DESC:RELATED PATENT APPLICATION:

This application claims the priority to, and benefit of Indian Patent Application No. 202311045686 filed on July 07, 2023; the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION:

The present invention relates to a novel composition for aquaculture feed. Particularly, the present invention relates to a composition for aquaculture feed comprising novel isolated bacterial strains exhibiting probiotic properties. More particularly, the present invention relates to developing a combination of novel endophytic bacteria and natural nutrients for aquaculture feed or a feed for aquarium or ornamental fishes with desirable characteristics.

BACKGROUND OF THE INVENTION:

The growth of aquaculture industry has been increasing tremendously in the recent past. The need for increased disease resistance, growth of healthy aquatic organisms and feed efficiency has led to the development of many techniques.

In fish feed, nutrition is critical as feed typically represents approximately 50% of the variable production cost. In recent years fish feed has advanced in the development of new, commercial diets which promotes optimal fish growth. Some of the fish feeds available in the prior art are described below:

CN105767594A discloses a High-protein-content growth-promoting environment-friendly fish feed and preparation method thereof. The high-protein-content growth-promoting environment-friendly fish feed comprises the following raw materials: bean cakes, cotton meal, corns, bran, rapeseed cakes, spirulina powder, seaweed powder, wheat middling, polygonatum sibiricum powder, fish oil, calcium lactate, calcium dihydrogen phosphate, lysine, methionine, aspartic acid, glutamic acid, a vitamin mixture, minerals, phytase, synbiotics, a traditional Chinese medicine compound additive and a food attractant.

CN103402353A discloses a vegetarian feeding method for carnivorous fish and shrimp with spirulina and chlorella algae using electrolyzed water and sodium thiosulfate, guar and oligo-fructans as additives. The food is produced from Spirulina and Chlorella algae that are cultured and produced by cell proliferation in culture tanks in electrolyzed nutrient water that has been treated with sodium thiosulfate to neutralize chlorine, and are subsequently dried and, with the addition of preferably 0.3% of guar gum powder and preferably 1-2% of oligofructan powder and with the addition of 10% electrolyzed water for sterilization, are pelleted and packaged.

Probiotic bacteria are the subject of WO2019135009A1, which addresses their application in fish farming to cure and/or prevent microbial infections in fish as well as to increase fish weight. The bacteria used are species belonging to the genera Pseudomonas, Psychrobacter, and Aliivibrio.

CN104522416A discloses Granular feed for juvenile fish of Bostrichthys sinensis and preparation method of granular feed. The granular feed comprises the following components in parts by weight: 40-60 parts of animal nutritional ingredients, 20-40 parts of plant nutritional ingredients, 15-30 parts of functional ingredients, 0.3-3 parts of special vitamins, 0.3-3 parts of special trace elements, 0.2-2 parts of casein hydrolysate and 0.2-2 parts of lysine. A preparation method comprises the following steps: (1) manufacturing raw materials; (2) synthesizing a biological material; (3) converting to a cooked material; (4) puffing and granulating; (5) drying; (6) spraying and coating; and (7) performing vacuum drying.

The prior art CN105104847A is related to aquatic product culture feed, specifically to fermented fish feed that may accelerate growth rate and is inexpensive and widely available. The following raw components, divided into parts by weight, are used to create the fermented feed. The ingredients are as follows: 20–25 parts maize flour, 15-20 parts wheat bran, 5-8 parts peanut bran, 5–10 parts fish meal, 5–10 parts bagasse pith, 3-5 parts leaven, 2-3 parts molasses, and 1-2 parts microelements.

The nutritional content of the fish feed is decided based on the factors like species of fish selected and life stage of the fish. Complete diet supply includes ingredients like proteins, carbohydrates, fats, vitamins, and minerals. In general, protein requirement is lower for herbivorous fish, and omnivorous fish species than carnivorous fish. Protein requirements are generally higher for smaller as well as early life stage fish.

In year 1900 the concept of probiotics was introduced by Nobel laureate Elie Metchnikoff, who discovered that the consumption of live bacteria (Lactobacillus bulgaricus) in yogurt or fermented milk had shown improved results in biological features of gastrointestinal track. Furthermore, probiotics have their use in the management of health conditions. As awareness of probiotics has increased, significant improvement in health conditions has been observed. Probiotic bacteria have been used widely in the form of food supplements like dairy products and juices, capsules, drops and powders.

As per the prior art the first use of probiotic in aquaculture has been started in year 1986. As the modification of the original word “probiotika”, the term probiotic was introduced by Lilly and Stillwill in the year 1965.

The most common commercially available probiotic bacterial strain are Lactobacillus and Bifidobacterium species such as Bifidobacterium (adolescentis, animalis, bifidum, breve, and longum) and Lactobacillus (acidophilus, casei, fermentum, gasseri, johnsonii, reuteri, paracasei, plantarum, rhamnosus, and salivarius).

Generally, in the manufacturing process of probiotics immediately after fermentation, the broth culture is concentrated (often from 1:5 to 1:10) by continuous centrifugation which is further followed by freeze drying. Thus, lyophilized microbial biomass which was used to prepare a probiotic product contains residual growth medium including microbial metabolites produced or secreted during cell growth. Some of the microbial metabolites include lactate, acetate, propionate, bacteriocins, reuterin, or other secreted molecules (e.g., immunomodulatory secreted peptides) which are also known as postbiotics. The actual contribution of these molecules to the interaction of probiotic formulations on host health is not known but, in light of data obtained from in vitro experiments, cannot be excluded.

Commercially available probiotics products are usually divided into two types they are mono strain, which is defined as containing single strain of a well-defined microbial species and multi-strain containing more than one strain of the same species or genus. The term multispecies is also used for the products which comprise strain from more than one genus.

As the consumption of large quantities of probiotic bacteria is used for treatment, safety is the primary concern in such products. Thus, to ensure and minimize the adverse effects of mono strain or multi strain probiotic bacteria, high quality standards must be followed in preparation to make certain viable strains which are compatible to each other, and also free from any contamination in the product.

The use of probiotics, prebiotics, and aquaculture synbiotics is widely applied in aquaculture specifically in manufacturing of fish feed. Probiotic bacteria play an important and beneficial role in the fish ecosystem and gastrointestinal track, which in turn enhances the growth of the fish.

Thus, in recent years the use of probiotic bacteria in fish feed composition has been increasing significantly.
Specific strains of probiotic organisms have gained importance in health benefits However, the concept of “probiotic umbrella” promoted by probiotic industry has been confused by consumers.

The umbrella concept seeks to take advantage of results obtained with a specific probiotic by extending them to others, blurring the specificity of the product, dose, duration of intake, combination of strains, and methods used to manufacture the formulation with which the benefits were obtained. Because of the relatively unregulated nature of the probiotic market, such transferal of claims from the tested product to one that has material differences in its formulation or manufacture opens the door to many problems and questions.

The problem follows:
The probiotic bacteria cannot survive in the cooked material during the manufacturing process of floating fish feed.

Bacterial compatibility is a major issue for multi-strain-based products.
Compatibility between strains plays a major role in avoiding antagonism which might lead to detrimental effect on host (Puvanasundram et al 2021)

Most commercial fish feed uses ubiquitous ingredients such as fish meal or fish oil from residues from fish factories or other animal origin which are varying the quality.

Microbiological malfunction is often seen, the most common is presence of bacteria, fungi, parasites, virus. (https://www.bankom.rs/doc/ogledi/en_fishmealbad.pdf)

In the case of vegans, fish feeds are majorly developed with chemicals (CN103402353A).

Lactobacillus bacteria, being a known probiotic organism, suffers from several drawbacks such as poor pH tolerance, poor bile tolerance and indifferent survival in acid bile salts and juices and lacks characteristics like stability in acid conditions, higher temperature, high pH tolerance.

There is a need for probiotic bacteria, which can overcome above discussed limitations of prior art and which comprises all the desired characteristics.

To solve all the problems of the existing aquaculture feed, a probiotic composition comprising all the desired characteristics such as compatibility with each other, acidic stability, intestinal stability, stability in presence of acid bile salts, and adhesion property, improved pH tolerance, improved viability, functional property at higher temperature and salinity conditions, is highly required.

