GRAS Notice (GRN) No.

595
http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/default.htm
ORIGINAL SUBMISSION

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 PHARMA FOODS INTERNATIONAL CO.,LTD.

1-49 Goryo-Ohara, Nishikyo-ku,
Kyoto 615-8245 Japan
PHONE : +81-75-394-8605
FAX : +81 -75-394-8889

August 3, 2015

Office of Food Additive Safety (HFS-200)

Center for Food Safety and Applied Nutrition
Food and Drug Administration
5100 Paint Branch Parkway
College Park, MD 20740-3835

Dear Sir or Madam:

Re: GRAS Notice for gamma-Aminobutyric Acid (GABA)

In accordance with proposed 21 CFR §170.36 [Notice of a claim for exemption based on a
Generally Recognized as Safe (GRAS) determination] published in the Federal Register
[62 FR18938 (17 April1997)], I am submitting, as the notifier [Pharma Foods International
Co., Ltd ., 1-49 Goryo-Ohara, Nishikyo-Ku, Kyoto, 61 5-8245,Japan], a Notice of the
determination, on the basis of scientific procedures, that gamma-Aminobutyric Acid (GABA)
derived from L-glutamate via Lactobacillus hilgardii K-3 fermentation, produced by Pharma
Foods Internationa l (PFI), as defined in the enclosed documents and CD-ROM, is GRAS
under specific conditions of use as a food ingredient, and therefore, is exempt from the
premarket approval requirements of the Federal, Food, Drug and Cosmetic Act. Information
setting forth the basis for the GRAS determination, which includes detailed information on
the notified substance, a summary of the basis for the GRAS determination, as well as a
consensus opinion of an independent panel of experts in support of the safety of GABA
under the intended conditions of use, also are enclosed for review by the agency.

I hereby certify that the enclosed electronic files for the GRAS Notice .f or gamma­
Aminobutyric Acid (GABA) entitled, "GRAS Exemption Claim for gamma-Aminobutyric acid
(GABA) for Use as a Food Ingredient in the United States (U.S.)" were scanned for viruses
prior to submission and is thus certified as being virus-free using McAfee VirusScan 8.8.

Should you have any questions or concerns regarding this GRAS Notice , please do not
hesitate to contact me at any point during the review process so that we may provide a
response in a timely manner.

Sincerely,
(b) (6)

Noriko Horie
Director of Sales & Marketing
Pharma Foods International Co ., ltd.
1-49 Goryo-Ohara, Nishikyo-Ku ,
Kyoto, 615-8245
Japan
Email: n-tani@pharmafoods.co.jp
Office phone: +81 75 394 8600
Office fax: +81 75 394 0009 [PJ~~~u~~!Q)
AUG 6 2015
OFHCE OF
FOOD ADDITIVE SAFETY
(b) (6)
GRAS Exemption Claim for gamma-Aminobutyric
acid (GABA) for Use as a Food Ingredient in the
United States (U.S.)

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GRAS Exemption Claim for a gamma-Aminobutyric

acid (GABA) for Use as a Food Ingredient in the
United States (U.S.)

Table of Contents
Page

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List of Appendices

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List of Figures and Tables

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E. Basis for GRAS Determination

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F. Availability of Information

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II. Detailed Information Regarding the Identity of the Substance

A. Identity

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Common or Usual Name: *$%$

Chemical Name: gammaDPLQREXW\ULFDFLG

Chemical Abstracts Service (CAS) Number: 

Empirical Formula: &+12

Molecular Weight: 

Structural Formula: 6HH)LJXUH,,$

Figure II.A-1 Structure of gamma-Aminobutyric Acid (GABA)

Physical Properties

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Table II.A-1 Physical Properties of gamma-Aminobutyric Acid (GABA)

Physical Property Value Temperature (°C) Source
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B. Method of Manufacture

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Figure II.B-1 Schematic Overview of the Manufacturing Process for gamma-

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Table II.B-1 List of Raw Materials and Processing Aids Used in the Manufacture of
GABA
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C. Specifications and Analytical Data

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DQGDVKUHVSHFWLYHO\ 6SHFLILFDWLRQVIRUKHDY\PHWDOVDQGPLFURELDOFRQWDPLQDWLRQDUH
DSSURSULDWHIRUXVHDVDIRRGLQJUHGLHQW $HURELFEDFWHULDDQG\HDVWDQGPROGDUHOLPLWHGE\
TXDOLW\FRQWUROSURFHGXUHVDQGDUHEHORZWKHHVWDEOLVKHGVSHFLILFDWLRQVRIDQG
&)8JUHVSHFWLYHO\

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Table II.C-1 Product Specifications for GABA

Specification Parameter Specification Method of Analysis
Identity
$SSHDUDQFH /LJKW\HOORZWR 9LVXDODSSHDUDQFH
OLJKWEURZQ
SRZGHU
gamma$PLQREXW\ULFDFLG 1/7 $PLQRDFLGDQDO\VLVZLWK+3/&EDVHGRQ%LDQFKLet al.
*$%$ 
0RLVWXUH  +HDWHGDLUGU\LQJDWQRUPDOSUHVVXUHPHWKRGƒ&KRXUV
EDVHGRQ0+/:S
$VK  'LUHFWDVKLQJPHWKRGWRƒ&KRXUVZLWKRXWVXOIXULF
DFLGEDVHGRQ0+/:S
Heavy Metals
7RWDO+HDY\0HWDOV —JJ 6RGLXPVXOILGHFRORULPHWU\EDVHGRQ0+/:S
/HDG —JJ $WRPLF$EVRUSWLRQ6SHFWURVFRS\EDVHGRQ0+/:
S
Microbiological Analysis
7RWDO$HURELF&RXQWV &)8J 0HWKRGVHVWDEOLVKHGE\)RRG+\JLHQH*XLGDQFHHGLWHGE\
-DSDQ)RRG+\JLHQH$VVRFLDWLRQ0+/:S
<HDVWDQG0ROG &)8J
&ROLIRUPEscherichia coli 1HJDWLYH

&)8 FRORQ\IRUPLQJXQLWV''7&$J VLOYHUGLHWK\OGLWKLRFDUEDPDWH+3/& KLJKSHUIRUPDQFHOLTXLG

FKURPDWRJUDSK\0+/: 0LQLVWU\RI+HDOWK/DERUDQG:HOIDUH-DSDQ-DSDQ¶V6SHFLILFDWLRQVDQG
WK
6WDQGDUGVIRU)RRG$GGLWLYHV HG1/7 QRWOHVVWKDQ

$GGLWLRQDOO\3),¶V*$%$ZLOODOVREHXVHGDVDUHIRUPXODWHGSURGXFWLQZKLFK*$%$QRWOHVV
WKDQSXULW\ZLOOEHGLOXWHGDSSUR[LPDWHO\IROGZLWK)/20$;Œ6WDUFKWRSURGXFHD
*$%$SURGXFWRIQRWOHVVWKDQSXULW\*$%$ 7KHSURGXFWVSHFLILFDWLRQVIRU*$%$
DUHSUHVHQWHGLQ7DEOH,,&

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Table II.C-2 Product Specifications for GABA 20

Specification Parameter
Identity
$SSHDUDQFH
gamma$PLQREXW\ULFDFLG
Specification Method of Analysis
:KLWHWROLJKW\HOORZ 9LVXDODSSHDUDQFH
SRZGHU
1/7 $PLQRDFLGDQDO\VLVZLWK+3/&EDVHGRQ%LDQFKLet al.
*$%$ 
0RLVWXUH  +HDWHGDLUGU\LQJDWQRUPDOSUHVVXUHPHWKRGƒ&
KRXUVEDVHGRQ0+/:S
$VK  'LUHFWDVKLQJPHWKRG WRƒ&KRXUVZLWKRXW
VXOIXULFDFLGEDVHGRQ0+/:S
Heavy Metals
7RWDO+HDY\0HWDOV —JJ 6RGLXPVXOILGHFRORULPHWU\ EDVHGRQ0+/:S
Microbiological Analysis
7RWDO$HURELF&RXQWV &)8J 0HWKRGVHVWDEOLVKHGE\)RRG+\JLHQH*XLGDQFHHGLWHGE\
-DSDQ)RRG+\JLHQH$VVRFLDWLRQ0+/:S
<HDVWDQG0ROG &)8J
&ROLIRUPEscherichia coli 1HJDWLYH
&)8 FRORQ\IRUPLQJXQLWV''7&$J VLOYHUGLHWK\OGLWKLRFDUEDPDWH+3/& KLJKSHUIRUPDQFHOLTXLG
FKURPDWRJUDSK\0+/: 0LQLVWU\RI+HDOWK/DERUDQG:HOIDUH-DSDQ-DSDQ¶V6SHFLILFDWLRQVDQG
WK
6WDQGDUGVIRU)RRG$GGLWLYHV HG1/7 QRWOHVVWKDQ

