Feed additive for improving performance of bred animals

Abstract

The present invention is in the field of feed additives for animals (e.g., bred animals), in particular feed additives added to animal feed to achieve improved performance (e.g., increasing daily weight gain and / or increasing feed efficiency (i.e., reducing feed conversion)) of such animals. In addition, the invention also relates to a culture medium and a cell culture method.

Description

Technical Field

[0001] This invention relates to the field of feed additives for animal feed, particularly for improving the performance of farmed animals. Furthermore, this invention also relates to cell culture, such as muscle cell culture, for example, in the preparation of cultured meat. Background Technology

[0002] Antimicrobial agents have been used as antibiotic growth promoters in livestock feed for decades. However, resistance to these antimicrobial agents is now considered a serious threat to human health. Since January 2006, the European Union has completely banned the use of antimicrobial agents as growth promoters in livestock feed. Simultaneously, driven by population growth, the demand for animal-derived protein is increasing. Furthermore, food production needs to be more sustainable, requiring more output with less input. In short, animal production needs to become increasingly efficient.

[0003] These developments have sparked a surge in the development of feed additives that improve the performance of farmed animals without inducing bacterial resistance. Plant-derived feed additives, also known as plant-based products, are substances of plant origin added to animal diets for the purpose of improving animal performance. Essential oils, herbs, and spices are all used as sources of bioactive components such as phenols, polyphenols, flavonoids, terpenes, and others.

[0004] The object of this invention is to provide compositions that enhance glucose uptake in cells, increase cell proliferation, and / or enhance the performance of farmed animals. Another object of this invention is to provide methods for increasing body weight gain, increasing daily weight gain, increasing feed intake, and / or reducing feed conversion ratio (FCR) (i.e., increasing feed efficiency) in farmed animals. Further, the object of this invention is to provide compositions that increase glucose uptake in muscle cells and / or increase cell proliferation in muscle cells. Such compositions can be part of a cell culture medium, particularly for culturing muscle cells to prepare cultured meat. Summary of the Invention

[0005] This disclosure provides animal feed comprising blackberry leaves or extracts thereof. The blackberry leaves may be dried and optionally pulverized.

[0006] Blackberry leaves, or an equivalent amount of extract, are included in amounts ranging from about 1 mg / kg of feed to about 10 g / kg of feed.

[0007] The extract may be selected from the group consisting of free water extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, acetone extract, hexane extract or supercritical CO2 extract, or any mixture thereof.

[0008] This disclosure also provides the use of blackberry leaves or extracts thereof as a feed additive.

[0009] The feed additive may be provided with written instructions to include the feed additive in animal feed in an amount of blackberry leaves or an equivalent amount of extract, in a quantity of about 1 mg / kg feed to about 10,000 mg / kg feed, preferably 5 mg / kg feed to about 5,000 mg / kg feed.

[0010] Feed additives may be included in premixes.

[0011] This disclosure further provides the use of blackberry leaves or extracts thereof, or compositions comprising blackberry leaves or extracts thereof, or animal feed as taught herein, for increasing animal performance.

[0012] This disclosure also provides the use of blackberry leaves or extracts thereof, or compositions comprising blackberry leaves or extracts thereof, or animal feed as taught herein, for increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, and / or increasing specific growth rate.

[0013] The extract may be selected from the group consisting of a free water extract, an ethanol extract, a methanol extract, an isopropanol extract, an ethyl acetate extract, an acetone extract, a hexane extract, or a mixture of these solvents or a supercritical CO2 extract, or any mixture thereof.

[0014] Feed additives may be intended to be included in the feed used for farmed animals.

[0015] Farmed animals may be selected from the following groups: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia and tuna; and crustaceans, such as shrimp.

[0016] This disclosure also provides the use of blackberry leaf extract for culturing cells (e.g., muscle cells and / or adipocytes), for increasing glucose uptake in cells (e.g., muscle cells and / or adipocytes), and / or for increasing cell proliferation (e.g., muscle cell proliferation and / or adipocyte proliferation).

[0017] Cells can be cultured for the purpose of preparing cultured meat (including cultured fish).

[0018] This disclosure further provides a cell culture medium comprising an extract of blackberry leaves.

[0019] Furthermore, this disclosure relates to a method for culturing cells (e.g., muscle cells and / or adipocytes), the method comprising the steps of: providing a cell culture medium as taught herein, inoculating the culture medium with cells to be cultured, and proliferating the cells.

[0020] Conventional definition

[0021] Numerous terms are used in the following description and examples. The following definitions are provided to provide a clear and consistent understanding of the specification and claims (including the scope of such terms). 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 pertains. All disclosures of publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety.

[0022] When referring to plants, the term "leaf" refers to any typically flat, green growth that emerges from the stem of a vascular plant. As the primary site of photosynthesis, leaves produce nutrients for the plant, which in turn ultimately nourishes and sustains all terrestrial animals. Botanically, leaves are an integral part of the stem system. They are connected to the rest of the plant via a continuous vascular system, allowing for the free exchange and transport of nutrients, water, and the end products of photosynthesis, particularly oxygen and carbohydrates, throughout the plant.