OBJECTS OF THE INVENTION:

The primary object of present invention is to provide a novel composition for aquaculture feed comprising novel isolated bacterial strains exhibiting probiotic properties.

Another object of the present invention is to develop a novel combination of endophytic bacteria and natural nutrients for aquaculture feed or a feed for aquarium fishes.

Another object of the present invention is to provide a composition comprising natural proteins, carbohydrates and fats completely derived from plants and other microorganisms and not from animal sources.

Yet another object of the invention is to develop a fish feed composition in which bacteria is stable at higher temperatures and has high tolerance towards higher pH and bile salts.

A further object of the present invention is to develop fish feed in which multi strain bacteria are compatible with each other and shows a promising result in terms of weight gain of fish and increase in fish activity.

SUMMARY OF THE INVENTION:

In one aspect, present invention discloses a novel combination of endophytic bacteria and natural nutrients for aquaculture feed or a feed for aquarium fishes, which is stable at high pH, high tolerance to bile acids, biofilm formation in adherence, and viable at high temperature and saline conditions. Wherein, feed does not comprise any material such as protein, carbohydrate, fat etc obtained from animal source.

According to one aspect of the present invention, the raw material for preparing the novel composition for aquaculture feed comprises:
i. a combination of probiotic microorganisms;
ii. protein source;
iii. carbohydrate source;
iv. fat source; and
v. optionally, an herbal mixture.

Wherein, the combination of the probiotic organisms is selected from the species comprising Bacillus licheniformis, Bacillus amyloliquefaciens and Lactobacillus sp. The said probiotic composition comprises 107 to 1010 CFU/g of the combination bacteria.

In one aspect of the invention, protein source is one of more component/ingredient selected from the group comprising of soybean, mustard de oiled cake (DOC), De Oiled Rice Bran (DORB), black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.
In one embodiment of the present invention, the protein source is selected from the group comprising soybean, mustard DOC and DORB.

Wherein, as a protein source, soyabean may be used in a concentration of 20-25% w/w, Mustard DOC may be used in a concentration of 5-10% w/w and DORB may be used in a concentration of 22-25% w/w.

In an exemplified embodiment of the invention, the concentration of the protein in the novel aquaculture feed obtained by this invention is 25-40% w/w.

In one aspect of the invention, the carbohydrate source is one or more component/ingredient selected from the group comprising of wheat, barley, maize, rice, oats, potato, millet, sorghum, cassava and quinoa or combination thereof.

In one of the embodiments, the carbohydrate source is Maize used in a concentration of 20-30% w/w.

In an exemplified embodiment of the invention, the concentration of the carbohydrate in the novel aquaculture feed obtained by this invention is 30-50%.

In one aspect of the invention, the fat source is one of more component/ingredient selected from the group comprising of ground nut, palm, linseed, sunflower, algal, rapeseed, coconut, hempseed, olive and safflower or combination thereof.

In one embodiment, the fat source is ground nut used in a concentration of 1-5% w/w.

In an exemplified embodiment of the invention, the concentration of the fat in the novel aquaculture feed obtained by this invention is 10-20%.

In one aspect of the invention the composition may optionally also comprise a herbal mixture, wherein the herbal mixture is selected from black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.

In one optional embodiment of the invention, the herbal mixture is used in a concentration of 3-5% w/w.

Endophytic strains of the present invention exhibit improved pH and bile tolerance, biofilm formation as well as viability at high temperature and high saline conditions. These novel isolates also form biofilm and having good adhesion property. The probiotic composition of the present invention can be produced and used in the form of solid powder or granulated form.

DETAILED DESCRIPTION OF THE INVENTION:

Feed used in aquaculture has a finite shelf life, like many edible products. It is essential to store and handle them carefully to attain and utilize the economic and nutritional value of these feeds. Fish feeds are formulated with many ingredients, which are intended to supply nutritional requirements and help in healthy growth of the fishes. Generally, fish additives vary in different forms and compositions. Particular feed additive aims at the feed quality, including pellet binders, antioxidants, and feed preservative.

Aquaculture operations across the world have increased from small ponds to an enormous business producing millions of livestock. Nutrition also plays a crucial role in encouraging reproduction of fish. The nutrients provided by the aquaculture feed mainly comprises of lipids, and fats, carbohydrates, proteins, vitamins, and minerals.

In general Aquaculture feed are of two types, they are as follows:
a) Extruded bits
b) Pressure pelleted feed.

Extruded bits will float on the surface of the water. Whereas pressure pelleted feed would sink in the water. Though many types of fish would feed on both types of feed.

The use of probiotic bacteria in fish feed has led to extreme beneficial effects to the fishes. Probiotic diets are being incorporated into feed composition along with other active nutrients. Probiotic bacteria comprising feed plays a vital role in fish gastrointestinal tract in turn enhances the growth performance. However, existing fish feed comprising probiotic bacteria suffers many disadvantages.

Therefore, there is a continuous quest for organisms with improved properties such as improved pH tolerance and better survival in acid bile salts and juices. Further, the organisms with improved viability and functional property at higher temperature and salinity conditions will be an added benefit while proposing probiotic compositions for aquaculture feed.

Accordingly, in one aspect present invention discloses novel isolated bacterial strains that can survive the external conditions, manufacturing process, different pH, salinity, high temperature with synergistic and compatibility property and deliverability to the gut to provide the desired benefit through bio-fermentation of plant-based ingredients maintaining all nutritional profile.

The present invention discloses novel isolated endophytic bacterial strains exhibiting probiotic properties. Particularly present invention discloses a novel combination of three isolated probiotic bacteria (Bacterial Code: TB0125, TB0134, TB0144). These isolates along with other probiotic organisms are useful as probiotics for fish feed.

The novel combination of the present invention comprises of three (3) isolated probiotic bacteria belonging to genus Bacillus and Lactobacillus.

Said Endophytic strains can survive the external conditions, manufacturing process, different pH, high salinity, high temperature with synergistic and compatibility property, and deliverability to the gut to provide the desired benefit through bio-fermentation of plant-based ingredients maintaining all nutritional profile.

Said isolated probiotic bacteria in extruded fish feed of the present invention are compatible to each other. No antagonistic effect has been developed.

Said Endophytic strains of the present invention exhibit improved pH and bile tolerance, biofilm formation as well as viability at high temperature and high saline conditions. These novel isolates also form biofilm and have good adhesion property.

The said bacteria have acid-tolerant property, i.e., the stability of probiotic bacteria in the presence of acids and bile salt, adhesion of bacteria in presence of bile salt tolerance.

The Endophytic bacterial strains of the present invention are viable and remains functional at higher water temperature ranges like 40°C±2°C and up to 10-15 % of saline conditions as well as normal water temperatures in tropical and temperate conditions.

The said isolated probiotic bacteria of the present invention are resistant to acid bile salt up to 0.2 to 2.5%. These isolates show good adhesion property even in presence of bile salt from 0.2 to 2.5%.

In another aspect, present invention discloses a novel combination of endophytic bacteria and natural nutrients for aquaculture feed or a feed for aquarium fishes, which is stable at high pH, high tolerance to bile acids, biofilm formation in adherence, and viable at high temperature and saline conditions.

In one aspect, the present invention provides a novel composition for aquaculture feed or a feed for aquarium fishes. According to the present invention, a novel composition for aquaculture feed comprises:

a combination of probiotic microorganisms;
protein source;
carbohydrate source;
fat source; and
optionally a herbal mixture.

The novel combination of the present invention comprises of three (3) isolated probiotic bacteria belonging to genus Bacillus and Lactobacillus.

According to the present invention, the combination of the probiotic microorganisms is selected from the species comprising Bacillus licheniformis, Bacillus amyloliquefaciens and Lactobacillus sp. which are deposited under Budapest treaty in International Depository, Microbial Type Culture Collection and Gene Bank (MTCC), India, on 24th August 2023, which has been accorded the Accession number MTCC 25670, MTCC 25671 and MTCC 25672 respectively.

The said probiotic composition comprises 107 to 1010 CFU/g of the combination bacteria.

In one aspect of the invention, protein source is one of more component/ingredient selected from the group comprising of soybean, mustard de oiled cake (DOC), De Oiled Rice Bran (DORB), black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.
In one embodiment of the present invention, the protein source is selected from the group comprising soybean, mustard DOC and DORB.

