FIELD OF INVENTION
[001] The present disclosure broadly relates to field of bio-preservatives, and particularly relates to a liposomal composition comprising nisin, xanthohumol, and at least one polymer.
BACKGROUND OF INVENTION
[002] Food spoilage being a major economic problem, food manufacturers always look for cost-effective options to deliver products with an extended shelf life. At the same time ensuring food safety is an even greater concern worldwide, primarily with the growing trend for convenience foods such as ready-to-eat meals, juices, preserves or snacks. Moreover, today's consumers prefer high quality food products with lesser preservatives/no preservatives which is a major challenge for food manufacturers.
[003] Bacteriocins are antimicrobial peptides, few examples of bacteriocins are nisin, pediocin, lacticin, sakacin, lactococcin, enterococin, plantaricin, leucocin that are produced by certain bacterial species and have potential to inhibit the growth of closely related bacterial species. (Boualem K et al., 2013, Journal of Food Research, 36-46). Amongst all bacteriocins, nisin (NS), a colorless, tasteless hydrophobic cationic peptide (3.5 kDa, listed under GRAS category) produced by Lactococcuslactis subsp. Lactis, has been used as a food preservative worldwide. [004] Over the past two decades application of NS has advanced beyond its role as being only a food bio-preservative. It exhibits anti-biofilm as well as immune-modulatory properties. Moreover, increasing evidence shows that NS can influence the growth of tumors and exhibit selective cytotoxicity towards cancer cells (Jae M. Shin et al., 2016, J Appl. Microbiol. 120, 1449-1465).
[005] Mechanisms involved in controlling bacterial growth by NS
involves inhibiting the peptidoglycan synthesis and destructing the membrane potential by specifically binding to phospholipids of cytoplasmic membrane. The binding takes place via electrostatic interactions leading to increase in the efflux of small molecules through pore formation. NS effectively inhibits Gram-positive

bacteria such as Listeria monocytogenes, Staphylococcus aureus, spores of Bacillus cereus and Clostridium botulinum. Whereas, NS is not effective against Gram-negative bacteria, yeast or molds unless a chelating agent is used simultaneously (Boualem K et al., 2013, Journal of Food Research, 36-46). Overall, the usage of other bacteriocins such as pediocin, lacticin, sakacin, lactococcin, enterococin, plantaricin, leucocin is restricted since the growth of the producer organism was required within the food to secrete bacteriocin (WO2005104878A1). [006] In spite of its extensive usage, NS is poorly soluble, highly sensitive to heat, pH and food enzymes. Undesirable interaction of NS with food compounds and their subsequent activity loss is unavoidable when it is added directly as a free form into food items. In order to overcome the activity loss, very high concentration of NS is added to the food products. Addition of high levels of NS into the food products may affect/change the organoleptic and textural properties of the food (P. da Silva Malheiros et al. 2010, Food Research International, 1198-1203). [007] Xanthohumol (2',4',4-trihydroxy-6'-methoxy-3 '-prenyl chalcone, XN), a characteristic component present in female inflorescences of hops {Humulus lupulus L.) plant has long been used in medical and brewing industry. It has been well documented that XN holds a broad range of health benefits such as antioxidant, anti-inflammatory, antimicrobial, immune modulatory activity etc. (Legette L et al. Mol Nutr Food Res. 2014; 58: 248-255) and also finds application in hair and scalp care (US 2008/0207928 Al). But still the usage of XN is very less due to its poor aqueous solubility, instability, and bioavailability (Legette L et al. Mol. Nutr. Food Res. 2014; 58:248-255).
[008] The foodborne pathogens may find difficult in establishing their colonies in the food products when preserved using different preservative strategies. For a desired biological benefit, actives should be more stable and must have the ability to readily solubilize at any condition and pass through cell membrane. Therefore, it is essential to improve the solubility and stability of NS as well as XN in order to increase its antimicrobial potential even at low concentration. [009] US20070042184A1 relates to microcapsules, and the aqueous beads comprising the active ingredient is encapsulated by hydrophobic shell matrix.

