Germinated Brown Rice

UNOFFICIAL TRANSLATION

THAI AGRICULTURAL STANDARD

TAS 4003-2012

GERMINATED BROWN RICE

National Bureau of Agricultural Commodity and Food Standards Ministry of Agriculture and Cooperatives

ICS 67.060 ISBN

THAI AGRICULTURAL STANDARD

TAS 4003-2012

GERMINATED BROWN RICE

National Bureau of Agricultural Commodity and Food Standards Ministry of Agriculture and Cooperatives 50 Phaholyothin Road, Chatuchak, Bangkok 10900 Telephone (02) 561 2277 Facsimile (02) 561 3357 www.acfs.go.th

Published in the Royal Gazette, Announcement and General Publication Volume 129, Special Section 144ง(Ngo),

Dated 19 September B.E. 2555(2012) (2) Technical Committee on the Elaboration of the Thai Agricultural Standard for Germinated Brown Rice

1. Mrs. Samlee Boonyaviwat Chairperson Rice Department 2. Mr. Ittipong Kunakonbodin Member Department of Internal Trade, Ministry of Commerce 3. Mr. Kriangkrai Krutthai Member Cooperative Promotion Department 4. Mrs. Chuensuke Methakullawat Member Office of the Consumer Protection Board, Prime Minister’s Office 5. Miss Warunee Sensupa Member Food and Drug Administration, Ministry of Public Health 6. Mr. Pisan Pongsapitch Member National Bureau of Agricultural Commodity and Food Standards 7. Mrs. Wilailuck Sukprakarn Member Bureau of Rice Product Development, Rice Department 8. Miss Sunanta Wongpiyachon Member Bureau of Rice Research and Development, Rice Department 9. Professor Onanong Naivikul Member Excellent Center of Rice Innovation, Kasetsart University 10. Mr. Aree Nachaidee Member Doi Khum Food Products Co., Ltd. 11. Mr. Samak Yingyong Member Community Enterprise of Rice Production and Processing Development for Health 12. Mr. Boonmee Surakhot Member Du Community Rice Center (Kasettip) 13. Mrs. Patcharee Tungtrakul Member Expert 14. Assoc. Prof. Voranuch Srijesdaruk Member Expert 15. Miss Ing-orn Panyakit Member and Secretary Office of Standard Development, National Bureau of Agricultural Commodity and Food Standards (3)

Germinated brown rice is a nourishing food especially containing gamma-aminobutyric acid or GABA which acts as an important neurotransmitter in the central nervous system of the body. With recent increase in numbers of germinated brown rice producers, the Agricultural Standards Committee considers that it is appropriate to establish the agricultural standard for Germinated Brown Rice to improve its product quality and safety for the consumer including contain sufficient nutrition for claims. This standard is based on the following document: TAS 4001-2008. Thai Agricultural Standard: Thai Aromatic Rice. National Bureau of Agricultural Commodity and Food Standards. Ministry of Agriculture and Cooperatives.

NOTIFICATION OF THE MINISTRY OF AGRICULTURE AND COOPERATIVES ON THE ESTABLISHMENT OF THAI AGRICULTURAL STANDARD: GERMINATED BROWN RICE UNDER THE AGRICULTURAL STANDARDS ACT, B.E. 2551 (2008)

Whereas the Agricultural Standards Committee deems it necessary to establish an agricultural standard on Germinated Brown Rice as a voluntary standard in accordance with the Agricultural Standards Act, B.E. 2551 (2008) to promote such agricultural commodity to meet its standard on quality and safety.

By virtue of Sections 5, 15 and 16 of the Agricultural Standards, Act B.E.2551 (2008), the Minister of Agriculture and Cooperatives hereby issues this Notification on the Establishment of Thai Agricultural Standard on Germinated Brown Rice (TAS 4403-2012) as a voluntary standard, details of which are attached herewith.

Notified on 6 July B.E. 2555(2012)

(Mr. Theera Wongsamut) Minister of Agriculture and Cooperatives

TAS 4003-2012

THAI AGRICULTURAL STANDARD

GERMINATED BROWN RICE

1. SCOPE This Thai Agricultural Standard covers germinated brown rice produced from non-glutinous and glutinous rice of Oryza sativa L., Gramineae family, which is undergone germination process and sold as rice kernels packed in a sealed package. This standard does not apply to other types of germinated brown rice products.

