TR2021022266A2 - INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA - Google Patents

INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA

Info

Publication number
TR2021022266A2
TR2021022266A2 TR2021/022266A TR2021022266A TR2021022266A2 TR 2021022266 A2 TR2021022266 A2 TR 2021022266A2 TR 2021/022266 A TR2021/022266 A TR 2021/022266A TR 2021022266 A TR2021022266 A TR 2021022266A TR 2021022266 A2 TR2021022266 A2 TR 2021022266A2
Authority
TR
Turkey
Prior art keywords
fermentation
ultrafiltration
hyaluronic acid
feature
microfiltration
Prior art date
Application number
TR2021/022266A
Other languages
Turkish (tr)
Inventor
Turhan İrfan
Özcan Ali̇
Yatmaz Ercan
Original Assignee
Irfan Turhan
Turhan İrfan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Irfan Turhan, Turhan İrfan filed Critical Irfan Turhan
Priority to TR2021/022266A priority Critical patent/TR2021022266A2/en
Publication of TR2021022266A2 publication Critical patent/TR2021022266A2/en
Priority to PCT/TR2022/051663 priority patent/WO2023129082A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14Ultrafiltration; Microfiltration
    • B01D61/149Multistep processes comprising different kinds of membrane processes selected from ultrafiltration or microfiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D69/00Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/02Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/04Specific process operations in the feed stream; Feed pretreatment
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/06Specific process operations in the permeate stream
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/12Addition of chemical agents
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/25Recirculation, recycling or bypass, e.g. recirculation of concentrate into the feed
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/26Further operations combined with membrane separation processes
    • B01D2311/2688Biological processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2325/00Details relating to properties of membranes
    • B01D2325/20Specific permeability or cut-off range

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Water Supply & Treatment (AREA)
  • Sustainable Development (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Bu buluş, Hyaluronik asit üretim fermentasyonlarında oluşan ürünün fermentasyonu durdurmadan ortamdan geri kazanılması ve kısmi saflaştırılması ile üretim veriminin artırılması ve ortamdaki mikroorganizmaların tekrar kullanılabilirliği ile ilgilidir.This invention relates to the recovery and partial purification of the product formed in hyaluronic acid production fermentations from the environment without stopping the fermentation, increasing the production efficiency and the reusability of microorganisms in the environment.

