CN112638168A - Method for feeding fish - Google Patents

Abstract

The present invention provides a method of feeding a low-feeding fish with a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria. Also described are methods of improving the appeal or palatability of a feed product to a subfamily fish, stimulating an increase in food intake, increasing the growth rate or food intake of a subfamily fish, and the use of a biomass comprising a mixed microbial population comprising microalgae and bacteria as a feed attractant or a feeding stimulant.

Description

Method for feeding fish

Priority of australian provisional application No. 2018902685 (the entire contents of which are hereby incorporated herein by reference in their entirety) entitled "feeding method" filed in 2018, 7, 25/7.

Technical Field

In general, the present invention relates to the use of a feed product comprising dried mixed microbial biomass as food for fish (e.g., low-grade feeding fish). The invention also relates to the use of the dried mixed microbial biomass as a feed attractant or feed stimulant for fish.

Background

Aquaculture worldwide is expanding to produce aquatic animals such as fish, mollusks and crustaceans as food to meet the increasing demand of growing populations. There is also a need to alleviate the pressure on populations of high-feeding fish, such as salmon, barramundi, tuna and cod, which has led to efforts to include higher proportions of low-feeding fish species in the human diet. Therefore, there is an increasing demand for lower-feeding fish (e.g., tilapia, catfish, and carps), and the world-wide aquaculture production thereof is rapidly expanding.

In particular, fish of the Tilapia family of the Pacifidae (Tilapia cichlid), such as the mouth-hatched non-crucian genus (Oreochromyis), the scaphoid non-crucian genus (Sarotherodon) and the Tilapia genus (Tilapia) are examples of commercially important low-feeding layered fishes. These fish are the subject of major aquaculture efforts on a world scale, particularly in tropical waters. Tilapia has become one of the most important fishes following carp and salmon. In 2015, tilapia family fish (tilapiine) had a global production of 567 million tons worth 89 billion dollars (FAO, 2018). They are among the easiest and most profitable fish for farms, as they tolerate high stocking densities and grow rapidly. The tilapia family of fish is omnivorous and may feed on a vegetable or grain based diet; higher feeding fish such as salmon, however, require high protein feed content to provide effective growth. The GIFT strain (Genetically Improved Farmed Tilapia) is a selectively bred nile Tilapia (Oreochromis niloticus) that accounts for 80% of the total Tilapia fingerling yield in china in 2010, 75% in thailand, and 40% in the philippines (Sukmanomon et al, 2012).

There is a need for a cost-effective sustainable formula feed to support increasing world-wide production of low-food fish. For example, fish meal and fish oil are currently included in commercial tilapia family fish diets in amounts of 0 to 20% and 0 to 10%, respectively. Fish meal is expensive and the supply may be unreliable. The replacement of fish meal and fish oil with more sustainable and cheaper alternatives in the diet for fish of the tilapia family has been the focus of research. Several studies have evaluated the replacement of fish meal with less expensive, locally available plant and animal meal in tilapia fish diets (El-Saidy and Gaber, 2002; Herath et al, 2016; Koch et al, 2016; Hg and Romano, 2013; Shiau et al, 1989). The replacement of fish meal with soy flour, soy protein concentrate and poultry by-product meal supplemented with essential amino acids (e.g. methionine, lysine) and/or phosphorus has been studied in several short-term studies on tilapia fries (Ng and Romano, 2013). Tilapia performance by means of diets containing soybean meal and essential amino acids as the only protein source has been shown to be worse than by means of diets also containing 15-30% poultry by-product meal and a control diet containing 20% fish meal (Koch et al, 2016). Fish meal can be replaced in tilapia diets by carefully selected plant and/or animal by-product powders, but the resulting growth is similar to or lower than in the presence of fish meal, with detrimental effects on other metrics such as feed conversion and protein retention (Herath et al, 2016; Koch et al, 2016).

Thus, plant-derived protein sources and animal by-products are considered to have low quality or low nutritional value, or to be less attractive to fish, when compared to aquatic protein sources. When fed as a protein component of a fish diet, the resulting performance is lower than a diet comprising proteins derived from aquatic organisms. Suitable alternatives to aquatic organism protein sources (e.g., fish meal) need to address one or more of the disadvantages of reducing or eliminating aquatic organism-derived proteins from fish feed products.

Studies have been conducted to identify sources of dietary protein for replacing fish meal in prawn diets (Richard et al, 2011; Sookying and Davis, 2011; Su a rez et al, 2009; Xie et al, 2016). Some success has been noted in replacing fish meal in the diet of Litopenaeus vannamei (sotopenaeus vannamei) (Sookying and Davis,2011), but Penaeus monodon (Penaeus monodon) has been found to benefit from the inclusion of fish meal (Glencross et al, 2014). This study also demonstrates that Novacq^TM(a natural feed ingredient consisting essentially of dried mixed microbial biomass) can provide a sustainable solution to completely eliminate the need for fish meal in penaeus monodon feeds. Comprises 5% and 10% Novacq^TMGrowth was also increased by as much as 60% at a range of protein levels (Glencrosss et al, 2015). The use of microbial biomass produced by using different systems, such as biological floc (biofloc) technology, has been shown to have varying degrees of success in shrimp (Anand et al, 2014; Kuhn et al, 2010) and tilapia (avimmelech, 2007; Azim and Little, 2008; Caldini et al, 2015). Caldini et al evaluated the effect of supplying dried biological floc biomass to farmed Nile tilapia. The conclusion in Caldini et al states that any level of substitution of artificial feed with dried biological flocks impairs growth performance in tilapia; and there is no legitimate reason for drying the biological floc biomass to provide it to farmed tilapia.

There remains a need for alternative sustainable feeds for feeding low-food fish (e.g. fish of the tilapia family of the family limidae) that address one or more of the disadvantages of known feed products.

Brief description of the invention

The inventors have found that a fish feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria promotes increased growth rate and/or improved health when provided to fish, particularly low-feeding fish, such as fish of the tilapia family of the family povidae. Surprisingly, the inventors have also found that the dried hybrid biomass functions as a feed attractant and/or a feeding stimulant in fish.

The inclusion of dried biomass including bacteria and microalgae in a feed product further comprising other nutritional components has been found to increase food intake when fed to fish and thus result in improved weight gain. The dried mixed biomass is considered to function as a feed attractant or a feed stimulant for fish (e.g., low-food fish, such as fish of the tilapia family of the family limnodidae). This finding is applicable to provide improved feed products for lower feeding fish. Without being bound by theory, it is believed that feeding stimulation may be associated with an improvement in feed palatability or attraction. However, feeding stimulation may also be the result of the dried biomass acting on different taste or metabolic pathways than those typically associated with nitrogen-enriched feed attractants or stimulants. For example, the feeding stimulation may be at least partially a result of increased gastrointestinal emptying rate or nutrient absorption rate.

Fish meal and krill meal are highly nutritional protein sources that are often incorporated into fish feed products. They are attractive to fish and, in addition to providing nutritional benefits, typically also function as feed attractants when incorporated into feed products. The use of dried biomass comprising bacteria and microalgae provides an alternative to the use of aquatic biomass as an attractant. The inventors have surprisingly found that the dried mixed microbial biomass may be more effective as a feed attractant or feed stimulant than an aquatic organism resource, as it has been found that inclusion of the biomass is more effective at lower concentrations in the feed product than are required for aquatic organism resources such as fish meal.