2. DEFINITIONS For the purpose of this standard, definitions given in TAS 4001 and the following shall apply: 2.1 Germinated brown rice means rice kernels undergone germination processes by soaking paddy or brown rice in water until embryo germinate to the length of0.5- 1.0 mm and heating (steam, oven or boil ) and/or drying to reduce moisture. In case of germinating from paddy, husk shall be removed after drying. 2.2 Whole germinated kernel means germinated brown rice of whole grain without any broken part. 2.3 Gamma-aminobutyric acid (GABA) means a non-protein amino acid produced from rice germination. Quantities of GABA produced are varied depending on raw material and process used in the production. 2.4 Discolour germinated kernel means germinated rice that has different color from the typical color of its variety. 2.5 Damaged kernel means germinated brown rice kernel with obvious damages as visible caused by moisture, heat, molds, insects etc. 2.6 Broken kernel means fragment of germinated brown rice with the length of five or more parts out of ten parts of the whole kernel.

2.7 Foreign matter means any substance other than the germinated brown rice, which may be presented in the germinated brown rice product such as husk, straw.

3. QUALITY

3.1 General requirements 3.1.1 characteristics of the variety with normal color; 3.1.2 free of foreign smell; 3.1.3 free of rice weevils, other insects or insect fragments; 3.1.4 free of harmful foreign objects to consumers such as stone and glass fragments.

TAS 4003-2012 2

3.2 Specific requirements 3.2.1 Contain whole germinated brown rice kernel not less than 90% by weight; 3.2.2 Moisture content not exceeding 14% by weight; 3.2.3 GABA content not less than 5 mg per 100 g on dry basis.

3.3 Defects Allowance for defects is shown in Table 1. However, total defects of germinated brown rice shall not exceed 10% by weight.

Table 1 Defective Allowance (Section 3.3)

Defects Percentage by weight (%) 1. Damaged kernels ≤ 1.00 2. Foreign matter ≤ 0.05 3. Damaged kernels, foreign matter, discoloured ≤ 10 germinated kernels, and broken kernels

4. CONTAMINANTS

Contaminants in germinated brown rice shall comply with the relevant laws and Thai Agricultural Standard on Contaminants. 5. PESTICIDES

Pesticide residues in germinated brown rice shall comply with the relevant laws and regulations and the requirements according to the Thai Agricultural Standards on Pesticide Residues: Maximum Residues Limits (TAS 9002) and Pesticide Residues: Extraneous Maximum Residues Limits (TAS 9003).

6. HYGIENE Production and handling of germinated brown rice at each step including storage and transportation shall follow hygienic practices that comply with TAS 9023; Thai Agricultural Standard on Code of Practice: General Principles of Food Hygiene and TAS 4404: Good Manufacturing Practices for Germinated Brown Rice so as to prevent contamination that may be harmful to consumers.

7. PACKAGING Germinated brown rice shall be packed in a dry, clean, and sealed package or by vacuum packing to prevent contamination harmful to consumers and able to keep its quality during storage and transportation. 3 TAS 4003-2012 8. LABELLING Labelling shall comply with the relevant laws. At least the following information shall appear on accompanying document or package. They shall be easily and clearly visible without false or deceptive information.

8.1 Product name shall be identified as “germinated brown rice”, “germinated rice”, germinated rice from brown rice”, “germinated rice from paddy” or “ Kao-Hang-Ngok”; 8.2 Net weight in gram or kilogram; 8.3 Name and address of the producer, packer and/or distributor, and product’s code number (if any); 8.4 Date of production and/or statement “best before date” (dd/mm/yy)”; 8.5 Lot number (can be specified by code); 8.6 Instruction for storage before and after opening the package, including preparation; 8.7 Thai language shall be used. In case of production for export, label in foreign languages with the same meaning as mentioned above can be used.

9. METHODS OF SAMPLING Sampling methods of germinated brown rice for analysis according to Section 3 appear in Appendix A. Sampling methods other than those specified in Appendix A shall be in accordance with the relevant laws.

10. METHODS OF ANALYSIS

10.1 Methods of analysis shall be as indicated in Table 2 (referring to the latest edition) shall apply:

Table 2 Methods of analysis (Section 10.1)

Provision Method Principle

1. General requirements Visual and sensory inspection - (Section 3.1) (Appendix B, Section 1.1) 2. Moisture content AOAC 925.10 or other Gravimetry (Section 3.3.2) equivalent methods (Appendix B, Section 2) 3. Defects (Section3.3) Sieving germinated brown 1. Subjective measurement rice sample to separate the 2. Gravimetry defects and weighing (Appendix B, Section 1.2) 4. GABA content Sample preparation, 1. High Performance (Section 3.2.3) amino acid extraction; Liquid hromatography analysis with HPLC (HPLC) (Appendix B, Section 3) 2. Fractionating or other equivalent methods 5. Net weight Weighing with a scale Gravimetry 10.2 Besides those mentioned above, other analytical methods required shall be in accordance with the provisions of the relevant laws and the requirements of the Thai Agricultural Standards on sampling and analysis. TAS 4003-2012 4 APPENDIX A METHOD OF SAMPLING (Section 9)

A.1 Definitions Definitions of Terms used in this standard are as the following: A.1.1 Sampling means the procedure of drawing samples. A.1.2 Lot means a set of commodity delivered at one time and known, or presumed by the sampling officer to be uniform characteristics such as source of origin, producer, type of product packer, type of package, mark and transporter.

