Mainly uneaten foods and excreta create biological oxygen demand and the concentration of dissolved nitrogen and phosphate (White, 2013). In order to avoid the impacts, incorporated SCP feed is choice for aquaculture. SCP it is an excellent source of protein which can be produced by algae, bacteria and fungi in spite of these advantages, only one fungal SCP product approved for human consumption has reached the market which is the mycoprotein obtained from Fusar'tum venenatum. This fungus is found to contain high protein content and hence used commercially for the production of single cell protein mycoprotein. The strain Fusarium venenatum A3/5 (IMI 145425, ATCC PTA-2684) was developed commercially by an ICI and Rank Hovis McDougall joint venture lu derive a mycoprotein used as a food. The hyphae of the fungus are similar in length and width to animal muscle fibers and hence the mycoprotein is used as an alternative to meat and is marketed to vegetarians as Quorn. It is also suitable as a substitute for fat in dairy products and for cereal in breakfast and snacks (GRAS notification 2001).

The present invention has overcome the drawbacks in the prior art, wherein biomass derived from the fungal strain as mycoprotein is incorporated in the plant based fish feed followed by administration to the tested fishes. Treated fishes were evaluated for notable growth parameters changes like length, weight, food conversion rate/specific growth rate and survival rate.
OBJECT OF THE INVENTION
Accordingly, the primary object of the present invention is to provide higher growth rate at all concentrations during the culture period.
The primary object of the present invention is to formulate mycoprotein based feed as an effective feed for commercially important aquatic species.
SUMMARY OF THE INVENTION
It will be understood that this disclosure is not limited to the particular
methodologies described, as there can be multiple possible embodiments of the present disclosure which are not expressly illustrated in the present disclosure. It is also to be understood that the terminology used-in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present disclosure.

The present invention relates to a formulation offish feed using fusarium venenatam and its applications on ariopsis seemanni growth.
It is an aspect of the present invention, wherein lyophilized fungal strain Fusarium venenatum is activated, wherein the said strain is inoculated into Oatmeal media culture, whereby the activated pure culture is maintained on Oatmeal agar slant at 4°C.
It is another aspect of the present invention to prepare inoculum, wherein said inoculum is prepared in oatmeal broth, whereby fungal slant culture is flooded with sterile distilled water containing few drops of Tween-20 and filtering the homogenous suspension through syringe filter under aseptic condition and using the collected filtrate as the source of inoculum.
It is yet another aspect of the present invention to produce biomass, wherein the biomass used for the fish formulation is produced in the Dates extract medium, wherein one litre of media is sterilized by autoclaving followed by inoculation of 1.0 ml of inoculum, further the seeded flasks are incubated at ambient temperature (28°C) for 3 days in an orbital shaker and after the incubation period, the broth treated for 70°C for one hour to remove the nucleic acid content and the broth is filtered through sterile cheese cloth and transferred to the sterile petri plate and kept at 70°C for 3 hours in a hot air oven to obtain the dried biomass.
It is further another aspect of the present invention to prepare fish feed, wherein the fish feed consists of 30% wheat flour, 30% ragi flour, 30% groundnut oil cake, 10%

dry fish powder and 3% dried biomass, wherein the said fish feed is grinded into fine powder and steam sterilized for 15 minutes and further pelletized, air dried, packed and stored at dry place at room temperature.
DETAILED DESCRIPTION OF THE INVENTION:
The following detailed description illustrates by way of example and not by way of
limitations.
Fungal strain collection and maintenance
Fungal strain Fusarium venenatum (CBS 458.93) used in the present study was obtained from Netherland Fungal Biodiversity Centre in lyophilized form. Activation of the lyophilized culture was carried out by inoculation into Oatmeal media as described in our earlier studies (Prakash and Karthick Raja Namasivayam, 2013). Activated pure culture was maintained on Oatmeal agar slant at 4°C.
Inoculum preparation
Inoculum used for biomass production was prepared in oatmeal broth. Fungal slant culture was flooded with sterile distilled water containing few drops of Tween-20 using sterile glass rod. Homogenous suspension thus obtained was filtered through syringe filter under aseptic condition and the collected filtrate was used as the source of inoculum.
Biomass production
Biomass used for the fish formulation was produced in the Dates extract medium. One litre of media thus prepared was sterilized by autoclaving followed by inoculation of 1.0 ml of inoculum thus prepared. Seeded flasks were incubated at

