Patent Application: US-201213983585-A

Abstract:
a nanoparticle includes a carbohydrate carrier and a bacteriocin . a method for prolonging efficacy of a bacteriocin against a food pathogen includes providing the bacteriocin in a delivery system , and inhibiting the food pathogen by the bacteriocin . a duration of efficacy of the bacteriocin against the food pathogen when the bacteriocin is provided in the delivery system exceeds a duration of efficacy of the bacteriocin when the bacteriocin is provided without the delivery system .

Description:
as further described herein , soluble nanocarriers can reduce the depletion of active compounds during storage without sacrificing their availability in times of need ( i . e . in the presence of pathogenic contamination ). a number of colloidal assemblies have been explored , such as polymersomes , particle - stabilized emulsions and colloidosomes , and layer - bylayer microcapsules . over a century ago , pickering ( 1907 ) indicated that colloidal particles could be used to stabilize emulsions , forming so - called “ pickering emulsions .” recently , there has been a resurgence of interest in micro - and nanoparticle - stabilized emulsions , mostly due to the use of the interfaces as templates for nano - construction . the distinct properties of these emulsions are attributable to the very large free energy of adsorption of the particles , which usually leads to highly stable emulsions ( aveyard et al ., 2003 ). associated with particle - stabilized emulsions , the concept of “ colloidosomes ” was proposed by velev et al . ( 1996 ) ( the term was coined by dinsmore et al . ( 2002 )) as selectively permeable capsules composed of colloidal particles . the colloidal particles used in emulsions thus far have been either inorganic or synthetic polymer - based “ hard ” particles such as silica particles and barium sulfate , calcium carbonate , bentonite , polystyrene , polytetrafluoroethylene ( aveyard et al ., 2003 ; binks et al ., 2007 ), and au -, ag -, or fe 3 o 4 - based nanoparticles ( wang et al ., 2005 ). due to the necessity of cross - linking individual particles for colloidosomes , synthetic materials such as polystyrene derivatives , poly -( divinylbenzene - alt - maleic anhydride ), and poly -( methylmethacrylate ) have also been used ( rossier - miranda et al ., 2009 ). however , it would likely be difficult to use these materials to construct colloidal assemblies for orally delivered systems such as food , nutraceuticals , and drugs . the present inventors have discovered that carbohydrate nanoparticles can prolong the efficacy of antimicrobial peptides against pathogens , and describe a novel methodology for improved food safety that allows controlled delivery of a broad variety of bioactive compounds . in addition , the present inventors have discovered that the use of all manner of emulsions ( e . g ., emulsions stabilized by pg - os , wcs - os , modified starch , gum arabic , whey protein and casein , phospholipids , and the like )— particularly emulsions with a negative charge at the interface ( i . e . the surface of oil droplets )— is effective . without wishing to be bound by a particular theory or to in any way limit the scope of the appended claims or their equivalents , it is presently believed that this negative charge at the surface of oil droplets is used to interact with positively charged bacteriocin , such as nisin . in some embodiments , the present inventors prepared an amphiphilic carbohydrate nanoparticle , phytoglycogen octenyl succinate ( pg - os ), and used pg - os - stabilized emulsion to deliver functional peptides with prolonged efficacy . pg - os was prepared through octenyl succinate ( os ) substitution ( an fda - approved reaction for food usages ) of phytoglycogen ( pg ), a major carbohydrate nanoparticle in the su1 - containing plants such as maize . throughout this description and the appended claims , the phrase “ phytoglycogen or glycogen - type material ” refers to dendritic ( i . e ., highly branched ) α - d - glucan and carbohydrate nanoparticles . the term “ phytoglycogen ” generally refers to material that is derived from plants while the term “ glycogen ” generally refers to material that is derived from microbials and / or animals . in previous studies , the present inventors have shown that pg - os is partially digestible and can form emulsions with outstanding physical and oxidative stability ( scheffler et al ., 2010a , b ). in the current study , the pg - os interfacial layer was used to adsorb nisin through electrostatic and hydrophobic interactions for extended efficacy against listeria monocytogenes . in some embodiments , the nanocarriers used in accordance with the present teachings are negatively charged , phytoglycogen - based dendritic polysaccharides that adsorb positively charged nisin molecules via electrostatic interactions . phytoglycogen ( pg ) was isolated from mutant maize , followed by an enzymatic modification and succinylation or octenyl succinylation . for nisin binding , phytoglycogen octenyl succinate ( pg - osa ) and phytoglycogen succinate ( pg - sa ) were dissolved in 0 . 05 m ph 5 . 5 naac buffer at 0 . 5 % and added with 0 . 1 % of nisin . after 24 hr incubation , the mixture was evaluated for the physical binding of nisin and the anti - listerial activity profile in a model bhi - agar system . the results indicated effective pg modifications , substantial nisin binding , and prolonged release of anti - listerial activity . tem images showed that pg particles ranged from 30 to 100 nm . zeta - potential of substituted pg reached up to − 39 . 5 mv . ultrafiltration assay showed that up to 76 % of nisin was bonded to pg derivatives . when nisin preparations were stored in the deep wells in the bhi - agar gel , free nisin displayed the quickest reduction of activity and the retained activity was negligible at day 5 . in contrast , binding with pg derivatives prolonged nisin activity to up to 15 days . in addition , the type and the degree of substitution of pg derivatives affected the binding and release properties of nisin . overall , enzymatic modification was beneficial to improved binding affinity and prolonged release , and the pg - osa nanocarriers were superior compared with pg - sa . the methodology developed by the present inventors has the potential to prolong the inhibition effect of nisin on the growth of listeria monocytogenes on the surface of foods , such as deli meats . pg - based nanocarriers may have unique benefits for the safety and quality of food . in principle , amphiphilic nisin molecules can be enriched at the oilwater interface and protected by the emulsifier layer from a quick depletion . without wishing to be bound by a particular theory or to in any way limit the scope of the appended claims or their equivalents , it is presently