[0023] The term "farmed animal" refers to animals raised or bred for agricultural purposes, such as for consumption or to generate income through products like meat, eggs, or dairy. Farmed animals can be classified based on their digestive systems. This classification includes monogastric animals, ruminants, and pseudoruminants.

[0024] As used herein, the term "pellet" or "feed pellet" refers to small particles or objects typically produced by pressing raw materials, such as feed ingredients (usually fermentable feed components like grains, cereals, legumes, and roughage). Feed pellets may also include other feed components, such as meat meal, fish meal, bone meal, by-products, oils, fats, fillers, or any mixture thereof, as well as minerals, vitamins, trace elements, and other substances. Animal feed pellets vary in their composition and structural properties (e.g., hardness, density, durability, shape, size, etc.) depending on the nutritional needs, diet, digestive system (monogastroparous, ruminant, etc.) and living environment (e.g., aquatic, terrestrial, domesticated, etc.) of the animal to which the feed pellets are intended. Animal feed pellets can have any size, shape (e.g., round, rectangular, cylindrical, etc.), weight, and / or length. It should be understood that the weight of a feed pellet will depend on the feed pellet components themselves (e.g., some components have a greater weight or density than others) and the shape, size, and length of the finished feed pellet product. It is known that the size, shape, weight, and / or length of feed pellets affect pellet durability. This holds true for any method used to manufacture feed pellets, including those taught herein. It is also widely accepted in the fields of agriculture and animal nutrition that animals (e.g., young and adult livestock, such as beef cattle (e.g., beef calves), dairy cattle (e.g., milk calves), and pigs) derive more benefits or achieve better gains (e.g., weight gain, height gain, enhanced growth curve) from pelleted feed compared to meal rations because pelleted feed is a more concentrated, palatable, and palatable form compared to meal rations. Pelleted feed has been shown to facilitate feed intake and minimize feed waste during consumption. It has also been demonstrated that, given the choice between the same feed in pellet or meal form, most animals prefer the pellet form. Animal feed pellets are typically produced on an industrial scale using processes such as pelleting. Processes used for producing animal feed pellets are well known to those skilled in the art.

[0025] As used herein, "secondary plant components" refers to specific compounds present in plants that do not contribute to plant growth and development but are essential for the plant's survival in its environment. These secondary plant components can be necessary for communication with other organisms in symbiotic interactions (e.g., attracting beneficial organisms such as pollinators) or antagonistic interactions (e.g., deterring herbivores and pathogens). They can further help cope with abiotic stresses (e.g., increased UV radiation). The broad functional scope of these specific metabolisms remains not fully understood.

[0026] As used herein, the term "premix" refers to a complex mixture of vitamins, minerals, trace elements, and other feed additives typically (but not always) incorporated into animal feed at levels between 0.2 wt% and 0.5 wt%. Adequate animal nutrition requires an animal feed composition consisting of macro-components ("main components") and sometimes micro-components (e.g., vitamins, (trace) minerals, organic acids, short-chain fatty acids, pigments, enzymes, probiotics, etc.) incorporated at ppm levels. To ensure that these micro-components are mixed uniformly with the macro-components, an intermediate dilution step is required via so-called premixes (or "pre-mixtures"). Premixes are legally defined as mixtures of feed additives or mixtures of one or more feed additives with feed material or water used as a carrier, not intended for direct feeding to animals ((EC) Regulation 1831 / 2003).

[0027] As used herein, the term "cell culture medium" refers to an aqueous solution of nutrients that can be used to grow cells over a long period of time. Typically, a cell culture medium comprises the following components: an energy source, usually carbohydrates such as glucose; amino acids, preferably a basic amino acid group including all essential amino acids; vitamins and / or other organic compounds required in low concentrations; free fatty acids; and inorganic compounds, including trace elements, inorganic salts, buffer compounds, and nucleosides and bases.

[0028] As used herein, the term “about” indicates a range of common tolerances in the art, such as within two standard deviations of the mean. The term “about” can be understood to encompass values ​​that deviate from the indicated value by up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%.

[0029] The terms “comprising” or “to comprise” and their conjugations, as used herein, refer to situations where the terms are used in their non-restrictive sense to mean including the items immediately following the word, but not excluding items not specifically mentioned. This also encompasses the more restrictive verbs “consistently of” and “comprises of”.