[0010] US8337923B2 relates to an antimicrobial material in an encapsulated form, wherein the encapsulated form comprises an antimicrobial material and a shell of encapsulating material.
SUMMARY OF THE INVENTION
[0011] In an aspect of the present invention, there is provided a liposomal composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1. [0012] In an aspect of the present invention, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition.
[0013] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0014] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0015] Figure 1 illustrates stability of NS: XN loaded liposomes, in accordance with an embodiment of the present disclosure.

[0016] Figure 2 illustrates the effect of liposome compositions in inhibiting
Staphylococcus aureus growth, in accordance with an embodiment of the present
disclosure.
[0017] Figure 3 illustrates the effect of liposome compositions in inhibiting
Escherichia coli growth, in accordance with an embodiment of the present
disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features, compositions,
and compounds referred to or indicated in this specification, individually or
collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0019] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
below.
[0020] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0021] The terms "comprise" and "comprising" are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
construed as "consists of only".
[0022] Throughout this specification, unless the context requires otherwise the
word "comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or
steps.

[0023] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably. [0024] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a ratio of 1:5 to 4:1 is intended to cover not only the specified limits of 1:5 and 4:1, but to cover the inner ranges such as 1:4.1 to 1:1.2, and 1 A3 to 1:1. It is also intended to cover fractional values such as 1:1.5, and 3.5:1 so on. [0025] An excipient is an inactive substance that serves as the vehicle or medium for a drug or other active substance. Excipients can also include non-active chemicals that provide textural, functional and sensorial features. Excipients include colouring agents, humectants, diluting agent, preservatives, emollients, other known cosmetically suitable additives, and combinations thereof. [0026] For the purpose of the present disclosure, active components of the present composition refer to nisin and xanthohumol. The composition has a weight ratio of the active components to the at least one polymer in a range of 1:500 to 1:4000. [0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0028] Research is active in the field of preservatives, and especially increasing the efficacy of well-established products is a smart approach in the present field. At present, some novel encapsulation methods are available to overcome the limitations of the preservative as discussed in the previous sections. [0029] Encapsulation of antimicrobial compounds using liposomes can offer a potential solution to protect antimicrobials, enhance their efficacy and stability in

food application. Liposomes are spherical bi-layer colloidal structures formed by self-assembly that may contain one or more phospholipid bilayers with an aqueous internal core.
[0030] Lysophosphatydilcholine (LPC) is obtained by hydrolysis of one fatty acyl residue from naturally abundant sources of phosphatidylcholine (PC). PC are easily available from cheap sources, such as egg yolk or soybeans, lecithin. LPC has two distinctly different regions: a long hydrocarbon chain of non-polar, lipophilic character; and a phosphoric acid-choline moiety, polar in nature. LPC can be found in low concentrations in cell membrane, where it plays an essential biological role as membrane-derived bioactive lipid mediator (Chavez-Zamudio R et al., 2017 Food Funct.20, 3346-3354). LPC is a good emulsifying and solubilizing agent, and finds application in food, cosmetics, agrochemicals and pharmaceutical industries. [0031] The present disclosure provides compositions and method for co-encapsulating nisin and xanthohumol at a definite ratio. Surprisingly, specific ratios of nisin and xanthohumol show enhanced antimicrobial activity against Gram-positive and Gram-negative bacteria when compared to free nisin and xanthohumol.
[0032] The present disclosure is to provide compositions comprising microscopic liposomes containing nisin and xanthohumol and process for obtaining said microscopic liposomes respectively, at a weight ratio (w/w) of nisin and xanthohumol in a range of 1:5 to 4:1. The liposomes within the specified range shows an enhanced stability and potential to interact with bacterial system and inhibits its growth.
[0033] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0034] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1. In another embodiment of the present disclosure, nisin to xanthohumol weight ratio is in a range of 1:4 to 4:1.

