Feed additive for improving performance of farming animals

Abstract

The present invention is in the field of feed additives for animals, e.g., farming animals, in particular feed additives to be added to animal feed to obtain an improved performance, e.g., increased average daily gain, and / or increased feed efficiency (i.e., decreased feed conversion ratio), of such animals. Moreover, it relates to a culture medium and a method of culturing cells.

Description

[0001] Title: Feed additive for improving performance of farming animals

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of feed additives for use in animal feed, in particular for use in improving performance of farming animals. Moreover, the present invention is in the field of cull culturing, e.g., of cell culturing of muscle cells, e.g., for preparing cultured meat.

[0004] BACKGROUND OF THE INVENTION

[0005] Antimicrobial agents have been used as antibiotic growth promoters in livestock feed for many decades. However, resistance to such antimicrobial agents is now considered a serious threat to human health. The use of antimicrobial agents as a growth promoter in livestock feed has been fully banned in the European Union since January 2006. At the same time, the demand for protein from animal origin is increasing, fuelled by the human population increase. Additionally, the food production needs to be more sustainable, requiring that more is produced with less. Taken together, animal production needs to be more and more effective.

[0006] These developments have initiated a surge of exploring feed additives that improve performance of farming animals yet do not induce resistance to bacteria. Phytogenic feed additives, also known as botanicals, are substances of plant origin added to animal diets with the aim of improving animal performance. Essential oils, herbs and spices all serve as sources for bioactive ingredients, e.g. phenols, polyphenols, flavonoids, terpenoids and others.

[0007] It is an object of the present invention to provide a composition that enhances glucose uptake in cells, that increases cell proliferation and / or that enhances performance of farming animals. It is also an object of the present invention to provide a method of increasing body weight gain, increasing average daily gain, increasing feed intake and / or of decreasing Feed Conversion Ratio (FCR), i.e. , of improving feed efficiency, in a farming animal. Further, it is an object of the present invention to provide a composition that increases glucose uptake in muscle cells and / or increases cell proliferation in muscle cells. Such composition may be part of a cell culture medium, particularly for use in culturing muscle cells for cultivated meat.

[0008] SUMMARY OF THE INVENTION

[0009] The present disclosure provides an animal feed comprising Rubus fruticosus leaves, or an extract thereof. The Rubus fruticosus leaves may be dried, and optionally ground.

[0010] The Rubus fruticosus leaves may be included in an amount of about 1 mg / kg to about The extract may be selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethylacetate extract, acetonic extract, hexane extract, or a supercritical CO2 extract, or a mixture of any of these.

[0011] The present disclosure also provides use of Rubus fruticosus leaves, or an extract thereof, as a feed additive.

[0012] Said feed additive may be supplied with written instructions to include it into animal feed in an amount of Rubus fruticosus leaves, of about 1 mg / kg to about 10000 mg / kg, preferably 5 mg / kg to about 5000 mg / kg, of feed, or an equivalent amount of extract.

[0013] The feed additive may be included in a premix.

[0014] The present disclosure further provides use of Rubus fruticosus leaves, or an extract thereof, or a composition comprising Rubus fruticosus leaves, or an extract thereof, or an animal feed as taught herein for increasing performance of an animal.

[0015] The present disclosure also provides use of Rubus fruticosus leaves, or an extract thereof, or a composition comprising Rubus fruticosus leaves, or an extract thereof, or an animal feed as taught herein for increasing feed intake, increasing average daily gain, increasing feed efficiency (i.e., decreasing feed conversion ratio), increasing relative growth rate, and / or increasing specific growth rate.

[0016] The extract may be selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethylacetate extract, acetonic extract, hexane extract or mixtures of these solvents, or a supercritical CO2 extract, or a mixture of any of these.

[0017] The feed additive may be intended for inclusion in feed for farming animals.

[0018] The farming animals may be selected from the group consisting of poultry, swine, ruminants, e.g., beef cattle and dairy cattle, fish, e.g. salmon, trout, seabream, sea brass, tilapia, tuna, and the like, and crustaceans, e.g., shrimp.

[0019] The present disclosure further provides use of an extract of Rubus fruticosus leaves, for culturing cells, e.g., muscle and / or fat cells, for increasing glucose uptake in cells, e.g., muscle and / or fat cells, and / or for increasing cell proliferation, e.g., muscle cell proliferation and / or fat cell proliferation.

[0020] The cells may be cultured for the purpose of preparing cultured meat, including cultured fish.

[0021] The present disclosure further provides a cell culture medium comprising an extract of Rubus fruticosus leaves.

[0022] Additionally, the present disclosure is concerned with a method of culturing cells, e.g., muscle and / or fat cells, said method comprising the step of providing a cell culture medium as taught herein, inoculating said culture medium with cells to be cultured, and allowing said cells to proliferate. GENERAL DEFINITIONS

[0023] In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references cited herein are incorporated herein in their entirety by reference.

[0024] The term “leaves” when referring to a plant refers to any usually flattened green outgrowth from the stem of a vascular plant. As the primary sites of photosynthesis, leaves manufacture food for plants, which in turn ultimately nourish and sustain all land animals. Botanically, leaves are an integral part of the stem system. They are attached by a continuous vascular system to the rest of the plant so that free exchange of nutrients, water, and end products of photosynthesis (oxygen and carbohydrates in particular) can be carried to its various parts.

