Nut Species And Walnut Milk Beverage Authentication Based On DNA Barcoding

Walnut(Juglans regia L)is one of the world's "four dried fruits".It has multiple values such as nutrition,intelligence,medicine and food therapy.Walnut milk is a natural vegetable protein beverage made from high-quality walnut kernel,which is popular among consumers.However,the adulteration of walnut milk in the market is emerging in an endless stream.It is urgent to establish a practical,fast and accurate identification method for walnut milk and other nut drinks.Therefore,in this study,ten kinds of nuts and non-nut soybeans,nine different brands of walnut milk and one pecan milk were selected as the research objects and three methods including DNA barcode method,high resolution melting analysis(HRM)and loop-mediated isothermal amplification(LAMP)are adopted to quickly identify the authenticity of commercially available walnut milk and pecan milk.The main research results are as follows:(1)Comparison and optimization of high quality DNA extraction methods for nuts and walnut milk: Nut DNA was extracted by three methods,and the best quality of the nut DNA extracted by the traditional reagent method of CTAB was screened.The concentration range was 122-1024 ng·?L-1,the purity OD value was between 1.8-2.0 and the fragment was complete.Three pretreatment methods and six different DNA extraction methods were compared for walnut milk samples,and it was concluded that vacuumed freeze-drying method was better than the other two pretreatment methods.The combination of CTAB and Axy Gen kit could extract high-quality walnut milk DNA with a concentration value of 65-135 ng·?L-1,a purity value closing to 1.80.(2)Construction of optimal DNA barcode sequence of nuts and identification of walnut milk authenticity: ITS2,rbc L and psb A-trn H were selected as candidate DNA barcode sequences.Based on the bioinformatics analysis of the success rate of amplification,sequencing and comparison,as well as the base content,the genetic distance between species,and the phylogenetic tree,ITS2 was found to be the most suitable DNA barcode to distinguish different nuts such as walnut and peanut.Based on ITS2 sequence,PCR amplification,sequencing and database comparison were performed for nine pecan milk and one pecan milk samples.It was found that among the nine different brands of walnut milk,the result of sequencing comparison of 8 walnut milk samples was walnut.However,the sequencing result of one walnut milk(NO.25-27)was peanut,and one pecan milk(NO.28-30)was walnut,which indicated that the principal components of the two products were inconsistent with the labels.(3)To identify the authenticity of walnut milk by combining DNA barcode and HRM analysis method: HRM analysis primers were designed based on ITS2 and psb Atrn H sequences of plant DNA barcodes,and the components of commercially available walnut milk and pecan milk(walnut and pecan)and common adulterated species(peanut and soybean)were identified by real-time PCR-HRM analysis.The HRM analysis result shows that curves of eight walnut milk samples and No.28-30 pecan milk samples were highly similar or overlapped with those of standard walnut,and the system was matched as walnut.However,the HRM curve and melting temperature of walnut milk sample No.25-27 overlapped with that of the standard peanut,and the matching results of the two gene systems were peanut.The adulteration concentration of mixed standard products showed that the adulteration concentration of walnut milk No.25-27 was higher than 90%,which indicated that No.25-27 pecan milk and No.28-30 pecan milk belonged to the adulterated products.(4)To identify the walnut milk by combining DNA barcode and LAMP technology: Walnut specific LAMP amplification primers group was designed based on ITS2 sequence.The primers were characterized with good specificity and sensitivity detection of 1%.The LAMP identification of nine kinds of commercially available walnut milk and one type of pecan milk were conducted.The results showed that except for No.9 brand walnut milk without LAMP characteristic bands,all the other samples had clear bands,including No.10 pecan milk,which indicated that one type of walnut milk had purposelessness provenance walnut ingredients,and another type of pecan milk contained walnuts.LAMP analysis based on specific barcode sequence can realize thermostatic,fast and accurate identification of walnut milk and provide scientific basis for plant protein beverage label identification.