Wherein, as a protein source, soyabean may be used in a concentration of 20-25% w/w, Mustard DOC may be used in a concentration of 5-10% w/w and DORB may be used in a concentration of 22-25% w/w.

In an exemplified embodiment of the invention, the concentration of the protein in the novel aquaculture feed obtained by this invention is 25-40% w/w.

In one aspect of the invention, the carbohydrate source is one or more component/ingredient selected from the group comprising of wheat, barley, maize, rice, oats, potato, millet, sorghum, cassava and quinoa or combination thereof.

In one of the embodiments, the carbohydrate source is Maize used in a concentration of 20-30% w/w.

In an exemplified embodiment of the invention, the concentration of the carbohydrate in the novel aquaculture feed obtained by this invention is 30-50%.

In one aspect of the invention, the fat source is one of more component/ingredient selected from the group comprising of ground nut, palm, linseed, sunflower, algal, rapeseed, coconut, hempseed, olive and safflower or combination thereof.

In one embodiment, the fat source is ground nut used in a concentration of 1-5% w/w.

In an exemplified embodiment of the invention, the concentration of the fat in the novel aquaculture feed obtained by this invention is 10-20%.

In one aspect of the invention the composition may optionally also comprise a herbal mixture, wherein the herbal mixture is selected from black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.

In one optional embodiment of the invention, the herbal mixture is used in a concentration of 3-5% w/w.

The probiotic composition of the present invention can be blended with balanced nutritional supplement for aquaculture by maintaining crude protein (30%), crude fat (3%), carbohydrates (4%), and moisture (10%).

Said Endophytic strains of the present invention exhibit improved pH and bile tolerance, biofilm formation as well as viability at high temperature and high saline conditions. These novel isolates also form biofilm and have good adhesion property. The probiotic composition of the present invention can be produced and used in the form of solid powder or granulated form.

Considering the disadvantages faced by the existing aquaculture feed, this combination product is required since no single organism possesses desirable characteristics for probiotics namely acidic stability, intestinal stability, stability in presence of bile, and good adhesion property.

Said novel combination of bacteria and natural nutrients in a specific formulation acts as highly nutritive feed for future fish farming.

The present invention provides compositions comprising novel endophytic bacteria in combination with natural proteins, carbohydrate and fat derived from plant and microbial sources. The present composition also contains protein, carbohydrates and fat, which have been obtained only from plant sources, and not from any other animal sources and prepared by bio fermentation technique.

The multi-strain composition of the present invention has probiotic properties, such as sustaining bile duct juices, and stability in variable pH and enzymatic condition.

The said multi-strain composition of the present invention is stable in which the organisms are compatible with each other.

The said probiotic composition comprises the agent that enhances the probiotic activity after encapsulation with hydrocolloid(s).

The probiotic composition of the present invention can be produced and used in the form of solid powder or granulated form.

The probiotic composition remains functional in saline water (sea water) and ensures the fish activity, improved weight biomass and vigour.

The instant probiotic compositions comprising acid-tolerant property, i.e. the stability of probiotic bacteria in the presence of acids and bile salt, adhesion of bacteria in presence of bile salt tolerance.

Experimental results with the present composition in comparison with the existing products reveal that there is increase in weight gain of about 30% or more than that of commercial product and also there is an increase in fish activity when these three bacteria have been used in the composition, in comparison with the existing products.

Following are the key features of the present invention:

The novel bacterial strains remain viable and functional at higher temperature 40±2? and 10-15% salinity conditions, as well as normal water temperature in tropical and temperate conditions, as shown in table no. 1. The optimum temperature of fish to grow is in between 24-32? [https://www.globalseafood.org/advocate/water-temperature-in-aquaculture/].

The existing multi-strain probiotic composition comprising three isolated bacterial stains having compatibility with each other, no antagonistic effect is found which is illustrated in table no. 2.

Growth kinetics of novel probiotic bacteria are illustrated in table no. 3.

Acid tolerance properties of said novel probiotic bacteria are illustrated in table no. 4.

Said novel strains are resistant to wide range of acid bile salts 0.2 to 2.5% as shown in table no. 5.

These novels probiotic bacteria also form biofilm and have good adhesion property, as illustrated in table no. 6.

The sources of entire protein, carbohydrate and fat are from plant materials and organisms which exhibit higher nutritive value as mentioned in table no. 7B.

Comparisons to commercial products, feed composition of present invention show more promising results in terms of weight gain (above 30% higher than commercial products) and activity when these novel three bacteria of the present invention are used in the extruded fish feed, illustrated in table no. 8.

This probiotic composition screened a stable sporulating bacterial endophytes with less reproducible time and could promote the growth of host and enhance their resistance toward various environmental stresses, which is illustrated in table no 8.

Viability during storage of feed is calculated at a frequency of 2 months till 6 months, Colony forming unit (CFU/ml) at 24hrs is calculated. The viability test of feed is illustrated in table no. 9.

Growth and biomass of fishes improved (table no. 10)

Apparent digestibility Coefficient (ADC) and protein dispersibility index (PDI) is higher than the competitive products Improves absorption of essential nutrient and bio-available protein (Refer to table no. 13-14).

Filter load of aquarium is reduced (table no. 12) by reducing the feces and thereby positively impacting overall water quality (table no. 15-16).

Designed to support fresh and marine water fishes (both hard and soft type)

Probiotics reduce the faeces due to highly assimilative diet and thereby
positively impacting overall water quality

The unique vegan ingredients used in the present product make this product further robust for performance in varied environment conditions including saline and fresh-water ecosystems as well as recognize as organic product.

Advantages of the present invention
Organism is resistant to acid, bile salt (0.2 to 2.5%)
Organisms are capable of functioning at higher temperature 40°C ± 2°C and 10-15% salinity conditions as well as normal water temperatures in tropical and temperate conditions.
The probiotic composition wherein the agent that enhances the probiotic activity after encapsulation with hydrocolloid(s).
The probiotic composition can be produced and used in solid powder or granulated form.
The probiotic composition comprises 107 to 1010 CFU/g.
The probiotic composition remains functional in saline water (sea water).
The probiotic composition can be blended with balanced nutritional supplement for aquaculture by maintaining crude protein (25-30%), crude fat (10-20%), crude fibre (3-5%), moisture (8-10%).
The probiotic composition ensures the fish activity, improved weight biomass and vigor.
Probiotics reduce the faeces due to highly assimilative diet and thereby positively impacting overall water quality.

The present invention demonstrated in examples shows versatility, highlighting wide-ranging possibilities, applications and offering valuable insights.

Certain specific aspect and embodiment of the present invention will be explained in detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the invention in any manner.

EXAMPLES:

The novel endophytic bacteria used in the below examples are Bacillus licheniformis having accession no. MTCC 25670 and Bacillus amyloliquefaciens having accession no. MTCC 25671 and Lactobacillus species having accession no. MTCC 25672.

These bacteria cultures were isolated from natural fish environment in Haryana, India,
Example 1: Growth study of bacteria

Samples of bacteria were serially diluted to 10-2, 10-4, 10-6 and 10-8 and 100µl from each serial dilution was spread on MRS agar Media (Hi-media) supplemented with 10 & 15% of NaCl in the medium plates. Plates were incubated at 30±2°C and 40±2°C for 2 days. After every 24 hrs interval, colony morphotype on each plate was observed.

Higher temperature resistance at 40±2°C and salinity resistance ranging from 10-15%, of endophytic bacteria were illustrated in below Table 1.

Table 1: Growth in salinity and temperature
Bacterial Code Media Plant Part Gram Stain Growth with 10% NaCl Growth with 15% NaCl Growth at 30±2°C Growth at 40±2°C
TB0144 NB LEAF SURFACE +VE + + + +
TB0134 NB ROOT +VE + + + +
TB0125 NB ROOT +VE + + + +

Example 2: Compatibility test of probiotic bacteria

The Potential Probiotic organisms were selected based on antagonistic and synergetic study in dual culture methods (Geria and Caridi, 2014). The bacteria as shown in table 2 were found to be compatible with each other.