Description

TARIFNAME FERMENTASYON YOLUYLA ÜRETILEN HYALURONIK ASIT'IN FERMENTASYON ORTAMINDAN ÇEVRIMIÇI GERI KAZANIMI iLE ÜRETIM VERIMININ ARTIRILMASI TEKNIK ALAN Bu bulus, Hyaluronik asit üretim fermentasyonlarinda olusan ürünün fermentasyonu durdurmadan ortamdan geri kazanilmasi ve kismi saflastirilmasi ile üretim veriminin artirilmasi ve ortamdaki mikroorganizmalarin tekrar kullanilabilirligi ile ilgilidir. BULUSUN ALT YAPISI Hyaluronik asit üretimi sirasinda karsilasilan viskozite artisi, hücre gelisiminin sinirlanmasi, hücrelerin üretim ve gelisim metabolizmalarinin yarismasi ve yan ürün olusmasi gibi problemler üretim verimini ciddi sekilde düsürmektedir. Ayrica her fermentasyon islemi sterilizasyon, ön kültür gelisimi ve besiyeri pH ayarlamasi gibi islemler sonucunda gerçeklestirilmektedir. Bu durumda üretim maliyetleri yükselmekte ve bahsedilen sorunlar nedeniyle verim önemli ölçüde azalmaktadir. Mevcut bulus ortamdan hyaluronik asitin uzaklastirilmasini saglayarak olasi kisitlamalarin önüne geçmekte ve bu sayede verimi artirmaktadir. Bununla birlikte çevrimiçi geri kazanim sayesinde farkli dilüsyon oranlarinda sürekli bir üretim prosesi olusturulmakta ve fermentasyon öncesi maliyetler en aza indirilmektedir. Hyaluronik asit çok genis kullanim alani olan hyaluronik asit 2019 yilinda küresel pazarda 9.1 milyar Amerikan Dolari hacminde paya sahip olmustur ve ekonomik yönündedir. Hyaluronik asit molekül agirligina ve saflik derecesine bagli olarak 4.000 ila 17.000 Amerikan Dolari arasinda fiyatlara sahip olabilmektedir. Onlarca kullanim alanina sahip olmakla birlikte Uluslararasi Estetik Plastik Cerrahi Dernegi (ISAPS) verilerine göre 2019 yilinda cerrahi olmayan uygulamalar arasinda 4.315.859 uygulama ile en büyük ikinci paya sahip olmustur. Kozmetik, gida, tip ve diger alanlar hesaba katilmaksizin yalnizca estetik uygulamalar düsünüldügünde bile global ölçekte ne denli genis bir pazari oldugu görülmektedir. asit uygulamasi yapilmistir. Bu deger ülke çapinda yapilan cerrahi olmayan uygulamalarin %35'ini olusturmakta ve dünyada en çok hyaluronik asit uygulamasi yapilan ülkeler arasinda Türkiye'nin 7. Sirada yer almasini saglamaktadir. Görüldügü üzere dünyada ve ülkemizde ciddi miktarlarda hyaluronik asit kullanimi gerçeklesmektedir. Ancak neredeyse 10 milyar Amerikan Dolar'lik hacme sahip piyasada ülkemiz hiçbir sekilde üretici konumunda yer almamaktadir. Hali hazirda ülkemizde kullanilan bütün hyaluronik asit ürünleri ya ithal edilmekte ya da saf hyaluronik asit alinip ülkemizde çesitli formlarda hazirlanarak satisa sunulmaktadir. Bu denli katma degeri yüksek bir ürünün ülkemizde üretilmesi ve mevcut bulusun patent altina alinmasinin ülkemizin bu pazarda güçlü bir sekilde yer almasina yapacagi katki bulusun ortaya çikarilmasinda olmustur. Bununla birlikte mevcut üretimlerde karsilasilan sorunlarin giderilmesi ve verimin yükseltilerek farkli dilüsyon oranlarinda sürekli sistemlerde hyaluronik asit üretilebilmesini saglamak adina bulus gelistirilmistir. Fermentasyonla hyaluronik asit üretimi sirasinda birçok sorunla karsilasilmakta ve üretim sinirlanmaktadir. Bunlar arasinda en önemlileri olarak; fermentasyon sirasinda artan hyaluronik asit konsantrasyonu nedeniyle viskozite artisi, bunun sonucunda yetersiz karistirma ve düsük oksijen transferi nedeniyle hücre gelisiminin sinirlanmasi, fermentasyonun ilerleyen süreçlerinde hücre gelisimi ve hyaluronik asit üretiminin öncül bilesikler için yarismasi, glikolitik metabolik yola gidis, olusan yan ürün (laktik asit) sebebiyle üretimin durmasi ve düsük molekül agirlikli hyaluronik asit üretimi gösterilebilir. Bu problemleri asmak adina söz konusu bulus gelistirilmistir. Çevrimiçi geri kazanim yöntemiyle, fermentasyon sirasinda üretilen hyaluronik asit'in üretimle es zamanli olarak fermentasyon ortamindan ayrilmasi saglanacaktir. Yine ayni ayirma asamasinda fermentasyon ortamini etkileyen ikincil metabolitler de uzaklastirilmis olacaktir. Nihayetinde ise sürekli bir sistemle üretiminin artirilmasi ve üretilen ürünün çevrim içi (on-line) olarak ortamdan uzaklastirilmasi hedeflenmektedir. Fermentasyon ortamindan çevrimiçi olarak sürekli sekilde geri kazanilan hyaluronik asit'in düsük, orta ve yüksek molekül agirliklarina sahip olacak sekilde siniflandirilmasi saglanacaktir. Böylelikle farkli molekül agirliklarindaki (düsük, orta, yüksek) hyaluronik asit birçok kullanim alanlarina uygulanabilen yüksek saflikta bir ürün haline gelecektir. Bulus hyaluronik asit üretiminde ortamdan ilgili bilesenin fermentasyon durdurulmaksizin uzaklastirilmasini saglayan bir sistemi tarif etmektedir. Biyoreaktör ve membran sistemlerinin entegre hale getirilmesiyle fermentasyon ortaminda olusabilecek viskozite artisi, ikincil metabolit olusumu, oksijen transfer hizi düsüsü, düsük molekül agirligi gibi problemlerin asilmasi saglanmis olacak ve sürekli sistemle farkli dilüsyon oranlarinda maliyetlerin azaltilarak daha yüksek miktarda ve tek düze ürün üretimi gerçeklestirilebilecektir. Söz konusu avantajlar farkli amaçlarla kullanilan filtrasyon sisteminin biyoreaktöre entegrasyonu ve sistemin durdurulmadan üretim ve saflastirmanin es zamanli gerçeklestirilmesi yönüyle bulus hyaluronik asit üretiminde avantaj saglamaktadir. Patent basvuru numarasi: CN205164514U Patent basligi: Hyaluronik asit üretiminde membran filtrasyon ekipmanlari kullanimi Faydali model, hyaluronik asidin verimli ve hizli bir sekilde ekstrakte edilebilmesi için bir membran filtrasyon cihazinin tasarlanmasini açiklamaktadir. Belirtilen faydali model hyaluronik asit'in saflastirilmasi için mikrofiltrasyon ve ultrafiltrasyon islemlerini içeren bir tasarimi içermektedir. Mevcut sistem bulus içinde yer alan kismi saflastirma basamagi ile benzesmekle birlikte; sundugumuz bulusta bu sistemlerin biyoreaktöre entegre sekilde olmasi ve saflastirma isleminin fermentasyon sirasinda