[0025] The term ‘farming animal’ refers to animals that are kept or raised for agricultural purposes, like for consumption or to generate income by for example meat, eggs or milk products. Farming animals can be grouped based on their digestive system. Such groups include monogastric, ruminant and pseudo-ruminant animals.

[0026] The term “pellets” or “feed pellets” as used herein refers to small particles or a body typically created by compressing an original material, for instance a mixture of raw feed material, typically fermentable feed ingredients such as grains, cereals, legumes, roughage, and the likes. Feed pellets may also comprise other feed ingredients such as meat meal, fish meal, bone meal, by-process products, oil, fat, fillers or any mixture thereof, etc., as well as minerals, vitamins and trace elements and others. Animal feed pellets vary in their composition as well as structural properties (e.g. hardness, density, durability, shape, size, etc.) depending on the nutritional needs, eating habits, digestive system (monogastric system, ruminant digestive system, etc.) and habitat (e.g., aquatic, terrestrial, domestic, etc.) of the animal for which the feed pellet is intended. Animal feed pellet may have any size, shape (e.g. round, rectangular, cylindrical, etc), weight and / or length. It is understood that the weight of the feed pellet will depend on the feed pellet composition per se (e.g. some ingredients have a greater weight or density than others) as well as the shape, size and length of the finished feed pellet product. It is known that the size, shape, weight and / or length of the feed pellet will influence pellet durability. This is true for any method for making feed pellets, including method as taught herein. It is further commonly agreed in the field of agriculture and animal nutrition that animals (e.g. young and adult livestock animals like beef (e.g. beef calf), dairy cows (e.g. dairy calf), and pigs, etc.) benefit more or make better gains (e.g. weight gain, increased height, enhanced growth curve) on pelleted feed than a meal ration because pelleted feed is in a more concentrated, readily edible and palatable form than meal or mash ration. Pelleted feed has been shown to ease food intake and minimize feed waste during the eating process. It was shown that most animals, if given the choice between the same feed in a pellet or a mash form, will prefer the pellet form. Animal feed pellets are typically produced on an industrial scale using for example a pelleting process. The skilled person is well-acquainted with processes for producing animal feed pellets.

[0027] The term “secondary plant constituents” as used herein refers to specialized compounds present in plants that do not aid in the growth and development of plants but are required for the plant to survive in its environment. Such secondary plant constituents may be essential for communicating with other organisms in mutualistic (e.g. attraction of beneficial organisms such as pollinators) or antagonistic interactions (e.g. deterrent against herbivores and pathogens). They may further assist in coping with abiotic stress such as increased UV- radiation. The broad functional spectrum of specialized metabolism is still not fully understood.

[0028] The term “premix” as used herein refers to a complex mixture of vitamins, minerals, trace elements and other feed additives that are typically (but not always) incorporated at levels between 0.2 and 0.5 wt% in the animal feed. Adequate animal nutrition requires animal feed compositions consisting of macro-ingredients (“bulk ingredients”) and micro-ingredients, sometimes incorporated at ppm levels (e.g., vitamins, (trace) minerals, organic acids, short chain fatty acids, pigments, enzymes, probiotics, etcetera). To secure that these microingredients are mixed with macro-ingredients in a homogeneous way, an intermediate dilution step is required via a so-called premix (or “premixture”). Premixtures are legally defined as mixtures of feed additives or mixtures of one or more feed additives with feed materials or water used as carriers, not intended for direct feeding to animals (Regulation (EC) No 1831 / 2003).

[0029] The term “cell culture medium” as used herein refers to an aqueous solution of nutrients which can be used for growing cells over a prolonged period of time. Typically, cell culture media include the following components: A source of energy, which will be usually a carbohydrate compound, e.g., glucose, amino acids, preferably the basic set of amino acids, including all essential amino acids, vitamins and / or other organic compounds which are required at low concentrations, free fatty acids, and inorganic compounds including trace elements, inorganic salts, buffering compounds and nucleosides and bases.

[0030] The term ‘about’, as used herein indicates a range of normal tolerance in the art, for example within 2 standard deviations of the mean. The term “about” can be understood as encompassing values that deviate at most 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, or 0.01% of the indicated value.

[0031] The terms “comprising” or “to comprise” and their conjugations, as used herein, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb “to consist essentially of” and “to consist of”.

[0032] Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] This patent application has been drafted into sections. However, these sections should not be read in isolation. Unless otherwise specified, each section is to be read in combination with the other sections. This means, for example, that all of the animal feeds and / or feed additive compositions described in the “Animal feed and / or additive composition” section are intended to be read with the “Methods of using the animal feed and / or feed additive” section (i.e. , all animal feeds recited in the first section are suitable for the methods described in the latter section). The various optional and preferred features can also be combined, even when taken from different parts of the specification. Likewise, all “aspects” and “embodiments” can be combined. No separation of embodiments is intended, unless explicitly stated.