Table 2: Compatibility test of probiotic bacteria
Bacterial Code OD in the MRS broth after 18 hrs CFU/ml at 18 hrs Remarks
TB0144 1.7 8.60 Single colony
TB0134 1.8 8.48 Single colony
TB0125 1.9 8.30 Single colony
TB0144 +
TB0134 +
TB0125 2.0 9.95 Multiple colony

Example 3: Growth kinetics of probiotic bacteria

Growth curve studies were conducted on three isolated bacteria using spectrometric (OD) measurement and CFU count, at specific intervals of the time starting from 24 hrs, 30 hrs, 36 hrs to 48 hrs. These growth curves along with CFU counts generated at different phases of an isolate’s growth would be used to accurately conduct functional and biochemical characterization tests. This process was done to inoculate all the isolates in a particular test at the same phase of their life cycle as well as with the same cell count. This would provide an accurate and reliable comparison of functional activity among the isolated bacterial strains.

Growth Kinetics of probiotics bacteria have been illustrated in the below Table 3:

Table 3: Growth kinetics of probiotic bacteria
Bacterial Code CFU/ml at 24 hrs CFU/ml at 30 hrs CFU/ml at 36 hrs CFU/ml at 48 hrs
TB0144 10.70 10.85 10.62 9.28
TB0134 10.54 10.77 10.73 9.18
TB0125 10.48 10.6 10.52 8.92
TB0144 +
TB0134 +
TB0125 10.80 9.95 9.96 9.98

Media and culture conditions (Khalil et al., 2007)

Prior to use, strains were sub-cultured at 1% v/v in MRS broth. Bacteria were incubated at 37°C for 24-48h to obtain a concentration of approximately 107 cfu/ml.

Example 4: Acid tolerance test of probiotic bacteria

Overnight cultures of the test isolates were inoculated into MRS broth previously adjusted to pH values 2-3.5 and 7 with 1N NaOH or HCL. The cultures were incubated aerobically at 37°C for 4h and the turbidity was measured at 650 nm at 30 minutes interval after two hours. Control broth was maintained at a pH 7 (Adnan et al., 2017; Allameh et al., 2012) .

Results of acid tolerance test of probiotic bacteria have been shown in below Table 4:
Table 4: Acid tolerance test of probiotic bacteria
Bacterial Code OD at O.D at 600 nm pH 2 OD at O.D at 600 nm pH 4 OD at O.D at 600 nm pH 7
TB0144 0.2 0.9 1.4
TB0134 0.3 1.1 1.4
TB0125 0.3 0.8 1.0
TB0144 +
TB0134 +
TB0125 0.5 0.9 1.5

Example 5: Bile tolerance test of probiotic bacteria

Overnight cultures were inoculated into MRS broth containing 0.2 - 1.5% (w/v) of ox-gall and incubated aerobically at 37°C for 4 h. The turbidity of the culture was determined at 650nm and at 30 minutes interval after two hours. Control was maintained m MRS broth without bile (Allameh et al., 2012; Adnan et al., 2017).

Organisms in the present invention are resistant to acid bile salt of the range 0.2 to 2.5 %.

The Bile Tolerance test (Hyronimus et al., 2000; Allameh et al., 2012; Adnan et al., 2017) of probiotic bacteria is illustrated below in Table 5.

Table 5: Bile Tolerance test of probiotic bacteria
Bacterial Code OD at Bile (0.2%) OD at Bile (2.5%)
TB0144 1.4 1.4
TB0134 1.5 1.0
TB0125 1.2 1.1
TB0144 +
TB0134 +
TB0125 1.5 1.7

Example 6: Adhesion Properties of the probiotic bacteria

Plain adhesion properties of probiotic bacteria: 100 µL of a 10 mg/mL Solution of Ox Bile (Himedia) was immobilized in 96-well microtiter plates by incubation overnight at 30°C with MRS broth at pH 2, 4, 7 (Chu et al., 2014). Then freshly grown cultures were incubated for 24 hrs which were later used for adhesion assay. 100 µL of culture were added to each well. The plates were then incubated for 3 hrs 37° C. Each well was washed five times with 200 µL of sterile phosphate buffered saline (PBS) to remove unbound bacteria and then treated with 200 µL of a 0.05% (v/v) tween 20 solution to desorb the bound bacteria. Aliquots were taken from the solution for enumeration CFU/ml.

Table 6: Adhesion properties of probiotic bacteria
Bacterial Code pH2 pH 4 pH 7
No. Initial cell (CFU/ml) No. Final cell (CFU/ml) % Adhered No. Initial cell (CFU/ml) No. Final cell (CFU/ml) % Adhered No. Initial cell (CFU/ml) No. Final cell (CFU/ml) % Adhered
TB0144 6.10 5.4 88.5 8.01 7.03 87.8 10.70 9.10 85.0
TB0134 6.23 5.7 91.5 8.08 6.06 75.0 10.54 9.31 88.3
TB0125 6.28 4.11 65.4 8.02 7.01 87.4 10.48 9.22 88.0
TB0144 +
TB0134 +
TB0125 6.71 7.82 91.1 8.03 7.05 87.8 10.80 9.91 91.8

Example 7: Composition of the present invention and Nutrient analysis of formulation:

Below table provides composition for aquaculture feed of the present invention. The fish feed composition is prepared with the major ingredients along with their concentration as exemplified in table 7A.

Table 7A: Fish Feed Composition
Components Concentration
Soybean 20-25.0%
Maize 20-30.0%
DORB 22-25.0%
Mustard DOC 5-10%
Ground nut 5-5%
Probiotic mix Qs
Herbal mix 3-5%

The sources of entire protein, carbohydrate and fat are from plant materials.

Table 7B illustrates Nutrient analysis of present formulation prepared under this invention

Table 7B: Nutrient analysis of the final formulation prepared under this invention
Nutrient analysis
Energy, kcal/100g 441.08
Protein, g/100g 29.25
Carbohydrates, g/100g 46.91
Fat, g/100g 15.16
Crude fibre % 3-5
Crude Ash % 3-5
Saturated Fatty Acid, g/100g 1.15
Monounsaturated Fatty Acid, g/100g 9.19
Polyunsaturated Fatty Acid, g/100g 4.82
Moisture % 10

Example 8: In-vivo evaluation of probiotic bacteria

In-vivo evaluation of probiotic bacteria, involves following steps:
In-vivo Experimental Set-up
Acclimatization of Fishes
Experimental Design
In-vivo Evaluation of Probiotics

In-vivo experimental set-up
The laboratory was well ventilated with an ambient temperature (28-30 °C). Two identical aquariums of 70 L capacity were used for the experiment. All the aquariums were connected to an air pump for proper and continuous aeration during the study with a constant flow rate. The aquariums were maintained under natural light/dark photoperiod. On average 6 fish fingerlings were transferred to each aquarium. The experiments were conducted for 180 days.

Acclimatization of fishes
Fish were transferred in oxygenated polythene bags to the research site and were transferred to the aquarium. The number of fish was equally maintained (6 each) with initial average weight was recorded. The fish were fed with basal feed on daily basis and continuous aeration was provided. Fish were acclimatized for a period of 14 days. The water in aquarium was changed on every 3rd day to remove waste and fecal matter.

Experimental design
The experiment was designed to evaluate the efficacy of the selected probiotic bacteria in the in-vivo condition. The experiment was carried out in two groups, as control (I) recommended commercial feed, and with probiotic formulated feed (II). Sample fish were weighed and counted at regular intervals.

In-vivo evaluation of probiotics
Growth and Survival Indices Growth of fish and the effect of probiotics on the growth were monitored by measuring the length (milli-meter gradings) and weight of sample fish at regular intervals. Different growth parameters as mean weight, % weight gain, % survival and specific growth rate (SGR) were calculated by using the following formula (Hamdan et al., 2016; Liu et al., 2017).

Mean weight, weight gain, survival percentage and specific growth percentage have been calculated based on the following formulae:

Mean weight=(Total weight)/(Total no.of fishes)
Weight gain (%)=(Final mean weight-Initial mean weight)/(initial mean weight) x100
Survival (%)=(No.of fish survived)/(No.of fish stocked) x100
Specific growth rate=(In (Wf)-In (Wi))/(No.of fish days) x100

Below table 8 illustrates In-vivo Evaluation of fishes fed with probiotic feed.

