Patent Application: US-201514658303-A

Abstract:
the present invention relates to the field of food technology and delivery of hydrophobic biologically active compounds , particularly nutrients , via food products and beverages . in particular the present invention provides isolated casein micelles useful for the encapsulation of hydrophobic nutrients , therapeutic and cosmetic compounds , compositions thereof and methods of preparing the micelles .

Description:
the present invention provides isolated casein micelles and methods for encapsulation of hydrophobic or poorly water - soluble biologically active compounds in cm , with minimal changes to the functional properties of both the micelles and the active compound . the present invention now discloses that adsorption of hydrophobic biologically active compounds , for example nutraceuticals , onto the hydrophobic domains of caseins , and the reformation of the micelles stabilize the hydrophobic compound in aqueous surrounding and protect them from degradation . such casein micelles - hydrophobic compound system facilitates the enrichment of low fat and fat free dairy and other food products with the bioactive molecules , while minimizing the effect of the compound incorporation on the food properties in general and during processing . encapsulation of biologically active compounds within casein micelles is advantageous over hitherto known encapsulation methods as the micelles are a natural component of milk products and their nanometric size minimizes their effect on the food product , including dairy as well as non dairy foods . in addition , when the active compound possesses undesirable attributes , the encapsulation in the micelles diminishes such unwanted features ( e . g . in the case of omega 3 fatty acids ). another important potential benefit is the improved bioavailability of the enclosed compound due to its distribution , at a molecular level , over a very large surface area of the caseins in the nanoscopic micelles , and the fact that caseins are evolutionally optimized for ease of digestion and absorption . the open tertiary molecular structure of casein also facilitates effective proteolysis . specific embodiments include a method for incorporation of vitamin d2 into cm , and evaluation of the encapsulation process by : ( a ) evaluation of the efficiency of encapsulation , i . e . the percent of added vitamin d2 which was incorporated into the micelles , ( b ) preservation of micelle properties : diameter as determined by dynamic light scattering ( dls ) and morphology ( as determined by cryo - tem ), ( c ) evaluation of the protective effect of the micelles over vitamin d2 from photochemical degradation induced by uv exposure . for convenience and clarity certain terms employed in the specification , examples and claims are described herein . as used herein , the term “ casein ” refers to the predominant protein in non - human mammals and human milk , comprising the subgroups α s1 , α s2 , β and κ . the term “ biologically active compound ” encompasses a compound having a therapeutic , nutritional and / or cosmetic activity . biologically active compounds according to the teaching of the invention include , but are not limited to peptides , proteins , amino acids , lipids , proteoglycans , polysaccharides , vitamins , hormones , drugs , steroids , phytochemicals , polynucleotides , flavorants , sweeteners , an anti - microbials , and preservatives . a “ nutraceutical ”, also known as a functional food ( or its component ), is generally any one of a class of dietary supplements , vitamins , minerals , herbs , healing or disease - preventative foods that have medical or pharmaceutical effects on the body . exemplary non - polar or hydrophobic nutraceuticals include , but are not limited to fatty acids ( e . g ., omega - 3 fatty acids , dha and epa ); fruit and vegetable extracts ; vitamins a , d , e and k ; phospholipids , e . g . phosphatidyl - serine ; certain proteoglycans such as chondroitin ; certain amino acids ( e . g ., iso - leucine , leucine , methionine , phenylanine , tryptophan , and valine ); various food additives , various phytonutrients , for example lycopene , lutein and zeaxanthin ; certain antioxidants ; plant oils ; and fish and marine animal oils and algae oils . it is to be understood that certain nutraceuticals can be also referred to as therapeutics as well as cosmetic compounds . the following examples are presented in order to more fully illustrate some embodiments of the invention . they should , in no way be construed , however , as limiting the broad scope of the invention . one skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention . sodium caseinate ( miprodan 30 , 93 . 