USE OF SOPHOROLIPIDS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of United States Provisional Patent Application No. 62/536,815 filed July 25, 2017, the disclosure of which is hereby incorporated herein by reference.

BACKGROUND

[0002] 1 . Field

[0003] The present invention relates to a method for improving gut health comprising treating a food product or supplement with one or more sophorolipids. The present invention also relates to a method for reducing gut inflammation comprising treating a food product or supplement with one or more sophorolipids.

[0004] 2. Descri tion of Related Art

[0005] Sophorolipids are surface-active glycolipid compounds that can by synthesized by a select number of non-pathogenic yeast species. Sophorolipids consist of a hydrophobic fatty acid tail of 16 or 18 carbon atoms and a hydrophilic carbohydrate head, sophorose. Sophorose is a glucose di-saccharide with an unusual β-1 ,2 bond and can by acetylated on the 6'- and/or 6"-positions. One terminal or sub-terminal hydroxylated fatty acid is β-glycosidically linked to the sophorose molecule. The carboxylic end of this fatty acid is either free (acidic or open form) or internally esterified at the 4"- or in some cases at the 6'- or 6"-position (lactonic form). The hydroxyl fatty acid itself counts in general 16 or 18 carbon atoms and can have one of more unsaturated bonds.

[0006] Due to their biodegradability and low eco-toxicity, the use of sophorolipids as bio-surfactants in industrial applications has been increasingly explored. Sophorolipids have also been shown to have anti-microbial properties.

[0007] A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one of a limited number of bacteria in the colon, and thus improves health. Prebiotics mostly are short-chain carbohydrates that alter the composition, or metabolism, of the gut microflora in a beneficial manner. The short-chain carbohydrates are also referred to as oligosaccharides, and usually contain between 3 and 10 sugar moieties or simple sugars. When oligosaccharides are consumed, the undigested portion serves as food for the intestinal microflora. Depending on the type of oligosaccharide, different bacterial groups are stimulated or suppressed.

[0008] Candidate oligosaccharides, of which the prebiotic potential has been investigated limitedly, are derived from germinated barley, dextrans, pectins, polygalacturonan, rhamnogalacturonan, mannan, hemicellulose, arabinogalactan, arabinan, arabinoxylan, resistant starch, melibiose, chitosan, agarose, alginate (Van Loo, 2005, Food Science and Technology Bulletin: Functional Foods 2: 83-100; Van Laere et al., 2000, J Agric Food Chem 48: 1644-1652; Lee et al. 2002, Anaerobe 8: 319-324; Hu et al, 2006, Anaerobe 12: 260-266; Wang et al, 2006, Nutrition Research 26: 597-603). All of these oligosaccharides are produced using enzymatic processes involving either the hydrolysis of polysaccharides or the synthesis starting from smaller carbohydrates using transglycosylation reactions. In some cases, hydrothermal treatment or autohydrolysis is applied to depolymerize lignocellulosic materials, such as xylans (Vazquez et al, 2006, Industrial Crops and Products, 24, 152-159).

[0009] Galacto-oligosaccharides are non-digestible carbohydrates and versatile food ingredients that possess prebiotic properties (Angus, F., Smart, S. and Shortt, C. 2005. In Probiotic Dairy Products ed. Tamine, A. pp. 120-137. Oxford: Blackwell Publishing). In addition, many other health benefits have been reported for these oligosaccharides including: improvement of defecation, stimulation of mineral absorption, elimination of ammonium, colon cancer prevention, as well as protection against certain pathogenic bacteria infections (Hopkins, M. J. and Macfarlane, G. T. 2003 Appl Environ Microbial 69: 1920-1927; Shoaf, K., G. L. Mulvey, G.D. Armstrong, and R. W. Hutkins. 2006 Infect Immun 74: 6920-6928; Macfarlane, G. T., Steed H., Macfarlane S. 2008 Journal of Applied Microbiology 104: 305-344).

[0010] Prebiotic oligosaccharides have been shown to confer a variety of health- promoting effects. Although not all of them have been fully demonstrated, the following beneficial effects have been postulated (Swennen et al, Grit. Rev. Food Sci Nutr. 2006, 46: 459-471 ): alleviation of constipation, improvement of mineral absorption, regulation of lipid metabolism, decrease in risk of colon cancer, beneficial in treatment of hepatic encephalopathy, positive effect on glycemia / insulinemia and modulation of the immune system of the intestine.

[0011] Non-digestible carbohydrates such as inulin, fructo-oligosaccharides and galacto-oligosaccharides are now widely used as prebiotics in order to manipulate the composition of the gut microbiota. A range of other naturally occurring oligosaccharides, and also synthetic products, have selective effects in vitro (Manderson et al, 2005, Appl Environ Microbial 71 : 8383-8389). Prebiotic effects are likely to be influenced by many features of the substrate, including solubility, the distribution of chain lengths, branching and substituents (Rossi et al, 2005, Appl Environ Microbial 71 : 6150-6158). Tests of the ability of isolated bacteria to utilize purified carbohydrates in vitro can provide a preliminary indication of substrate preferences in mixed eco-systems like the gut. It is expected that responses to prebiotics will depend on the dietary context and the gut environment and will be influenced by variations in the species composition and the resident gut microbiota between individuals.

[0012] Thus, there is a need for additional prebiotics that improve gut health which can provide a combination of activities including prebiotic, anti-microbial, and antiinflammatory. The solution to this technical problem is provided by the embodiments characterized in the claims.

BRIEF SUMMARY

[0013] The present application relates to methods for improving gut health by administration of one or more sophorolipids. In a preferred embodiment, the one or more sophorolipids are administered orally. The one or more sophorolipids may be in the form of a supplement. In another embodiment, the one or more sophorolipids are added to a food product.