believed that that negatively charged emulsifiers are superior to neutral emulsifiers to retain nisin ( positively charged ) against l . monocytogenes . waxy corn starch octenyl succinate ( wcs - os ) and phytoglycogen octenyl succinate ( pg - os ) were used as models of negatively charged emulsifiers , and tween 20 was used as a model of neutral emulsifier . wcs - os , pg - os , and tween 20 were dispersed in buffer and added with oil . the mixtures were subjected to homogenization and thereafter added with the same amount of nisin . to evaluate the depletion of nisin activity , each preparation was added to bhi - agar wells , aliquoted after various storage periods , and measured for the retention of inhibitory activity against l . monocytogenes . the preliminary data indicated that the retention of nisin activity was much higher in pg - os and wcs - os - stabilized emulsions than in the free nisin dispersion and tween 20 - stabilized emulsion . thus , in some embodiments , nisin and listeria monocytogenes were used as the peptide and pathogen models , respectively , and phytoglycogen ( pg )- based nanoparticles were developed as carriers of nisin . pg from su1 mutant maize was subjected to β - amylolysis as well as subsequent succinate or octenyl succinate substitutions . the goal was to minimize the loss of peptide during storage and meanwhile realize an effective release in the presence of bacteria . the capabilities of pg derivatives as carriers of nisin were evaluated using centrifugal ultrafiltration , zeta - potential , and the initial availability of nisin against l . monocytogenes . all methods indicated that nisin loading was favored by a high degree of substitution ( ds ), presence of hydrophobic octenyl moiety , and β - amylolysis of pg nanoparticles . to evaluate the prolonged nisin efficacy , preparations containing nisin and pg derivatives were loaded into a bhi - agar deep - well model ( mimicking nisin depletion at the nutrient - containing surface ). the residual inhibitory activities of preparations against l . monocytogenes were monitored during 21 days of storage at 4 ° c . the results showed that all pg derivatives led to the prolonged retention of nisin activity and the longest retention was associated with high ds , β - amylolysis , and octenyl succinate . evidently , both electrostatic and hydrophobic interactions are the driving forces of nisin adsorption , and the glucan structure at the nanoparticle surface also affects nisin loading and retention during storage . l . monocytogenes is a gram - positive food borne microorganism [ 1 ] that grows widely in environments , even at refrigerated temperatures , and survives for a long period of time in manufacturing plants and on food surfaces . it is responsible for outbreaks and a number of recent usda recalls [ 2 , 3 ]. according to the center for disease control and prevention ( cdc ), listeriosis is a serious infection and an important public health problem . listeriosis causes hundreds of deaths each year in the u . s . and there is zero tolerance policy for l . monocytogenes in ready - to - eat foods . an effective strategy to reduce the risk of listeriosis will have a profound impact on society and may help save lives . nisin is produced from lactococcus lactis fermentation . it is a positively charged lantibiotic peptide [ 4 - 7 ] that is able to bind to negatively charged cytoplasmic membranes . nisin contains 34 amino acids and has a molecular weight of 3 . 4 kd . it has been approved as a food preservative and is effective in suppressing gram - positive bacteria such as l . monocytogenes . nisin kills bacteria by forming pores on cell membranes [ 8 ] and can be used broadly in food [ 9 ]. the antibacterial efficacy of nisin during storage is governed by multiple factors . migration of nisin to a food mass reduces its effect at the food surface [ 10 ]. components such as proteases and glutathione [ 11 ], titanium dioxide , and sodium metabisulphite can adversely affect nisin stability [ 9 ]. in order to prolong its efficacy , nisin has been incorporated in packaging films or coatings [ 10 , 12 - 15 ]. the challenges for this strategy lie in the cost of film - making on an industrial scale and in the tailoring of nisin release . recently , liposome - encapsulated nisin has been constructed and tested in milk fermentation [ 16 ] and the ripening of cheddar cheese [ 17 ]. the stability and entrapment efficiency of nisin in liposome has also been studied [ 18 , 19 ]. phytoglycogen ( pg ) is a water - soluble glycogen - like α - d glucan in plants . the largest source of pg is the maize mutant su1 , a major genotype of sweet corn . the su1 mutation leads to a deficiency in su1 , an isoamylasetype starch debranching enzyme ( dbe ) [ 20 ]. in amyloplasts , starch synthases , branching enzymes , and dbe work in concert to synthesize starch [ 21 ]. the role of dbe is to trim abnormal branches that inhibit the formation of starch granules [ 22 , 23 ]. in the absence of dbe , the highly branched pg is formed to replace starch . chemical modifications have been used to bring functionalities to pg nanoparticles [ 24 , 25 ]. among food - related reactions , succinate substitution is used to bring negative charges , and octenyl succinate substitution is used to bring negative charges and hydrophobicity [ 26 ]. for both , the properties of pg derivatives can be controlled by the degree of substitution . in this study , pg was subjected to β - amylolysis and subsequent succinate or octenyl succinate substitution . pg derivatives were evaluated for their capability for loading nisin and prolonging nisin efficacy against l . monocytogenes . the goal was to minimize the loss of peptide during storage and meanwhile realize an effective release in the presence of bacteria . the objective was to reveal the relationship between the structure of pg - based nanoparticles and prolonged antimicrobial efficacy . by this work , novel carbohydrate nanomaterials for enhanced performance of bioactive peptides for food were discovered . due to their biodegradability and functionality , the carbohydrate nanoparticles studied in this work may also contribute to the delivery of therapeutic proteins and peptides . in general , the efficacies of protein therapeutics are limited by their instability , immunogenicity , and shorter half lives [ 27 ]. to address these issues , a number of delivery systems have been designed , including covalent attachment of polyethylene glycol ( and other biodegradable polymers ) and adsorption or encapsulation with colloidal systems [ 27 - 34 ]. recently , poly ( lactic - co - glycolic acid ) microspheres containing base or divalent cations were used as adjuvant of vaccines or to maintain the stability of encapsulated peptides [ 28 , 29 ], and poly ( lactic acid )- polyethylene glycol microspheres