This finding predicts the use of dried biomass comprising bacteria and microalgae in fish feed products comprising low quality nutritional components or components having low nutritional value to stimulate the fish to consume increased amounts of the feed product. It has been found that the incorporation of the biomass results in increased feeding and greater weight gain and growth when compared to traditional feed formulations and feed formulations without aquatic biological resources. The dried mixed microbial biomass can also be applied in situations where it is desirable to enhance the feed appeal or palatability of a feed product. Palatability of ingredients for fish is important as this can have a significant impact on the utility of feed products regardless of nutrient composition and digestibility. Using the dried biomass in the absence of aquatic organism resources such as fish meal to improve palatability or appeal of feed products provides the opportunity to incorporate an expanded range of locally available feed grade raw ingredients (e.g., plant-derived protein sources or animal by-products) to replace aquatic organism-derived protein resources (e.g., fish meal or trash fish). This can improve the cost-effectiveness of fish diets by reducing the cost of obtaining and transporting premium ingredients (e.g., fish meal) in situations where local supply of premium raw ingredients is limited.

Accordingly, in a first aspect, the present invention provides a method of feeding a low-feeding fish, comprising feeding the fish a feed product comprising a dried biomass comprising a mixed microbial population comprising microalgae and bacteria.

In another aspect, the invention also provides a method of feeding a low-food fish comprising the step of feeding the fish a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

In another aspect, the invention also provides the use of a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria in an amount effective to provide nutrition to a fish having lower feeding habits.

It has surprisingly been found that the inclusion of the dried hybrid biomass (which includes bacteria and microalgae) in a feed product further comprising other nutritional components stimulates increased food intake when fed to low-feeding fish and thus results in improved weight gain. It is believed that the dried mixed biomass can function as a feed attractant or a feed stimulant for fish such as low-grade feeding fish (e.g., fish of the tilapia family of the family limidae).

Thus, in a further aspect, the present invention provides the use of dried biomass comprising a mixed population of microorganisms including microalgae and bacteria as a feed attractant or feed stimulant for low-grade predatory fish.

In yet a further aspect, the invention provides a method of improving the appeal and/or palatability of a feed product to a low-priority fish, comprising feeding said fish said feed product in the presence of dried biomass comprising a mixed microbial population comprising microalgae and bacteria.

In another aspect, the invention provides a method of stimulating a low-grade fish to increase its food intake, comprising providing to the fish a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

In another aspect, the invention also provides a method of increasing the growth rate or food intake of a low-priority fish, comprising providing to the fish a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

It is also believed that the dried mixed microbial biomass can be used to increase the attractiveness and efficacy of fishing lures for aquatic animals due to its attractiveness to fish.

Thus, in a further aspect, the invention further provides the use of a dried biomass comprising a mixed microbial population comprising microalgae and bacteria as a bait for aquatic animals.

In yet a further aspect, the invention provides a bait comprising dried biomass comprising a mixed type microbial population comprising microalgae and bacteria.

In some embodiments, the dried biomass is a hybrid microbial biomass according to WO 2009/132392 a1 (the contents of which are included herein in their entirety), or a hybrid microbial biomass prepared according to the methods described in WO 2009/132392 a 1. In some embodiments, the dried biomass preferably comprises bacteria in an amount of 5% w/w to 25% w/w. In some embodiments, the dried biomass comprises microalgae in an amount of 10% w/w to 80% w/w. In a preferred embodiment, the dried mixed microbial biomass is Novacq^TM. Preferably, the feed product comprises 2% w/w to 15% w/w dried biomass. Preferably, the feed product comprises one or more further nutritional ingredients, preferably selected from a protein source, a carbohydrate source and/or a lipid source. Preferably, the feed product is a nutritionally balanced product. Preferably, the feed product is nutritionally balanced for a particular fish species.

Brief Description of Drawings

FIG. 1 is a diagrammatic representation of a representation shown in Novacq^TMThe relationship between inclusion rate and daily gain in body weight and daily feed intake of GIFT tilapia.

FIG. 2 is a graphical representation showing the results for different Novacq during the course of the experiment^TMAverage daily dry matter feed intake (expressed as% body weight) of inclusion rate(s).

FIG. 3 is a graphical representation showing the relationship between body weight gain and fish meal content of a meal, with and without Novacq^TMComprises the following steps. Significant differences were marked by different letters [ two-way ANOVA, Novacq Effect, P<0.05; fish meal effect, P<0.05; interaction of P>0.05]。

Detailed Description

1. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

Throughout this specification the terms "comprises," "comprising," "includes," "including," or any other variation thereof, are to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout this specification, unless otherwise indicated, reference to a numerical value is to be understood as meaning "about" that numerical value. The term "about" is used to indicate that a value includes the inherent variation of error for the device or method employed to determine the value, or the variation that exists among experimental values.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

As used herein, the term "dried biomass comprising a mixed microbial population comprising microalgae and bacteria" or "dried mixed microbial biomass" and the like refers to dried biomass, such as solid biomass, comprising a mixed microbial population comprising bacteria and microalgae. Typically, other microorganisms may be present, such as yeasts, protists and fungi. The biomass may also include cellulosic organic matter. The mixed microbial biomass is prepared by culturing a mixed or heterogeneous population of microorganisms under controlled conditions in which the growth of both microalgae and bacteria is stimulated. The bacteria are stimulated to grow by the addition of a carbon source that is utilized by the bacteria. This co-cultivation results in a mixed microbial biomass comprising a significant amount (e.g., 5% w/w to 25% w/w) of biomass derived from the bacteria. Methods for producing mixed microbial biomass are described in the following documents: PCT/AU2009/00053 published as WO 2009/132392A 1 on 5.11.20099 (Commonwelth Scientific and Industrial Research organization); and PCT/AU 2014/000419A 1 (Commonwelth Scientific and Industrial Research organization) disclosed in WO 2014/165936A 1 on 16/10 of 2014. In a preferred embodiment, the dried mixed microbial biomass is used as a prawn feed supplement and is under the name Novacq^TMCommercially produced biomass for sale. Novacq as used herein^TMManufactured by Ridley agri products (www.ridley.com.au) in australia and comprising dried mixed microbial biomass prepared according to the processes described in WO 2009/132392 a1(CSIRO) and WO2014/165936 a1(CSIRO), the contents of which are incorporated herein in their entirety.

As used herein, the term "food grade" refers to the classification of fish by a food grade of 2 to 5 for different species depending on what food they eat and to what food grade to which their food is classified. As used herein, the terms "low-feeding" or "lower-feeding" and the like refer to fish that are lower in the food chain and have a feeding hierarchy of, for example, 2.0-2.5 or 3. Low-food fish, which are typically produced on a commercial scale by aquaculture, include fish of the tilapia family of the family poyotidae, such as the mouth hatched non-crucian genus, the brook non-crucian genus and the tilapia genus, including GIFT (genetically modified farmed tilapia) lines. Other tilapia family fishes of the family Pacifidae include the genera Carassius (Alcolapia), Danafiu (Danailia), Iranochilia (Iranociichia) and Pseudosciaena (Steatocrus). In some embodiments, the fish of the tilapia family of the family liidae is nile tilapia, also known as nile hatch non-crucian. In particular, one line of nile mouth hatch non-crucian is the GIFT line of nile tilapia. Other low-food fish that are typically produced by aquaculture on a commercial scale include, but are not limited to: a carp class; and catfishes including Sharak catfish,

(pandasius), basha (basa) and chai (tra).