A.1.3 Sampling plan means a design of sampling scheme which includes sampling techniques and sample sizes to be drawn from the lot for inspection to obtain adequate information to accept the lot.

A.2 Sampling Procedure Sampling of germinated brown rice shall be carried out in order to obtain the representative of the lot. Samples shall be randomly and distributely drawn throughout the lot. All samples are thoroughly mixed and reduced to the proper laboratory sample size sufficient for quality determination. The number of samples shall be in accordance with Table A1, A2, and A3. However, the laboratory samples shall be kept for verification in case of any problem arises.

A.3 Sampling and acceptance number

Sampling and acceptance number shall conform to the sampling plan designed as the or other sampling plan with the same technical equivalence..

A.3.1 Sampling and acceptance number for quality determination (Section 3): (1) Random sampling from the same lot with the sample size specified in Table A.1; (2) The lot of germinated brown rice shall be considered to meet the requirements if the nonconforming samples in section 3 not exceeding the specified acceptance number in Table A.1. A.3.2 Sampling and acceptance number for analysis of contaminants, pesticide residues and gamma-amino butyric acid:

(1) equally take product samples from each package drawn in accordance with A.3.1, then mix thoroughly to obtain total weight not less than 1 kg; (2) the lot of germinated brown rice shall be considered to meet the requirements if the results of sample analysis conformed with Sections 3.2.3, 4 and 5.

5 TAS 4003-2012

Table A.1 Sample sizes and acceptance numbers for the product package with net weight of less than 1 kg. (Section A.3.1) Lot Size Sample Size Acceptance Number (units of package) (units of package) (units of nonconforming package) 4 800 or less 6 1 4 801 to 24 000 13 2 24 001 to 48 000 21 3 48 001 to 84 000 29 4 84 001 to 144 000 38 5 144 001 to 240 000 48 6 more than 240 000 60 7

Table A.2 Sample sizes and acceptance numbers for the product package with net weight of over 1 kg but less than 4.5 kg.

(Section A.3.1) Lot Size Sample Size AcceptanceNumber (units of packages) (units of packages) (units of nonconforming packages) 2 400 or less 6 1 2 401 to 15 000 13 2 15 001 to 24 000 21 3 24 001 to 42 000 29 4 42 001 to 72 000 38 5 72 001 to 120 000 48 6 more than 120 000 60 7

TAS 4003-2012 7

Table A.3 Sample sizes and acceptance numbers for the product package with net weight of more than 4.5 kg.

(Section A.3.1) Lot Size Sample Size Acceptance Number (units of packages) (units of packages) (units of nonconforming packages) 600 or less 6 1 601 to 2 000 13 2 2 001 to 7 200 21 3 7 201 to 15 000 29 4 15 001 to 24 000 38 5 24 001 to 42 000 48 6 more than 120 000 60 7

Source : CODEX STAN 233-1969: CODEX SAMPLING PLAN FOR PREPACKAGED FOODS (AQL 6.5).

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APPENDIX B METHOD OF ANALYSIS (Section 9)

B.1 Determination of Defects B.1.1 Discolour kernels, damaged kernels, and broken kernels (1) Weigh the laboratory sample of germinated brown rice at least 100 g. (2) Select discolour kernels, damaged kernels and broken kernels from the sample and weigh. B.1.1.1 Calculate percentage of discolour kernels and broken kernels as follows Discolour and broken kernels (%) = (weight of discolour kernels+weight of broken kernels) x 100 total weight of germinated brown rice sample

B.1.1.2 Calculate percentage of damaged kernels as follows: Damaged kernels (%) = weight of damaged kernels x 100 total weight of germinated brown rice sample B.1.2 Foreign matter B.1.2.1 Apparatus (1) Blower (2) Sieve B.1.2.2 Determination (1) Weigh the laboratory sample of germinated brown rice at least 100 g; (2) Sieve the sample and separate the light foreign matter such as husk, straw, pedicel, and undeveloped kernels from the sample by using blower. (3) If foreign matter remains, separate by visual inspection with naked eyes, record the weight, and calculate the percentage of foreign matter as follows:

Foreign matter (%) = total weight of foreign matter x 100 total weight of germinated brown rice sample B.2 Moisture Content B.2.1 Apparatus (1) Hot air oven (2) Balance with 4 decimal digits (measurable down to 0.0001 g.) (3) Desiccator (4) Grinding machine 80-100 mesh (5) Aluminum dish with cover

Remark: In case of determination by other methods such as moisture analyzer, the analyzer has to be calibrated or approved by the Ministry of Commerce or other competent authorities. 9 TAS 4003-2012 B.2.2 Determination B.2.2.1 Grind sample to obtain flour. B.2.2.2 Place lid under aluminum dish, dry in a hot air oven at 130 ± 3C for 1 h, cool in a desiccator, then weigh the dish and lid. B.2.2.3 Weigh flour approximately 1 g and put into the aluminum dish and weigh the dish with lid. B.2.2.4 Put the aluminum dish containing flour with the lid under in the oven to dry at 130 ± 3C for 1 h (start recording time when oven temperature reached 130C) or until having constant weight, then close the lid and cool to room temperature in a desiccator, and weigh.

B.2.3 Calculate percentage of moisture content as follows: Moisture content (%) = (B – C) x 100 (B – A) Where: A = weight of the aluminum dish with lid B = weight of the aluminum dish with lid containing flour, before drying C = weight of the aluminum dish with lid containing flour, after drying

B.3 GABA Content by High Performance Liquid Chromatography (HPLC) B.3.1 Reagents and apparatus (1) Methanol (2) Acetonitrile (3) Chloroform AR grade (4) Trifluoroacetic acid (TFA), 0.05%concentration (5) Di-sodium tetraborate buffer 0.01 M (6) 9-Fluorenylmethyl chloroformate (FOMC) solution, conc 1,000 mg/l in acetonitrile solution (HPLC grade) (7) Standard amino acids e.g. histidine, arginine, glutamine, serine, glutamic acid, GABA, alanine, proline, valine, and leucine (8) Deionized water (9) Centrifuge tube, 50 ml (10) Centrifuge (11) Vortex mixer (12) Vacuum oven (13) pipette, 10 and 20 ml (14) Micropipette; p 10, p 100, p 200, and p 1 000 (15) Nylon membrane syringe filter, 0.2 micron (16) Syringe, 5 ml (17) HPLC column: water Symmetry C18, 5 mm, 3.9 x 150 length (18) Fluorescence Detector Jasco FP-920 TAS 4003-2012 10

(19) Waters 485 Tunable Absorbance Detector (20) Waters 600 controller pump (21) Waters 717 plus Auto-sampler (22) Waters SAT/IN Module (23) Grinding machine 80-100 mesh B.3.2 Protein extraction for amino acid (1) Finely grind germinated brown rice with grinding machine and weigh approximately 2 g put into a centrifuge tube. (2) Add 8 ml solution of methanol, chloroform and water at the ratio of 12:5:3 into the tube. (3) Shake with vortex mixer for 2 min, then centrifuge at 5,700 rpm for 15 min, repeat 3 times. (4) Transfer supernatant into a flask and evaporate, add 8 ml solution of chloroform and water at the ratio of 3:5 to the sediment in the centrifuge tube, shake with vortex mixer for 2 min, centrifuge at 5,700 rpm for 15 min, then transfer the supernatant into a flask and evaporate together with the first supernatant. (5) Evaporate the supernatant collected in the flask in the vacuum oven at 60 C until dried. (6) Dissolve the dried extract in the flask with 5 ml of deionized water. (7) Pipette 1 ml of the extracted amino acid solution in (6) and 1 ml of FOMC solution into a 5 ml volumetric flask, make up to 5 ml with sodium borate buffer, shake the flask and leave for 15 min, filter through 0.2 µm syringe nylon membrane filter into the HPLC vial, then inject 10 µl of the filtrate into the HPLC to determine the amino acid profile and content by comparison with the chromatogram of the standard amino acids.