[0035] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2.
[0036] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:4.
[0037] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:1.5.
[0038] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:1.
[0039] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1.5:1.
[0040] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 4:1.
[0041] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein nisin and
xanthohumol are active components of the composition and the active components
to the at least one polymer weight ratio is in a range of 1:500 to 1:4000. In another
embodiment of the present disclosure the active components to the at least one
polymer weight ratio is in a range of 1:2000 to 1:3900. In yet another embodiment
of the present disclosure, the active components to the at least one polymer weight
ratio is 1:3800.
[0042] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2, and wherein nisin and
xanthohumol are active components of the composition and the active components

to the at least one polymer weight ratio is in a range of 1:500 to 1:4000. In another embodiment of the present disclosure the active components to the at least one polymer weight ratio is in a range of 1:2000 to 1:3900. In yet another embodiment of the present disclosure, the active components to the at least one polymer weight ratio is 1:3800.
[0043] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein the at least one
polymer is selected from a group consisting of L-a-lysophosphatidylcholine, 1,2-
didecanoyl-sn-glycero-3-phosphocholine (DDPC), l,2-dierucoyl-sn-glycero-3-
phosphocholine (DEPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-glycero-3-
phosphocholine(DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine(DPPC), l,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC), Egg-PC (EPC), hydrogenated Egg PC (HEPC),
hydrogenated Soy PC (HSPC), l-myristoyl-2-palmitoyl-sn-glycero 3-
phosphocholine (Milk Sphingomyelin MPPC), l-myristoyl-2-stearoyl-sn-glycero-
3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine
(PMPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-
2-stearoyl-sn-glycero-3-phosphocholine (PSPC), l-stearoyl-2-myristoyl-sn-
glycero-3-phosphocholine (SMPC), l-stearoyl-2-oleoyl-sn-glycero-3-
phosphocholine (SOPC), 1 -stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine (SPPC), and combinations thereof. In another embodiment of the present disclosure, the at least one polymer is L-a-lysophosphatidylcholine [0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein nisin has a weight percentage in a range of 0.005 to 0.03% with respect to the composition, xanthohumol has a weight percentage in a range of 0.005 to 0.03% with respect to the composition. In another embodiment of the present disclosure, nisin has a weight percentage in a range of 0.005-0.010%) with respect to the composition,

xanthohumol has a weight percentage in a range of 0.015-0.025% with respect to the composition. In one of the embodiment of the present disclosure, the at least one polymer has a weight percentage in a range of 99.94-99.99% with respect to the composition.
[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein the composition has an active loading in a range of 0.2-6%, and the composition has an encapsulation efficiency in a range of 20-62%. In another embodiment of the present disclosure, the composition has an active loading in a range of 2.40-5.80%), and the composition has an encapsulation efficiency in a range of 45-62%>. [0046] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition has an active loading in a range of 0.2-6%), and the composition has an encapsulation efficiency in a range of 20-62%). [0047] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1. In another embodiment of the present disclosure, the at least one excipient is selected from a group consisting of colouring agents, humectants, diluting agent, preservatives, emollients, other known cosmetically suitable additives, and combinations thereof. In yet another embodiment of the present disclosure, weight percentage range of 99.94-99.99%) of the at least one polymer can be changed as per weight percentage of the at least one excipient. It can be contemplated that apart from weight percentage range of nisin and xanthohumol, weight percentage of the at least one polymer is flexible to the addition of the at least one excipient subject to the conditions that the characteristics of the composition as described in the present disclosure is not compromised.
[0048] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1.

[0049] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein
nisin and xanthohumol are active components of the composition and the active
components to L-a-lysophosphatidylcholine weight ratio is in a range of 1:500 to
1:4000.
[0050] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2.
[0051] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is 1:4.
[0052] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is 1:1.5.
[0053] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is 1:1.
[0054] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is 1.5:1.
[0055] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) L-a-lysophosphatidylcholine,
wherein nisin to xanthohumol weight ratio is 4:1.
[0056] In an embodiment of the present disclosure, there is provided a process for
preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least
one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1,
said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii)
obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the
at least one polymer, to obtain the composition.