[0014] The present invention also relates to a method for improving gut health comprising treating a food product or supplement with one or more sophorolipids. [0015] The present invention also relates to a method for reducing gut inflammation comprising treating a food product or supplement with one of more sophorolipids.

[0016] Compositions, preferably prebiotic compositions, comprising sophorolipids are also provided. Additionally, food products and supplements treated with sophorolipids are provided. Such compositions, food products, and supplements can be used to improve gut health in an animal in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.

[0018] FIG. 1 shows an illustration of the development of the SCID mouse model of colitis.

[0019] FIG. 2 shows the experimental design to study the effect of sophorolipid on gut health in SCID mice. LSL(6'Ac,6"Ac) was administered via diet supplementation in the amounts detailed. Each group included 10 mice unless noted otherwise.

[0020] FIG. 3 shows the effect of sophorolipid diet supplementation on gut inflammation as measured by the ratio of colon length to colon weight. A. The effect of all treatment groups on gut inflammation. B. The effect of LSL(6'Ac,6"Ac) on gut inflammation.

[0021] FIG. 4 shows the effect of sophorolipid diet supplementation on gut histopathology.

[0022] FIG. 5 shows the effect of sophorolipid diet supplementation on organ weight.

DETAILED DESCRIPTION

[0023] Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0024] Throughout the present specification and the accompanying claims, the words "comprise", "include" and "having" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0025] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. By way of example, "a sophorolipid" may mean one sophorolipid or more than one sophorolipid.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0027] The subject disclosure features, in one aspect, a method for improving gut health by administration of one or more sophorolipids. In a preferred embodiment, the one or more sophorolipids are administered orally. The one or more sophorolipids may be in the form of a supplement. In another embodiment, the one or more sophorolipids are added to a food product.

[0028] Sophorolipids contain a fatty acid tail and a carbohydrate moiety, sophorose, which is a glucose disaccharide with a 13-1 ,2 bond. The fatty acid tail is β-glycosidically linked to the sophorose molecule. The carboxylic end of this fatty acid may be free (the acidic or open form) or internally esterified at the 4" or at the 6' or 6"-position (the lactonic form). The fatty acid tail may have from 6 to 26 carbon atoms. Generally, the fatty acid tail has 16, 18, or 20 carbon atoms and can have one or more unsaturated bonds. For general overviews and terminology relating to sophorolipids, see Van Bogaert et al, Appl Microbiol Biotechnol (2007) 76:23-34; Van Bogaert et al, Process Biochemistry (201 1 ) 46:821-833; and Lang et al, Fat Sci. Technol. 1989 (91 ), vol. 9, 363-366. WO2004/044216 relates to the antimicrobial properties of sophorolipids and their use. [0029] Sophorolipids can be naturally produced by certain types of yeast strains, notably Starmerella bombicola (also referred to as Candida bombicola) and Candida apicola. Such sophorolipids are referred to as natural sophorolipids. WO2004/044216 and WO2012/0801 16 describe the fermentation of natural sophorolipids in C. bombicola. WO2012/0801 16 also describes the isolation of sophorolipids. Such sophorolipids may be used in a method of the invention. Based on any of these publications, the skilled person understands how to produce such sophorolipids.

[0030] The term "sophorolipids" herein also encompasses modified sophorolipids. Bisht et al (J. Org. Chem. 1999, 64:780-789) describes enzyme-mediated acylation and esterification of sophorolipids. WO2004/044216 describes the chemical synthesis of several modified sophorolipids. Asmer et al (Journal of the American Oil Chemists' Society (1988), vol. 65, no. 9, 1460-1466) disclose the microbial production of sophorolipids. Sophorolipids can also be chemically modified. WO2006/069175 discloses several modified sophorolipids. The carbohydrate moiety can be alkylated, e.g. on the 6' and/or 6" positions. For example, the 6' and 6" position may be acetylated. The 6' position is identical to the 6" position except that the 6' position is closest to the fatty acid tail. Such modified sophorolipids may be used in a method of the invention. Based on any of these publications, the skilled person understands how to produce modified sophorolipids.

[0031] In some embodiments, the sophorolipid may be in the form of a free acid or ester thereof. The sophorolipid may be a sophorolipid of formula (I), wherein Ri and R2 are, independently, H or acetyl; R3 is H or a Ci - Cs alkyl group; and R ₄ is a linear or branched, saturated or unsaturated, alkane unit comprising from 6 to 24 carbon atoms.

[0032] Ri may be H or acetyl. [0033] R2 may be H or acetyl.

[0034] R3 may be H; methyl; ethyl; propyl or isopropyl; n-butyl or isobutyl; n-pentyl, isopentyl, fe/f-pentyl, 2,2-dimethylpropyl; n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2 di- methylbutyl, or 2,3 di-methylbutyl; n-heptane, 2-methylhexane, 3-methylhexane, 2,2- dimethylpentane, 2,3- dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3- ethylpentane, or 2,2,3-trimethylbutane; n-octyl, 2-methylheptane, 3-methylheptane, 4- methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,4- dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethylhexane, 3-ethyl- 2-methylpentane, 3-ethyl-3-methylpentane, 2,2,3-trimethylpentane, 2,2,4- trimethylpentane, 2,3,3-trimethylpentane, 2,3,4-trimethylpentane, or 2,2,3,3- tetramethylbutane.

[0035] In a preferred embodiment, R3 is an alkylester. In a preferred embodiment, the alkylester is an ethyl group or a butyl group.