[0030] The use of the indefinite articles "a" or "an" to refer to an element does not preclude the possibility of more than one element, unless the context clearly requires that there be exactly one element. Therefore, the indefinite articles "a" or "an" generally mean "at least one". Detailed Implementation

[0031] This patent application has been divided into several parts. However, these parts should not be read in isolation. Unless otherwise stated, each part should be read in conjunction with the other parts. This means, for example, that all animal feed and / or feed additive compositions described in the "Animal Feed and / or Feed Additive Compositions" section are intended to be read in conjunction with the "Methods of Using Animal Feed and / or Feed Additives" section (i.e., all animal feeds set forth in the first part are applicable to the methods described in the latter part). Even when extracted from different parts of the specification, various optional and preferred features can be combined. Similarly, all "Aspects" and "Descriptions" can be combined. Unless explicitly stated otherwise, the descriptions of the embodiments are not intended to be separated.

[0032] The inventors have surprisingly discovered that compositions comprising blackberry leaf extract enhance cellular glucose uptake in vitro. Furthermore, they have found that blackberry leaf extract increases cell proliferation in vitro.

[0033] Furthermore, the inventors surprisingly discovered that adding blackberry leaves to broiler feed in relatively small amounts increases its feed efficiency performance. Further, the inventors have found that adding blackberry leaves to salmon feed increases feed intake.

[0034] Animal feed and / or additive compositions

[0035] Therefore, this disclosure provides animal feed comprising blackberry leaves or extracts thereof. This disclosure also provides feed additives comprising blackberry leaves or extracts thereof.

[0036] The leaves can be used fresh (i.e., directly after harvest) or dried by any method known in the art. Those skilled in the art are familiar with suitable drying techniques. For example, drying can be performed using a drum dryer, belt dryer, dehydration drying, vacuum drying, microwave drying, infrared drying, or natural drying (sun drying). Therefore, the feed additive compositions or animal feeds taught herein may include dried blackberry leaves.

[0037] Before or after drying (preferably after drying), the leaves can be ground to any suitable size. For example, fresh or dried leaves can be ground to an average particle size of less than 2 mm, preferably less than 1.5 mm, even more preferably less than 1 mm, even more preferably less than 0.8 mm, even more preferably less than 0.6 mm, and most preferably less than 0.5 mm.

[0038] Animal feed or feed additives may consist of leaves only, leaf extracts only, or a combination of leaves and leaf extracts.

[0039] The blackberry used in this article can be any variant of the corresponding species.

[0040] Animal feed can be in any form suitable for the animals to consume. For example, poultry feed can be in the form of crushed pellets, powder, or pellets. Pig feed can be in the form of coarse powder or pellets. Feed for fish and crustaceans is typically in the form of extruded or compressed pellets, but may also be in the form of flakes. In embodiments, animal feed is in the form of crushed pellets, powder, coarse powder, flakes, or pellets, which may be extruded or compressed.

[0041] Animal feed typically includes protein, fat, carbohydrates, minerals, and vitamins. It may further include organic acids and other feed additives. Those skilled in the art know how to formulate appropriate animal feeds for the target animal.

[0042] Blackberry leaves (preferably in dried and ground form) may be included in animal feed in amounts ranging from about 1 mg / kg feed to about 10,000 mg / kg feed, preferably about 5 mg / kg feed to about 8,000 mg / kg feed, more preferably about 10 mg / kg feed to about 6,000 mg / kg feed, even more preferably about 15 mg / kg feed to about 4,000 mg / kg feed, even more preferably about 20 mg / kg feed to about 3,000 mg / kg feed, even more preferably about 25 mg / kg feed to about 2,000 mg / kg feed, such as about 30 mg / kg feed to about 1,500 mg / kg feed, about 40 mg / kg feed to about 1,200 mg / kg feed, about 50 mg / kg feed to about 1,000 mg / kg feed, or about 60 mg / kg feed to about 800 mg / kg feed.

[0043] Optionally or additionally, an equivalent amount of blackberry leaf extract may be used. Plant extraction is a process aimed at extracting certain components present in a plant (so-called secondary phytochemicals, including alkaloids, terpenes, saponins, phenolic compounds, flavonoids, and / or tannins). Plant extraction is a solid / liquid separation operation: a solid object (in this document, blackberry leaves, optionally dried and / or ground) is contacted with a fluid (solvent). The target plant component is then dissolved and contained within the solvent. The resulting solution is the desired extract. The solvent may eventually be removed, although this is not mandatory. The solvent may be any solvent suitable for preparing plant extracts for animal feed. Suitable solvents include, but are not limited to, polar solvents (e.g., water, alcohols such as ethanol, methanol, and isopropanol), moderately polar solvents (e.g., acetone, dichloromethane), and nonpolar solvents (e.g., ethyl acetate, hexane, diethyl ether, chloroform). Those skilled in the art may choose a single solvent or two or more solvents to prepare the extract according to this disclosure. Generally, extraction processes include maceration, steaming, boiling, extraction, percolation, Soxhlet extraction, surface extraction, ultrasound-assisted and microwave-assisted extraction, supercritical fluid extraction (e.g., using supercritical carbon dioxide), accelerated solvent extraction, and solid-phase extraction. The fractionation and purification of phytochemicals can be achieved using various techniques (e.g., liquid-liquid partitioning, cross-flow filtration, and chromatography (e.g., paper chromatography, thin-layer chromatography, gas chromatography, high-performance liquid chromatography, centrifugal partition chromatography)). Finally, the compounds obtained from the extracts can be characterized using a variety of identification techniques (e.g., mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy, or any combination thereof). Those skilled in the art can select appropriate extraction methods and, optionally, appropriate characterization methods.

