Person: Alfonso Ruiz, Leopoldo
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Alfonso Ruiz
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Leopoldo
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Agronomía, Biotecnología y Alimentación
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IS-FOOD. Research Institute on Innovation & Sustainable Development in Food Chain
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0000-0002-5662-9997
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1974
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Publication Open Access DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein(Elsevier, 2022) Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaBovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in the dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify the A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. The results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10−5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for the A1 allele in A2 samples at 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify the A1 allele absence in “A2 milk” by screening commercial products on the market.Publication Open Access Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection(Springer Nature, 2022) Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Azparren Domínguez, Leire; Urrutia Vera, Olaia; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOODMilk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.Publication Open Access Worldwide research trends on milk containing only A2 β-casein: a bibliometric study(MDPI, 2022) Jiménez Montenegro, Lucía; Alfonso Ruiz, Leopoldo; Mendizábal Aizpuru, José Antonio; Urrutia Vera, Olaia; Institute on Innovation and Sustainable Development in Food Chain - ISFOODThe protein fraction of β-casein may play a key role in the manifestation of a new intolerance: milk protein intolerance. The most common forms of β-casein among dairy cattle breeds are A1 and A2 β-casein. During gastrointestinal digestion of A1 β-casein, an opioid called peptide β-casomorphin-7 (BCM-7) is more frequently released, which can lead to adverse health outcomes. For that reason, novel products labelled as "A2 milk"or "A1-free dairy products" have appeared on the market. In this context, a bibliometric analysis on A2 β-casein research was carried out through the Web of Science (WoS) database. The main objective of this work was to provide an overview of the state of the art in the field of β-casein A2 by analyzing the number of publications per year, trends in thematic content, the most frequently used terms, and the most important institutions and countries in the field. This bibliometric study showed that a greater effort is needed to determine the possible implications of this novel product for human health and the market.