[0036] R ₄ may be linear or branched. R ₄ may be fully saturated or may have one or more carbon-carbon double bonds. R ₄ forms part of the fatty acid tail of the sophorolipid. A preferred R ₄ has 15 carbon atoms. An example of a fatty acid tail having 18 carbon atoms is oleate. Eicosapentaenoic acid (EPA) is another suitable fatty acid tail, with 20 carbon atoms. A preferred fatty acid tail is 9-octadecenoate.

[0037] In some embodiments, the sophorolipid is 17-L-[(2'-0-p-D-glucopyranosyl-p-D- glucopyranosyl)oxy]-cis-9-octadecenoic acid 6' -acetate (17S-[[6'-0-acetyl-2'-0-(6"-0- hydroxy- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoic acid; ASL(6'Ac,6"OH)), 17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9 - octadecenoic acid 6" -acetate (17S-[[6'-0-hydroxy-2'-0-(6"-0-acetyl- β-D- glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoic acid; ASL(6'OH,6"Ac)), 17-L- [(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octa decenoic acid 6'-6"- diacetate (17S-[[6'-0-acetyl-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoic acid; ASL(6'Ac,6"Ac)), 17-Ι_-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octadecenoic acid (17S-[[6'-0-hydroxy-2'- 0-(6"-0-hydroxy- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoic acid; ASL(6OH,6"OH)), butyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9- octadecenoate-6 -acetate (butyl-17S-[[6'-0-acetyl-2'-0-(6"-0-hydroxy- β-D- glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoate; BSL(6'Ac,6"OH)), butyl-17- L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-o ctadecenoate-6"-acetate (butyl-17S-[[6'-0-hydroxy-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoate; BSL(6OH,6"Ac)), butyl-17-ί-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-octadecenoate- 6'-6"-diacetate (butyl- 17S-[[6'-0-acetyl-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z- octadecenoate; BSL(6'Ac,6"Ac)), butyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D- glucopyranosyl)-oxy]-cis-9-octadecenoate (butyl-17S-[[6'-0-hydroxy-2'-0-(6"-0-hydroxy- P-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoate ; BSL(6OH,6OH)), ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9-octadecenoate-6'- acetate (ethyl-17S-[[6'-0-acetyl-2'-0-(6"-0-hydroxy- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoate; Ε8Ι_(6Άΰ,6"ΟΗ)), ethyl-17-ί-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-octadecenoate- 6"-acetate (ethyl-17S- [[6'-0-hydroxy-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z- octadecenoate; ESL(6OH,6"Ac)), ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D- glucopyranosyl)-oxy]-cis-9-octadecenoate-6'-6"-diacetate (ethyl-17S-[[6'-0-acetyl-2'-0- (6"-0-acetyl- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z-octadecenoate ; ESL(6'Ac,6"Ac), ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9- octadecenoate (ethyl-17S-[[6'-0-hydroxy-2'-0-(6"-0-hydroxy- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoate; ESL(6OH,6"OH)), 17-Ι_-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octadecenoic acid 1 ',4"-lactone-6'- acetate (17S-[[6'-0-acetyl-2'-0-(6"-0-hydroxy- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoic acid, 1 ,4" ester; LSL(6'Ac,6OH)), 17-Ι_-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octadecenoic acid 1 ',4"-lactone-6"- acetate (17S-[[6'-0-hydroxy-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoic acid, 1 ,4" ester; LSL(6OH,6"Ac)), 17-Ι_-[(2'-0-β-ϋ- glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octadecenoic acid 1 ',4"-lactone 6'-6"- diacetate (17S-[[6'-0-acetyl-2'-0-(6"-0-acetyl- p-D-glucopyranosyl)-p-D- glucopyranosyl]oxy]-9Z-octadecenoic acid, 1 ,4" ester; LSL(6'Ac,6"Ac)), and 17-L-[(2'-0- P-D-glucopyranosyl-p-D-glucopyranosyl)oxy]-cis-9-octadecenoi c acid 1 ',4"-lactone (17S- [[6'-0-hydroxy-2'-0-(6"-0-hydroxy- p-D-glucopyranosyl)-p-D-glucopyranosyl]oxy]-9Z- octadecenoic acid, 1 ,4" ester; LSL(6OH,6"OH)).

[0038] In a preferred embodiment, the sophorolipid is 17-L-[(2'-0-p-D-glucopyranosyl- P-D-glucopyranosyl)oxy]-cis-9-octadecenoic acid 1 ',4"-lactone 6'-6"-diacetate, ethyl-17- L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-o ctadecenoate-6'-acetate, ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9-octadecenoate-6"- acetate, ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9- octadecenoate-6'-6"-diacetate, ethyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D- glucopyranosyl)-oxy]-cis-9-octadecenoate, butyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D- glucopyranosyl)-oxy]-cis-9-octadecenoate-6'-acetate, butyl-17-L-[(2'-0-p-D- glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-octadecenoate- 6"-acetate, butyl-17-L- [(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy]-cis-9-oct adecenoate-6'-6"- diacetate, and/or butyl-17-L-[(2'-0-p-D-glucopyranosyl-p-D-glucopyranosyl)-oxy ]-cis-9- octadecenoate.

[0039] In some embodiments, the sophorolipid may be a 6"-mono-acetylated ethyl sophorolipid (ESL(6OH,6"Ac), a de-acetylated ethyl sophorolipid (ESL(6OH,6"OH) or ESL), a di-acetylated ethyl sophorolipid (ESL(6'Ac,6"Ac) or Di-acetyl-ESL), a de- acetylated butyl sophorolipid (BSL(6OH,6"OH) or BuSL), a di-acetylated sophorolipid (LSL(6'Ac,6"Ac) or LSL), and/or a di-acetylated butyl sophorolipid (BSL(6'Ac,6"Ac) or Di- acetyl-BuSL).