[0044] The amount of one or more extracts to be incorporated into the animal feed and / or feed additives taught herein can be determined by making the content of one or more secondary plant components in the extract equal to the content of one or more secondary plant components in blackberry leaves. Therefore, an amount of extract providing the content of one or more secondary plant components equivalent to a specified amount of said one or more secondary plant components in blackberry leaves can be incorporated into the animal feed.

[0045] In suitable embodiments, the extract can be prepared using a polar solvent (such as water, ethanol, or a mixture thereof).

[0046] In this embodiment, the extract is selected from the group consisting of a water extract, an ethanol extract, a methanol extract, an isopropanol extract, an ethyl acetate extract, a propanol extract, a hexane extract, or a supercritical CO2 extract, or any mixture thereof. Optionally, as stated above, extraction can be performed using a mixture of solvents.

[0047] Furthermore, this disclosure provides for the use of blackberry leaves or extracts thereof as a feed additive. The extracts are available as taught herein.

[0048] The feed additives taught herein may further include vitamins, minerals, organic acids, antioxidants, and / or pigments. These feed additives may be provided in the form of premixes. The feed additives or premixes are preferably intended for inclusion in feeds intended for farmed animals or companion animals.

[0049] The feed additive may be provided with written instructions to include in animal feed the amount of the composition taught herein in the form of about 1 mg / kg feed to about 10,000 mg / kg feed, preferably about 5 mg / kg feed to about 8,000 mg / kg feed, more preferably about 10 mg / kg feed to about 6,000 mg / kg feed, even more preferably about 15 mg / kg feed to about 4,000 mg / kg feed, even more preferably about 20 mg / kg feed to about 3,000 mg / kg feed, even more preferably about 25 mg / kg feed to about 2,000 mg / kg feed, such as about 30 mg / kg feed to about 1,500 mg / kg feed, about 40 mg / kg feed to about 1,200 mg / kg feed, about 50 mg / kg feed to about 1,000 mg / kg feed, or about 60 mg / kg feed to about 800 mg / kg feed, or, in the case of using blackberry leaf extract, an equivalent amount of the extract.

[0050] Methods of using animal feed and / or feed additives

[0051] In one aspect, this disclosure provides a method for feeding animals (e.g., farmed animals or companion animals, preferably farmed animals) with blackberry leaves or extracts thereof or animal feed as taught herein.

[0052] In a further aspect, this disclosure relates to the use of blackberry leaves or extracts thereof, or animal feed as taught herein, for increasing or improving the performance of animals (preferably farmed animals).

[0053] In another aspect, this disclosure relates to the use of blackberry leaves or extracts thereof, or animal feed as taught herein, for increasing feed intake, increasing average daily weight gain, increasing weekly weight gain, increasing biomass yield, and / or increasing feed efficiency (i.e., reducing feed conversion ratio).

[0054] This disclosure further provides a method for improving animal performance (e.g., increasing feed intake, increasing average daily weight gain, increasing weekly weight gain, increasing biomass yield, and / or increasing feed efficiency (i.e., reducing feed conversion ratio)), the method comprising the step of administering blackberry leaves or extracts thereof, a composition comprising blackberry leaves or extracts thereof, or an animal feed as taught herein (e.g., an animal feed comprising blackberry leaves or extracts thereof) to the animal.

[0055] Animal feeds and / or feed additives used in the above methods may be as described above.

[0056] The extract can be as described above.

[0057] Animals may be monogastric, proruminant, or ruminant. Animals may be selected from farmed animals and companion animals, with farmed animals being preferred. Farmed animals may be selected from the group consisting of: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia, and tuna; and crustaceans, such as shrimp. Companion animals may be selected from the group consisting of: ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters.

[0058] In practice, the performance benefits obtained in animals fed with the feed additives or animal feeds taught herein are relative to the results obtained in animals (of the same species) fed the same animal feed but without such feed additives or animal feeds.

[0059] Cell culture medium / methods for culturing cells

[0060] In one aspect, this disclosure provides a cell culture medium comprising an extract of blackberry leaves. The extract may be as taught herein.

[0061] In another aspect, this disclosure provides a method for culturing cells, the method comprising the steps of: providing a cell culture medium as taught herein, inoculating the culture medium with cells to be cultured, and proliferating the cells.

[0062] This disclosure also relates to the use of blackberry leaf extracts 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).

[0063] In this implementation, the cells are cultured for the purpose of preparing cultured meat or cultured fish.

[0064] The cells are preferably muscle cells and / or fat cells.

