ANIMAL FEED, USES OF AERIAL PARTS OF GLYCYRRHIZA GLABRA AND OF AERIAL PARTS OF GLYCYRRHIZA GLABRA OR OF AN ANIMAL FEED, METHOD OF FEEDING AN ANIMAL, AND COMPOSITION

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., an increasing feed intake, increasing average daily gain, increasing feed efficiency (i.e., decreasing feed conversion ratio), and / or increasing milk fat yield, of such animals.

Description

55 ANIMAL FEED, USES OF AERIAL PARTS OF GLYCYRRHIZA GLABRA AND OF AERIAL PARTS OF GLYCYRRHIZA GLABRA OR OF AN ANIMAL FEED, METHOD OF FEEDING AN ANIMAL, AND COMPOSITION FIELD OF THE INVENTION

[001] The present invention is in the field of feed additives for animals, for example, farm animals, in particular feed additives to be added to animal feed to obtain improved performance, for example, increased feed intake, increased average daily gain, increased feed efficiency (i.e., decreased feed conversion ratio) and / or increased milk fat production in these animals. FUNDAMENTALS OF THE INVENTION

[002] Antimicrobial agents have been used as antibiotic growth promoters in livestock feed for many decades. However, resistance to these 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 completely banned in the European Union since January 2006. At the same time, the demand for animal protein is increasing, driven by the growing human population. Furthermore, food production needs to be more sustainable, requiring that more be produced with less. Taken together, animal-based food production needs to become increasingly efficient.

[003] These developments have sparked a wave of exploration into feed additives that improve the performance of farm animals but do not induce bacterial resistance. Phytogenic feed additives, also known as botanicals, are plant-derived substances added to animal diets to improve animal performance. Essential oils, herbs, and spices serve as sources. Petition 870250079705, dated 05 / 09 / 2025, page 8 / 74 / 55 of bioactive ingredients, for example, phenols, polyphenols, flavonoids, terpenoids and others.

[004] Glycyrrhiza glabra is a Fabaceae plant in the family Leguminosae, native to Eurasia, Central and Southwest Asia, and the Mediterranean region. Specifically, the roots (with stolon and rhizome) of Glycyrrhiza glabra are called licorice. They have been used by humans for at least 4,000 years in various food applications. The roots of Glycyrrhiza glabra are used as a medicinal plant for a variety of effects (El-Saber Batiha et al. 2020. Biomolecules, 10): treatment of digestive system disorders, respiratory tract disorders, epilepsy, fever, sexual debility, paralysis, rheumatism, leucorrhea, psoriasis, prostate cancer, malaria, hemorrhagic diseases, and jaundice. In the roots, glycyrrhizic acid (also known as glycyrrhizin, glycyrrhetinic acid glycoside, or 18-β glycyrrhetic acid) is the main constituent. Glycyrrhizic acid represents about 10% of the dry weight of licorice root (Pastorino et al. 2018. Phytother Res, 32: 2323-39).

[005] In the most recent literature reviews that consider licorice, the authors discuss only the root part of the plant (Bisht et al. 2022. Phytomed Plus, 2: 100206; Hejazi et al. 2021. Food Chem Toxicol, 150: 112057). For example, in a review summarizing the anti-inflammatory effect of licorice, especially at the intestinal level, Leite et al. (2022. Int J Mol Sci, 23) focused on glycyrrhizin and glycyrrhetinic acid, both compounds found only in the roots of Glycyrrhiza glabra.

[006] Licorice powder and licorice essential oils have been used previously in livestock. Alagawany et al. (2019. Animals (Basel), 9) summarized the interest in licorice powder for improving the performance of poultry, focusing on glycyrrhizic acid, a compound found exclusively in the roots. Thus, in the technique, licorice is equivalent to the roots of the Glycyrrhiza glabra plant. Petition 870250079705, dated 05 / 09 / 2025, page 9 / 74 / 55

[007] Vlaisavljevic et al. (2018. Industrial Crops and Products, 112: (217-24) compared 2 root samples and 2 leaf samples of Glycyrrhiza glabra using different chromatography techniques and showed that glycyrrhizic acid was detected only in root extracts, not in leaves. They also reported radically different compositions between roots and leaves. Siracusa et al. (2011. Phytotherapy, 82: 546-56) demonstrated that compounds from Glycyrrhiza glabra leaves are present in small traces or are not present in the roots of the same plant. The aerial parts of Glycyrrhiza glabra, such as the leaves, are considered an agrochemical residue (Pastorino et al. 2018. Supra. Siracusa et al. 2011. Supra. Gowthaman et al. 2021. Letters in Animal Biology 01(2): 14 - 20). They do not contain glycyrrhizic acid, which is considered the most important compound present in the roots. Zamiri et al. (Jan 2015. Trop Anim Health Prod) used Glycyrrhiza glabra leaves as forage as a source of so-called "condensed tannins" for lambs.Ashraf et al. (2017. Pak J Pharm Sci, 30: 567-72) injected Glycyrrhiza glabra leaf extract into eggs to study its effect on Newcastle disease virus. The authors suggested that Glycyrrhiza glabra leaf extract could be used to control Newcastle disease virus. Abarghuei and Salem (2021. Environ Sci Pollut Res Int) aimed to evaluate the effect of Glycyrrhiza glabra pulp and leaves on reducing ruminal biogas production in sheep.

[008] One object of the present invention is to provide a feed additive and / or animal feed to improve the performance of animals, preferably farm animals, preferably of many different species of farm animals, for example, to increase feed intake, increase body weight gain, increase average daily gain, increase feed efficiency (i.e., decrease feed conversion ratio), increase specific growth rate, increase relative growth rate, increase milk fat production and / or to Petition 870250079705, dated 05 / 09 / 2025, p. 10 / 74 / 55 to prevent, reduce the severity of and / or treat microbial infections, for example, bacterial infection, viral infection, parasitic infection, fungal infection and / or yeast infection, in such animals, and / or to prevent, reduce the severity of and / or treat liver toxicity or liver damage due to exposure to mycotoxins. SUMMARY OF THE INVENTION

[009] The present description provides an animal feed comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof.

[0010] In one embodiment, the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are dried and, optionally, ground.

[0011] In one embodiment, the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are included in an amount of about 1 mg / kg to about 10,000 mg per kg of feed, or an equivalent amount of extract.

[0012] In one embodiment, the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetonic extract, hexane extract or a supercritical CO2 extract, or a mixture of any of these.

[0013] In a further aspect, the present description refers to the use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as a food additive.

[0014] In one embodiment, said feed additive is provided with written instructions for including it in animal feed in an amount of Glycyrrhiza glabra aerial parts, preferably Glycyrrhiza glabra leaves, from about 1 mg / kg to about 10,000 mg per kg of feed, or an equivalent amount of extract. Petition 870250079705, dated 05 / 09 / 2025, p. 11 / 74 / 55

[0015] In one embodiment, the food additive is included in a premix.

[0016] In another aspect, the present description provides for the use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, to increase the performance of an animal, for example, to increase feed intake, increase average daily gain, increase feed efficiency (i.e., decrease feed conversion ratio), increase relative growth rate, increase specific growth rate and / or increase milk fat production.

[0017] The present description further refers to a composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed as taught in this document for use in reducing mortality and / or for use in preventing, alleviating the severity and / or reducing microbial infection. In one embodiment, the microbial infection is caused by a Gram-negative bacterium, for example, of the genus Vibrio, for example, of the species Vibrio parahaemolyticus.

[0018] In one embodiment, the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetonic extract, hexane extract or a supercritical CO2 extract, or a mixture of any of these.

[0019] In one embodiment, the feed additive is intended for inclusion in feed for livestock or pets.

[0020] In one modality, farm animals are selected from the group consisting of farmed poultry, swine, ruminants, for example, beef cattle and dairy cattle, fish, for example, salmon, trout, sea bream, sea bass, tilapia, tuna and the like, and crustaceans, for example, shrimp. Petition 870250079705, dated 05 / 09 / 2025, p. 12 / 74 / 55

[0021] In another modality, the pet is selected from the group that consists of ornamental fish, cats, dogs, horses, rabbits, guinea pigs and hamsters. GENERAL DEFINITIONS

[0022] In the description and examples that follow, several terms are used. To provide a clear and consistent understanding of the descriptive report and the claims, including the scope to be determined by such terms, the following definitions are provided. Unless defined otherwise in this document, all technical and scientific terms used have the same meaning commonly understood by one skilled in the art to which this invention pertains. Descriptions of all publications, patent applications, patents, and other references cited in this document are incorporated herein in their entirety by reference.

[0023] The term “licorice” is the common name for Glycyrrhiza glabra. In prior art, the term was used to refer to the roots of the plant.

[0024] The term “aerial part,” when referring to a plant, refers to anything that exists in the air or in the space above a solid surface. The aerial part of plants simply denotes the structures of a plant that are above the ground, including stems, leaves, petioles, flowers, fruits, and seeds. The term includes any aerial part alone, for example, only leaves, or only stem, or the entire aerial part (i.e., all parts of the plant above the ground). In one embodiment, when the term “aerial parts” is used in this document, it may be replaced by the term “leaves.”

[0025] The term “leaves,” when referring to a plant, refers to any green, usually flattened, protuberance on the stem of a vascular plant. As the primary sites of photosynthesis, leaves produce food for plants, which in turn nourish and sustain all the Petition 870250079705, dated 05 / 09 / 2025, page 13 / 74 / 55 terrestrial animals. Botanically, leaves are an integral part of the stem system. They are connected by a continuous vascular system to the rest of the plant, so that the free exchange of nutrients, water and end products of photosynthesis (oxygen and carbohydrates in particular) can be transported to its various parts.

[0026] The term “farm animal” refers to animals that are kept or raised for agricultural purposes, such as for consumption or to generate income through, for example, wool, meat, eggs, or dairy products. Farm animals can be grouped based on their digestive system. These groups include monogastric animals, ruminants, and pseudoruminants. Alternatively or additionally, they can be grouped based on their ecosystem. These groups include aquaculture animals (when referring to animals that are raised in an aquatic ecosystem, such as fish and shrimp) and farm animals (when referring to animals raised on land, such as poultry, pigs, and cattle).

[0027] The term “pet” refers to domesticated animals or animals raised in domestic environments whose physical, emotional, behavioral, and social needs can be readily met as companions at home or in a close daily relationship with humans. Suitable species for being pets include dogs, cats, horses, rabbits, ferrets, birds, guinea pigs and other selected small mammals, small reptiles, and ornamental fish.