As used herein, the term "hyperphagic" or "hyperphagic" refers to fish that are at high levels in the food chain and have a hyperphagic stratification of, for example, 3.5-4.5. Specifically, the high-feeding fish produced by aquaculture includes Rachycentron canadum, trout, cod, salmon, king fish (Kingfish), and Palmata.

Unless otherwise indicated, it is to be understood that all percentages described herein are weight percentages [% w/w ]. When referring to the composition of the mixed microbial biomass, the percentages refer to% w/w on an air dried mass/mass basis.

As used herein, the term "feed product" refers to a feed composition comprising dried mixed microbial biomass and, preferably, one or more additional nutritional ingredients selected from a carbohydrate source, a protein source, and a lipid source. The feed product may further comprise one or more additional nutritional ingredients selected from vitamins and minerals; and/or excipients such as binders. The composition is preferably substantially homogeneous and may be in any suitable form known in the art for use in aquaculture, such as powders, pastes, cakes, granules, pellets and the like. In some embodiments, preferably, the food product is in the form of pellets. Preferably, the feed product composition is nutritionally balanced for feeding to low-feeding fish. In some embodiments, the feed product is substantially free of aquatic organism-derived resources, such as aquatic organism-derived proteins and/or oils.

As used herein, the term "nutritionally balanced" means that the feed product has particular selected components, such as carbohydrates, proteins and lipids, in appropriate proportions that will effectively support the growth of the relevant low-feeding fish. The skilled person will be readily able to determine the amounts and proportions of the various nutritional components necessary for a given fish species based on the teachings of the examples herein and the general knowledge in the art.

The term "aquatic organism resource" should be understood to include elements, products, compositions or resources derived from an aquatic ecosystem or environment, including strains, species, populations and populations derived from an aquatic ecosystem or environment, including resources belonging to fish, shellfish or other aquatic animals, such as protein sources derived from aquatic animals, including "trash fish" or "worthless fish," fish meal, squid meal or krill meal, and lipid sources derived from aquatic animals, including fish oil, squid oil or krill oil, as will be understood by those skilled in the art. "trash fish" or "worthless fish" refers to fish that are considered to have little utility or value as food fish and are therefore typically discarded.

When used herein with respect to nutritional ingredients, terms such as "low quality" or "low nutritional value" refer to ingredients, for example plant-derived nutritional sources, particularly plant-derived protein sources, which are generally less nutritional or less acceptable for fish than aquatic organism-derived resources. Consumption of low quality or low nutritional value foods results in reduced growth rates, when based on the amount consumed. The term "unpalatable" nutritional composition refers to a composition that is less attractive to fish or is objectionable to fish. The presence of unpleasant ingredients in fish feed results in less food being consumed by the fish and therefore the growth rate is low. Examples of low quality or low nutritional value ingredients include plant derived protein sources such as soybean meal, lupin (kernel) meal and gluten meal.

2. Method of the invention

The present invention relates to a method of feeding or rearing a low-food fish under aquaculture conditions. Non-limiting examples of low-food fish include fish of the tilapia family of the family Pacific; catfish species, e.g. basha, crabapple and

and a carp species; although it is contemplated that the invention is applicable to any low-food fish, particularly those that are raised commercially. Preferably, the fish is from the family Tilapia of the family Limonidae and includes the mouth-hatched non-crucian genus, the brook's teethFish of the genera other than crucian and tilapia, including GIFT (genetically modified farmed tilapia) strains. One particular fish is nile tilapia, also known as nile hatch non-crucian. In particular, one strain of nile hatchery non-crucian is the GIFT strain nile tilapia.

Preferably, the dried biomass used in the methods herein is prepared from a mixed or heterogeneous population of microorganisms comprising a mixed population of microorganisms including microalgae and bacteria under controlled conditions in which the growth of both microalgae and bacteria is stimulated. The bacteria are stimulated to grow by the addition of a carbon source that is utilized by the bacteria. This results in a mixed microbial biomass comprising a significant amount (e.g., about 1% w/w to about 50% w/w; about 5% w/w to about 40% w/w; or 5% w/w to about 25% w/w) of the biomass derived from the bacteria.

Preferably, the mixed population of microorganisms comprises microalgae and bacteria, wherein the bacteria are present in an amount of about 5% w/w to about 25% w/w on a dry matter basis and the microalgae is present in an amount of about 10% w/w to about 80% w/w on a dry matter basis. Preferably, the dry weight ratio of bacteria to microalgae is between about 20:1 to about 0.4: 1. More preferably, the bacteria are present in the microbial biomass in an amount of from about 5% w/w to about 10% w/w on a dry matter basis. Preferably, the mixed microbial biomass comprises a minimum bacterial content of 5% w/w of the dried biomass.

Mixed microbial biomass and methods for its preparation are described in WO 2009/132392 a 1. The microbial biomass is suitably dried prior to incorporation into the feed additive. An example of a dried mixed microbial biomass is under the name Novacq^TMThe following are commercially available.

The inventors have demonstrated that the inclusion of the dried mixed microbial biomass in a feed product fed to a low-feeding fish results in a significant weight gain in the fish. The results demonstrated herein show that the average growth improvement relative to the control diet without biomass was 7.8%, 23.6% and 34.6% when included at 2.5%, 5% and 10%, respectively. Thus, daily feed intake increases with biomass inclusion and closely reflects growth trends. Feed intake in absolute amounts and ratio to fish body weight was significantly higher in the diet containing 10% w/w biomass than the corresponding control diet.

Preferably, the mixed microbial biomass is contained in a feed product such as a powder, granules or pellets. In a preferred embodiment, the feed product is in the form of pellets, e.g. extruded pellets. Preferably, the mixed microbial biomass is included in a feed product at a level of about 5% w/w to about 15% w/w or about 5% w/w to about 10% w/w. In some embodiments, the dried mixed microbial biomass may be included at a level of about 5% w/w or about 10% w/w. Preferably, the feed product further comprises one or more further nutritional ingredients, preferably selected from the group consisting of a protein source, a carbohydrate source and a lipid source. Preferably, the feed product is nutritionally balanced.

In the method of the invention, it will be appreciated that the fish should be provided with sufficient feed product to allow them to support growth. In some preferred embodiments, the fish are allowed to feed to satiety. The skilled person will readily be able to determine the amount of food needed depending on the nutrient composition of the feed product and the species and size of the fish, based on general knowledge in the aquaculture field and the teachings in the examples herein.

In view of its ability to function as a feed attractant or a feeding stimulant in fish, the present invention also provides a method of attracting aquatic animals, such as fish, that includes the use of a bait comprising dried mixed microbial biomass. Also provided is the use of the dried hybrid microbial biomass as a fishing bait.