[0040] The sophorolipids of the invention may be produced by any microorganism that naturally produces sophorolipids. Microorganisms, such as yeast, have been demonstrated to produce high levels of sophorolipids. Yeast that produce sophorolipids include, but are not limited to, Starmerella {Candida) bombicola, Candida floricola, Candida riodocensis, Candida rugosa, Candida kuoi, Candida stellata, Candida tropicalis, Candida apicola, Torulopsis petrophilum, Rhodotorula (Candia) borgoriensis, Rhodotorula muciliginosa, Candida batistae, Torulopsis gropengiesseri, Cryptococcus sp. , Cyberlindnera samutprakarnensis, Pichia anomala, Wickerhamiella domercqiae, and Yarrowia lipolytica. [0041] Sophorolipids can be easily produced by, for example, inoculating a sophorolipid-producing yeast on a liquid medium containing carbon sources, such as vegetable oil and fat, and sugars such as glucose, and stirring the medium while aerating the medium at a mild temperature and under pressure. In a preferred embodiment, the sophorolipids are isolated and/or purified from the fermentation medium to remove fermentation by-products prior to use. Isolation and/or purification methods are known in the art. Any suitable isolation and/or purification method may be used to obtain substantially purified sophorolipids.

[0042] "Substantially free" means preferably that the corresponding impurities are present only in trace amounts, e.g. in less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1 % by weight, less than 0.5% by weight, less than 0.2% by weight, less than 0.1 % by weight, less than 0.01 % by weight, less than 0.001 % by weight or less than 0.0001 % by weight, in relation to the complete weight of the corresponding dry extract or compound of the formula I or mixture of compounds of the formula I.

[0043] In some embodiments, a microorganism that naturally produces sophorolipids may be modified to increase production of the sophorolipids of the invention.

[0044] In other embodiments, the sophorolipids of the invention may also be produced recombinantly or may be synthesized chemically.

[0045] The food product of the invention includes, but is not limited to, any food, beverage, or animal feed.

[0046] The term "food products" as used herein is to be understood in a very broad sense and includes, but is not limited to, dairy products, meat products, fish products, beverage products, baking products, unpasteurized food products, salads, and sauces, marinades, salsas and seasonings.

[0047] A "dairy product" is understood to include any food product made using milk or milk products, including, but not limited to, milk, yoghurt, ice cream, cheese, skimmed milk, acidified milk, butter milk, condensed milk, spreads, margarines, milk powder, butter, EMC (Enzyme Modified Cheese), Dulce de leche, coffee whitener; coffee creamer, cream, sour cream, ghee, and dairy analogue. Cheese may be any kind of cheese, e.g. fresh cheese, hard cheese, curd cheese, cream cheese, white mould cheese, blue mould cheese and process cheese. The term 'analogue of a dairy product' or 'dairy analogue' refers to a dairy-like product which contains a dairy composition as defined herein and which composition comprises at least one analogue of a dairy ingredient. In various embodiments, the milk is raw milk or milk that has been pasteurized.

[0048] A "meat product" is understood to include any food product, which contains animal tissue, including, but not limited to, beef, pork, and poultry. The term "ready-to-eat meat product" is intended to include any meat product, which does not require cooking prior to consumption, including, but not limited to, pates, hot dogs, bologna, ham, salami, sausages, deli meats, and cold cuts.

[0049] A "fish product" is understood to include any food product which contains tissue from an aquatic animal, including, but not limited to, lobster, crab, fresh water, smoked salmon, smoked other fish, salted fish, saltwater fish and other seafood.

[0050] A "baking product" is understood to include any product prepared from a dough or a batter. The product may have a soft or a crisp character and may be of a white, light, or dark type. Baked products include, but are not limited to, bread (such as white, whole-meal, or rye bread), French baguette-type bread, laminated dough products (such as (Danish) pastry, croissants, or puff pastry), pita bread, tortillas, tacos, cakes, pancakes, biscuits, cookies, doughnuts, bagels, pie crusts, muffins, steamed bread, and crisp bread. Types of baked products, methods to characterize and to produce them are known to those skilled in the art; see, for example, "Baking Science and Technology", by E.J. Pyler, L.A. Gorton, 2008, (2 volumes) Sosland Publishing Company, Kansas, USA, or "Baked Products: Science, Technology and Practice" by S.P. Cauvain, L.S. Young, 2006, Blackwell Publishing Ltd, Oxford, UK.

[0051] "Unpasteurized food product" is understood to include any food product, whereby at least one ingredient is unpasteurized and which does not undergo a final heat treatment.

[0052] A "salad" is understood to include any food product, which contains vegetables, fruits or mixtures thereof. Examples include, but are not limited to, products that are presented for consumers to choose from in a display commonly referred to as a "salad bar", deli salads, processed fruit and vegetables, cut salads and cut vegetables (such as cut lettuce, cut romaine lettuce, cut spinach and cut endive). Of course, the salads can also be uncut.

[0053] In some embodiments, the food product is a liquid food product, preferably a beverage such as iced tea, flavoured water, fruit drinks and fruit juice, carbonated drinks, and lemonade.