[0028] The term “pellets” or “feed pellets” as used in this document refers to small particles or a body normally created by compressing an original material, for example, a mixture of feed raw materials, typically fermentable feed ingredients such as grains, cereals, legumes, forage and the like. Feed pellets may also comprise other feed ingredients such as meat meal, fish meal, bone meal, by-products, oil, fat, Petition 870250079705, dated 05 / 09 / 2025, page 14 / 74 / 55 fillers or any mixture thereof, etc., as well as minerals, vitamins, trace elements and others. Animal feed pellets vary in their composition as well as in their structural properties (e.g., hardness, density, durability, shape, size, etc.), depending on the nutritional needs, feeding 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 pellets can be of 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 composition of the feed pellet itself (for example, some ingredients have a higher 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 the pellet's durability. This is true for any method of manufacturing feed pellets, including the method taught in this document. It is also a consensus in the field of agriculture and animal nutrition that animals (e.g., young and adult farm animals such as beef cattle (e.g., beef calves), dairy cows (e.g., dairy calves), and pigs, etc.) benefit more or achieve greater gains (e.g., weight gain, height increase, improved growth curve) with pelleted feed than with meal feed, because pelleted feed is in a more concentrated, readily edible, and palatable form than meal or bran feed. Pelleted feed has been shown to facilitate feed intake and minimize feed waste during the feeding process.It has been shown that most animals, given the choice between the same feed in pellet or meal form, will prefer the pellet form. Animal feed pellets are typically produced on an industrial scale using, for example, a pelleting process. Petition 870250079705, dated 05 / 09 / 2025, page 15 / 74 / 55, the expert is well acquainted with the processes of producing animal feed pellets.

[0029] The term “premix,” as used in this document, refers to a mixture of ingredients designed to be mixed with other ingredients, usually raw materials to be used for feed, before use. The ingredients within the ingredient mixture in the premix are generally ingredients that must be added to animal feed in small amounts, such as vitamins and minerals and, optionally, also antioxidants, pigments, and / or organic acids. For example, a premix for fish feed may comprise vitamins, minerals, antioxidants, and / or pigments, but generally does not comprise organic acids. In contrast, a premix for poultry or swine feed may comprise vitamins, minerals, organic acids, and antioxidants, but not pigments.

[0030] The term “feed conversion rate” or “FCR”, as used in this document, is a ratio or rate that measures the efficiency with which the bodies of farm animals convert animal feed into the desired output, for example, weight. The term “feed efficiency”, as used in this document, is the inverse of FCR.

[0031] The term “relative growth rate” or “RGR” as used in this document is expressed as a percentage and is calculated as follows: (Final weight - initial weight) / initial weight.

[0032] The term “specific growth rate” or “SGR” is a coefficient that measures the percentage increase in fish weight per day: SGR=(Ln(Wt)-Ln(W0))*100 / t(d), where W0[g] = weight in grams at the beginning of the period, Wt [g] = weight in grams at the end of the period, t[d] = period, expressed in number of days; and Ln = natural logarithm.

[0033] The term “secondary plant constituents”, as used in this document, refers to specialized compounds present in plants. Petition 870250079705, dated 05 / 09 / 2025, page 16 / 74 / 55, which do not aid in plant growth and development, but are necessary for the plant to survive in its environment. These secondary plant constituents may be essential for communication with other organisms in mutualistic interactions (e.g., attraction of beneficial organisms such as pollinators) or antagonistic interactions (e.g., deterrence against herbivores and pathogens). They may further help to cope with abiotic stress, such as increased UV radiation. The broad functional spectrum of specialized metabolism is not yet fully understood.

[0034] The term “about”, as used in this document, indicates a normal tolerance range in the technique, for example, within 2 standard deviations of the mean. The term “about” can be understood as encompassing values ​​that deviate by a maximum of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% from the indicated value.

[0035] The terms “comprising” or “comprises” and their conjugations, as used in this document, refer to a situation where said terms are used in their non-limiting sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb “consist essentially of” and “consist of”.

[0036] Referring to an element with 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” generally means “at least one”. BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 compares the chromatograms of the Glycyrrhiza glabra root sample (clearly showing the glycyrrhizic acid peaks) with the Glycyrrhiza glabra leaf sample where the acid peaks Petition 870250079705, dated 05 / 09 / 2025, page 17 / 74 / 55 glycyrrhizic acid is absent.

[0038] Figure 2 shows the excretion of fecal oocysts from chickens without (NC) or with (PC, MO, GG) E. maxima infection in the absence of additional treatment (PC) or with treatment using monensin (MO), 55 ppm of Glycyrrhiza glabra leaves (55 ppm GG) or 160 ppm of Glycyrrhiza glabra leaves (160 ppm GG). DETAILED DESCRIPTION OF THE INVENTION

[0039] This patent application has been drafted in sections. However, these sections should not be read in isolation. Unless otherwise specified, each section should be read in combination with the other sections. This means, for example, that all animal feeds and / or feed additive compositions described in the section “Animal feed and / or feed additive composition” should be read with the section “Methods of use of animal feed and / or feed additive” (i.e., all animal feeds cited in the first section are suitable for the methods described in the last section). The various optional and preferred features may also be combined, even when taken from different parts of the descriptive report. Similarly, all “aspects” and “embodiments” may be combined. There is no intention to separate embodiments unless explicitly stated.

[0040] The present inventors have surprisingly found that the inclusion of a relatively small amount of Glycyrrhiza glabra aerial parts, preferably leaves, in broiler chicken feed, shrimp feed or salmon feed, increases their performance, for example, in terms of body weight gain or average daily gain and feed efficiency. Animal feed composition and / or feed additive

[0041] Thus, the present description provides an animal feed comprising aerial parts of Glycyrrhiza glabra, or an extract of Petition 870250079705, dated 05 / 09 / 2025, page 18 / 74 / 55 the same, preferably leaves of Glycyrrhiza glabra, or an extract thereof.

[0042] Animal feed may be in any form suitable for ingestion by the animal in question. For example, poultry feed may be in the form of ground feed, bran, or pellets. Pig feed may be in the form of flour or pellets. Fish and crustacean feed usually comes in the form of extruded pellets, for example, extruded for cooking or pressed, but may also come in other forms, for example, in the form of flakes. In one embodiment, animal feed may be in the form of ground feed, bran, flour, flakes, or pellets, which pellets may be extruded, for example, extruded for cooking, or may be pressed. In a suitable embodiment, animal feed is in the form of a pellet.

[0043] Animal feed generally comprises protein, fat, carbohydrates, minerals, and vitamins. It may additionally contain antioxidants, pigments, organic acids, enzymes, phytogenic compounds, and / or other feed additives. The expert knows how to formulate an appropriate animal feed depending on the target animal, the target animal's life stage, and / or the target animal's physical condition.

[0044] The aerial parts of Glycyrrhiza glabra, preferably leaves, may be of any variety or subspecies of Glycyrrhiza glabra. Optionally, the aerial parts or leaves may have been processed, for example, fermented, before their use in animal feed or as a feed additive, as taught in this document.

[0045] The aerial parts of Glycyrrhiza glabra, preferably Glycyrrhiza glabra leaves, can be used fresh or dried by any means known in the art. Those skilled in the art are familiar with suitable drying technologies. For example, drying can occur using a drum dryer, belt dryer, dehydrator drying, vacuum drying, micro-drying. Petition 870250079705, dated 05 / 09 / 2025, page 19 / 74 / 55 waves, infrared drying or natural drying (sun drying). In a preferred embodiment, the aerial parts of Glycyrrhiza glabra or the leaves of Glycyrrhiza glabra are dried.

[0046] Before or after drying, preferably after drying, the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, can be ground to any suitable size. For example, fresh or dried leaves can be ground to an average particle size smaller than 2 mm, preferably smaller than 1.5 mm, even more preferably smaller than 1 mm, even more preferably smaller than 0.8 mm, even more preferably smaller than 0.6 mm, most preferably smaller than 0.5 mm, smaller than 0.4 mm or smaller than 0.3 mm, or even smaller than 0.2 mm or 0.150 mm.

[0047] The aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, may be included in an amount of about 1 mg / kg to about 10,000 mg / kg of feed, preferably about 5 mg / kg to about 8,000 mg / kg of feed, more preferably about 10 mg / kg to about 6,000 mg / kg of feed, even more preferably about 15 mg / kg to about 4,000 mg / kg of feed, even more preferably about 20 mg / kg to about 3,000 mg / kg of feed, again more preferably about 25 mg / kg to about 2,000 mg / kg of feed, as well as about 30 mg / kg to about 1,500 mg / kg of feed, about 40 mg / kg to about 1,200 mg / kg of in feed, doses range from approximately 50 mg / kg to approximately 1,000 mg / kg of feed, or from approximately 60 mg / kg to approximately 800 mg / kg of feed.

[0048] In one embodiment, the animal feed taught in this document may be a feed suitable for ingestion by farm animals, for example, poultry, swine or ruminants, such as dairy cows, beef cattle, sheep, goats and the like. The expert knows how to formulate a suitable feed for each species of livestock, as well as for each life stage for each species of livestock. Petition 870250079705, dated 05 / 09 / 2025, page 20 / 74 / 55

[0049] In one embodiment, the animal feed taught in this document may be an aquaculture feed, that is, a food suitable for ingestion by fish or crustaceans. Said feed may preferably be in the form of a pellet, more preferably an extruded pellet, for example, extruded for cooking, or pressed. Said feed may comprise protein, fat, carbohydrates, vitamins, minerals and water, and may optionally further comprise pigments, for example, astaxanthin and / or antioxidants. The exact amounts of protein, fat, carbohydrates, vitamins, minerals and water may depend on the species of fish or crustacean, as well as the life stage of the fish or crustacean to which the feed will be provided. The skilled person knows how to formulate a fish or crustacean feed with a suitable composition.

[0050] Feed, when produced to meet the nutritional requirements of marine fish such as cod, pollock, sea bass and gilthead seabream, may contain at least 10%, at least 12.5% ​​or at least 15% fat by weight. Alternatively, feed may contain at least 18%, at least 20%, at least 23%, at least 25%, at least 26% or at least 28% fat by weight when intended to be fed to salmonids. Preferably, the fat used in feed suitable for fish or crustaceans may comprise ω3 fatty acids such as DHA and / or EPA.

[0051] In one embodiment, the animal feed taught in this document may be a suitable feed for a pet, for example, dog, cat, horse, etc. The expert knows how to formulate a pet feed depending on the type of pet as well as its stage of life.

[0052] As an alternative to the aerial parts or leaves, or additionally, an equivalent amount of extract of Glycyrrhiza glabra aerial parts or Glycyrrhiza glabra leaves may be used.

[0053] Plant extraction is a process that aims to extract certain Petition 870250079705, dated 05 / 09 / 2025, page 21 / 74 / 55 components (the so-called secondary plant constituents, including, without limitation, alkaloids, terpenoids, saponins, phenolic compounds, flavonoids and / or tannins) present in plants. This is a solid / liquid separation operation: a solid object (in this case, aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, optionally dried and / or ground) is placed in contact with a fluid (the solvent). The plant components of interest are then solubilized and contained in the solvent. The solution thus obtained is the desired extract. The solvent may eventually be eliminated, although this is not necessary. 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 solvents (e.g., acetone, dichloromethane), and non-polar solvents (e.g., ethyl acetate, hexane, ether, chloroform). The skilled individual may select a single solvent or two or more solvents to prepare an extract according to the present description. In general, extraction procedures include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, supercritical extraction, ultrasound-assisted and microwave-assisted extractions, supercritical fluid extraction (e.g., with supercritical carbon dioxide), accelerated solvent extraction, and solid-phase extraction.The fractionation and purification of phytochemical substances can be achieved through the application of various techniques, including, but not limited to, liquid-liquid partitioning, cross-flow filtration techniques, and chromatographic methods such as paper chromatography, thin-layer chromatography, gas chromatography, high-performance liquid chromatography, and centrifugal partition chromatography. Finally, the compounds obtained are distinguished using various identification techniques, such as mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, and resonance spectroscopy. Petition 870250079705, dated 05 / 09 / 2025, page 22 / 74 / 55 nuclear magnetic, or any combination thereof. The expert is able to select a suitable extraction method.