The invention also provides fishing lures comprising dried mixed microbial biomass. The fish bait may be used to attract aquatic animals, such as fish, molluscs or crustaceans. In some embodiments, the fish is a high-or low-feeding fish. Depending on the species of aquatic animal targeted by the fishing lure, the fishing lure may further comprise other bait components selected from, for example, natural bait components such as earthworms; leeches; grubs; maggots; caterpillar; frogs; fish meal; fish, such as bait fish, salmon (chum) or minox (minnows); frogs; shrimp; and insects such as grasshoppers or ants. The bait of the invention may also include any attractant known in the art of commercial or recreational fishing, such as cheese, bread, and the like.

3. Material of the invention

The mixed microbial biomass for use in the methods of the invention is prepared from a mixed microbial population under controlled conditions in which the growth of both microalgae and bacteria is stimulated. The bacteria are stimulated to grow by the addition of a carbon source that is utilized by the bacteria. This results in a mixed microbial biomass comprising significant amounts of biomass derived from the bacteria. Biomass comprising a mixed population of microorganisms including microalgae and bacteria and methods for producing the same are disclosed in the following references: PCT/AU2009/000539 (Commonwelth Scientific and Industrial Research organization) published as WO 2009/132392A 1 on 5.11.2009; and PCT/AU2014/000419 (Commonwelth Scientific and Industrial Research organization) disclosed in WO 2014/165936A 1 on 16/10 of 2014, the contents of each of which are incorporated herein in their entirety. The source of the microorganisms may occur naturally in the water used in the cultivation system for producing the microbial biomass and may include raw, unfiltered seawater, wastewater from an aquaculture pond, or recycled water from a previous culture. In addition to bacteria and microalgae, the hybrid biomass used in the present invention may further include yeast, fungi, and/or protists.

Methods for producing mixed microbial biomass generally include:

a) providing a mixed microbial population comprising microalgae and bacteria, preferably the bacteria are present in an amount of about 5 wt% to about 25 wt% on a dry matter basis, and the microalgae are present in an amount of about 10 wt% to about 80 wt% on a dry matter basis;

b) adding a carbon source to the mixed biological population;

c) adding a nitrogen source to the mixed biological population;

d) culturing the mixed microbial population under conditions suitable for the growth of both microalgae and bacteria to form a microbial biomass; and

e) harvesting the microbial biomass.

Methods for culturing, harvesting and drying the mixed microbial biomass are described in WO2014/165936 a1 and WO 2009/132392 a 1. In some embodiments, the carbon source is preferably derived from waste, large-volume, low-value agricultural materials and agricultural wastes. Preferably, the carbon source is locally produced. The low value agricultural material may include products, by-products or waste streams from sugar cane processing, such as filter mud, sugar cane tips, molasses or bagasse. Other sources include products, by-products or waste streams from rice, wheat, triticale, corn, sorghum, tapioca, oilseed (including canola meal and lupin hulls) processing, and elevator dust from grain processing facilities.

The mixed microbial biomass includes a significant amount (e.g., about 1% w/w to about 50% w/w, about 5% w/w to about 40% w/w, or 5% w/w to about 25% w/w) of a bacteria-derived biomass. More preferably, the bacteria are present in the microbial biomass in an amount of from about 5% w/w to about 10% w/w on a dry matter basis. Preferably, the mixed microbial biomass comprises a minimum bacterial content of 5% w/w of the dried biomass.

In some embodiments, the mixed population of microorganisms comprises greater than 50% w/w of the mixed population of bacteria; e.g., greater than 60% w/w, greater than 70% w/w, or greater than 80% w/w or 85% w/w of the mixed population of bacteria.

In some embodiments, the microalgae is present in the mixed microbial biomass in an amount of from about 0.1% w/w to about 50% w/w, from about 0.1% w/w to about 40% w/w, from about 0.1% w/w to about 30% w/w, from about 0.1% w/w to about 25% w/w, from about 0.1% w/w to about 20% w/w, from about 0.1% w/w to about 15% w/w, from about 0.1% w/w to about 10% w/w, or from about 0.1% w/w to about 5% w/w on a dry matter basis. In some embodiments, the biomass comprises a mixed microbial population comprising microalgae and bacteria, wherein the bacteria are present in an amount of about 5% w/w to about 20% w/w on a dry matter basis, and the microalgae is present in an amount of about 10% w/w to about 80% w/w on a dry matter basis.

In a preferred embodiment, the biomass comprises a mixed population of microorganisms including microalgae and bacteria, wherein the bacteria are present in an amount of about 5% w/w to about 20% w/w or about 25% w/w on a dry matter basis, and the microalgae are present in an amount of about 10% w/w to about 80% w/w on a dry matter basis. In some embodiments, the bacteria are present in an amount of about 5% w/w to about 20% w/w or about 5% w/w to about 10% w/w. Preferably, the mixed population of microorganisms comprises microalgae and bacteria, wherein the bacteria are present in an amount of about 5% w/w to about 25% w/w on a dry matter basis and the microalgae is present in an amount of about 10% w/w to about 80% w/w on a dry matter basis. Preferably, the dry weight ratio of bacteria to microalgae is between about 20:1 to about 0.4: 1.

It is well within the knowledge of the skilled person to determine the composition of the mixed microbial biomass. Quantification of microalgae content can be based on chlorophyll-a content of the microbial biomass; and quantification of bacteria can be based on muramic acid content by using conventional methods known in the art.

Preferably, the harvested biomass is dried to form a dried mixed microbial biomass. The biomass may be dried by using any suitable means known in the art, but rapid drying at moderate temperatures (e.g. 40 to 80 ℃, e.g. about 40 ℃) under high air flow is preferred. Preferably, the dried product comprises less than 10 wt% moisture.

Although it can be used in its dried form as a feed material without further processing or addition of other ingredients, for the purposes of the present invention, it is advantageous to combine the dried mixed microbial biomass with additional nutritional ingredients to form a feed product.

In some preferred embodiments, the feed product comprises: a dried biomass comprising a mixed microbial population comprising microalgae and bacteria; and one or more further nutritional ingredients.

In a preferred embodiment, the mixed microbial biomass comprises from about 2% to about 25% by weight of the feed product. Preferably, the biomass comprises about 5% or more by weight of the feed product. In some embodiments, the biomass comprises from 2 wt% to 15 wt%, from 5 wt% to 25 wt%, from 5 wt% to 20 wt%, from 5 wt% to 15 wt%, or from 5 wt% to 10 wt%. Suitably, the feed product comprises nutritional ingredients selected from a carbohydrate source, a protein source and a lipid source. Preferably, the feed product is nutritionally balanced. Preferably, the additional nutritional ingredients are selected such that the feed product is nutritionally balanced for low-food fish, such as fish of the tilapia family of the family limidae.

In some embodiments, the feed product comprises from about 30% to about 40% protein; from about 45% to about 55% carbohydrate; and about 4% to about 6% lipid.

Suitable nutritional sources are well known in the art of fish nutrition and aquaculture. Protein sources include, but are not limited to, aquatic sources such as trash or worthless fish, krill meal, squid meal, and fish meal, and mixtures thereof. Protein sources derived from non-aquatic biological sources include, but are not limited to, soybean meal, poultry meal, lupin (kernel) meal, and gluten meal, and mixtures thereof. In some embodiments, the protein source is derived from a non-aquatic biological source.