were used to deliver insulin [ 30 ]. at the micro - to nano - scales , both liposome and solid lipid particulates have been used to deliver peptides [ 31 , 32 ], and the peptide loading was affected by factors including the surface charge and hydrophobicity . at the nano - scale , the medusa system was commercially designed for delivering proteins and peptides [ 33 ]. this system consists of a poly l - glutamate backbone grafted with α - tocopherol , and the sustained drug release is based on reversible drug interactions with hydrophobic nanodomains of the nanoparticles [ 33 ]. recently , amphiphilic copolymers of polylactic acid grafted onto hyperbranched polyglycerol were prepared to form a corona - core nanostructure and used to deliver protein [ 34 ]. conceivably , the carbohydrate nanoparticles prepared in this study , such as negatively charged , amphiphilic phytoglycogen octenyl succinate , may have potential in the delivery of therapeutic proteins and peptides . in some embodiments , an amphiphilic , negatively charged carbohydrate nanoparticle , phytoglycogen octenyl succinate ( pg - os ), was used to form oil - in - water emulsion for delivering bacteriocin nisin against the food pathogen listeria monocytogenes . dynamic light scattering test showed that in emulsion , all pg - os nanoparticles were adsorbed at the surface of oil droplets . zeta - potential analysis indicated an effective adsorption of positively charged nisin molecules at the surface of pg - os interfacial layer . nisin depletion model showed that , during 50 days of storage , the anti - listerial activity of nisin - containing pg - os - stabilized emulsion was substantially greater than that of nisin solution . in contrast , the emulsion stabilized with a neutral , small - molecule surfactant ( tween 20 ) or negatively charged , hyperbranched carbohydrate polymer ( modified starch ) was either ineffective or less effective than the nanoparticle - stabilized emulsion to retain nisin activity during storage . the following examples and representative procedures illustrate features in accordance with the present teachings , and are provided solely by way of illustration . they are not intended to limit the scope of the appended claims or their equivalents . sweet corn silver queen ( a su1 hybrid ) was purchased from burpee co . ( warminster , pa .). bradford protein assay kit was purchased from bio - rad ( hercules , calif .). waxy corn starch was obtained from national starch food innovation ( bridgewater , n . j .). succinic anhydride , nisin , tween 20 , and isopropyl alcohol were purchased from sigma - aldrich ( st . louis , mo .). 1 - octenyl succinic anhydride was obtained from dixie chemical co . ( houston , tex .). beta - amylase , pullulanase , and isoamylase were purchased from megazyme ( wicklow , ireland ). brain heart infusion ( bhi ) and agar were purchased from bd ( franklin lakes , n . j .). sweet corn kernels were ground into grits and then mixed with six weights of deionized water . the suspension was homogenized using a high - speed blender ( waring laboratory , torrington , conn .) and then centrifuged at 8000 g for 20 min . the supernatant was collected and passed through a 270 - mesh sieve . three volumes of ethanol were added to the supernatant to precipitate polysaccharides . after centrifugation and decanting , the precipitate was suspended using ethanol and filtrated to dehydrate for three cycles . the solid material obtained after removing the residual ethanol was pg . sweet corn kernels were ground into grits and then mixed with four weights of deionized water . the suspension was homogenized using a high - speed blender ( waring laboratory ), and the solids were removed with a 270 - mesh sieve . the liquid was adjusted to ph 4 . 8 to precipitate proteinaceous material . after centrifugation ( 10 , 000 g , 20 min ), the supernatant was placed at 4 ° c . for 24 h and subjected to centrifugation ( 10 , 000 g , 20 min ) to remove amylose . this procedure was repeated once . the collected supernatant was adjusted to ph 6 . 9 , autoclaved ( 121 ° c ., 20 min ), and centrifuged ( 10 , 000 g , 20 min ) after cooling . three volumes of ethanol were added to the liquid collected to precipitate polysaccharides . the solid was further dehydrated with three cycles of ethanol dispersion - filtration and dried in a fume hood . twenty grams of pg was dissolved in 400 ml ph 6 . 0 , 50 mm sodium acetate buffer . one hundred microliters of β - amylase ( 1 , 800 u / ml ) was added to the solution . the reaction was conducted in a shaking water bath ( 60 ° c ., 70 rpm ) for 10 h . three volumes of ethanol were added to the reactant . after the centrifugation and decanting , the precipitate was suspended using ethanol and filtrated for three cycles . the solid obtained after removing the residual ethanol was pg β - dextrin ( pgb ). to prepare non - granular waxy corn starch ( wcs ), 20 g of wcs was dispersed in 400 ml of sodium hydroxide solution ( 2 %, w / v ) by heating in a boiling - water bath for 10 min . after cooling , the dispersion was adjusted to ph 7 . 0 using hydrogen chloride ( 10 %, w / w ). three volumes of ethanol were added to the dispersion to precipitate polysaccharides . the solid was further dehydrated with three cycles of ethanol dispersion - filtration and dried in a fume hood . weight - average molecular weight ( m w ) and z - average root mean square radius ( r z ) of pg and pgb were determined using the procedure described by scheffler [ 24 ]. the chain length distribution of pg and pgb was characterized using the procedure described by shin et al . [ 35 ]. substitution of pg and pgb with octenyl succinate group was described by scheffler et al . ( 24 ). substitution with succinate group was essentially the same except that succinic anhydride was used in the replacement of 1 - octenyl succinate anhydride . the materials collected were pg succinate ( pg - s ), pg octenyl succinate ( pg - os ), pg β - dextrin succinate ( pgb - s ), and pg β - dextrin octenyl succinate ( pgb - os ). degree of substitution ( ds ) values pg - s , pg - os , pgb - s , and pgb - os materials were determined using a method described by scheffler et al . [ 24 ]. for pg and wcs , the octenyl succinate ( os ) substitution and determination of the degree of substitution ( os ) were conducted as described by scheffler et al . ( 2010a ). the materials prepared were pg - os and wcs - os . tem imaging and determination of molecular mass , root mean square ( rms ) radius , and dispersed molecular density of both pg - os and wcs - os were conducted as described by scheffler et al . ( 2010b ). tem imaging of pg , pgb , and selected pg - os and pgb - os was conducted as described by scheffler et al . [ 25 ]. commercial nisin solid contains 2 . 