TR2022016347A2

TR2022016347A2 - USE OF EGGSHELL MEMBRANE PROTEINS AS FOOD SUPPLEMENT

Info

Publication number: TR2022016347A2
Application number: TR2022/016347
Authority: TR
Inventors: Akdemi̇r Evrendi̇lek Gulsun; Karakaya Nusret; Demi̇r İrem
Original assignee: Bolu Abant İzzet Baysal Üni̇versi̇tesi̇ Strateji̇ Geli̇şti̇rme Dai̇re Başkanliği
Filing date: 2022-10-28
Publication date: 2022-11-21

Abstract

Buluş, yumurtanın soyulmasıyla atık olarak çıkan yumurta kabuğu zarında bulunan proteinlerin gıda takviyesi olarak kullanılması ile ilgilidir. Buluş özellikle; kuru yumurta kabuklarının EDTA ile 30-150 dak arasında muamele edilmesi, % 0.1, 0.5 ve 1 SDS içeren lizis solüsyonu ile 10-14 saat 4°C?de muamele edilmesi, süpernatant kısımlardan farklı eppendorf tüpüne aktarım yapılıp ve aseton eklenerek 10-14 saat 4°C?de bekletilmesi, santrifüj uygulandıktan sonra protein tortusu temiz bir tüpe alınarak ultra saf suyun içinde çözünmesinin sağlanması, %1 SDS/0.1 N NaOH eklendikten sonra Folin reaktifi eklenerek farklı sürelerde 0-1440 dak arasında 37??de bekletilerek spektrofotometrede 660 nm?de okumaları yapılması, methanol ve ultra saf su ile yıkanarak kimyasalların uzaklaştırılması işlem adımlarını içeren yumurta kabuğunu zarında bulunan proteinlerin gıda takviyesi ve besin takviyesi olarak kullanılması ile ilgilidir.The invention is about using the proteins found in the egg shell membrane, which is released as waste when peeling the egg, as a food supplement. The invention especially; Treating dry egg shells with EDTA for 30-150 minutes, treating them with lysis solution containing 0.1, 0.5 and 1% SDS for 10-14 hours at 4°C, transferring the supernatant sections to a different Eppendorf tube and adding acetone for 10-14 hours. It was kept at 4°C, after centrifugation, the protein residue was taken into a clean tube and dissolved in ultrapure water, after adding 1% SDS/0.1 N NaOH, Folin reagent was added and kept for different periods between 0-1440 min at 37° and 660 nm in the spectrophotometer. It is related to the use of proteins in the eggshell membrane as food supplements and nutritional supplements, which includes the process steps of taking ? de readings and removing chemicals by washing with methanol and ultrapure water.

Description

TARIFNAME YUMURTA KABUGU ZARI PROTEINLERININ GIDA TAKVIYESI OLARAK KULLANIMI TEKNIK ALAN Bulus, yumurtanin soyulmasiyla atik olarak çikan yumurta kabugunu zarinda bulunan proteinlerin gida ve besin takviyesi olarak kullanilmasi ile ilgilidir. Bulus özellikle; kuru yumurta kabuklarina EDTA, HCL ve asetik asit ile 30-150 dak arasinda muamele edildikten sonra kurutularak toz formuna getirilmesi, % 0.1, 0.5 ve 1 SDS veya ayni oranlarda Triton X-100 içeren ve içlerinde 2-merkaptoetanol bulunan lizis solüsyonu ile 10-14 saat 4°C'de muamele edilmesi, süpernatant kisimlardan farkli eppendorf tüpüne aktarim yapilip ve aseton eklenerek 10-14 saat 4°C'de bekletilmesi, santrifüj uygulandiktan sonra protein tortusunun temiz bir tüpe alinarak ultra saf suyun içinde çözünmesinin saglanmasi, %1 SÜS/0.1 N NaOH eklendikten sonra Folin reaktifi eklenerekfarkli sürelerde 0-1440 dak arasinda 37°C'de bekletilerek spektrofotometrede 660 nm'de okumalari yapilmasi, methanol ve ardindan ultra saf su ile yikanarak kimyasallarin uzaklastirilmasi islem adimlarini içeren yumurta kabugunu zarinda bulunan proteinlerin gida takviyesi olarak kullanilmasi ile ilgilidir. TEKNIGIN BILINEN DURUMU Gida endüstrisinde oldukça fazla kullanilan ürünlerden birisi yumurtadir. Yumurta ekmekçilik, firincilik, pastacilik, hazir yemek vb. is kollarinda oldukça yaygin bir sekilde kullanilir. Yumurtanin her geçen yil üretimi ve tüketimi de artmaktadir. Yumurtanin iç kismi alindiktan sonra yumurta kabugu ve kabuga yapismis olarak yer alan iç membran çabuk bozularak kötü koku yaymaktadir. Bu nedenle, yumurta kabugu genel olarak çabuk bozulan ve kötü koku yayan bir atik olarak siniflandirilir ve degerlendirilemez. Yumurta dis kabuk membrani yapisinda bulundurdugu proteinlerden dolayi oldukça iyi bir besin maddesi iken degerlendirilememektedir. Bu nedenle yumurta kabugu membraninda bulunan proteinlerin elde edilmesi ve farkli ürünlere dönüstürülmesi ve bu sayede çevreye kirliliginin önlenmesini saglayarak atiklarin degerlendirilmesi büyük önem arz etmektedir. Yumurtanin kendisi protein açisindan zengin birgida olmakla birlikte herhangi bir sekilde degerlendirilemeyen dis kabuk zar membrani da içerdigi proteinler sayesinde önemli bir besin kaynagi olma potansiyelini tasimaktadir. Yumurta zari kabuk alti dis zar (kabuk zari), kabuk alti iç zar (yumurta zari) ve sinirlayici zar olarak 3'e ayrilmaktadir. Bu yapilar birbirlerine zar lifi yardimiyla baglanarak bir bütün halini almistir. Zar lifleri zara protein yapisini kazandiran ana bilesendir. Yumurta kabugu zari yaklasik olarak %60 zar lifleri, yaklasik olarak %80-85 oraninda proteinden olusmaktadir. Bu proteinlerin %10'unu kolajen, %70-75'ini ise lizin türevi çapraz bag içeren protein ve glikoproteinler olusturmaktadir. Kabuk ve kabuk alti zarlarda bulunan protein sayisi 500'den fazladir. Sadece kabuk alti zarlarinda ise 62 adet protein bulunmaktadir. Bu proteinlere lizozim, ovotransferrin, ovalbumin, ovokaliksin-36 ve keratin gibi proteinler örnek olarak verilebilir. Zarlarda en çok bulunan proteinlerden bazilari ise prolin, glutamik asit ve glisin proteinleridir. Ayni zamanda dis zarlar tip I ve iç zarlar tip I ve tip V yapidaki kolajen proteinini içermektedir. Kolajen tip X ise hem iç hem dis zarda bulunur. Kolajen bag dokularda bulunan temel yapi proteinidir. Tip I kolajen insan vücudunda en çok bulunan ve en dayanikli olan kolajen tipidir. Baslica kemik dokuda bulunur. Tip V ve X kolajen basta eklemler olmak üzere kemik ve kikirdak gibi bag dokuda bulunan kolajen tipleridir Bu bilgiler isiginda gida olarak tüketilen yumurtanin içinde bulunan proteinlerin yani sira, tüketilmeden çöpe atilan zar kisimlarinin da kolajen gibi çok degerli ve yapisal proteinlerce zengin oldugu görülmektedir. Fakat YKZ'inin protein kaynagi olarak kullanila bilinmesi için temel iki problemin çözülmesi gerekmektedir. Yumurta zari dis kabuga sikica bagli oldugu için zarin kabuktan ayrilmasi çözülmesi gereken ilk problem iken, ayrilan zarin sindirilebilir protein kaynagi olarak kullanimi çözülmesi gereken ikinci problemdir. YKZ fazla miktarda disülfüt bagi içerdiginden oldukça kararli bir yapisi bulunmaktadir ve disülfit baglari zarin sulu sistemlerde çözünürlügünü engellemekte ve dolayisiyla protein kaynagi olarak kullanilamamaktadir. Günümüzde yumurta kabugunun organik zar matriksi ile olan kuvvetli etkilesimlerinden