gerçeklesmesi ile ilgili faydali modelden fa rklilasma ktadir. Patent basligi: Yüksek Moleküler Agirlikli Hyaluronik Asitin Saflastirilmasi Için Verimli Proses Bu bulus, hyaluronik asit ve tuzunu üretmek ve saflastirmak için gelistirilmis tekniklerle ilgilidir. Mevcut bulus ayni sekilde, biyomedikal uygulamalara sahip olan hyaluronik asit ve tuzunun üretilmesi, saflastirilmasi ve prosesin optimizasyonu ile ilgilidir. Ilgili bulus, hyaluronik asit'in çöktürme ve diafiltrasyon islemleri ile besiyeri sivisindan saflastirilmasini tariflemektedir. Mevcut bulus etanol, silika jel ve aktif karbon gibi kimyasallari kullanarak safsizliklari uzaklastirmakta ve ileri saflastirma için ultrafiltrasyon membranlarindan yararlanmaktadir. Sundugumuz bulusta bu sistemlerin biyoreaktöre entegre sekilde olmasi ve saflastirma isleminin fermentasyon sirasinda gerçeklesmesi ile ilgili faydali modelden farklilasmaktadir. Patent basligi: Tibbi sinif hyaluronik asidi arindirma islemi Mevcut bulus, biyolojik kaynaklardan üretilen hyaluronik asit'i medikal uygulamalar için gereken saflik derecesinde elde edilmesini tariflemektedir. Bulusta hyaluronik asit'in saflastirilmasi için; fermentasyon sivisinin çöktürme, mikrofiltrasyon, diyafiltrasyon, ultrafiltrasyon ve ters osmoz sistemi kullanilmistir. Sunulan bulusta kullanilmasi planlanan filtrasyon sistemi bulus ile benzerlik gösterse de belirlenen ayirma sinirlari ve filtrasyon sisteminin çevrimiçi geri kazanim sistemiyle entegre olmasi ile patent kapsaminda tarif edilen bulustan ayrilmaktadir. Patent basligi: HYALURONIKASIT ARlTMA YÖNTEMI Bu bulus, yüksek saflikta hyaluronik asit elde etmek için öncelikle düsük moleküler agirliga ve pigmentlere sahip hiyalüronik asidin ultrafiltrasyon ile uzaklastirilmasi ve ardindan proteinler, nükleik asitler, endotoksinler vb. safsizliklari aktif kömür ve istege bagli olarak gama alümina ilave edilerek uzaklastirmayi tarif eden yöntem ile ilgilidir. Ilgili bulus, filtrasyonun düsük molekül agirlikli hyaluronik asit'in uzaklastirilmasi için kullanilmasi nedeniyle sunulan bulustaki yöntemle iliskilendirilebilir. Ancak sunulan bulusta filtrasyon sistemi çevrimiçi geri kazanim prosesi içerisinde tercih edilmekte ve seçilen ayirma sinirlari ile molekül agirliklarina göre kategorize edilen hyaluronik asit üretimine katki saglamaktadir. Prensip olarak ayni temellere dayansa da sunulan bulusta kullanimi yönüyle filtrasyon sistemi ilgili bulustan fa rklilasma ktadir. REFERANS LISTESI . Biyoreaktör . Mikrofiltrasyon sistemi . Ultrafiltrasyon sistemi . Ultrafiltrasyon besleme . Kismi saflastirilmis Hyaluronik asit . Besiyeri sivisi . Mikrofiltrasyon retentat . Mikrofiltrasyon permeat . Ultrafiltrasyon retentat mÜOUJJU'IJ-wl\-` . Ultrafiltrasyon permeat ÇIZIMLERIN KISA AÇIKLAMASI Sekil 1. Söz konusu bulus çevrimiçi geri kazanim semasi temsili genel görünümü BULUSUN AÇIKLAMASI Bu bulus, fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi olup, semasi asagidaki sekil 1'deki gibi referans listesi unsurlari ile sinirlandirilamayacak sekilde tanimlanabilir; Biyoreaktör (1), Mikrofiltrasyon sistemi (2) Ultrafiltrasyon sistemi (3) Ultrafiltrasyon besleme (4) Kismi saflastirilmis Hyaluronik asit (5), Besiyeri sivisi (A) Mikrofiltrasyon retentat (B) Mikrofiltrasyon permeat (C), Ultrafiltrasyon retentat (D) Ultrafiltrasyon permeat (E). Kosullari optimize edilmis fermentasyonlar sonucunda üretilen hyaluronik asit, mikrofiltrasyon sistemi (2) ve Ultrafiltrasyon sistemi (3) ile kismi olarak saflastirilacaktir. Sunulan bulusta paketinde planlanan hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanilmasi için öncelikli olarak hücrelerin fermentasyon sivisindan uzaklastirilmasi gerekmektedir. Bu nedenle besiyerisi sivisi (A) ilk olarak 0.45 um gözenek çapina sahip filtrelerden geçirilecek ardindan Ultrafiltrasyon sistemine (3) verilecektir. Hücrelerin uzaklastirilmasi sonrasinda besiyeri sivisinda (A) kalan diger bilesenlerin (Hücresel protein, seker kalintilari, ikincil metabolitler) ortamdan ayrilmasi ve hyaluronik asit'in kismi olarak saflastirilmasi için Ultrafiltrasyon islemi uygulanacaktir. Hyaluronik asit'in kismi saflastirilmasi ve molekül agirliklarina göre düsük-orta-yüksek olarak ayrilmasi saflastirma islemleri gerçeklestirilecektir. Denemeler sonrasinda hyaluronik asit miktari, molekül agirligi ve kalinti protein miktari ölçülerek saflastirma katsayisi belirlenecektir. Membran sistem sartlarinin optimizasyonu sonrasinda mikrofiltrasyon-ultrafiltrasyon sistemi (2,3) kullanilarak bir sonraki is paketi olan çevrimiçi geri kazanim sistemi olusturulacaktir. Belirlenen mikrofiltrasyon-uItrafiltrasyon sistem (2,3) kombinasyonunu Içeren membran sistemi ile biyoreaktör (1) kombine edilerek sürekli bir sistem kurulacaktir. Bu sayede sürekli fermentasyon hyaluronik asit üretiminin gerçeklestirilmesi ve ayni zamanda üretilen hyaluronik asit'in çevrimiçi filtrasyon sistemiyle ortamdan alinmasi saglanacaktir. Sistemden hyaluronik asit içeren fermente sivinin bir kismi belirli bir üretim miktarina ulasinca ortamdan alinacaktir. Ortamdan alinan besiyeri sivisi (A) yerine biyoreaktöre (1) taze ve steril besiyeri eklenecektir. Ayni zamanda hyaluronik asit içeren besiyeri sivisi (A) hücrelerin ayrilmasi için mikrofiltrasyon sisteminden (2) geçirilecektir. Filtrasyon sonrasinda retentat olarak ayrilan bakteri hücreleri yeniden biyoreaktöre (1) döndürülecek, hyaluronik asit içeren permeat sivisi ise ultrafiltrasyon sistemine (3) gönderilecektir. Bu sayede bakteriler üretim fazinda tutularak farkli dilüsyon oranlarinda sürekli hyaluronik asit üretimi saglanmis olacaktir. Hücrelerin ayrilmasinin ardindan ultrafiltrasyon sisteminde (3) protein kalintilari ve diger safsizliklarin yüksek miktarda uzaklastirilmasi saglanacaktir. Ultrafiltrasyon asamasinda permeat olarak ayrilan sivi yeniden filtrasyon sistemine beslenecektir. Geri besleme