Compositional Analysis of Genetically Engineered GR2E “Golden Rice” in Comparison to That of Conventional Rice

Swamy et al. (2019) Compositional Analysis of Genetically Engineered GR2E “Golden Rice” in Comparison to That of Conventional Rice. Journal of Agricultural and Food Chemistry

Abstract (to see full article, click on title)

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of β-carotene and other provitamin A carotenoids in the grain. Except for β-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89−113% and 57−99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

Compositional parameters analyzed in the paddy rice, rice straw, and bran of GR2E and control rice:

• Proximates and fiber: moisture, crude protein, crude fat, ash, acid detergent fiber (ADF), neutral detergent fiber fire (NDF), crude fiber fiber, total dietary fiber (TDF), and carbohydrate
• Minerals: calcium, phosphorus, magnesium, potassium, zinc, manganese, copper, iron, and sodium
• Vitamins: thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9), and α-tocopherol (E)
• Polysaccharides: total starch and amylose
• Fatty acids: caprylic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), pentadecanoic (C15:0), palmitic (C16:0), palmitoleic (C16:1 Δ9), heptadecanoic (C17:0), stearic (C18:0), oleic (C18:1 Δ9), linoleic (C18:2 Δ9,12), α-linolenic (C18:3 Δ9,12,15), arachidic (C20:0), eicosenoic (C20:1), eicosadienoic (C20:2Δ11,14), eicosatrienoic (C20:3 Δ11,14,17), arachidonic (C20:4 Δ5,8,11,14), behenic (C22:0), erucic (C22:1 Δ13), lignoceric (C24:0), and nervonic (C24:1 Δ15)
• Amino acids: lysine, arginine, glycine, histidine, isoleucine, leucine, phenylalanine, threonine, valine, alanine, aspartic acid, glutamic acid, proline, serine, tyrosine, cysteine, methionine, and tryptophan
• Carotenoids: β-cryptoxanthin, all-trans-α-carotene, all-trans-β-carotene, 9′-cis-β-carotene, and total carotenoids
• Bioactive compounds: phytic acid and trypsin inhibitor

Compositional parameters analyzed in the rice straw and bran of GR2E and control rice:

• Proximates and fiber: moisture, crude protein, crude fat, ash, ADF, NDF, crude fiber, and carbohydrate
• Minerals: calcium and phosphorus

Testing the performance of Golden Rice

Yes, it's still rice. But more nutritious!

A question that worries many people when dealing with genetically modified foodstuffs is whether unintended changes have taken place in the final product. For many, this eventuality —very much engrained in the imagination of people who ignore the science and the experience acquired over the years— has been the major stepping stone to the acceptance of transgenic produce. But without a concrete fear, there can't be concrete answers, and the bogeyman remains a figment in the minds of technophobes. Detailed molecular analyses have failed to find new allergens showing up as a consequence of having introduced a new gene into a plant, and determination of the expression levels of ten-thousands of genes have also shown that the only changes encountered are related to the introduced genes and those involved in related metabolic pathways. Gene expression levels vary probably less after the introduction of a foreign gene than after the traditional crossing of two varieties, where thousands of regulatory factors and pathways interact in unexpected ways.

Genetically modified plants destined for field release are some of the most meticulously examined organisms in the world, and this is not different for Golden Rice. In fact, it is one of the reasons why it has not reached the people who need it, yet. Many tests have been performed on Golden Rice and many are still being performed. A number of tests require kilogram amounts of seed, which is difficult to achieve when it has to be done in the glasshouse.

Does it taste as good? This and many other questions must be answered before Golden Rice is released at the farm level.

In this section we report on various tests conducted on Golden Rice and also on pending analyses. From the nutritional point of view the most important question is that of bioavailability. Terms like bioavailability and other are explained in the corresponding sections, but see also in the FAQs section. Not less important are retention studies, taste and cooking tests, agronomic performance, storability of the seed and other tests.

Bioavailability

Bioavailability relates to the fraction of carotene that your digestive system can extract out of a given foodstuff and make available to the organism for its utilisation. It is largely determined by the food matrix in which the nutrient is embedded. However, carotenoids can be stored in several ways in plant tissues:

• Protein-bound, stoichiometrically (in light-harvesting complexes, in green vegetables); similarly in lobsters.
• Protein-associated; by a protein that associates a proteolipid-carotenoid complex with a protein called fibrillin and homologs thereof, as in red pepper.
• Free in membranes; in chromoplasts, like in the petals of daffodils (probably etioplasts and amyloplasts).
• In plastoglobules, frequently met in flowers and algae (probably the most bioavailable form).
• In crystals, as in tomato and carrot (this is probably the least available form).
• Additional determinants may act as promoters or inhibitors of resorption.

This again means that a person can, in principle, obtain more utilisable β-carotene from an orange-fleshed sweet potato which has ca. 18 μg/g β-carotene than from carrots that have around 60 μg/g.

In a given human population there are also genetic factors that determine bioavailability of carotenoids. The expression or modification of the β-carotene receptor determines availability and transport into the blood serum (and probably also into cells).

It has now been clearly demonstrated that, contrary to what detractors from the technology would have liked us to believe, Golden Rice is more than capable of providing the necessary β-carotene to the target population. A group of 68 Chinese children aged 6-8 years were fed Golden Rice, spinach, or administered a given amount of β-carotene in oil. Blood samples were analysed to determine how much of the β-carotene was actually being extracted from the food or the oil. The latter is known to be the most effective way to present this vitamin to the human organism. The result was that β-carotene in Golden Rice is as effective as pure β-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of about 100 to 150 g cooked Golden Rice (50 g dry weight) can provide about 60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-year-old children. The detailed account of this trial can be read in a paper published in the American Journal of Clinical Nutrition .