dolayi olusan kompleks yapisi zarin kabuktan ayrilmasini zorlastirmaktadir. YKZ'nin kabuktan ayrilmasi amaciyla farkli yöntemler test edilmistir. Mevcut teknikte bilinen U56176376 numarali basvuruda, yumurta kabuklarini zardan ayirmak için gelistirilen tank içerisine alinan kabuklarin parçalanarak boyutlarin küçültülmesi (0.5-0.5 mm) ve bir solvent -tercihen su- içerisinde bekletilerek zarin kabuktan ayrilmasi hedeflenmistir. Bu yöntem zarlar çok parçalandigi, su sarfiyati fazla oldugu ve islatilan zarlarin kurutulmasi için ekstra enerji gerektigi için tercih edilmemektedir. Mevcut teknikte asit, alkali ve asit-alkali kombinasyonlarinin kullanilmasi ve asitle kaynatma gibi yöntemler denenmis olmasina ragmen bu yöntemlerde pahali olmalari ve çok zaman gerektirdigi için pratikte kullanilmamaktadirlar. Kavitasyon uygulayarak yumurta kabugunun sulu sistemlerde zardan ayrilmasi da rapor edilen çalismalar arasindadir. Farkli frekans ( uygulanan mikrodalga enerjisinin yumurta kabugu ve zar arasindaki baglarin kirilmasinda etkili oldugu rapor edilmistir. Mevcut teknikte zarin kabuktan ayrilmasi amaciyla manuel olarak ayirma, kabuktaki kalsiyum karbonat tabakasini eritmek amaciyla seyreltik asit (asetik asit, hidroklorik asit, EDTA, sülfirik asit) kullanimi, hava içeren basinçli su kullanarak hava akiminda zar ve kabugun ayrilmasi ve buharla isitilan sulu ortamda seri halde bulunan matkap vasitasiyla zarin ayrilmasi yapilan denemeler arasinda yer almaktadir. Mevcut teknikte bulunan bir çalismada yumurta kabugu zarlarinin kabuklardan ayrildiktan sonra çesitli islemler sonucunda ultrasonik reaktör ile frekans uygulamasiyla 100 g'da 73 g protein elde edilmistir. Chen-Lee ve Huang (2019) "Çözünür yumurta kabugu zari protein yüklü kitosan/fucoidan kusurlu bagirsak epitel hücrelerinin tedavisi için nanopartiküller" konulu çalismalarinda çesitli islemlerden geçirilmis yumurta kabugu zarlari ayrilarak bir dizi karisimda bekletildikten sonra enzim (proteaz) ve ardindan santrifüj uygulamasiyla protein ekstraksiyonu gerçeklestirilmistir. I no ve ark (2006) yapmis olduklari çalismada çesitli islemlerden geçirilerek yumurta kabugundan ayrilmasi saglanan yumurta kabugu zarina farkli bir enzim (pepsin) kullanimiyla santrifüj islemi uygulanarak protein ekstraksiyonu gerçeklestirilmistir. Mevcut teknikte bulunan bir çalismada YKZ proteinlerinin çözünebilmesi amaciyla asetik asit varliginda 3-merkaptopropiyonik asit ile muamele edildikten sonra karisimin pH'si 'e ayarlanmis ve yumurta zarinin yapisi ve morfolojisi incelenmistir. TYZ 3- merkaptopropiyonik asit ve asetik asit içeren solüsyon ile muamele edildikten sonra karisim 90 °C'ye getirilmis ve 12 saat süresince zarin çözünürlügünün saglanmasi için muamele edilmistir. Solüsyonun pH'si NaOH kullanarak ayarlanmis ve zar metanolle yikandiktan sonra kurutulmustur Mevcut teknikte YKZ proteinlerinin ekstraksiyonu amaciyla farkli lizis solüsyonlarinin test edildigi bir çalismada ise en yüksek ekstraksiyon % 0.1 oraninda 2-merkaptoetanol ve % 1 SDS içeren ve pH si 8.8 olan 5 mM TRIS-HCL solüsyonununun 37°C sicaklikta 21 gün süresince uygulamasi sonrasinda elde edilmistir. Uygulama sonrasinda zarlar yikandiktan sonra kurutulmustur. Mevcut teknikte YKZ'nin kabuktan ayrilmasi için asetik asit, EDTA ve fosfat solüsyonunun denendigi çalismada kabuklarindan kalsiyumun uzaklastirilmasi (decalcification) için modifiye edilmis EDTA yöntemi tavsiye edilmistir. Bu yöntemde 250 mL TRIS-HCL (50 mM) ile karistirilan 50 g yumurta kabugu tozu 4 M guanidin hidroklorid içerisine aktarilarak 72 saat süreyle bekletilmistir. Santifüjlenen karisimda çökelti 40 saat süresince ile karistirilmis ve santrifüjlenen karisimdan elde edilen supernatant diyaliz edildikten sonra liyofilize edilmistir. Mevcut teknikte bulunan JP628868682 nolu Egg shell membrane protein solubilized product and method for producing the same, composition containing the eggshell membrane protein solubilized product, composition for hair cosmetics, and hair treatment method using the composition for hair cosmetics baslikli patentte yumurta zari proteinlerinin (YZP) çözünürlügünü saglamak için pH'si 6-12 arasina ve sicaklik enzim uygulamasina 30-60C arasi sicakliklarda 24 saat süresince devam edilmis ve çözelti kurutulmustur. Bu yöntemde büyük molekül agirligina sahip YZP elde etmek mümkün olmadigi gibi katalist görevi gören enzimde oldukça pahalidir. substance and treating method baslikli patentte yumurta zari proteinlerinin (YZP) kozmetik amaçli olarak saçlarin bakiminda kullanimi amaçlanmaktadir. Bu amaçla saç bakiminda kullanilan keratine YZP ilave edilmistir. Bu amaçla soluble (çözünür) YZP °C de 5 saat bekletilmistir. Elde edilen karisim filtre kagidindan süzülmüs ve nötralize edildikten sonra tuzu uzaklastirilmis ve 26 g çözünür YKP elde edilmistir. solubilization method using enzymes baslikli patentte proteolitik enzimler ve indirgen (reducing) ajan kombinasyonunun kullanimini içermektedir. Bu amaçla yumurta zari kimotripsin, subtilisin, papain, basilolizin, stem bromelain, lösilaminopeptidaz, pepsin ve tripsin gibi proteolitik enzimler (ticari olarak mevcut biosoak, Proleather FG-F, Papain W40, Protease N, Bromelain F, Ummamizyme G, Samoaase Y100, ProteAX, Protease S, Sumiteam LP500, Deskin C, Protin NY10, Protin PC10, Sumiteam MP gibi enzimler) ve indirgeyici ajanlar (sulfite, bisulfite, L-cysteine, N-acetyl-L-cysteine, 2-mercaptoethanol, glutathione, ve DTT vb) kullanilmasini içermektedir. Reaksiyon için pH 45-95 arasi ve tercihen nötr pH lar test edilmis olup 10 dak-24 saat süren reaksiyon genel olarak 30- 80°C'ler arasi ve tercihen 40-70°C araliginda gerçeklestirilmistir. Reaksiyonun etkinligini arttirmak için karistirma ve çalkalama islemleri uygulanmistir. Karisimda 0.1% enzim (w/v) 1.0% yumurta zarina ilave edilmis ve reaksiyon 60°C'de 72 saat süresince devam etmis, fakat görsel olarak çözünürlük gözlenmemistir. Mevcut teknikte bulunan U510526423BZ numarali ve Eggshell membrane solubilization Agustos 2010) numarali patent ile iliskilendirilmis olup TKZ çözüne bilirligini saglamak amaciyla indirgeme (reducing) ajanlari ve proteaz kombinasyonu kullanimini içermektedir. Enzim karisimi 10% (w/v) YKZ içeren reaksiyon çözeltisine (pH 7.4 de eklenip çözünürlük saglanmaktadir. Enzim karisimina PROTEINASE K (Roche), BIOSORK, PROLEATHER FG-F veya PAPAIN W40 ve PROTEINASE K eklenerek son konsantrasyonun % 0.01 (w/v) olmasi saglanmis ve diger enzim karisimlari eklenerek son konsantrasyon % 0.1% (w/v) olarak ayarlanmistir. Elde edilen karisim 50° C de 12 saat süreyle karistirilarak inkübasyona birakilmistir. Karisima n-butylamine eklenip son konsantrasyon 10 mM'a ayarlandiktan sonra 37° C de 60 dak inkübasyona birakilmistir. Ikinci asamada TKZ indirgeyici ajan ile muamele edilmis ve proteaz ile muamele edilmistir. Bu amaçla 500 mg TKZ na 5 mL (pH 7.4 de 100 mM PBS buffer ve eklenip 37° C de 1 saat inkübasyona birakilmistir. Daha sonra TKZ solusyondan uzaklastirilip 50 mL distile su ile 5 kez yikanip sodyum sülfit uzaklastirilmistir. Daha sonra distile su ortamdan uzaklastirilmis ve 5 mL % 0.1 (w/v) BIOSORK ( solüsyonu eklenip 37° C de inkübasyona birakilmistir. 