Suitable carbohydrate sources include, but are not limited to, wheat flour, rice bran, tapioca starch, rice flour, corn or corn flour, or mixtures thereof.

Suitable lipid sources include, but are not limited to, lipids derived from aquatic organisms such as fish oil, krill oil or squid oil or mixtures thereof, or non-aquatic organism lipids. Non-aquatic lipids include, but are not limited to, plant lipids such as vegetable oils, canola (rapeseed) oil, linseed oil, hemp seed oil, soybean oil, pumpkin seed oil, or mixtures thereof.

In some embodiments, the feed product can be substantially free of aquatic biological resources, wherein the aquatic biological resources can be a protein source derived from an aquatic animal, such as trash fish, fish meal, squid meal, or krill meal; or a lipid source such as krill oil, fish oil or squid oil.

The feed product may further comprise ingredients such as: binders, such as gluten, alginate or starch; vitamin mixtures suitable for the intended aquatic species; a mineral mixture suitable for the intended aquatic species; and other nutritional, pharmaceutical or growth supplements. The selection of suitable additional ingredients and the amounts to be included in the feed ingredients will be well within the knowledge of those skilled in the art. The skilled person will appreciate that the ingredients should be non-toxic to the fish in the amounts present.

In one embodiment, the feed product comprises a dried mixed microbial biomass; one or more protein sources; one or more sources of carbohydrates; and one or more lipid sources; for example, dried microbial biomass, soybean meal, fish meal, wheat flour, wheat gluten, fish oil, and soybean oil. In addition to carbohydrate, protein and lipid sources, the feed product may also comprise additional nutritional components selected from the group consisting of: mineral sources, such as calcium diphosphate (calcium source); and amino acids such as L-lysine or DL-methionine. The feed product may also comprise micronutrients in the form of minerals and/or vitamins.

Preferably, the ingredients of the feed product are combined to provide a homogeneous composition. The homogeneous mixture may then be further processed, if desired. The feed product may be in any form suitable for feeding fish in an aquaculture environment. Conveniently, the feed product is in the form of a paste, powder, granule, cake or pellet. In a preferred embodiment, the feed product is in pellet form, and preferably is a nutritionally balanced pellet-formed feed product. Methods of preparing pellets (e.g., extruded pellets) are well known in the art and are described in the examples herein and in WO 2009/132392 a1 and WO2014/165936 a 1.

Methods for preparing feed products for fish are well known in the art of aquaculture. Typically, the desired ingredients are selected and combined in the desired proportions. Preferably, the dry ingredients are milled and combined by mixing to provide a homogeneous composition. Typically, the ingredients are combined by mixing (e.g., via the use of a planetary mixer) to provide a homogeneous mixture. The dry ingredients may be combined with liquid ingredients (e.g., lipid ingredients) prior to extruding the resulting mixture. In some embodiments, the dry ingredients are combined with water in an extruder. The mixture is then extruded and dried, after which the resulting pellets are impregnated with a lipid source (if desired). The water content of the pellets can be controlled to provide floating pellets. Extrusion of the mixture through a 2.5-3.0mm die (e.g., a 2.8mm die) provides pellets having a diameter suitable for low-grade feeding fish (e.g., fish of the tilapia family of the family liidae). In some embodiments, the pellets are cut at the die face to a length of about 4mm to about 5mm, for example 4.5mm in length, giving pellets having a diameter of about 2.5mm to about 3.0mm and a length of about 4mm to about 5 mm. In some embodiments, the pellet is about 2.8mm by 4.5mm in size.

In order that the invention may be readily understood and put into practical effect, there shall now be described by way of the following non-limiting examples specific preferred embodiments.

Examples

Materials and methods

Culture system and fish population

Growth experiments were performed in the laboratory of the world fish headquarters (world fish farms, marysian champagne) using GIFT (genetically modified farmed tilapia) nile tilapia (nilo hatch non-crucian). A total of 54x 10L transparent aquarium (tank) on a recirculating fresh water system was used. The water flow was regulated at 2 exchanges per hour and recycled at 95% through a series of filter media and protein skimmers. Stored rain water is used as make-up water. Water quality was maintained throughout the experiment as follows: average value ± s.d., water temperature 27.1 ± 0.8 ℃; D.O.7.0. + -. 1.1mg L^-1；pH＝6.8±0.3；NH₃<0.5ppm；NO₂<1.0ppm；NO₃<80 ppm. A total of 1500 fries were obtained from the world fish GIFT breeding center in ridela, malaysia. The population was graded to include only fish within one standard deviation of the mean for the experiment. A total of 10 fish/water tanks (average weight 11.43. + -. 1.75 g. + -. SD) were randomly allocated. Three samples (initial fish with 10 pools) were taken for subsequent composition analysis.

Dietary treatment and composition

A total of eight nitrogen-iso and energy-iso experimental diets were extruded. Milling each of said ingredients to<750 μm, the compositional chemical composition can be found in table 1. A vertical planetary mixer (BakerMix, artarmeron, NSW, Australia) was used to mix thoroughly a total of 10kg of each of the experimental diets (without oil component) described. The meal was then extruded through a laboratory scale 24mm twin screw extruder (MPF24:25, Baker Perkins, Peterborough, UK) with intermeshing co-rotating screws. The extruder consists of a series of intermeshing Feed Screws (FS), advancing paddles (FP) and Lead Screws (LS) arranged according to a prescribed barrel diameter (D) such that the overall construction is 16D FS, 2D FP, 1D LS, 1D FP, 2D LS, to the die, starting from the drive end. Using a profiled strip having a length of 3mmA single 2.8mm diameter cylindrical die tapered at a 67 ° angle. The barrel consisted of four temperature zones set at 70, 80, 100 and 110 ℃ from drive to die respectively and the machine was run at c.160rpm. Mixing the paste at a volume of c.4.2-4.9kg h^-1Is delivered into the barrel and is 2.1 to 3.0L h^-1Water was pumped into the bucket by peristaltic means (Thermoline, wetherl Park, NSW, Australia). Water addition was varied between meals to yield-4.2 mm

The floating feed. The pellets were cut to a length of 4.5mm at the die face with a 2-blade variable speed cutter. The pellets were then allowed to dry at 60 ℃ for 24 hours, after which each meal was vacuum impregnated with a specific oil profile. The pellets were then allowed to cool before bagging and labeling. All feeds were kept frozen (-20 ℃), except when the feed was fed or weighed.

The dietary treatment consisted of: four containing 10% fish meal and increasing levels of Novacq^TM(0, 2.5, 5 and 10%) of the diet, and four diets with and without (respective controls) 10% Novacq^TMWith decreasing levels of fish meal (5% and 0%). Incorporation of Novacq by substitution of equal amounts of wheat flour^TMWhile fish meal was replaced with soy flour and increasing levels of methionine, lysine and fish oil, while soy oil was reduced to maintain equal total lipids (table 2). A baseline commercial diet was also used in this experiment. The measured chemical composition confirmed that all diets showed similar protein and energy content (table 2).

Table 1. composition of commercial diets and ingredients used in experimental feeds, on a dry matter basis.

Table 2 diet formulation and feed composition on a dry matter basis.