5 % protein , md foods ingredients amba , videbaek , denmark ). vitamin d2 ( sigma - aldrich , rehovot , israel ). ethanol ( absolute ), hydrochloric acid ( concentrated ), ( bio - lab , jerusalem , israel ). tripotassium citrate , sodium hydroxide , ( merck , darmstadt , germany ). calcium chloride ( carlo erba , rodano , italy ). dipotassium hydrogen phosphate ( spectrum , ca , usa ). petroleum ether and diethyl ether ( bio - lab , jerusalem , israel ). potassium hydroxide and pyrogallol ( merck , darmstadt , germany ). ethylene diamine tetraacetic acid ( edta ) ( acros , nj , usa ). methanol and acetonitrile ( both hplc grade ) ( lab scan , dublin , ireland ). non covalent binding of vitamin d2 to sodium caseinate was achieved by dropwise addition of 1 - 100 mm , typically 2 - 50 mm , more typically 5 - 20 mm , more typically 15 mm solution of the vitamin in absolute ethanol into a 1 - 20 % caseinate , typically 3 - 8 %, more typically 5 % caseinate solution , while stirring , to a final concentration of about 5 - 50 , 000 μm , typically about 10 - 5000 μm , more typically about 25 - 1000 , more typically 25 - 100 , more typically about 65 μm . preparation of re - assembled cm ( rcm ) was done based on the method described by knoop et al . ( 8 ). however , unlike knoop et al . the casein is provided as a rehydrated commercial sodium caseinate powder , rather than a freshly prepared caseinate , in order to extend the commercial applicability of the method . to 2 - 10 %, more typically 5 % non - enriched caseinate solution and to vitamin d2 enriched caseinate solution ( 200 ml each ), 0 . 5 - 1 . 5m , more typically 1 m tri - potassium citrate ( 4 ml ), 0 . 05 - 0 . 5m , more typically 0 . 2 m k 2 hpo 4 ( 24 ml ) and 0 . 05 - 0 . 5m , more typically 0 . 2 m cacl 2 ( 20 ml ) were added . eight consecutive additions of the k 2 hpo 4 solution ( 2 . 5 ml ) and of the cacl 2 ( 5 ml ) were performed , at 15 - minute intervals . during this process , samples were stirred in a thermostated bath at 25 - 42 ° c ., more typically 37 ° c . the ph was maintained between 6 . 5 - 7 . 0 , more typically between 6 . 7 - 7 . 0 , using 0 . 1n hcl or 1n naoh . the volume was then adjusted to 400 ml with water , the ph was corrected to 6 . 5 - 7 . 0 , more typically to 6 . 7 , and the final dispersions were stirred moderately for one hour ( 8 ; 9 ). alternatively , phosphate and optionally also citrate are added to the aqueous casein solution containing the hydrophobic molecule and the mixture is blended to make solution a . for example , this solution contains 400 ml 5 % sodium caseinate , 2 ml 5 mg / ml vitamin d2 in absolute ethanol , 88 ml 0 . 4m k2hpo4 and 10 ml 0 . 8m sodium citrate . a calcium ion source solution ( b ) is prepared separately . the calcium solution comprises , for example , 300 ml 0 . 08m cacl2 . solutions a and b are then combined during a high pressure homogenization process . ultra - high pressure homogenization may be done by any method known in the art . for example , ultra - high pressure homogenization is performed using a micro debee ultra high pressure homogenizer , at 20 - 25 kpsi , using a 0 . 1 mm orifice and 1 mm reactor cylinders , without back pressure , at a temperature of ˜ 40 ° c . flow rates are adjusted so that the main stream ( solution a ) and the added stream ( b ) are processed simultaneously throughout the process . micelle preparations were centrifuged at 20 ° c . and 25 , 000 × g for one hour and the supernatant was separated from the pellet by decantation . the supernatant was then ultra - filtered using amicon 8050 stirred ultrafiltration cell with a 10 , 000 da nominal molecular weight limit membrane ( millipore ). all fractions were collected and analyzed for vitamin d2 content . evaluation of micelle protection against uv light induced degradation of vitamin d2 : samples containing vitamin d2 - enriched rcm ( d2 - rcm ) were placed in a wooden light - proof cabinet , and exposed to a 254 nm uv light , at 200 μw / cm 2 intensity for 3 , 6 , 12 , and 24 hours . at each exposure time , three 20 ml samples were compared : a micelle dispersion preparation , a negative control ( an identical sample covered with an aluminum foil to completely block the uv ), and another control containing only serum from the d2 - rcm preparation , which was exposed to uv . the serum samples were obtained by centrifuging d2 - rcm dispersion and collecting the supernatant . samples of caseinate and vitamin d2 at concentrations similar to their concentrations in the rcm suspension ( 2 . 5 % and 31 . 75 μm respectively ) were prepared . uv absorbance spectra of the samples were analyzed by absorbance scan at wavelengths between 220 nm and 360 nm , using a pharmacia biotech ultrospec 3000 spectrophotometer . pellets were resuspended in a 100 mm edta solution of same weight as the removed supernatant , and equilibrated for 6 hours at 4 ° c . both pellet , serum and permeate from each sample underwent saponification and extraction procedures based on renken and warthesen ( jo ): five ml of each sample were placed into a 25 ml glass stoppered round bottom flask wrapped with aluminum - foil . 3 ml of 5 % koh and 1 . 5 ml of 1 % ethanol pyrogallol solutions were added . the samples were flushed with nitrogen , capped and then left to stir slowly in the dark for 12 hours at room temperature . each sample was then poured into a separatory funnel . the round bottomed flask was washed with 2 ml of water , then 0 . 75 ml of ethanol , and lastly 5 ml of petroleum ether : diethyl ether ( 90 : 10 v / v ), adding each wash liquid into the separatory funnel . the mixture was gently mixed and the phases were allowed to separate . the hydrophilic phase was then poured into a second separatory funnel adding 0 . 75 ml ethanol and 5 ml of the ether mixture . after gentle mixing the phases were allowed to separate . the hydrophobic phase was put into the first separatory funnel . 4 . 5 ml water was used to wash the hydrophobic phase four times ( 10 ). the hydrophobic phase was collected and the solvents were evaporated using nitrogen . the dried sample was re - suspended in 1 ml solution of methanol : water ( 93 : 7 v / v ) ( 11 ). vitamin d2 analysis was done by reverse phase hplc ( rp - hplc ). all samples were analyzed for vitamin d2 using a 4 . 6 × 100 mm c18 - c2 rp - hplc column and a uv detector at 265 nm . the gradient used was zero to 75 % acetonitrile as eluent b , while methanol : water ( 93 : 7 v / v ) serve as eluent a ( 11 ). calibration curve was prepared using vitamin d2 standard in methanol : water ( 93 : 7 v / v ) solution at 7 concentrations ranging from 5 to 250 μg / ml . vitamin d2 fractions were collected during rp - hplc and analyzed for uv absorbance spectrum from 220 nm to 360 nm for further identity validation , using a pharmacia biotech ultrospec 3000 spectrophotometer . size and morphology determination of rcm : for both rcm and d2 - rcm , average size was measured by dynamic light scattering ( dls ) ( bic 90plus , brookhaven instruments corp .). morphology was determined using cryogenic transmission electron microscopy ( cryo - tem ): specimens were prepared in a controlled environment vitrification system ( cevs ) at controlled temperature and humidity to avoid loss of volatiles . the samples were brought to a desired temperature ( 25 ° c . and 35 ° c .) and allowed to equilibrate in the cevs for an hour . then , a 7 μl drop of the examined dispersion was placed on a tem copper grid covered with a perforated carbon film , and blotted with a filter paper to form a thin liquid film of the sample ( 100 - 200 nm thick ). the thinned sample was immediately plunged into liquid ethane at its freezing temperature (− 183 ° c .) to form a vitrified specimen , and then transferred to liquid nitrogen (− 196 ° c .) for storage until examination . the vitrified specimens were examined in a philips cm120 transmission electron microscope operating at an accelerating voltage of 120 kv . an oxford ct3500 cryo - specimen holder was used to maintain the vitrified specimens below − 175 ° c . during sample transfer and observation . specimens were recorded digitally on a cooled gatan multiscan 791 ccd camera using the digital micrograph 3 . 