B.3.3 Free amino acids by HPLC B.3.3.1 Reagent and apparatus (1) Methyl alcohol solution (2) Acetonitrile solution

(3) Trifluoroacetic acid solution, 0.05% concentration

(4) Di-sodium tetraborate 0.1 M adjusted to pH 10 with 8 M Sodium hydroxide

(5) 9-Fluorenylmethyl chloroformate (FOMC) solution, conc 1,000 mg/l in acetonitrile solution (HPLC grade)

(6) Standard Amino acids such as histidine, arginine, glutamine, serine, glutamic acid, GABA, alanine, proline, valine, and leucine

(7) Micropipettes; 50, 100, 200, and 1,000 µl

(8) Volumetric flasks; 5, 50, 100, 1,000, and 2,000 ml

(9) Vial with cap for HPLC 11 TAS 4003-2012

(10) Syringe Nylon Membrane Filter, 0.2 µm pore size with 13 mm diameter

(11) Nylon Membrane Filter, 0.2 µm pore size with 47 mm diameter

(12) Plastic syringe, 5 mm (13) Volumetric flasks; 5, 50, 100, 500, 1,000, and 2,000 ml (14) Guard cartridge for HPLC mobile phase (15) Analytical balance (16) HPLC (17) Fluorescence detector

B.3.3.2 HPLC running conditions (1) Symmetry reverse-phase C18 column (5 µm, 3.9 x 150 mm) (2) Control column temperature at 40C (3) Apply fluorescence detector with  excited at 270 nm and  emission at 315 nm (4) Inject sample of 10 µl (5) Conditions for gradient elution based on 3 mobile solutions with the flow rate of 1 ml/min as shown in the Table B.1.

Conditions for gradient elution based on mobile phase consist of 3 solvents/eluents with the flow rate of 1 ml/min as shown in the Table B.1.

Table B.1 Conditions for standard GABA analysis of germinated brown rice

Time (min) Flow Rate 0.05% TFA (%) Acetonitrile (%) Methanol (%) 0 1.0 65 35 0 1.5 1.0 65 35 0 3.0 1.0 80 15 5 10 1.0 55 20 25 15 1.0 55 20 25 21 1.0 35 30 35 23 1.0 40 30 30 27 1.0 0 75 25 33 1.0 65 35 0

TAS 4003-2012 12

B.3.4 Preparation of standard amino acids and standard curve Standard curve production B.3.4.1 Prepare mixed 10 standard amino acids solution from histidine, arginine, glutamine, serine, glutamic acid, GABA, proline, alanine, valine, and leucine at concentrations of 2, 5, 10, 15, 20, 25, and 30 mg/l.

B.3.4.2 Pipette 1 ml of mixed standard amino acids solution prepared in B.3.4.1 and 1 ml of FOMC solution into a 5 ml volumetric flask, make up the volume to 5 ml with sodium borate buffer solution, shake the flask and leave for 15 min, filter through 0.2µm syringe nylon membrane filter into HPLC vial, then inject the sample into HPLC to produce chromatogram of each standard amino acid against its concentration, providing peak area of each amino acid as shown in Figure B.1

Figure B.1 Retention time (RT) of the standard GABA and other free amino acids.

From Figure B.1, when inject other standard amino acids solution in cooperated with GABA analysis. It is found that RT range differently in sequencing as follows:

Histidine 2.219 min Arginine 2.644 min Glutanine 5.978 min Serine 9.520 min Glutamate 10.622 min FMOC 17.023 min GABA 22.022 min Alanine 22.474 min Proline 24.061 min Valine 26.754 min Leucine 29.779 min

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B.3.4.3 From HPLC analysis, the content of amino acids in the sample can be calculated from retention time and peak area of the standard amino acids at various concentrations. Peak areas of the standard GABA at various concentrations are presented in Table B.2 and the standard curve is illustrated in Figure B.2. ภาษาไทย

Table B.2 Peak area of the standard GABA at various concentrations. (Section B.3.4.3) Concentration Peak Area (mg/l) 5 1,817,094.00 10 4,759,386.20 15 8,532,576.70 20 140,417,115.58 30 200,970,697.00 40 800,338,636.00 50 1,200,017,351.00 60 1,400,300,548.00 70 1,700,592,202.00

Figure B.3 Standard curve of GABA at concentrations of 2, 5, 10, 15, 20, 25, and 30 mg/l from HPLC analysis, with correlation coefficient (R2) of 0.9993. TAS 4003-2012 14

Figure B.2 Standard curve of GABA at various concentrations from HPLC analysis, with correlation (R2) of 0.953.

15 TAS 4003-2012

APPENDIX C UNIT

Units and symbols used in this standard and units allowed by International System of Units or Le Systéme International d’ Unités (SI) are in the following table:

Measurement Unit Symbol nanometer nm Length micrometer µm millimeter mm centimeter cm Volume millimeter ml microliter µl Concentration milliliter per liter ml/l Mass milligram mg gram g kilogram kg Temperature degree Celsius C Time minute min hour h

NOTIFICATION OF THE MINISTRY OF AGRICULTURE AND COOPERATIVES ON THE ESTABLISHMENT OF THAI AGRICULTURAL STANDARD: GERMINATED BROWN RICE UNDER THE AGRICULTURAL STANDARDS ACT, B.E. 2551 (2008)

Notified on 6 July B.E. 2555(2012)

(Mr. Theera Wongsamut) Minister of Agriculture and Cooperatives

TAS 4003-2012

THAI AGRICULTURAL STANDARD

GERMINATED BROWN RICE

2.7 Foreign matter means any substance other than the germinated brown rice, which may be presented in the germinated brown rice product such as husk, straw.

