Monitoring and traceability of genetically modified soya bean event GTS 40-3-2 during soya bean protein concentrate and isolate preparation

To evaluate DNA fragmentation and GMO quantification during soya bean protein concentrate and isolate preparation, genetically modified soya bean event GTS 40-3-2 (Roundup ReadyTM soya bean, RRS) was blended with conventional soya beans at mass percentages of 0.9%, 2%, 3%, 5% and 10%. Qualitative PCR and real-time PCR were used to monitor the taxon-specific lectin and exogenous cp4 epsps target levels in all of the main products and by-products, which has practical significance for RRS labelling threshold and traceability. Along the preparation chain, the majority of DNA was distributed in main products, and the DNA degradation was noticed. From a holistic perspective, the lectin target degraded more than cp4 epsps target during both of the two soya bean proteins preparations. Therefore, the transgenic contents in the final protein products were higher than the actual mass percentages of RRS in raw materials. Our results are beneficial to the improvement of GMO labelling legislation and the protection of consumer rights.

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Recommendation?
Accept as is Qualitative PCR and real-time PCR were used to monitor the taxon-specific lectin and exogenous cp4 epsps target levels in all of the main products and by-products, which has practical significance for RRS labelling threshold and traceability. Along the preparation chain, the majority of DNA was distributed in main products, and the DNA degradation was noticed. From a holistic perspective, the lectin target degraded more than cp4 epsps target during both of the two soybean proteins preparations. Therefore, the transgenic contents in the final protein products were higher than the actual mass percentages of RRS in raw materials. Our results are beneficial to the improvement of GMO labelling legislation and the protection of consumer rights. We hope this manuscript is suitable for "Royal Society Open Science". We would like to express our great appreciation to you and the reviewers for comments on our manuscript. Looking forward to hearing from you.   Table S1 as a supplementary material. Please refer to Table S1 for more details. Thanks again for the kind suggestions.

Reviewer #2:
The manuscript reported the investigation of genetically modified soybean during protein concentration and isolate preparations. The study needs some minor revisions: Authors' response: We appreciate the reviewer very much for the constructive comments and useful guidances on our work. According to your suggestions, we have addressed the following points seriously and amended the relevant parts in the revised manuscript. A list of response to each point was raised below carefully. Please refer to the responses to Comment 1-4 for more details.  Thanks again for the useful comments.

Comment 3:
The authors miss the controls made of only GTS 40-3-2 and conventional beans.
Authors' response: Thanks for reviewer's useful and insightful suggestions. We agree with the reviewer that the samples made of pure GTS 40-3-2 and conventional soybeans should be detected, which have been investigated previously by our team [8]. In our previous research, the mass variations of genomic DNA and length distributions of DNA fragments in pure GTS 40-3-2 and conventional soybeans and the variations in transgenic contents during soybean protein concentrate (SPC) and soybean protein isolate (SPI) preparation were monitored. The results showed that the distribution of DNA in the samples differed, and the lectin and cp4 epsps targets degraded to some extent along the preparation chains. Meanwhile, the DNA distribution and degradation thereby affected GMO quantification.
On the basis of our previous research, genetically modified soybeans with RRS mass percentages of 0.9%, 2%, 3%, 5%, and 10% were selected to further clarify the variations of RRS proportions during the SPC and SPI preparation started from raw soybean materials with different transgenic contents. The RRS mass percentages (0.9%, 2%, 3%, 5%, and 10%) used in this research were chosen according to the labelling rules worldwide. For example, the rules in Japan, Thailand, Taiwan, South Africa, and Indonesia state that any food product should be clearly labelled if it contains 5% or more GMO components [9]. The corresponding threshold values are 3% in South Korea [10] and 0.9% in the European Union and Russia [11].
Therefore, the samples made of pure GTS 40-3-2 and conventional beans were not included in this research. Thanks again for the kind suggestion.  As revealed in Fig. 1, the integrities of genomic DNAs extracted from raw soybean material (1), soybean kernel (3), and defatted soybean kernel (4) were high. While, the bands of genomic DNAs