[0057] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition.
[0058] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is 1:4, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition.
[0059] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is 1:1.5, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition.
[0060] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition, wherein nisin and xanthohumol are active components of the composition and the active components to the at least one polymer weight ratio is in a range of 1:500 to 1:4000.
[0061] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the

at least one polymer, to obtain the composition, wherein nisin and xanthohumol are active components of the composition and the active components to the at least one polymer weight ratio is 1:3800.
[0062] In an embodiment of the present disclosure, there is provided a process for
preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least
one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1,
said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii)
obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the
at least one polymer, to obtain the composition, wherein the at least one polymer is
selected from a group consisting of L-a-lysophosphatidylcholine, 1,2-didecanoyl-
sn-glycero-3 -phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine
(DEPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-
glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-glycero-3-
phosphocholine(DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine(DPPC), l,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC), Egg-PC (EPC), hydrogenated Egg PC (HEPC),
hydrogenated Soy PC (HSPC), l-myristoyl-2-palmitoyl-sn-glycero 3-
phosphocholine (Milk Sphingomyelin MPPC), l-myristoyl-2-stearoyl-sn-glycero-
3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine
(PMPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-
2-stearoyl-sn-glycero-3-phosphocholine (PSPC), l-stearoyl-2-myristoyl-sn-
glycero-3-phosphocholine (SMPC), l-stearoyl-2-oleoyl-sn-glycero-3-
phosphocholine (SOPC), 1 -stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine (SPPC), and combinations thereof. In another embodiment of the present disclosure, the at least one polymer is L-a-lysophosphatidylcholine. [0063] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition, wherein nisin has a weight

percentage in a range of 0.005 to 0.03% with respect to the composition, xanthohumol has a weight percentage in a range of 0.005 to 0.03% with respect to the composition.
[0064] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; and (iv) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition, wherein the composition has an active loading in a range of 0.2-6%, and the composition has an encapsulation efficiency in a range of 20-62%.
[0065] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; (iv) obtaining the at least one excipient; and (v) contacting nisin, xanthohumol, the at least one polymer, and the at least one excipient, to obtain the composition. [0066] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; (iv) obtaining the at least one excipient; and (v) contacting nisin, xanthohumol, the at least one polymer, and the at least one excipient, to obtain the composition. [0067] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; (iv) obtaining the at least one excipient; and (v) contacting nisin, xanthohumol, the at least one

polymer, and the at least one excipient, to obtain the composition, wherein nisin and xanthohumol are active components of the composition and the active components to the at least one polymer weight ratio is in a range of 1:500 to 1:4000.
[0068] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; (iv) obtaining the at least one excipient; and (v) contacting nisin, xanthohumol, the at least one polymer, and the at least one excipient, to obtain the composition, wherein nisin and xanthohumol are active components of the composition and the active components to the at least one polymer weight ratio is 1:3800. [0069] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) nisin; (b) xanthohumol; (c) at least one polymer; and (d) at least one excipient, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, said process comprising: (i) obtaining nisin; (ii) obtaining xanthohumol; (iii) obtaining the at least one polymer; (iv) obtaining the at least one excipient; and (v) contacting nisin, xanthohumol, the at least one polymer, and the at least one excipient, to obtain the composition, wherein nisin has a weight percentage in a range of 0.005 to 0.03% with respect to the composition, xanthohumol has a weight percentage in a range of 0.005 to 0.03% with respect to the composition.
[0070] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1, and wherein the composition exhibits antimicrobial effect.
[0071] In an embodiment of the present disclosure, there is provided a composition comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2, and wherein the composition exhibits antimicrobial effect.