Fish meal and fish oil: south America sources, supplied by Ridley, Narangba, QLD, Australia. Soybean meal: KEWPIE Stock feeds, Kingaroy, QLD, Australia; wheat flour and wheat gluten: manildra, Auburn, NSW, Australia. Wheat bran: allora Grain and Milling, Allora, QLD, Australia; novacq: CSIRO bind production, commercial secret. Micronutrients (mg/kg diet): choline (60% choline chloride), 5; vitamin C (Stay-C), 2; fish vitamin and mineral premix, 6¹；

¹The vitamin and mineral premix content in the final feed (IU or g/kg feed) was: vitamin a, 15 KIU; vitamin D3, 1.5 KIU; 0.1g of vitamin E; vitamin K3, 0.01 g; vitamin B1, 0.025 g; vitamin B2, 0.03 g; vitamin B3, 0.15 g; vitamin B5, 0.05 g; vitamin B6, 0.01 g; vitamin B9, 5 mg; vitamin B12, 0.03 mg; biotin, 1 mg; 0.45g of vitamin C; choline chloride, 1 g; inositol, 0.35 g; ethoxyquin, 0.125 g; 0.015g of copper; ferrous ion, 0.04 g; 0.1g of magnesium; manganese, 0.09 g; 0.15g of zinc.

Feeding and growth measurements

The fish were fed manually twice a day to satiation. Each tank was fed in a minimum of three separate feeding events, 120 minutes in the morning and 120 minutes in the afternoon, during which time the fish were fed until 10 minutes of uneaten pellets were observed in the last time, indicating that the fish reached satiety. The uneaten pellets were collected daily on a 300 μm sieve and dried in a pre-weighed aluminum foil until a constant dry weight (4 hours at 105 ℃). Daily delivered feed was obtained from the differential weights before and after feeding for the tank-specific feed container and expressed as dry matter. Feed intake was calculated daily as the difference between delivered dry matter and collected dry matter. Feed Intake (FI) is expressed as "grams of feed dry matter/fish/day" (accounting for any daily mortality), and as Specific Feed Intake (SFI) in weight percent (i.e., SFI ═ FI/BW)_e x 100). Average expected Body Weight (BW) per day of fish was calculated based on two weeks of fish weight and measured linear growth rate achieved over all treatments_e)：

The fish were individually weighed on days 0 and 42 and the tank as a whole was weighed on days 14 and 28 to minimize fish handling stress. The fish were anesthetized with clove oil before weighing and allowed to recover in their assigned water tank after weighing. This procedure was performed every check-weighing day. Any observed mortality within the first week was simply replaced with equivalent fish from the storage tank of the same weight.

For all treatments, fish growth was found to be linear over the experimental period (on average, body weight 1.76x days +7.49 g). Thus, fish growth is expressed in the figures as weight gain or daily weight gain. Growth is also expressed as percentage difference relative to the control diet.

Feed conversion rates account for mortality and are also expressed in a corrected manner for feed dry matter as follows:

for protein and energy, nutrient retention efficiency was calculated from feed intake and carcass composition as follows:

analysis of chemical composition

Fish carcasses were frozen at-20 ℃ until analysis. The dry matter content was determined gravimetrically after drying at 105 ℃ for 4 hours. The samples were then ground, transported to a CSIRO analytical laboratory, and analyzed for chemical composition. The ash content was determined based on the change in mass after 6 hours of combustion in a muffle furnace at 550 ℃. The total lipid content was determined gravimetrically after extraction in 2:1:0.4 chloroform: methanol: water using a modification to the method proposed by Folch et al (1957). The measurement of total nitrogen content was determined by the Dumas method using a Flash 2100 elemental analyzer (Thermo Fisher scientific, Waltham, MA, USA) and used to calculate the sample protein content based on N x 6.25.25. The total energy was determined by isoconpheric bomb calorimetry in a Parr 6200 oxygen bomb calorimeter, with 1108CL bomb for ingredients and diet, and 1109A semimicroscale bomb (Par Instrument Company, Moline, IL, USA) for feces. Carbohydrates are calculated by the difference.

Statistical analysis

Differences in breeding performance index and carcass composition were examined by one-way ANOVA followed by post hoc comparisons using Tukey-Kramer test. Repeated measures ANOVA were used to test the differences in daily and specific feed intake between treatments for the entire duration and the first 7 days of the experiment. Testing for fish meal and Novacq by two-way ANOVA^TMSignificant differences in daily weight gain and interactions were involved. Prior to all analyses, the ANOVA hypothesis of residual normality and homogeneity of variance was examined using the Shapiro-Wilk and Levene tests, respectively. Subjecting survival data to arcsine square root transformation and data log₁₀Or square root transform, when a Levene test is required to satisfy homogeneity with respect to variance (p.0.05). All statistical analyses were performed by using NCSS 11.

Results

Novacq in actual tilapia diet^TMEffect of inclusion rate

Tilapia grew well on all experimental diets, with weight gain and survival>80% of the total weight of the meal, which is not different from the reference Commercial Diet (CD)(Table 3). Novacq^TMIncluded significantly increased body weight gain (table 3 and figure 1). When included at 2.5%, 5%, and 10%, respectively, relative to the absence of Novacq^TMThe mean growth improvement of the control diet was 7.8%, 23.6% and 34.6% (table 3).

Daily feed intake is dependent on Novacq^TMIncrease in inclusion and closely reflect the growth trend (figure 1). Feed intake in absolute amounts (FI) and ratio to fish body weight (SFI) in a diet containing 10% Novacq^TMWas significantly higher in the diet of (a) than the control diet (table 3). Obtained in the whole experiment with Novacq^TMIncluded trend for higher specific intake (figure 2).

Feed conversion ratio (fed and expressed as feed DM) did not follow Novacq^TMSignificantly varied in inclusion and was significantly better than baseline CD on all experimental feeds containing 10% FM (table 3). The retention efficiency of protein and energy did not differ significantly between diets (table 3).

Carcass composition analysis showed no significant differences between the fish except for a reduction in protein for fish fed with 10FM-5NQ relative to CD and 10FM-2.5NQ diet (table 4).

TABLE 3 after 42 days on baseline Commercial Diet (CD) and with increasing Novacq^TMThe actual diet included average initial body weight, body weight gain, survival, daily feed intake, FCR and nutrient retention efficiency in tilapia fed (average ± s.e., n ═ 6). Marked differences by different letters [ one-way ANOVA, P<0.05; repeated measures ANOVA, P for FI and SFI<0.05]。

TABLE 4 basic Commercial Diet (CD) and increasing Novacq after 42 days^TMMean carcass composition of tilapia fed the actual diet included (mean ± s.e., n ═ 6). Data are on a dry matter basis. Marked differences by different letters [ one-way ANOVA, P<0.05]。

With and without Novacq^TMReplacement of fish meal

Significantly lower weight gain was achieved when the fish were fed a feed with 0% fish meal compared to 10% fish meal (figure 3). However, 10% Novacq^TMHas a positive effect on the overall body weight gain with all fish meal inclusions (table 5). When fish meal was reduced to 5% and 10%, respectively, the mean growth relative to control 10FM-0NQ was reduced by 3% and 14.5%. When fish meal is reduced to 5% and 10%, respectively, 10% Novacq is added^TMIn this case, the average growth of FM-0NQ was increased by 19.5% and 15.5% relative to the control (Table 5).