Table 8: In-vivo evaluation of fishes fed with probiotic feed
Parameters Control (commercial fish feed) Treated (present product) Differences
Mean weight 11.2 g 13.3g 2.1 g
Weight gain (%) 60% 90.5% 30.5%
Survival (%) 83.33% 100% 16.6%
Specific growth rate (%) 84% 105% 21%

Example 9: Viability during storage of probiotic feed

100 mg of probiotic formulation were inoculated into MRS broth and incubated at 30°C for 24 h. Subsequently serial dilutions were made to enumerate the colonies using the serial plate technique at two months interval. Result shows 97.5% viability during storage at room temperature.

Table 9 illustrates the storage of probiotic feed for frequency of every 2 months up to 6 months.
Table 9: Viability during storage of probiotic feed
Time Log10CFU/ml at 24 hrs
Zero days 10.66
2 Months 10.51
4 Months 10.40
6 Months 9.80

Example 10: In-vivo Evaluation of Probiotics against commercial and vegan feed and comparison of weight among several fish species:

The laboratory was well ventilated with an ambient temperature (28-30 °C). Six identical aquariums of 70 L capacity were used for the experiment. All the aquariums were connected to an air pump for proper and continuous aeration during the study with a constant flow rate. Six identical filters were also installed in the aquariums. The aquariums were maintained under natural light/dark photoperiod. The experiment was conducted for 150 days.

Initially, the fish (18 koi fish, 18 goldfish, 6 parrot fish and 6 cichlid fish) were transferred in oxygenated polythene bags to the research site and were transferred to the aquarium. The initial average weight was recorded. The fish fed at the rate of 2% of their body weight on daily basis and continuous aeration was provided. Fish were acclimatized for a period of 14 days. The water in aquarium was changed on every 15th day to remove waste and faecal matter.

The experiment was designed to investigate the impact of different feeding regimes, including the use of probiotic bacteria, on the growth performance of various fish species over a 5-months period. The treatments followed was:

Treatment 1 Treatment 2 Treatment 3
Soft fishes
Aquarium 1: Stocked with 6 koi and 6 goldfish, receiving vegan feed supplemented with probiotic bacteria. Aquarium 2: Stocked with 6 koi and 6 goldfish, receiving vegan feed without probiotic supplementation. Aquarium 3: Stocked with 6 koi and 6 goldfish, receiving commercial feed only.
Hard fishes
Aquarium 4: Stocked with 2 parrot fish and 2 cichlid fish, receiving vegan feed supplemented with probiotic bacteria. Aquarium 5: Stocked with 2 parrot fish and 2 cichlid fish, receiving vegan feed
without probiotic supplementation. Aquarium 6: Stocked with 2 parrot fish and 2 cichlid fish, receiving commercial
feed only.

The feeding plans were implemented consistently throughout the experiment, with feed provided daily according to the specified regimen. Fish weights and lengths were measured at the beginning and end of the 5-months period to assess growth performance.

Daily Weight Gain (DWG):

Daily weight gain was calculated as per procedure of Wang (2010). DWG was studied to know the effect of formulated functional feed on growth performance during 150 days of feed experiment study.
DWG (g/day) = W2 - W1/t, where W1 was the initial average weight of the fish, W2 was the average weight of the fish at the termination of treatment and t is the total number of days of the experiment.

Relative Growth Rate (RGR):
RGR was studied as per the procedure mentioned in Wang (2010)
RGR= final wt-intital wt/initial wt x100

Specific Growth Rate (SGR):
SGR was studied as per the procedure mentioned by Dharmaraj and Dhevendaran (2010). SGR is defined as the increase in cell mass per unit time.
SGR=1ogw2-logw1/tX100
Where W1 = Initial Weight (g), W2 = Final Weight (g), t = Total no. of experiment days

Table 10: In-vivo Evaluation for weight gain
No. of fish Mean weight
Relative Growth Rate (RGR) (%) Daily weight gain Total weight gain

Initial Final
Goldfish
Treatment 1: Aquarium 1: Vegan feed + Probiotic 6.0 2.2±0.1 9.2±0.32 314.3 0.05 41.81
Treatment 2: Aquarium 2: Vegan feed 6.0 2.1±0.08 9.2±0.3 342.1 0.05 42.76
Treatment 3: Aquarium 3: Commercial feed 6.0 2.2±0.11 8.2±0.03 277.8 0.04 36.12
Koi fish
Treatment 1: Aquarium 1: Vegan feed + Probiotic 6.0 17.3±0.8 25.0±0.14 44.4 0.05 46.22
Treatment 2: Aquarium 2: Vegan feed 6.0 17.4±0.7 21.8±0.29 25.1 0.03 26.16
Treatment 3: Aquarium 3: Commercial feed 6.0 17.2±0.65 19.9±0.31 15.7 0.02 16.17
Parot fish
Treatment 1: Aquarium 4: Vegan feed + Probiotic 2.0 21.0±1 51.8±0.58 146.6 0.21 61.56
Treatment 2: Aquarium 5: Vegan feed 2.0 21.5±0.5 45.2±1.05 110.0 0.16 47.30
Treatment 3: Aquarium 6: Commercial feed 2.0 22.0±0.0 39.6±0 80.0 0.12 35.20
Cichlid fish
Treatment 1: Aquarium 4 Vegan feed + Probiotic 2.0 8.5±0.5 15.5±0.5 82.4 0.05 14.00
Treatment 2: Aquarium 5: Vegan feed 2.0 8.5±0.5 12.5±0.5 47.1 0.03 8.00
Treatment 3: Aquarium 6: Commercial feed 2.0 8.0±0.0 10.5±0.5 31.3 0.02 5.00

Table 11: In-vivo Evaluation for length gain
No. of fish Mean length
Relative length Rate (RLR) (%)
Initial Final
Goldfish
Treatment 1: Aquarium 1: Vegan feed + Probiotic 6.0 4.83±0.1 8.65±0.2 78.88
Treatment 2: Aquarium 2: Vegan feed 6.0 4.90±0.16 6.62±0.26 35.10
Treatment 3: Aquarium 3: Commercial feed 6.0 4.87±0.19 5.84±0.22 20.00
Koi fish
Treatment 1: Aquarium 1: Vegan feed + Probiotic 6.0 10.88±0.27 19.46±0.48 78.80
Treatment 2: Aquarium 2: Vegan feed 6.0 10.68±0.15 14.53±0.35 36.09
Treatment 3: Aquarium 3: Commercial feed 6.0 10.72±0.14 13.40±0.18 25.00
Parot fish
Treatment 1: Aquarium 4: Vegan feed + Probiotic 2.0 8.90±0.3 21.96±0.06 146.70
Treatment 2: Aquarium 5: Vegan feed 2.0 9.10±0.3 19.11±0.63 110.00
Treatment 3: Aquarium 6: Commercial feed 2.0 9.20±0.4 16.56±0.72 80.00
Cichlid fish
Treatment 1: Aquarium 4 Vegan feed + Probiotic 2.0 8.95±0.15 20.57±0.55 129.83
Treatment 2: Aquarium 5: Vegan feed 2.0 9.25±0.05 19.43±0.11 110.00
Treatment 3: Aquarium 6: Commercial feed 2.0 8.80±0.1 15.84±0.18 80.00

Results:
The data from the experiment clearly indicated that probiotic supplementation in fish feed significantly enhanced both the growth and length of the fish. Fish fed with vegan feed supplemented with probiotics exhibited higher weight gains and relative growth rates (RGR) compared to those fed with vegan feed alone or commercial feed. For example, goldfish in the probiotic-supplemented group showed a final weight of 9.2 grams with an RGR of 314.3%, while those in the vegan feed and commercial feed groups had RGRs of 342.1% and 277.8%, respectively. Similarly, koi fish on the probiotic-supplemented diet achieved a final weight of 25.0 grams with an RGR of 44.4%, compared to 21.8 grams (RGR of 25.1%) and 19.9 grams (RGR of 15.7%) for the vegan feed and commercial feed groups, respectively.