[0072] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:4, and wherein the composition exhibits
antimicrobial effect.
[0073] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:1.5, and wherein the composition exhibits
antimicrobial effect.
[0074] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1:1, and wherein the composition exhibits
antimicrobial effect.
[0075] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 1.5:1, and wherein the composition exhibits
antimicrobial effect.
[0076] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) nisin; (b) xanthohumol; and (c) at least one polymer, wherein nisin
to xanthohumol weight ratio is 4:1, and wherein the composition exhibits
antimicrobial effect.
[0077] Although the subject matter has been described in considerable detail with
reference to certain examples and implementations thereof, other implementations
are possible.
EXAMPLES
[0078] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. 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. Although methods and materials similar or equivalent to those

described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. [0079] The present section discloses the materials and methods used in the present study along with the working and non-working compositions of the present disclosure.
Material and Methods
[0080] Nisin A (Nisaplin®) was obtained from Danisco, Copenhagen, Denmark, xanthohumol (XN) from hop (Humulus lupulus) (the purity more than 96%), L-a-lysophosphatidyl choline and phosphate buffered saline (PBS) were purchased from Sigma, USA, ethanol and hydrochloric acid were purchased from Fisher Scientific, India.
[0081] Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, reference strains were obtained from American Type Culture Collection, USA cultured and maintained using Eosin methylene blue agar and Baird Parker Agar Base (Hi Media, India) respectively.
Example 1
Preparation of liposomes
[0082] The co-encapsulation of nisin and xanthohumol loaded liposomes were
prepared as described below.
[0083] Briefly, adequate amount of L-a-lysophosphatidylcholine (polymer; 76
mg) was dissolved in appropriate volume of ethanol (3.98 ml) which contains
different ratios of NS and XN under mild stirring. The ratios of NS: XN (w/w) are
represented in the preparations as follows: Prep 1-1:4- (0.004 mg: 0.016 mg);
Prep 2 - 1 : 1.5 (0.008 mg: 0.012 mg); Prep 3-1:1 (0.01 mg: 0.01 mg); Prep 4 -
1.5:1 (0.012 mg : 0.008 mg); Prep 5-4:1 (0.016 mg : 0.004 mg); Prep 6 - NS
only (0.02mg); and Prep 7 - XN only (0.02 mg) as described in Table 1. Then, the
organic solvent was evaporated by incubating at 50°C to obtain liposomes loaded

with NS and XN. The resulting liposomes were dispersed in buffered phosphate
saline (5 ml, 10 mM, and pH 6.4). The mixture was then exposed to five cycles of
sonication for 1 min and filtered using syringe filter.
Table 1: Liposomal compositions comprising different weight ratios of nisin and
xanthohumol.
Experiment code Nisin Xanthohumol Weight ratio Polymer
(NS)(mg) (XN)(mg) (NS: XN) (mg)
Prepl 0.004 0.016 L4 76
Prep 2 0.008 0.012 1:1.5 76
Prep 3 O01 (X01 LI 76
Prep 4 0.012 0.008 L5il 76
Prep 5 0.016 0.004 4:1 76
Prep 6 0.02 0 - 76
Prep 7 0 O02 - 76
[0084] Result: Liposomes containing various concentration of NS and XN at definite ratio were successfully prepared and dissolved in phosphate buffered saline pH 6.8 (Table 1). Then the excess of unincorporated NS-XN molecules were removed by filtration using 0.22um syringe filter. The above-mentioned preparations were used for further studies.
Example 2
Stability studies
[0085] The stability studies with different preparations were performed to assess
the performance of respective preparations.
[0086] Appropriate amount of NS-XN loaded liposomes were dissolved in Milli
Q water. Then the aqueous solution of NS-XN loaded micelles were stored at room
temperature and monitored periodically (once a week; for 2 months) for any
changes in appearance. When the hydrophobic drugs are completely solubilized in