[0065] In a further aspect, this disclosure provides a method for preparing cultured meat, the method comprising the steps of: providing a cell culture medium as taught herein, inoculating the culture medium with muscle cells and / or adipocytes, and proliferating the muscle cells and / or adipocytes.

[0066] In another aspect, this disclosure provides a method for increasing glucose uptake in cells (e.g., muscle cells) and / or increasing cell proliferation (e.g., muscle cell proliferation), the method comprising the step of culturing the cells in a culture medium (e.g., a culture medium as taught herein) comprising an extract of blackberry leaves.

[0067] The cell culture media taught in this article can be used in a variety of cell culture processes. Cell culture can be carried out in adherent culture (e.g., monolayer culture or preferably suspension culture).

[0068] Cell culture media typically contain a mixture of amino acids, carbohydrates (preferably in the form of one or more sugars), (inorganic) salts, vitamins, buffer systems, other nutrients, and compounds that regulate the cell cycle. These media are available from commercial suppliers in powder form (reconstituted with water) or in liquid form. The media can be in liquid form (optionally obtained by reconstitution from powder) or solid form. The requirements for the various components of a cell culture medium can vary depending on the cell type and / or cell line. Those skilled in the art know how to select an appropriate cell culture medium for culturing a specific cell type or cell line.

[0069] Amino acids are the building blocks of proteins and are therefore essential components of all known cell culture media. Essential amino acids must be included in the culture medium because cells cannot synthesize them themselves. They are necessary for cell proliferation, and their concentration determines the maximum achievable cell density. Non-essential amino acids can also be added to the culture medium to replace amino acids that have been depleted during growth. Supplementing the culture medium with non-essential amino acids can stimulate growth and prolong cell viability. Amino acids can be provided in the form of single amino acids or in the form of dipeptides, oligopeptides, polypeptides, or mixtures thereof.

[0070] In embodiments, 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-cysteine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine in free form or in dipeptide or oligopeptide form.

[0071] Carbohydrates in the form of sugars are usually the primary energy source. Most culture media contain glucose and / or galactose. Cell culture media may further include maltose and fructose.

[0072] Inorganic salts in cell culture media can help maintain osmotic balance and regulate membrane potential by providing sodium, potassium, and calcium ions. In embodiments, the cell culture medium includes sodium, potassium, calcium, magnesium, zinc, copper, iron, and / or selenium ions, preferably in the form of salts, such as NaCl, KCl, CaCl2·2H2O, NaH2PO4, MgSO4, ZnSO4·7H2O, CuSO4·5H2O, Fe(NO3)3·9H2O, and / or Na2SeO3.

[0073] Many vitamins are essential for cell growth and proliferation. Cells cannot synthesize sufficient amounts of vitamins, and therefore vitamins are an important supplement required in tissue culture. In some embodiments, serum may be the primary source of vitamins in cell culture. In other embodiments, the cell culture medium may include synthetic vitamins. In embodiments, the culture medium includes one or more of vitamin B12, vitamin A, vitamin E, riboflavin (vitamin B2), thiamine (vitamin B1), vitamin B6, pantothenic acid, folic acid, niacin, cyanocobalamin, pyridoxine, nicotinamide, and / or biotin. Vitamins may be present in any suitable form. For example, vitamin A may be present as retinyl acetate or any other retinyl ester. Those skilled in the art will be able to select suitable forms of one or more of the described vitamins.

[0074] Trace elements (such as copper, zinc, and selenium) can be present in the cell culture media taught herein, for example, in the form of inorganic salts as described above.

[0075] pH adjustment can be important for optimal cell culture. Therefore, in embodiments, the culture media taught herein further include suitable buffers to adjust the pH of the medium. Those skilled in the art can select suitable buffer systems. Suitable buffer systems include, but are not limited to, Tris buffer, Tris-EDTA buffer, Tris-acetate-EDTA buffer, PBST buffer, HEPES buffer, and citrate buffer.

[0076] The cell culture media taught in this paper may include pH indicators (such as phenol red) to allow for continuous pH monitoring.

[0077] In embodiments, the cell culture media taught herein comprise blackberry leaf extract at concentrations between 25 µg / ml and 2000 µg / ml, preferably between 50 µg / ml and 1500 µg / ml, such as between 75 µg / ml and 1000 µg / ml, or between about 80 µg / ml and 800 µg / ml, obtained by extracting about 1 g of dried and ground blackberry leaves with about 8 g of solvent (preferably a polar solvent, even more preferably a solvent consisting of ethanol and water). Of course, the same cell culture media can be obtained using different extraction grades of blackberry leaves and different amounts of extract. Such cell culture media are included within the scope of this disclosure.

[0078] The cell culture media taught in this article may further include albumin, transferrin, and / or fibronectin. These proteins can be particularly important in serum-free media.