200 uL örnekler farkli inkübasyon zamanlarinda alinip (0, 1, 2, 3, 4, ve 6 saat) lisil oksidaz (lysyl oxidase) aktivitesi ölçülmüstür. Bu amaçla son konsantrasyonun mM olmasi için 20 uL 100 mM n-butylamin örneklere eklenmis ve örnekler 37°C de 45 dak inkübasyona birakilmistir. Daha sonra 220 uL Nash reagent eklenip 50° C de 30 dak inkübasyona birakilip 388 nm de ölçüm yapilmistir. membrane protein soluble product, and composition containing eggshell membrane protein soluble product baslikli patentte YKZ pH'si 11'e ayanlanip alkaline proteaz eklendikten sonra hidrolize devam edilmistir. Alkaline proteaza ilave olarak ikinci enzim eklendikten sonra asamada çözeltinin pH'si 9'a ayarlanip hidrolize devam edilmis ve solüsyon isitilip YKZ çözünebilir forma getirilip alkaline proteaz inaktif hale getirilmistir. shell membrane protein solubilized product, and cosmetic composition, pharmaceutical composition and functional food Which contain egg-shell membrane protein solubilized product baslikli patentte TKZ süspansiyon solüsyonunun pH'si 1-3.5 arasina ayarlanip bes dak içerisinde süspansiyon sicakligi hizli bir isitma ile asit çözeltisi içerisine alinan YKZ'inda bulunan kalsiyum komponentleri çözündürülmekte ve daha sonra sivi faz uzaklastirilmaktadir. Yapilan islemde YKZ' nin çözünebilir hale gelmesi yüksek sicaklikta isitma ile çözünebilir hale gelmesi ve aggregate olusturmasi sayesinde gerçeklesmektedir. YKZ proteinleri izolelektrik noktanin altinda asidik bölgede pozitif yüklüdürler ve hizli bir sekilde isitildiginda çözünür proteinler pozitif yük nedeniyle birbirlerini itmekte ve aggregate olusturmadan çözünür formda kalmaktadirlar. Diger taraftan izolelektrik noktadan alkali bölgeye gidildikçe YKZ'inda bulunan kalsiyum iyonlari pozitif yükleri nötralize etmektedir ve yeterli çözünürlük elde edilememektedir. comprising eggshell membrane hydrolysate, and use thereof baslikli patent çözünür forma getirilen YKZ'nin kaplama amaciyla biyokompetible nanofilm üretiminde kullanimini içermektedir. Enzimatik hidroliz (papain, bromelain, cathepsin K, separase, chymotrypsin ve subtilisin) veya enzimatik hidroliz ve disülfit baglarinin nötr sartlarda kirilmasi gerçeklestirilmekte ve çözelti haline getirilen TKZ proteinlerinin 0.1- 10 g/L konsantrasyonlarda çözeltisi elde edilmekte ve çözünür formdaki proteinler ambalaj film olarak kullanilmaktadir. Mevcut teknikte bulunan JPH05253285A numarali ve Eggshell film-collagen artificial membrane baslikli patentte yapay membran olusturulmadan önce YKZ çözünebilir hale getirilmis ve bu amaçla alkali alkol veya disülfit baglarini koparan indirgeyici ajanlar kullanilmistir. Patentte S-S baglarini kirmak için performik asitin de kullanilabilecegini ve bu amaçla hidrojen peroksit (% 30) ve formik asit (% 99) solüsyonlarinin da kullanildigi belirtilmistir. Formik asit solüsyonuna % 01-10 oraninda TKZ eklenmis ve karisim 0-50 °C'de 10 dak- 5 saat bekletilerek S- baglari kirilmistir. Formik asit solüsyonu santrifüjlenerek veya filtre edilerek uzaklastirildiktan sonra çözünebilirligi saglamak amaciyla enzim (pepsin, tripsin, kimotripsin vb) uygulamasina maruz birakilmistir. Enzim uygulamasinda optimum parametreler tespit edilmistir. Mesela pepsin kullanildiginda sicaklik -40 °C ve pH 2-4 olarak rapor edilmistir. Çözünür forma getirilen YKZ proteinleri çözeltisi içerisinde çözündürüldükten sonra uygun kaliplara dökülüp jellesmesi birakilip membran olusturulmustur. Mevcut teknikte bulunan JPH06192443A numarali ve Production of re-formed egg shell membrane baslikli patentte 1 g YKZ 20 mL 5M ß-thiopropiyonik asit çözeltisine eklenip 80°C de 48 saat bekletilip çözünebilir YZK elde edilmis ve filtre edilip çözünmeyen fraksiyonlar uzaklastirildiktan sonra 1.5 mLfiltrat 20x50 cm lik cam kaliba alinip metanol içerisinde termostatik odada basinç altinda kurumaya birakilmis ve ß-thiopropiyonik asitin uzaklastirilmasi saglanmistir. Kuruyan film oda sicakligina alinip metanolün uzaklastirilmasi saglanmistir. Mevcut teknikte bulunan EP3653721Bl numarali ve Method and composition for hydrolyzing eggshell membrane baslikli patentte YKZ farkli konsantrasyonlarda (5-155, sodyum klorid içeren %1 papain solusyonu kullanilmistir. YKZ'nin çözünürlügü için 10 mg YKZ , sodyum hidroksmetansülfinat (HOCHzSOzNa), sodyum metabisulfit (Na25205), ve dithiothreitol (DTT) 1 mL% 0.5-5 SDS içeren solusyonlarda basarili bir sekilde test edilmistir. Son olarak -50mM konsantrasyonu olan çözeltinin pH sini ayarlamak içim buffer eklenmis ve 1.12, ve 14 saat inkübasyona birakilmistir. Hazirlanan çözeltilerden pH si 6.2 olan enzim olarak papain ve bromelain (cysteine protease endopeptidase), denatürasyon ajani olarak SDS ve indirgeme ajani olarak HOCHzSOzNa ve Na25205 içeren çözeltinin 12 saatlik inkübasyonu, ve karisimin ayni indirgeme ajani olarak Na25205 içeren çözeltinin 1 saatlik inkübasyonu sonucunda YKZ'inda %100 çözünürlük elde edilmistir. Benzer sekilde papain veya bromlain SDS ve HOCHzSOzNa içeren ve pH si 6.2 olan karisimin 12 saatlik inkübasyonu yine %100 çözünürlük saglamistir. Ilave olarak ayni çözeltide indirgeme ajani olarak DTT kullanildiginda 1 saatlik inkübasyon sonunda yine %100 çözünürlük elde edilmistir. Mevcut teknikte bulunan U58173174BZ numarali ve Solubilized protein composition obtained from avian eggshell membrane baslikli patentte YKZ farkli süre ve sicakliklarda sodyum hidroksit, potasyum hidroksit ve kalsiyum hidroksit gibi bazik solüsyonlara 50 °C nin üzerine çikarildiktan sonra sicakliga bagli olarak 3 ile 24 saat arasinda bekletilip hidrolizi saglanmis ve karisim sogutulduktan sonra santrifüjlenip supernatant alinmistir. Santrifüjlenen karisimdan çözünmeyen fraksiyonlar uzaklastirilmis ve sivi faz membrandan süzülerek 100 kDa dan daha büyük olan çözünebilir protein karisimi elde edilmistir. Sonuç olarak, mevcut teknikte var olan yukarida belirtilen problemlerin çözümü için, yeni bir ekonomik, kullanisli, yumurta kabugu zari proteinlerinin ayristirma yöntemine duyulan gereksinim ve mevcut çözümlerin yetersizligi ilgili teknik alanda bir gelistirme yapmayi zorunlu kilmistir. BULUSUN AMACI Mevcut