SCREENING FOR GMO IN FERMENTED SOY SAUCE

Soy sauce is worldwide popular condiment of Asian origin. With the advent of GM soybean production, soy sauce drew the interest of food safety control. Samples collected for inspection are generally of industrial grade soy sauce type, which is produced from hydrolyzed soybean and grain. Following the failure to perform RealTime PCR based GMO screening on a number of submitted samples we tested our screening system on soy sauce produced following traditional method based on fermentation. Four batches of soy sauce were produced and DNA extracted. DNA concentration ranged from 32,68 to 65,36 ng/μl. Amplification of taxon specific target was successful with rather high Ct ( > 30). Promoter P-35S sequence was not detected, but T-NOS was detected in three samples with values reaching or exceeding LOD of the method. The results show that it is possible to detect transgenic elements in traditionally produced soy sauce while DNA extraction from industrial grade soy sauce is not possible.


Introduction
Soy sauce is an Asian condiment that originated in China about 2500 years ago.Its use was later spread to the other parts of Asia while, it was brought to Europe in the 17th century.Its recognition in the Western societies followed the rise in popularity of Asian cuisine.Traditional soy sauce is mature, fermented product, made from soybeans, grains and salt.The production process is slow and does not achieve industrial scale.As current cumulative consumption greatly surpasses the capacities of traditional production, industrial grade soy sauce is produced from hydrolyzed soy protein and grain.While this process results in affordable product of satisfactory sensory qualities, acid hydrolyses renders this type of product inaccessible for DNA based analysis such as those applied in food safety sector.With the increasing presence of GM soybean in food supply, soy sauce became an object of scrutiny from food safety perspective.
According to ISAAA report 83% (92.1 million hectares) of the 111 million hectares of the globally planted soy are biotech (James, 2015).Legal framework on GMO of the European Union and Law on GMO in Bosnia and Herzegovina (2009) require labeling of products that contain more than 0.9 % GM content per species.Successful implementation of any legal framework depends on the abilities of analytical laboratories to provide correct information.Therefore, official laboratories utilize validated analytical methods and DNA based methods are the methods of choice when it comes to GMO analysis (Anklam et al., 2002).Unfortunately, validation is generally performed on general type of matrix such as grain or flour and does not guarantee comparable performance on different type of matrix.Thus, when DNA extraction from industrial grade soy sauce proved to be challenging, we decided to explore the matrix rather than alter the method.We hypothesized that the DNA extraction method validated for soybean would provide sufficient quantity of PCR grade DNA from soy sauce produced by traditional method.

Materials and methods
We prepared soy sauce by traditional method in order to test the capacity of CTAB DNA extraction procedure validated for soybean to yield sufficient quantity of PCR grade DNA.
The production method was derived by analyzing a number of traditional recipes and extrapolating common ingredients and steps.All ingredients were procured locally.Soybean, being the target ingredient, was obtained from different manufacturers (Tab.1).Raw soybeans were soaked in water for 15 hours and then boiled for about an hour.Cooked and smashed soybeans were mixed with ca.half the amount of wheat flour to form a homogeneous mixture.The obtained mixture was divided in smaller cakes and left for about 20 days in a warm and humid place and allowed for mold to develop.Following the development of mold, the cakes were dried in a warm place and soaked in salt brine (6 % NaCl solution).Fermentation was allowed to proceed slowly with occasional agitation.The fully fermented mixture was filtered to remove the solids while liquid soy sauce was used for DNA extraction and GMO screening.

Results and discussion
Following traditional recipe we prepared four batches of soy sauce originating from different sources.Some physical and chemical characteristics of the obtained soy sauces are listed in Tab. 2. While soy sauces made by industrial procedure have liquid consistency, dark color and sour or salty flavor our traditional products have thicker consistency and lighter color.High Ct values for lectine (30.69-34.53)indicated low concentration of target DNA but still within limit of detection (LOD) of the method.All samples tested negative for p-35S, while 3 samples tested positive for t-NOS.High Ct values for t-NOS reactions (34.49-36.30)indicated that concentration of the target DNA approaches LOD of the method (LOD≥4C).Considering LOD of the t-NOS method only sample 1/16 tested positive.
According to high Ct values for lectine we can conclude that concentration of target DNA was low which reflected to Ct values for t-NOS.Additional optimization of DNA extraction protocol such as adjustment of amount of the starting material and additional purification step with chloroform are necessary to increase target DNA yield.(34.49-36.30)indicated that concentration of the target DNA approaches LOD of the method which is bellow the labeling threshold and only sample 1/16 can be considered as positive.The results show that it is possible to detect transgenic elements in soy sauce made following traditional recipe while DNA extraction from industrial soy sauce is not possible.

Table 1 .
List of analyzed samples

Table 2 .
Physical and chemical characteristics of soy sauces produced following the traditional recipe

Table 3 .
Ct values of RealTime PCR reactions All analyzed samples tested negative for p-35S, while t-NOS was detected in three samples.However, high Ct values for t-NOS reactions