Validation of a method employing liquid chromatography to identify and quantify glycomacropeptide of casein in raw milk / Validación de un método mediante cromatografía líquida para identificar y cuantificar glicomacropéptido de caseína en la leche cruda

Background: The information on official testing methods, or regulatory methods in Colombia to test whey in milk is limited; this restriction of information goes against the possibility of mitigating the risk of food fraud. Objectives: The validation of an HPLC method to determine casein glycomacropeptide (c-GMP), a protein that countries such as Brazil, Spain, and Ecuador have used as an indicator of raw milk adulteration with whey, was carried out. Methods: A 10mL sample of raw milk is precipitated with 24% TCA using ultrasound, a process followed by filtration. The collected fraction ensured the separation of c-GMP and then injected into the liquid chromatography. Results: A 30 minutes analysis allowed the determination of c-GMP with a retention time of 12.9 ± 0.5 minutes. The performance characteristics method in the validation exercise were: recovery percentage 99.97%, linearity R2> 0.95; % RSD accuracy <5.3%. Conclusion, the method exhibits desirable attributes for the intended purpose

Antecedentes: En Colombia la información de dominio público en metodologías de análisis de lactosuero en leche es limitada, restringiendo la posibilidad de acceder a ellas para mitigar el riesgo de fraude alimentario. Objetivos: Se realizó validación de un método por HPLC para determinar en leche cruda c-GMP, proteína usada como indicador de adulteración en países como Brasil y Ecuador. Metodos: Una muestra de 10mL de leche cruda es precipitada con TCA al 24% empleando ultrasonido, proceso seguido por filtración. La fracción recolectada aseguró la separación del c-GMP para luego inyectar al cromatógrafo líquido. Resultados: La determinación de c-GMP permitió el análisis en 30 minutos con tiempo de retención de 12,9 ± 0,5 minutos. Las características de desempeño del método en el ejercicio de validación fueron: porcentaje de recuperación 99,97%, linealidad R2>0,95; precisión %RSD< 5,3%. Conclusión: el método al final del ejercicio exhibe atributos para el fin previsto

Milk fraud by the addition of whey using an artificial neural network / Identificação de fraude de leite por adição de soro de leite usando redes neurais artificiais

ABSTRACT: The adulteration of milk by the addition of whey is a problem that concerns national and international authorities. The objective of this research was to quantify the whey content in adulterated milk samples using artificial neural networks, employing routine analyses of dairy milk samples. The analyses were performed with different concentrations of whey (0, 5, 10, and 20%), and samples were analyzed for fat, non-fat solids, density, protein, lactose, minerals, and freezing point, totaling 164 assays, of which 60% were used for network training, 20% for network validation, and 20% for neural network testing. The Garson method was used to determine the importance of the variables. The neural network technique for the determination of milk fraud by the addition of whey proved to be efficient. Among the variables of highest relevance were fat content and density.

RESUMO: A adulteração do leite pela adição de soro de leite é um problema que diz respeito às autoridades nacionais e internacionais. O objetivo deste trabalho foi quantificar o teor de soro em amostras de leite adulterado por meio de redes neurais artificiais, usando como variáveis de entrada os resultados de análises rotineiras em amostras de leite. As análises foram realizadas com diferentes concentrações em relação à adição de soro de leite (0, 5, 10 e 20%), e as amostras foram analisadas quanto à gordura, sólidos não gordurosos, densidade, proteína, lactose, minerais e ponto de congelamento, totalizando 164 ensaios, dos quais 60% foram utilizados para treinamento em rede, 20% para validação de rede e 20% para teste de rede neural. O método de Garson foi utilizado para determinar a importância das variáveis. A técnica de redes neurais para a determinação da fraude ao leite por adição de soro provou ser eficiente. Entre as variáveis de maior relevância estavam o teor de gordura e a densidade.

The Promising Efficacy of Probiotics, Casein Phosphopeptide and Casein Macropeptide as Dental Anticariogenic and Remineralizing Agents Part I; An In vitro Study