islemi sistem stabil hale gelene kadar (yaklasik 90 dk.) devam ettirilecek ve sürenin filtrasyon sonunda kismi saflastirilmis hyaluronik asit retentat olarak ayrilacaktir. Ayirma islemi sirasinda fermentasyon sonlandirilmadan taze besiyeri sivisi (A) biyoreaktöre (1) beslenecektir. Ayni zamanda üretim fazinda bulunan ve mikrofiltrasyon sisteminde (2) ayrilan üretici mikroorganizmalar biyoreaktöre (1) geri döndürülecektir. Detayli açiklama; Filtrasyon sistemi: Mikrofiltrasyon (2) (0.45 pm ve 0.22 um gözenek çapina sahip (cut-off) degerine sahip Polietersülfon) birimlerinden olusan sistemlerdir. Içerisinde hyaluronik asit yer alan fermentasyon sivisi ilk olarak mikrofiltrasyon sisteminden (2) geçirilecektir. Hücre ayrimi bu islem sirasinda gerçeklestirilmektedir. Mikrofiltrasyon sisteminden (2) ayrilan sivi ultrafiltrasyon sistemine (3) verilerek hyaluronik asit ayrimi gerçeklestirilecektir. Bu Islem sirasinda fermentasyon durdurulmayacak ve taze besiyeri sivisi (A) beslemesi gerçeklestirilecektir. Ayni zamanda mikroofiltrasyon sisteminde (2) ayrilan üretici mikroorganizmalar taze besiyeri sivisi (A) ilavesi ile birlikte ortama geri beslenerek üretimin devamliligi saglanacaktir. Fermentasyon ortamindan peristaltik pompalar yardimiyla hortumlar içerisinde alinan fermentasyon sivisi ilk olarak 0.45 pm ve/veya 0.22 pm gözenek çapindaki filtrelerden geçirilecektir. Mikrofiltrasyon sisteminde (2) 1.0-5.0 bar araliginda belirtilen gözenek çapindaki membranlarda oda sicakliginda %90 permeat ve ml/saat besleme hizi degerlerinde hücre ayirma islemleri gerçeklestirilecektir. Sistemin retantat hacmi baslangiç hacminin %10tuna düstügünde sistem durdurulacaktir. Mikrofiltrasyon permeat (C) akisindan alinan sivi peristaltik pompalar yardimiyla ultrafiltrasyon sistemine (3) beslenecektir. Mikrofiltrasyon Retentat (B) akisinda biriken hücreler ise biyoreaktöre (1) geri beslenerek taze besiyeri sivisi (A) ile fermentasyona devam edilecektir. Fermentasyon sivisi ultrafiltrasyon sistemi (3) içerisinde ilk olarak 1000 kDa ayirma sinirindaki filtreden geçirilecektir. ultrafiltrasyon Retentat (D) sivisinda bulunan 1000 kDa üzerinde molekül agirligina sahip hyaluronik asit yüksek molekül agirlikli ürün kategorisindeki uygulamalar için ayrilacaktir. ultrafiltrasyon Permeat (E) akisindaki fermentasyon sivisi 500 kDa ayirma sinirindaki filtrelerden geçirilerek yeniden molekül agirliklarina göre 500 kDa üstü ve alti olmak üzere ayrilacaktir. Bu adim sonrasinda 100 kDa ayirma sinirina sahip filtreler ile filtrasyon islemine devam edilecek ve retentat permeat (ultrafiltrasyon permeat (E) ile ultrafiltrasyon retentat (D)) akilari düsük molekül agirlikli Kismi saflastirilmis Hyaluronik asit (5) elde edilmesi için ayrilacaktir. Ilâhî` I." H TR DESCRIPTION INCREASING THE PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED THROUGH FERMENTATION FROM THE FERMENTATION ENVIRONMENT TECHNICAL FIELD This invention aims to increase the production efficiency by recovering and partial purification of the product formed in hyaluronic acid production fermentations from the environment without stopping the fermentation and preventing microorganisms in the environment. It is about the reusability of the items. BACKGROUND OF THE INVENTION Problems encountered during the production of hyaluronic acid, such as increased viscosity, limitation of cell development, competition in the production and development metabolism of cells, and by-product formation, seriously reduce production efficiency. In addition, each fermentation process is carried out as a result of processes such as sterilization, pre-culture development and medium pH adjustment. In this case, production costs increase and efficiency decreases significantly due to the mentioned problems. The present invention prevents possible restrictions by removing hyaluronic acid from the environment and thus increases the efficiency. In addition, thanks to online recovery, a continuous production process is created at different dilution rates and pre-fermentation costs are minimized. Hyaluronic acid, which has a very wide usage area, had a share of 9.1 billion US dollars in the global market in 2019 and is economical. Hyaluronic acid can be priced between 4,000 and 17,000 US dollars, depending on its molecular weight and purity level. Although it has dozens of areas of use, according to the data of the International Society of Aesthetic Plastic Surgery (ISAPS), it had the second largest share among non-surgical applications with 4,315,859 applications in 2019. Even when only aesthetic applications are considered, without taking into account cosmetics, food, medicine and other fields, it can be seen how large a market it has on a global scale. acid application was made. This value constitutes 35% of non-surgical applications performed nationwide and makes Turkey rank 7th among the countries where the most hyaluronic acid applications are performed in the world. As can be seen, significant amounts of hyaluronic acid are used in the world and in our country. However, our country is in no way a producer in the market with a volume of almost 10 billion US dollars. Currently, all hyaluronic acid products used in our country are either imported or pure hyaluronic acid is purchased and prepared in various forms in our country and offered for sale. The contribution of the production of such a high value-added product in our country and the patenting of the current invention to our country's strong presence in this market has been contributed to the discovery of the invention. In addition, the invention was developed in order to eliminate the problems encountered in current production and to increase the efficiency and enable the production of hyaluronic acid in continuous systems at different dilution rates. Many problems are encountered during the production of