5 % pure nisin , balanced with sodium chloride and denatured milk solids . to prepare nisin solution , 120 mg nisin solid was dissolved in 3 . 0 ml sodium acetate buffer ( 50 mm , ph 5 . 5 ), gently agitated for 15 h , and centrifuged at 5 , 000 g for 5 min at 15 ° c . the supernatant was collected as 1 , 000 μg / ml nisin solution . zeta - potential was used to evaluate the surface charge density of the nanoparticles . to measure the zeta - potential , pg derivatives ( 1 . 0 mg / ml ) were dissolved in sodium acetate buffer ( 50 mm , ph5 . 5 ) and loaded to zetasizer nano ( malvern , westborough , mass .) at room temperature . to evaluate the effect of nisin on the zeta - potential of nanoparticles , a 0 . 3 ml diluted nisin solution ( 200 μg / ml in sodium acetate buffer ) was mixed with 2 . 7 ml solution of each pg derivative ( 1 . 0 mg / ml ). after 24 h incubation at room temperature , the zeta - potential was measured for each mixture . a centrifugal ultrafiltration device ( microsep , pall life sciences ) with molecular weight cut - off of 300 kd was used to evaluate the nisin loading to nanoparticles . in principle , non - loaded nisin molecules can pass through the membrane , whereas those loaded cannot . for the test , a 2 . 7 ml solution of pg or each of its derivatives ( 1 . 0 mg / ml ) and 0 . 3 ml nisin solution ( 200 pg / ml ), both in sodium acetate buffer ( 50 mm , ph5 . 5 ), were mixed and incubated for 30 min at room temperature . for each mixture , an aliquot of 2 . 7 ml was transferred to a microsep tube and centrifuged ( 1 , 000 g at 15 ° c . for 2 h ). from the filtrate , an aliquot of 800 μl was used to test the amount of nisin using the bradford assay kit ( bio - rad ). nisin solutions ( 2 , 4 , 8 , 10 , 12 , 14 , 16 , 18 , and 20 μg / ml ) were used as the standards . to prepare emulsions , pg - os and wcs - os were each dissolved in sodium acetate buffer ( 50 mm , ph 5 . 5 , 22 ° c .) to form a solution of 10 mg / ml . as a reference , 1 . 0 mg / ml of tween 20 solution was also prepared using the sodium acetate buffer . vegetable oil was added to each emulsifier solution , at twice ( for pg - os and wcs - os ) or 20 times ( tween 20 ) the weight of the emulsifier . the mixtures were first subjected to high - speed homogenization ( 18 , 000 rpm for 1 min , t25 ultra - turrax , ika ) and then high - pressure homogenization ( 103 mpa , two cycles , nano debee , bee international ). subsequently , 4 ml of nisin solution ( 1500 or 2000 μg / ml ) was added to a 16 - ml - aliquot of collected emulsion . each mixture was further diluted with the same volume of sodium acetate buffer . using this procedure , emulsions were prepared to contain 150 ( or 200 ) μg / ml nisin and 4 . 0 mg / ml pg - os or wcs - os or 0 . 40 mg / ml tween 20 . these emulsions were sterilized using a boiling - water bath for 3 min before further tests . the distributions of particle size ( denoted by hydrodynamic diameter ) of the pg - os and wcs - os solutions ( before and after homogenization at 103 mpa , two cycles ) and the emulsions containing 150 μg / ml nisin were determined using a zetasizer nano ( zs90 , malvern instruments ) at 25 ° c . using the automatic setting with 1 min of equilibration . to determine zeta - potentials , emulsions containing 0 , 150 , and 200 μg / ml nisin were diluted to 20 volumes using 50 mm ph 5 . 5 sodium acetate buffer . the measurement was conducted at 25 ° c . using the automatic setting with 1 min of equilibration . nisin activity , either for those freshly prepared or stored in bhi agar deep - well , was determined as described by pongtharangkul and demirci ( 2004 ) with modifications . agar diffusion bioassay was used to determine the nisin activity against l . monocytogenes . bhi ( 3 . 7 %) solution containing 0 . 75 % agar and 1 . 0 % tween 20 was prepared and autoclaved . after cooling to approximately 40 ° c ., the solution was inoculated by a 1 . 0 % volume of bhi broth containing l . monocytogenes v7 ( ca . 108 colony - forming units / ml ). to each square petri - dish plate ( 10 × 10 cm ), a 32 ml inoculated bhi agar solution was added and allowed to solidify . thereafter , holes with 7 . 0 mm diameter were made using a cork borer , and 100 μl of each nisin preparation was added to each agar well . the petri - dish plates were then incubated for 24 h at room temperature , and the size of inhibitory ring was measured to indicate the activity against l . monocytogenes . to correlate the nisin dose with its activity against l . monocytogenes , solutions with a series of nisin concentrations ( 20 , 40 , 60 , 80 , and 100 μg / ml ) were prepared and subjected to the agar diffusion bioassay . to prepare the bhi - agar deep - well model for nisin depletion test , a solution containing bhi ( brain heart infusion ) solids ( 3 . 7 %) and agar ( 1 . 0 %) was autoclaved for 20 min at 121 ° c . the hot solution ( 225 ml ) was poured into a 600 - ml beaker to a height of 40 mm . after gel solidification , four wells ( from gel surface to bottom ) were made in each beaker using a 7 . 0 - mm borer . subsequently , 1 . 0 ml of nisin preparation was added to each well . immediately after loading ( day 0 ) and after 5 , 10 , 15 , 20 , 30 , 40 , and 50 days of storage at 4 ° c ., a 100 - μl aliquot of each nisin preparation was transferred from the well to a bioassay plate to determine the residual nisin activity . in this study , a bhi - agar deep - well model was established to mimic the depletion of nisin at nutrient - containing surfaces , such as a food surface ( fig1 ). bhi is a nutritious culture medium that supplies protein and other nutrients necessary to support the growth of fastidious and nonfastidious microorganisms . it contains infusions from calf brains and beef hearts , proteose and peptone , dextrose , sodium chloride , and disodium phosphate . in our earlier work , it was found that bhi - containing broth and gel always led to a rapid reduction or elimination of nisin activity , suggesting a nisin depletion effect . therefore , bhi is an ideal nutrient model for studying nisin depletion and retention . our experiments have consistently shown that bhi - containing broths and gels lead to rapid depletion of peptide activity . in a deep well filled with a liquid peptide preparation , peptide molecules diffuse from the solution into the gel ( causing diffusion - based depletion ), and bhi components diffuse from the agar gel into the solution ( causing irreversible peptide adsorption or degradation ). for continuous sampling at various stages of storage , nisin preparations were added to the deep wells of the bhi - agar gel . for the deep - well storage , the inner surface of a well was used to mimic the outer surface of solid food . at the inner surface , nisin molecules diffuse from the solution toward the bulk of the gel , and bhi components diffuse from the agar gel to the solution . this process is comparable to what happens at the surface of gel - like foods applied with antimicrobial peptide : peptide molecules diffusing into food mass and food components diffusing to the aqueous layer at the surface . to prepare bhi - agar gel with deep wells , a solution containing bhi solid ( 3 . 