LC-MS/MS kullanılarak optimize edilen metot ile inek sütünün amino asit komposizyonunun belirlenmesi
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2022
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info:eu-repo/semantics/openAccess
Özet
Hayvansal kaynaklı proteinlerin biyoyararlılığı yüksektir. Süt insan beslenmesinde vazgeçilmez olan ve biyoyararlılığı yüksek proteinler içeren hayvansal kaynaklı bir gıdadır. Sütün amino asit bileşiminin belirlenmesi, sütün besin değerini etkileyebilecek peptitlerin ve proteinlerin kalitatif değerlendirmesi için çok önemlidir. Gıdaların amino asit profillerinin belirlenmesinde HPLC metodu yaygın olarak kullanılsa da, son zamanlarda amino asitlerin türevlendirme olmadan analiz edilebilmesine olanak sağlamasından dolayı LC-MS/MS metodu ön plana çıkmaktadır. Bu çalışmanın amacı, hidroliz sonrası LC-MS/MS ile türevlendirme olmaksızın sütteki amino asitlerin tayini için bir metot optimize etmek ve optimize edilen bu metot ile Türkiye'de yetiştirilen beş sığır ırkından (Siyah Alaca, Simental, Esmer, Yerli Kara ve Boz Irk) elde edilen sütlerin amino asit profillerini araştırmaktır. Süt örneklerine asidik hidrolizin yanı sıra alkali hidroliz de uygulanmış ve triptofan içerikleri de çalışmanın sonuçlarına dahil edilmiştir. Sonuç olarak sütlerde lisin, histidin, sistin, aspartik asit, treonin, alanin, glutamik asit, prolin, valin, izolösin, tirozin, lösin, feni¬lalanin, metiyonin, glisin, serin, arjinin ve triptofan olmak üzere toplam 18 adet amino asidin komposiyonunun belirlenmesi gerçekleştirilmiştir. Daha önce inek sütlerinde yapılan çalışmalarla uyumlu olarak, LC-MS/MS kullanılarak optimize edilen metot ile yapılan bu çalışmada da inek sütlerinde en bol bulunan amino asit glutamik asit, en bol bulunan esansiyel amino asit lösin, en az miktarda bulunan esansiyel amino asit ise triptofan olarak bulunmuştur. Farklı ırklardan elde edilen inek sütlerinin lisin, histidin, sistin, aspartik asit, treonin, alanin, glutamik asit, prolin, valin, izolösin, tirozin, lösin, feni¬lalanin, metiyonin ve triptofan konsantrasyonları için önemli farklılıklar (P<0,05) gözlenirken, glisin, serin, arjinin konsantrasyonları açısından önemli bir fark olmadığı görülmüştür (P>0,05). Farklı ırkların sütlerinin amino asit kompozisyonu karşılaştırıldığında, yerli ırk olan Boz Irk ve kültür ırkı Siyah Alaca sütlerinde toplam esansiyel amino asitlerin toplam amino asitlere oranının çalışılan diğer ırkların sütlerine göre daha yüksek olduğu görülmüştür. Yine iki ırkın sütlerinin lisin, valin, izolösin, lösin, treonin, feni¬lalanin, triptofan, histidin, glisin, sistin, serin, arjinin, aspartik asit, alanin, glutamik asit, prolin ve tirozin konsantrasyonlarında istatiksel olarak anlamlı bir fark olmadığı görülmüştür (P>0,05). Süt örneklerinin amino asit komposizyonunun yanı sıra kimyasal komposizyonunun da incelendiği çalışmada, Boz Irk sütünün yüksek protein içeriği ile diğer ırkların sütlerinden önemli ölçüde farklı olduğu, yağ içeriğinin ise en düşük olduğu görülmüştür. Laktoz içeriği açısından değerlendirildiğinde ise Boz Irk ve Yerli Kara sütlerinin diğer ırklardan istatiksel olarak önemli oranda (P <0,05) yüksek olduğu tespit edilmiştir. Simental ırkı sütünün ise çalışılan ırkların sütleri arasında en yüksek yağ içeriğine sahip olduğu belirlenmiştir. Çalışmada ısıl işlemin sütler üzerinde etkisini değerlendirmek amacıyla tez kapsamında çalışılan beş sığır ırkından elde edilen sütlere, aynı normlarda ev tipi kaynatma ve pastörizasyon işlemi uygulanmış ve sütlerde ısıl işlem öncesi ve sonrası hidroksimetilfurfural (HMF), lisin ve renk analizleri değerlendirilmiştir. Süte uygulanan ısıl işlem şiddetinin ve kalitenin belirlenmesinde kullanılan ve Maillard reaksiyonu sonucunda ortaya çıkan en bilinen ve en yaygın ara ürünlerden biri olan HMF'yi tespit etmek amacıyla yüksek hassasiyet ve seçiciliğe sahip LC-MS/MS'te bir yöntem geliştirilmiş ve valide edilmiştir. Analizler sonucunda pastörize edilen örneklerde ölçüm limitinin altında HMF oluştuğu gözlenirken kaynatılan süt örneklerinde ise 7,14±0,89-9,39±3,35 µg/kg aralığında HMF oluştuğu gözlemlenmiştir.