5HSUHVHQWDWLYHORWVDUHURXWLQHO\DVVD\HGWRHQVXUHFRPSOLDQFHZLWKILQDOSURGXFWFKHPLFDO
SK\VLFDODQGPLFURELRORJLFDOVSHFLILFDWLRQV $QDO\VLVRIQRQFRQVHFXWLYHORWVRI*$%$DQG
*$%$GHPRQVWUDWHVWKDWWKHPDQXIDFWXULQJSURFHVVSURGXFHVDFRQVLVWHQWSURGXFWWKDW
FRQIRUPVWRWKHHVWDEOLVKHGVSHFLILFDWLRQV 7KHUHVXOWVRIWKHEDWFKDQDO\VHVDUHSUHVHQWHGLQ
7DEOHV,,&DQG,,&IRU*$%$DQG*$%$UHVSHFWLYHO\DQGWKH&HUWLILFDWHVRI$QDO\VLV
DUHSURYLGHGLQ$SSHQGL[%$WWDFKPHQW%DQG%UHVSHFWLYHO\

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Table II.C-3 Analysis of 3 Manufactured Lots of GABA

Specification Parameter Specification Lot Number/Date of Manufacture
2J10/ 4B06/ 4E21/
10/10/2012 2/6/2014 5/21/2014
Identity
$SSHDUDQFH /LJKW\HOORZWROLJKWEURZQ &RQIRUPV &RQIRUPV &RQIRUPV
SRZGHU
*$%$ 1/7   
0RLVWXUH    
$VK    
Heavy Metals
7RWDO+HDY\0HWDOV—JJ    
/HDG—JJ    
$UVHQLF—JJ    
Microbiological Quality
7RWDO$HURELF&RXQWV    
&)8J
<HDVWDQG0ROG&)8J    
&ROLIRUPEscherichia coli 1HJDWLYH 1HJDWLYH 1HJDWLYH 1HJDWLYH
&)8 FRORQ\IRUPLQJXQLWV*$%$ gammaDPLQREXW\ULFDFLG1/7 QRWOHVVWKDQ

Table II.C-4 Analysis of 3 Manufactured Lots of GABA 20

Specification Parameter Specification Lot Number/Date of Manufacture
6A31/ 6B07/ 7F15/
1/31/2006 2/7/2006 6/15/2007
Identity
$SSHDUDQFH :KLWHWROLJKW\HOORZSRZGHU &RQIRUPV &RQIRUPV &RQIRUPV
*$%$ 1/7   
0RLVWXUH    
$VK    
Heavy Metals
7RWDO+HDY\0HWDOV—JJ    
$UVHQLF —JJ    
Microbiological Quality
7RWDO$HURELF&RXQWV    
&)8J
<HDVWDQG0ROG&)8J    
&ROLIRUPEscherichia coli 1HJDWLYH 1HJDWLYH 1HJDWLYH 1HJDWLYH
&)8 FRORQ\IRUPLQJXQLWV*$%$ gammaDPLQREXW\ULFDFLG1/7 QRWOHVVWKDQ

&RPSRVLWLRQDODQDO\VLVRIRQHORWRI*$%$ZDVFRQGXFWHGDVVKRZQLQ7DEOH,,&LQGLFDWLQJ
WKDW3),¶VLQJUHGLHQWFRPSULVHVQRWOHVVWKDQ*$%$ZLWKWKHUHPDLQLQJPDWHULDOFRPSRVHG
RIVPDOODPRXQWVRIIUHHDPLQRDFLGVDVZHOODVPRLVWXUHDVK

3KDUPD)RRGV,QWHUQDWLRQDO&R/WG 
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FDUERK\GUDWHVDQGOLSLGV” 6RGLXPFKORULGHDFFRXQWVIRUDSSUR[LPDWHO\RIWKH

WRWDODVKDQGQRPLQHUDOVZHUHSUHVHQWDWOHYHOVWKDWZRXOGEHRIWR[LFRORJLFDOFRQFHUQ

Table II.C-5 Compositional Analysis of GABA

a
Analysis Parameter Results (g/100 g) Method of Analysis
Compositional Parameters
gamma$PLQREXW\ULFDFLG  $PLQRDFLGDQDO\VLVZLWK+3/&
7RWDODPLQRDFLGV  $FLGK\GURO\VLVZLWK+3/&
$VK  'LUHFWLQFLQHUDWLRQPHWKRG
0RLVWXUH  +HDWHGDLUGU\LQJDWQRUPDOSUHVVXUH
'LHWDU\ILEHU  (Q]\PHZHLJKWPHWKRG
7RWDOVDFFKDULGH  3KHQROVXOIXULFDFLGPHWKRG
/LSLGV ” $FLGGHFRPSRVLWLRQPHWKRG
0RQRJO\FHULGHV 1' *DVFKURPDWRJUDSK\
TOTAL 101
Minerals
6RGLXPFKORULGH  $WRPLFDEVRUSWLRQVSHFWURPHWHUPHWKRG
6RGLXP  $WRPLFDEVRUSWLRQVSHFWURPHWHUPHWKRG
3RWDVVLXP  $WRPLFDEVRUSWLRQVSHFWURPHWHUPHWKRG
3KRVSKRUXV  ,&3PHWKRG
&DOFLXP  ,&3PHWKRG
0DJQHVLXP  ,&3PHWKRG
,URQ  ,&3PHWKRG
=LQF  ,&3PHWKRG
0DQJDQHVH  ,&3PHWKRG
&RSSHU 1' ,&3PHWKRG
TOTAL MINERAL CONTENT 3.52
*$%$ gammaDPLQREXW\ULFDFLG+3/& KLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\

$QDO\VLVRIWKHWRWDODFLGK\GURO\]HGIUHHDPLQRDFLGFRQWHQWWKURXJKXVLQJKLJKSHUIRUPDQFH
OLTXLGFKURPDWRJUDSK\+3/&IRXQGWKDWWKHLQJUHGLHQWW\SLFDOO\FRQWDLQVDYDULHW\RIIUHHDPLQR
DFLGVDQGGLSHSWLGHVWKDWRULJLQDWHIURPWKHIHUPHQWDWLRQPHGLXPWRZKLFKWKHPDMRULW\ZDV
FRPSULVHGRIXQPHWDEROL]HGJOXWDPDWHRULJLQDWLQJIURPWKHIHUPHQWDWLRQSURFHVV
7DEOH,,& 6PDOODPRXQWVRIDGGLWLRQDODPLQRDFLGVIURPWKHIHUPHQWDWLRQPHGLXPZHUH
DOVRSUHVHQWHDFKDWOHYHOVEHORZ )XUWKHUDQDO\VLVRIWKHWRWDODFLGK\GURO\]HGDPLQR
DFLGFRQWHQWRI*$%$FRUUHVSRQGHGRQDZHLJKWWRZHLJKWEDVLVWRWKHWRWDOIUHHDPLQRDFLGDQG
GLSHSWLGHFRQWHQWRIWKHLQJUHGLHQWLQGLFDWLQJWKDWWKHSURGXFWLVIUHHRIFRQWDPLQDWLQJSURWHLQV