[0054] A "beverage" is understood to include ready-to-drink compositions as well as concentrates comprising water and at least one other ingredient and includes, but is not limited to, carbonated and non-carbonated soft drinks, carbonated and non-carbonated water compositions, fountain beverage compositions, frozen ready-to-drink beverage compositions, coffee beverage compositions, decaffeinated coffee beverage compositions, tea beverage compositions (from regular tea, tea derived from fruit products, tea derived from herb products, or decaffeinated tea), dairy beverage compositions, beverage compositions comprising milk derived from soy, rice, coconut or other plant material, powdered soft drinks, vitamin-enhanced soft drinks, liquid concentrated beverage compositions, flavored water compositions, enhanced water compositions, juice compositions (juice derived from any fruit or any combination of fruits and/or juice derived from any vegetable or any combination of vegetables), juice-flavored drinks (juice derived from any fruit or any combination of fruits, juice derived from any vegetable or any combination of vegetables), nectar beverage compositions, sport drinks, highly caffeinated high energy drinks, non-alcoholic beer or wine compositions, and alcoholic beverage compositions (e.g. wine, champagne, malt liquor, rum, gin, vodka, other hard liquors, beer, reduced calorie beer-type beverages, and other beer-type beverages obtained from a cereal solution such as beer, ale, stout, lager, porter, low alcoholic beer, kvass, rye-bread beer, shandy, and malt drinks). If in the form of a concentrate, beverage products suitable for consumption can be prepared by adding volumes of water to the concentrate. Typically, beverage products suitable for consumption can be prepared from the concentrates by combining approximately 1 -part concentrate with between approximately 3- to approximately 7-parts water. In general, water is the basic ingredient of the beverage products disclosed herein, typically being the vehicle or liquid portion in which the remaining ingredients are dissolved, emulsified, suspended or dispersed. Purified water can be used in the manufacture of certain embodiments of the beverages disclosed here, and water of a standard beverage quality can be employed in order not to adversely affect beverage taste, odor, or appearance. The water typically will be clear, colorless, free from objectionable minerals, tastes and odors, free from organic matter, low in alkalinity and of acceptable microbiological quality based on industry and government standards applicable at the time of producing the beverage product. Moreover, beverage products may comprise one or more additional additives selected from anti-foam ing agents, flavors, clouding agents, coloring agents, thickening agents, vitamins, amino acids, minerals, foaming agents, hydrocolloids, herbs, nutraceutical compounds, acidity regulators, preservatives, polysaccharides, sweetening agents, emulsifiers, antioxidants, dietary fibers, mono- and polynucleotides, polypeptides, enzymes and mixtures thereof. Each of these materials may be a single component or a mixture of two or more components.

[0055] The pH of the liquid food product is not crucial. The method is effective at pH between 2 and 8, or between pH 3 and 7.

[0056] The invention further provides a composition comprising a sophorolipid and at least one additional compound including, but not limited to, water, a solvent (such as ethanol or DMSO), an acidity regulator (such as citric acid), an anticaking agent (such as isomalt), an antifoaming agent (such as methylethylcellulose, or mono- or di- glycerides of fatty acids), an antioxidant (such as vitamin C or sulphite), a binder (such as e.g. cyclodextrin, cross-linked sodium carboxymethyl cellulose, ethyl-, methyl-, hydroxypropyl- hydroxypropylmethyl- or methylethylcellulose), a bulking agent (such as cellulose, methylcellulose, or carnauba wax), a carrier (such as alginate), a colour, a surfactant, a colour retention agent, an anti-microbial (such as natamycin pediocin, nisin, levulinic acid, propionic acid, acetic acid, hops acids, and/or lauric arginate), an emulsifier (such as polyethylene glycol, triacetin, triethyl citrate, castor oil, choline salts such as choline tartrate or -lactate, xylitol, lactitol, maltitol, polydimethylsiloxane, sodium laurylsulfate, and lecithin), a preservative (such as natamycin), a dispersant (such as polyoxyethylene compounds such as polyoxyethylene sorbitan monolaurate / - monooleate / -monopalmitate / -monostearate / -tristearate, cellulose, polyvinylpyrrolidone, or propylene glycol), and a thickener (such as alginates or carrageenan).

[0057] According to the invention, the composition comprising a sophorohpid and at least one additional compound is also referred to as "the sophorohpid composition". Examples of a sophorohpid composition are an aqueous sophorohpid solution, an aqueous sophorohpid suspension, an aqueous sophorohpid emulsion, and an ethanolic sophorohpid suspension.

[0058] In some embodiments, the sophorohpid composition is a liquid composition. The advantage of a liquid composition is that it can be conveniently added to a food product, particularly to a liquid food product. The desired amount can be measured, e.g. using a measuring flask or cylinder, instead of weighed. Using a liquid sophorohpid composition allows for the sophorohpid to be dissolved more quickly or more efficiently, and to be distributed over the product more evenly. A liquid sophorohpid composition may be less prone to cake formation. A preferred liquid sophorohpid composition may comprise an emulsifier or an antifoaming agent.

[0059] Alternatively, the sophorohpid composition is a solid composition. An advantage of a solid composition is that such composition is lighter in weight and may be more stable than a liquid composition. A solid sophorohpid composition can be dispersed in or onto a food product. A preferred solid sophorohpid composition comprises a carrier and/or a dispersant, which components may improve the mixing properties or may facilitate dosage.

[0060] In some embodiments, the sophorohpid can be administered as a prebiotic formulation (i.e., without bacteria) or as a probiotic formulation (i.e., with desirable bacteria such as bifidobacteria as described herein). In general, any food or beverage that can be consumed by human infants or adults or animals may be used to make formulations containing the prebiotic and probiotic compositions of the present invention. Exemplary foods include those with a semi-liquid consistency to allow easy and uniform dispersal of the prebiotic and probiotic compositions of the invention. However, other consistencies (e.g., powders, liquids, etc.) can also be used without limitation. Accordingly, such food items include, without limitation, dairy-based products such as cheese, cottage cheese, yogurt, and ice cream. Processed fruits and vegetables, including those targeted for infants/toddlers, such as apple sauce or strained peas and carrots, are also suitable for use in combination with the sophorolipids of the present invention. Both infant cereals such as rice- or oat-based cereals and adult cereals such as Musilix are also be suitable for use in combination with the sophorolipids of the present invention. In addition to foods targeted for human consumption, animal feeds may also be supplemented with the prebiotic and probiotic compositions of the invention.