7 %) and agar ( 1 . 0 %) was autoclaved for 20 min at 121 ° c . the hot solution ( 225 ml ) was poured into a 600 - ml beaker to form a 40 - mm height of liquid . after gel solidification , four wells ( from gel surface to bottom ) were made in each beaker using a 7 . 0 - mm borer . to each well , a 1 . 0 ml nisin preparation containing 100 μg / ml nisin , either with or without pg derivatives ( 5 . 0 mg / ml ), was added . immediately after loading ( 0 day ) and after 3 , 5 , 7 , 10 , 15 , and 21 days of 4 ° c . storage , a 100 ijl aliquot of each nisin preparation was transferred from the well to the bioassay plate to determine the residual nisin activity against l . monocytogenes . for each preparation , one portion was applied in the model and another portion was stored in a regular test tube as a reference . the references were used to evaluate the stability of nisin in the presence of nanoparticles only . during β - amylolysis of α - d - glucan , two connected glucosyl units ( i . e . one maltosyl unit ) are continuously released from the non - reducing ends of external linear chains , producing β - dextrin . fig2 shows the chain length distribution of pg and pgb . for pg , there is a large chain population at about dp 8 - 10 ( dp : degree of polymerization ) and a small one at about dp 16 . there were also minor amounts of maltose ( dp 2 ) and maltotriose ( dp 3 ) but no maltotetraose ( dp 4 ). for pgb , there was a substantial increase of dp 2 , dp 3 , and dp 4 , suggesting the shortening of external chains due to β - amylolysis . tem images of pg and pgb indicate the presence of nanoparticles with sizes from 30 - 100 nm in diameter ( fig3 ). most nanoparticles were 60 - 90 nm , which is comparable with the root mean square radius ( rz ) of about 45 nm for both pg and pgb nanoparticles ( table 1 ). as shown in table 1 , the impact of β - amylolysis on the particle size ( rz ) of pg was negligible , whereas the weight - average molecular weight ( m w ) was slightly reduced from 7 . 9 × 10 7 for pg to 7 . 5 × 10 7 mol / g for pgb . fig4 a - b depict the impact of β - amylolysis on pg structure . at the surface of a pg nanoparticle , there are long linear chains and newly formed branch units ( fig4 a ). after β - amylolysis , the long linear chains can be substantially shortened , whereas the branch units at the surface remained nearly intact ( fig4 b ). this resulted in essentially the same r z value for pg and pgb . due to β - amylolysis , m w was reduced and the dispersed molecular density ( p ) was reduced accordingly . conceivably , β - amylolysis had a thinning effect at the surface of nanoparticles , which would affect the loading capacity of modified nanoparticles ( discussed later ). the degrees of substitution ( os ) of pg derivatives are shown in table 2 . evidently , higher doses of succinic anhydride ( sa ) or octenyl succinic anhydride ( osa ) led to higher os values . while the substitution efficiency was usually under 60 %, the high doses ( 12 % for sa and 26 % for osa ) correlated with the high substitution efficiency ( 60 %). moreover , substrates ( pg or pgb ) and substitution reagents ( sa and osa ) had negligible impact on the substitution efficiency . in this work , the following abbreviations are used to denote different pg and pgb derivatives : pg - s ( 0 . 05 ) and pg - s ( 0 . 12 ) for pg succinate with os of 0 . 050 and 0 . 121 respectively , pgb - s ( 0 . 05 ) and pgb - s ( 0 . 12 ) for pgb succinate with os of 0 . 050 and 0 . 120 respectively , pg - os ( 0 . 05 ) and pg - os ( 0 . 12 ) for pg octenyl succinate with os of 0 . 049 and 0 . 120 , respectively ; and pgb - os ( 0 . 05 ) and pgb - os ( 0 . 12 ) for pgb octenyl succinate with os of 0 . 048 and 0 . 119 , respectively . tem images of pg - os ( 0 . 12 ) and pb - os ( 0 . 12 ) are shown in fig3 . it appears that the particle sizes of both derivatives were a little smaller than those of pg and pgb . compared with pg and pgb , there was less aggregation among the substituted nanoparticles possibly due to the electrostatic repulsion caused by the negative charges from substitution groups . zeta - potential is the electrostatic potential between the plane of shear ( within the interfacial double layer ) and the bulk fluid away from the interface . it is a very useful parameter for evaluating the stability of colloidal dispersion and the interactions among charged molecules . in this study , zeta potential was used in understanding the interactions between the negatively charged nanoparticles and the positively charged nisin molecules in the solution . fig5 shows the zeta - potentials of pg derivatives with and without added nisin . the zeta - potentials of pg and pgb at ph 5 . 5 were slightly negative (− 5 . 1 for pg and − 3 . 6 for pgb ), suggesting the presence of a trivial amount of anionic compounds . usually , purified pg contains approximately 1 % protein , which could have contributed to the negative charges observed . with the grafting of succinate or octenyl succinate groups , the zeta - potential was substantially decreased ( increased absolute value ), indicating the negative charges introduced by carboxylate groups . in general , a ds of 0 . 05 led to a zeta - potential ranging from − 22 to − 24 mv , regardless of the involvement of pg , pgb , succinate , or octenyl succinate ( fig5 ). in contrast , a ds of 0 . 12 led to a zeta - potential around − 33 to − 38 mv for each type of pg derivative . adding 20 μg / ml nisin led to a significant increase ( decrease in the absolute value ) in zeta - potential for nanoparticles ( fig5 ). for pg - s ( 0 . 05 ), pg - os ( 0 . 05 ), pgb - s ( 0 . 05 ), and pgb - os ( 0 . 05 ), the addition of nisin changed the zeta - potential to − 7 . 4 , − 7 . 1 , − 8 . 8 , and − 9 . 3 mv , respectively . for pg - s ( 0 . 12 ), pg - os ( 0 . 12 ), pgb - s ( 0 . 12 ), and pb - os ( 0 . 12 ), the addition of nisin changed the zeta - potential to − 10 . 4 , − 9 . 4 , − 10 . 5 , and − 10 . 7 mv , respectively . conceivably , the decrease in the absolute value of zeta - potential was due to the reduction in negative charge at the surface of nanoparticles caused by the adsorption of positively charged nisin molecules . in this study , the loading of nisin to the nanoparticles was evaluated by measuring the concentration of nisin in the filtrate of ultrafiltration . the total nisin concentration in the original preparation was 20 μg / ml . for pg and pgb , the nisin concentration in the filtrate was 19 μg / ml ( fig6 ), suggesting negligible capability of non - substituted nanoparticles for loading nisin . for the substituted nanoparticles , their nisin - loading capability was affected by ds , substitution groups , and substrates . the impact of ds on nisin loading was the most evident . when ds increased from 0 . 