bulus yukarida bahsedilen dezavantajlari ortadan kaldirmak ve ilgili teknik alana yeni avantajlar getirmek üzere gelistirilen, yumurta kabugunu zarinda bulunan proteinlerin gida takviyesi olarak kullanilmasi ile ilgilidir. Bulusun en önemli özelligi, proteinlerin çözünebilirligini 60 dak gibi kisa süre içerisinde gerçeklesmis olmasidir. Bulusta YKZ proteinlerinin çözünürlügünü saglamak için farkli yöntemler denenmis olmakta birlikte bu çalisma kapsaminda kullanilan yöntem daha önce rapor edilen yöntemlerden farklidir. Protein içerigi zenginlestirilmis ürünler (meyve suyu vb.) ve soft kapsüller incelendiginde bu ürünlerin bezelye, pirinç, kolajen gibi farkli protein kaynaklari içerdigi görülmüstür. Bezelye, pirinç, kolajen gibi protein kaynaklari ile karsilastirildiginda yumurta kabugu zari proteinleri nadir olarak görülen bazi amino asitleri içermekte oldugundan en iyi protein takviyesi olabilecek kaynaklar arasinda yer almaktadir. Çözünür yumurta kabugu zari proteinleri ile formüle edilen benzer ürünler bulunmamaktadir. Bulusun yapisal ve karakteristik özellikleri ve tüm avantajlari asagida verilen detayli açiklama sayesinde daha net olarak anlasilacaktir ve bu nedenle degerlendirmenin de bu detayli açiklama göz önüne alinarak yapilmasi gerekmektedir. BU LUSUN DETAYLI AÇIKLAMASI Bu detayli açiklamada, yumurta kabugu zari proteinlerinin zardan ayristirma yönteminin tercih edilen yapilanmalari, sadece konunun daha iyi anlasilmasina yönelik olarak ve hiçbir sinirlayici etki olusturmayacak sekilde açiklanmaktadir. Literatürde yer alan çalismalarda yumurta zarinin kabuktan ayrilmasi için kullanilan yöntemlerin çok uzun süre gerektirdigi veya yeterli derecede etkin olmadigi görülmektedir. Benzer sekilde zar proteinlerinin çözünürlügü amaciyla gelistirilen yöntemlerin etkinligi belirtilmemistir. Bu nedenle yeni yöntemlerin gelistirilmesine ihtiyaç duyulmaktadir. Literatür verileri kapsaminda ultrasonikasyon, mikrodalga, su içinde yüzdürme gibi yöntemler test edilmis ve bu yöntemlerin YKZ'ini ayirmada etkili olmadigi görülmüstür. Yumurta kabugunun küçük parçalara ayrilip farkli islemler uygulamasi literatürde tavsiye edilmekle birlikte ufalanmis zarlarin kabuktan ayrilmasi mümkün olmamistir. Bu nedenle bulus kapsaminda yaklasik olarak 3 g temizlenmis ve oda sicakliginda kurutulmus olan kuru yumurta kabuklari 50, 100, ve 150 mM EDTA (etilen diamin tetra asit) ile 30-150 dak arasinda muamele edilmistir. Bütün uygulamalarda yumurta oraninda yumurta kabugu/su kullanilmistir. Bütün örneklerde pH, iletkenlik, bulaniklik, optik yogunluk ve Kjeldahl protein tayin yöntemi ölçümleri yapilmistir. En yüksek 0.185 HCL ile 1/30 oraninda tespit edilmistir. Fakat, bu protein çözünür olmadigi için spektrofotometrik olarak tespit edilememis dolayisiyla Kjeldahl yakma yöntemiyle tespit edilebilmistir. Ikinci asamada örneklerin çözünürlügü için Kjejdahl yöntemiyle protein orani yüksek çikan örnekler 50mM EDTA (, %1.25 asetik asit ( ve su ile hazirlanan kontrol örnekleri içinde 100 uL 2-merkaptoetanol (ayni zamanda ß-merkaptoetanol, BME, 2BME, 2-ME veya ß-met, HOCHzCHzSH formüllü bir tiyol bilesigidir. Disülfüt baglarinin indirgenmesinde biyolojik anti-oksidant görevi yapar ve bunu hidroksil radikallerini birbirlerinden ayirarak basarir) bulunan pH % 0.1, 0.5 ve 1 Triton X-100 (Triton X-100, protein veya organelleri çikarmak için hücreleri parçalamak veya canli hücrelerin zarlarini geçirgen hale getirmek için yaygin olarak kullanilmaktadir) ve diger solüsyon için ayni oranlarda SDS olacak sekilde lizis solüsyonu ile muamele edilerek 1 gece boyunca buzdolabi sicakliginda bekletilmistir. Ardindan süpernatant kisimlardan farkli eppendorf tüpüne aktarim yapilarak ve aseton eklenerek 1 gece boyunca buzdolabi sicakligindan bekletilmis ve böylece proteinlerin çökmesi saglanmistir. 10000 rpm'de 30 dak boyunca santrifüj uygulandiktan sonra protein tortusu temiz bir tüpe alinarak ultra saf suyun içinde çözünmesi saglanmis (50 uL) ve elde edilen soluble (çözünür) proteinler Lowry reaktifleri ve 150 uL %1 SÜS/0.1 N NaOH eklendikten sonra 0.1 mL Folin reaktifi eklenerek farkli sürelerde (0, 30, 60, yapilmistir. YKZ proteinlerinin çözünürlügü üzerinde SDS, Triton X-100 ve bekletme + %1 SDS uygulamasinda 60 ve 240 dak ve EDTA+%1 SDS uygulamasinda 60 dak bekleme sonunda elde edilmistir. Solüsyonda bulunan kimyasallar methanol ve ardindan su ile yikanarak uzaklastirilmis ve elde edilen protein fraksiyonu soft kapsül ve protein içerigi zenginlestirilmis meyve suyu formülasyonunda kullanilmistir. YKZ proteinlerinin çözünürlügünü saglamak için farkli yöntemler denenmis olmakla birlikte; çalisma kapsaminda kullanilan yöntem, daha önce rapor edilen yöntemlerden farklidir. Ayrica, çalisma kapsaminda proteinlerin çözünebilirligi 60 dak gibi kisa süre içerisinde gerçeklesmistir. Söz konusu bulus: - kuru yumurta kabuklarinin EDTA, HCL ve asetik asit ile 30-150 dak arasinda muamele edildikten sonra kurutularak toz formuna getirilmesi, - % 0.1, 0.5 ve 1 SDS veya ayni oranlarda Triton X-100 içeren ve içlerinde 2- merkaptoetanol bulunan lizis solüsyonu ile 10-14 saat 4°C'de muamele edilmesi, - süpernatant kisimlardan farkli eppendorf tüpüne aktarim yapildiktan sonra aseton eklenerek 10-14 saat 4°C'de bekletilmesi, - santrifüj uygulandiktan sonra protein tortusunun temiz bir tüpe alinarak ultra saf suyun içinde çözünmesinin saglanmasi, - %1 SÜS/0.1 N NaOH eklendikten sonra Folin reaktifi eklenerek farkli sürelerde okumalarinin yapilmasi, - methanol ve ultra saf su ile yikanarak kimyasallarin uzaklastirilmasi Soft kapsül eldesinde dolgu maddesi seklinde ticari olarak satilan pektin ve/veya laktoz (Starch 1500 ve Fast-Flo Laktoz); glidant olarak misir nisastasi, lübrikant olarak Mg- stearat; dagitici olarak AcDiSol ve yüzey etkin madde olarak Na-lauril sülfat ve parlatici olarak jelatin ve gliserin kullanilmistir. Farkli yas ve cinsiyet gruplarina göre renk ve aroma maddeleri eklenen soft kapsüllere antimikrobiyel madde olarak metil paraben eklenmistir. Protein içerigi artirilmis meyve suyu üretiminde seftali ve kayisi nektari gibi meyve nektarlari elma, üzüm gibi meyve sulari kullanilmis ve yüzey etkin madde olarak Na- lauril sülfat eklenmistir. %15-20 oraninda eklenen YKZ proteinleri en az %15 protein orani olacak sekilde formülasyona eklenmistir. Protein içerigi zenginlestirilmis ürünler (meyve suyu vb.) ve soft kapsüller incelendiginde bu ürünlerin bezelye, pirinç, ve kolajen gibi farkli protein kaynaklari içerdigi görülmüstür. Çözünür yumurta kabugu zari proteinleri ile formüle edilen benzer ürünler bulunmamaktadir. Bulus kapsaminda elde edilen proteinin liyofilize edilerek, enkapsüle