the core of the liposomes, then a clear solution is observed. Therefore, any change in the appearance of the solution indicates instability.
[0087] Result: On performing the stability studies, no change was observed in the appearance of the aqueous solution even after 3 months, which clearly indicates that NS-XN loaded were retained inside the liposomes without disruption (Figure 1). Thus, all the preparations were found to be stable at room temperature in aqueous medium even after 3 months of preparation.
Example 3
Determination of drug loading entrapment efficiency of NS: XN
The drug loading efficiency of the different preparations were performed by the
methods as described.
Quantification of nisin (NS) using spectrophotometer
[0088] The quantification of NS encapsulated liposomes were estimated using
BCA Protein Assay Kit (Thermofisher Scientific). Briefly, 25ul of liposomes
dissolved in methanol were mixed with 200ul of BCA reagent (50 parts of Reagent
A + 1 parts of Reagent B;) in microplate wells. Then the plate was incubated at 37°
for 30 min and the absorbance was measured at 562nm using plate reader (Thermo
MultiSkan GO Microplate Reader). The concentration of nisin were then calculated
according to the manufactures formula.
Quantification of xanthohumol using HPLC
[0089] Quantification of XN encapsulated liposomes were estimated using high
performance liquid chromatography (HPLC; Agilent 1200 with PDA Detector).
Briefly, an appropriate amount of liposomes was dissolved in required volume of
methanol. Then 20uL of the mixture was injected automatically in the injection
port and analyzed using the mobile phase consisting of 1% acetic acid/ methanol.
The flow rate was set at 1 mL/min with Inertsil ODS C-18, 250*4.6mm, 5um
column. The detection wave-length for XN was set at 370 nm, and the elution time
for XN was 21 mins under chromatographic conditions described above.
[0090] Drug loading (DL %) and encapsulation efficiency (EE %) of the NS: XN
liposomes were calculated using the equation (a) and (b) below.

Active Ann
Active- loading (DL%) = x 100 (a)
Polymer + Active
Experimental Active loading „ ,.
Encapsulation efficiency (EE%) = x 100 v3)
Theoretical Active loading
[0091] Results: DL% and EE % of the NS-XN-loaded liposomes were determined and the results have been tabulated in Table 2 below.
[0092] It can be observed that highest active loading of 5.72 % and encapsulation efficiency of 61.24% is depicted by preparation-2 having a weight ratio of NS: XN of 1:1.5 when compared to other preparations. The second highest percentage of active loading (2.49%) and encapsulation efficiency (49.72%) is depicted by preparation-1 having a weight ratio of NS: XN of 1:4. Table 2:

Example 4
Bacterial growth inhibition assay
[0093] Percentage of bacterial inhibition by NS: XN loaded liposomes were
estimated using Alamar blue assay (Thermo Fisher Scientific, USA). Isolated
colonies of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC
25923 from Eosin methylene blue agar and Baird Parker Agar Base plates
respectively were used to prepare mid log phase inoculum. Assays were performed
in flat bottom, polystyrene, non-tissue culture treated micro titer plates using a

mixed culture of Escherichia coli ATCC 25922 (representing Gram negative; 0.5 x 10 ) and Staphylococcus aureus ATCC 25923 (representing Gram Positive; 0.5 x
7 7
10 ) containing lx 10 CFU/ml in peptone broth. Then 20ul of liposomes containing various concentration of Prep 1-1 : 4 - (0.004 mg: 0.016 mg); Prep 2 -1 : 1.5 (0.008 mg: 0.012 mg); Prep 3-1:1 (0.01 mg: 0.01 mg); Prep 4 - 1.5 : 1 (0.012 mg : 0.008 mg); Prep 5-4:1 (0.016 mg : 0.004 mg); Prep 6 - NS only (0.02mg); and Prep 7 - XN only (0.02 mg) were added to final well volumes of 100 ul and incubated for 4h at 37°C. Then appropriate amount of Alamar blue was added to wells (105 ul total volume), the plates shaken gently and the absorbance was measured at 570 nm immediately using Varioscan microplate reader (Thermo fisher scientific). The percentage inhibition was calculated and all experiments were performed in triplicates.
[0094] Results: The results obtained for the present example for S. aureus and E. coli have been depicted in Figure 2 and Figure 3 respectively. Referring to Figure 2, it can be observed that the liposome consisting only nisin (prep 6) inhibits around 8% of bacterial growth, whereas the liposome consisting only xanthohumol (prep 7) does not exhibit any effective inhibition. In view of the individual effects, it can be appreciated that preparations 1, 2, and 5 exhibit synergistic inhibition of bacterial growth, and preparation 2 (1:1.5; NS:XN) exhibits the highest inhibition of 19.9% followed by preparation 5 (4:1; NS:XN) - 14.38% inhibition, which is followed by preparation 1 (1:4; NS:XN) - 12.9%.
[0095] Referring to Figure 3, it can be observed that individually neither nisin alone nor xanthohumol alone is able to exhibit any significant inhibition of E. coli. Whereas, all the preparations comprising a combination of nisin and xanthohumol exhibit a synergistic inhibition of E. coli. Preparation-2 (6.32%) and preparation-1 (4.66%)) exhibit highest inhibition followed by preparations 5, 4, and 3. [0096] Thus, it can be asserted that NS: XN loaded liposomes inhibit the growth of both Gram-positive and Gram-negative bacteria, as compared to the individual effects of both the components independently.