Feed intake in absolute amounts (FI) and ratio to fish body weight (SFI) in a diet containing 10% Novacq^TMCompared to the diet without Novacq^TMThe diet of (a) was significantly higher (table 5).

Feed conversion (fed and expressed as feed DM) tended to increase with decreasing fish meal, but not with fish meal and/or Novacq^TMIncluding significantly (table 5). The retention efficiency of protein and energy did not differ significantly between diets (table 5). Carcass composition analysis showed no significant differences between the fish (table 6).

TABLE 5 after 42 days with and without 10% Novacq^TMThe 50% and 100% fish meal replacement administered was the average initial weight, weight gain, survival, daily feed intake, FCR and nutrient retention efficiency (mean ± s.e., n ═ 6) for the tilapia fed. Marked differences by different letters [ one-way ANOVA, P<0.05; repeated measures ANOVA, P for FI and SFI<0.05]。

TABLE 6 after 42 days with and without 10% Novacq^TMImplementation50% and 100% fish meal replaced the average carcass composition of the tilapia fed (average ± s.e., n ═ 6). Data are on a dry matter basis. No significant differences were found [ one-way ANOVA, P<0.05]。

	5FM-0NQ	5FM-10NQ	0FM-0NQ	0FM-10NQ
					Dry matter (%)	27.93±1.78	24.11±0.43	22.48±0.15	23.92±0.62
Crude protein (%)	57.45±1.68	60.26±0.78	62.34±0.59	58.09±1.79
					Total energy (KJ/g)	23.14±0.51	23.14±0.53	23.93±0.28	24.07±0.11
Total lipid (%)	25.13±1.83	26.15±1.34	28.28±2.54	26.84±1.47
					Ash (%)	14.08±1.06	14.58±0.67	12.48±0.52	13.76±0.28

The results clearly demonstrate the growth benefits present in actual extruded tilapia diets at various inclusion rates including dried mixed microbial biomass; and the dried mixed microbial biomass improves the growth performance of tilapia in diets with low or zero fish meal. When included at 10% the biomass significantly increased the growth performance of tilapia in diets with 10%, 5% and 0% fish meal up to 35%. The meal is an extruded meal, which demonstrates that the benefit of the dried mixed microbial biomass is not diminished when an extrusion heat treatment is applied. All extruded diets containing the biomass outperformed the benchmark commercial diet containing similar amounts of protein and energy, and therefore the growth results achieved in this study were commercially relevant. The body weight gain increased in parallel with increasing mixed microbial biomass inclusion rates with 7.8%, 23.6% and 34.5% body weight gain relative to an isoenergetic and an isonitrogenous control diet with 2.5%, 5% and 10% dried mixed microbial biomass inclusion, respectively. Although the weight gain response slowed towards the highest inclusion, the response plateau was not reached, indicating that greater weight gain could be achieved with higher inclusion rates in the tilapia diet. With respect to the amount needed in tilapia diets to achieve a response, inclusion of 5% maximizes the benefit of the biomass.

Previous successful fish meal replacement studies have achieved a body weight gain consistent with experimental controls (see Furuya et al, 2004; Koch et al, 2016). The inventors have found that a complete replacement of 10% fish meal with soy meal and an increased inclusion of methionine, lysine and fish oil to compensate for nutrient deficiency still resulted in a significant reduction in daily weight gain of about 14.5%. Moderate levels of fish meal (5%) did not significantly affect body weight gain. These results are consistent with several previous studies showing that tilapia growth performance decreases as fish meal is replaced by individual plant proteins, possibly due to the presence of anti-nutritional factors (especially those from soybean meal) (Borgeson et al, 2006; Koch et al, 2016). Replacing soy flour with a complex mixture of processed plants or discarded protein components results in superior growth. The reduction in growth observed in this study on a 0% fish meal diet could not be correlated with a significant effect of fish meal removal on feed intake (4.8% BW/day), FCR (1.06) or protein retention (45%), suggesting that other mechanisms for nutrient intake or digestion play a role.

The results herein have demonstrated that dried mixed microbial biomass can increase tilapia body gain by as much as 35% on a 0% fish meal diet, which results in a net improvement in breeding performance of 15.5% relative to a 10% fish meal control. As such, the use of the biomass as a feed additive can not only compensate for the adverse effects of fish meal removal in tilapia meal, but also improve the sustainability value of the meal in countries with limited opportunities for using high quality primary ingredients.

The feed intake on the experimental diet reflects the growth trend achieved. Characterizing the palatability of an ingredient is important because, regardless of its nutrient composition and digestibility, it can have a significant impact on its usefulness as a feed. In order for an animal to exhibit an accurate feed intake response, it must be given the opportunity to reject the feed (glencrosss et al, 2007). In this study, careful manual feeding (which allows sufficient time and effort to feed the fish to satiation) was performed to collect uneaten food from all tanks twice daily following the feeding event. To unhook growth and feed intake, feed dry matter intake was expressed as a percentage of daily expected body weight based on the measured linear growth rate, to account for fish body weight change. Fish fed on a commercial benchmark diet showed a relatively high dry matter intake, which in relation to their poorer growth rate (when compared to current diets) resulted in significantly poorer food conversion, probably due to the use of less digestible ingredients. Fish fed on a diet incorporating biomass consumed significantly more food (not only in absolute terms, but also in percentage of their body weight) during the course of the experiment than fish fed on their respective controls, and the response was directly related to inclusion rate. The 10% inclusion of the dried mixed microbial biomass resulted in an approximately 33% increase in feed intake at all fish meal inclusion levels, indicating a significant effect of the dried mixed microbial biomass on tilapia meal palatability regardless of fish meal content. Fish meal is often used as a palatability enhancer in fish feed, but it is apparent from these studies that feed intake can be stimulated by inclusion of dried mixed microbial biomass, beyond the effects of fish meal.

The effect of dried mixed microbial biomass as a feeding stimulant was rapid and was observed within the first few days of feeding (fig. 2). Fish fed with a diet comprising the biomass often exhibit greedy feeding behavior and greater appetite (observations) than corresponding fish fed with a control diet. The feed intake measurements measured in these studies, along with similar feed conversion rates and REs, strongly directed the dried mixed microbial biomass to a feed stimulant or feed inducer in tilapia. This is surprising because the most common physicochemical properties of feed attractants and stimulants for fish are non-volatile, low molecular weight, nitrogen containing, amphoteric, water soluble, stable to heat treatment, and have a wide biological profile. These properties are consistent with those of free amino acids and related nitrogen-containing species such as nucleotides, nucleosides, and quaternary ammonium bases (de la higurera, 2007). Thus, strong feeding stimulation of dried mixed microbial biomass was unexpected in view of its low nitrogen content (i.e., N ═ 0.6%), which may indicate stimulation of different taste pathways (Lamb, 2007).

The disclosure of each patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety.

Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Thus, those of skill in the art will, in light of the present disclosure, appreciate that various modifications and changes can be made in the specific embodiments illustrated without departing from the scope of the invention. All such modifications and variations are intended to be included herein within the scope of the appended claims.