edilerek farkli ürünlerde (krem, sampuan vb.) ve özel amaçli besinsel takviyelerde protein kaynagi olarak farkli formülasyonlarda kullanilmasi mümkündür. Bulusta YKZ proteinlerinin ekstraksiyonu veya çözünebilirligi asamalari modifiye edilebilmekle birlikte yine farkli formülasyonlarda kullanimi söz konusu olabilmektedir. Bu basvurunun koruma kapsami istemler kisminda belirlenmis olup yukarida kesinlikle örnekleme amaciyla anlatilanlarla sinirli tutulamaz, teknikte uzman bir kisinin bulusta ortaya konan yeniligi, benzer yapilanmalari kullanarak ortaya koyabilecegi ve/veya bu yapilanmayi ilgili teknikte kullanilan benzer amaçli diger alanlara da uygulayabilecegi açiktir. Dolayisiyla böyle yapilanmalarin yenilik ve özellikle teknigin bilinen durumunun asilmasi kriterinden yoksun olacagi da asikârdir. TR TR DESCRIPTION USE OF EGG SHELL MEMBRANE PROTEINS AS FOOD SUPPLEMENT TECHNICAL FIELD The invention relates to the use of the proteins found in the egg shell membrane, which is removed as waste when peeling the egg, as food and nutritional supplements. In particular, the invention; After treating dry egg shells with EDTA, HCL and acetic acid for 30-150 minutes, drying them and turning them into powder form, lysis solution containing 0.1, 0.5 and 1% SDS or Triton hour at 4°C, transferring the supernatant parts to a different Eppendorf tube and adding acetone and keeping it at 4°C for 10-14 hours, after centrifugation, transferring the protein residue into a clean tube and ensuring that it dissolves in ultrapure water, 1% SUNS/ Using the proteins in the eggshell membrane as a food supplement involves the following steps: adding Folin reagent after adding 0.1 N NaOH, keeping it at 37°C for different periods of time between 0-1440 minutes, taking readings at 660 nm on the spectrophotometer, removing chemicals by washing with methanol and then ultrapure water. It is related to. KNOWN STATE OF THE TECHNOLOGY One of the most used products in the food industry is egg. Egg baking, baking, pastry, ready meals etc. It is used quite widely in business lines. The production and consumption of eggs is increasing every year. After the inner part of the egg is removed, the egg shell and the inner membrane attached to the shell deteriorate quickly and emit a bad odor. For this reason, eggshells are generally classified as a waste that spoils quickly and emits a bad odor and cannot be evaluated. Although egg is a very good nutrient due to the proteins it contains in its outer shell membrane structure, it cannot be evaluated. For this reason, it is of great importance to obtain the proteins found in the eggshell membrane and convert them into different products and thus to evaluate the waste by preventing pollution to the environment. Although the egg itself is a protein-rich food, its outer shell membrane, which cannot be evaluated in any way, also has the potential to be an important food source, thanks to the proteins it contains. The egg membrane is divided into 3 parts: outer membrane under the shell (shell membrane), inner membrane under the shell (egg membrane) and limiting membrane. These structures have become a whole by connecting to each other with the help of membrane fibers. Membrane fibers are the main component that gives the membrane its protein structure. The eggshell membrane consists of approximately 60% membrane fibers and approximately 80-85% protein. 10% of these proteins consist of collagen, and 70-75% consist of proteins and glycoproteins containing lysine-derived cross-links. The number of proteins in the shell and subshell membranes is more than 500. There are 62 proteins in the subshell membranes alone. Proteins such as lysozyme, ovotransferrin, ovalbumin, ovocalyxin-36 and keratin can be given as examples of these proteins. Some of the most abundant proteins in membranes are proline, glutamic acid and glycine proteins. At the same time, the outer membranes contain type I and the inner membranes contain type I and type V collagen protein. Collagen type X is found in both the inner and outer membranes. Collagen is the basic structural protein found in connective tissues. Type I collagen is the most abundant and durable type of collagen in the human body. It is mainly found in bone tissue. Type V and However, in order for YKZ to be used as a protein source, two basic problems must be solved. Since the egg membrane is tightly attached to the outer shell, separating the membrane from the shell is the first problem to be solved, while the use of the separated membrane as a digestible protein source is the second problem to be solved. Since YKZ contains a large amount of disulfide bonds, it has a very stable structure and disulfide bonds prevent the solubility of the membrane in aqueous systems and therefore cannot be used as a protein source. Today, the complex structure of the egg shell due to its strong interactions with the organic membrane matrix makes it difficult to separate the membrane from the shell. Different methods have been tested to separate YKZ from the shell. In the application numbered U56176376, which is known in the current technique, it is aimed to reduce the size (0.5-0.5 mm) by shredding the shells taken into the tank developed to separate the egg shells from the membrane and to separate the membrane from the shell by keeping them in a solvent - preferably water. This method is not preferred because the membranes are very fragmented, water consumption is high, and extra energy is required to dry the wetted membranes. Although methods such as using acid, alkali and acid-alkali combinations and boiling with acid have been tried in the current technique, these methods are not used in practice because they are expensive and require a lot of time. Separation of the egg shell from the membrane in aqueous systems by applying cavitation is also among the reported studies. It has been reported that microwave energy applied at different frequencies is effective in breaking the bonds between the egg shell and the membrane. In the current technique, manual separation is used to separate the membrane from the shell, and the use of dilute acid (acetic acid, hydrochloric acid, EDTA, sulfuric acid) to dissolve the calcium carbonate layer in the shell. Separation of the membrane and shell in air flow using pressurized water containing air and separation of the membrane by means of a drill placed in series in an aqueous environment heated by steam are among the experiments carried out. In a study in the current technique, egg shell membranes were separated from the shells after various processes and as a result of various processes, 100 g with the application of frequency with an ultrasonic reactor. 