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Table II.C-6 Detailed Amino Acid Analysis of GABA

a,
Analysis Parameter Results (g/100 g)
Total Amino Acids Free Amino Acid Content Polypeptide Content
(Acid Hydrolysis)
$UJLQLQH   1'
/\VLQH   
+LVWLGLQH   1'
3KHQ\ODODQLQH   1'
7\URVLQH   1'
/HXFLQH   1'
,VROHXFLQH   
0HWKLRQLQH   
9DOLQH   1'
$ODQLQH   1'
*O\FLQH   
3UROLQH   1'
*OXWDPLFDFLG   
6HULQH   
7KUHRQLQH   
$VSDUWLFDFLG   
7U\SWRSKDQ 1' 1' 1'
&\VWHLQH  1' 
TOTAL 7.00 4.12 2.88
Total Amino Acid Content 7.00 7.00
*$%$ gammaDPLQREXW\ULFDFLG1' QRWGHWHFWHG
D
$PLQRDFLGDQGSRO\SHSWLGHDQDO\VLVZDVFRQGXFWHGXVLQJKLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\

(OLPLQDWLRQRIWKHVRXUFHRUJDQLVPL. hilgardii .IURPWKHILQDOSURGXFWLVDFKLHYHGWKURXJK

WKHDSSHUWL]DWLRQSURFHVVDQGVXEVHTXHQWILOWHUVWHULOL]DWLRQVWHSV 0RUHRYHUODFWLFDFLGEDFWHULD
VXFKDVL. hilgardii DUHKLJKO\VHQVLWLYHWRR[LGDWLYHHQYLURQPHQWVDQGWKXVLWLVXQOLNHO\WKDWWKH
RUJDQLVPZRXOGVXUYLYHWKHR[LGDWLYHFRQGLWLRQVRIVSUD\GU\LQJIXUWKHUHQVXULQJWKDWPLFURELDO
FRQWDPLQDWLRQRIL. hilgardii .LQWKHILQDOSURGXFWLVSUHYHQWHG $QDO\VLVRIWKHILQDO*$%$
LQJUHGLHQWFRQILUPHGWKHDEVHQFHRIL. hilgardii .VHH$SSHQGL[&$WWDFKPHQW&
)XUWKHUPRUHLWLVQRWH[SHFWHGWKDWWKHIHUPHQWDWLRQFRQGLWLRQVZRXOGEHFRQGXFLYHWR
FRQWDPLQDWLRQRIWKHILQDOSURGXFWZLWKL. hilgardii .GHULYHGELRJHQLFDPLQHVDVPDQ\RIWKH
ELRJHQLFDPLQHSUHFXUVRUVKLVWLGLQHW\URVLQHDQGSKHQ\ODODQLQHZHUHGHWHFWHGDWPLQLPDO
FRQFHQWUDWLRQVRI”DQGDQDO\VLVRIWKH*$%$LQJUHGLHQWFRQILUPHGWKHDEVHQFHRI
KLVWDPLQHVHH$SSHQGL[&$WWDFKPHQW& $GGLWLRQDOO\DQDO\VLVRIL. hilgardii .
DVVRFLDWHGPLFURELDOE\SURGXFWVLQFOXGLQJHWK\OFDUEDPDWHDQGFLWUXOOLQHGHPRQVWUDWHGWKDWLW
ZDVQRWGHWHFWHGLQ3),¶VILQDO*$%$LQJUHGLHQWVHH$SSHQGL[&$WWDFKPHQW&&
UHVSHFWLYHO\

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$QDO\VLVRIWKHEXONVWDELOLW\RI*$%$LQWHUPVRIWKH*$%$FRQWHQWDQGPRLVWXUHGHPRQVWUDWHG
WKDWWKHILQDOSURGXFWZDVVWDEOHIRUDSHULRGRIXSWRZHHNVDWURRPWHPSHUDWXUH
7DEOH,,&

Table II.C-7 GABA Stability Following Storage at Room Temperature (20°C) for 11, 17
and 18 Weeks
Lot Number Specification (%) Initial (%) After stored at 20°C (%) Difference (%)
GABA Content
% !  DWZHHNV 
/  DWZHHNV 
.  DWZHHNV 
0RLVWXUH
%   DWZHHNV 
/   DWZHHNV 
.   DWZHHNV 
*$%$ gammaDPLQREXW\ULFDFLG

%XONVWDELOLW\VWXGLHVDOVRLQGLFDWHWKDWWKH*$%$IRUPXODWLRQLVVWDEOHIRUXSWRPRQWKV
XQGHUDPELHQWFRQGLWLRQV7DEOH,,& %DVHGRQWKLVWKHVKHOIOLIHZDVVHWDW\HDUV

Table II.C-8 Bulk Stability of GABA 20 Powder (Lot No. 4H11) Following Storage at
Room Temperature for a Period of 27 Months
Months 0 6 12 18 24 27
gamma$PLQREXW\ULF$FLG*$%$      

*$%$ZDVDOVRGHWHUPLQHGWREHVWDEOHXQGHUFRQGLWLRQVUHSUHVHQWDWLYHRILWVLQWHQGHGXVHLQ
IRRGVLQFOXGLQJDFRQFHQWUDWLRQRILQDTXHRXVVROXWLRQXQGHUYDULHGS+FRQGLWLRQVUDQJLQJ
IURPWRDQGHOHYDWHGWHPSHUDWXUHVRIWRƒ&)LJXUHV,,&WR,,& 7KXV*$%$LV
H[SHFWHGWREHVWDEOHXQGHUWKHSURSRVHGFRQGLWLRQVRIXVHLQIRRGVDQGEHYHUDJHV

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Figure II.C-1 Bulk Stability of GABA Powder (Lot No. 4H11) Under Heated Conditions
Over a Period of 60 Minutes

Figure II.C-2 Stability of a 5% GABA Solution (Lot No. 4H11) Under Heated Conditions
for a Period of 120 Minutes

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Figure II.C-3 Stability of a 5% GABA Solution (Lot No. 4H11) Under Varied pH Conditions

III. Self-Limiting Levels of Use

7KHOHYHOVRIXVHRI*$%$DUHOLPLWHGGXHWRIRRGIRUPXODWLRQLVVXHVDVWKHLQJUHGLHQWLV
H[SHFWHGWRLPSDUWDQXQGHVLUDEOHIODYRUWRWKHIRRGZKHQLQFRUSRUDWHGDWKLJKFRQFHQWUDWLRQV
6SHFLILFWKUHVKROGVIRUWKHVHHIIHFWVKDYHQRWEHHQGHWHUPLQHG

IV. Basis for GRAS Determination

'HWHUPLQDWLRQRIWKH*5$6VWDWXVRI*$%$LVRQWKHEDVLVRIVFLHQWLILFSURFHGXUHV ,QIRUPDWLRQ
VXSSRUWLQJWKHJHQHUDOUHFRJQLWLRQRIWKHVDIHXVHRI*$%$LQFOXGHV

x 'DWDSHUWDLQLQJWRWKHLGHQWLW\LQWHQGHGXVHDQGHVWLPDWHGLQWDNHRI*$%$

x %DFNJURXQGRFFXUUHQFHRI*$%$DVDQDWXUDORUDGGHGFRPSRQHQWRIYDULRXVIRRGV
FRQVXPHGLQWKH86DQG-DSDQLQFOXGLQJHVWLPDWHVRIEDFNJURXQGGLHWDU\H[SRVXUHV

x ,QIRUPDWLRQFKDUDFWHUL]LQJWKHNLQHWLFVDQGPHWDEROLFIDWHRI*$%$LQFOXGLQJGDWDRQWKH
FDSDFLW\RI*$%$WRFURVVWKHEORRGEUDLQEDUULHUIURPWKHV\VWHPLFFLUFXODWLRQ

x 7KHHQWLUHW\RIDQLPDODQGKXPDQVWXGLHVDVVHVVLQJWKHVDIHW\RI*$%$FRQVXPSWLRQ
DQG

x 'DWDSHUWDLQLQJWRWKHVDIHW\RIL. hilgardii .IRUXVHLQWKHSURGXFWLRQRIDIRRG

LQJUHGLHQW

0RUHRYHUWKHVHGDWDZHUHUHYLHZHGE\D3DQHORI([SHUWVTXDOLILHGE\VFLHQWLILFWUDLQLQJDQG
H[SHULHQFHWRHYDOXDWHWKHVDIHW\RI*$%$DVDFRPSRQHQWRIIRRGZKRFRQFOXGHGWKDWWKH