Bibliography

Andd, p.s.s., Kohli, m.p.s., Kumar, s., sundary, j.k., Roy, s.d., Venkateshwarlu, g., Sinha, a. and Pailan, G.H, (2014) Effect of biological on growth for and geographic enzyme activities in Penaeus monodon, aqua 418 419, 108-.

Avnimelech,Y.(2007).Feeding with microbial flocs by tilapia in minimal discharge bio-flocs technology ponds.Aquaculture 264,140-147.

Azim, M.E. and Little, D.C. (2008). The bioflo technology (BFT) in inductor ranks: Water quality, bioflo composition, and growth and welfare of Nile tirapia (Oreochris nilotius). Aquaculture 283,29-35.

Borgenson, T.L., Racz, V.J., Wilkie, D.C., White, L.J., and Drew, M.D. (2006.) Effect of reproducing fixed and oil with simple or complex geometries of capturing and reproducing in diodes fed to Nile tirapina 149. Aquaculture Nutrition 12,141-149.

Caldini, N.N., Cavalcante, D.d.H., Rocha Filho, P.R.N. and S.a, M.V.d.C.e. (2015) Feeding Ni tilapia with associated diodes and dried biologics biomass. acta scientific. animal Sciences 37, 335-.

de la Higuera,M.(2007).Effects of Nutritional Factors and Feed Characteristics on Feed Intake.In Food Intake in Fish,pp.250-268:Blackwell Science Ltd.

El-Saidy, D.M.S.D. and Gaber, M.M.A. (2002) Complete Replacement of Fish Metal by Soybean Metal with dimension L-Lysine Supplementation for Nile titanium Oreochromys niloticus (L.) indexing. journal of the ld Aquaculture Society 33,297-306.

Folch, J., Lees, M, and Sloane-Stanley, G. (1957). A simple method for the isolation and purification of total lipids from animal tissues.J.biol.chem 226,497-509.

Furuya, W.M., Pezzato, L.E., Barros, M.M., Pezzato, A.C., Furuya, V.R.B., and Miranda, E.C, (2004). Use of ideal protein concept for precision formation of amino acid levels in real-medium-free for judgment purposes Nile titanium (Oreochris nilotius L.). Aquaculture Research 35, 1110-.

Glencross, b., Arnold, S. and Irvin, S. (2015.) Bioactive factors in microbial biological have to offset reductions in the level of protein in the di of black tiger shrim, Penaeus monodon.

Glencross, b., Irvin, s., Arnold, s., Blyth, d., Bourne, N, and Preston, n. (2014) Effective use of a microbial biological product to a defect the complete replacement of fisher resources in the experiments for the black tiger shock 431,12-19.

Glencross, b.d., Booth, m. and alan, g.l. (2007). a feed is only as a good as an entity of entries-a review of entry evaluation constructs for an approach feed 13,17-34.

Herath, S.S., Haga, Y, and Satoh, S. (2016.) A tenseal use of corn co-products in fisher-free cuts for juvene nitrile titanium, Oreochromyis niloticus, Fisheies Science 82, 811-.

Koch, j.f., Rawles, s.d., Webster, c.d., Cummins, v., Kobayashi, y., Thompson, k.r., Gannam, a.l., twinell, r.g., and Hyde, N.M, (2016) (Optimizing mesh-free commercial units for nickel titanium, orochromyis niloticus, aqua culture 452, 357-.

Kuhn, D.D., Lawrence, A.L., Boardman, G.D., Patnaik, S., Marsh, L, and Flick, G.J, (2010) Evaluation of two types of biology of biological derived from biological specimen of fish effect as fed materials for Pacific white shock, Lipopeus vannamei.Aquaculture 303,28-33.

Lamb,C.F.(2007).Gustation and Feeding Behaviour.In Food Intake in Fish,pp.108-130:Blackwell Science Ltd.

Ng, W.K. and Romano, N. (2013). A review of the circulation of degraded tilt through the culture cycle. reviews in Aquaculture 5,220- & 254.

Richard, L., target, A., Rigolet, V., Kaushik, S.J. and Geurden, I. (2011.) Availability of essential amino acids, nutrient customization and growth in great black maker shrimp, Penaeus monodon, following fibrous replacement by protein acquisition 322, 109-.

Shiau, S.Y., Kwok, C.C., Hwang, J.Y., Chen, C.M., and Lee, S.L. (1989). Replacing of Fishmeal with Soybean mean in Male titanium (Oreochromys niloticus) fingerprint at a Suboptical Protein level.journal of the World Aquaculture Society 20, 230-.

Sookying, D. and Davis, D.A. (2011). Pond production of Pacific white shock (Litopenaeus vannamei) fed high levels of sobeban medium in variance combinations 319, 141-.

Su a rez, J.A., Gaxila, G., Mendoza, R., Cadavid, S., Garcia, G., Alanis, G., Su a rez, A., Failleace, J., and Cuzon, G. (2009) Substistition of real time with plant resources and energy budget for white stub kiln limb Lipopeaeus vannamei (Boone,1931) Aquulture 289, 118-.

Sukmanomon, S., Kamonra, W., Poompuang, S., Nguyen, T.T.T., Bartley, D.M., May, B, and Na-Nakorn, U.S. (2012). Genetic changes, intra-and inter-specific transcription in failed palapia (Oreochrosis nilotius) in Thailand, Aquaculture 324-.

Xie, S.,. w., Liu, Y.,. j., Zeng, S.,. Niu, J., and Tian, L. -x. (2016.). Partial replacement of fish-medium by protein concentrate and protein based protein blend for a jravenie Pacific white shred, Litopenaeus vannamei.Aquulture 464,296-302.

Claims (14)

1. A method of feeding a low-feeding fish, comprising feeding the fish a feed product comprising a dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

2. A method of feeding a low-feeding fish, comprising the step of feeding the fish a feed product comprising a dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

3. Use of a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria in an amount effective to provide nutrition to a low-feeding fish.

4. Use of dried biomass comprising a mixed microbial population comprising microalgae and bacteria as a feed attractant or feed stimulant for a low-grade feeding fish.

5. A method of improving the appeal and/or palatability of a feed product to a subfamily fish, comprising feeding the feed product to the fish in the presence of dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

6. A method of stimulating a subfamily fish to increase its food intake, comprising providing to the fish a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

7. A method of increasing the growth rate or food intake of a hypophagic fish, comprising providing to the fish a feed product comprising a dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

8. The method or use according to any one of claims 1 to 7 wherein the feed product further comprises one or more nutritional ingredients.

9. The method or use according to any one of claims 1 to 8, wherein the feed product comprises the dried biomass in an amount of 5% w/w to about 15% w/w.

10. The method according to any one of claims 1, 2, 5, 6, 8 or 9 or the use according to any one of claims 3, 4, 8 or 9, wherein the low-grade fish is fish of the tilapia family of the family limidae.

11. The method or use according to claim 10, wherein the fish of the tilapia family of the Pacifidae is Nile tilapia (Nile mouth hatch non-crucian).

12. A method of fishing fish using a bait, wherein the bait comprises a dried biomass comprising a mixed microbial population comprising microalgae and bacteria.

13. Use of a dried biomass comprising a mixed population of microorganisms including microalgae and bacteria as a bait for aquatic animals.

14. A bait comprising a dried biomass comprising a mixed microbial population comprising microalgae and bacteria.

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