73 g of protein was obtained in. Chen-Lee and Huang (2019) in their study titled "Nanoparticles for the treatment of defective intestinal epithelial cells from soluble eggshell membrane protein-loaded chitosan/fucoi", eggshell membranes that have gone through various processes were separated and kept in a series of mixtures and then enzyme ( Protein extraction was carried out by applying protease) and then centrifugation. In their study, I no et al (2006) carried out protein extraction by applying centrifugation using a different enzyme (pepsin) to the egg shell membrane, which was separated from the egg shell by going through various processes. In a study within the current technique, after treatment with 3-mercaptopropionic acid in the presence of acetic acid in order to dissolve YKZ proteins, the pH of the mixture was adjusted to 0 and the structure and morphology of the egg membrane was examined. After TYZ was treated with a solution containing 3-mercaptopropionic acid and acetic acid, the mixture was brought to 90 °C and treated for 12 hours to ensure the solubility of the membrane. The pH of the solution was adjusted using NaOH and the membrane was dried after washing with methanol. In a study in which different lysis solutions were tested for the extraction of YKZ proteins in the current technique, the highest extraction was achieved with 5 mM TRIS-containing 0.1% 2-mercaptoethanol and 1% SDS and a pH of 8.8. It was obtained after the application of HCL solution at 37°C for 21 days. After application, the membranes were washed and dried. In the study where acetic acid, EDTA and phosphate solution were tried to separate YKZ from the shell in the current technique, a modified EDTA method was recommended to remove calcium from the shell (decalcification). In this method, 50 g of eggshell powder mixed with 250 mL TRIS-HCL (50 mM) was transferred into 4 M guanidine hydrochloride and kept for 72 hours. The precipitate was mixed with the centrifuged mixture for 40 hours, and the supernatant obtained from the centrifuged mixture was lyophilized after being dialyzed. In the current state of the art patent titled Egg shell membrane protein solubilized product and method for producing the same, composition containing the eggshell membrane protein solubilized product, composition for hair cosmetics, and hair treatment method using the composition for hair cosmetics, egg membrane proteins (YZP) are mentioned. To ensure its solubility, the pH was kept between 6-12 and the enzyme application was continued at temperatures between 30-60C for 24 hours and the solution was dried. In this method, it is not possible to obtain HZP with high molecular weight, and the enzyme that acts as a catalyst is also quite expensive. The patent titled substance and treating method aims to use egg membrane proteins (YZP) for cosmetic purposes in hair care. For this purpose, YZP has been added to keratin used in hair care. For this purpose, soluble YZP was kept at °C for 5 hours. The resulting mixture was filtered through filter paper and neutralized, then the salt was removed and 26 g of soluble YKP was obtained. The patent titled solubilization method using enzymes includes the use of a combination of proteolytic enzymes and reducing agents. For this purpose, egg membrane proteolytic enzymes such as chymotrypsin, subtilisin, papain, bacillolysin, stem bromelain, leucylaminopeptidase, pepsin and trypsin (commercially available biosoak, Proleather FG-F, Papain W40, Protease N, Bromelain F, Ummamizyme G, Samoaase Y100, ProteAX , enzymes such as Protease S, Sumiteam LP500, Deskin C, Protin NY10, Protin PC10, Sumiteam MP) and reducing agents (sulfite, bisulfite, L-cysteine, N-acetyl-L-cysteine, 2-mercaptoethanol, glutathione, and DTT etc. ) includes the use of For the reaction, pH between 45-95 and preferably neutral pH were tested, and the reaction, which lasted 10 minutes-24 hours, was generally carried out between 30-80°C and preferably between 40-70°C. Mixing and shaking processes were applied to increase the efficiency of the reaction. In the mixture, 0.1% enzyme (w/v) was added to 1.0% egg membrane and the reaction continued for 72 hours at 60°C, but no visual solubility was observed. It is associated with the current state of the art patent numbered U510526423BZ and Eggshell membrane solubilization (August 2010) and involves the use of a combination of reducing agents and proteases to ensure TKZ solubility. The enzyme mixture is added to the reaction solution containing 10% (w/v) YKZ (pH 7.4) to ensure solubility. PROTEINASE K (Roche), BIOSORK, PROLEATHER FG-F or PAPAIN W40 and PROTEINASE K are added to the enzyme mixture to reach a final concentration of 0.01% (w/v). ) was ensured and other enzyme mixtures were added and the final concentration was adjusted to 0.1% (w/v). The resulting mixture was stirred and incubated at 50° C for 12 hours. After n-butylamine was added to the mixture and the final concentration was adjusted to 10 mM, the final concentration was adjusted to 10 mM at 37° C. It was incubated for 60 minutes at C. In the second stage, TKZ was treated with a reducing agent and treated with protease. For this purpose, 5 mL of 100 mM PBS buffer (pH 7.4) was added to 500 mg TKZ and incubated for 1 hour at 37° C. TKZ was removed from the solution and washed 5 times with 50 mL of distilled water to remove sodium sulfite. Then, the distilled water was removed from the medium and 5 mL of 0.1% (w/v) BIOSORK ( solution was added and incubated at 37° C. 200 uL samples were taken at different incubation times (0, 1, 2, 3, 4, and 6 hours) and lysyl oxidase activity was measured. For this purpose, 20 uL of 100 mM n-butylamine was added to the samples to make the final concentration mM and the samples were incubated at 37°C for 45 minutes. Then, 220 uL Nash reagent was added and incubated at 50° C for 30 min, and measurements were made at 388 nm. In the patent titled membrane protein soluble product, and composition containing eggshell membrane protein soluble product, hydrolysis continued after the YKZ pH was adjusted to 11 and alkaline protease was added. After adding the second enzyme in addition to the alkaline protease, the pH of the solution was adjusted to 9 and hydrolysis continued and the solution was heated and YKZ was brought into soluble form and the alkaline protease was inactivated. In the patent titled shell membrane protein solubilized product, and cosmetic composition, pharmaceutical composition and functional food Which contains egg-shell