[0079] Due to safety and contamination concerns, the use of cell culture media in the pharmaceutical or food industries (e.g., for the preparation of therapeutically active recombinant peptides or for the preparation of cultured meat) typically does not permit the use of any biologically derived materials. Therefore, the cell culture media taught herein are preferably serum-free and / or protein-free media. The term "serum-free and / or protein-free media" refers to a media with a fully defined chemical composition, free from additives of animal origin, such as tissue hydrolysates, like fetal bovine serum, etc.

[0080] The osmolality of the cell culture medium taught herein can be set at the beginning of culture between about 280 mOsm / kg and about 365 mOsm / kg, but can be gradually increased during culture. Preferably, the initial osmolality of the cell culture medium taught herein is between about 280 mOsm / kg and about 365 mOsm / kg, for example, between about 285 mOsm / kg and about 360 mOsm / kg, between about 290 mOsm / kg and about 355 mOsm / kg, between about 295 mOsm / kg and about 350 mOsm / kg, or between 300 mOsm / kg and 345 mOsm / kg.

[0081] The temperature for cell culture is selected within the range where cells can survive and grow. Typical temperatures used for cell culture are in the range of about 30°C to about 38°C. For example, cells are initially grown at a temperature of about 36°C 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 culture to allow for optimal viability, growth, or production. Those skilled in the art are able to select appropriate culture conditions based on cell type.

[0082] The invention is further illustrated by the following embodiments, but not limited thereto. Based on the above discussion and these embodiments, those skilled in the art can determine the essential features of the invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from its teachings and scope. Therefore, in addition to those shown and described herein, those skilled in the art will clearly understand various modifications to the invention from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0083] Example

[0084] Example 1

[0085] In this embodiment, the effect of 250 ppm dietary supplementation of dried blackberry leaves on the production performance of broiler chickens was investigated.

[0086] Blackberry leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting practices. Dried blackberry leaves are ground to a particle size of <0.3 mm.

[0087] The following diets are provided in powder form according to the age of the chickens: starter feed from 0 to 14 days old, grower feed from 15 to 28 days old, and finisher feed from 29 to 42 days old. The composition of the basal diet is described in Table 1.

[0088] Table 1. Composition of the basic daily ration

[0089]

[0090] The experimental treatments were conducted as follows. The control group chickens received a basal starter feed, a basal growing feed, and a basal finishing feed. The experimental group chickens received a basal diet supplemented with 250 grams of dried, ground blackberry leaves per metric ton of feed.

[0091] Each experimental treatment was assigned 12 columns per treatment.

[0092] Each pen (4' x 5') contained one drinker and one feed tube. Chickens were raised on fresh wood chips. Thirty-four 0-day-old Ross 308 chicks were assigned to each pen. Chickens were assigned to pens on Day 1 to ensure even distribution by weight across the 108 study pens. Continuous lighting was provided from Day 0 to Day 42.

[0093] When the chickens were 0, 14, 28, and 42 days old, all broilers were weighed in their respective pens. Feed was added as needed and removed when the chickens were weighed. The following data were collected during the study: broiler body weight (BW) and feed intake (FI) at 14, 28, and 42 days of age.

[0094] The feed conversion ratio of a pen is calculated by dividing the total feed consumed by (the total body weight of all animals in the pen + the total body weight of all animals that died in the pen).

[0095] The corrected feed conversion ratio for 42-day-old infants using the final BW correction is calculated as follows: Feed conversion ratio + (2.22 kg - actual body weight) / 7.

[0096] The statistical analysis was a one-way ANOVA using the following model: Y ij =µ+processing i +ε ij , where Y is the production response, the mean is µ, the treatment is a categorical variable with 3 levels (control group, treatment group 1, or treatment group 2), and ε is the residual error. Fisher's test is used to compare the means of the 3 levels of the treatment at a 95% confidence level.

[0097] The results are presented in the table below.

[0098]

[0099] The results showed that adding 250 grams of blackberry leaves per metric ton of feed to broiler diets improved feed intake and weight gain at all stages of the production process.

[0100] Example 2

[0101] In this embodiment, the effects of dietary additions of 250 ppm and 500 ppm of dried blackberry leaves on broiler production performance were investigated.

[0102] Blackberry leaves are cultivated. Cultivation and drying follow good agricultural and harvesting practices. Dried blackberry leaves are ground to a particle size of <0.3 mm.

[0103] The following feed rations are provided in powder form according to the age of the chickens: starter feed from 0 to 14 days old, growing feed from 15 to 28 days old, and finishing feed from 29 to 42 days old. The composition of the basal diet is as follows, expressed in kg / 100kg.

[0104]

[0105] The experimental treatments were conducted as follows. Control group chickens received a basal starter feed, a basal growing feed, and a basal finishing feed. Treatment group 1 chickens received a basal diet supplemented with 250 grams of dried, ground blackberry leaves per metric ton of feed. Treatment group 2 chickens received a basal diet supplemented with 500 grams of dried, ground blackberry leaves per metric ton of feed.