[0054] The amount of Glycyrrhiza glabra aerial parts extract or Glycyrrhiza glabra leaf extract to be incorporated into the animal feed and / or feed additive described in this document can be determined by the equation of the content of one or more secondary plant constituents in the extract to the content of said one or more secondary plant constituents in the indicated quantities of Glycyrrhiza glabra aerial parts or Glycyrrhiza glabra 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 quantity of Glycyrrhiza glabra aerial parts or leaves can therefore be incorporated into said animal feed.For example, secondary plant constituents present in Glycyrrhiza glabra leaves include, but are not limited to, phenolic compounds, for example, flavanones, prenylated dihydrostilbenes, prenylisoflavones, prenylated flavanones, dihydrochalcones, chalcones, flavone-C-glycosides, flavonol-O-glycosides, hydroxycinnamic acids and coumarins in their glycosidic form or as aglycone.

[0055] In one embodiment, the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetone 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.

[0056] The present description further provides a food additive comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as taught in this document. In addition, the present description provides the use of aerial parts of Glycyrrhiza glabra, preferably leaves of Petition 870250079705, dated 05 / 09 / 2025, page 23 / 74 / 55 Glycyrrhiza glabra, or an extract thereof, as a food additive.

[0057] Said food additive may additionally comprise vitamins, minerals, organic acids, antioxidants and / or pigments. Said food additive may be provided in the form of a premix. The food additive is preferably intended for inclusion in feed for livestock or pets.

[0058] The said feed additive may be supplied with written instructions for its inclusion in animal feed in an amount of Glycyrrhiza glabra aerial parts, preferably Glycyrrhiza glabra leaves, of about 1 mg / kg to about 10,000 mg / kg of feed, preferably about 5 mg / kg to about 8,000 mg / kg of feed, more preferably about 10 mg / kg to about 6,000 mg / kg of feed, even more preferably about 15 mg / kg to about 4,000 mg / kg of feed, even more preferably about 20 mg / kg to about 3,000 mg / kg of feed, again more preferably about 25 mg / kg to about 2,000 mg / kg of feed, as well as about 30 mg / kg to about 1,500 mg / kg of feed, about 40 mg / kg to approximately 1,200 mg / kg of feed, approximately 50 mg / kg to approximately 1.000 mg / kg of feed or about 60 mg / kg to about 800 mg / kg of feed or, if an extract of Glycyrrhiza glabra aerial parts or Glycyrrhiza glabra leaves is used, an equivalent amount of extract as instructed above. Methods of using animal feed and / or feed additives

[0059] In one aspect, the present description provides a method of feeding an animal, for example, a farm animal such as a chicken, a pig, a cow, a fish, a shrimp or the like, or a pet such as a dog or a cat, with an animal feed as taught in this document.

[0060] In a further aspect, the present description refers to the use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or animal feed or feed additive. Petition 870250079705, dated 05 / 09 / 2025, page 24 / 74 / 55 as taught in this document, to increase an animal's performance.

[0061] In another aspect, the present description refers to the use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed or feed additive as taught in this document, to increase feed intake, increase body weight gain, increase average daily gain, increase feed efficiency (i.e., decrease feed conversion ratio), increase relative growth rate, increase specific growth rate and / or increase milk fat production.

[0062] In one embodiment, the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or a food additive or animal feed as taught in this document, can be used as an antibacterial agent, as an antiviral agent, as an antifungal agent, or as an antiparasitic agent.

[0063] In another embodiment, the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or a food additive as taught in this document, may be used as a preservative or as a cytoprotective agent.

[0064] In addition, the present description provides for the use of a composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as taught in this document, or an animal feed or feed additive as taught in this document, in reducing mortality, for example, in animals challenged by a disease, for example, in shrimp challenged by a disease, for example, challenged with acute hepatopancreatic necrosis disease (AHPND), for example, by bacteria of the genus Vibrio, for example, of the species Vibrio parahaemolyticus.

[0065] This description further provides for the use of a Petition 870250079705, dated 05 / 09 / 2025, page 25 / 74 / 55 composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as taught in this document, or an animal feed or feed additive, as taught in this document, for the prevention, relief of severity and / or reduction of microbial infection and / or diseases caused by such microbial infection.

[0066] The present description also provides a method for preventing, alleviating and / or reducing microbial infection in an animal, said method comprising the step of administering to said animal a composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed or feed additive as taught in this document. Microbial infection can be caused by any microbe, such as a bacterium, virus, parasite, yeast or fungus.

[0067] Microbial infection can, for example, be caused by Gram-positive or Gram-negative bacteria.

[0068] Non-limiting examples of bacteria that may be causing microbial infection include Escherichia species, Salmonella species, Vibrio species, Flavobacterium species, Campylobacter species, Clostridium species, Streptococcus species, Piscirickettsia species, such as Piscirickettsia salmonis, causing Salmon Rickettsial Syndrome (SRS), and others, such as Escherichia coli, Salmonella typhimurium, Salmonella dublin, Salmonella enterica, Salmonella enteritidis, Clostridium perfringens, Pasteurella multocida, Listeria monocytogenes, Mycoplasma bovis, Haemophilus somnus, Campylobacter jejunum, Campylobacter hepaticus, Campylobacter bilis and the like.

[0069] In one embodiment, the microbial infection is caused by a Gram-negative bacterium, for example, of the genus Vibrio.

[0070] In aquaculture, several Vibrio spp. are currently considered Petition 870250079705, dated 05 / 09 / 2025, p. 26 / 74 / 55 pathogens or opportunistic pathogens of fish, crustaceans and shrimp raised in captivity, including V. anguillarum, V. ordalii, V. vulnificus, V. alginolyticus, V. parahaemolyticus, Aliivibrio salmonicida, V. harveyi, V. tubiashii and V. cholerae. Vibriosis caused by the species mentioned above is a common and devastating bacterial disease in fish larviculture and aquaculture.

[0071] In one embodiment, the microbial infection is caused by a microbe of the species V. anguillarum, V. ordalii, V. vulnificus, V. alginolyticus, V. parahaemolyticus, Aliivibrio salmonicida, V. harveyi, V. tubiashii and / or V. cholerae. In this case, the infected animal may be a fish or crustacean, for example, a shrimp. In another embodiment, the microbial infection is caused by V. parahaemolyticus. The infected animal may be a crustacean, for example, a shrimp. Diseases caused by V. parahaemolyticus infection may include Vibriosis.

[0072] Other Gram-negative bacteria that may be causing microbial infection may be members of the Flavobacteriaceae family, for example, of the genus Flavobacterium, for example, those selected from the group consisting of F. columnaris, F. johnsoniae, Tenacibaculum maritimum (formerly known as Flexibacter maritimus), F. psychrophilum, F. branchiophilum, Tenacibaculum ovolyticum (formerly Flexibacter ovolyticus) and Chryseobacterium scopthalmum (formerly F. scopthalmum). Diseases caused by infection with these bacteria may be Flavobacteriosis.

[0073] In another modality, the microbial infection is caused by a Gram-positive bacterium, for example, of the genus Streptococcus, for example, Streptococcus agalactiae.

[0074] Members of the genus Streptococcus cause mild to severe bacterial diseases in animals. These organisms typically colonize one or more species as commensals and can cause infections. Petition 870250079705, dated 05 / 09 / 2025, p. 27 / 74 / 55 opportunistic in these hosts. S. agalactiae is a common cause of subclinical mastitis in cattle. In some regions, it also appears to be quite common in clinical or subclinical mastitis in sheep and goats. S. canis is an opportunistic pathogen that primarily affects dogs and cats. It can cause a variety of diseases, including skin and soft tissue infections, arthritis, reproductive diseases, mastitis, pneumonia, septicemia, and streptococcal toxic shock syndrome, as well as cervical lymphadenitis in kittens aged 3 to 6 months and otitis externa in dogs. In cats, this organism sometimes causes neonatal septicemia. S. dysgalactiae subsp. Dysgalactiae is generally associated with clinical or subclinical mastitis in cattle, but has also been detected in other conditions in this species, including severe cellulitis and toxic shock syndrome. Some reports of S. dysgalactiae subsp.Studies of Dysgalactiae in other hosts describe mastitis in sheep and goats, suppurative polyarthritis in lambs, septicemia in fish and dogs, and septicemia and encephalitis in vampire bats.

[0075] In one embodiment, bacteria of the genus Streptococcus are selected from the group consisting of Streptococcus agalactiae, Streptococcus canis, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus equi subsp. zooepidemicus, Streptococcus halichoeri, Streptococcus iniae, and Streptococcus suis. Diseases caused by infection with these bacteria can include skin and soft tissue infections, arthritis, reproductive diseases, mastitis, pneumonia, septicemia, and streptococcal toxic shock syndrome.

[0076] Non-limiting examples of viruses that can cause microbial infection include, depending on the type of farm animal or pet, the white spot syndrome virus (WSSV) which causes white spot syndrome or white spot disease, the infectious hypodermic and hematopoietic necrosis virus (IHHNV) which causes Infectious Hypodermic and Hematopoietic Necrosis, the Syndrome Virus of Petition 870250079705, dated 05 / 09 / 2025, page 28 / 74 / 55 Taura virus (TSV) which causes Taura syndrome, Infectious Myonecrosis Virus (Myo / IMNV) which causes infectious myonecrosis, Fish Myocarditis Virus (PMCV) which causes fish myocarditis, viral hemorrhagic septicemia virus which causes viral hemorrhagic septicemia, infectious hematopoietic necrosis virus which causes infectious hematopoietic necrosis, salmon infectious anemia virus (ISAV) which causes salmon infectious anemia, fish orthoreovirus (PRV) which causes skeletal and cardiac muscle inflammation (HSMI), jaundice syndrome, proliferative browning syndrome and red blood cell inclusion syndrome in fish, lake tilapia virus, occult mortality Nodavirus, shrimp iridescent hemocyte virus and abalone herpesvirus, porcine reproductive and respiratory syndrome virus (PRRSV) which causes porcine reproductive and respiratory syndrome,porcine epidemic diarrhea virus (PEDV) that causes porcine epidemic diarrhea, African swine fever virus that causes African swine fever, classical swine fever virus that causes classical swine fever, Nipah virus, swine vesicular disease virus that causes swine vesicular disease, transmissible gastroenteritis virus that causes transmissible gastroenteritis, avian influenza virus that causes avian influenza, infectious bursal disease virus that causes infectious bursal disease, Marek's disease virus that causes Marek's disease, avian metapneumovirus, avian infectious bronchitis virus that causes avian infectious bronchitis, infectious laryngotracheitis virus that causes infectious laryngotracheitis, duck hepatitis virus that causes duck hepatitis, pseudorabies virus that causes pseudorabies, bluetongue virus that causes bluetongue virus, foot-and-mouth disease virus (serotypes A, O, C, SAT1, SAT2, SAT3, Asia1) which causes foot-and-mouth disease,Japanese encephalitis virus that causes Japanese encephalitis, rabies virus that causes rabies, Rift Valley fever virus that causes Rift Valley fever, rinderpest virus that causes rinderpest, vesicular stomatitis virus that causes vesicular stomatitis. Petition 870250079705, dated 05 / 09 / 2025, page 29 / 74 / 55 West Nile fever virus that causes West Nile fever and others.