membrane protein solubilized product, the pH of the TKZ suspension solution was adjusted between 1-3.5 and the suspension temperature was heated within five minutes and taken into acid solution. The calcium components in the YKZ are dissolved and then the liquid phase is removed. In the process, the solubility of YKZ is achieved by heating at high temperatures and forming aggregates. YKZ proteins are positively charged in the acidic region below the isoleelectric point, and when heated rapidly, soluble proteins repel each other due to the positive charge and remain in soluble form without forming aggregates. On the other hand, as we move from the isoelectric point to the alkaline region, the calcium ions in the YKZ neutralize the positive charges and sufficient resolution cannot be achieved. The patent titled Composing eggshell membrane hydrolysate, and use thereof covers the use of YKZ brought into soluble form in the production of biocompatible nanofilm for coating purposes. Enzymatic hydrolysis (papain, bromelain, cathepsin K, separase, chymotrypsin and subtilisin) or enzymatic hydrolysis and the breaking of disulfide bonds under neutral conditions are carried out and the solution of the TKZ proteins in solution is obtained at concentrations of 0.1- 10 g/L and the proteins in soluble form are used as packaging films. . In the current state of the art patent numbered JPH05253285A and titled Eggshell film-collagen artificial membrane, YKZ was made soluble before creating the artificial membrane, and for this purpose alkaline alcohol or reducing agents that break the disulfide bonds were used. It is stated in the patent that performic acid can also be used to break S-S bonds and that hydrogen peroxide (30%) and formic acid (99%) solutions are also used for this purpose. 01-10% TKZ was added to the formic acid solution and the S-bonds were broken by keeping the mixture at 0-50 °C for 10 minutes - 5 hours. After the formic acid solution was removed by centrifugation or filtering, it was subjected to enzyme (pepsin, trypsin, chymotrypsin, etc.) application to ensure solubility. Optimum parameters were determined for enzyme application. For example, when pepsin was used, the temperature was reported as -40 °C and pH 2-4. After the YKZ proteins brought to soluble form were dissolved in the solution, they were poured into appropriate molds and allowed to gel, and a membrane was formed. In the current state of the art patent numbered JPH06192443A and titled Production of reformed egg shell membrane, 1 g of YKZ was added to 20 mL of 5M ß-thiopropionic acid solution and kept at 80°C for 48 hours to obtain soluble YZK. After filtering and removing the insoluble fractions, 1.5 mL of the filtrate was 20x50 cm. It was taken into a glass mold and left to dry in methanol under pressure in a thermostatic room, and ß-thiopropionic acid was removed. The dried film was placed at room temperature and methanol was removed. In the current state of the art patent numbered EP3653721Bl and titled Method and composition for hydrolyzing eggshell membrane, YKZ 1% papain solution containing sodium chloride in different concentrations (5-155) was used. The solubility of YKZ has been successfully tested in solutions containing 10 mg of YKZ, sodium hydroxmethanesulfinate (HOCH2SO2Na), sodium metabisulfite (Na252O5), and dithiothreitol (DTT) in 1 mL of 0.5-5% SDS. Finally, buffer was added to adjust the pH of the solution with a concentration of -50mM and incubation was allowed for 1, 12, and 14 hours. As a result of the 12-hour incubation of the solution containing papain and bromelain (cysteine protease endopeptidase) as enzymes with a pH of 6.2, SDS as the denaturation agent, and HOCH2SO205 and Na25205 as reducing agents, and the 1-hour incubation of the same solution containing Na25205 as the reducing agent of the mixture, YKZ was obtained from the prepared solutions. 100% resolution was achieved. Similarly, 12-hour incubation of the mixture containing papain or bromolain SDS and HOCH2SO2Na with a pH of 6.2 again provided 100% solubility. Additionally, when DTT was used as a reducing agent in the same solution, 100% solubility was again achieved after 1 hour of incubation. In the current state-of-the-art patent numbered U58173174BZ and titled Solubilized protein composition obtained from avian eggshell membrane, YKZ was placed in basic solutions such as sodium hydroxide, potassium hydroxide and calcium hydroxide at different times and temperatures, above 50 °C, and then kept for 3 to 24 hours depending on the temperature and hydrolysis was achieved. and after the mixture was cooled, it was centrifuged and the supernatant was taken. Insoluble fractions were removed from the centrifuged mixture and the liquid phase was filtered through the membrane to obtain a soluble protein mixture larger than 100 kDa. As a result, in order to solve the above-mentioned problems existing in the current technique, the need for a new economical, useful method of separation of eggshell membrane proteins and the inadequacy of existing solutions have made it necessary to make a development in the relevant technical field. PURPOSE OF THE INVENTION The present invention is about the use of proteins found in the eggshell membrane as a food supplement, which was developed to eliminate the above-mentioned disadvantages and bring new advantages to the relevant technical field. The most important feature of the invention is that the solubility of proteins was achieved in a short time of 60 minutes. Although different methods have been tried to ensure the solubility of YKZ proteins in the invention, the method used within the scope of this study is different from previously reported methods. When protein-enriched products (fruit juice, etc.) and soft capsules were examined, it was seen that these products contained different protein sources such as peas, rice and collagen. Compared to protein sources such as peas, rice and collagen, eggshell membrane proteins are among the sources that can be the best protein supplement as they contain some rarely seen amino acids. There are no similar products formulated with soluble eggshell membrane proteins. The structural and characteristic features and all the advantages of the invention will be more clearly understood thanks to the detailed explanation given below, and therefore the evaluation should be made taking this detailed explanation into consideration. DETAILED DESCRIPTION OF THE INVENTION In this detailed description, preferred embodiments of the method of separation of eggshell membrane proteins from the membrane are explained only