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SURSRVHGXVHVRI*$%$DUHVDIHDQGVXLWDEOHDQGZRXOGEH*5$6EDVHGRQVFLHQWLILF
SURFHGXUHV>VHH$SSHQGL[$Expert Panel Report Concerning the Generally Recognized as
Safe (GRAS) Status of gamma-Aminobutyric Acid (GABA) for Use as a Food Ingredient@.
$VXPPDU\RIWKHGDWDLVSUHVHQWHGKHUHLQ

A. Estimated Intake of GABA

Background Dietary Consumption of GABA

$VVKRZQLQ7DEOH,9$*$%$H[LVWVQDWXUDOO\LQPDQ\GLIIHUHQWIRRGVDWORZOHYHOVDQGLQ
KLJKHUOHYHOVLQIHUPHQWHGIRRGSURGXFWV ([DPSOHVRI*$%$ULFKIRRGVLQFOXGHWRPDWRHV
SRWDWRHVPHORQVDQGWKHWUDGLWLRQDO.RUHDQDQG-DSDQHVHIRRGNLPFKLDQGPLVR 7KHVHIRRGV
KDYHEHHQUHSRUWHGWRFRQWDLQIURPWRPJ*$%$JIRRGSURGXFW6WHZDUGet al.,
)R[/RLXGLFHet al., +D\DNDZDet al., 0DWXPRWRet al., 1RPXUD
et al., $NDVWX0XUFLDet al1DNDPXUDet al ,QDGGLWLRQVLQFHWKH
FRQYHUVLRQRIJOXWDPDWHWR*$%$LVUHSRUWHGIRUPDQ\ODFWLFDFLGEDFWHULDRWKHUIRRGVDUHOLNHO\
WRFRQWDLQDSSUHFLDEOHDPRXQWVRI*$%$DVDUHVXOWRIWKHSURFHVVLQJPHWKRGVXVHGWRSURGXFH
WKHPe.g.ODFWLFDFLGIHUPHQWHGIRRGVVXFKDVFXUHGPHDWVDQGFKHHVHV )RUH[DPSOH
FHUWDLQFKHHVHVVXFKDV.RPSDQVLWLFKHHVHKDYHEHHQUHSRUWHGWRFRQWDLQOHYHOVRI*$%$DV
KLJKDVPJJ

Table IV.A-1 Reported GABA Content of Various Foodsa

Food Item GABA Content Food Item GABA Content
(mg/100 g) (mg/100 g)
*UHHQ7HD/HDIGULHG WR &XFXPEHU 
0HORQ  7RIX 
7RPDWR  6R\EHDQJUHHQ 
6DQ0DU]DQR7RPDWR   &DUURW 
.LPFKLIHUPHQWHG  5DGLVK 
9HJHWDEOH-XLFH  3HUVLPPRQ 
7RPDWR-XLFH  *UHHQSHSSHU 
0LVRVR\SDVWH  *UHHQEHDQ 
3RWDWR   .LZL 
.LPFKLOLJKWO\SLFNOHG  8QSROLVKHGULFH 
*UDSHV  /HWWXFH 
(JJ3ODQW  3HDFK 
0DQGDULQ2UDQJH  2QLRQ 
5LFH9LQHJDU  6SLQDFK 
&KRFRODWH  &KLQHVHFDEEDJH 
$PDQDWVXRUDQJH  5LFHSROLVKHG 
1DYHORUDQJH  0LON 
*HUPLQDWHGULFH  &DQQHGFRIIHH 

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Table IV.A-1 Reported GABA Content of Various Foodsa

Food Item GABA Content Food Item GABA Content
(mg/100 g) (mg/100 g)
3XPSNLQ  &KHGGDU 
&DEEDJH  *RXGD 
6KLWDNHPXVKURRP  3DUPLJLDQRFKHHVH 
/RTXDW  .RPSDQLVWLFKHHVH 
%URFFROL  3RWDWR7XEHU 
D
$GDSWHGIURP6WHZDUGet al. )R[+D\DNDZDet al. 1DNDPXUDet al0DWXPRWRet al.
$NDVWX1RPXUDet al. /RLXGLFHet al. 0XUFLDet alLQGHSHQGHQWDQDO\VLV
FRQGXFWHGE\3KDUPD)RRGV,QWHUQDWLRQDO&R/WG

8VLQJDQQXDOper capita FRQVXPSWLRQGDWDIRUWKHDYDLODEOHIRRGVDERYH6WRIEHUJDQG

*UXQGVFKREHUDQGSXEOLVKHGLQIRUPDWLRQRQWKH*$%$FRQWHQWRIYDULRXVIRRGVWKH
DYHUDJH86LQWDNHRI*$%$IURPLWVQDWXUDORFFXUUHQFHZDVHVWLPDWHGWREH
PJSHUVRQGD\HTXLYDOHQWWRPJNJERG\ZHLJKWGD\IRUDNJLQGLYLGXDO:KLOHWKH
FRQVXPSWLRQGDWDLVROGWKLVYDOXHLVOLNHO\WREHDQRYHUHVWLPDWLRQRI*$%$LQWDNHVDVPRUH
UHFHQWHVWLPDWHVRIWKHEDFNJURXQGLQWDNHRI*$%$IURPQDWXUDOGLHWDU\VRXUFHVXVLQJ
-DSDQHVHVXUYH\GDWD-DSDQ1DWLRQDO+HDOWKDQG1XWULWLRQ([DPLQDWLRQ6XUYH\0+/:
DEZHUHGHWHUPLQHGWREHORZHUPJSHUVRQGD\GHVSLWH-DSDQHVHGLHWV
FRQWDLQLQJPRUHIHUPHQWHGIRRGVUHODWLYHWR1RUWK$PHULFDQGLHWV

Food Uses of GABA World-Wide

0DQ\*$%$HQULFKHGIRRGVKDYHEHHQODXQFKHGRQWRWKH-DSDQHVHPDUNHWRYHUWKHSDVW
\HDUVZLWKSRVLWLYHFRQVXPHUUHFHSWLRQ 3URGXFWVWKDWFXUUHQWO\DUHHQULFKHGZLWK*$%$LQ
-DSDQLQFOXGHWHDJHUPLQDWHGULFHSURGXFWVSLFNOHVIHUPHQWHGPLONDQGFKRFRODWHDVGHWDLOHG
LQ7DEOH,9$ 7KH*$%$FRQWHQWLQWKHVHSURGXFWVUDQJHVIURPDSSUR[LPDWHO\PJJ
LQJHUPLQDWHGULFHWRPJJLQFKRFRODWH

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Table IV.A-2 Examples of GABA-Enriched Foods and Beverages Currently Sold in Japan
Food Product Name Manufacturer GABA Content Year Launched
7HD *$%5217($ 26.,.7 PJJ 
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Table IV.A-3 Examples of Dietary Supplement Products in the U.S. Identified as

Containing GABA as a Dietary Ingredient
Product Ingredient(s) [Form] Directions for Amount of Daily Intake of
Use GABA (mg)/ GABA (mg)
serving
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Table IV.A-4 Summary of the Individual Proposed Food-Uses and Use-Levels for
gamma-Aminobutyric Acid (GABA) in the U.S. (NHANES 2011-2012 Data)
a
Food Category Food-Uses Serving Size Proposed Use Level
(per serving) (%)
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Table IV.A-5 Summary of the Estimated Daily Intake of gamma-Aminobutyric Acid

(GABA) from Proposed Food-Uses in the United States by Population
Group (NHANES 2011-2012 Data)
Population Group Age All-Person Consumption All-Users Consumption
Group (mg/person/day) (mg/person/day)
(Years) th th
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Table IV.A-6 Summary of the Estimated Daily Per Kilogram Body Weight Intake of
gamma-Aminobutyric Acid (GABA) from Proposed Food-Uses in the United
States by Population Group (NHANES 2011-2012 Data)
Population Group Age All-Person Consumption All-Users Consumption
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Family: Lactobacillaceae
Genus: Lactobacillus
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 2014/5/ 19
Pharma Foods International Co., Ltd.
1-49 Goryo-Ohara, Nishil,yo-lw,
Kyoto 615-8245, Japan
Phone: +81-75-394-8605
FAX : +81-75-394-8889