[0077] In one embodiment, the microbial infection is caused by a parasite, for example, an endoparasite or an ectoparasite. The parasite may be a member of the phylum Apicomplexa which includes Cryptosporidium spp., Plasmodium spp., Eimeria spp., Neospora, Babesia and Theileria.

[0078] Non-limiting examples of parasites taught in this document include those of the genus Eimeria that cause coccidiosis in a variety of animal species, those of the genus Cryptosporidium that cause cryptosporidiosis, and those of the genus Enterocytozoon that cause, among others, early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp (Enterocytozoon hepatopenaei).

[0079] Parasites of the genus Eimeria include, without limitation, Eimeria tenella, Eimeria acervulina, Eimeria praecox, Eimeria mitis, Eimeria necatrix and Eimeria maxima, which cause coccidiosis in poultry, Eimeria zuernii, Eimeria alabamensis and Eimeria bovis, which cause coccidiosis in ruminants such as cattle, as well as any other species of Eimeria that causes coccidiosis in various animal species.

[0080] Parasites of the genus Cryptosporidium include, without limitation, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, and Cryptosporidium hominis, all causing cryptosporidiosis.

[0081] The present description further provides a method for improving the performance of animals, for example, farm animals, for example, by increasing feed intake, increasing average daily gain, increasing feed efficiency (i.e., decreasing the feed conversion ratio) and / or increasing milk production, said method comprising the step of administering aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or Petition 870250079705, dated 05 / 09 / 2025, page 30 / 74 / 55 an animal feed or food additive, as instructed in this document, for said animals.

[0082] In addition, the present description provides a method for reducing mortality in an animal, said method comprising the step of administering to said animal a composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed or feed additive as taught in this document.

[0083] Furthermore, the composition described in this document has been found to provide cells, particularly liver cells, with protection against mycotoxins such as Aflatoxin B1. It is known that absorbed mycotoxins, like all toxins, are detoxified in the liver. Even short-term exposure to uncontrolled mycotoxins is sufficient to cause significant liver damage and loss of liver function. Therefore, the composition described in this document can also be used as a preservative or as a cytoprotective agent. The animal feed or composition described in this document can be used to prevent, reduce the severity of, and / or treat liver toxicity or liver damage due to mycotoxin exposure.

[0084] The animal feed and / or feed additive for use in the above methods may be as described above.

[0085] The extract may be as described above.

[0086] Animals can be selected from between farm animals and pets. Farm animals can be selected from the group consisting of poultry, pigs, ruminants, for example, beef cattle and dairy cattle, fish, for example, salmon, trout, sea bream, sea bass, tilapia, tuna and the like, and crustaceans, for example, shrimp. Pets can be selected from the group consisting of ornamental fish, cats, dogs, horses, rabbits, guinea pigs and Petition 870250079705, dated 05 / 09 / 2025, p. 31 / 74 / 55 hamsters.

[0087] Animals can be animals affected by a microbial infection (also called “infected animals”) or that are at risk of being infected by a microbe (also called “animals at risk of infection”). The infected animal, or the animal at risk of infection, can be an aquatic species, for example, a fish or a shrimp, or it can be a livestock species, for example, chicken, pig or cow, or any other animal species mentioned in this document.

[0088] In one embodiment, the performance benefits obtained in animals fed the feed additive or animal feed taught in this document are relative to the results obtained with animals (of the same species) fed an identical animal feed, but without such feed additive.

[0089] The present invention is further illustrated, but not limited to, by the following examples. From the above discussion and examples, one skilled in the art can determine the essential features of the present invention and, without departing from the teaching and scope thereof, can make various alterations and modifications to the invention to adapt it to various uses 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 preceding description. Such modifications must also be within the scope of the appended claims. Suitable options:

[0090] 1. Animal feed comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, wherein the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are included in an amount of about 1 mg / kg to about 10 g per kg of feed, or an equivalent amount of extract. Petition 870250079705, dated 05 / 09 / 2025, page 32 / 74 / 55

[0091] 2. Animal feed according to embodiment 1, wherein the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are dried and optionally ground.

[0092] 3. Animal feed according to either embodiment or 2, wherein the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetonic extract, hexane extract or a supercritical CO2 extract, or a mixture of any of these.

[0093] 4. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as a feed additive, wherein said feed additive is supplied with written instructions for including it in animal feed in an amount of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, of about 1 mg / kg to about 10,000 mg per kg of feed, or an equivalent amount of extract.

[0094] 5. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, as an antibacterial, antiviral, antifungal, antiyeast, antiparasitic agent or feed preservative.

[0095] 6. Use according to embodiment 4 or 5, wherein the feed additive, antibacterial, antiviral, antifungal, antiyeast, antiparasitic agent or feed preservative is included in a premix.

[0096] 7. Method of feeding an animal, said method comprising the step of administering to said animal an animal feed in accordance with any of the modalities 1 to 3.

[0097] 8. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed in accordance with any of the embodiments 1 to 3 to increase the Petition 870250079705, dated 05 / 09 / 2025, p. 33 / 74 / 55 performance of an animal.

[0098] 9. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed according to any of the embodiments 1 to 3 to increase feed intake, increase average daily gain, increase feed efficiency (i.e., decrease feed conversion ratio), increase relative growth rate, increase specific growth rate and / or increase milk fat production.

[0099] 10. A composition comprising aerial parts of Glycyrrhiza glabra, preferably Glycyrrhiza glabra leaves, or an extract thereof, or an animal feed in accordance with any of embodiments 1 to 3 for use in reducing mortality.

[00100] 11. A composition comprising aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed according to any of the embodiments 1 to 3 for use in the prevention, relief of severity and / or reduction of microbial infection.

[00101] 12. A composition for use according to the modality 11, wherein said microbial infection is caused by a microbe selected from the group consisting of a bacterium, a virus, a fungus, a yeast or a parasite.

[00102] 13. A composition for use according to any of embodiments 11 or 12, wherein the microbial infection is caused by a Gram-negative bacterium, for example, of the genus Vibrio, for example, of the species Vibrio parahaemolyticus.

[00103] 14. A composition for use in accordance with either embodiment 11 or 12, wherein the microbial infection is caused by a virus, for example, the white spot syndrome virus.

[00104] 15. A composition for use in accordance with any of Petition 870250079705, dated 05 / 09 / 2025, page 34 / 74 / 55 of modalities 11 or 12, in which the microbial infection is caused by a parasite, for example, of the genus Eimeria, of the genus Cryptosporidium or of the genus Enterocytozoon.

[00105] 16. A composition comprising aerial parts of Glycyrrhiza glabra, preferably Glycyrrhiza glabra leaves, or an extract thereof, or an animal feed in accordance with any of embodiments 1 to 3 for use in the prevention, reduction of severity and / or treatment of liver toxicity or liver damage due to exposure to mycotoxins, for example, exposure to Aflatoxin B1.

[00106] 17. Use according to any of embodiments 4 to 6, or composition for use according to any of embodiments 10 to 16, wherein the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetone extract, hexane extract or mixtures of these solvents or a supercritical CO2 extract, or a mixture of any of these.

[00107] 18. Use in accordance with any of the embodiments 4 to 6 or composition for use in accordance with any of the embodiments 10 to 17, wherein the feed additive is intended for inclusion in feed for livestock or pets.

[00108] 19. Use according to modality 18, wherein the farm animals are selected from the group consisting of farmed poultry, swine, ruminants, for example, beef cattle and dairy cattle, fish, for example, salmon, trout, sea bream, sea bass, tilapia, tuna and the like, and crustaceans, for example, shrimp.

[00109] 20. Use according to modality 18, where the pet is selected from the group consisting of ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters. EXAMPLES Petition 870250079705, dated 05 / 09 / 2025, p. 35 / 74 29 / 55 • EXAMPLE 1

[00110] In this example, a sample of Glycyrrhiza glabra roots and a sample of Glycyrrhiza glabra leaves were analyzed for the presence of 18-beta glycyrrhizic acid, which is typical of Glycyrrhiza glabra roots and the main reason for the use of licorice in food and feed applications.

[00111] The material used for the analysis was an Atlantis T3 3.5 pm 4.6 x 150 mm column (no. 1.7) with acetonitrile and 3% acetic acid in water as solvent.

[00112] A reference solution was made by dissolving 10.5 mg of monoammonium glycyrrhizate in 0.8% ammonium hydroxide in water and then diluting to 50.0 ml. 25 ml of this stock solution were diluted to 50 ml with 0.8% ammonium hydroxide solution. The stock solution was prepared as follows. Weigh 0.4 g of powdered roots and leaves (laboratory mill, 2.0 mm sieve) and extract them with 50.0 ml of 0.8% ammonium hydroxide in water, then ultrasonically extract them for 30 minutes and centrifuge for 5 minutes at 4,400 rpm. The supernatant creates the stock solution. The diluted sample solution was prepared as follows. 2 ml of this stock solution were diluted with 0.8% ammonium hydroxide in water to 10 ml and create the diluted sample solution.

[00113] The results of the analysis are summarized in Table 1 below. Table 1. 18-beta-glycyrrhizic acid content in roots and leaves._______________ Sample Name 18-beta glycyrrhizic acid LoD [%] Peak Area Content related to dry matter [%] Glycyrrhiza glabra roots 8.2 1046 4.13 Glycyrrhiza glabra leaves, diluted 9.1 Not detected Not quantifiable Glycyrrhiza glabra leaves, stock solution 9.1 11 0.009

[00114] Figure 1 compares the chromatograms of the root sample of Glycyrrhiza glabra (top graph, clearly showing the acid peaks) Petition 870250079705, dated 05 / 09 / 2025, page 36 / 74 / 55 glycyrrhizic acid) with the leaf sample of Glycyrrhiza glabra (lower graph), in which the glycyrrhizic acid peaks are absent. Table 1 summarizes the results of Figure 1 and shows that no glycyrrhizic acid or traces of glycyrrhizic acid were detected in the leaves of Glycyrrhiza glabra. On the other hand, 4.13% glycyrrhizic acid was found in the root sample of the same plant.

[00115] This experiment was repeated three times with leaves from different Glycyrrhiza glabra plants, and the same results were obtained. The typical glycyrrhizic acid peaks were absent. • EXAMPLE 2

[00116] The effect of dietary inclusion doses of Glycyrrhiza glabra leaves on the productive performance of shrimp was investigated.