The bioavailability of proteins of animal origin is high. Milk is an animal originated food that is indispensable in human nutrition. Determination of the amino acid composition of milk is very important for the qualitative evaluation of peptides and proteins that may affect the nutritional value of milk. Although the HPLC method is widely used in the determination of the amino acid profiles of foods, the LC-MS/MS method has recently come to the fore because it allows the analysis of amino acids without derivatization. The aim of this study was to optimize a method for the determination of amino acids in milk without derivatization by LC-MS/MS after hydrolysis and to investigate the amino acid profiles of milk obtained from five cattle breeds (Holstein, Simmental, Brown Swiss, Anatolian Black and Turkish Grey) by using the optimized method. In addition to acidic hydrolysis, alkaline hydrolysis was also applied to milk samples and tryptophan contents were also included in the results of the study. As a result, the composition of 18 amino acids in milk, including lysine, histidine, cystine, aspartic acid, threonine, alanine, glutamic acid, proline, valine, isoleucine, tyrosine, leucine, phenylalanine, methionine, glycine, serine, arginine and tryptophan determination was carried out. Consistent with previous studies in cow milk, in this study conducted with the optimized method using LC-MS/MS, the most abundant amino acid was glutamic acid, the most abundant essential amino acid leucine and the least abundant essential amino acid was tryptophan in cow milk. While significant differences (P<0.05) were observed for lysine, histidine, cystine, aspartic acid, threonine, alanine, glutamic acid, proline, valine, isoleucine, tyrosine, leucine, phenylalanine, methionine and tryptophan concentrations of cow's milk obtained from different breeds, there was no significant difference in glycine, serine and arginine concentrations (P>0.05). When the amino acid composition of the milks of different breeds was compared, it was seen that the ratio of total essential amino acids to total amino acids in the milk of the domestic breed Turkish Grey and the culture breed Holstein was higher than the milk of the other studied breeds. There was no significant difference in the concentrations of lysine, valine, isoleucine, leucine, threonine, phenylalanine, tryptophan, histidine, glycine, cystine, serine, arginine, aspartic acid, alanine, glutamic acid, proline and tyrosine in the milks of the two breeds (P>0.05). In the study, the chemical composition of the milk samples were examined as well as the amino acid composition. It was observed that the milk of the Turkish Grey was significantly different from the milk of other breeds with its high protein content, while the fat content was the lowest. In terms of lactose content, it was determined that milk of Turkish Grey and Anatolian Black were statistically significantly higher (P <0.05) than the other breeds. It was determined that the milk of Simmental breed had the highest fat content among the milks of the studied breeds. In the study, in order to evaluate the effect of heat treatment on milk, domestic boiling and pasteurization processes were applied to the milk obtained from five cattle breeds within the scope of the thesis, and HMF, lysine and color analyzes were evaluated before and after heat treatment in milk. HMF is one of the most well-known and most common intermediate marker, which is used to determine the severity and quality of heat treatment applied to milk and is produced as a result of the Maillard reaction. An easy, practical and inexpensive method in LC-MS/MS with high sensitivity and selectivity to detect HMF has been developed and validated. As a result of the analysis, it was observed that HMF was formed below the limit of quantification in pasteurized samples, while HMF was formed in the range of 7.14±0.89-9.39±3.35 µg/kg in boiled milk samples.