1 software , in the low - dose imaging mode to minimize beam exposure and electron - beam radiation damage . brightness and contrast adjustments were done using photoshop 7 . 0 me . samples of rcm and d2 - rcm suspensions , as well as a sample of skim milk reconstituted from powder , were homogenized using a micro debee ultra high pressure homogenizer , by 1 pass at the single - reversed - flow mode at 185 ± 10 mpa , using a 0 . 1 mm orifice , and a back - pressure of 10 ± 3 mpa . average diameter of rcm and d2 - rcm was measured before and after homogenization process by dls ( see method details above ). relative average diameter changes were then determined for each sample . fig1 presents the results of the analysis of vitamin d2 in preparations of micelles enriched with vitamin d2 ( d2 - rcm ) and control rcm preparations without the vitamin . both analyses of the micelle pellets obtained by centrifugation and of their respective serum fractions are presented . in the chromatograms of the control rcm preparation fractions ( pellet — fig1 a , and serum — fig1 b ) vitamin d2 peaks were absent , while in both d2 - rcm fractions those peaks were observed . uv absorbance spectra of the peaks identified as vitamin d2 indicated good matching between vitamin d2 standard and vitamin d2 eluted at the same position in the sample runs . during the analysis , 45 - 95 %, more typically 65 - 85 % of total vitamin d2 added were recovered by the extraction procedure from the serum and the pellet together . 25 - 75 %, more typically 45 - 60 % of the recovered vitamin d2 were found to be incorporated into the micelles , which accounted for 2 - 15 %, more typically 8 % by weight of the total d2 - rcm suspension prepared . it was determined that vitamin d2 concentration in the rcm was about 2 - 22 times , more typically 5 - 10 times greater than its concentration in serum : 44 - 57 μg / ml vs . 2 - 8 μg / ml respectively . therefore , fortification of milk using such vitamin d2 - enriched rcm accounting for only 0 . 001 - 1 %, more typically 0 . 1 - 1 %, more typically 0 . 5 - 0 . 6 % of the total milk casein , would provide about one third of the vitamin d2 recommended daily allowance ( rda ) for adults in a single glass of milk ( 200 ml ). the re - assembled micelles had average diameters of 146 and 152 nm without and with vitamin d2 respectively ( fig2 ). as mentioned hereinabove , the normal size range of cm in milk is ˜ 50 - 500 nm , and the average is ˜ 150 nm . d2 - rcm and rcm had similar morphology , which was also typical to naturally occurring cm , as may be judged by the available resolution of the tem micrographs ( fig3 ). these micrographs suggest that the incorporation of vitamin d2 has minimal effect over the morphology of the cm . following an ultra - high - pressure homogenization process the average diameter of rcm was reduced to 122 nm ( 26 % reduction ) and that of d2 - rcm was reduced to 125 nm ( 27 % reduction ). the reference micelles from reconstituted skim milk showed a 9 % reduction in diameter during the homogenization . while this shows that the reformed micelles are expectedly somewhat weaker than the original micelles , their durability through such extreme shear suggests they could well withstand typical processing shear which is seldom that high . the similar extent of reduction in size for rcm and d2 - rcm suggests that the incorporation of vitamin d2 into rcm did not weaken their structure as reflected by shear stability . fig4 shows the size distribution of cm obtained by in - line merging of two streams , just before the high pressure chamber of a high - pressure homogenizer . one stream was the aqueous casein solution containing the hydrophobic molecule ( s ), as well as phosphate and optionally also citrate , and the other stream was a calcium ion source solution . the average size of the cm obtained was ˜ 100 nm fig6 shows a cryo - tem image of the vitamin d2 - containing cm obtained this way . analysis of vitamin d2 in the micelle - pellet and supernatant of the centrifuged micelle preparation showed 4 - 5 times higher vitamin concentration in the micelle pellet compared to the supernatant ( serum ). the pellet contained 30 . 