ambient lemperature-(28°C) for 3 days in an orbital.shaker.__After_Jhe incubation period, the broth treated for 70°C for one hour to remove the nucleic acid content. Then the broth was filtered through sterile cheese cloth and transferred to the sterile petri plate and kept at 70°C for 3 hours in a hot air oven. Thus, dried biomass was used for the further studies.
Preparation offish feed
The essential nutrient requirements of fishes are protein, lipid, carbohydrate, vitamins and minerals. Plant based fish feed as reported by Thyagarajan et al (2014) was used in this study composed of soybean, groundnut oilcake and wheat flour which were mixed in 1:1:1 ratio and grounded into fine powder and sieved. The modified fish feed was prepared using three feed ingredients such as wheat flour (30%), ragi flour (30%), groundnut oil cake (30%), and dry fish powder (10%) along with 3% of dried biomass. Respective ingredient was mixed completely with enough volume of water followed by grinding into fine powder form. Processed material was steam sterilized for 15 minutes. After heat treatment, the dried material was pelletized. Collected pellets were allowed to air dry and dried pellets were packed in airtight container, stored in dry place at room temperature for further use.
Evaluation of growth of Ariopsis seemanni
Treatment
Mycoprotein supplemented fish feed was administrated to the tested fishes based on their age in the range of 12% to 5% of its body weight twice daily (dawn and dusk) and the treatment lasted for 90 days. Every day, 20% of the water in the tank was replaced with fresh water. Every week, the length and weight of the fishes was measured to calculate their growth.

Growth parameters
Effect of mycoprotein supplemented diet on the growth parameters of Ariopsis
reema>Mj'was~stii'died by "determination of-body-weight-changes^ofJ'ishes.duringJhe
experimental period. The other growth parameters like weight gain (%), feed conversion ratio (FCR) and specific growth rate was studied (Fayeolori &Manuel 2014). The growth parameters were calculated following the methods described by Paiboon Panase and Kriansak Mengumphan (2015).

In the present invention, fish feed is formulated with various sources like wheat flour (30%), ragi flour (30%), groundnut oil cake (30%), and dry fish powder (10%) along with 3% mycoprotein and control feed prepared with without mycoprotein. Fusarium venenatum was selected as the best organism Tor "" mycoprotein production as it has rich nutritive value like protein content (>41%), fat content (13%), dietary fiber (25%), minerals and antioxidants (Anderson & AOAC 2005).
the level of growth and significance offish feed is carried out by statistical analysis. In the present invention, one way ANOVA is used to find the significance of formulated fish feed with respect to control feed. The alternative protein sources are replaced with fungal biomass derived from Fusarium venenatum.
The fish feed is prepared with 3% of mycoprotein and treated for 20 fishes and without mycoprotein uses as control feed. The trail was studied with 90 days and results were observed in terms of weight and length (Table 3.1).

Table 3.2 shows the result of fish growth study was observed in terms of weight, length, average weight, specific growth rate, feed conversion rate and survival rate. Among the treatment mycoprotein incorporated fish feed showed significant difference when compared with control.

Table 3.3 shows the statistical descriptive analysis based on the growth of the fish treated with mycoprotein. The results of control group and treated group are depicted in the above table (Table 3.3). The control group is fish (Ariopsis seemanni) treated with commercial fish feed. The test group is fish (Ariopsis seemanni) fed with mycoprotein. At the end of the experiment ie, on 90th day, length and weight of Ariopsis seemanni is observed. The weight and length of fish in mycoprotein treated group is increased by 7.88% and 3.19 % on an average respectively when compared with control group. The raw data collected were analyzed by one way ANOVA in SPSS. It is evident from the above table that the results of treated group are highly significant (p< 0.001**) in between the groups compared to within the group.

Table 3.4 shows the statistical descriptive analysis based on the growth of the fish treated with mycoprotein. From Table 3.4, it is observed that the mean for fish consuming commercial feed is 10.76 and standard deviation is 1.81 and the mean for the fish given formulated feed given 17.88 and standard deviation is 1.02. Hence, it is interpreted that there is difference in the weight offish consuming commercial feed and the formulated feed.
Table 3.5 shows the statistical descriptive analysis based on the growth of the fish treated with mycoprotein.' From Table 3.5, it is observed that the mean for fish consuming commercial feed is 6.52 and standard deviation is 0.88 and the mean for the fish given formulated feed given 9.71 and standard deviation is 0,78. Hence, it is

interpreted that there is difference in the length of fish consuming commercial feed and the formulated feed.
Table 3.6 shows the statistical descriptive analysis based on the weight difference of the fish groups and within group treated with mycoprotein.
Table 3.7 shows the statistical descriptive analysis based on the length of the fish treated with mycoprotein.