[00117] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Collection Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. The composition of the basal diet is presented in Table 2. Table 2. Composition of the basal diet of extruded shrimp feed. Ingredients | Inclusion Levels (kg / 100kg) | Soy Products / By-products | 35 to 40 | Wheat Products / By-products | 35 to 40 | Fish Meal / By-product | 8 to 15 | Macromineral | 4 to 6 | Lecithin / Soybean Oil | 1.5 to 2.5 | Fish Oil NA | 1.5 to 2 | Crustacean-Based Attractant | 1.5 to 3 | Hydrolysates | 1.5 to 3 | Amino Acid Mix | 0.5 to 1.2 | Vitamin and Mineral Premix | 0.3 to 0.5

[00118] The diets were produced as extruded pellets of 1.4 ± 0.1 mm.

[00119] The feed production procedure followed normal guidelines for shrimp feed extrusion. Firstly, all the Petition 870250079705, dated 05 / 09 / 2025, page 37 / 74 / 55 dry raw materials (except micro-ingredients) were ground through a 1 mm grinding sieve (50 Hz). Then, the ingredients were weighed with an accuracy of 0.01 g on an electronic scale following the formula specifications. After extrusion, the pellets were dried until the moisture content was below 10%. The final rations were bagged and stored at 4°C.

[00120] The experimental treatments were carried out as follows. The “Control” shrimp received basal shrimp feed. The “Treated” shrimp received the basal diet supplemented with 520 g per metric ton of feed (520 ppm) of ground dried Glycyrrhiza glabra leaves.

[00121] Control and Treatment were each assigned to 1 plastic tank. Each tank with a capacity of 290L was equipped with an individual filtration system and filled with artificial seawater with a salinity of 20 g / L. 75 shrimp were introduced into each tank. The shrimp were post-larvae of Penaeus vannamei.

[00122] The total weight of the groups was measured at the beginning (day 0) and at the end of the trial (day 18). The average body weight of the shrimp was calculated at the beginning and end of each group. The daily feeding rate for each group was calculated based on the average body weight of the shrimp and adjusted daily according to the expected growth and mortality of the shrimp. Feed was automatically dispensed 6 times a day for a period of 18 days. Water quality was maintained by the filtration system and regular water changes. The water temperature was maintained at 24°C. The room where the tanks were located was illuminated for 12 hours a day.

[00123] Growth performance was evaluated using the following parameters: weight gain (WG) and feed conversion ratio (FCR). Table 3. Shrimp performance Productive Response (unit) Control Treatment Treatment Effect (%) Weight Gain (g) 2.12 2.30 +8.3% FCR 0.95 0.88 -7.4% Petition 870250079705, dated 05 / 09 / 2025, page 38 / 74 / 55

[00124] The results presented in Table 3 show that the addition of Glycyrrhiza glabra leaves to the shrimp diet improved productive performance. The addition of Glycyrrhiza glabra leaves increased weight gain by 8.3%. The feed conversion ratio was reduced (meaning that feed efficiency was improved) by the addition of Glycyrrhiza glabra leaves by 7.4%. EXAMPLE 3

[00125] The effect of 2 doses of dietary inclusion of dried Glycyrrhiza glabra leaves on the productive performance of broiler chickens was investigated.

[00126] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Collection Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. The diets, provided in meal form, were supplied according to the age of the bird: starter feed provided from 0 to 14 days, grower feed provided from 15 to 28 days, and finisher feed provided from 29 to 42 days. The compositions of the basal diets are presented in Table 4. Table 4. Compositions of the basal starter feed, grower feed, and finisher feed._________________________________________________________________________________________ Ingredients, in kg / 100kg Starter feed Grower feed Finisher feed Corn 58.44 60.36 64.56 Soybean meal 30.469 23.82 19.87 Dried distillers grains with solubles 4.00 8.00 8.00 Meat and bone meal 4.00 4.00 3.821 Soybean oil 0.972 2.024 2.256 Limestone 0.309 0.287 0.221 Dicalcium phosphate 0.609 0.281 0.110 Salt 0.391 0.375 0.379 Choline 0.064 0.069 0.069 Lysine 0.218 0.273 0.259 Methionine 0.287 0.266 0.236 Threonine 0.107 0.106 0.085 Mixture that provides other minerals and vitamins 0.143 0.143 0.143 Petition 870250079705, dated 05 / 09 / 2025, page 39 / 74 / 55

[00127] The experimental treatments were carried out as follows. Birds in the Control treatment received basal starter feed, basal grower feed, and basal finisher feed. Birds in treatment 1 received basal diets supplemented with 39 g per metric ton of feed of ground dried Glycyrrhiza glabra leaves. Birds in treatment 2 received basal diets supplemented with 78 g per metric ton of feed of ground dried Glycyrrhiza glabra leaves.

[00128] Each experimental treatment was assigned to 12 pens per treatment. Each pen (the dimensions of each pen are 10.16 cm (4') X 12.7 cm (5')) contained 1 drinker and a feeding tube. The birds started on fresh wood shavings. 34 Ross 308 day 0 chicks were allocated to each pen. The birds were distributed equally by body weight among the 108 study pens. Continuous lighting was provided from day 0 to day 42.

[00129] All broiler chickens were weighed per pen when the birds were 0, 14, 28, and 42 days old. Feed was weighed as needed and weighed when the birds were weighed. The following data were collected during the study: body weight of the broiler chickens on day 0 and day 42 (to calculate body weight gain), feed intake on day 42, feed conversion ratio, and mortality on day 42. Based on these results, feed conversion and adjusted feed conversion were calculated.

[00130] Feed conversion is calculated for a pen as the total feed consumed divided by (sum of the total body weights in the pen + the sum of all mortality weights in the pen).

[00131] The adjusted feed conversion ratio with final BW adjustment on day 42 is calculated as follows: Feed conversion + (2.22 kg - actual body weight) / 7. Petition 870250079705, dated 05 / 09 / 2025, page 40 / 74 / 55 Table 5. Broiler chicken performance Productive Response (unit) Treatments Control Treatment 1 Treatment 2 Feed intake (kg) 3.763 3.769 3.792 Body weight gain (kg) 2.334a 2.409b 2.411B FCR1 1.548a 1.505b 1.495B Adjusted FCR 1.565a 1.501b 1.489B 1FCR = feed conversion ratio

[00132] The results presented in Table 5 show that the addition of ground Glycyrrhiza glabra leaves to the diet of broiler chickens improved productive performance. The addition of ground Glycyrrhiza glabra leaves increased final body weight (P = 0.053) by 3.2% for both inclusion levels of Glycyrrhiza glabra leaves. The feed conversion ratio was reduced by the addition of Glycyrrhiza glabra leaves. For example, the adjusted feed conversion ratio was reduced (P = 0.005) by 4.1% with 39 ppm of Glycyrrhiza glabra leaves and by 5% with 78 ppm of Glycyrrhiza glabra leaves. Mortality was the same for all treatments. EXAMPLE 4

[00133] In this experiment, the effect of dietary inclusion of dried Glycyrrhiza glabra leaves on the response of shrimp to a disease challenge was investigated. The disease challenge was an acute hepatopancreatic necrosis disease (AHPND) challenge produced by the bacterium Vibrio parahaemolyticus.

[00134] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Collection Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. The composition of the basal diet is shown in Table 6 below. Table 6. Composition of the basal diet of extruded shrimp feed. Ingredients | Inclusion levels (in kg / 100kg) | Soy products / by-products | 35 to 40 | Wheat products / by-products | 35 to 40 | Fish meal / by-product | 8 to 15 | Macromineral | 4 to 6 Petition 870250079705, dated 05 / 09 / 2025, page 41 / 74 / 55 Lecithin / soybean oil 1.5 to 2.5% Fish oil NA 1.5 to 2% Crustacean-based attractant 1.5 to 3% Hydrolysates 1.5 to 3% Amino acid blend 0.5 to 1.2% Vitamin and mineral premix 0.3 to 0.5%

[00135] The diets were produced as extruded pellets of 1.4 ± 0.1 mm.

[00136] The feed production procedure followed standard guidelines for shrimp feed extrusion. First, all dry raw materials (except micro-ingredients) were ground through a 1 mm grinding sieve (50 Hz). Then, the ingredients were weighed with an accuracy of 0.01 g on an electronic scale following the formula specifications. After extrusion, the pellets were dried until the moisture content was below 10%. The final feeds were bagged and stored at 4°C.

[00137] During the first part of the experiment, the shrimp were housed in groups and fed either the “Control” diet or the “Treatment” diet. These 2 diets were obtained as follows: the Control group received the basal diet; the Treatment group received the control diet supplemented with 1,040 g of dried Glycyrrhiza glabra leaves per metric ton of feed (1,040 ppm).

[00138] The Control and Treatment groups were each assigned to 1 plastic tank. Each tank, with a capacity of 290L, was equipped with an individual filtration system and filled with artificial seawater with a salinity of 20 g / L. 75 shrimp were introduced into each tank. The shrimp were post-larvae of Penaeus vannamei.

[00139] After 18 days of growth, the shrimp from these 2 groups were transferred to the disease challenge facility and housed in the infection unit (1 shrimp per tank) for acclimation. The animals were housed individually in 10 L tanks and fed with the Petition 870250079705, dated 05 / 09 / 2025, p. 42 / 74 / 55 same diet they received during the first 18 days. The tanks were filled with artificial seawater with a salinity of 20 g / L and equipped with a mechanical / biological filter that ensured water quality. The water temperature was maintained at 27°C and the room where the tanks were kept was illuminated for 12 hours a day. They were allowed to acclimate for 3 days.

[00140] Each treatment replicate (Control or Treatment) corresponds to 3 blocks of 10 individual shrimp per treatment. The shrimp were hand-fed 4 times a day (9:00, 11:30, 14:00, 16:30) and additional feeding was provided at 20:00 and 12:00 on the days of AHPND inoculation. The shrimp were monitored twice a day for clinical signs of disease and mortality.

[00141] The challenge was an acute hepatopancreatic necrosis disease (AHPND) challenge. The bacterium used was Vibrio parahaemolyticus isolated from shrimp carrying AHPND. A stock of this bacterium is kept permanently frozen at -70°C. After thawing, the stock is aseptically inoculated into culture medium and grown using standard conditions. The optical density of the resulting bacterial suspension was determined spectrophotometrically. These data were used to determine the concentration of bacteria in the suspension in colony-forming units per millimeter. Quantified suspensions of the bacteria were used to inoculate shrimp by immersion in all phases of this experiment. Each tank was inoculated with the same dose of bacteria.

[00142] The mortality rate was measured and the cumulative mortality at 14 days post-infection (%) was calculated. The results are shown in Table 7 below. Petition 870250079705, dated 05 / 09 / 2025, pages 43 / 74 / 55 Table 7. Mortality of shrimp subjected to infection by Vibrio parahaemolyticus. Cumulative mortality at 14 days post-infection (%) Control 59.5% Treatment 50% Treatment vs. Control Effect (%) -16%

[00143] The addition of Glycyrrhiza glabra leaves to the diet of shrimp significantly reduced mortality due to Vibrio parahaemolyticus. EXAMPLE 5

[00144] The effect of dried Glycyrrhiza glabra leaves on the productive performance of broiler chickens was investigated.