SPECIFICATION

Commodity: PharmaGABATM
Shelf Life : 2 years fr·om manufacturing date

Specification Methods

Appearance light yellow to light Visual appearance

brown powder

GABA more than 80.0% Amino acid analysis with

HPLC

Moisture less than 5.0%, Heated-air drying at normal

pressure method
Ash less than 15.0% Direct ashing method

Arsenic less than 2flg/g DDTC-Ag method

Heavy Metals less than 10flg/g Sodium sulfide colorimetry

Lead less than 0.5flg/g Atomic absot·ption method

Total Aerobic Counts less than 1000 CFU/g Methods established by Food
Hygiene Guidance (Edited by
Japan Food Hygiene Association)
Yeast and Mold less than 300 CFU/g Methods estnblished by Food
Hygiene Guidance (Edited by
Japan Food Hygiene Associntio!J)
Colifonn I E. coli negative Methods established by Food
Hygiene Guidance (Edited by
Jnpan Food Hygiene Association)

*Storage temperature

(b) (6)

Production Control Div.

 2014/6/9

Pharma Foods International Co., Ltd.

1-49 Gor-yo-Ohara,Nishilcyo-lm,
Kyoto 615-8245 Japan
Phone : +81-75-394-8605
F A X: +81-75-394-8889

CERTIFICATE OF ANALYSIS

Commodity: PharmaGABA™
Lot No.: 2J10
Manufacturing Date: 2012/10/10
Measurement Date: 2012/10/26
Shelf-Life: 2 years from manufacturing date

Specification Results
Appearance light yellow to light brown conformed
powder
GABA more than 80.0% 87.2°/o

Moisture less than 5.0% 2.8°/o

Ash less than 15.0°/o 3.0%

Arsenic less than 2Jlg/g less than 2Jlg/g

Heavy Metals less than lOJlglg less than lOJlg/g

Lead less than 0.5Jlg/g less than 0.5Jlg/g

Total Aerobic Counts less than lOOOCFU/g 20CFU/g

Yeast and Mold less than 300CFU/g less than 1OCFU/g

Coliform I E. coli negative negative

-
(b) (6)

Froduction Control Div.

 2014/6/9

Pbarma Foods International Co., Ltd.

1-49 Goryo-Ohara,Nishikyo-lm,
Kyoto 615-8245 Japan
Phone : +81-75-394-8605
F A X: +81-75-394-8889

CERTIFICATE OF ANALYSIS

Commodity:
PharmaGABATM
Lot No.:
4B06
Manufacturing Date:
2014/2/6
Measurement Date:
2014/2/14
Shelf-Life:
2 years from manufacturing date

Specification Results
Appearance light yellow to light brown conformed
powder
GABA more than 80.0°/o 84.6°/o

Moisture less than 5.0% 3.0o/o

Ash less than 15.0°/o 3.1 °/o

Arsenic less than 2f.lg/g less than 2f.lg/g

Heavy Metals less than lOf.lg/g less than lOf.tg/g

Lead less than 0.5f.tg/g less than 0.5f.tg/g

Total Aerobic Counts less than lOOOCFU/g less than lOCFU/g

Yeast and Mold less than 300CFU/g less than lOCFU/g

Coliform I E. coli negative negative

(b) (6)
r
Production Control Div.
 2014/ 6/2

Pharma Foods International Co., Ltd.

1-49 Goryo-Ohar·a,Nishil{yo-Im,
Kyoto 615-8245 Japan
Phone : +81-75-394-8605
F A X: +81-75-394-8889

CERTIFICATE OF ANALYSIS

Commodity:
PharmaGABATM
Lot No.:
4E21
Manufacturing Date:
2014/5/21
Measurement Date:
2014/6/2
Shelf-Life:
2 years from manufacturing date

Specification Results
Appearance light yellow to light brown conformed
powder
GABA more than 80.0% 89.8°/o

Moisture less than 5.0% 3.0%

Ash less than 15.0°/o 3.1%

Arsenic less than 2J.tg/g less than 2J..Lg/g

Heavy Metals less than lOJ..Lg/g less than lOJ..Lg/g

Lead less than 0.5J..Lg/g less than 0.5J..Lg/g

Total Aerobic Counts less than lOOOCFU/g 30 CFU/g

Yeast and Mold less than 300CFU/g less than lOCFU/g

Coliform I E. coli negative negative

(b) (6)

-P'h>duction Control Div.

 2014/8/5

Pl1a rma Foods International Co., L td.

1-49 Goryo-Ohara, Nishikyo-Jm,
Kyoto 615-8245, J apan
P hon e: +81-75-394-8605
FAX : +81-75-394-8889

Sl?ECJIJF:U:CAT TION

Commodity: J?ha:rma GAJBA 20-D

Shelf L ife : 2 years from manufacturing date

Specification Methods

Appearance white to light yellow Visual appearance

powder
GABA more than 20.0°/o Amino acid analysis with
HPLC
Moisture less than 10.0°/o Heated-air drying at
normal pressure method
Ash less than 18.0°/o Direct ashing method

Arsenic less than 2f.lg/g DDTC-Ag method

Heavy Metals less than lO,..,g/g Sodium sulfide colorimetry

Total Aerobic Counts less than 1000 CFU/g Methods established by F ood
Hygiene Guidance (Edited by
.Japan Food Hygiene Association)
Yeast and Mold less than 300 CFU/g Methods established by F ood
Hygiene Guidance (Edited by
J apan F ood Hygiene Association)
Coliform I E. coli negative Methods established by F ood
H ygiene G uidance (Edited by
.Japa n Food Hygiene Association)

Sto.-age temperature (b) (6)

__J. ....­
Production Control Div.
 201417/28

Pharma F oods I ntemational Co., Ltd.

1-49 Goryo-Oha ra,N ishikyo-ku,
Kyoto 615-8245 Japan
Phone: +81-75-394-8605
FA X : +81-75-394-8889

CERT:U:JFICATJE OlF ANALYS][§

Commodity:
PharmaGABA 20-D
Lot No.:
6A31
Manufacturing Date:
2006/1/31
Measurement Date:
2006/2/14
Shelf-Life:
2 years from manufacturing date

Specification Results

Appearance
white to light yellow conformed
powder
GABA more than 20.0% 22.9%

Moisture less than 10.0 % 4.8°/o

Ash less than 18.0 % 15.0%

Arsenic less than 2 ).!g/g less than 2 ).!g/g

Heavy Metals less than 10 ).!g/g less than 10 ).!g/g

Total Aerobic Counts less than 1000 CFU/g less than 10 CFU/g

Yeast and Mold less than 300 CFU/g less than 10 CFU/g

Coliform I E. coli negative negative

(b) (6)
I

Productio'ii Control Div.

 2014/7/28

Pharma Foods Internationa l Co., Ltd.

1-49 Goryo-Oha•·a,Nishikyo-Im,
Kyoto 615-8245 Japan
Phone : +81 -75-394-8605
FAX : +81-75-394-8889

CERT:U:JF:n:CATE OF ANALYS:n:S

Commodity: PharmaGABA 20-D

Lot No.: 6B07
Manufacturing Date: 2006/2/7
Measurement Date: 2006/2/27
Shelf-Life: 2 years f..om manufacturing date

Specification Results

Appearance white to light yellow conformed

powder
GABA mot·e than 20.0o/o 22.9°/o

Moisture less than 10.0 °/o 5.6%

Ash less than 18.0 % 14.7°/o

Arsenic less than 2 ).!g/g less than 2 ).!g/g

Heavy Metals less than 10 ).!g/g less than 10 ).!g/g

Total Aerobic Counts less than 1000 CFU/g 10 CFU/g

Yeast and Mold less than 300 CFU/g less than 10 CFU/g

Coliform I E. coli negative negative

(b) (6)

I
Pt1i"ducti~tcontt·ol Div.
(b) (6)

000086
GAMMA-AMINOBUTYRIC ACID GRAS NOTICE

APPENDIX C

ANALYSES RELATED TO THE SOURCE ORGANISM

x $WWDFKPHQW&$QDO\VLVRIL. hilgardii .

x $WWDFKPHQW&$QDO\VLVRI+LVWDPLQH
x $WWDFKPHQW&$QDO\VLVRI(WK\OFDUEDPDWH
x $WWDFKPHQW&$QDO\VLVRI&LWUXOOLQH

000087

TchnoSuruga Laboratory Co., Ltd. SIID16618-01

Specific-Primer PCR analysis report

Identification No. SIID16618-01

Date this report was written March 30, 2015

Client PHARMA FOODS INTERNATIONAL CO., LTD.