[0035] The present inventors have surprisingly found that a composition comprising extracts of Rubus fruticosus leaves enhanced glucose uptake by cells in vitro. Moreover, it was found that extracts of Rubus fruticosus leaves increased cell proliferation in vitro.

[0036] Moreover, the present inventors have surprisingly found that inclusion of Rubus fruticosus leaves in a relatively small amount in broiler feed, increases their performance in terms of feed efficiency. Further, the present inventors have found that inclusion of Rubus fruticosus leaves in salmon feed increased feed intake.

[0037] Animal feed and / or additive composition

[0038] Thus, the present disclosure provides an animal feed comprising Rubus fruticosus leaves, or an extract thereof. The present disclosure also provides an feed additive comprising Rubus fruticosus leaves, or an extract thereof.

[0039] The leaves may be used fresh, i.e., as they are after harvest, or may be dried by any means known in the art. The skilled person knows suitable drying technologies. For example, drying may take place using a drum drier, belt dryer, dehydrator drying, vacuum drying, microwave drying, or infrared-drying, or natural drying (sun drying). Thus, the feed additive composition or animal feed taught herein may comprise dried Rubus fruticosus leaves.

[0040] Prior to or after drying, preferably after drying, the leaves may be ground to any suitable size. For example, the fresh or dried leaves may be ground to an average particle size of smaller than 2 mm, preferably smaller than 1.5 mm, even more preferably smaller than 1 mm, yet more preferably smaller than 0.8 mm, even more preferably smaller 0.6 mm, most preferably smaller than 0.5 mm.

[0041] The animal feed or feed additive may comprise leaves only, may comprise an extract of leaves only, or may comprise a combination of leaves and extract of leaves.

[0042] The R. fruticosus used herein may be any variety of the respective species.

[0043] The animal feed may be in any form suitable for ingestion by the relevant animal. For example, poultry feed may be in the form of crumble, mash, or pellets. Swine feed may be in the form of meal or pellets. Feed for fish and crustaceans is usually in the form of extruded or pressed pellets but may also be in the form of flakes. In an embodiment, the animal feed is in the form of a crumble, mash, meal, flakes, or pellets, which pellets may be extruded or may be pressed.

[0044] The animal feed generally comprises protein, fat, carbohydrates, minerals and vitamins. It may further comprise organic acids, and other feed additives. The skilled person knows how to formulate a suitable animal feed depending on the target animal.

[0045] The R. fruticosus leaves, preferably in dried and ground form, may be included in animal feed in an amount of about 1 mg / kg to about 10000 mg / kg of feed, preferably about 5 mg / kg to about 8000 mg / kg of feed, more preferably about 10 mg / kg to about 6000 mg / kg of feed, even more preferably about 15 mg / kg to about 4000 mg / kg of feed, yet more preferably about 20 mg / kg to about 3000 mg / kg of feed, again more preferably about 25 mg / kg to about 2000 mg / kg of feed, such as about 30 mg / kg to about 1500 mg / kg of feed, about 40 mg / kg to about 1200 mg / kg of feed, about 50 mg / kg to about 1000 mg / kg of feed, or about 60 mg / kg to about 800 mg / kg of feed.

[0046] Alternatively, or additionally, an equivalent amount of extract of R. fruticosus leaves may be used. Plant extraction is a process that aims to extract certain components (so-called secondary plant constituents, including alkaloids, terpenoids, saponins, phenolic compounds, flavonoids, and / or tannins) present in plants. It is a solid / liquid separation operation: a solid object (in the present case leaves of R. fruticosus, optionally dried and / or ground) is placed in contact with a fluid (the solvent). The plant components of interest are then solubilised and contained within the solvent. The solution thus obtained is the desired extract. The solvent may eventually be eliminated, although this is not required. The solvent may be any solvent suitable for preparing plant extracts for use in animal feed. Suitable solvents include, without limitation, polar solvents (e.g., water, alcohols such as ethanol, methanol and isopropanol), intermediate polar solvent (e.g., acetone, dichloromethane), and nonpolar solvents (e.g., ethyl acetate, hexane, ether, chloroform). The skilled person may select a single solvent or two or more solvents to prepare an extract in accordance with the present disclosure. In general, extraction procedures include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, superficial extraction , ultrasound-assisted, and microwave-assisted extractions, supercritical fluid extraction (e.g. with supercritical carbon dioxide), accelerated solvent extraction, and solidphase extraction. Fractionation and purification of phytochemical substances may be achieved through application of various techniques such as liquid-liquid partitioning, cross-flow filtration techniques and chromatographic methods, such as paper chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography, centrifugal partitioning chromatography. Finally, compounds obtained in the extract may be characterized using diverse identification techniques such as mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy, or any combination thereof. The skilled person is capable of selecting a suitable extraction method, and optionally a suitable characterization method.

[0047] The amount of extract or extracts to be incorporated into the animal feed and / or feed additive taught herein may be determined by equating the content of one or more secondary plant constituents in the extract to those in the R. fruticosus leaves. An amount of extract that provides a content of one or more secondary plant constituents that is equivalent to the content of said one or more secondary plant constituents in the indicated amount of R. fruticosus leaves may thus be incorporated into said animal feed.