The improved growth performance can be attributed to the enhanced nutrient absorption and digestion facilitated by probiotics. Probiotics are known to promote a healthy gut microbiome, which enhances the digestive efficiency of fish, leading to better utilization of the nutrients present in the feed. This improved nutrient uptake translates into more efficient growth, as evidenced by the higher daily weight gains (DWG) observed in the probiotic-treated groups.

In terms of length gain, the data also showed significant improvements for fish fed with probiotic-supplemented diets. For instance, goldfish in the probiotic group grew from an initial length of 4.83 cm to a final length of 8.65 cm, achieving an RGR in length of 78.88%, compared to 35.10% and 20.00% in the vegan feed and commercial feed groups, respectively. Koi fish in the probiotic group grew from 10.88 cm to 19.46 cm, with an RGR of 78.80%, outperforming the vegan feed group (36.09%) and the commercial feed group (25.00%).
The significant increases in both weight and length highlight the overall effectiveness of probiotics in enhancing the growth metrics of fish. By improving the efficiency of nutrient digestion and assimilation, probiotics not only support better weight gain but also promote more substantial skeletal growth, resulting in increased length. These findings underscore the dual benefits of probiotics in aquaculture: enhanced growth performance and improved environmental sustainability through reduced waste production. Consequently, the inclusion of probiotics in fish feed is a promising strategy for optimizing fish health and growth while maintaining a healthier and more sustainable aquatic ecosystem.

Example 11: Evaluation of filer load

The filter load in aquaculture systems is a critical parameter that indicates the efficiency of feed utilization and digestion. It represents the combination of uneaten feed and fecal matter accumulated in the filtration system. High filter loads suggest poor feed efficiency and increased waste, which can lead to deteriorating water quality and a stressed aquatic environment. Evaluating filter load is essential for understanding the impact of different feed formulations, including probiotic supplementation, on feed efficiency and environmental sustainability. Probiotics in fish feed can enhance nutrient absorption and digestion, reducing waste output and thereby improving water quality and ecosystem health.

At the beginning of the experiment and at monthly intervals, filter loads were collected from each aquarium's filtration system. The filter loads were carefully removed to ensure all accumulated debris and organic matter were collected. This regular collection was essential for tracking changes in waste production over time.

The collected filter loads were washed thoroughly to remove any soluble substances and contaminants. The cleaned filter loads were then dried at a controlled temperature of approximately 70°C to remove all moisture. This drying process ensured accurate weight measurements by eliminating any water content from the samples.

The dried filter loads were weighed using a precision scale to obtain accurate measurements of the accumulated waste. The weights were recorded for each aquarium at each collection interval, allowing for detailed monitoring of changes in filter load over the course of the experiment. This data provided insights into the efficiency of the different feeding regimes and the impact of probiotics on feed utilization and waste production.

Table 12: Amount of Filter load generated during the experiment
1st months 2nd months 3rd
months 4th months 5th months
Soft fish (Koi and Gold)
Treatment 1: Aquarium 1 (Vegan feed + Probiotic) in Grams 9.5±0.36 11±0.18 10.5±0.18 12±0.26 12±0.14
Treatment 2: Aquarium 2 (Vegan feed) in Grams 13±0.2 13.4±0.2 16±0.3 17±0.1 18±0.3
Treatment 3: Aquarium 3 (Commercial feed) in Grams 17±0.3 18.5±0.27 20±0.38 21±0.29 23.4±0.35
% of less amount in comparison to aquarium 3 and 1 44.12 40.54 47.50 42.86 48.72
% of less amount in comparison to aquarium 2 and 1 9.07 12.58 8.42 9.33 11.08
Hard fish (Parot and Cichlid)
Treatment 1: Aquarium 4 (Vegan feed + Probiotic) in Grams 5±0.06 5.6±0.22 6.7±0.1 7.1±0.18 7.7±0.35
Treatment 2: Aquarium 5 (Vegan feed) in Grams 7±0.37 7.5±0.38 7.9±0.54 8.3±0.76 9.6±0.11
Treatment 3: Aquarium 6 (Commercial feed) in Grams 8±0.41 8.8±0.63 9.4±0.21 10±0.23 11±0.35
% of less amount in comparison to aquarium 4 and 6 37.50 36.36 28.72 29.00 30.00
% of less amount in comparison to aquarium 5 and 6 28.57 25.33 15.19 14.46 19.79

Soft Fish (Goldfish and Koi): The filter load for soft fish fed with vegan feed with probiotics was consistently lower across all months compared to those fed with vegan feed alone and commercial feed. For example, in the 5th month, the filter load for fish fed with vegan feed and probiotics was 12.0 g, compared to 18.0 g for vegan feed and 23.4 g for commercial feed. This indicates that probiotic supplementation reduced waste production significantly.

Hard Fish (Parrot and Cichlid): Similarly, hard fish fed with vegan feed with probiotics showed lower filter loads. In the 5th month, the filter load for fish fed with vegan feed with probiotics was 7.7 g, compared to 9.6 g for vegan feed and 11.0 g for commercial feed. This suggests improved feed utilization and digestion with probiotics.

Therefore, reduced filter load means less waste accumulation, leading to better water quality in the aquariums. Cleaner water is essential for the health of the fish and the overall ecosystem.

Example 12: Apparent digestibility Coefficient (ADC)

The Apparent Digestibility Coefficient (ADC) is a crucial metric in aquaculture nutrition studies as it measures the efficiency with which fish digest and absorb nutrients from their feed. The higher ADC indicates that the feed is more digestible, meaning that the fish can utilize a greater proportion of the nutrients provided, which can lead to better growth performance and reduced waste. Understanding the digestibility of different feed formulations, including those with probiotic supplements, helps in optimizing feed efficiency, reducing feed costs, and minimizing environmental impacts.

The different diets (vegan feed, vegan feed with probiotics, commercial feed) were analyzed to get the percentage of nutrients, mainly protein which was 40%. The fish were fed according to the experimental design with ensured consistent feeding times and amounts to maintain uniform conditions.

All fecal samples were collected from each aquarium to ensure an adequate sample size for analysis using a siphon and net to avoid contamination with uneaten feed.
The fecal samples were dried at a controlled temperature (e.g., 70°C) to remove moisture content, ensuring accurate weight measurements.

The nutrient content (protein) was analyzed in both the diet and the fecal samples with the standard protocol of protein estimation (Lowrey).

The ADC was calculated to determine the digestibility of the feeds Cho, et al. (1982). The ADC was calculated using the formula ADC = 1 - [(F/D) x (Di/Fi)],
D = Percentage of nutrient in the diet.
F = Percentage of nutrient in the feces.
Di = Percentage of the digestion indicator in the diet.
Fi = Percentage of the digestion indicator in the feces.

Table 13: Apparent digestibility Coefficient (ADC)
commercial feed given (g) feces collected (g) protein of the feces protein of feeds (29.25%) ADC (%)
Soft fish (Koi and Gold)
Treatment 1: Aquarium 1 (Vegan feed + Probiotic) in Grams 210 12 0.48 359.78
0.999

Treatment 2: Aquarium 2 (Vegan feed) in Grams 210 18 1.08 359.78
0.997

Treatment 3: Aquarium 3 (Commercial feed) in Grams 210 23.4 3.51 359.78
0.990

Hard fish (Parot and Cichlid)
Treatment 1: Aquarium 4 (Vegan feed + Probiotic) in Grams 615 7.7 0.308 179.89
0.998

Treatment 2: Aquarium 5 (Vegan feed) in Grams 615 9.6 0.576 179.89
0.997

Treatment 3: Aquarium 6 (Commercial feed) in Grams 615 11 1.98 179.89
0.989

Soft Fish (Goldfish and Koi): The ADC values for soft fish fed with vegan feed with probiotics (0.999) were higher than those fed with vegan feed alone (0.998) and commercial feed (0.993). This indicates that the probiotic-supplemented diet was more digestible, allowing the fish to absorb more nutrients efficiently.

Hard Fish (Parrot and Cichlid): Similarly, the hard fish showed higher ADC values when fed with vegan feed + probiotics (0.999) compared to vegan feed alone (0.998) and commercial feed (0.992). The higher ADC in probiotic-supplemented diets suggests enhanced nutrient absorption.