[0061] Alternatively, the prebiotic and probiotic compositions of the invention may be used to supplement a beverage. Examples of such beverages include, but are not limited to, infant formula, follow-on formula, toddler's beverage, milk, fermented milk, fruit juice, vitamin water, fruit-based drinks, and sports drinks. Many infant and toddler formulas are known in the art and are commercially available, including, for example, Carnation Good Start (Nestle Nutrition Division; Glendale, CA) and Nutrish A/B produced by Mayfield Dairy Farms (Athens, TN). Other examples of infant or baby formula include those disclosed in U.S. Pat. No. 5,902,617. Other beneficial formulations of the compositions of the present invention include the supplementation of animal milks, such as cow's milk.

[0062] Alternatively, the prebiotic and probiotic compositions of the present invention can be formulated into pills or tablets or encapsulated in capsules, such as gelatin capsules. Tablet forms can optionally include, for example, one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge or candy forms can comprise the compositions in a flavor, e.g., sucrose, as well as pastilles comprising the compositions in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The inventive prebiotic or probiotic formulations may also contain conventional food supplement fillers and extenders such as, for example, rice flour. [0063] In some embodiments, the prebiotic or probiotic composition will further comprise a non-human protein, non-human lipid, non-human carbohydrate, or other non- human component. For example, in some embodiments, the compositions of the invention comprise a bovine (or other non-human) milk protein, a soy protein, a rice protein, beta-lactoglobulin, whey, soybean oil or starch.

[0064] The dosages of the prebiotic and probiotic compositions of the present invention will be varied depending upon the requirements of the individual and will take into account factors such as age (infant versus adult), weight, and reasons for loss of beneficial gut bacteria (e.g., antibiotic therapy, chemotherapy, disease, or age). The amount administered to an individual, in the context of the present invention should be sufficient to establish colonization of the gut with beneficial bacteria over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that may accompany the administration of a prebiotic or probiotic composition of the present invention. In some embodiments, the dosage range will be effective as a food supplement and for reestablishing beneficial bacteria in the intestinal tract.

[0065] The amount of the sophorolipid in the food product may be from about 1 to about 1000 ppm, more preferably from about 5 to about 500 ppm, even more preferably from about 10 to about 200 ppm, all based on the total weight of the food product. Exemplary Bifidobacteria dosages include, but are not limited to, 10 ⁴ to 10 ¹² colony forming units (CFU) per dose. A further advantageous range is 10 ⁶ to 10 ¹⁰ CFU.

[0066] According to the invention, "treating" may include contacting, for example, contacting the food product with a sophorolipid. This may be done by adding a sophorolipid to a food product, or adding a food product to a sophorolipid. The food product may be treated such that the sophorolipid is in or on the product, or both. In liquid food products, the sophorolipid will generally be in the food product. In solid food products, the sophorolipid can be in or on the product, or both.

[0067] The sophorolipid can be added to the food product when it is ready for consumption. For example, a sophorolipid can be added to cookies, cheese, yoghurt, a dressing, fruit juice, etc. Alternatively, a sophorolipid can be added to a food product in the making, i.e. during its production process. For example, a sophorolipid may be added to a dough before it is baked to become a bread, a cake, etc. It may also be added to milk before curd formation or to curd before cheese making. It may also be added to a juice concentrate, which has to be diluted before it is ready for consumption. A combination is also possible. For example, a sophorolipid may be added to milk before curd formation, and once the cheese is ready for consumption, additional sophorolipid can be added onto the cheese (onto the surface).

[0068] The sophorolipid may be used in the method of the invention as such, for example, as a powder or as a solution. The sophorolipid may also be in the form of a composition comprising a sophorolipid and at least one additional compound, such as the sophorolipid composition according to the invention, see the following sections. The sophorolipid may comprise two or more different types of sophorolipid.

[0069] In the context of the invention "treating" includes contacting the sophorolipid with the food product and bringing the sophorolipid and the food product together. Treating may be done by adding, spraying, brushing, injecting, dispersing, dissolving. The method may include stirring, mixing, and shaking after the sophorolipid and the food product have been brought together, e.g. to improve the efficacy or distribution of the sophorolipid in the food product.

[0070] The sophorolipid compositions, such as, for example, prebiotic or probiotic formulations of the invention, can be administered to any individual in need thereof. In some embodiments, the individual is an infant or toddler. For example, in some embodiments, the individual is less than, e.g., 3 months, 6 months, 9 months, one year, two years or three years old. In some embodiments, the individual is an adult. For example, in some embodiments, the individual is over 50, 55, 60, 65, 70, or 75 years old. In some embodiments, the individual is immuno-deficient (e.g., the individual has AIDS or is taking chemotherapy).

[0071] Exemplary Bifidobacteria that can be included in the probiotic compositions of the invention include, but are not limited to, B. longum bv infantis, B. longum bv longum, B. breve, and B. adoiescentis. The Bifidobacterium used will depend in part on the target consumer. For example, in some embodiments, B. longum bv infantis is administered with the sophorolipid compositions of the invention to an infant or young child (e.g., under 5 years old). In some embodiments, B. longum bv infantis is included in, or in conjunction with, an infant formula or follow-on formula. In some embodiments, the compositions are administered to an adult or an elderly person. In some embodiments, the person is at least 50, 60, 70, or 80 years old.