[0048] In a suitable embodiment, the extract may be prepared using a polar solvent such as water, ethanol, or a mixture thereof.

[0049] In an embodiment, the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethylacetate extract, acetonic extract, hexane extract, or a supercritical CO2 extract, or a mixture of any of these. Alternatively, a mixture of solvents may be used for extraction, as set forth above.

[0050] Moreover, the present disclosure provides use of R. fruticosus leaves or an extract thereof as a feed additive. The extract may be obtained as taught herein.

[0051] The feed additive taught herein may further comprise vitamins, minerals, organic acids, antioxidants, and / or pigments. Said feed additive may be provided in the form a premix. The feed additive or premix is preferably intended for inclusion in feed for farming animals or companion animals.

[0052] Said feed additive may be supplied with written instructions to include it into animal feed in an amount of the composition taught herein of about 1 mg / kg to about 10000 mg / kg of feed, preferably about 5 mg / kg to about 8000 mg / kg of feed, more preferably about 10 mg / kg to about 6000 mg / kg of feed, even more preferably about 15 mg / kg to about 4000 mg / kg of feed, yet more preferably about 20 mg / kg to about 3000 mg / kg of feed, again more preferably about 25 mg / kg to about 2000 mg / kg of feed, such as about 30 mg / kg to about 1500 mg / kg of feed, about 40 mg / kg to about 1200 mg / kg of feed, about 50 mg / kg to about 1000 mg / kg of feed, or about 60 mg / kg to about 800 mg / kg of feed, or, in case an extract of R. fruticosus leaves is used, an equivalent amount of extract. Methods of using the animal feed and / or feed additive

[0053] In an aspect, the present disclosure provides a method of feeding an animal, e.g., a farming animal or a companion animal, preferably a farming animal, with R. fruticosus leaves or an extract thereof, or an animal feed as taught herein.

[0054] In a further aspect, the present disclosure relates to use of R. fruticosus leaves or an extract thereof, or an animal feed as taught herein, for increasing or improving performance of an animal, preferably a farming animal.

[0055] In another aspect, the present disclosure is concerned with use of R. fruticosus leaves or an extract thereof, or an animal feed as taught herein, for increasing feed intake, increasing average daily gain, increasing weekly gain, increasing biomass yield, and / or increasing feed efficiency (i.e. , decreasing feed conversion ratio).

[0056] The present disclosure further provides a method of improving performance of animals, e.g., increasing feed intake, increasing average daily gain, increasing weekly gain, increasing biomass yield, and / or increasing feed efficiency (i.e., decreasing feed conversion ratio), said method comprising the step of administering to said animals R. fruticosus leaves, or an extract thereof, a composition comprising R. fruticosus leaves, or an extract thereof, or an animal feed as taught herein, e.g., comprising R. fruticosus leaves, or an extract thereof.

[0057] The animal feed and / or feed additive for use in the above methods may be as described hereinabove.

[0058] The extract may be as described hereinabove.

[0059] The animal may be a monogastric animal, a pre-ruminant animal, or a ruminant animal. The animals may be selected from farming animals and companion animals, and are preferably farming animals. The farming animals may be selected from the group consisting of poultry, swine, ruminants, e.g., beef cattle and dairy cattle, fish, e.g. salmon, trout, seabream, sea brass, tilapia, tuna, and the like, and crustaceans, e.g., shrimp. The companion animals may be selected from the group consisting of ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters.

[0060] In an embodiment, the benefits in performance obtained in animals fed the feed additive or animal feed taught herein are relative to results obtained with animals (of the same species) fed an identical animal feed yet without such feed additive or animal feed.

[0061] Cell culture medium I method of culturing cells

[0062] In an aspect, the present disclosure provides a cell culture medium comprising an extract of Rubus fruticosus leaves. The extract may be as taught herein.

[0063] In another aspect, the present disclosure provides a method of culturing cells, said method comprising the step of providing a cell culture medium as taught herein, inoculating said culture medium with cells to be cultured, and allowing said cells to proliferate. The present disclosure is also concerned with use of an extract of Rubus fruticosus leaves, for culturing cells, e.g., muscle cells, for increasing glucose uptake in cells, e.g., muscle cells, and / or for increasing cell proliferation, e.g., muscle cell proliferation.

[0064] In an embodiment, the cells are cultured for the purpose of preparing cultured meat or cultured fish.

[0065] The cells are preferably muscle and / or fat cells.

[0066] In a further aspect, the present disclosure provides a method of preparing cultured meat, said method comprising the step of providing a cell culture medium as taught herein, inoculating said culture medium with muscle and / or fat cells, and allowing said muscle and / or fat cells to proliferate.

[0067] In yet another aspect, the present disclosure provides a method of increasing glucose uptake in cells, such as muscle cells, and / or of increasing cell proliferation, e.g., muscle cell proliferation, said method comprising the step of culturing said cells in a culture medium comprising an extract of Rubus fruticosus leaves, e.g., a culture medium as taught herein.

[0068] The cell culture medium taught herein can be used in various cell culture processes. Cultivation of cells can be carried out in adherent culture, for instance in monolayer culture or preferably in suspension culture.