Example 13: Protein dispersibility index of fish feed (PDI)

The Protein Dispersibility Index (PDI) is a critical metric used to measure the solubility of protein in fish feed under controlled conditions. It is particularly important in assessing the degree of protein denaturation, which can significantly affect the nutritional quality and digestibility of the feed. Higher PDI values generally indicate better protein quality and solubility, leading to improved nutrient absorption and growth performance in fish. Probiotic supplementation in fish feed can enhance PDI by promoting better protein breakdown and solubility, thus maximizing the nutritional benefits of the feed.

Sample Preparation
To ensure the homogeneity of the fish feed samples, the initial step involved grinding or crushing the samples into fine particles. This homogeneity was crucial for obtaining consistent and reliable results. Precisely 2.5 grams of each fish feed sample were weighed using an analytical balance for accuracy. The weighed samples were then transferred into labeled centrifuge tubes to prevent any mix-up during subsequent steps.

Extraction
To extract the soluble proteins from the fish feed samples, 50 mL of distilled water was added to each centrifuge tube containing the ground feed. The pH of the solution was adjusted to a standardized value, typically pH 7.0, using a pH meter to ensure optimal protein solubility. The mixture was then homogenized using a blender or homogenizer for a predetermined time, such as 1 minute, to ensure complete dispersion of the feed particles in the water. This step was critical to ensure that the proteins were fully extracted into the solution.

Centrifugation
The homogenized samples were centrifuged at a predetermined speed, such as 3000 rpm, for a specified duration, like 10 minutes. This process separated the soluble protein fraction from the insoluble particles. The centrifugation step was essential to clarify the extract by sedimenting the non-soluble components, allowing the soluble proteins to remain in the supernatant.

Protein Determination
After centrifugation, the supernatant, which contained the soluble protein fraction, was carefully transferred from each centrifuge tube to a clean tube, ensuring no sediment was included. The total protein content of the supernatant was then measured using a standard protein assay method, such as the Bradford assay or the Lowry assay. The absorbance or concentration values obtained from these assays were recorded and compared against a protein standard curve to determine the exact protein content. This final step was crucial for quantifying the soluble protein, which was used to calculate the Protein Dispersibility Index (PDI) of the fish feed samples.

Sample Preparation
To ensure the homogeneity of the fish feed samples, the initial step involved grinding or crushing the samples into fine particles. This homogeneity was crucial for obtaining consistent and reliable results. Precisely 2.5 grams of each fish feed sample were weighed using an analytical balance for accuracy. The weighed samples were then transferred into labeled centrifuge tubes to prevent any mix-up during subsequent steps.

Extraction
To extract the soluble proteins from the fish feed samples, 50 mL of distilled water was added to each centrifuge tube containing the ground feed. The pH of the solution was adjusted to a standardized value, typically pH 7.0, using a pH meter to ensure optimal protein solubility. The mixture was then homogenized using a blender or homogenizer for a predetermined time, such as 1 minute, to ensure complete dispersion of the feed particles in the water. This step was critical to ensure that the proteins were fully extracted into the solution.

Centrifugation
The homogenized samples were centrifuged at a predetermined speed, such as 3000 rpm, for a specified duration, like 10 minutes. This process separated the soluble protein fraction from the insoluble particles. The centrifugation step was essential to clarify the extract by sedimenting the non-soluble components, allowing the soluble proteins to remain in the supernatant.

Protein Determination
After centrifugation, the supernatant, which contained the soluble protein fraction, was carefully transferred from each centrifuge tube to a clean tube, ensuring no sediment was included. The total protein content of the supernatant was then measured using a standard protein assay method, such as the Lowry assay. The absorbance or concentration values obtained from these assays were recorded and compared against a protein standard curve to determine the exact protein content. This final step was crucial for quantifying the soluble protein, which was used to calculate the Protein Dispersibility Index (PDI) of the fish feed samples.

Calculation:
PDI is calculated using the formula: PDI (%): (soluble protein/total protein) x 100
This provides a percentage value that indicates the proportion of protein in the feed that is soluble under the test conditions.

Table 14: Protein dispersibility index of fish feed (PDI)
PDI%
Soft fish (Koi and Gold)
Treatment 1: Aquarium 1 (Vegan feed + Probiotic) in Grams 0.011±0.2
Treatment 2: Aquarium 2 (Vegan feed) in Grams 0.0088±0.4
Treatment 3: Aquarium 3 (Commercial feed) in Grams 0.0038±0.2
Hard fish (Parot and Cichlid)
Treatment 1: Aquarium 4 (Vegan feed + Probiotic) in Grams 0.0091±0.3

Treatment 2: Aquarium 5 (Vegan feed) in Grams 0.0075±0.4
Treatment 3: Aquarium 6 (Commercial feed) in Grams 0.0033±0.1

Soft Fish (Goldfish and Koi): The PDI values for soft fish fed with vegan feed with probiotics (0.011) were higher than those fed with vegan feed alone (0.0088) and commercial feed (0.0038). This indicates that the probiotic-supplemented diet resulted in better protein solubility and, therefore, higher nutritional quality.

Hard Fish (Parrot and Cichlid): Similarly, hard fish showed higher PDI values when fed with vegan feed + probiotics (0.0091) compared to vegan feed alone (0.0075) and commercial feed (0.0033). The higher PDI in probiotic-supplemented diets suggests improved protein quality and potential digestibility.

Example 14: Chemical analysis of sludges and water quality

Chemical analysis of sludges and water quality in aquaculture systems is essential for understanding the environmental impact of different feeding regimes. The composition of sludge and water quality parameters such as ammonia, nitrate, nitrite, carbonate, phosphate, Biological Oxygen Demand (BOD), and Chemical Oxygen Demand (COD) provide critical information on the effectiveness of feed utilization and the efficiency of waste management. Probiotic supplementation in fish feed can significantly reduce harmful waste compounds, leading to improved water quality and a healthier aquatic ecosystem. This not only benefits the fish by providing a cleaner environment but also promotes sustainable aquaculture practices.

Sludge Collection and Preparation
Sludge samples were collected from the filter loads and bottom residues of each aquarium after every month. The collected sludge samples were stored in labeled containers to avoid contamination and ensure accurate identification. The collected samples of five months were dried.

Sludge Chemical Analysis
Key chemical parameters in the sludge, including ammonia, nitrate, nitrite, carbonate, phosphate, BOD, and COD, were measured using standardized methods such as colorimetric assays, titration, and spectrophotometry. The analysis were done by their party. The concentrations of these parameters were recorded in parts per million (ppm) to allow for precise comparisons across different treatments.

Water Quality Assessment
Water quality parameters were evaluated after three months using commercially available test kits. These kits measured parameters such as pH, nitrate, nitrite, carbonate, chlorine, and alkalinity. Water samples were collected from each aquarium at consistent depths and times to ensure reliability. The samples were analyzed according to the test kit instructions, and the values obtained for each parameter were recorded.

Table 15: Chemical parameters of sludges in ppm after 5 months
Ammonia Nitrate Nitrite Carbonate Phosphate BOD COD
Soft fish (Koi and Gold)
Treatment 1: Aquarium 1 (Vegan feed + Probiotic) in Grams 299 96 14 1148 280 1150 9600
Treatment 2: Aquarium 2 (Vegan feed) in Grams 461 120 21 1340 300 1770 14800
Treatment 3: Aquarium 3 (Commercial feed) in Grams 911 224 42 1720 320 3504 29200
Hard fish (Parot and Cichlid)
Treatment 1: Aquarium 4 (Vegan feed + Probiotic) in Grams 336 120 16 1580 200 1680 14000
Treatment 2: Aquarium 5 (Vegan feed) in Grams 673 136 20 1530 220 2592 23420
Treatment 3: Aquarium 6 (Commercial feed) in Grams 730 280 34 1610 320 2810 21600

Table 16: Chemical parameters of sludges in ppm after one months
pH Nitrate Nitrite Carbonate Chorine Alkalinity
Soft fish (Koi and Gold)
Treatment 1: Aquarium 1 (Vegan feed + Probiotic) in Grams 8.4 40 0 75 0 180
Treatment 2: Aquarium 2 (Vegan feed) in Grams 8.4 90 1 75 0 300
Treatment 3: Aquarium 3 (Commercial feed) in Grams 7.8 160 10 300 0 300
Hard fish (Parot and Cichlid)
Treatment 1: Aquarium 4 (Vegan feed + Probiotic) in Grams 7.2 20 0 25 0.5 80
Treatment 2: Aquarium 5 (Vegan feed) in Grams 7.2 20 0 75 0.5 120
Treatment 3: Aquarium 6 (Commercial feed) in Grams 7.8 160 0 150 0.5 300