CONFIDENTIAL

Confirmation

1. This report is authorized and approved by the scientist responsible on behalf of

TechnoSuruga Laboratory Co., Ltd.

2. Unless otherwise divert to research presentation (prepublication paper) or patent

description, when you wish to duplicate all or part of this report and provide to

third parties, this shall not be reproduced without prior approval of TechnoSuruga

Laboratory Co. , Ltd.

3. No prior agreement of TechnoSuruga Laboratory Co., Ltd. is necessary for

reproduction if the name of the company is specified as the data source.

TechnoSuruga Laboratory Co., Ltd.

330 Nagasaki Shimizu-ku Shizuoka-shi, Shizuoka 424-0065, Japan

TEL: (+81)54-349-6155 FAX : (+81)54-349-6121

000088

PF{I)
TchnoSuruga Laboratory Co., Ltd. SIID16618-01

Sample Information
Sample code SliD Date culture received Source of sample

PharmaGABA Lot 7809 16618-01 March 11 , 2015 Lactic acid ferm ented

extract

Notes

2
000089

p
TchnoSuruga Laboratory Co., Ltd. SIID16618-01

PURPOSE
In order to investing whether Lactobacillus species exists in a sample or not, Lactobacillus

species Specific-Primer-PCR analysis was performed.

METHODS
1. DNA extraction

• Method MORA-EXTRACT kit (Kyokuto Pharmaceutical, Tokyo)

2. PCR

Primer set LactoR'F 1) - LBFR 1) (16S rRNA gene targeted Lactobacillus

species-specific primer set)

PCR condition 3 step PCR on a Rotor-Gene™ Q (QIAGEN) cycler using

SYBR® Premix Ex Tag™ II (TaKaRa Bio, Shiga)

" Company and product names are trademarks or registered trademarks of each company

or its subsidiaries in Japan and/or other countries.

3
000090

TchnoSuruga Laboratory Co., Ltd. SIID16618-01

RESULTS

PCR detection using Lactobacillus Species-Specific-Primer

PCR amplification was performed with extracted DNA from the sample. The Lactobacillus

species-specific sequences was amplified using PCR method with Lactobacillus

species-specific-primer set, Lacto R'F-LBFR. The estimated fragment size of amplification

product was about 360 base pairs (bp) when this primer set was used. The presence of PCR

product (amplicon) was checked by agarose gel electrophoresis. The 16S rRNA gene sequence

fragment of Lactobacillus species was not amplified (Figure 1).

<J

Figure 1. Agarose gel image of PCR product (Lactobacillus species-specific-primer set Lacto

R'F-LBFR, Arrow, the fragment size of PCR product bout 360 bp in size.

M: size marker (100bp ladder), 1:16381-03.

000091

p
TchnoSuruga Laboratory Co., Ltd. SI ID16618-01

CONCLUSION

Table 1. The result of Lactobacillus species-specific-primer PCR

SliD Lactobacillus spp.

16618-01

(PharmaGABA Lot. 7809) -

+: PCR amplicon was observed

-: PCR amplicon was not observed.

Specific-Primer-PCR analysis was performed to investigate whether Lactobacillus species

exists in this sample or not. From the result mentioned above, 16S rRNA gene fragment of the

Lactobacillus species was not amplified using sample DNA for PCR by Lactobacillus

species-specific primer set. Therefore, it was considered that the Lactobacillus species did not

exist or lower than the detection limit of PCR in this sample.

000092

TchnoSuruga Laboratory Co., Ltd. 8 11016618-01

REFERENCES
1) SONGJ IDA (P.), NAKAYAMA (J .), TATEYAMA (A.), TANAKA (S.) , TSUBOUCH I (M .),

KIYOHARA (C.), SHIRAKAWA (T.), and SONOMMOTO (K.): Differences in developing

intestinal microbiota between allergic and non-allergic infants; a pilot study in JAPAN. Biosci.

Biotechno/, Biochem., 2007, 71, 2338-2342.

000093

 I)
TchnoSuruga Laboratory Co., Ltd. SIID1 6618-01

This analysis report was translated by Pharma Foods International., Co., Ltd., based on the test

results provided by TechnoSuruga Laboratory Co., Ltd . We, Pharma Foods International Co. ,

Ltd. hereby confirm that the translation and original test results are consistent.

(b) (6)

Translated by Kyung Hwa Moon

Deputy Manager, Sales Department, Global Strategy Group

Pharma Foods International Co., Ltd.

000094

 I)
TchnoSuruga Laboratory Co., Ltd. SIID16381-02

Specific~Primer PCR analysis report

Identification No. SIID16381-02

Date this report was written March 9, 2015

Client PHARMA FOODS INTERNATIONAL CO., LTD.

CONFIDENTIAL

Confirmation

1. This report is authorized and approved by the scientist responsible on behalf of

TechnoSuruga Laboratory Co., Ltd.

2. Unless otherwise divert to research presentation (prepublication paper) or patent

description, when you wish to duplicate all or part of this report and provide to

third parties, this shall not be reproduced without prior approval of TechnoSuruga

Laboratory Co. , Ltd.

3. No prior agreement of TechnoSuruga Laboratory Co., Ltd. is necessary for

reproduction if the name of the company is specified as the data source.

TechnoSuruga Laboratory Co., Ltd.

330 Nagasaki Shimizu-ku Shizuoka-shi, Shizuoka 424-0065, Japan

TEL: (+81)54-349-6155 FAX: (+81)54-349-6121

000095

 TchnoSuruga Laboratory Co., Ltd. SIID16381 -02

Sample Information
Sample code SliD Date culture received Source of sample

PharmaGABA Lot 4806 16381 -02 February 6, 2015 -

Notes

2
000096

 Rl)
TchnoSuruga Laboratory Co., Ltd. 8 11016381 -02

PURPOSE
In order to investing whether Lactobacillus species exists in a sample or not, Lactobacillus

species Specific-Primer-PCR analysis was performed.

METHODS
1. DNA extraction

Method MORA-EXTRACT l<it (Kyokuto Pharmaceutical , Tokyo)

2. PCR
1
Primer set LactoR'F > - LBFR 1> (168 rRNA gene targeted Lactobacillus

species-specific primer set)

PCR condition 3 step PCR on a Rotor-Gene™ Q (QIAGEN) cycler using

SYBR® Premix Ex Tag™ II (TaKaRa Bio, Shiga)

• Company and product names are trademarks or registered trademarks of each company

or its subsidiaries in Japan and/or other countries.

3
000097

TchnoSuruga Laboratory Co., Ltd. SII D16381 -02

RESULTS

PCR detection using Lactobacillus Species-Specific-Primer

PCR amplification was performed with extracted DNA from the sample. The Lactobacillus

species-specific sequences was amplified using PCR method with Lactobacillus

species-specific-prim er set, Lacto R'F-LBFR. The estimated fragment size of amplification

product was about 360 base pairs (bp) when this primer set was used. The presence of PCR

product (amplicon) was checked by agarose gel electrophoresis. The 16S rRNA gene sequence

fragment of Lactobacillus species was not amplified (Figure 1).

<J

Figure 1. Agarose gel image of PCR product (Lactobacillus species-specific-primer set Lacto

R'F-LBFR, Arrow, the fragment size of PCR product bout 360 bp in size.

M: size marker (1 OObp ladder}, 1:16381 -03.