[0106] Each experimental treatment group was assigned 12 columns.

[0107] Each pen (4' x 5') contains one drinker and one feed tube. Chickens are raised on sawdust. Thirty-four 0-day-old Ross 308 chicks are assigned to each pen. Chicks are assigned to pens on Day 1 to ensure even distribution by weight across all study pens. Continuous lighting is provided from Day 0 to Day 42.

[0108] At 0, 14, 28, and 42 days of age, all broilers were weighed by pen. Feed was added as needed and removed as the chickens were weighed. The following data were collected during the study: broiler body weight and feed intake (FI) at 14, 28, and 42 days of age. This data was collected to calculate the 42-day feed conversion ratio (D42 FCR) for a pen as total feed consumed divided by (total body weight of all chickens in that pen + total body weight of all chickens that died). The corrected feed conversion ratio (D0-42 MFCR) at 42 days of age, corrected for the final BW, was calculated as: Feed conversion ratio + (2.22 kg - actual body weight) / 7.

[0109] The results are presented in Table 2.

[0110] Table 2. FCR and weight-corrected FCR after treatment with 250 ppm or 500 ppm dried and ground blackberry leaves

[0111]

[0112] The results showed that adding 250 ppm and 500 ppm of dried and ground blackberry leaves improved broiler performance by more than 4.5% in improving their feed efficiency.

[0113] Example 3

[0114] This study investigated the effects of dried blackberry leaves on the production performance of salmon.

[0115] Blackberry leaves were obtained from cultivation. Cultivation and drying followed good agricultural and harvesting practices. Blackberry leaves were ground to a particle size of <0.3 mm. A basal diet was formulated to meet the nutritional requirements of Atlantic salmon with approximately 42% crude protein, 32% crude fat, 8% moisture, 24.630 kJ / g energy, and 5.4% ash.

[0116] The experimental treatments were conducted as follows. Control group salmon received a basal diet. Treatment group salmon received a basal diet supplemented with 290 grams of dried, ground blackberry leaves per metric ton of feed instead of wheat. All macro and micro components (including the dried, ground blackberry leaf component) were first dry-mixed, and then the entire mixture was extruded into 7mm pellets.

[0117] This experiment involved two treatments (control group and treatment group) using Atlantic salmon with an average weight of 750g. Each treatment was replicated in three separate 3-meter tanks, with 75 fish per tank. The water temperature was maintained at 12°C and the salinity at 31 ppt. The salmon were fed three times daily. The lighting protocol was 24-hour illumination. The experiment lasted 141 days. The salmon were weighed on day 1 (providing the average initial weight in g) and on day 3 (providing the average final weight in g). Other results were measured: final biomass (in g), total weight of dead fish (in g), and total feed intake (in g). Specific growth rate (SGR) and feed intake, all expressed as % / day, were calculated below.

[0118]

[0119]

[0120] Table 3 shows the results of the experiment.

[0121] Table 3. Feed intake of the control group (no blackberry leaves added) and the treatment group (290 ppm dried and ground blackberry leaves added).

[0122]

[0123] Adding 290 ppm of blackberry leaves increased salmon feed intake by 4.2%.

[0124] Example 4: In vitro muscle growth

[0125] In this embodiment, the effects of ground blackberry leaves on the proliferation of cultured muscle cells and the uptake of glucose by muscle cells were determined.

[0126] 1 g of powdered blackberry leaves was extracted with 8 g of 50% (w / w) ethanol / water to provide blackberry leaf extract.

[0127] Two results were used: protein content used to assess muscle cell proliferation and glucose uptake by muscle cells.

[0128] Mouse muscle cells (C2C12) were seeded and grown to confluence (3 days). Cells differentiated for 4 days to form myotubes, then were treated with a plant extract for 24 hours. After supplementation, glucose was depleted for 24 hours, followed by serum depletion overnight. After washing, the fluorescent glucose analog 2-NBDG (2-(N-(7-nitrobenzene-2-oxa-1,3-diazol-4-yl)-amino)-2-deoxyglucose, used to monitor glucose uptake by live cells, was added to the culture medium. Fluorescence was measured after washing and lysing cells 30 minutes later. Metformin was used as a positive control; culture medium and solvent controls were used as negative controls.

[0129] Protein content determination was used to identify cell proliferation in the applied in vitro model. The C2C12 mouse myoblast cell line was first supplemented with plant extract 24 hours post-inoculation. Cell differentiation was initiated five days later. A second supplementation with plant extract was performed 24 hours after the onset of differentiation. A final supplementation was performed on day 3 of the differentiation phase. Cells were lysed on day 7 after the onset of differentiation, and protein content was determined using the Bradford assay. Culture medium and solvent controls were performed for each batch. Insulin-like growth factor (IGF) was used as a positive control. The assay was repeated three times.

[0130] Table 4 shows the effect on glucose uptake, expressed as a percentage of the control group, with each mean being the average of three replicates.