Objectives: To investigate the effect of different mixtures from nano casein phosphopeptides (CPP), amorphous calcium phosphate (ACP), probiotic Lactobacillus rhamnosus B-445 (L. rhamnosus) and casein glycomacropeptide (GMP) against Streptococcus mutans (S. mutans) growth and its adhesion. Methods: CPP was prepared from the tryptic digest of bovine casein and GMP by the action of chymosin on casein solution. Four mixtures namely: Group 1) Nano CPP; Group 2) Nano CPP+ ACP; Group 3)Nano CPP+ ACP + L. rhamnosus; and Group 4)Nano CPP+ ACP+ GMP were prepared and tested for its inhibitory activity against S. mutans growth and its adhesion to saliva-treated glass surfaces in comparison with a commercial product (GC MI paste plus) and chlorhexidine (0.2%) as a positive control.  The particle size and zeta potential of nano CPP and its complex with ACP were evaluated. Furthermore, the viability of L. rhamnosusin its mixture was determined during two weeks of storage at pH 6.8 and 8 respectively. Results: Revealed that Nano CPP had an average particle size (7.75 nm) and zeta potential (-8.43 mV) lower than that of CPP+ACP mixture. Probiotic containing mixture exhibited inhibitory activity slightly less than the positive control at pH 6.8. All tested mixtures reduced the adhesion of S. mutans to saliva-treated glass surfaces and the highest was that containing probiotic and GMP. L. rhamnosus showed acceptable stability in CPP+ACP mixture during storage period. Conclusions: All these findings suggest the use of probiotic, CPP+ACP mixture as a dental anticariogenic and remineralizing agents.

New advances in phenylketonuria therapy research / 国际儿科学杂志

Phenylketonuria is a most common group of genetic metabolic diseases.Phenylketonuria is caused by enzymatic defects in the metabolic pathway,which is characterized by high blood phenylalanine concentration.Patients need early,reasonable treatment once diagnosis,otherwise there will be serious nervous system sequelae.Available treatments aim to decrease the blood phenylalanine concentration,reduce nervous system symptoms.The current primary treatment of phenylketonuria is the limitation of dietary phenylalanine intake.Considering the poor compliance with long-term eating restrictions and the heavy family burden,the application of new medicine such as trahydropterina cofactor,glycomacropeptide,large neutral amino acids can improve the therapeutic effect and living condition of phenylketonuria patients.In addition,recombinant phenylalanine ammonia lyase,hepatocyte transplantation,gene therapy,probiotics and other new treatments also seem to be a promising approach in the near future.

Alternative Therapies for PKU

Abstract The phenylalanine (PHE)-restricted diet has improved in quality and diversity over time and has proven to be effective in all patients. Nevertheless, this treatment imposes a heavy social and economic burden to patient and family and impacts quality of life. Sustained adherence to PHE restriction is difficult to maintain. Moreover, even patients with phenylketonuria (PKU) with normal intelligence quotient (IQ) have lower IQ than matched individuals without PKU and can have deficits in multiple other aspects of neuropsychological function, including cognitive and executive function, working memory. They can also have behavior problems, depression, and low self-esteem. In recent years, alternative treatments for PKU have been developed and their use has been indicated for some patients who are candidates for options besides traditional treatment. Sapropterindihydrochloride, large neutral amino acids, and glycomacropeptide are alternative treatment options in use for selected patients. The aim of this article is to review the current knowledge of these new approaches to PKU treatment.

Anti-Helicobacter pylori activity of crude N-acetylneuraminic acid isolated from glycomacropeptide of whey / 한국실험동물학회지

Helicobacter pylori colonizes the gastric mucosa of about half of the world's population, causing chronic gastritis and gastric cancer. An increasing emergence of antibiotic-resistant H. pylori arouses demand on alternative non-antibiotic-based therapies. In this study, we freshly prepared crude N-acetylneuraminic acid obtained from glycomacropeptide (G-NANA) of whey through a neuraminidase-mediated reaction and evaluated its antibacterial ability against H. pylori and H. felis. Overnight cultures of the H. pylori were diluted with fresh media and different concentrations (1-150 mg/mL) of crude G-NANA were added directly to the culture tube. Bacterial growth was evaluated by measuring the optical density of the culture medium and the number of viable bacteria was determined by a direct count of the colony forming units (CFU) on agar plates. For the in vivo study, mice were orally infected with 100 µL (5×108 cfu/mL) of H. felis four times at a day's interval, accompanied by a daily administration of crude G-NANA or vehicle. A day after the last infection, the mice were daily administered the crude G-NANA (0, 75, and 300 mg/mL) for 10 days and euthanized. Their stomachs were collected and bacterial colonization was determined by quantitative real-time PCR. Crude G-NANA inhibited H. pylori's growth and reduced the number of viable bacteria in a dose-dependent manner. Furthermore, crude G-NANA inhibited bacterial colonization in the mice. These results showed that crude G-NANA has antibacterial activity against Helicobacter and demonstrated its therapeutic potential for the prevention of chronic gastritis and gastric carcinogenesis induced by Helicobacter infection in humans.