[00145] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Collection Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. The diets, provided in meal form, were supplied according to the age of the bird: starter feed provided from 0 to 14 days, grower feed provided from 15 to 28 days, and finisher feed provided from 29 to 42 days. The compositions of the basal diets are presented in Table 8 below. Table 8. Compositions of the basal starter feed, grower feed, and finisher feed._________________________________________________________________________________________ Ingredients, in kg / 100kg Starter feed Grower feed Finisher feed Corn 57.02 58.49 58.189 Soybean meal 29.693 23.505 18.654 dried distillers grains Soluble 6.00 10.00 15.000 Meat and bone meal 4.00 4.00 4.000 Soybean oil 1.105 2.213 2.833 Limestone 0.333 0.309 0.212 Dicalcium phosphate 0.567 0.236 Salt 0.433 0.391 0.342 Choline 0.062 0.064 0.054 Lysine 0.238 0.277 0.275 Methionine 0.285 0.260 0.213 Threonine 0.110 0.103 0.074 Mixture providing other minerals and vitamins 0.154 0.152 0.154 Petition 870250079705, dated 05 / 09 / 2025, page 44 / 74 / 55

[00146] The experimental treatments were carried out as follows. The birds in the Control treatment received basal starter feed, basal grower feed and basal finisher feed. The “Treatment” was created by supplementing the basal diets with 55 g per metric ton of feed of ground dried Glycyrrhiza glabra leaves.

[00147] Each experimental treatment was assigned to 12 pens per treatment. Each pen (the dimensions of each pen are 10.16 cm (4') X 12.7 cm (5')) contained 1 drinker and a feeding tube. The birds started on fresh wood shavings. 34 Ross 308 day 0 chicks were allocated to each pen. The birds were distributed equally by body weight among the 24 study pens. Continuous lighting was provided from day 0 to day 42.

[00148] All broiler chickens were weighed per pen when the birds were 0, 14, 28, and 42 days old. Feed was weighed as needed and weighed when the birds were weighed. The following data were collected during the study: body weight of broiler chickens on day 0 and day 42 (to calculate body weight gain), feed intake on day 42, feed conversion ratio, and mortality on day 42. Based on these results, feed conversion and adjusted feed conversion were calculated.

[00149] Feed conversion is calculated for a pen as the total feed consumed divided by (sum of the total body weights in the pen + the sum of all mortality weights in the pen).

[00150] The adjusted feed conversion ratio with final BW adjustment on day 42 is calculated as follows: Feed conversion + (2.22 kg - actual body weight) / 7. Table 9. Broiler chicken performance at 42 days._____________________ Productive Result Control Treatment Effect, % Feed intake (kg) 4.11 4.16 +1.2% Body weight gain (kg) 2.37 2.44 +2.9% Petition 870250079705, dated 05 / 09 / 2025, page 45 / 74 / 55 Adjusted FCR 1.69 1.65 -2.4%

[00151] The results presented in Table 9 show that the addition of Glycyrrhiza glabra leaves to the diet of broiler chickens improved productive performance: it increased the final body weight by 2.9%. The feed conversion ratio was reduced by the addition of Glycyrrhiza glabra leaves by 2.4%. EXAMPLE 6

[00152] The effect of dried Glycyrrhiza glabra leaves on the productive performance of salmon was investigated.

[00153] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Harvesting Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. A basal diet was formulated to meet the nutritional requirements of Atlantic salmon with levels of approximately 41% crude protein, 28% crude lipids, 7% moisture, 24 kJ / g energy and 5% ash.

[00154] The experimental treatments were performed as follows. Control treatment salmon received the basal diet. Treatment salmon received the basal diet supplemented with 130 g per metric ton of feed of ground dried Glycyrrhiza glabra leaves in place of wheat. All macro and micro ingredients, including the ground dried Glycyrrhiza glabra leaves, were dry-mixed before the complete mixture was extruded to create a 4 mm pellet.

[00155] Atlantic salmon with an average weight of 420g were used in the experiment with 2 treatments (Control and Treatment) in triplicate in 1-meter tanks with 40 fish per tank. The water temperature was maintained at 12°C with a salinity of 31 ppt. The salmon were fed 3 times a day. The light regime was 24 hours. The experiment lasted 54 days. The salmon were weighed on the first day (and provided the average initial weight, expressed in g), as well as on the last day of the experiment. Petition 870250079705, dated 05 / 09 / 2025, page 46 / 74 40 / 55 experiment (which provided the average final weight, expressed in g). The number of feeding days was 54. Other results were measured: the final biomass (expressed in g) and the total weight of dead fish (expressed in g) and the total feed intake (expressed in g). The specific growth rate (SGR) and feed intake, both expressed as % per day, were calculated as follows. "D_ / Average final weight .. d VnumberOe «ias «e o Cr íí — L i1_ .,,.,.,1 -LUUvAverage initial weight7, ~ Total ration ingested j Feed intake = SGR x fr , (Final biomass - average initial weight + Total weight of dead fish)

[00156] Table 10 shows the result of the experiment. Table 10. Performance of salmon fed with Glycyrrhiza leaves. Control Treatment Effect (%) Feed intake, % / d 0.527 0.566 7.4% RGR1 0.906 0.934 3.1% SGR 1.169 1.206 3.2% 1RGR is the relative growth rate.

[00157] The inclusion of 130 ppm of Glycyrrhiza glabra leaves increased feed intake by 7.4% and improved the specific growth rate by 3.2%. EXAMPLE 7

[00158] In this example, the effect of Glycyrrhiza glabra leaves in mitigating the impact of Aflatoxin BI on liver cells was studied.

[00159] Alpha 12 mouse liver cells (AML12) were cultured and differentiated in DMEM / F12 medium supplemented with 10% FBS, 1% insulin-transferrin-selenium, 100 IU / mL penicillin, 100 pg / mL streptomycin, and 40 ng / mL dexamethasone at 37°C in a humidified atmosphere with 5% CO2. The cell line was maintained by serial passages.

[00160] 0.1 grams of dried Glycyrrhiza glabra leaves were suspended in 500 µL of DMSO, an aqueous solution of DMSO (DMSO: H2O = Petition 870250079705, dated 05 / 09 / 2025, page 47 / 74 / 55 1:1) or sterile water to obtain stock solution. After vigorous shaking, the completely dissolved suspensions were filtered using a 0.22 μm filter membrane and stored at -20°C. Purified Aflatoxin B1 was dissolved in basic medium or DMSO stored at -20°C before dilution in cell culture medium. Control samples were treated with medium.

[00161] The cytotoxic effect of Aflatoxin B1 and various concentrations of PE solution on AML-12 cells was evaluated using the cell counting kit-8 (CCK-8) reagent. In summary, after treatment with 8 mg / L of Aflatoxin B1 solutions with or without Glycyrrhiza glabra (GG) leaf extract (at 10 µg / mL, 15 µg / mL and 20 µg / mL) for 24 h, 10 µL of CCK-8 solution were added according to the manufacturer's instructions. A control without Aflatoxin B1 was used, and at the indicated time, the absorbance at 450 nm was determined using a microplate reader.

[00162] The results are shown in Table 11 below. Table 11. Cell viability (%) of Treatment Cell viability (%) [1] Control 100 [2] = [1] + 8 mg / L Aflatoxin B1 51.8 [3] = [2] + 10 ug / mL GG 55.9 [4] = [2] + 15 ug / mL GG 64.7 [5] = [2] + 20 ug / mL GG 74.0

[00163] It can be concluded that Glycyrrhiza glabra leaves attenuate the effects of Aflatoxin B1 in cells. EXAMPLE 8

[00164] Newborn chicks from the Longnecker hatchery in Elizabethtown, PA, were housed in Petersime incubator units and provided with feed and water ad libitum. All chicks were weighed and allocated to a cage. Sixty chicks were used per treatment (5 birds / cage = 12 replicates). Initial body weight was the same across treatments. Petition 870250079705, dated 05 / 09 / 2025, pages 48 / 74 / 55

[00165] For E. maxima infection, newly propagated E. maxima (ARS strain) was used to infect the birds. On day 14 after hatching, the birds were infected with 10,000 E. maxima oocysts by oral gavage.

[00166] The experimental treatments were as follows: [1] = Negative control (NC), uninfected and untreated chicken; [2] = Positive control (PC) = [1] + infection; [3] = [2] + 90 mg / kg of feed monensin (MO); [4] = [2] + 55 ppm of dried Glycyrrhiza glabra leaves; [5] = [2] + 160 ppm of dried Glycyrrhiza glabra leaves. Monensin is the current standard used to control coccidiosis.

[00167] The birds were weighed individually on days 7, 14, 18, 20 and 23 to calculate weight gain.

[00168] Oocyst excretion: Feces from each group were collected separately 6 to 8 days post-infection (dpi) for coccidiosis. To count coccidial oocysts, several dilutions were initially made to determine the ideal dilutions for oocyst enumeration for each sample. Oocysts were counted microscopically using a McMaster counting chamber using a sodium chloride flotation method. The total number of oocysts excreted per hen was calculated using the formula: total oocysts / bird = (oocyst count x dilution factor x fecal sample volume / counting chamber volume) / number of birds per cage. Results

[00169] Performance results are shown in Table 12 below. Petition 870250079705, dated 05 / 09 / 2025, page 49 / 74 / 55 Table 12. Performance data of chickens without (NC) or with (PC, MO, GG) E. maxima infection in the absence of additional treatment (PC) or with treatment using monensin (MO), 55 ppm of Glycyrrhiza glabra leaves (55 ppm GG) or 160 ppm of Glycyrrhiza glabra leaves (160 ppm GG). NC PC MO 55 ppm ADG, gd 15 to 19 (0 to 4 dpi) 69.3 59.2 76.3 68.8 63.9 d 15 to 21 (0 to 6 dpi) 71.8 30.3 72.9 50.5 48.2 d 15 to 23 (0 to 8 dpi) 74.3 24.6 79.3 46.4 43.7 ADFI, gd 15 to 19 (0 to 4 dpi) 90.9 83.4 90 84.2 82.8 d 15 to 21 (0 to 6 dpi) 94.5 75.6 93.5 84.8 80.2 d 15 to 23 (0 to 8 dpi) 95.4 70.7 94.3 79.2 75.3 FCR d 15 to 19 (0 to 4 dpi) 1.32 1.49 1.191 1.23 1.33 d 15 to 21 (0 to 6 dpi) 1.32 3.35 1.31 1.76 1.68 d 15 to 23 (0 to 8 dpi) 1.29 2.19 1.21 1.92 1.88 dpi means days after infection; BW means body weight; ADG means average daily gain; ADFI means average daily feed intake; FCR means feed conversion ratio.

[00170] Monensin showed excellent effects in preserving growth performance in chickens infected with E. maxima. Dietary supplementation with dried Glycyrrhiza glabra leaves was also found to improve growth performance in chickens infected with E. maxima.