[0131] Table 4. Effects of blackberry leaf extract on glucose uptake in C2C12 mouse myoblast cell line, expressed as a percentage relative to the control group (without blackberry leaf extract).

[0132]

[0133] In a separate experiment, following the same protocol, the effect of 300 µg / mL blackberry leaf extract on glucose uptake in muscle cells was measured again. This second experiment confirmed that 300 µg / mL blackberry leaf extract enhanced glucose uptake in muscle cells by 34.8%.

[0134] Table 5 shows the average muscle cell proliferation, expressed as a percentage of the control group. Each mean is the average of 6 values.

[0135] Table 5. Mean muscle cell proliferation, expressed as a percentage of the control group. Each mean is the average of 6 values.

[0136]

[0137] Adding at least 250 µg / mL of blackberry leaf extract increased muscle cell proliferation by 3.8%.

[0138] Example 5

[0139] This study investigated the effects of dietary supplementation with dried and ground blackberry leaves on shrimp growth.

[0140] Blackberry leaves were obtained from cultivation. Cultivation and drying followed good agricultural and harvesting practices. Blackberry leaves were masked to a particle size of <0.3 mm. The basal diet composition is as stated in Table 6.

[0141] Table 6. Composition of the basic diet for extruded shrimp feed.

[0142]

[0143] The diet is made into 2.0 ± 0.1 mm extruded pellets.

[0144] The feed production process follows standard guidelines for shrimp feed extrusion. First, all dried raw materials (except trace components) are ground through a 1mm (50Hz) mill sieve. Next, each component is weighed according to the formulation specifications. After extrusion, the pellets are dried until the moisture content is below 10%.

[0145] The experimental treatments were conducted as follows: The "control group" shrimp received a basal shrimp diet. "Treatment group 1" shrimp received a basal diet supplemented with 0.75 kg of dried and ground blackberry leaves per metric ton of feed. "Treatment group 2" shrimp received a basal diet supplemented with 1 kg of dried and ground blackberry leaves per metric ton of feed.

[0146] Litopenaeus vannamei shrimp with an initial weight of 4 grams were reared in 225-liter glass tanks at a stocking density of 32 shrimp per square meter. Artificial seawater at a concentration of 20 g / L was prepared by adding a commercial salt mixture to purified water. Each tank was equipped with an independent mechanical / biological filter. Water was not shared between tanks. Water quality was maintained through filtration, aeration, and regular water changes. The water temperature was maintained at 30°C. The space was provided with 12 hours of light per day. The shrimp were fed six times daily. They were fed either the control group's diet or the diets of different treatment groups.

[0147] The performance response measured in this experiment is:

[0148] - Specific growth rate (SGR), expressed as % / day

[0149] - Weekly growth, calculated on a weekly basis

[0150] Feed conversion ratio (FCR)

[0151] The results are shown in Table 7 below. Compared with the control group, the SGR, weekly growth and FCR of shrimp treated with blackberry leaves were all improved.

[0152] Table 7. Performance Response of Shrimp

[0153]

Claims

1. An animal feed comprising blackberry leaves.

2. The animal feed according to claim 1, wherein the blackberry leaves are dried and optionally ground.

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

4. Uses of blackberry leaves as a feed additive.

5. The use according to claim 4, wherein the feed additive is provided with written instructions to include the feed additive in animal feed at an amount of about 1 mg / kg feed to about 10,000 mg / kg feed, preferably 5 mg / kg feed to about 5,000 mg / kg feed.

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

7. Use of blackberry leaves or extracts thereof, or compositions comprising blackberry leaves or extracts thereof, or animal feed according to any one of claims 1-3, for increasing animal performance.

8. Use of blackberry leaves or extracts thereof, or compositions comprising blackberry leaves or extracts thereof, or animal feed according to any one of claims 1-3, for feeding animals, increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, and / or increasing a specific growth rate.

9. The use according to claim 7 or 8, wherein the extract is selected from the group consisting of a water extract, an ethanol extract, a methanol extract, an isopropanol extract, an ethyl acetate extract, an acetone extract, a hexane extract, or a mixture of these solvents or a supercritical CO2 extract, or any mixture thereof.

10. The use according to any one of claims 4-9, wherein the feed additive is intended to be included in feed for farmed animals.

11. The use according to claim 10, wherein the farmed animal is selected from the group consisting of: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia and tuna; and crustaceans, such as shrimp.

12. Use of blackberry leaf extract for culturing muscle cells and / or adipocytes, for increasing glucose uptake in cells (e.g., muscle cells and / or adipocytes), and / or for increasing cell proliferation (e.g., muscle cell proliferation and / or adipocyte proliferation).

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

14. A cell culture medium comprising an extract of blackberry leaves.

15. A method for culturing muscle cells and / or adipocytes, the method comprising the following steps: Provide a cell culture medium according to claim 14, inoculate the cells to be cultured into the culture medium, and proliferate the cells.

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