Gamma-linolenik asit ile zenginleştirilmiş anne sütü yağına benzer yapılandırılmış yağların üretimi

Lipase-catalyzed production of human milk fat substitutes (HMFS) containing gamma-linolenic acid

Özet

Anne sütü yağına benzer yapılandırılmış yağların (YY) bitkisel kaynaklı yağlardan spesifik lipaz enzimlerinin katalizlediği interesterifikasyon (asidoliz) tepkimeleri ile üretilmeleri mümkün olmaktadır. Bu çalışmada gamma-linolenik asit (GLA) ile zenginleştirilmiş anne sütü yağına benzer YY’ların tripalmitin, fındık yağı serbest yağ asitleri (SYA) ve hodan (boraj) yağından elde edilen GLA konsantresi arasında gerçekleştirilen enzimatik asidoliz tepkimeleri ile üretilmesi ve tepkiyüzey yöntemi (TYY) ile reaksiyon koşullarının optimizasyonu amaçlanmıştır. Enzimatik asidoliz tepkimelerinde Rhizomucor miehei’den elde edilen ve bir sn-1,3 spesifik lipaz enzimi olan Lipozyme® RM IM kullanılmıştır. Tepki-yüzey yönteminde seçilmiş farklı faktörlerin etkilerinin incelenmesi ve optimum koşulların belirlenmesi amacıyla, 5 seviyeli Merkezil Bileşik Deney Tasarımı (CCD) kullanılmış ve "substrat mol oranı (Toplam yağ asitleri/Triaçilgliserol, Sr)", "reaksiyon sıcaklığı (T, ºC)" ve "reaksiyon süresi (t, saat)" değişken faktörler olarak seçilmiştir. Reaksiyonlar sonucunda seçilen tepkiler [oleik asit miktarı (%) ve GLA miktarı (%)] için, “çoklu regresyon” ve “geriye dönük eleme” yöntemleri uygulanarak başarılı kuadratik modeller elde edilmiştir. Hedeflenen özellikte ürün (%10 oranında GLA, %45 oranında oleik asit içeren) elde etmek için gerekli optimum koşullar [Sr: 14.8 mol/mol, T: 55ºC ve t: 24 saat] olarak bulunmuştur. Bu koşullarda modelin doğruluğu deneysel olarak da kontrol edilmiş ve çalışma sonucunda bebek beslenmesi ve sağlığı açısından önemli etkileri olan GLA ile zenginleştirilmiş ve anne sütü yağı ile benzer absorpsiyon özelliklerine ve yağ asidi kompozisyonuna sahip bir YY’ın üretimi mümkün olmuştur.

Abstract

Breast milk is the main and most preferred source of nutrients for infants and human milk fat (HMF) is its component which supplies the highest fraction of the infant's required dietary energy. Human milks are characterized by the dominance of triacylglycerols (TAG) (more than 98% of HMF) where the saturated 16-carbon palmitic acid (C16:0) (20-30%) is in the sn-2 position (60-70%) of the glycerol backbone, the sn-1 and sn-3 positions are being taken by unsaturated fatty acids (FA). This unique structure is different from most vegetable oils and animal fats. Previous reports provided convincing information that the higher fatty acid (FA) and calcium absorption and efficient use of dietary energy was the result of this specific position of these fatty acids in triacylglycerol (TAG) moiety. Structured lipids (SL) resembling TAGs of human milk fat can be produced by interesterification from vegetable oils, using sn-1,3 specific lipases as biocatalyst and such TAG can be used in infant food formulations. There has also been a great interest for the supplementation of infant formulas with FA such as gamma-linolenic acid (GLA). In infant formulae, GLA-containing oil, especially borage oil is used for its health benefits and for its antagonist action on arachidonic acid (AA) metabolism. Since GLA is rapidly elongated to di-homo gamma-linolenic acid (DGLA) and subsequently $Delta$5-desaturated to AA and is also cheaper and easier to produce than AA, an alternative way for supplementation of infant formulas with AA is to use GLA instead of highly active AA. Human milk fat substitutes (HMFS) containing GLA can be produced by enzymatic interesterification reactions. These formulae are also helpful where $Delta$6-desaturase enzyme is insufficient. The purpose of the present work was to synthesize SLs resembling HMF enriched with GLA by enzymatic acidolysis reactions between tripalmitin, hazelnut oil FA and GLA in n-hexane. Commercially immobilized 1,3 specific lipase, Lipozyme® RM IM, obtained from Rhizomucor miehei, was used as the biocatalyst for the acidolysis reactions. Moreover it was aimed to model and optimize the reaction conditions via response surface methodology (RSM). For this purpose central composite design (CCD) with five levels and three factors; substrate molar ratio (Total FA/TAG, $S_r$)", "reaction temperature (T, ºC)" and "reaction time (t, hour)" were used. The reactions were optimized considering target GLA and oleic acid incorporation. Good quadratic models were obtained for the incorporation of GLA (response 1) and oleic acid (response 2) by "multiple regression" and "backward elimination". The determination coefficient $(R^2)$ values for the models were found to be 0.92 and 0.94. Based on the experimental results, the regression coefficients $(beta)$ and significance (P) values were calculated. Among first order parameters substrate molar ratio had negative effect on the oleic acid incorporation. Time was the most significant first order parameter followed by temperature and substrate molar ratio for GLA incorporation. For both responses, second order parameters, temperature* temperature and time*time had negative effects and were found to be significant. The predicted values obtained from the models had a linear relationship with the observed values which indicates that the generated models adequately represent the relationship between the response and reaction parameters. The optimal conditions generated from the models for the targeted GLA (10%) and oleic acid (45%) incorporation were: 14.8 mol/mol, 55°C and 24 h for substrate molar ratio, reaction temperature and reaction time, respectively. Models were verified using the optimal conditions obtained with RSM. The SL resembling HMFS contained palmitic acid at 73.9 %, which is also close to that of HMF. In this study, a HMFS product containing GLA was successfully produced having both the associated health benefits of GLA and similar fatty acid composition as well as similar absorption characteristics with human milk fat. This SL may be an important ingredient for commercial use in infant formula and contribute to the infant’s nutrition and development.