[00171] Figure 2 shows the results of fecal oocyst excretion. Monensin was found to reduce lesion score and oocyst count, corresponding to the growth results. Glycyrrhiza glabra leaves also significantly reduced the oocyst count. With the addition of 160 ppm of Glycyrrhiza glabra leaves to the feed, the oocyst count was even lower than with monensin. EXAMPLE 9

[00172] The effect of dietary inclusion of Glycyrrhiza glabra leaves on the response of shrimp to a viral disease (white spot disease or white spot syndrome) was investigated. Petition 870250079705, dated 05 / 09 / 2025, p. 50 / 74 / 55

[00173] The leaves of Glycyrrhiza glabra are from cultivation. Cultivation and drying followed Good Agricultural and Collection Practices. The Glycyrrhiza glabra leaves were ground to a particle size < 0.3 mm. The composition of the basal diet is presented in Table 13. Table 13. Composition of the basal diet of extruded shrimp feed. Ingredients | Inclusion Levels (kg / 100kg) | Soy Products / By-products | 35 to 40 | Wheat Products / By-products | 35 to 40 | Fish Meal / By-product | 8 to 15 | Macromineral | 4 to 6 | Lecithin / Soybean Oil | 1.5 to 2.5 | Fish Oil NA | 1.5 to 2 | Crustacean-Based Attractant | 1.5 to 3 | Hydrolysates | 1.5 to 3 | Amino Acid Mix | 0.5 to 1.2 | Vitamin and Mineral Premix | 0.3 to 0.5

[00174] The diets were produced as extruded pellets of 1.4 ± 0.1 mm.

[00175] The feed production procedure followed normal guidelines for shrimp feed extrusion. After extrusion, the pellets were dried until the moisture content was below 10%. The final feeds were bagged and stored at 4°C.

[00176] The experimental treatments were carried out as follows. The “Control” shrimp received basal shrimp feed. The “Treatment 1” shrimp received the basal diet supplemented with 1,040 g per metric ton of feed (1,040 ppm) of dried and ground Glycyrrhiza glabra leaves. The “Treatment 2” shrimp received the basal diet supplemented with 1,040 g per metric ton of feed (1,040 ppm) of dried and ground Glycyrrhiza glabra leaves and 150 g per metric ton of capsicum oleoresin.

[00177] The shrimp were housed individually in 10 L glass tanks filled with artificial seawater composed of the addition of Petition 870250079705, dated 05 / 09 / 2025, page 51 / 74 / 55 a commercial salt mixture was added to purified water at 20 g / L. The tanks were equipped with individual mechanical / biological filters. No water was shared between tanks. Water quality was maintained through filtration, aeration, and regular water changes. The water temperature was maintained at 27°C. The room was illuminated for 12 hours a day. The shrimp were fed 3 times a day. The shrimp were monitored twice a day for clinical signs of disease and mortality.

[00178] The treatment replicates consisted of 3 blocks of 10 individual shrimp per treatment. The blocks were placed in different locations of the challenge setup.

[00179] Three days before initiating the disease challenge, the shrimp were transferred to the disease challenge setup. The shrimp were fed different treatment diets. A white spot syndrome virus (WSSV) was used in this experiment to confer the disease challenge. A virus stock was kept frozen at -70°C in the facility. A solid WSSV inoculum was created and used in the oral infection experiment. The parameter evaluated was mortality (%). The results are shown in Table 14 below. Table 14. Treatment Final Mortality (%) Effect compared to control, % Control 46.7 - Treatment 1 40.0 -14% Treatment 2 33.3 -29%

[00180] The addition of Glycyrrhiza glabra leaves improved survival in response to WSSV virus infection in shrimp. The addition of products containing capsicum oleoresin further improved resistance to the virus. EXAMPLE 10

[00181] This experiment aimed to test the effectiveness of dried Glycyrrhiza glabra leaves in transition dairy cows, with a Petition 870250079705, dated 05 / 09 / 2025, page 52 / 74 / 55 first dose tested of 260 mg / head / day.

[00182] 30 multiparous dairy cows (parity 2+) blocked by BW, MY previous and expected parturition, and assigned to one of the following treatments during weeks -3 to +6 of lactation (n = 15 / trt). The treatments were as follows: 1) control; 2) 260 mg / head / day of dried Glycyrrhiza glabra leaves.

[00183] Measurements taken throughout the study: i) Individual feed intake (daily); Individual milk production (daily); Milk composition (weekly; fat, protein, lactose); Individual body weight (weekly).

[00184] Daily data were subjected to two-way ANOVA. Weekly data were subjected to one-way ANOVA with repeated measures. Results and conclusion

[00185] The results are shown in Table 15 below. Table 15. Performance parameters of dairy cows. Treatment Values ​​of p CON GG DMI, kg 19.0 19.1 0.95 Milk production, kg / d 41.4 40.8 0.87 BW, kg 617 637 0.05 Milk fat, % 4.27 4.48 0.05 Milk protein, % 3.28 3.31 0.80

[00186] It was found that supplementing dried Glycyrrhiza glabra leaves in the diet of dairy cows during the transition period increased the percentage of milk fat (p = 0.05) and increased the body weight of the cows (p = 0.05). EXAMPLE 11 Objective

[00187] The overall objective of the project was to determine the effectiveness of a composition comprising cinnamon oil, tea extract and pomegranate extract (CO / TE / PE; 1:1:1) in attenuating the load and excretion of parasites, in Petition 870250079705, dated 05 / 09 / 2025, page 53 / 74 / 55 development and pathogenesis of diseases and in increasing the protective innate mucosal immunity of the animal host during Cryptosporidium parvum infection using a transgenic mouse infection model. Materials and methods

[00188] Twenty-four (24) 8-week-old male interferon-gamma knockout (IFN-γ KO) mice (B6.129S7-IfngTM1Ts / J) were acquired and quarantined and acclimatized for 1 week prior to the start of the experiments. There were three treatment groups, with 8 mice housed individually per treatment. The treatments consisted of: Group 1, uninfected-untreated; Group 2, C. parvum infected-untreated (Control, DMSO reconstitution reagent only); Group 3, infected-dried Glycyrrhiza glabra leaves. Each mouse in the infected groups was infected by oral gavage administration of 104 isolated C. parvum AUCP-1 oocysts on day 3 after the start of the experiment. 1. Parasites

[00189] The Cryptosporidium parvum AUCP-1 isolate was maintained and propagated in male Holstein calves. C. parvum oocysts were extracted and purified from freshly collected calf feces by sequential sieve filtration, Sheather sugar flotation, and discontinuous centrifugation in a sucrose density gradient. The purified oocysts were washed and stored in phosphate-buffered saline (PBS) at 4°C and used within 3 months to ensure maximum viability assessed by excystation. 2. Animals

[00190] Male knock-out mice for IFN-γ (B6.129S7Ifngtm1Ts / J), 7 weeks old, were acquired from The Jackson Laboratory, USA, and housed under biosafety level 2 conditions. Petition 870250079705, dated 05 / 09 / 2025, page 54 / 74 / 55 Commercial rat food and water were provided ad libitum. The animals were allowed to acclimate for 4 days before the experiments began. 3. Preparation of Glycyrrhiza glabra

[00191] Dried powdered Glycyrrhiza glabra leaves were reconstituted in molecular grade dimethyl sulfoxide (DMSO), followed by sterilization by filtration using a 0.22 μm filter and stored at room temperature in the dark until use. 4. Treatment, infection, sample collection and determination of physical parameters

[00192] The mice were divided into three groups of 8 mice per group. Within each group, each individual mouse was housed in an individual cage. The groups were as follows: • Uninfected untreated group (UU) • Infected DMSO-treated group (DMSO) • Infected group treated with GG

[00193] Three days prior to C. parvum infection, mice in the various treatment groups began receiving daily oral gavage administration of compounds or DMSO as follows: GG = 55 mg / kg; DMSO = 0.05 ml DMSO; UU = 0.05 ml DMSO. On the third day after the start of treatment, mice in the infected groups (DMSO, GG) each received 10⁴ C. parvum oocyst suspensions in PBS by oral gavage, and the respective daily treatments per group continued for 14 days post-infection. In addition to treatment, mice were provided with regular commercial food and water ad libitum. Throughout the treatment period, fecal samples were collected daily and the physical consistency of the feces was determined. The animals were also scored for physical status and activity, body weight, and appetite. In addition, mice were sacrificed and their distal small intestines were extracted. Petition 870250079705, dated 05 / 09 / 2025, page 55 / 74 / 55 were used for histopathological examination. Results 1. Physical parameters:

[00194] In general, the Uninfected and Untreated (UU) mice were active, healthy, had a good appetite, and gained weight. The weight gain and activity observed are typical of healthy mice at this age (7 weeks), as they would still be in the growth phase. The infected mice treated with DMSO (DMSO) showed typical cryptosporidiosis disease progression, with diarrhea, weight loss, poor body condition, and reduced activity. Overall, they lost weight during the experimental period, despite still growing.

[00195] Mice infected and treated with GG had better physical outcomes than the DMSO group. They did not develop diarrhea, remained active and healthy-looking throughout the period, and gained weight, although not as much as uninfected mice. At autopsy, they had healthy-looking intestines without hemorrhages. From these observations, it was evident that treatment with GG attenuated cryptosporidiosis and improved the health of the infected animals. 2. Effect of the composite treatment on the oocyst load of C. parvum in the feces of infected mice:

[00196] As expected, infected mice in the control group treated with DMSO showed a progressive increase in oocyst excretion, with the first peak load of approximately 2 x 10⁸ oocysts per gram of feces (EPG) being reached on day 10 post-infection, with a second, higher peak of 2.2 x 10⁸ oocysts observed 15 days post-infection. Treatment with GG resulted in oocyst loads that were consistently and significantly (P < 0.05) lower than those of infected mice treated with DMSO throughout the Petition 870250079705, dated 05 / 09 / 2025, page 56 / 74 / 55 treatment period. These results corroborated the physical parameters that indicated that treatment with GG attenuated the progression of the disease and the outcome. 3. Attenuation of histopathological lesions:

[00197] At the end of the treatments, the mice were sacrificed and their distal small intestines used for histopathological examination. As expected, the uninfected control group mice showed healthy intestinal mucosa with prominent villi. On the other hand, the infected mice treated with DMSO had intestinal lesions distinguished by mucosal erosion, villous atrophy, crypt hypertrophy, and accumulation of inflammatory cells. The mice treated with GG showed better results than the infected mice treated with DMSO, presenting virtually intact mucosa with prominent villi. Although the mice treated with GG showed an accumulation of inflammatory cells in the mucosa, there was no evident hypertrophy of the intestinal crypts, indicating that these mice still had the capacity to replace their intestinal epithelial cells from the intact crypts.These findings are consistent with the significantly reduced number of oocysts detected in mice treated with GG and therefore indicate that this treatment attenuated parasite proliferation and intestinal pathology in infected mice. EXAMPLE 12

[00198] The objective of this experiment was to determine the effectiveness of Glycyrrhiza glabra leaves in attenuating parasite load and excretion, disease development and pathogenesis, and increasing the protective innate mucosal immunity of the animal host during Cryptosporidium parvum infection using a bovine calf infection model. Experimental approach Petition 870250079705, dated 05 / 09 / 2025, page 57 / 74 / 55

[00199] Determination of efficacy in attenuating cryptosporidiosis

[00200] GG (dried and ground Glycyrrhiza glabra leaves, reconstituted in molecular-grade dimethyl sulfoxide (DMSO) and stored at room temperature in the dark during use) was administered individually to calves by oral gavage once daily for 3 days before infection and continued for 14 days after infection with C. parvum. Calves were also provided with a milk replacer diet and water ad libitum. Throughout the treatment period, fecal samples were collected daily for measurement of C. parvum oocyst excretion by quantitative real-time PCR and determination of fecal physical consistency. Animals were also scored for physical status and activity, body weight, feeding, body temperature, coat condition, and body posture.