[0069] Cell culture media generally contain a mixture of amino acids, carbohydrates, preferably in the form of one or more sugars, (inorganic) salts, vitamins, buffering systems, other nutrients, and compounds which regulate the cell cycle. They are available either in a powder form, to be reconstituted in water, or as a liquid form from commercial suppliers. The culture medium may be in a liquid, optionally after reconstitution from a powder form, or solid form. The requirements for the various components of cell culture media may vary among cell types and / or cell lines. The skilled person knows how to select a suitable cell culture medium for culturing specific cell types or cell lines.

[0070] Amino acids are the building blocks of proteins, and thus are obligatory ingredients of all known cell culture media. Essential amino acids must be included in the culture media as cells cannot synthesize these by themselves. They are required for the proliferation of cells and their concentration determines the maximum achievable cell density. Nonessential amino acids may also be added to the medium to replace those that have been depleted during growth. Supplementation of media with non-essential amino acids may stimulate growth and prolongs the viability of the cells. The amino acids may be provided in the form of single amino acids, or may be provided in the form of dipeptides, oligopeptides, polypeptides, or a mixture of these.

[0071] In an embodiment, the amino acids are selected from the group consisting of L-arginine, L-leucine, L-isoleucine, glycine, L-phenylalanine, L-alanine, L-asparagine, L-serine, L-valine, L-histidine, L-proline, L-tryptophan, L-methionine, L-lysine, L-glutamic acid, and L-cysteine, or any combination thereof. In a preferred embodiment, the cell culture medium comprises at least L-Arginine, L-Cysteine, L-Cystine, L-Glutamine, L-Histidine, L-lsoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, either in free form or in the form of dipeptides or oligopeptides.

[0072] Carbohydrates in the form of sugars are typically the major source of energy. Most of the media contain glucose and / or galactose. Cell culture media may further comprise maltose and fructose.

[0073] Inorganic salt in the cell culture media may help to retain the osmotic balance and help in regulating membrane potential by providing sodium, potassium, and calcium ions. In an embodiment, the cell culture medium comprises sodium ions, potassium, ions, calcium ions, magnesium ions, zinc ions, copper ions, ferric ions, and / or selenium ions, preferably in the form of salts, e.g., in the form of NaCI, KCI, CaCI2-2H2O, NaH2PO4, MgSO4, ZnSO4-7H2O, CuSO4-5H2O, Fe(NO3)3-9H2O, and / or Na2SeO3.

[0074] Many vitamins are essential for growth and proliferation of cells. Vitamins cannot be synthesized in sufficient quantities by cells and are therefore important supplements required in tissue culture. In some embodiments, serum may be the major source of vitamins in cell culture. In other embodiments, the cell culture medium may comprise synthetic vitamins. In an embodiment, the culture medium comprises one or more of vitamin B12, vitamin A, vitamin E, riboflavin (vitamin B2), thiamine (vitamin B1), vitamin B6, pantothenic acid, folic acid, niacin, cyanocobalomin, pyridoxine, nicotinamide, and / or biotin. The vitamins may be present in any suitable form. For example, vitamin A may be present in the form of vitamin A acetate or any other vitamin A ester. The skilled person is capable of selecting suitable forms of the one or more recited vitamins.

[0075] Trace elements like copper, zinc, and selenium may be present in the cell culture medium taught herein, e.g., in the form as inorganic salts as recited above.

[0076] Regulating pH may be important for optimum cell culturing. Therefore, in an embodiment, the culture medium taught herein further comprises a suitable buffering agent to regulate pH of the culture medium. The skilled person is capable of selecting a suitable buffering system. Suitable buffering systems include, without limitation, Tris buffer, Tris-EDTA buffer, Tris-acetate-EDTA buffer, PBST buffer, HEPES buffer, citrate buffer, and the like.

[0077] A pH indicator such as phenol red may be included in the cell culture medium taught herein to allow constant monitoring of pH.

[0078] In an embodiment, the cell culture medium taught herein comprises between 25 and 2000 pg / ml, preferably between 50 and 1500 pg / ml, such as between 75 and 1000 pg / ml, or between about 80 and 800 pg / ml, of a Rubus fruticosus leaves extract obtainable by extracting about 1 g of dried and ground Rubus fruticosus leaves with about 8 g of solvent, preferably a polar solvent, even more preferably a solvent consisting of ethanol and water. Of course, an identical cell culture medium may be obtained using a different extraction grade of Rubus fruticosus leaves and different amounts of the extract. Such cell culture media are included in the scope of the present disclosure.

[0079] The cell culture medium taught herein may further comprise albumin, transferrin, and / or fibronectin. These proteins may be particularly important in serum-free media.

[0080] The use of cell culture media in the field of pharmaceutical or food industry, for instance for the production of therapeutically active recombinant polypeptides or for preparing cultured meat, does generally not allow the use of any material of biological origin due to safety and contamination issues. Therefore, the cell culture medium taught herein is preferably a serum- and / or protein-free medium. The term “serum- and / or protein-free medium” represents a fully chemically defined medium, containing no additives from animal source like tissue hydrolysates, e.g. fetal bovine serum or the like.