05 to 0 . 12 , the amount of non - loaded nisin was significantly reduced for all pg - s , pgos , pgb - s , and pgb - os nanoparticles . considering the high zeta - potential absolute value associated with high ds ( fig5 ), it is believed that the electrostatic interaction between nanoparticles and nisin played an essential role in nisin adsorption . at equivalent ds , octenyl succinate substitution usually led to a greater nisin loading than succinate . for example , the non - loaded nisin for pg - os ( 0 . 05 ) ( 7 . 7 μg / ml ) was lower than that for pg - s ( 0 . 05 ) ( 12 . 5 μg / ml ), and 5 . 2 μg / ml for pg - os ( 0 . 12 ) was lower than 7 . 5 μg / ml for pg - s ( 0 . 12 ). therefore , in addition to the electrostatic interaction , the hydrophobic interaction between octenyl moieties and nisin also contributed to peptide adsorption . the type of substrate ( pg or pgb ) also affected nisin loading . at equivalent ds , the amount of non - loaded nisin for pgb - s ( 0 . 05 ) was much lower than that for pg - s ( 0 . 05 ). similar result was observed between pgb - s ( 0 . 12 ) and pg - s ( 0 . 12 ). in contrast , the differences between pg - os and pgb - os were less significant . as mentioned earlier , β - amylolysis has a thinning effect at the surface of nanoparticles ( fig4 b ) that may improve nisin loading . however , this effect seems to be interrelated with the type of substitution . in this study , the inhibitory activity of nisin was evaluated using a diffusion test against l . monocytogenes . fig7 shows the relationship between the size of inhibition ring and the concentration of free nisin . using the equation shown in fig7 , the size of inhibition ring for each nisin preparation can be converted to the “ availability of nisin ”, i . e . the concentration of free nisin that offers the same inhibitory capability in the diffusion bioassay . fig8 shows the initial availability of nisin for nanoparticle solutions containing 100 μg / ml nisin . for the non - substituted nanoparticles , pg and pgb , the initial availability of nisin was 94 . 8 and 99 . 4 μg / ml , respectively . this indicates that the initial inhibitory behavior of nisin in both pg and pgb solutions was essentially the same as that of the 100 μg / ml free nisin solution . in contrast , for pg derivatives , the initial availability of nisin was much lower than that for 100 μg / ml . for example , for pg - os ( 0 . 12 ) and pgb - os ( 0 . 12 ), the initial availability of nisin was 43 . 8 and 32 . 9 μg / ml , respectively . evidently , the loading of nisin to nanoparticles was the primary factor in the reduction of the initial availability of nisin . in general , the availability of nisin was affected by os , substitute groups , and substrates . specifically , octenyl succinate substitution and β - amylolysis were more effective than high os for reducing the initial availability of nisin . in this study , the residual activity of each nisin preparation stored in the bhi - agar deep - well model was evaluated using the size of inhibitory ring in the bioassay . fig9 compares free nisin and preparations containing nisin and pg - os ( 0 . 12 ) or pgb - os ( 0 . 12 ) nanoparticles . for each preparation , inhibitory rings for both the model and reference are shown . for the reference groups , the size of inhibitory ring remained essentially the same over the 21 - day storage , suggesting a high stability of nisin regardless of the presence of nanoparticles . overall , the size of inhibitory ring was in the order of free nisin & gt ; pg - os ( 0 . 12 )& gt ; pgb - os ( 0 . 12 ), reflecting the availability of nisin of individual preparations . deep - well model tests demonstrated the effectiveness of using nanoparticles to prolong the efficacy of nisin against l . monocytogenes . for the initial test at 0 day , the solution of free nisin showed the highest nisin activity . after 7 days , the activity of free nisin was negligible , whereas the activities of pg - os ( 0 . 12 ) and pgb - os ( 0 . 12 ) preparations were evident . after 15 days , the residual nisin activity was clearly retained for pgb - os ( 0 . 12 ), whereas for pg - os ( 0 . 12 ) the nisin activity was almost lost . antimicrobial activity during the 21 - day storage at 4 ° c . is compared among various nisin preparations ( fig1 a - b ). in general , nanoparticle - containing preparations showed reduced depletion of nisin activity compared to the solution of free nisin . the effect of pg and pgb was rather low , corresponding to their lack of capability to adsorb nisin ( fig6 and 8 ). for substituted nanoparticles , octenyl succinate substitution correlated to a greater effect than succinate in reducing nisin depletion . this shows that the hydrophobic interaction played an important role in the retention of nisin activity , which was consistent with the high nisin loading ( fig6 ) and low initial availability of nisin ( fig8 ) associated with octenyl succinate substitution . in addition , pgb - based nanoparticles had a greater capability to retain nisin activity than did pg - based , regardless of the substitution with succinate or octenyl succinate . this shows the effect of nanoparticle surface structure on nisin loading and retention . conceivably , the surface thinning of nanoparticles due to β - amylolysis resulted in a greater nisin loading , which led to a longer period of activity retention . overall , ds , hydrophobicity , and glucan structure all affect nisin loading and release , and these factors can be used to design pg - based carbohydrate nanoparticles for prolonged efficacy of nisin . compared with film and liposome - based peptide carriers [ 10 , 12 - 19 ], carbohydrate nanoparticles can be conveniently applied to target systems and easily manipulated for desirable loading and retention of antimicrobial peptide . similar concepts have been proposed in drug delivery . for instance , nanoparticles made from poly ( lactic - co - glycolic acid ) ( plga ) were used to deliver anti - hiv - 1 peptide [ 36 ]. however , nanocarriers used in drug delivery are mostly synthetic or inorganic , which are not suitable for food uses . in contrast , carbohydrate nanoparticles used in the current work are digestible [ 25 ] and abundant , showing potentials in both the food and drug areas . apart from the electrostatic interaction , the hydrophobicity of peptides is a major factor of loading and release . recently , bysell et al . [ 37 ] reported that by end - tagging antimicrobial peptides using oligotryptophan groups , the binding of peptides with poly ( acrylic acid ) microgels can be