[0072] It will be appreciated that it may be advantageous for some applications to include other Bifidogenic factors in the formulations of the present invention. Such additional components may include, but are not limited to, fructoligosaccharides such as Raftilose (Rhone-Poulenc, Cranbury, NJ), inulin (Imperial Holly Corp., Sugar Land, TX), and Nutraflora (Golden Technologies, Westminister, CO), as well as lactose, xylooligosaccharides, soyoligosaccharides, lactulose/lactitol, among others. In some applications, other beneficial bacteria, such as Lactobacillus, can be included in the formulations.

[0073] In some embodiments, the compositions of the invention are administered to a human or animal in need thereof. For example, in some embodiments, the compositions of the invention are administered to a person or animal having at least one condition selected from the group consisting of inflammatory bowel syndrome, constipation, diarrhea, colitis, Crohn's disease, colon cancer, functional bowel disorder (FBD), irritable bowel syndrome (IBS), excess sulfate reducing bacteria, inflammatory bowel disease (IBD), and ulcerative colitis. Irritable bowel syndrome (IBS) is characterized by abdominal pain and discomfort, bloating, and altered bowel function, constipation and/or diarrhea. There are three groups of IBS: Constipation predominant IBS (C-IBS), Alternating IBS) and Diarrhea predominant IBS (D-IBS). The compositions of the invention are useful, e.g., for repressing or prolonging the remission periods on Ulcerative patients. The compositions of the invention can be administered to treat or prevent any form of Functional Bowel Disorder (FBD), and in particular Irritable Bowel Syndrome (IBS), such as Constipation predominant IBS (C-IBS), Alternating IBS) and Diarrhea predominant IBS (D-IBS); functional constipation and functional diarrhea. FBD is a general term for a range of gastrointestinal disorders which are chronic or semi-chronic and which are associated with bowel pain, disturbed bowel function and social disruption. [0074] In another embodiment of the invention, the compositions of the invention are administered to those in need stimulation of the immune system and/or for promotion of resistance to bacterial or yeast infections, e.g., Candidiasis or diseases induced by sulfate reducing bacteria.

[0075] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0076] EXAMPLES

[0077] Sophorolipids can be analyzed by, e.g., HPLC, LC-MS or NMR spectrometry. A suitable NMR spectrometry method is as follows: approximately 5 mg of the sophorolipid and internal standard (dimethoxy benzene) can be weighed (to within 0.001 mg, with microbalance) in a 4 ml vial. The samples can be dissolved in 2 ml MeOD. 1 H NMR spectra can be recorded on a 700 MHz NMR equipped with a cryo probe, measured with a probe temperature of 300K with an interpulse delay of 30 seconds and 16 scans. A suitable LC-MS methodology is as follows: Column: a reversed phase (C18) UPLC- column (1 .7 pm, 100x2.1 mm (LxlD) and gradient elution. The column is kept at 50°C. Gradient elution is performed by mixing 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile in the following way: 0-14 min, 40% B to 100% B; 14-17 min, 100% B; 17-17.1 min, 100% B to 40% B and 17.1 -20 min, 40% B. The flowrate is 400ul/min. Positive ion APCI mode is chosen as ionization mode for these compounds, identification is performed by high-resolution mass spectrometry. Quantification is performed by external calibration curves in combination with internal standard correction

[0078] EXAMPLE 1 : Evaluation of toxicity of sophorolipids on human peripheral blood leukocytes (PBLs).

[0079] Cell viability was measured by Alamar Blue at various concentrations (1 .25pm, 2.5μηπ, 5μηπ, Ι Ομητι, and 20μηπ) of six sophorolipids: LSL(6'Ac,6"Ac); ESL(6OH,6"Ac); ESL(6OH,6"OH); ESL(6'Ac,6"Ac); BSL(6OH,6"OH); and BSL(6'Ac,6"Ac).

[0080] Results: None of the sophorolipids induced toxicity at any concentration tested. [0081] EXAMPLE 2: Evaluation of effect of sophorolipids on inflammatory mediators in human PBLs.

[0082] PBLs were isolated from human blood. PBLs were treated with LPS to induce inflammatory response in the presence of different concentrations (1 pm, 5pm, and 10pm) of six sophorolipids: LSL(6'Ac,6"Ac); ESL(6OH,6"Ac); ESL(6OH,6"OH); ESL(6'Ac,6"Ac); BSL(6OH,6"OH); and BSL(6'Ac,6"Ac).

[0083] Results: ESL(6OH,6"OH) decreased IL-1 β secretion, IL-6 secretion, IL-8 secretion, TNF-a secretion, and ΜΙΡ-1 β secretion. ESL(6'Ac,6"Ac) decreased IL-1 β secretion, IL-6 secretion, IL-8 secretion, TNF-a secretion, and ΜΙΡ-1 β secretion. BSL(6OH,6"OH) decreased IL-1 β secretion, IL-6 secretion, IL-8 secretion, TNF-a secretion, and ΜΙΡ-1 β secretion. LSL(6'Ac,6"Ac) decreased IL-8 secretion, TNF-a secretion, and ΜΙΡ-1 β secretion. ESL(6'OH,6"Ac) decreased TNF-a secretion and MIP- 1 β secretion. BSL(6'Ac,6"Ac) decreased TNF-a secretion.

[0084] These results demonstrate that sophorolipids have anti-inflammatory and anti- cytokine effects in PBLs.

[0085] EXAMPLE 3: Evaluation of effect of sophorolipids on inflammatory mediators in microglia.

[0086] Microglial cells were obtained from primary microglial cultures from E22 rats. On day 1 , microglial cells were seeded in 96-well plates and allowed to adhere for 24 hours. On day 3, the microglial cells were pre-treated with sophorolipid for 24 hours (n = 1 1 ). On day 4, the microglial cells were stimulated with lipopolysaccharide (LPS). On day 5, supernatants were collected and analysed for cytokines of interest.