3. QUALITY

TAS 4003-2012 2

3.3 Defects Allowance for defects is shown in Table 1. However, total defects of germinated brown rice shall not exceed 10% by weight.

Table 1 Defective Allowance (Section 3.3)

Defects Percentage by weight (%) 1. Damaged kernels ≤ 1.00 2. Foreign matter ≤ 0.05 3. Damaged kernels, foreign matter, discoloured ≤ 10 germinated kernels, and broken kernels

4. CONTAMINANTS

Contaminants in germinated brown rice shall comply with the relevant laws and Thai Agricultural Standard on Contaminants. 5. PESTICIDES

10. METHODS OF ANALYSIS

10.1 Methods of analysis shall be as indicated in Table 2 (referring to the latest edition) shall apply:

Table 2 Methods of analysis (Section 10.1)

Provision Method Principle

A.1.3 Sampling plan means a design of sampling scheme which includes sampling techniques and sample sizes to be drawn from the lot for inspection to obtain adequate information to accept the lot.

A.3 Sampling and acceptance number

Sampling and acceptance number shall conform to the sampling plan designed as the or other sampling plan with the same technical equivalence..

5 TAS 4003-2012

Table A.2 Sample sizes and acceptance numbers for the product package with net weight of over 1 kg but less than 4.5 kg.

TAS 4003-2012 7

Table A.3 Sample sizes and acceptance numbers for the product package with net weight of more than 4.5 kg.

Source : CODEX STAN 233-1969: CODEX SAMPLING PLAN FOR PREPACKAGED FOODS (AQL 6.5).

8 TAS 4003-2012

APPENDIX B METHOD OF ANALYSIS (Section 9)

B.3.3 Free amino acids by HPLC B.3.3.1 Reagent and apparatus (1) Methyl alcohol solution (2) Acetonitrile solution

(3) Trifluoroacetic acid solution, 0.05% concentration

(4) Di-sodium tetraborate 0.1 M adjusted to pH 10 with 8 M Sodium hydroxide

(5) 9-Fluorenylmethyl chloroformate (FOMC) solution, conc 1,000 mg/l in acetonitrile solution (HPLC grade)

(6) Standard Amino acids such as histidine, arginine, glutamine, serine, glutamic acid, GABA, alanine, proline, valine, and leucine

(7) Micropipettes; 50, 100, 200, and 1,000 µl

(8) Volumetric flasks; 5, 50, 100, 1,000, and 2,000 ml

(9) Vial with cap for HPLC 11 TAS 4003-2012

(10) Syringe Nylon Membrane Filter, 0.2 µm pore size with 13 mm diameter

(11) Nylon Membrane Filter, 0.2 µm pore size with 47 mm diameter

(12) Plastic syringe, 5 mm (13) Volumetric flasks; 5, 50, 100, 500, 1,000, and 2,000 ml (14) Guard cartridge for HPLC mobile phase (15) Analytical balance (16) HPLC (17) Fluorescence detector

Conditions for gradient elution based on mobile phase consist of 3 solvents/eluents with the flow rate of 1 ml/min as shown in the Table B.1.

Table B.1 Conditions for standard GABA analysis of germinated brown rice

TAS 4003-2012 12

Figure B.1 Retention time (RT) of the standard GABA and other free amino acids.

From Figure B.1, when inject other standard amino acids solution in cooperated with GABA analysis. It is found that RT range differently in sequencing as follows:

13 TAS 4003-2012

Figure B.3 Standard curve of GABA at concentrations of 2, 5, 10, 15, 20, 25, and 30 mg/l from HPLC analysis, with correlation coefficient (R2) of 0.9993. TAS 4003-2012 14

Figure B.2 Standard curve of GABA at various concentrations from HPLC analysis, with correlation (R2) of 0.953.

15 TAS 4003-2012

APPENDIX C UNIT

Units and symbols used in this standard and units allowed by International System of Units or Le Systéme International d’ Unités (SI) are in the following table:

TAS 4003-2012

THAI AGRICULTURAL STANDARD

GERMINATED BROWN RICE

3. QUALITY

This translation is made by The National Bureau of Agricultural Commodity and Food Standards (ACFS) to establish correct understanding about this standard to foreigners. Reference to Thai Agricultural Standard in any jurisdiction shall be made to the Thai version only.