[0081] The osmolality of the cell culture medium taught herein may be set between about 280 and about 365 mOsm at the beginning of culturing but may gradually increase during culturing. Preferably the cell culture medium taught herein has an initial osmolality of between about 280 and about 365 mOsm / kg, such as between about 285 and about 360 mOsm / kg, between about 290 and about 355 mOsm / kg, between about 295 and about 350 mOsm / kg, or between 300 and 345 mOsm / kg.

[0082] The temperature of the cell culture is selected in a range where cells are viable and grow. A typical temperature for cell culturing is in the range between about 30° C. and about 38° C. For instance, the cells are initially grown at temperatures of about 36 to about 37° C. which is optimal for CHO cells. However, the exact temperature can be adapted to the needs of the cells and can be changed during culturing to allow their optimal viability, growth or production. The skilled person is capable of selecting suitable culturing conditions depending upon the cell type.

[0083] The present invention is further illustrated, but not limited, by the following examples. From the above discussion and the examples, one skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the teaching and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0084] EXAMPLES

[0085] EXAMPLE 1

[0086] In this example, the effect of 250 ppm dietary inclusion of dried Rubus fruticosus leaves on the productive performance of broiler chicken was investigated. Rubus fruticosus leaves came from cultivation. Cultivation and drying followed Good Agricultural and Collecting practices. The dried Rubus fruticosus leaves were grinded to a particle size of < 0.3 mm.

[0087] Diets, fed in mash form, were provided by age of bird: Starter feed fed from 0 day to 14 days, Grower feed fed from 15 days to 28 days and Finisher feed fed from 29 days to 42 days. The basal diets compositions were as set forth in Table 1.

[0088] Table 1. Basal diet compositions.

[0089] The experimental treatments were made as follow. The Control treatment birds received basal Starter feed, basal Grower feed and basal Finisher feed. The experimental treatment, birds received the basal diets, all supplemented with 250 g per metric ton of feed of dried ground Rubus fruticosus leaves.

[0090] Each experiment treatment was assigned to 12 pens per treatment.

[0091] Each pen (dimensions of each pen are 4' X 5') contained 1 water fountain and a feed tube. Birds started on new wood shavings. 34 Ross 308 day 0 chicks were allocated to each pean. Birds were distributed to pens on day 1 so that equal distribution of birds by body weight among the 108 study pens. Continuous lighting was provided Od to 42 days.

[0092] All broilers were weighed by pen when bird ages are 0, 14, 28 and 42d. Feed was weighed in as needed and weighed out when birds are weighed. The following data were collected during the study: broiler body weights (abbreviated BW) and feed intakes (abbreviated Fl) at day 14, day 28 and day 42.

[0093] Feed conversion is calculated for a pen as the total feed consumed divided by (sum of total of all body weight for the pen + sum total of all mortality weight for the pen).

[0094] Adjusted feed conversion ratio with final BW adjustment at day 42 is calculated as follows: Feed conversion + (2.22 kg - actual body weight) / 7. The statistical analysis was a one-way ANOVA with the following model Yy = p + Treatment + £y in which Y is the productive response which average is p, Treatment is a categorical variable with 3 levels (Control or Treatment 1 or Treatment 2) and E is the residual error. Means for the 3 levels of the Treatment were compared using the Fisher test with 95% confidence.

[0095] The results are presented in the following table.

[0096] The results show that the addition of 250 g in a metric ton of feed of Rubus fruticosus leaves in the diet of broiler chicken improve feed intake and body weight gain at all stages of the production process.

[0097] EXAMPLE 2

[0098] In this example, the effect of 250 ppm and 500ppm of dietary inclusion of dried Rubus fruticosus leaves on the productive performance of broiler chicken was investigated.

[0099] Rubus fruticosus leaves came from cultivation. Cultivation and drying followed Good Agricultural and Collecting practices. The dried leaves were grinded to a particle size of < 0.3 mm.

[0100] Diets, fed in mash form, were provided by age of bird: Starter feed fed from 0 day to 14 days, Grower feed fed from 15 days to 28 days and Finisher feed fed from 29 days to 42 days. The basal diets compositions were the following, expressed in kg / 100kg. The experimental treatments were made as follow. The Control treatment birds received basal Starter feed, basal Grower feed and basal Finisher feed. The treatment 1 birds received the basal diets, all supplemented with 250 g per metric ton of feed of dried ground Rubus fruticosus leaves. The treatment 2 birds received the basal diets, all supplemented with 500 g per metric ton of feed of dried ground Rubus fruticosus leaves.

[0101] Each experiment treatment was assigned to 12 pens per treatment.

[0102] Each pen (dimensions of each pen are 4' X 5') contained 1 water fountain and a feed tube. Birds started on wood shavings. 34 Ross 308 day 0 chicks were allocated to each pean. Birds were distributed to pens on day 1 so that equal distribution of birds by body weight among all the study pens. Continuous lighting was provided 0 d to 42 days.