Bioconversion is the fraction of available β-carotene converted to the active form, ie, retinol or vitamin A.

Bioefficacy is the capability of an individual —governed by individual internal factors— to convert β-carotene (provitamin A) into retinal, retinol (vitamin A) and retinyl esters. It is expressed as the amount of β-carotene required to yield one retinol activity equivalent (1 RAE).

The best way to determine bioavailability is by feeding human subjects and measuring the levels of retinol (vitamin A) in the blood. For an excellent review on the determination of bioavailability and bioconversion see Yeum and Russell (2002).

• Yeum K-J and Russell RM (2002) Carotenoid bioavailability and bioconversion. Annual Review of Nutrition 22:483-504.
• Tanumihardjo SA (2002) Factors influencing the conversion of carotenoids to retinol: bioavailability to bioconversion to bioefficacy. Interntional Journal of Vitamins and Nutriets Research 72:40-45.

Taste

Uncooked Golden Rice has an attractive yellow to orange colour, which fades after cooking due to the loss of translucency of the starch the grain. The β-carotene should not affect the taste at the concentration levels present in Golden Rice. This will have to be confirmed by sensory panels before commercial release.

Taste trials not only involve an elaborate sensory analysis using its own descriptive language, but it also makes use of advanced technologies, such as gas chromatography and mass spectrometry to identify volatile components. For more details you may consult the cited literature below. Trials have also been delayed by the reduced availability of Golden Rice seed and the ethical and safety requirements involved in the testing of genetically modified foodstuffs using human subjects.

• Champagne ET, Bett KL, Vinyard BT, McClung AM, Barton FE, Moldenhauer K, Linscombe S, McKenzie KS (1999) Correlation between cooked rice texture and rapid visco-analyser measurements. Cereal Chemistry 76:764-771.
• Goodwin HL, Jr, Koop LA, Rister ME, Miller RK, Maca JV, Chambers E, Hollingsworth M, Bett K, Webb BD, McClung AM (1996) Developing a common language for the US rice industry: Linkages among breeders, producers, processors and consumers. TAMRC Consumer Product Market Research CP2-96. Texas A&M: College Station, TX.
• Lyon BG, Champagne ET, Vinyard BT, Windham WR, Barton FE, Webb BD, McClung AM, Moldenhauer KA, McKenzie KS, Kohlwey DE (1999) Effects of degree of milling, drying condition, and final moisture content on sensory texture of cooked rice. Cereal Chemistry 76:56-62.
• Meilgaard M, Civille GV, Carr BT (1999) Sensory Evaluation Techniques. CRC Press: Boca Raton, FL.
• Surles RL, Weng N, Simon PW, Tanumihardjo SA (2004) Carotenoid profiles and consumer sensory evaluation of specialty carrots (Daucus carota, L) of various colors. Journal of Agricultural Food Chemistry 52:3417-3421.

Agronomic Performance

It would be very hard to convince a farmer to adopt Golden Rice just because of improved nutritional quality unless yield and other agronomic characteristics were at least as good or better than their best varieties. This is especially understandable in developing countries, where population density is high and farms are small. Moreover, consumers in those countries should not and would not be able to pay a premium for Golden Rice.

Carotenoids are isoprenoid derivatives, as are vitamin E, chlorophyll, and the plant hormones abscisic acid and gibberellic acid. A diversion of the pathway could affect the production of these compounds and thus a number of plant functions. Further, carotenoids are photosensitive compounds involved in protecting the plant from excessive sun radiation. For these reasons, it is very important to determine whether the expression of the introduced genes has any deleterious effects on the agronomic performance of the rice plants.

The first Golden Rice field trial carried out in 2004 in Louisiana, gave us the first opportunity to determine its agronomic performance under real field conditions. Previous experiments in the glasshouse had not given any indication of reduced or altered performance. But real field conditions could have led to unexpected outcomes. As opposed to the controlled conditions encountered in a glasshouse, in the field several factors can fluctuate, including temperature, humidity, light intensity, as well as other biotic and abiotic interactions.

In the Louisiana field trial, the Golden Rice plants stood side by side with control plants of the same variety. Reassuringly, Golden Rice was not distinguishable from its progenitors and other controls in a number of agronomic parameters measured (plant height, days to flowering, seed set, 100-seed weight, total biomass, etc). Meanwhile, this has been confirmed in several field trials and in different genetic backgrounds. Thus, we can feel reassured that the trait is not affecting the agronomic performance of the rice plants.

Retention Studies

The question to be answered here is, how much β-carotene is left intact after various typical ways of preparing rice, eg, boiling or frying. It has been determined that, for most vegetables, the best way to make β-carotene bioavailable, is by cooking and adding some oil. Carotenoids are highly soluble in oil, therefore addition of oil or butter to boiled vegetables makes it easier for them to be extracted from the food matrix and taken up by the intestine. In general, carotenoids are quite heat stable, hence most of them —80% or more— remain utilisable after most typical cooking procedures applied to various vegetable sources. Any losses are generally compensated by the increased bioavailability.

Beta-carotene content and stability

The first Golden Rice version (SGR1) produced an average of 1.6 μg β-carotene per gram dry-seed weight. The level of β-carotene production in the field is very efficient, possibly thanks to higher photosynthetic activity in the sunlight. Because of their chemical nature —several conjugated double bonds— carotenoids are susceptible to light and oxidation. A number of studies have been conducted to determine the effects of light and air (oxygen) of harvested, stored Golden Rice. Results are highly encouraging. Carotenoid content usually stabilises after some initial losses, leaving enough to guarantee high nutritive value even after prolonged storage.