2 TAS 4003-2012

3.3 Defects Allowance for defects is shown in Table 1. However, total defects of germinated brown rice shall not exceed 10% by weight.

Table 1 Defective Allowance (Section 3.3)

Defects Percentage by weight (%) 1. Damaged kernels ≤ 1.00 2. Foreign matter ≤ 0.05 3. Damaged kernels, foreign matter, discoloured ≤ 10 germinated kernels, and broken kernels

4. CONTAMINANTS

Contaminants in germinated brown rice shall comply with the relevant laws and Thai Agricultural Standard on Contaminants.

5. PESTICIDES

8. LABELLING Labelling shall comply with the relevant laws. At least the following information shall appear

3 TAS 4003-2012

on accompanying document or package. They shall be easily and clearly visible without false or deceptive information.

10. METHODS OF ANALYSIS

10.1 Methods of analysis shall be as indicated in Table 2 (referring to the latest edition) shall apply:

Table 2 Methods of analysis (Section 10.1)

Provision Method Principle 1. General requirements Visual and sensory inspection - (Section 3.1) (Appendix B, Section 1.1) 2. Moisture content AOAC 925.10 or other Gravimetry (Section 3.3.2) equivalent methods (Appendix B, Section 2) 3. Defects (Section3.3) Sieving germinated brown 1. Subjective measurement rice sample to separate the 2. Gravimetry defects and weighing (Appendix B, Section 1.2) 4. GABA content Sample preparation, 1. High Performance (Section 3.2.3) amino acid extraction; Liquid hromatography analysis with HPLC (HPLC) (Appendix B, Section 3) 2. Fractionating or other equivalent methods 5. Net weight Weighing with a scale Gravimetry

4 TAS 4003-2012

10.2 Besides those mentioned above, other analytical methods required shall be in accordance with the provisions of the relevant laws and the requirements of the Thai Agricultural Standards on sampling and analysis.

5 TAS 4003-2012

APPENDIX A METHOD OF SAMPLING (Section 9)

A.1.3 Sampling plan means a design of sampling scheme which includes sampling techniques and sample sizes to be drawn from the lot for inspection to obtain adequate information to accept the lot.

A.3 Sampling and acceptance number

Sampling and acceptance number shall conform to the sampling plan or other sampling plan with the same technical equivalence..

6 TAS 4003-2012

Table A.2 Sample sizes and acceptance numbers for the product package with net weight of over 1 kg but less than 4.5 kg.

(Section A.3.1) Lot Size Sample Size Acceptance Number (units of packages) (units of packages) (units of nonconforming packages) 2 400 or less 6 1 2 401 to 15 000 13 2 15 001 to 24 000 21 3 24 001 to 42 000 29 4 42 001 to 72 000 38 5 72 001 to 120 000 48 6 more than 120 000 60 7

7 TAS 4003-2012

Table A.3 Sample sizes and acceptance numbers for the product package with net weight of more than 4.5 kg.

Source : CODEX STAN 233-1969: CODEX SAMPLING PLAN FOR PREPACKAGED FOODS (AQL 6.5).

8 TAS 4003-2012

APPENDIX B METHOD OF ANALYSIS (Section 9)

B.1 Determination of Defects B.1.1 Discolour kernels, damaged kernels, and broken kernels (1) Weigh the laboratory sample of germinated brown rice at least 100 g. (2) Select discolour kernels, damaged kernels and broken kernels from the sample and weigh. B.1.1.1 Calculate percentage of discolour kernels and broken kernels as follows Discolour and broken kernels (%) = (weight of discolour kernels + weight of broken kernels) x 100 total weight of germinated brown rice sample

Remark: In case of determination by other methods such as moisture analyzer, the analyzer has to be calibrated or approved by the Ministry of Commerce or other competent authorities.

9 TAS 4003-2012

B.2.2 Determination B.2.2.1 Grind sample to obtain flour. B.2.2.2 Place lid under aluminum dish, dry in a hot air oven at 130 ± 3C for 1 h, cool in a desiccator, then weigh the dish and lid. B.2.2.3 Weigh flour approximately 1 g and put into the aluminum dish and weigh the dish with lid. B.2.2.4 Put the aluminum dish containing flour with the lid under in the oven to dry at 130 ± 3C for 1 h (start recording time when oven temperature reached 130C) or until having constant weight, then close the lid and cool to room temperature in a desiccator, and weigh. B.2.3 Calculate percentage of moisture content as follows:

Moisture content (%) = (B – C) x 100 (B – A) Where: A = weight of the aluminum dish with lid B = weight of the aluminum dish with lid containing flour, before drying C = weight of the aluminum dish with lid containing flour, after drying

B.3.2 Protein extraction for amino acid

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(1) Finely grind germinated brown rice with grinding machine and weigh approximately 2 g put into a centrifuge tube. (2) Add 8 ml solution of methanol, chloroform and water at the ratio of 12:5:3 into the tube.