for a better understanding of the subject and without creating any limiting effect. Studies in the literature show that the methods used to separate the egg membrane from the shell require a very long time or are not effective enough. Similarly, the effectiveness of the methods developed for the solubility of membrane proteins has not been stated. Therefore, there is a need to develop new methods. Within the scope of literature data, methods such as ultrasonication, microwave, and water flotation have been tested and it has been observed that these methods are not effective in distinguishing YKZ. Although it is recommended in the literature to tear the egg shell into small pieces and apply different processes, it has not been possible to separate the crumbled membranes from the shell. Therefore, within the scope of the invention, approximately 3 g of cleaned and dried egg shells at room temperature were treated with 50, 100, and 150 mM EDTA (ethylene diamine tetra acid) for 30-150 minutes. In all applications, egg shell to water ratio was used. pH, conductivity, turbidity, optical density and Kjeldahl protein determination method were measured in all samples. The highest was detected at a rate of 1/30 with 0.185 HCL. However, since this protein was not soluble, it could not be detected spectrophotometrically, so it could be detected by the Kjeldahl digestion method. In the second stage, for the solubility of the samples, the samples with high protein content by the Kjejdahl method were mixed with 100 uL 2-mercaptoethanol (also known as ß-mercaptoethanol, BME, 2BME, 2-ME or ß-) in control samples prepared with 50mM EDTA (, 1.25% acetic acid () and water. met is a thiol compound with the formula HOCHzCHzSH. It acts as a biological anti-oxidant in the reduction of disulfide bonds and achieves this by separating hydroxyl radicals from each other) found at pH 0.1, 0.5 and 1% Triton X-100 (Triton X-100 is used to lyse cells to remove proteins or organelles or widely used to make the membranes of living cells permeable) and SDS in the same proportions for the other solution, and kept at refrigerator temperature for 1 night. Then, the supernatant was transferred to a different Eppendorf tube and acetone was added and kept at refrigerator temperature for 1 night. After centrifugation at 10000 rpm for 30 minutes, the protein residue was transferred to a clean tube and dissolved in ultrapure water (50 uL) and the resulting soluble proteins were mixed with Lowry reagents and 150 uL of 1% SU/0.1. After adding N NaOH, 0.1 mL of Folin reagent was added and the test was carried out for different periods of time (0, 30, 60). The solubility of YKZ proteins was obtained after 60 and 240 min in SDS, Triton The chemicals in the solution were removed by washing with methanol and then with water, and the resulting protein fraction was used in the formulation of soft capsules and fruit juice with enriched protein content. Although different methods have been tried to ensure the solubility of YKZ proteins; The method used within the scope of the study is different from previously reported methods. Additionally, within the scope of the study, the solubility of proteins was achieved in a short time of 60 minutes. The invention in question: - drying egg shells into powder form after being treated with EDTA, HCL and acetic acid for 30-150 minutes, - containing 0.1, 0.5 and 1% SDS or Triton X-100 in the same proportions and containing 2-mercaptoethanol. treatment with lysis solution for 10-14 hours at 4°C, - after transferring the supernatant parts to a different Eppendorf tube, adding acetone and keeping it at 4°C for 10-14 hours, - after applying centrifugation, transferring the protein residue to a clean tube and using ultrapure water. - Ensuring that it dissolves in water, - Adding Folin reagent after adding 1% SU/0.1 N NaOH and taking readings at different times, - Removing chemicals by washing with methanol and ultrapure water. Pectin and/or lactose (Starch 1500 and Starch 1500 and Fast-Flo Lactose); corn starch as glidant, Mg-stearate as lubricant; AcDiSol was used as a dispersant, Na-lauryl sulfate as a surfactant, and gelatin and glycerin were used as brighteners. Methyl paraben has been added as an antimicrobial agent to the soft capsules, to which color and flavoring substances are added according to different age and gender groups. In the production of fruit juice with increased protein content, fruit nectars such as peach and apricot nectars, apple and grape juices were used and Nalauril sulfate was added as a surfactant. YKZ proteins added at a rate of 15-20% were added to the formulation at a rate of at least 15% protein. When protein-enriched products (fruit juice, etc.) and soft capsules were examined, it was seen that these products contained different protein sources such as peas, rice, and collagen. There are no similar products formulated with soluble eggshell membrane proteins. It is possible to use the protein obtained within the scope of the invention in different formulations by lyophilizing and encapsulating it as a protein source in different products (cream, shampoo, etc.) and special-purpose nutritional supplements. In the invention, although the extraction or solubility stages of YKZ proteins can be modified, it is possible to use them in different formulations. The scope of protection of this application is determined in the claims section and cannot be limited to those explained above for exemplary purposes. It is clear that a person skilled in the art can demonstrate the innovation put forward in the invention by using similar structures and/or apply this structure to other areas with similar purposes used in the relevant technique. Therefore, it is obvious that such structures will lack the criteria of innovation and especially exceeding the known state of the technique.TR TR

Claims

1- The invention is related to the use of the proteins found in the egg shell membrane, which is removed as waste after peeling the egg, as a food supplement, and its feature is; Treating dry egg shells with EDTA for 30-150 minutes, treating them with lysis solution containing mercaptoethanol for 10-14 hours at 4°C, transferring the supernatant parts to a different Eppendorf tube and adding acetone and keeping it at 4°C for 10-14 hours. It includes the process steps of taking the protein residue into a clean tube after centrifugation and ensuring that it dissolves in ultrapure water, keeping it at 37°C for periods between 0-1440 minutes, and removing the chemicals by washing it with methanol and ultrapure water in the spectrophotometer.