The bioavailability of proteins of animal origin is high. Milk is an animal originated food that is indispensable in human nutrition. Determination of the amino acid composition of milk is very important for the qualitative evaluation of peptides and proteins that may affect the nutritional value of milk. Although the HPLC method is widely used in the determination of the amino acid profiles of foods, the LC-MS/MS method has recently come to the fore because it allows the analysis of amino acids without derivatization. The aim of this study was to optimize a method for the determination of amino acids in milk without derivatization by LC-MS/MS after hydrolysis and to investigate the amino acid profiles of milk obtained from five cattle breeds (Holstein, Simmental, Brown Swiss, Anatolian Black and Turkish Grey) by using the optimized method. In addition to acidic hydrolysis, alkaline hydrolysis was also applied to milk samples and tryptophan contents were also included in the results of the study. As a result, the composition of 18 amino acids in milk, including lysine, histidine, cystine, aspartic acid, threonine, alanine, glutamic acid, proline, valine, isoleucine, tyrosine, leucine, phenylalanine, methionine, glycine, serine, arginine and tryptophan determination was carried out. Consistent with previous studies in cow milk, in this study conducted with the optimized method using LC-MS/MS, the most abundant amino acid was glutamic acid, the most abundant essential amino acid leucine and the least abundant essential amino acid was tryptophan in cow milk. While significant differences (P<0.05) were observed for lysine, histidine, cystine, aspartic acid, threonine, alanine, glutamic acid, proline, valine, isoleucine, tyrosine, leucine, phenylalanine, methionine and tryptophan concentrations of cow's milk obtained from different breeds, there was no significant difference in glycine, serine and arginine concentrations (P>0.05). When the amino acid composition of the milks of different breeds was compared, it was seen that the ratio of total essential amino acids to total amino acids in the milk of the domestic breed Turkish Grey and the culture breed Holstein was higher than the milk of the other studied breeds. There was no significant difference in the concentrations of lysine, valine, isoleucine, leucine, threonine, phenylalanine, tryptophan, histidine, glycine, cystine, serine, arginine, aspartic acid, alanine, glutamic acid, proline and tyrosine in the milks of the two breeds (P>0.05). In the study, the chemical composition of the milk samples were examined as well as the amino acid composition. It was observed that the milk of the Turkish Grey was significantly different from the milk of other breeds with its high protein content, while the fat content was the lowest. In terms of lactose content, it was determined that milk of Turkish Grey and Anatolian Black were statistically significantly higher (P <0.05) than the other breeds. It was determined that the milk of Simmental breed had the highest fat content among the milks of the studied breeds. In the study, in order to evaluate the effect of heat treatment on milk, domestic boiling and pasteurization processes were applied to the milk obtained from five cattle breeds within the scope of the thesis, and HMF, lysine and color analyzes were evaluated before and after heat treatment in milk. HMF is one of the most well-known and most common intermediate marker, which is used to determine the severity and quality of heat treatment applied to milk and is produced as a result of the Maillard reaction. An easy, practical and inexpensive method in LC-MS/MS with high sensitivity and selectivity to detect HMF has been developed and validated. As a result of the analysis, it was observed that HMF was formed below the limit of quantification in pasteurized samples, while HMF was formed in the range of 7.14±0.89-9.39±3.35 µg/kg in boiled milk samples.
Açıklama
Anahtar Kelimeler
süt, amino asit, ırk, LC-MS/MS, HMF, lisin, milk, amino acid, breed, lysine