(3) Shake with vortex mixer for 2 min, then centrifuge at 5,700 rpm for 15 min, repeat 3 times. (4) Transfer supernatant into a flask and evaporate, add 8 ml solution of chloroform and water at the ratio of 3:5 to the sediment in the centrifuge tube, shake with vortex mixer for 2 min, centrifuge at 5,700 rpm for 15 min, then transfer the supernatant into a flask and evaporate together with the first supernatant. (5) Evaporate the supernatant collected in the flask in the vacuum oven at 60 C until dried. (6) Dissolve the dried extract in the flask with 5 ml of deionized water. (7) Pipette 1 ml of the extracted amino acid solution in (6) and 1 ml of FOMC solution into a 5 ml volumetric flask, make up to 5 ml with sodium borate buffer, shake the flask and leave for 15 min, filter through 0.2 µm syringe nylon membrane filter into the HPLC vial, then inject 10 µl of the filtrate into the HPLC to determine the amino acid profile and content by comparison with the chromatogram of the standard amino acids.

B.3.3 Free amino acids by HPLC B.3.3.1 Reagent and apparatus (1) Methyl alcohol solution (2) Acetonitrile solution (3) Trifluoroacetic acid solution, 0.05% concentration (4) Di-sodium tetraborate 0.1 M adjusted to pH 10 with 8 M Sodium hydroxide (5) 9-Fluorenylmethyl chloroformate (FOMC) solution, conc 1,000 mg/l in acetonitrile solution (HPLC grade) (6) Standard Amino acids such as histidine, arginine, glutamine, serine, glutamic acid, GABA, alanine, proline, valine, and leucine (7) Micropipettes; 50, 100, 200, and 1,000 µl (8) Volumetric flasks; 5, 50, 100, 1,000, and 2,000 ml (9) Vial with cap for HPLC (10) Syringe Nylon Membrane Filter, 0.2 µm pore size with 13 mm diameter (11) Nylon Membrane Filter, 0.2 µm pore size with 47 mm diameter (12) Plastic syringe, 5 mm (13) Volumetric flasks; 5, 50, 100, 500, 1,000, and 2,000 ml (14) Guard cartridge for HPLC mobile phase (15) Analytical balance (16) HPLC (17) Fluorescence detector

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Conditions for gradient elution based on mobile phase consist of 3 solvents/eluents with the flow rate of 1 ml/min as shown in the Table B.1.

Table B.1 Conditions for standard GABA analysis of germinated brown rice

B.3.4 Preparation of standard amino acids and standard curve

Standard curve production B.3.4.1 Prepare mixed 10 standard amino acids solution from histidine, arginine, glutamine, serine, glutamic acid, GABA, proline, alanine, valine, and leucine at concentrations of 2, 5, 10, 15, 20, 25, and 30 mg/l.

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Figure B.1 Retention time (RT) of the standard GABA and other free amino acids.

From Figure B.1, when inject other standard amino acids solution in cooperated with GABA analysis. It is found that RT range differently in sequencing as follows:

Table B.2 Peak area of the standard GABA at various concentrations. (Section B.3.4.3) Concentration (mg/l) Peak Area

5 1,817,094.00

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10 4,759,386.20 15 8,532,576.70 20 140,417,115.58 30 200,970,697.00 40 800,338,636.00 50 1,200,017,351.00 60 1,400,300,548.00 70 1,700,592,202.00

Figure B.3 Standard curve of GABA at concentrations of 2, 5, 10, 15, 20, 25, and 30 mg/l from HPLC analysis, with correlation coefficient (R2) of 0.9993.

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Figure B.2 Standard curve of GABA at various concentrations from HPLC analysis, with correlation (R2) of 0.953.

APPENDIX C UNIT

Units and symbols used in this standard and units allowed by International System of Units or Le Systéme International d’ Unités (SI) are in the following table:

Measurement Unit Symbol nanometer nm Length micrometer µm millimeter mm centimeter cm Volume millimeter ml microliter µl Concentration milliliter per liter ml/l Mass milligram mg

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gram g kilogram kg Temperature degree Celsius C Time minute min hour h