000098

p
TchnoSuruga Laboratory Co., Ltd. SIID16381 -02

CONCLUSION

Table 1. The result of Lactobacillus species-specific-primer PCR

SliD Lactobacillus spp.

16381-02

(PharmaGABA Lot. 4806) -

+: PCR amplicon was observed

-: PCR amplicon was not observed.

Specific-Primer-PCR analysis was performed to investigate whether Lactobacillus species

exists in this sample or not. From the result mentioned above, 16S rRNA gene fragment of the

Lactobacillus species was not amplified using sample DNA for PCR by Lactobacillus

species-specific primer set. Therefore, it was considered that the Lactobacillus species did not

exist or lower than the detection limit of PCR in this sample.

000099

PRI)
TchnoSuruga Laboratory Co., Ltd. SIID16381 -02

REFERENCES
1) SONGJIDA (P.), NAKAYAMA (J.), TATEYAMA (A.), TANAKA (S.), TSUBOUCHI (M.),

KIYOHARA (C.), SH IRAKAWA (T.), and SONOMMOTO (K.): Differences in developing

intestinal microbiota between allergic and non-allergic infants; a pilot study in JAPAN. Biosci.

Biotechnol, Biochem. , 2007, 71, 2338-2342.

000100

 TchnoSuruga Laboratory Co., Ltd. SI ID1 6381-02

This analysis report was translated by Pharma Foods International. , Co., Ltd ., based on the test

results provided by TechnoSuruga Laboratory Co , Ltd. We, Pharma Foods International Co.,

Ltd. hereby confirm that the translation and original test results are consistent.

Date: fvfc~V"cl') 3 I ' 20 f5"'

(b) (6)

Translated by Kyung Hwa Moon

Deputy Manager, Sales Department, Global Strategy Group

Pharma Foods International Co., Ltd.

000101

 •itfi*± 7?/AJilti·?;Ji s11016381-03

TechnoSuruga Laboratory Co.,Ltd.

Specific-Primer PCR ana I ys is report!

Identification No. SJJD1 6381-03

Date this rep01i was written March 20, 20 15

Client PHARMA FOODS INTERNATIONAL CO., LTD.

CONFIDENTIAL

Confirmation

I. This repmi is authorized and approved by the scientist responsible on behalf of

TechnoSumga Laboratory Co., Ltd.
2. Unless othe1wise dive1i to research presentation (prepublication paper) or patent
description, when you wish to duplicate all or part of this repmi and provide to third
parties, this shall not be reproduced without prior approval ofTechnoSumga Laboratory
Co.,Ltd.
3. No prior agreement ofTechnoSuruga Laboratmy Co., Ltd. is necessary for
reproduction of results if the name of the company is specified as the data source.

Scienlisl Responsible

r.:n 711

TechnoSuruga Laboratory Co., Ltd.

330 Nagasaki Shimizu-ku Shizuoka-shi, Shizuoka 424-0065, Japan
TEL: (+81)54-349-6 155 FAX: (+81)54-349-6 121

000102

 ait* *± 7~/AJiln ·7* SIID16381-03

TechnoSuruga Laboratory Co.,Ltd.

Sample Infm·mation

Sample code SliD Date culture received Source of sample

PharmaGABA Lot.4E21 16381-03 February 6, 20 15

----------·· -··-----------·------·--- ........----- - - - --- - - · - - -···--

- - - - - - - -- -

t -- · - -·- -- --·1- - - - - - -1- - - - - - - ------------

1--- -·· - - ··- "-' - - -- -- - - - - - · - ___..._ ....... ,__________ . . ....._ ...____________ .............. ...........

- -··..-·-·---- - --·- ·1- - - - - - -·- - - - - - - -·- -·---· - - - · - - - -..··-··· ---·-

1-· - - - - - - - -· - 1- - - - - - - - - .----·--·-·- · - - · - - - - - - - - - - -

- - - - - · · - - · · - " -""'"'_ _____ _ _- --·- - - - - - - · - - -1

Notes

000103

•it~*± 7?/~Ji11i·7;Ji SIID16381-03

TechnoSuruga Laboratory Co.,Ltd.

PURPOSE
In order to investigate whether Lactobacillus species exists in a sample or not, Lactobacillus
species Specific-Primer-PCR analysis was performed.

METHODS
1. DNA extraction
Method MORA-EXTRACT kit (Kyokuto Pharmaceutical, Tokyo)

2.PCR
Primer set LactoR'FI) - LBFR 1l (16S rRN A gene targeted Lactobacillus
species-specific primer set)

PCR condition 3 step PCR on a Rotor-Gene™ Q (QIAGEN) cycler using SYBR®

Premix Ex Taq™ II (TaKaRa Bio, Shiga)

*Company and product names are trademarks or registered trademarks of each comoany or its
subs idi aries in Japan and/or other countries.

000104

 t~it~*± 7?/~JII1i·7~
TechnoSuruga Laboratory Co.,Ltd. SI ID1 6381-03

RESULTS

PCR detection using Lactobacillus Species- Specific-Primer

PCR amplification was pe1formed with extracted DNA from the sample. The Lactobacillus
species-specific sequence was amplified using PCR method with Lactobacillus
species-specific-primer set, LactoR'F- LBFR. T he estimated fragment size of amplification product
was about 360 base pairs (bp) when this primer set was used. The presence of PCR product
(amplicon) was checked by agarose gel electrophoresis. The 16S rRNA gene sequence fragment of
the Lactobacillus species was not amplified (Figure 1).

Figure 1. Agarose gel image of PCR product (Lactobaci//usspecies- specific- primer

set Lac toR' F - LBFR, Arrow, the fragment size of PCR product about 360 bp in size.
M: size marker (100bp ladder} , 1: 16381-03.

000105

 ait~*± 7~/AJ~1i·?*
TechnoSuruga Laboratory Co.,Ltd. SIID16381-03

CONCLUSION

Table 1. The result of Lactobacillus species-specific-primer PCR

SliD Lactobaci I Ius spp.

16389-03 -
(PharmaGABA Lot. 4E21)
+: PCR amp I icon was observed,
PCR amp I icon was not observed

Specific-.Primer-PCR analysis was performed to investigate whether Lactobacillus species exists

in this sample or not. From the result mentioned above, 16S rRNA gene fragment of the
Lactobacillus species was not amplified using sample DNA for PCR by Lactobacillus
species-specific-primer set. Therefore, it was considered that the Lactobacillus species did not
existed or lowers than the detection limit of PCR in this sample.

5
000106

 Gitfi*i 7? / AJIIjj • 7;Ji SIID16381-03

TechnoSuruga Laboratory Co.,Ltd.

REFERENCES
I) SONGJINDA (P.), NAKAYAMA (J.), TATEYAMA (A.), TANAKA (S.), TSUBOUCHI (M.),
KIYOHARA (C.), SHIRAKAWA (T.) and SONOMOTO (K.): Differences in developing
intestinal microbiota between allergic and non-allergic infants: a pilot study in Japan. Biosci.
Biotechnol. Biochem., 2007, 7L 2338-2342.