3 ± 0 . 4 and the supernatant contained 6 . 7 ± 0 . 7 micrograms / ml of the vitamin . quantification of the protective effect of the micelles against uv light - induced photochemical degradation of vitamin d2 table 1 presents vitamin d2 degradation as the remaining percent of the initial concentration in each fraction with exposure time , in d2 - rcm suspension exposed to uv light , d2 - rcm suspension unexposed to uv light ( control i ) and in d2 - rcm suspension serum exposed to uv light ( control ii ). ( ud = undetectable ). the data in table 1 merits several observations : first , the comparison of the uv exposed serum ( control ii ) to the serum of control i ( unexposed ) shows how relatively quickly photochemical degradation of unprotected vitamin d2 occurs . the vitamin in the serum is presumably bound to residual soluble casein molecules which did not aggregate into micelles . the main interesting observation is the comparison of the rate of degradation of the vitamin within the micelles in the exposed preparation to that of the uv exposed serum ( control ii ). this comparison demonstrates the significant relative protection conferred by the micelles to the encapsulated vitamin . the micelles also confer some protection to the vitamin in their surrounding serum , as the rate of degradation in the serum of the exposed micelle dispersion was lower than that in the exposed micelle - free serum ( control ii ). this may be explained by a “ shade ” effect of the micelles which block and absorb much of the light . lastly , it is observed that the degradation of the vitamin in the micelles of the unexposed preparation of control i ( although slightly obscured by experimental error ) was slower than in the serum of this preparation . this degradation may be due to chemical oxidation , ( e . g . by dissolved oxygen ) and this observation suggests that the micelles confer some protection against chemical degradation as well , however this remains to be verified by other experiments . the nature of the protective effect of the micelles against photo - degradation of the vitamin was examined by comparing the absorbance spectra for both caseinate and vitamin d2 components in the rcm suspension as is shown in fig5 , at the concentrations examined for each of the fractions ( caseinate and vitamin d2 ) in the rcm suspension , caseinate , being a protein with aromatic side groups and double - bonds , absorbs significantly more uv light than vitamin d2 . these data support the conclusion that casein micelles have protective effect for the vitamin d2 enclosed therein and to some degree also to vitamin d2 around the micelles . casein micelles were shown to be potential nano - vehicles for added nutraceuticals such as the fat - soluble vitamin d2 chosen here as a model . in terms of encapsulation efficiency , about 25 % to about 75 %, more typically 45 - 60 % of the vitamin retrieved from the micelle suspension was found in the reformed micelles — which contained about 2 to about 22 fold , more typically 4 - 10 fold higher concentration of the vitamin compared to the surrounding medium . some vitamin d may be lost by binding to hydrophobic domains of unaggregated proteins in the serum . the extraction - based analysis method allowed the retrieval of about 45 to about 95 %, more typically 65 - 85 % of the added vitamin . the micelles &# 39 ; morphology and size were similar to those of naturally occurring cm , in accord with the purpose to minimize modification of micelle properties . it was also shown that apart from their effectiveness in stabilizing oil - soluble compounds in aqueous environment , the rcm have an additional protective affect against photochemical degradation of the entrapped hydrophobic compound , for example the nutraceutical vitamin d2 . the foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can , by applying current knowledge , readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept , and , therefore , such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments . it is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation . the means , materials , and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention . 1 . dekruif , c . g . ; 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