substantially improved . in our work , the superiority of pgb - os with high ds value was related to the molecular characteristic of nisin , that is , the lysine residues offer positive charges and lanthionine and methyl lanthionine residues offer hydrophobicity [ 38 ]. in general , both electrostatic and hydrophobic interactions should be effectively utilized for designing carbohydrate nanocarriers of antimicrobial peptides . fig1 conceptually depicts the adsorption of peptides at the interface of the pg - os - stabilized oil droplets . this adsorption substantially reduces the number of free peptide molecules that are susceptible to quick depletion . to understand the impact of emulsifiers on the duration of peptide efficacy , in addition to pg - os we selected two other amphiphilic materials : tween 20 , a small - molecule , neutral surfactant , and waxy corn starch octenyl succinate ( wcs - os ), a hyperbranched carbohydrate polymer that can form stable emulsions . both pg - os and wcs - os are amphiphilic , negatively charged macromolecules , but they have drastically different structures . the tem images in fig1 show pg - os as dense nanoparticles and wcs - os as highly dispersed , worm - like macromolecules . titration analysis showed that the degree of substitution ( os ) was 0 . 013 for both pg - os and wcs - os . this is equivalent to an average of 13 octenyl succinate groups per 1 , 000 glucosyl units of pg or wcs . analysis using high performance size - exclusion chromatography ( hpsec ) combined with multi - angle laser light scattering ( malls ) indicated that the weight average molecular masses ( m w ) of pg - os and wcs - os were 1 . 74 ± 0 . 01 × 10 7 and 2 . 31 ± 0 . 14 × 10 7 g / mol , respectively . the z - average root mean square ( rms ) radii ( r z ) of pg - os and wcs - os were 25 . 83 ± 0 . 31 and 115 . 70 ± 4 . 10 nm , respectively . the dispersed molecular densities ( defined as ρ = mw / rz 3 ) ( wong et al ., 2003 ) of pg - os and wcs - os were 1011 . 9 and 15 . 0 g / mol · nm 3 , respectively . this unusually high density of the pg - os nanoparticles could lead to the formation of a thick , dense interfacial layer over the oil droplets in emulsions ( fig1 ). the particle size distribution of pg - os , wcs - os , and the emulsions stabilized by each was measured using dynamic light scattering . as shown in fig1 a , the hydrodynamic diameter ( d h ) distribution of pg - os was not affected by homogenization , showing resistance of the nanoparticles to the high shear induced by homogenization . in contrast , wcs - os was rather fragile , being reflected by a substantial particle size reduction following homogenization ( z - average oh decreasing from 196 to 118 nm ) ( fig1 b ). in the presence of nisin , the z - average d h of droplets in the pg - os - stabilized emulsion was 336 nm . notably , free nanoparticles in the emulsion were undetectable , suggesting full adsorption of pg - os at the oil - water interface ( described in fig1 ). in the wcs - os - stabilized emulsion , the z - average d h of droplets was 50 . 2 nm , much lower than that of homogenized wcs - os . this highlights the flexibility of homogenized wcsos molecules to attach at the interface and assume a “ shrunken ” conformation to accommodate the nanoscale oil droplets . fig1 shows the impact of nisin concentration on the zeta - potential of the emulsion droplets . without nisin , the zeta - potentials of the pg - os - and wcs - os - stabilized emulsions were − 15 . 5 and − 16 . 7 mv , respectively . the addition of nisin substantially changed the zeta - potential for both the pg - os and wcs - os emulsions , and these changes were strongly related to the amount of nisin added . evidently , the adsorption of nisin molecules occurred at the surface of the emulsion droplets . for the tween 20 - stabilized emulsion , the zeta - potential increased modestly from − 0 . 3 to 0 . 6 mv with 200 μg / ml of nisin added , suggesting very low nisin adsorption at the surface of the oil droplets . fig1 shows the retention of nisin activity against l . monocytogenes indicated by the size of the inhibitory ring ( defined in fig1 ) during storage tests . for the “ control ” groups , nisin preparations were stored in regular test tubes at 4 ° c . ; therefore , any change in ring size indicated a change in nisin availability caused only by the interaction between the peptide and the delivery system . as expected , the free nisin control did not show an appreciable change during storage , demonstrating the high stability of nisin . there was a minor reduction of ring size for both the pg - os - and tween 20 - stabilized emulsions , indicating slowly increasing adsorption during storage . in contrast , the ring size of the wcs - os - stabilized emulsion decreased rapidly to a negligible level after 10 days , which may have been caused by overly strong adsorption of peptide at the interface . as shown in fig1 , the pg - os - stabilized emulsion demonstrated the greatest ability to preserve nisin activity during extended storage . after 40 days , for the model groups the size of the inhibitory ring for the pg - os emulsion was the largest among all preparations , whereas the rings for free nisin and the tween 20 emulsion were undetectable . in fact , the ring size of the pg - os emulsion at 40 days was larger than those of free nisin and the tween 20 emulsion at 10 days . the ring size of the wcs - os emulsion was always smaller than that of the pg - os emulsion , particularly at 0 , 20 , and 40 days . in fig1 a and 16b , the preservation of nisin activity was quantitatively compared among the various preparations . two initial total nisin concentrations , 150 and 200 μg / ml , were used in efficacy tests . a 6 - order polynomial “ standard curve ” was also drawn ( fig1 c ) to correlate the size of the inhibitory ring with the amount of available nisin . in general , the initial activity of the free nisin preparation was the highest , but it decreased sharply in the first 5 days and continued to decrease in the later stages . with an initial nisin concentration of 150 μg / ml , the available nisin in the free nisin preparation was calculated to be 10 . 2 μg / ml after 5 days , & lt ; 1 μg / ml after 20 days , and negligible after 40 days . in contrast , the pg - os - stabilized emulsion showed substantial inhibitory effects during the extended storage period . due to interfacial adsorption , the available nisin was initially about 43 . 8 μg / ml and then decreased slowly during storage to about 25 . 3 μg / ml after 5 days , 19 . 4 μg / ml after 20 days , and 14 . 