[0103] All broilers were weighed by pen when bird ages are 0, 14, 28 and 42d. Feed was weighed in as needed and weighed out when birds are weighed. The following data were collected during the study: broiler body weights and feed intakes (abbreviated Fl) at day 14, day 28 and day 42. These data was collected to calculate feed conversion at day 42 (abbreviated D42 FCR), obtained for a pen as the total feed consumed divided by (sum of total of all body weight for the pen + sum total of all mortality weight for the pen). Adjusted feed conversion ratio with final BW adjustment at day 42 (abbreviated DO-42 MFCR) is calculated as follows: Feed conversion + (2.22 kg - actual body weight) 1 7.

[0104] The results are presented in Table 2.

[0105] Table 2. FCR and bodyweight-adjusted FCR upon treatment with 250 or 500 ppm dried and ground Rubus fruticosus leaves.

[0106] The results show that the addition of 250 ppm and 500 ppm of dried ground leaves of Rubus fruticosus improved the performance of broiler chicken by improving their feed efficiency with more than 4.5%.

[0107] EXAMPLE 3

[0108] The effect of dried Rubus fruticosus leaves on the productive performance of salmons was investigated.

[0109] Rubus fruticosus leaves came from cultivation. Cultivation and drying followed Good Agricultural and Collecting practices. The Rubus fruticosus leaves were grinded to a particle size of < 0.3 mm. A basal diet was formulated to meet the nutritional requirements of Atlantic salmon at ca. 42% crude protein, 32 % crude lipid, 8 % moisture, 24.630 kj / g Energy and 5.4 % ash levels. The experimental treatments were made as follows. The Control treatment salmons received the basal diet. The Treatment salmons received the basal diet supplemented with 290 g per metric ton of feed of dried ground Rubus fruticosus leaves in place of wheat. All macro and micro ingredients, including dried ground Rubus fruticosus leaves ingredients were dry mixed before the complete mix was extruded to create a 7 mm pellet.

[0110] Atlantic salmon of an average weight of 750g were involved in the experiment with 2 treatments (Control and T reatment) in triplicate of 3-meter tanks with 75 fish per tank. The water temperature was maintained at 12°C with a salinity of 31 ppt. Feed was provided to the salmons 3 times per day. Light regime was 24 hours with light. The experiment lasted for 141 days. Salmons were weighted on the first day (and provided mean initial weight, expressed in g), as well as on the last day of the experiment (which provided mean final weight, expressed in g). The number of feeding days was 141. Other outcomes were measured: the final biomass (expressed in g) and the total weight of dead fish (expressed in g), and the total feed eaten (expressed in g). Specific growth rate (SGR) and feed intake, both expressed in % per day were calculated as follows. cro - / Mean final weight / \(l / number of feeding days)1

[0111] “ I / Mean initial weight J

[0112] Table 3 shows the result of the experiment.

[0113] Table 3. Feed intake for control (no addition of Rubus fruticosus leaves) and Treatment (addition of 290 ppm dried and ground Rubus fruticosus leaves).

[0114] The inclusion of 290 ppm of Rubus fruticosus leaves increased feed intake by 4.2% in salmons.

[0115] EXAMPLE 4. MUSCLE GROWTH IN VITRO

[0116] In this example, the effect of ground Rubus fruticosus leaves on muscle cell proliferation and glucose uptake my muscle cells in vitro was determined.

[0117] 1 g of powdered Rubus fruticosus leaves were extracted in 8 g of 50% (w / w) ethanol / water to provide a R. fruticosus leaf extract.

[0118] Two outcomes were used: protein content to assess muscle cell proliferation and glucose uptake by the muscle cell. Mouse muscle cells (C2C12) were seeded and grown to confluency (3 days). Cells were differentiated for 4 days to form myotubes and were then treated with the plant extracts for 24 hours. After supplementation, glucose was depleted for 24 hours followed by serum depletion overnight. After washing, a fluorescent glucose analog for monitoring glucose uptake into living cells called 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1 ,3-diazol-4-yl)-Amino)-2-Deoxyglucose) was added to the medium. After 30 min fluorescence was measured after washing and lysing the cells. Metformin was used as positive control; medium and vehicle controls were used as negative controls.

[0119] The determination of the protein content was used as a mean to identify cell proliferation in the applied in vitro model. The first supplementation of the C2C12 mouse myoblast cell line with the plant extracts was performed 24 hours after seeding. After five days, differentiation of the cells was initiated. The second supplementation with the plant extracts was performed 24 hours after the beginning of the differentiation. At day 3 of the differentiation phase, the last supplementation was performed. Cells were lysed 7 days after the beginning of the differentiation and the protein content was determined using a Bradford assay. A medium and a vehicle control were performed with each batch. Insulin-like growth factor (IGF) was used as a positive control. The assay was carried out in triplicates.

[0120] Table 4 shows the effect on glucose uptake, expressed as percent of the control treatment, each mean is the average of 3 replicates.

[0121] Table 4. The effect of Rubus fruticosus leaves extract on glucose uptake in C2C12 mouse myoblast cell line, expressed as a percentage relative to the control (no addition of R. fruticosus leaves extract).

[0122] In a separate experiment, following the same protocol, glucose uptake by the muscle cell of 300 pg / ml of Rubus fruticosus leaves extract was measured again. This second experiment confirmed that 300 pg / mL of Rubus fruticosus leaves extract enhances glucose uptake by the muscle cell by 34.8%.