[0097] The present disclosure discloses a liposome composition comprising: nisin; xanthohumol; and at least one polymer. The composition of the present disclosure has been exclusively studied with L-a-lysophosphatidylcholine as the polymer. Liposomal compositions comprising different weight ratios of nisin and xanthohumol were found to be stable, and revealed surprising inhibitory effect on both Gram-positive and Gram-negative bacteria. The compositions of the present disclosure comprise ingredients which are safe to use, and easy to obtain. The method of preparing the composition is relatively simple and cost-effective. The compositions of the present disclosure can be used as a bio-preservative in various food products.

I/We Claim:
1. A liposomal composition comprising:
a) nisin;
b) xanthohumol; and
c) at least one polymer,
wherein nisin to xanthohumol weight ratio is in a range of 1:5 to 4:1.
2. The liposomal composition as claimed in claim 1, wherein nisin to xanthohumol weight ratio is in a range of 1:4.5 to 1:1.2.
3. The liposomal composition as claimed in claim 1, wherein nisin and xanthohumol are active components of the composition and the active components to the at least one polymer weight ratio is in a range of 1:500 to 1:4000.
4. The liposomal concentration as claimed in claim 3, wherein the active components to the at least one polymer has a weight ratio of 1:3800.
5. The liposomal composition as claimed in claim 1, wherein the at least one polymer is selected from a group consisting of L-a-lysophosphatidyl choline, 1,2-didecanoyl-sn-glycero-3 -phosphocholine (DDPC), l,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Egg-PC (EPC), hydrogenated Egg PC (HEPC), hydrogenated Soy PC (HSPC), l-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (Milk Sphingomyelin MPPC), l-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), l-stearoyl-2-myristoyl-sn-glycero-3-

phosphocholine (SMPC), l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), and combinations thereof.
6. The liposomal composition as claimed in claim 1, wherein nisin has a weight percentage in a range of 0.005 to 0.03% with respect to the composition, xanthohumol has a weight percentage in a range of 0.005 to 0.03% with respect to the composition.
7. The liposomal composition as claimed in claim 1, wherein the composition has an active loading in a range of 0.2-6%, and the composition has an encapsulation efficiency in a range of 20-62%.
8. The composition as claimed in any one of the claims 1-7, wherein the composition further comprises at least one excipient.
9. A process for preparing the composition as claimed in claim 1, said process comprising:

a) obtaining nisin;
b) obtaining xanthohumol;
c) obtaining the at least one polymer; and
d) contacting nisin, xanthohumol, and the at least one polymer, to obtain the composition.
10. A process for preparing the composition as claimed in claim 8, said process
comprising:
a) obtaining nisin;
b) obtaining xanthohumol;
c) obtaining the at least one polymer;
d) obtaining the at least one excipient; and
e) contacting nisin, xanthohumol, the at least one polymer, and the at least one excipient, to obtain the composition.

11. The composition as claimed in any one of the claims 1-8, wherein the composition exhibits antimicrobial effect.