000107

ait~~± 7?/AJII1i·?;F
TechnoSuruga Laboratory Co.,Ltd. SIID16381-02

SllD16381-02
13 f-;j· 2015 fF 3 fl 9 El
~~1] **it~t±7 y - 7 7 -;(

t~~11~~ fJJf ~ 1m§~ g~

m~~~ .:Eff{j)f-~ffij_ 7~11~ fiJ~ f.iR

1. Jis:¥1H'r1i~:if'~it~t:l:7:7 J 7-Jv:ff · 7# tJi~;I~Jf{f~I:J: Qlf<~2i:tiJJ. C'9o

2.W~~ft C-~~00 ~M~~~-~~~m~~~. ~-~~~-$~k~~

T~-RGTm - fi~Em9QW~~~.~it~tl7:7J7-~:ff·7*~•M0
~;\Vi~~,~ct~ IJ ~9a

3. m = :l!f~O)ijiiffltto/JI: rf*:rl:~t-':7:7 J 7-Jv:ff · 7*1:J:Qi\it1'iU1i*J O)\j?~fY1

~c Gk•~· t~it~t±7:7 J 7-Jv:ff · 7if~•iWO)-@J~~:FF~ct~ 0 ~9o

:f*j:\~tl?'-:7; 7-.Jvjj • 7~ ~~itm

'f424-0065 ~f!llfiJ J?..,IfltllQJTIHJ!i7J<!z::&iiJ16 330 -t§::l:-ili
TEL: 054-349-6155 FAX: 054-349-6121

000108

 ~itfi~ 7?/AJ!f:t.i·?;Ji
TechnoSuruga Laboratory Co.,Ltd. SIID16381-02

SliD
PharmaGABALot.4B06 16381-02 20 15 if: 2 ~ 6 R
- -- - - - - --- - - - - - - -- 1- - - - - ------ - - - - - - - - - - - - - - - -

- - - - - -- --1- - - --------------- --- -- - - - - - - - - - - -

·- - - - - - -1--···· - ---- - - - - - -·- -1- - - - - - - - - - ---

-·------·-·- ----- ---------------- - - - - - - - - - - t - - - - - - - - -- ·-·------ - - ·-- - - - - - - - - - - - - - ·

1----------------·- -- - - - - - ----+------------ - - - - - - - - - - ·- - -1

1-------1-----~------ +------------

-----------------------r-------·--------- ·- ----------- -- --- ----·-------·--------

000109

 ait~*± 7'?/AJ111l·7;Ji
TechnoSuruga Laboratory Co.,Ltd. SII 016381-02

(§189
Lactobacillus JrJ!,t?\~f$:~1 1:1¥1'±9 ~ iJ\ c' '5 iJ\1:-:J l.t~T¥-f~ 7°71 ~-~ ffi \t)t:::_ PCR ¥t
f:J: ~~tl:l~i'rv) ;t9o

MORA-EXTRACT (~~JR®!~, JtOy()

2. PCR
. 15fffl771 ? - LactoR'F 1>- LBFR 1l (Lactobacillus f{fl, 16S rDNA)

· PCR ~4H~ SYBR® Premix Ex Taq™ 11 (TaKaRa Bio, Shiga)

Rotor-Gene™ Q (QIAGEN) 1: J: ~ 3step PCR

000110

 a:«fr*± 7?/AJII1i·?;Ji SII D16381-02

TechnoSuruga Laboratory Co.,Ltd.

<J

~ 1. Lactobaci !Ius m\~~~ :15-1 ";("- I::J::.Q PCR il~j~~~<7)7 jj' IJ- .A ij Jl/ii~~j}<lt.Jf~.
v-/1§:-9-1 :SI ID16381-02, M:-tt-1;(";("-JJ- (1 00 bp)

000111

 ait~*i 7?/AJII11•7;Ji
TechnoSuruga Laboratory Co.,Ltd. SI ID16381-02

SIID16381-02

Lactobacillus HI
+ : PCR ±~~~L - : PCR 1~±~~m

l:~20)f.ti:!t~:, Lactobacillus ~:a:::M~c LJ-:.4\f/M 7'71' ? -PCR Ef:4~7'J11~ Gh*it Jv-r: l

t-:. (~ I : v- /~-ry I) o ; : 0);: ci"J' G, ~14>:~ ~;:: l:t Lactobacillus f(ii,O)Jj /.b. DNA 7'J"tf{­

{£ l t.t. ~), £!0 ~ lt) l:t~~tl:', ~.fU~J;J, r-r: £!0 Q c :1§- A. G~1.;t 9 o

000112

 -it~~ 7?/~)if1j·?;fi
TechnoSuruga Laboratory Co.,Ltd. SI ID16381-02

*Hi.@
*¥~1!f:i!n:&~9 ~ :t)Hf.ra<J t~ ~JJ:r.~ ~~:-:J~ ;t v--cl:t, t~:J:~1:1:7:? J 7-.Jvjj · 7 ~ H:~~r
tf~;t 'C'Sf~:~9f;bit <ff_~ V)o

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KlYOHARA (C.), SHlRAKAWA (T.) and SONOMOTO (K.): Differences in developing
intestinal mjcrobiota between allergic and non-allergic infants: a pilot study in Japan. Biosci.
Biotec/mol. Biochem., 2007, 71. 2338-2342.

000113

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000114

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000115

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Bioteclmol. Biochem., 2007, 71, 2338-2342.

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000120

 Japan Food Research Laboratories

Authorized by the Japanese Government
52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151-0062, Japan
No. 207060355-001
June 18, 2007

CERTIFICATE OF ANALYSIS
Client: PHARMA FOODS INTERNATIONAL CO., LTD.
1-49 Goryo-Ohara, Nishikyo-ku, Kyoto 615-8245, JAPAN

Sample name: PharmaGABA Lot.070209A

Received date: June 05, 2007

This is to certify that the following result(s) have been obtained from our analysis on the
above-mentioned sample(s) submitted by the client.

Test Result(s)
Test Item Result QL N M
Hjstamjne Not detected 0.5 mg I 100g 1
QL: Quantitation limit N: Notes M: Method
Method
1: HPLC

This test result was translated by Pharma Foods International Co., Ltd . Based on the
analysis results provided by Japan Food Research Laboratories and we, Pharma Foods
International Co., Ltd. hereby confirm that the translation and analysis result are consistent.

(b) (6)

Translated by Kyung Hwa Moon

Deputy Manager, Sales Department, Global Strategy Group
Pharma Foods International Co., Ltd.

1/1

000121

(b) (6)

000122
(b) (6)

000123
 J opon

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2008fF (SfL~20fF) OW 21B

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(Lo I. 7B09)

71 fJT 1\il: iW! lfi § *5 *~ tLi liN lft. 7t 15 tt;;

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rt"

000124

 Japan Food Research Laboratories

Authorized by the Japanese Government
52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151 -0062, Japan
No. 208021242-001
February 21, 2008

CERTIFICATE OF ANALYS IS
Client: PHARMA FOODS INTERNATIONAL CO., LTD.
1-49 Goryo-Ohara, Nishikyo-ku, Kyoto 615-8245, JAPAN

Sample name: PharmaGABA Lot.7B09

Received date: February 15, 2008

This is to certify that the following result(s) have been obtained from our analysis on the
above-mentioned sample(s) submitted by the client.

Test Result(s)
Test Item Result QL N M
Ethyl carbamate Not detected 0 01 ppm
QL: Quantitation limit N: Notes M: Method
Method
1: Gas chromatography-mass spectrometory

This test result was translated by Pharma Foods International Co., Ltd . Based on the
analysis results provided by Japan Food Research Laboratories and we , Pharma Foods
International Co., Ltd. hereby confirm that the translation and analysis result are consistent.

(b) (6)

k'
Translated by Kyung Hwa Moon
Deputy Manager, Sales Department, Global Strategy Group
Pharma Foods International Co., Ltd .

1/1

000125

(b) (6)

000126
(b) (6)

000127
(b) (6)

000128
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);/, J:

000129

 F*I J
Japan Food Research Laboratories
Authorized by the Japanese Government
52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151-0062, Japan
No. 20901538-001
January 23, 2009

CERTIFICATE OF ANALYSIS
Client: PHARMA FOODS INTERNATIONAL CO., LTD.
1-49 Goryo-Ohara, Nishikyo-ku, Kyoto 615-8245, JAPAN

Sample name: PharmaGABATM Lot.7809

Received date: January 9, 2009

This is to certify that the following result(s) have been obtained from our analysis on the
above-mentioned sample(s) submitted by the client.

Test Result(s)
Test Item Result QL N M
Free cjtrulline Not detected 0.02 qf100g
QL: Quantitation limit N: Notes M: Method
Method
1: Amino acid analyzer method

This test result was translated by Pharma Foods International Co., Ltd . Based on the
analysis results provided by Japan Food Research Laboratories and we, Pharma Foods
International Co., Ltd. hereby confirm that the translation and analysis result are consistent.

(b) (6)

Translated by Kyung Hwa Moon

Deputy Manager, Sales Department, Global Strategy Group
Pharma Foods International Co. , Ltd .

1/1

000130

(b) (6)

000131
SUBMISSION END

000132