Proteólise da κ-caseína em leite e bebida láctea UAT durante o período de validade comercial / κ-casein proteolysis during the shelf-life of UHT milk and milk beverage

A proteólise do leite e da bebida láctea UAT durante a estocagem é um dos fatores mais importantes na determinação da validade comercial desses. O teor de glicomacropeptídeos (GMP) é um marcador das ações proteolíticas no leite, pois é capaz de indicar indiretamente a integridade da κ-caseína. Assim, no presente trabalho, objetivou-se avaliar a proteólise da κ-caseína do leite e da bebida láctea UAT ao longo do período de validade. Foram avaliadas 60 amostras de cinco diferentes marcas comerciais de cada produto através do método espectrofotométrico para quantificação de ácido siálico. Para o leite UAT foi observado o intervalo entre 3,83 e 15,27 µg de ácido siálico/mL no início do período de validade (D0), 5,95 a 17,16 µg de ácido siálico/mL 60 dias após o início do período (D60) e 6,54 a 21,11 µg de ácido siálico/mL no final do período, ou seja, 120 dias após (D120) a produção. Já para as bebidas lácteas foram observados intervalos entre 11,12 e 24,25; 15,74 e 24,73 e 17,19 e 26,49 µg de ácido siálico/mL para o dia inicial, 60 e 120 dias do período de validade, respectivamente. Os valores obtidos para o índice proteolítico do leite e bebida láctea UAT aumentaram gradativamente ao longo do período de validade dos produtos, sendo tal diferença detectável a partir de sessenta dias. Não foi observada influência da abertura da embalagem na proteólise da k-caseína.

The UHT milk and milk beverage proteolysis during storage is the major factor that limits the shelf-life of these products. The GMP content has assumed importance because it can indirectly indicate the integrity of κ-casein in milk. So, this study focused on evaluates the κ-casein proteolysis in UHT milk and milk beverage during the shelf-life. 60 samples obtained of five different brands of each product were evaluated by spectrophotometric assay for quantifying acid sialic. For UHT milk was observed the values ranging from 3.83 to 15.27 µg acid sialic/ml at the begin of the shelf-life, from 5.95 to 17.16 mg sialic acid/ml at the middle of the period (60 days after the production) and 6.54 to 21.11 mg sialic acid/ml at the end of shelf-life (120 days). For milk beverages were observed ranges between 11.12 to 24.25, 15.74 to 24.73 µg of sialic acid/ml and from 17.19 to 26.49 mg sialic acid/ml respectively for the beginning, 60 and 120 days of the shelf-life. The proteolysis rate in UHT milk and milk beverage increased during the shelflife and is detectable after 60 days of the shelf-life. The practice of open the package did not influence the κ-casein proteolysis.

Detection of glycomacropeptide content in breast milk and comparison with varieties of newborn infant formula / 中华实用儿科临床杂志

Objective To detect the glycomacropeptide (GMP) content of human breast milk,and take it as reference of newborn infant formula milk powder to optimize the nutritional content of infant formulas.Methods Thirty primiparas who fit the following conditions were selected:healthy,no special diet habits,living stably,having adequate milk,aged between 25 to 39 years old,and term delivery,were selected.Then they were divided into 2 groups,the colostrum group and mature milk group,and each group had 15 cases.Each case had been collected of breast milk 5 mL(front milk).The gathering time of colostrum group was the 2nd day postpartum and mature milk group was the 42nd day postpartum.Hydrolyzing the breast milk by chymosin at 37 ℃,then the sialic acid(SA) content was detected by a sialic acid detection kit,and GMP content was represented by SA content.In addition,6 brands of formula milk powder were detected in the same way as breast milk after being made into standard liquid milk.The differences between groups were compared by analysis of variance (ANOVA).Results Best conditions for enzymolysis were:chymosin concentration 0.25 g/L,hydrolysis time 120 min.SA content of colostrum group was (3486.98 ± 406.70) mg/L,while mature milk group was (2687.95 ± 375.85) mg/L,as the former was significantly higher than the latter (P ＜ 0.01),but the differences between individuals within each group were small (CVco1 =0.12,CVma ilk =0.14).The average level of SA content of various infant formulas was (1196.93 ± 608.40)mg/L,which was significantly lower than colostrum and mature milk(all P ＜ 0.01).SA contents of various brands of formulas were various,and the difference among these brands was relatively big(CV =0.63).Conclusions The content of GMP in human colostrum was higher than that of the mature milk.The contents of the GMP in different brands of infant formula milk powder were uneven,and the measured values were quite different with human milk.However,because of the difference of the molecular weight of GMP and the number of amino acid residues between human milk and bovine milk,the amount of GMP required to achieve the same physiological effects may be different in breast milk and bovine milk.In order to optimize the nutritional content of infant formula,and make it even closer to human milk,it is necessary to further explore the best GMP content relative to human milk.