hyaluronic acid by fermentation and production is limited. The most important among these are; Increase in viscosity due to increasing hyaluronic acid concentration during fermentation, resulting in limitation of cell development due to insufficient mixing and low oxygen transfer, competition of cell development and hyaluronic acid production for precursor compounds in the later stages of fermentation, transition to the glycolytic metabolic pathway, due to the by-product (lactic acid) formed. Cessation of production and production of low molecular weight hyaluronic acid may be shown. In order to overcome these problems, the invention in question was developed. With the online recovery method, the hyaluronic acid produced during fermentation will be separated from the fermentation medium simultaneously with the production. During the same separation stage, secondary metabolites affecting the fermentation environment will also be removed. Ultimately, it is aimed to increase production with a continuous system and to remove the produced product from the environment online. Hyaluronic acid, which is continuously recovered online from the fermentation medium, will be classified as having low, medium and high molecular weights. Thus, hyaluronic acid in different molecular weights (low, medium, high) will become a high-purity product that can be applied to many areas of use. The invention describes a system that enables the removal of the relevant component from the medium in the production of hyaluronic acid without stopping the fermentation. By integrating the bioreactor and membrane systems, problems such as increased viscosity, secondary metabolite formation, decrease in oxygen transfer rate, and low molecular weight that may occur in the fermentation environment will be overcome, and higher amounts and uniform product production can be achieved by reducing costs at different dilution rates with the continuous system. The invention provides advantages in the production of hyaluronic acid in terms of the integration of the filtration system used for different purposes into the bioreactor and the simultaneous production and purification without stopping the system. Patent application number: CN205164514U Patent title: Use of membrane filtration equipment in the production of hyaluronic acid The utility model describes the design of a membrane filtration device so that hyaluronic acid can be extracted efficiently and quickly. The specified useful model includes a design that includes microfiltration and ultrafiltration processes for the purification of hyaluronic acid. Although the current system is similar to the partial purification step in the invention; The invention we present differs from the useful model in that these systems are integrated into the bioreactor and the purification process occurs during fermentation. Patent title: Efficient Process for Purifying High Molecular Weight Hyaluronic Acid This invention relates to improved techniques for producing and purifying hyaluronic acid and its salt. The present invention also relates to the production, purification and process optimization of hyaluronic acid and its salt, which have biomedical applications. The relevant invention describes the purification of hyaluronic acid from the broth by precipitation and diafiltration processes. The present invention removes impurities using chemicals such as ethanol, silica gel and activated carbon and utilizes ultrafiltration membranes for further purification. The invention we present differs from the beneficial model in that these systems are integrated into the bioreactor and the purification process occurs during fermentation. Patent title: Process of purifying medical grade hyaluronic acid The present invention describes obtaining hyaluronic acid produced from biological sources at the level of purity required for medical applications. In order to purify hyaluronic acid in the invention; Sedimentation, microfiltration, diafiltration, ultrafiltration and reverse osmosis systems of the fermentation liquid were used. Although the filtration system planned to be used in the presented invention is similar to the invention, it differs from the invention described in the patent with the determined separation limits and the integration of the filtration system with the online recovery system. Patent title: HYALURONIC ACID PURIFICATION METHOD This invention involves first removing hyaluronic acid with low molecular weight and pigments by ultrafiltration to obtain high purity hyaluronic acid, and then removing proteins, nucleic acids, endotoxins, etc. It is about the method that describes the removal of impurities by adding activated charcoal and optionally gamma alumina. The relevant invention can be related to the method of the presented invention since filtration is used to remove low molecular weight hyaluronic acid. However, in the present invention, the filtration system is preferred in the online recovery process and contributes to the production of hyaluronic acid, which is categorized according to the selected separation limits and molecular weights. Although it is based on the same principles in principle, the filtration system differs from the relevant invention in terms of its use in the presented invention. REFERANCE LIST . Bioreactor . Microfiltration system. Ultrafiltration system. Ultrafiltration feeding. Partially purified Hyaluronic acid. Medium broth . Microfiltration retentate. Microfiltration permeate. Ultrafiltration retentate mÜOUJJU'IJ-wl\-` . Ultrafiltration permeate BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Representative overview of the said invention online recovery scheme DESCRIPTION OF THE INVENTION This invention is the online recovery of hyaluronic acid produced by fermentation from the fermentation medium, the scheme of which cannot