[00201] The experiments consisted of 3 groups with 5 calves per group, as follows: • 5 calves infected with C. parvum treated with GG (13.75 g / kg of BW) • 5 calves infected with C. parvum, not treated • 5 uninfected and untreated calves Results and conclusion

[00202] In general, the uninfected and untreated calves were active, healthy, had a good appetite, and were gaining body weight. The weight gain and activity observed are typical of healthy calves at this age, as they would still be in a phase of rapid growth. The infected calves treated with DMSO (DMSO) showed typical cryptosporidiosis disease progression, with diarrhea, weight loss, poor body condition, and reduced activity. They became progressively sicker over time, and two calves died from the disease 10 and 12 days after infection, respectively. Overall, all calves in this group lost weight. Petition 870250079705, dated 05 / 09 / 2025, pp. 58 / 74 / 55 during the experimental period, despite being in the age range of rapid growth.

[00203] Calves infected and treated with GG had better physical outcomes than the untreated infected group. Initially, 2 animals in this group developed diarrhea 3 days after infection, but recovered in 2 to 3 days, although one calf remained somewhat lethargic for several days. The remaining calves in the GG group remained active throughout the period and gained weight, although not as much as the uninfected calves. From these observations, it was evident that treatment with GG attenuated cryptosporidiosis and improved the health of the infected animals.

[00204] As expected, infected control calves treated with DMSO showed a progressive increase in oocyst excretion, with a peak load of approximately 2.5 x 10⁸ oocysts per gram of feces (EPG) obtained on day 9 post-infection. Treatment with GG resulted in oocyst loads that were consistently significantly (P < 0.05) lower than those of infected calves treated with DMSO throughout the treatment period, with the first peak of 6.5 x 10⁴ oocysts per gram of feces observed 8 days post-infection, while the second wave of oocyst excretion peaked at 5.5 x 10⁴ oocysts per gram of feces on day 11 post-infection. These results corroborated the physical parameters that indicated that treatment with GG attenuated disease progression and outcome.

[00205] At the end of the treatments, the calves were sacrificed and their distal small intestines used for histopathological examination. As expected, the calves in the uninfected control group showed healthy intestinal mucosa with prominent villi. On the other hand, the infected calves treated with DMSO had intestinal lesions distinguished by villous atrophy. Notably, the calves treated with GG Petition 870250079705, dated 05 / 09 / 2025, pp. 59 / 74 / 55 showed better results than infected calves treated with DMSO, presenting virtually intact mucosa with prominent villi. On average, uninfected calves, infected calves treated with DMSO, and infected calves treated with GG had villi measuring 300, 260, and 285 μm, respectively. These findings are consistent with the significantly reduced number of oocysts detected in calves treated with GG and therefore indicate that treatment with GG attenuated parasite proliferation and intestinal pathology in infected calves. EXAMPLE 13

[00206] The objective of this experiment was to verify if treatment with dried Glycyrrhiza glabra would have a beneficial effect on parasitic infection by Enterocytozoon hepatopenaei (EHP), one of the most prevalent pathogens and a major challenge in shrimp aquaculture worldwide. It is an intracellular parasitic disease associated with stunted growth in farmed shrimp, high size variation, and numerous economic losses for shrimp farmers. Materials and methods

[00207] The trial design consisted of 3 groups: a negative control, a positive control, and treatment with dried Glycyrrhiza glabra leaves (“GG Treatment”). The trial was established as a completely randomized design (CRD) with 50 shrimp per tank. The trial was conducted for 63 days: including 14 days for pre-challenge, 7 days for EHP cohabitation challenge, and a 42-day post-challenge period.

[00208] The shrimp were fed their respective diets to satiety, during 4 meals per day during the test. The shrimp were fed 5% BW pre-challenge, 3% BW during the EHP infection challenge, and 5% BW post-challenge. The amount of feed was adjusted depending on the estimated biomass and feeding behavior of the shrimp in the tanks. Petition 870250079705, dated 05 / 09 / 2025, pp. 60 / 74 / 55

[00209] EHP-infected shrimp (stock inocula) having an average EHP load / density of 10⁸ to 10⁹ copies per gram of EHP-infected shrimp (quantified using qPCR) were used for the challenge. GG treatment and positive control tanks were challenged by the cohabitation method with 10 EHP-infected shrimp per tank. Negative control tanks were co-cultured with 10 specific pathogen-free (SPF) shrimp. The challenge lasted 7 days of cohabitation. Then, the inoculum (EHP-infected shrimp) and the SPF shrimp were removed from the tanks.

[00210] The SPF and the infected shrimp were separated by a dividing net that allowed free water exchange. The cohabitation challenge method is designed as follows: in each 350 L plastic tank, a rectangular net of 40 cubic liters is installed. During the challenge, 10 EHP-infected shrimp were stocked inside the suspended net, and the experimental shrimp were stocked outside at the same time. In addition, 20 L of water from the EHP-infected inoculum tank was supplied to the positive control and GG treatment tanks to facilitate the challenge.

[00211] Good water quality was maintained throughout the study to ensure optimal shrimp growth and survival over the test period. Results

[00212] Shrimp survival rates are shown in Table 16 below. Table 16. Shrimp survival rates at the end of the trial. Treatment Survival rate (%) Negative control 90.00 ±0.00 Positive control 49.00 ±1.15 Treatment GG 66.50 ±3.00

[00213] The higher survival rate of shrimp was verified with the Petition 870250079705, dated 05 / 09 / 2025, page 61 / 74 / 55 treatment GG, which was significantly higher than the positive control (P < 0.05).

[00214] At the end of the trial, the shrimp were harvested and weighed to calculate growth performance indices. The growth performance parameters of the shrimp on the day of termination are described in Table 17. Table 17. Growth performance parameters after 63 days of culture. Treatment Average initial weight (g) Average final weight (g) Average weight gain (g) ADG (g / day) SGR (% / day) Feed intake (g) of FCR Negative control 1.10 12.51 11.41 0.20 3.86 594.48 1.17 Positive control 1.10 5.43 4.33 0.09 2.53 288.55 3.97 Treatment 1.10 5.70 4.60 0.09 2.61 286.80 2.33 GG

[00215] EHP infection clearly affected shrimp performance (“Positive Control”). Final average body weight, average body weight gain, average daily gain, specific growth rate (SGR), feed intake, and feed conversion ratio (FCR) were all negatively affected by EHP infection. Treatment with GG was able to improve performance parameters compared to the Positive Control.

[00216] It was found that, at the end of the trial, the Positive Control had an average EHP load of 6.6 x 10⁸. The GG Treatment group had an average EHP load of 4.73 x 10⁵, which is significantly lower than the Positive Control. Therefore, based on the EHP load as a measure of effectiveness, the GG treatment reduced the EHP load in shrimp after infection.

[00217] In conclusion, treatment with Glycyrrhiza glabra leaves improved the outcome of EHP infection in terms of performance and mortality. Petition 870250079705, dated 05 / 09 / 2025, pp. 62 / 74

Claims

1 / 4 CLAIMS 1. Animal feed, characterized in that it comprises aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, wherein the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are included in an amount of about 1 mg / kg to about 10 g per kg of feed, or an equivalent amount of extract.

2. Animal feed according to claim 1, characterized in that the aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, are dried and, optionally, ground.

3. Animal feed according to claim 1 or 2, characterized in that the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetonic extract, hexane extract or a supercritical CO2 extract, or a mixture of any of these.

4. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, characterized by being a feed additive 5, wherein said feed additive is provided with written instructions for including it in animal feed in an amount of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, from about 1 mg / kg to about 10,000 mg per kg of feed, or an equivalent amount of extract.

5. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, characterized by being an antibacterial, antiviral, antifungal, antiyeast, antiparasitic agent or feed preservative.

6. Use according to claim 4 or 5, characterized by the fact that the feed additive, antibacterial, antiviral, antifungal, antiyeast, antiparasitic agent or feed preservative is included in a premix.

7. A method for feeding an animal, characterized in that said method comprises the step of administering to said animal an animal feed as defined in any one of claims 1 to 3.

8. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or of an animal feed as defined in any of claims 1 to 3, characterized in that it is for increasing the performance of an animal.

9. Use of aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or of an animal feed as defined in any of claims 1 to 3, characterized in that it increases feed intake, increases average daily gain, increases feed efficiency (i.e., decreases feed conversion ratio), increases relative growth rate, increases specific growth rate and / or increases milk fat production.

10. Composition, characterized in that it comprises aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed as defined in any of claims 1 to 3 for use in reducing mortality.

11. Composition, characterized in that it comprises aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed as defined in any of claims 1 to 3 for use in the prevention, relief of severity and / or reduction of microbial infection.

12. Composition for use according to claim 11, characterized in that said microbial infection is caused by a microbe selected from the group consisting of a bacterium, a virus, a fungus, a yeast or a parasite.

13. Composition for use according to claim 11 or 12, characterized in that the microbial infection is caused by a Gram-negative bacterium, for example, of the genus Vibrio, for example, of the species Vibrio parahaemolyticus.

14. Composition for use according to claim 11 or 12, characterized in that the microbial infection is caused by a virus, for example, the white spot syndrome virus.

15. Composition for use according to claim 11 or 12, characterized in that the microbial infection is caused by a parasite, for example, of the genus Eimeria, the genus Cryptosporidium or the genus Enterocytozoon.

16. Composition, characterized in that it comprises aerial parts of Glycyrrhiza glabra, preferably leaves of Glycyrrhiza glabra, or an extract thereof, or an animal feed as defined in any of claims 1 to 3 for use in the prevention, reduction of severity and / or treatment of liver toxicity or liver damage due to exposure to mycotoxins, for example, exposure to Aflatoxin B1.

17. Use according to any one of claims 4 to 6 or composition for use according to any one of claims 10 to 16, characterized in that the extract is selected from the group consisting of an aqueous extract, ethanolic extract, methanolic extract, isopropanolic extract, ethyl acetate extract, acetone extract, hexane extract or mixtures of these solvents or a supercritical CO2 extract, or a mixture of any of these.

18. Use in accordance with any of claims 4 to 6 or composition for use in accordance with any of claims 10 to 17, characterized in that the feed additive is intended for inclusion in feed for livestock or pets. Petition 870250079705, dated 05 / 09 / 2025, pp. 73 / 74 4 / 4 19. Use according to claim 18, characterized in that the farm animals are selected from the group consisting of farmed poultry, swine, ruminants, for example, beef cattle and dairy cattle, fish, for example, salmon, trout, sea bream, sea bass, tilapia, tuna and the like, and crustaceans, for example, shrimp.

20. Use according to claim 18, characterized in that the pet is selected from the group consisting of ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters. Petition 870250079705, dated 05 / 09 / 2025, pp. 74 / 74

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