[0087] Sophorolipid pre-treatment was effective in reducing both PGE2 (IC50 = 29.8 μΜ) and TNF-a (IC50 = 21 .2 μΜ) secretion. [0088] EXAMPLE 4: Evaluation of toxicity of sophorolipids on colon epithelial cells.

[0089] HT-29 cells (a colon adenocarcinoma cell line) were used because these cells resemble mature intestinal cells in vitro. Cell viability was measured by Alamar Blue at various concentrations (1 .25pm, 2.5pm, 5pm, 10pm, and 20pm) of six sophorolipids: LSL(6'Ac,6"Ac); ESL(6OH,6"Ac); ESL(6OH,6"OH); ESL(6'Ac,6"Ac); BSL(6OH,6"OH); and BSL(6'Ac,6"Ac).

[0090] Results: None of the sophorolipids induced toxicity at any concentration tested.

[0091] EXAMPLE 5: Evaluation of effect of sophorolipids on inflammatory mediators in colon epithelial cells (HT-29)

[0092] HT-29 cells were treated with TNF-a to induce inflammatory response in the presence of different concentrations (2.5pm, 5pm, 10pm, and 20pm) of six sophorolipids: LSL(6'Ac,6"Ac); ESL(6OH,6"Ac); ESL(6OH,6"OH); ESL(6'Ac,6"Ac); BSL(6OH,6"OH); and BSL(6'Ac,6"Ac). EGCG was used as a positive control.

[0093] Results: LSL(6'Ac,6"Ac) decreased GRO-a/CXCL1 secretion and IL-8 secretion. All six sophorolipids decreased IL-1 a secretion and RANTES secretion.

[0094] These results demonstrate that sophorolipids have anti-inflammatory effects in HT-29 cells.

[0095] EXAMPLE 6: Effect of sophorolipids on inflammatory mediators in RAW macrophage cell line.

[0096] RAW macrophages were exposed to various sophorolipids (LSL(6'Ac,6"Ac); ESL(6'Ac,6"Ac); ESL(6OH,6"OH); and ASL(6OH,6"OH)) and to sophorose at 1 - 100 μΜ concentrations for 12 hours and then inflammatory mediators were measured. Treatment for 12 hours with all sophorolipids tested were able to decrease IL-6. LSL(6'Ac,6"Ac) and ESL(6OH,6"OH) decreased TNF-a after 12 hours of treatment. [0097] EXAMPLE 7: Effect of sophorolipids in an in vivo model of human colitis.

[0098] The effect of sophorolipids on gut health was studied in SCID mice. SCID mice were developed from normal BALB/c mice in which CD4+ cells were transferred to result in a pathology that is similar to human colitis. IL-12 has been previously shown to reduce inflammation in this model as shown in FIG. 1.

[0099] Diets were introduced to female SCID-Balb mice at 45 days of age. Retro- orbital blood samples were collected 7 days after the diet was started (+7d) to obtain plasma samples and determine red blood cell counts. After 28 days on the diet (+28d), CD4+ T-cells were transferred into the mice intraperitoneally. The mice were weighed and food consumption was monitored weekly from +0d to +28d and from +70d to +77d. Necropsy was performed on +77d and organs and trunk blood were collected for analysis.

[00100] The treatment groups analysed are shown in FIG. 2. Two normal control groups were included (either Harlan Teklad 8640 mice or Sc/ci/Balb mice) without diet supplementation and without insult. One positive control was included without diet supplementation and treated with 0.5 mg/kg/wk anti-IL12 administered intraperitoneally. One negative control group did not receive diet supplement. Three groups received diet supplementation (100 mg/kg/d LSL(6'Ac,6"Ac)). Harlan Teklad 8640 mice fed a diet supplemented with 7% alpha-linolenic acid (18:3n3) and 55% linoleic acid (18:2n6) were also included as a negative control.

[00101] The results of this study are present in FIGS. 3-5. The mice tolerated 100 mg/kg/d LSL(6'Ac,6"Ac) with no loss of body weight and normal food consumption. LSL(6'Ac,6"Ac) supplementation reduced inflammation and its related histopathology.

[00102] EXAMPLE 8: Effect of dietary supplementation of sophorolipids on performance and digestibility in non-challenged broilers.

[00103] One day old male COB500 broilers were used for this study. The broilers were housed six birds per cage and six cages were treated for each group. Thus, 36 broilers were included in each treatment group. During the study, the broilers were fed ad libitum the diet presented in Table 1 in pelleted form for 28 days +/- supplementation. [00104] Table 1.

[00105] The diet was supplemented with one of the treatments described in Table 2. For each treatment, 150 kg feed was prepared. Titanium oxide (intestinal marker) was incorporated at 1 %. Each sophorolipid was solubilized in DMSO and mixed with 5. soybean oil. The resulting mixture was sprayed onto the mash diet and pelleted.

[00106] Table 2.

[00107] The broilers were weighed at d8, d14, d22, and d28. Feed consumption was measured for the same time periods. Daily weight gain (DWG) and daily food intake (DFI) were calculated for the following periods: d8-14, d 15-21 , d22-28, and d8-28.

[00108] Feces was collected at d20, d21 , d22, and d23. Total nitrogen and gross energy were measured for calculation of apparent total tract digestibility (ATTD) of protein and energy and apparent metabolizable energy (AMEn), respectively.

[00109] Cecal samples were collected at d28 for determination of the microbiota and short chain fatty acids (SCFA).

[00110] The effect of sophorolipid supplementation on cecal short chain fatty acids (SCFA) is shown in Table 3. [00111] Table 3.

[00112] Broilers fed the diet with LSL(6'Ac,6"Ac) at 100 ppm reduced cecal propionate and total SCFA concentrations in comparison to the control group.

[00113] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention. [00114] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.