be limited to the reference list elements as in figure 1 below. definable; Bioreactor (1), Microfiltration system (2) Ultrafiltration system (3) Ultrafiltration feed (4) Partially purified Hyaluronic acid (5), Medium liquid (A) Microfiltration retentate (B) Microfiltration permeate (C), Ultrafiltration retentate (D) Ultrafiltration permeate (E). Hyaluronic acid produced as a result of fermentations with optimized conditions will be partially purified by the microfiltration system (2) and the Ultrafiltration system (3). In order to recover the hyaluronic acid online from the fermentation medium, which is planned in the package of the present invention, the cells must first be removed from the fermentation liquid. For this reason, the medium liquid (A) will first be passed through filters with a 0.45 um pore diameter and then given to the Ultrafiltration system (3). After the cells are removed, the Ultrafiltration process will be applied to separate the other components (Cellular protein, sugar residues, secondary metabolites) remaining in the medium liquid (A) and to partially purify the hyaluronic acid. Partial purification of hyaluronic acid and separation into low-medium-high according to molecular weights will be carried out. After the trials, the purification coefficient will be determined by measuring the amount of hyaluronic acid, molecular weight and amount of residual protein. After optimizing the membrane system conditions, the next work package, an online recovery system, will be created using the microfiltration-ultrafiltration system (2,3). A continuous system will be established by combining the bioreactor (1) with the membrane system containing the specified microfiltration-uItrafiltration system (2,3) combination. In this way, continuous fermentation hyaluronic acid production will be achieved and at the same time the produced hyaluronic acid will be removed from the environment with the online filtration system. A portion of the fermented liquid containing hyaluronic acid will be removed from the system when it reaches a certain production amount. Fresh and sterile medium will be added to the bioreactor (1) instead of the medium liquid (A) taken from the environment. At the same time, the medium (A) containing hyaluronic acid will be passed through the microfiltration system (2) to separate the cells. After filtration, the bacterial cells separated as retentate will be returned to the bioreactor (1), and the permeate liquid containing hyaluronic acid will be sent to the ultrafiltration system (3). In this way, bacteria will be kept in the production phase and continuous hyaluronic acid production will be ensured at different dilution rates. After the cells are separated, a high amount of protein residues and other impurities will be removed in the ultrafiltration system (3). In the ultrafiltration stage, the liquid separated as permeate will be fed to the filtration system again. The feedback process will continue until the system becomes stable (approximately 90 minutes) and at the end of the filtration period, the partially purified hyaluronic acid will be separated as retentate. During the separation process, fresh medium liquid (A) will be fed to the bioreactor (1) without terminating the fermentation. At the same time, producer microorganisms present in the production phase and separated in the microfiltration system (2) will be returned to the bioreactor (1). Detailed explanation; Filtration system: These are systems consisting of microfiltration (2) (Polyethersulfone with a pore diameter (cut-off) value of 0.45 pm and 0.22 um) units. The fermentation liquid containing hyaluronic acid will first be passed through the microfiltration system (2). Cell separation is carried out during this process. The liquid separated from the microfiltration system (2) will be given to the ultrafiltration system (3) to separate hyaluronic acid. During this process, fermentation will not be stopped and fresh medium liquid (A) will be fed. At the same time, the producer microorganisms separated in the microfiltration system (2) will be fed back into the environment with the addition of fresh medium liquid (A), ensuring continuity of production. The fermentation liquid taken from the fermentation medium in hoses with the help of peristaltic pumps will first be passed through filters with a pore diameter of 0.45 pm and/or 0.22 pm. In the microfiltration system (2), cell separation processes will be carried out on membranes with a specified pore diameter between 1.0-5.0 bar, at room temperature, at 90% permeate and ml/hour feeding rate. The system will be stopped when the retentate volume of the system decreases to 10% of the initial volume. The liquid taken from the microfiltration permeate (C) flow will be fed to the ultrafiltration system (3) with the help of peristaltic pumps. The cells accumulated in the microfiltration retentate (B) flow will be fed back to the bioreactor (1) and fermentation will continue with fresh medium liquid (A). The fermentation liquid will first be passed through the filter at the 1000 kDa separation limit in the ultrafiltration system (3). Hyaluronic acid with a molecular weight over 1000 kDa found in ultrafiltration Retentate (D) fluid will be reserved for applications in the high molecular weight product category. The fermentation liquid in the ultrafiltration Permeate (E) flow will be passed through filters with a separation limit of 500 kDa and will be separated again according to their molecular weights, above and below 500 kDa. After this step, the filtration process will continue with filters with a separation limit of 100 kDa and the retentate permeate (ultrafiltration permeate (E) and ultrafiltration retentate (D)) flows will be separated to obtain low molecular weight Partially purified Hyaluronic acid (5). Ilahi` I." H TR