The data clearly demonstrated that probiotic supplementation in fish feed significantly reduced the concentrations of various harmful compounds in both sludge and water. In aquariums where fish were fed with vegan feed supplemented with probiotics, the levels of ammonia, nitrate, nitrite, carbonate, phosphate, BOD, and COD in the sludge were markedly lower compared to those in the vegan feed and commercial feed treatments. For instance, ammonia concentrations in the sludge from the probiotic treatment were reduced to 299 ppm, compared to 461 ppm in the vegan feed and 911 ppm in the commercial feed treatments. This substantial reduction suggests that probiotics enhance the efficiency of nitrogen utilization in fish, leading to less nitrogenous waste excreted into the environment.
Similarly, the probiotic-supplemented diets resulted in lower nitrate and nitrite levels, which were crucial for maintaining water quality. Nitrate levels in the water from the probiotic treatment were only 40 ppm for soft fish, in stark contrast to 90 ppm in the vegan feed and 160 ppm in the commercial feed treatments. Reduced nitrate and nitrite levels indicate that probiotics improve the nitrification process, facilitating better conversion of ammonia to less harmful compounds and thereby maintaining a healthier aquatic environment.

The data also showed a significant decrease in carbonate and phosphate levels in the sludge, which can be attributed to the enhanced breakdown and assimilation of these compounds due to probiotic activity. Probiotics likely facilitate the more efficient utilization of phosphorus, reducing its excretion as waste. This was evident in the lower phosphate concentrations (280 ppm in the probiotic treatment versus 300 ppm and 320 ppm in the vegan feed and commercial feed, respectively).

The reductions in BOD and COD were particularly noteworthy, with the probiotic treatment showing substantially lower values (e.g., 9600 ppm COD in the probiotic treatment versus 14800 ppm and 29200 ppm in the vegan feed and commercial feed treatments, respectively). Lower BOD and COD levels indicate a significant decrease in organic pollutants and overall waste load in the water, suggesting that probiotics improve the biodegradability of organic matter. This results in less oxygen demand for the decomposition of organic waste, thus maintaining higher dissolved oxygen levels, which is crucial for fish health and overall water quality.

In conclusion, the data underscore the effectiveness of probiotic supplementation in fish feed in reducing harmful waste compounds and maintaining superior water quality. Probiotics enhance nutrient absorption and digestion, reduce waste production, and facilitate better breakdown of organic matter, all of which contribute to a healthier and more sustainable aquatic ecosystem. This not only improves the growth and health of the fish but also supports more environmentally friendly aquaculture practices.

Example 15: Evaluation of Biochemical Properties

The biochemical tests conducted on the bacterial strains TB0144, TB0134, and TB0125 revealed distinct metabolic capabilities and characteristics for each strain with the help of Himedia test kit as per Bergey's manual of Systematic Bacteriology (Holt et al., 1989). These tests help in understanding the potential applications and functionalities of these strains, particularly in their roles as probiotics.

The results highlight that all three strains possess the ability to ferment a wide range of carbohydrates, which is advantageous for their role as probiotics. The positive results for glucose, lactose, arabinose, and other sugars suggest that these strains can thrive on various substrates available in fish feed, enhancing their probiotic efficacy.

TB0144 and TB0125 share similarities in their metabolic profiles, particularly in their inability to utilize citrate consistently and their mixed acid fermentation capabilities (methyl red positive). In contrast, TB0134 exhibits unique features such as urease and phenylalanine deamination activities, which might offer additional benefits in specific aquaculture applications.

Table 17: Biochemical properties of the probiotics

TEST TB0144 TB0134 TB0125 TEST TB0144 TB0134 TB0125
Indole - - - Citrate Utilization + + -
Methyl red + - + Lysine Utilization - - -
Voges Proskaure’s - - - Ornithine utilization - - -
Citrate utilization - - - Urase - + -
Glucose + + + Phenylalanine Deamination - + -
Adonitol + + + Nitrate Production + + +
Arabinose + + + H2S production - - -
Lactose + + + Glucose + + +
Sorbitol + + + Adonitol - - +
Mannitol + + + Lactose + + +
Rhamnose + + + Arabinose - + +
Sucrose + + + Sorbitol - + -

Conclusion:
The novel fish feed composition comprising isolated novel probiotic bacterial strains shows significant improvements in fish growth, health, and environmental sustainability. The novel isolates form biofilm that has good adhesion property and survives in extruded feeds. The probiotic strains exhibit high tolerance to salinity (10 to 15%), high temperature (40±2°C), pH, and bile (0.2 to 2.5%), ensuring their functionality in diverse aquaculture conditions. This probiotic composition is screened as sporulating bacterial endophytes with less reproducible time and promotes the growth of aquarium fish. The plant-based feed formulation enhances nutrient absorption, reduces waste production, and improves water quality, making it a promising solution for sustainable aquaculture practices. Unique vegan ingredients used makes this product further robust for performance in varied environment conditions including saline and freshwater ecosystems.

References:
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,CLAIMS:
1. A composition for aquaculture feed, comprising:
i. a combination of probiotic microorganisms;
ii. a protein source;
iii. a carbohydrate source;
iv. a fat source; and
v. optionally an herbal mixture.

2. The composition as claimed in claim 1, wherein the probiotic microorganisms are selected from Bacillus licheniformis, Bacillus amyloliquefaciens and Lactobacillus sp.

3. The composition as claimed in claim 1, wherein the concentration of probiotic microorganisms is 107 to 1010 CFU/g.

4. The composition as claimed in claim 1, wherein the protein source is selected from the group comprising of soybean, mustard de oiled cake (DOC), de oiled rice bran (DORB), black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.

5. The composition as claimed in claim 4, wherein protein source is selected from the group comprising of soybean, mustard DOC and DORB.

6. The composition as claimed in claim 5, wherein the concentration of soyabean is 20-25% w/w, Mustard DOC is 5-10% w/w and DORB is 22-25% w/w

7. The composition as claimed in claim 1, wherein the carbohydrate source is selected from the group comprising of wheat, barley, maize, rice, oats, potato, millet, sorghum, cassava and quinoa or combination thereof.

8. The composition as claimed in claim 7, wherein the concentration of carbohydrate source is 20-30% w/w.

9. The composition as claimed in claim 7, wherein the carbohydrate source is maize.

10. The composition as claimed in claim 1, wherein the fat source is selected from the group comprising of ground nut, palm, linseed, sunflower, algal, rapeseed, coconut, hempseed, olive and safflower or combination thereof.

11. The composition as claimed in claim 10, wherein the concentration of fat source is 1-5% w/w.

12. The composition as claimed in claim 10, wherein the fat source is ground nut.

13. The composition as claimed in claim 1, wherein the herbal mixture is selected from the group comprising of black cumin seeds, sunflower, cottonseed, canola, lupin, rapeseed, guar, almond and moringa oleifera or combination thereof.

14. The composition as claimed in claim 13, wherein the concentration of herbal mixture is 3-5% w/w.

15. The composition as claimed in claim 1 wherein the composition is formulated into aquaculture feed formulation selected from solid powder, granules and biofilm.

16. The composition as claimed in claim 15, wherein the aquaculture feed formulation obtained from the claimed composition comprises protein 25-40%.

17. The composition as claimed in claim 15, wherein the aquaculture feed formulation obtained from the claimed composition comprises carbohydrate 30-50%.

18. The composition as claimed in claim 15, wherein the aquaculture feed formulation obtained from the claimed composition comprises fat 10-20%.

19. The composition as claimed in claim 15 wherein the aquaculture feed formulation is free to any ingredients obtained from animal source.

20. The composition as claimed in claim 15, wherein the aquaculture feed formulation is stable at high pH, has high tolerance to bile acids and biofilm formation in adherence and is viable at high temperature and in saline conditions.