[0123] Table 5 shows the mean muscle cell proliferation, expressed as percentage of the control treatment. Each mean value is the average of 6 values. Table 5. Mean values for muscle cell proliferation, expressed as percentage of the control treatment. Each mean value is the average of 6 values.

[0124] The addition of at least 250 pg / mL of Rubus fruticosus leaves extract increased muscle cell proliferation by 3.8%.

[0125] EXAMPLE 5

[0126] The effect of the dietary inclusion of Rubus dried and ground fructicosus leaves on the growth of shrimps was investigated.

[0127] Rubus fructicosus leaves came from cultivation. Cultivation and drying followed Good Agricultural and Collecting practices. The Rubus fructicosus leaves were grinded to a particle size of < 0.3 mm. The basal diet composition was as set forth in Table 6.

[0128] Table. 6. Basal diet composition of extruded shrimp feed.

[0129] The diets were produced as 2.0 ± 0.1 mm extruded pellets.

[0130] The feed production procedure followed the normal guidelines for shrimp feed extrusion. First, all dried raw materials (except micro-ingredients) were ground through a 1 mm (50 Hz) grinder screen. Next, ingredients were weighed following the formula specifications. After the extrusion, the pellets were dried until the moisture content was below 10%.

[0131] The experimental treatments were made as follow. The ‘Control’ shrimps received basal shrimp feed. ‘Treatment T shrimps received the basal diet supplemented with 0.75 kg per metric ton of feed of dried and ground Rubus fruticosus leaves. ‘Treatment 2’ shrimps received the basal diet supplemented with 1 kg per metric ton of feed of dried and ground Rubus fructicosus leaves. L. vannamei shrimps with initial weight of 4g were housed in 225L glass tanks filled with stocking density of 32 shrimps per meter square. Artificial seawater was prepared by adding a commercial salt mixture to purified water at 20 g / L. The tanks were equipped with individual mechanical / biological filter. No water was shared between the tanks. Water quality was maintained by filtration, aeration and regular water changes. Water temperature was kept at 30°C. The room was illuminated 12h a day. Shrimps were fed 6 times a day. Shrimps were fed with the control or different treatment diets.

[0132] The performance responses measured in this experiment were:

[0133] Specific Growth Rate (SGR), in % / d - Weekly growth, in g per week

[0134] Feed Conversion Ratio (FCR)

[0135] The results are shown in Table 7 below. SGR, weekly growth and FCR were all improved in shrimp treated with Rubus fruticosus leaves compared to the control. Table 7. Performance responses in shrimp.

Claims

CLAIMS1. Animal feed comprising Rubus fruticosus leaves.

2. Animal feed according to claim 1, wherein the Rubus fruticosus leaves are dried, and optionally ground.

3. Animal feed according to claim 1 or 2, wherein the Rubus fruticosus leaves, are included in an amount of about 1 mg / kg to about 10 g / kg of feed.

4. Use of Rubus fruticosus leaves as a feed additive.

5. Use according to claim 4, wherein said feed additive is supplied with written instructions to include it into animal feed in an amount of about 1 mg / kg to about 10000 mg / kg, preferably 5 mg / kg to about 5000 mg / kg, of feed.

6. Use according to claim 4 or 5, wherein the feed additive is included in a premix.

7. Use of Rubus fruticosus leaves, or an extract thereof, or a composition comprising Rubus fruticosus leaves, or an extract thereof, or an animal feed according to any one of claims 1-3 for increasing performance of an animal.

8. Use of Rubus fruticosus leaves, or an extract thereof, or a composition comprising Rubus fruticosus leaves, or an extract thereof, or an animal feed according to any one of claims 1-3 for feeding an animal, increasing feed intake, increasing average daily gain, increasing feed efficiency (i.e., decreasing feed conversion ratio), increasing relative growth rate, and / or increasing specific growth rate.

9. Use according to any of claims 7 or 8, wherein the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethylacetate extract, acetonic extract, hexane extract or mixtures of these solvents, or a supercritical CO2 extract, or a mixture of any of these.

10. Use according to any one of claims 4-9, wherein the feed additive is intended for inclusion in feed for farming animals.

11. Use according to claim 10, wherein the farming animals are selected from the group consisting of poultry, swine, ruminants, e.g., beef cattle and dairy cattle, fish, e.g. salmon, trout, seabream, sea brass, tilapia, tuna, and the like, and crustaceans, e.g., shrimp.

12. Use of an extract of Rubus fruticosus leaves, for culturing muscle and / or fat cells, for increasing glucose uptake in cells, e.g., muscle and / or fat cells, and / or for increasing cell proliferation, e.g., muscle cell proliferation and / or fat cell proliferation.

13. Use according to claim 12, wherein the cells are cultured for the purpose of preparing cultured meat or cultured fish.

14. Cell culture medium comprising an extract of Rubus fruticosus leaves.

15. Method of culturing muscle and / or fat cells, said method comprising the step of providing a cell culture medium according to claim 14, inoculating said culture medium with cells to be cultured, and allowing said cells to proliferate.

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