Claims (1)

1.ISTEMLER . Bu bulus, fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; en az 1 adet 0.45 um ve/veya 0.22 um gözenek çapindaki membran bulunduran mikrofiltrasyon sistemi (2) ve 1000 bir ultrafiltrasyon sisteminden (3) olusmasidir. .Istem 1'de bahsedilen fermentasyon yoluyla üretilen hyaluronik asitiin fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; filtrasyon sistemleri (mikrofiltrasyon sistemi (2), ultrafiltrasyon sistemi (3)) arasindaki baglantinin peristaltik pompalar ve 2-10 mm çapindaki hortumlardan olusmasidir. .Istem 1'de bahsedilen fermentasyon yoluyla üretilen hyaluronik asitlin fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; mikrofiltrasyon sistemi (2) ve ultrafiltrasyon sistemi (3) arasinda 0.5 M NaCl içeren solüsyon bulunmasidir. . Bulus fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi yöntemi olup, özelligi; mikrofiltrasyon retentat (B) çikisinin 100 ml saf su içeren steril bir kaba alinmasi ardindan biyoreaktöre (1) geri beslenmesi, mikrofiltrasyon permeat (C) çikisinin ise ultrafiltrasyon sistemine (3) iletilmesidir. . istem 4'te bahsedilen fermentasyon yoluyla üretilen hyaluronik asit”in fermentasyon ortamindan çevrimiçi geri kazanimi yöntemi olup, özelligi; mikrofiltrasyon sisteminin (2) mikrofiltrasyon permeat (B) çikisindan alinan fermentasyon sivisi 0.5 M NaCl solüsyonu içerisine aktarilmasi ve buradan ultrafiltrasyon sistemine (3) beslenmesidir. . Istem 5'te bahsedilen fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi yöntemi olup, özelligi; ultrafiltrasyon sistemine (3) giren fermentasyon sivisinin önce 1000 kDa ayirma sinirindaki filtreden geçmesi ve ultrafiltrasyon permeat (E) ile ultrafiltrasyon retentat (D) akislari toplama kaplarinda biriktirilmesidir. . Istem 6'da belirtilen islem olup, özelligi; bu islemden Çikan ultrafiltrasyon permeat (E) akisinin 500 kDa ayirma sinirindaki filtreye beslenmesi ve ultrafiltrasyon retentat (D) akisi ise sistemden ayrimlandirilmasidir. . Istem 7'de belirtilen islem olup özelligi; bu islemden çikan ultrafiltrasyon permeat (E) akisi 100 kDa ayirma sinirindaki filtreye beslenmesi ve ultrafiltrasyon retentat (D) akisi ise sistemden ayrimlandirilmasidir. . istem 8'de belirtilen islem olup özelligi; bu islemden çikan ultrafiltrasyon permeat (E) akisi 50 kDa ayirma sinirindaki filtreye beslenmesi ve ultrafiltrasyon retentat (D) akisi ise sistemden ayrimlandirilmasidir. 10. Istem 'l'de bahsedilen fermentasyon yoluyla üretilen hyaluronik asit*in fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; mikrofiltrasyon sistemi (2) ve ultrafiltrasyon sistemi (3) çalisma basincinin 0.5 ile 10.0 bar arasinda olmasidir. Istem 1'de bahsedilen fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; mikrofiltrasyon sisteminde (2) ve ultrafiltrasyon sisteminde (3) toplanan retentat akisi miktari baslangiç hacminin %5-20'si, permeat akisi miktari %80-95i kadar olmasidir. 12.Istem 1lde bahsedilen fermentasyon yoluyla üretilen hyaluronik asit'in fermentasyon ortamindan çevrimiçi geri kazanimi sistemi olup, özelligi; 0.45 pm ve 0.22 pm gözenek çapina sahip selüloz asetat filtrelerden olusan mikrofiltrasyon Polietersülfon ultrafiltrasyon sistemi (3) içermesidir. TR1.CLAIMS. This invention is an online recovery system of hyaluronic acid produced by fermentation from the fermentation medium, and its feature is; It consists of a microfiltration system (2) containing at least one membrane with a 0.45 um and/or 0.22 um pore diameter and a 1000 ultrafiltration system (3). It is an online recovery system of hyaluronic acid produced by fermentation mentioned in claim 1 from the fermentation medium, and its feature is; The connection between filtration systems (microfiltration system (2), ultrafiltration system (3)) consists of peristaltic pumps and 2-10 mm diameter hoses. It is an online recovery system of hyaluronic acid produced by fermentation mentioned in Claim 1, from the fermentation medium, and its feature is; There is a solution containing 0.5 M NaCl between the microfiltration system (2) and the ultrafiltration system (3). . The invention is a method of online recovery of hyaluronic acid produced by fermentation from the fermentation medium, and its feature is; The microfiltration retentate (B) outlet is taken into a sterile container containing 100 ml of pure water and then fed back to the bioreactor (1), while the microfiltration permeate (C) outlet is transmitted to the ultrafiltration system (3). . It is a method of online recovery of hyaluronic acid produced by fermentation mentioned in claim 4 from the fermentation medium, and its feature is; The fermentation liquid taken from the microfiltration permeate (B) outlet of the microfiltration system (2) is transferred into 0.5 M NaCl solution and fed from there to the ultrafiltration system (3). . It is a method of online recovery of hyaluronic acid produced by fermentation from the fermentation medium as mentioned in claim 5, and its feature is; The fermentation liquid entering the ultrafiltration system (3) first passes through the filter with a separation limit of 1000 kDa and the ultrafiltration permeate (E) and ultrafiltration retentate (D) streams are collected in collection containers. . It is the process specified in claim 6 and its feature is; The ultrafiltration permeate (E) flow resulting from this process is fed to the filter at the 500 kDa separation limit and the ultrafiltration retentate (D) flow is separated from the system. . It is the process specified in claim 7 and its feature is; The ultrafiltration permeate (E) flow resulting from this process is fed to the filter at the 100 kDa separation limit, and the ultrafiltration retentate (D) flow is separated from the system. . It is the process specified in claim 8 and its feature is; The ultrafiltration permeate (E) flow resulting from this process is fed to the filter at the 50 kDa separation limit, and the ultrafiltration retentate (D) flow is separated from the system. 10. It is an online recovery system of hyaluronic acid produced by fermentation mentioned in claim '1' from the fermentation medium, and its feature is; The working pressure of the microfiltration system (2) and ultrafiltration system (3) is between 0.5 and 10.0 bar. It is an online recovery system of hyaluronic acid produced by fermentation mentioned in claim 1 from the fermentation medium, and its feature is; The amount of retentate flow collected in the microfiltration system (2) and ultrafiltration system (3) is 5-20% of the initial volume, and the amount of permeate flow is 80-95%. 12. It is an online recovery system of hyaluronic acid produced by fermentation from the fermentation medium as mentioned in claim 1, and its feature is; It contains a microfiltration Polyethersulfone ultrafiltration system (3) consisting of cellulose acetate filters with 0.45 pm and 0.22 pm pore diameters. TR
TR2021/022266A 2021-12-31 2021-12-31 INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA TR2021022266A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
TR2021/022266A TR2021022266A2 (en) 2021-12-31 2021-12-31 INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA
PCT/TR2022/051663 WO2023129082A1 (en) 2021-12-31 2022-12-29 Increasing the productivity of hyaluronic acid fermentation by online recovery from fermentation medium