Progress in biological function of glycomacropeptide / 国际儿科学杂志

Glycomacropeptide(GMP) is a polypeptide fragment derived from κ-casein after rennin treatment.At present,the structures of GMP in human beings,cows and goats have been established.The sialic acid structure contained in the polypeptide chain of GMP is very important for the exertion of the biological function.GMP has many biological functions,such as anti-infection,regulating immunity,anti-inflammation,nourishing and maintaining health,and so on.As a new-type bio-functional protein,it will be more and more widely used in the areas of medicine and foodstuff.

Aislamiento y rendimiento del GMP mediante precipitación de lactosuero con ácido tricloroacético / GMP isolation and yield by whey precipitation with trichloroacetic acid

Durante la elaboración del queso, la k-caseína es hidrolizada por la renina (Quimosina E.C.3.4.23.4) en el enlace peptídico Fen105-Met106 generando dos fracciones: la para-k-caseína y el glicomacropéptido (GMP) que se libera al lactosuero. El GMP presenta una estructura química particular donde predominan los aminoácidos con cadena lateral ramificada, no presenta aminoácidos aromáticos y contiene carbohidratos unidos a residuos de treonina; por esta razón se le ha atribuido una variedad de actividades biológicas. Se ha estimado que en Venezuela se generan alrededor de 713 toneladas de lactosuero anualmente. Un volumen considerable de este subproducto se produce en el estado Zulia, constituyéndose esto en una fuente de péptidos y proteínas de alta calidad nutricional que está siendo subutilizada. Con el propósito de evaluar el aislamiento y rendimiento del GMP a partir de la precipitación de lactosuero de ricotta con ácido tricloroacético 50 por ciento, se realizaron 6 extracciones con este ácido a 50 mL de cada tipo de suero analizado: suero ricotta, suero comercial resuspendido y suero ácido (control negativo). Se verificó mediante pruebas químicas y PAGE-SDS 15 por ciento de manera indirecta, la presencia de GMP en las preparaciones obtenidas. Se observaron bandas de 6,5; 18,3 y 19,0 kDa en suero ricotta y suero comercial resuspendido. Las bandas de 18,3 y 19,0 posiblemente correspondan a la forma trimérica del péptido. El rendimiento del GMP en términos de proteínas fue en promedio 1,17 mg/50mL (1,17 por ciento) y 4,51 mg/50mL (0,81 por ciento), para suero ricotta y suero comercial, respectivamente. Los resultados indican que es factible obtener preparaciones del GMP, sin embargo, para plantear la producción a escala industrial de este péptido para su aprovechamiento, se requiere evaluar otros procedimientos donde se obtenga a bajo costo una preparación purificada del GMP.

During cheese manufacturing k-casein is hydrolyzed by rennin (Quimosine E.C.3.4.23.4) on peptidic bond Fen105- Met106 releasing two fractions: para-k-casein and glycomacropeptide (GMP). GMP shows a particular chemical structure in which ramified lateral chain aminoacids prevail, without aromatics aminoacids, but with carbohydrates short chains linked to some threonine residues; because of this, a variety of biological activities have been attributed to molecule. It has been considered that in Venezuela, 713 tons of whey are generated annually. An important volume of this byproduct is produced in the Zulia State, becoming itself a source of peptides and proteins of high nutritional quality that has been subused. With the purpose of evaluating GMP isolation and yield from ricotta whey precipitation with 50 percent trichloroacetic acid treatment, 6 extractions where performed with this acid to 50 mL of each analyzed whey: ricotta whey, resuspended commercial whey and acid whey (negative control). By means of chemical tests and PAGE-SDS 15 percent, indirect presence of GMP was verified in all preparations. Bands of 6.5, 18.3 and 19.0 kDa were observed in ricotta and commercial whey. Bands of 18.3 and 19.0 possibly correspond to the peptide trimeric structure. GMP yield in terms of protein content was 1.17 mg/50mL (1.17 percent) and 4.51 mg/50mL (0.81 percent), for ricotta and commercial whey, respectively. Results show that it feasible to obtain preparations of GMP, however, in order to produce this peptide industrially for its use, evaluation of low cost procedures for GMP purification is required.