3 μg / ml after 40 days . the tween 20 - stablized emulsion showed minor improvement over free nisin after 10 days . without wishing to be bound by a particular theory or to in any way limit the scope of the appended claims or their equivalents , it is presently believed that the hydrophobic interaction between nisin and the surface of the oil droplets may lead to a minor level of adsorption as indicated by the zeta - potential data ( fig1 ). this interaction , however , was apparently not sufficient to successfully prolong nisin efficacy . the behavior of the wcs - os - stabilized emulsion is noteworthy . regardless of the initial amount of nisin , the initial ring size ( and , accordingly , the amount of available nisin ) was lower than that for the pg - os - stabilized emulsion . this implies that nisin adsorption was stronger in the wcs - os emulsion than in the pg - os emulsion . meanwhile , the retention of nisin activity observed for the wcs - os emulsion was lower than that for the pg - os emulsion throughout the storage period . the fact that nisin availability quickly declined in the control group ( fig1 ) but was somewhat retained in the model group suggests that certain components ( possibly protein molecules ) in the storage model may have reduced the over - adsorption of nisin at the interface of the droplets in the wcs - os emulsion . in the effort to retain the activity of antimicrobial compounds , most researchers have been focusing on the incorporation of active compounds to films . specifically , these researches mimicked the scenario in which the bacterial contamination occurs before packaging , and the goals were to reduce the microbial growth during the storage of food in the initial package . the films have been prepared using either synthetic polymers such as plastics or biopolymers such as polysaccharides and proteins . for example , polyethylene film was used to retain nisin activity in the 20 - day storage and a reduction from log 10 6 . 3 to log 10 3 . 6 was realized for b . thermosphacta ( siragusa et al ., 1999 ). nguyen et al . ( 2008 ) infused nisin into cellulose film to inhibit the growth of l . monocytogenes , achieving a 2 log cfu / g reduction compared with the non - nisin control after 14 days of storage . nisin - containing polylactic acid films prepared by jin and zhang ( 2008 ) were tested against l . monocytogenes and a reduction of 4 . 5 log cfu / ml over the controls was shown . similar studies have been conducted using films prepared from other materials , such as sodium caseinate ( kristo et al ., 2008 ), soy protein ( sivarooban et al . 2008 ), alginate ( millette et al ., 2007 ), and zein ( hoffman et al ., 2001 ). in most studies , the antimicrobial efficacy assay followed a procedure in which the food ( or food model ) were inoculated first with bacteria and then stored for a period of time during which the growth of bacteria was monitored . conceivably , this procedure was used to test an effective release of antimicrobial compounds from films . in most antimicrobial studies , the control group did not contain antimicrobial compounds . in one report free nisin was used as the control ( millette et al ., 2007 ), and the efficacy of nisin - containing alginate beads was shown to be lower than that of free nisin . this is not a surprise . with the same amount of nisin , the initial availability of peptide molecules of a free nisin preparation should be higher than that of a film . the comparison between free and film - incorporated nisin was not found in other publications , possibly due to an understanding that potential nisin depletion in food would justify the use of films for retaining nisin activity during storage . our study , on the other hand , directly compared the efficacy of nisin with and without a delivery system ( e . g . pg - os or wcs - os emulsion ). the design of our study is based on the concept that , a successful bacteriocin delivery system should not only show antimicrobial effect , but also show an effect greater than that of free nisin preparation . another issue is the timing of bacterial contamination , and subsequent intended protection strategy . for l . monocytogenes , it was recently identified that the contamination at the production and packaging stages has been largely under control , while 80 % of listeria - related deaths are attributed to ready - to - eat foods from deli counters ( houchins , 2008 ). therefore , it is important to prevent bacterial growth after the exposure of food to environment at retail and home storages . one approach to address this challenge is to apply antimicrobial compounds at the packaging stage and maintain the antimicrobial activity throughout the life of this product . another approach is to apply antimicrobial compounds right after opening the package . both approaches should be able to keep the food protected from bacterial contamination before consumption . from this perspective , this study was undertaken for developing a strategy to protect food from potential contamination in the later ( rather than earlier ) stages of product life . using pg - os emulsion as a delivery system , the nisin efficacy against listeria can be retained for as long as 50 days . the established strategy is novel , not only due to the use of carbohydrate nanoparticle - mediated assemblies , but also due to its potential to reduce a recently identified but major cause of pathogenic contamination . one advantage of using a dispersion system ( over a film - based strategy ) to deliver bacteriocin is to retain the majority of antimicrobial compounds with food after removing the package . the pg - os - stabilized emulsion showed an outstanding ability to prolong the efficacy of bacteriocin nisin against the food pathogen l . monocytogenes . the use of amphiphilic carbohydrate nanoparticle - mediated colloidal assembly for prolonged delivery of bioactive compounds has not been reported previously . a key finding of this work is that the interface of carbohydrate nanoparticle - stabilized emulsion can be used to deliver bioactive compounds . pg - os is a novel , digestible nanomaterial , and its potential benefits are beginning to be revealed ( scheffler et al ., 2010a , b ). importantly , the structure of phytoglycogen nanoparticles can be manipulated through biological , chemical , and enzymatic approaches , allowing the creation of a new class of nano - constructs and devices . this study was conducted from the perspective of food applications ; 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( 19 ) wong k ., kubo a ., jane j ., harada k ., satoh h ., nakamura y . 2003 . structures and properties of amylopectin and phytoglycogen in the endosperm of sugary - 1 mutants of rice . j . cereal sci . 37 : 139 - 149 . the foregoing detailed description and accompanying drawings have been provided by way of explanation and illustration , and are not intended to limit the scope of the appended claims . many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art , and remain within the scope of the appended claims and their equivalents .