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
TR2021/022266A TR2021022266A2 (en) 2021-12-31 2021-12-31 INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA

Publications (1)

Publication Number Publication Date
TR2021022266A2 true TR2021022266A2 (en) 2022-01-21

Family

ID=85117219

Family Applications (1)

Application Number Title Priority Date Filing Date
TR2021/022266A TR2021022266A2 (en) 2021-12-31 2021-12-31 INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA

Country Status (2)

Country Link
TR (1) TR2021022266A2 (en)
WO (1) WO2023129082A1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100137579A1 (en) * 2008-12-01 2010-06-03 Sustineo Biotechnology Co., Ltd. Process for purifying medical grade hyaluronic acid
IT201700081449A1 (en) * 2017-07-18 2019-01-18 Fidia Farm Spa PURIFICATION PROCEDURE OF HYALURONIC ACID
CN112375160A (en) * 2020-11-20 2021-02-19 广东双骏生物科技有限公司 Method for separating and purifying hyaluronic acid from microbial fermentation liquor

Also Published As

Publication number Publication date
WO2023129082A1 (en) 2023-07-06

Similar Documents

Publication Publication Date Title
AU2015217006B2 (en) Ultrafiltration unit for continuous buffer or media exchange from a protein solution
US20220325708A1 (en) Alternating tangential flow rapid harvesting
Sikder et al. Purification of lactic acid from microfiltrate fermentation broth by cross-flow nanofiltration
CN101503707B (en) Method and device for continuous fermentation and separation coupling of biomacromolecule product
CN106132977A (en) For effective purification from the method for the neutral human milk oligosaccharides (HMO) of fermentable
CN105017360B (en) A kind of preparation method of vitamin B12
TW201231674A (en) Method for preparing concentrated aqueous solution of sugar
CN103865792A (en) Circulating microbial fermentation reaction and feed liquid separation integrated equipment
Matsumoto et al. Influence of extracellular polysaccharides (EPS) produced by two different green unicellular algae on membrane filtration in an algae-based biofuel production process
CA3122178A1 (en) Method for separating biomass from a solution comprising biomass and at least one oligosaccaride
CN113842779A (en) Continuous membrane filtration system and filtration method for erythritol fermentation liquor
TR2021022266A2 (en) INCREASING PRODUCTION EFFICIENCY BY ONLINE RECOVERY OF HYALURONIC ACID PRODUCED BY FERMENTATION FROM THE FERMENTATION MEDIA
CN102838624A (en) Method for purifying clavulanic acid from fermentation liquor
CN106631854B (en) A method of inorganic salts in removal l-Alanine fermented feed liquid
CN103917496B (en) Make water system
CN106359603A (en) Method for producing diary product with determined lactose amount
CN106417612A (en) Process for making dairy products free of lactose
CN102276742A (en) Method for cleanly producing MMW (medium molecular weight) hydroxyethyl starches
AU2011304167B2 (en) Production method for chemicals by continuous fermentation
JP2023528657A (en) Improved desalting of fermentation broths and fine chemical purification of e.g. oligosaccharides
JPS62237906A (en) Method for separating and concentrating organic material having low boiling point from aqueous solution
WO2019107498A1 (en) Filter device
Gryta et al. Studies of polypropylene membrane fouling during microfiltration of broth with bacteria
CN216604772U (en) Continuous membrane filtration system of erythritol zymotic fluid
JPS61293380A (en) Cultivation of microorganism and apparatus therefor