Journal of Dairy Science and Biotechnology

Korean Society of Dairy Science and Biotechnology (한국낙농식품응용생물학회)
권호 표지

Bacterial Reverse Mutation Test Evaluation of Hydrolyzed GMP Powder Containing Highly Concentrated Sialic Acid (23%) produced by Enzyme Separation and Solvent Enrichment Method

효소분리 및 용매정제법으로 제조한 고농도 Sialic Acid(23%)가 함유된 GMP 가수분해분말의 미생물복귀돌연변이시험 연구

  • Received : 2016.06.08
  • Accepted : 2016.06.22
  • Published : 2016.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

The goal of this study was to develop hydrolyzed whey protein powder (23%-GNANA) manufactured with high content of sialic acid, a marker compound that is usually present at 7% concentration in GMP obtained from the milk protein. It is a safe food, used worldwide in infant and baby foods, etc. The test substance was prepared using (7% sialic acid containing) GMP as a raw material. Alcalase, an enzyme approved as a food additive, was used after separating sialic acid, with 100% efficiency, and 23%-GNANA (composed of 23% sialic acid and protein; product name: HELICOBACTROL-23), provided by MEDINUTROL Inc. (Korea), manufactured to have high (23%) content through ethanol soaking and enrichment. Bacterial reverse mutation (Ames) test was conducted in accordance with the GLP Guideline using the test substance specified above. To detect its mutagenicity potential in microorganisms, histidine auxotrophic strains of Salmonella typhimurium, TA98, TA100, TA1535, and TA1537, and tryptophan auxotrophic Escherichia coli strain, WP2uvrA, were used. The bacterial reverse mutation (Ames) test was performed using five concentrations of the test substances (0, 61.7, 185, 556, 1,670, $5,000{\mu}g/plate$). The evaluation did not reveal repetitive increase of colony generating values and positive criteria for reverse mutagenicity for any tested concentration in the five strains regardless of the presence of metabolic activation system, and no dose-dependency. In conclusion, the safety of 23%-GNANA test substance was verified by the bacterial reverse mutation test conducted before registration of 23%-GNANA as a food additive.

본 시험은 안전식품인 우유단백질에서 분리되어 영유아 식품 등에 세계적으로 사용되고 있는 GMP 내 기본적으로 7%로 결합되어 있는 지표성분인 sialic acid의 함유량이 높게 제조한 23%-GNANA를 기능성 식품 원료 개발함에 최종 연구 목표를 두었다. 시험물질은 23% sialic acid와 GMP 단백질로 구성(제품명: HELICOBACTROL-23)되어 있으며, (주)한일바이오메드사(한국)에서 공여 받아 미생물복귀돌연변이시험을 실시하였다. 미생물에 대한 돌연변이 유발성 유무를 검색하기 위해 히스티딘 요구성 균주인 Salmonella typhimurium TA98, TA100, TA1535 및 TA1537과 트립토판 요구성 균주인 Escherichia coli WP2uvrA를 이용하였다. 미생물복귀돌연변이시험은 시험물질을 5단계의 농도군(0, 61.7, 185, 556, 1,670, $5,000{\mu}g/plate$)으로 하여 평가하였다. 평가결과로서, 대사활성계 존재 유무와 관계없이 5균주 모두에서 시험물질의 각 농도에 의한 복귀돌연변이 유발원 양성기준인 콜로니 생성수치가 재현성있는 증가를 나타내지 않았으며, 용량의존성도 확인되지 않았다. 결론적으로, 시험물질인 23%-GNANA의 식품첨가물로서 등록을 위하여 수행한 미생물돌연변이시험에서 안전성이 확인되었다.

Keywords

References

  1. Aimutis, W. R. 2004. Bioactive properties of milk proteins with particular focus on anticariogenesis. J. Nutr. 134: 989-995. https://doi.org/10.1093/jn/134.4.989S
  2. Ernest, P. B. 2000. Biological activities of bovine glyco-macropeptide. British Journal of Nutrition 84:39-46.
  3. Gorog, P. and Kovacs, I. B. 1978. Anti-inflammatory effect of sialic acid. Agents and Actions 8:543-545. https://doi.org/10.1007/BF02111443
  4. Iijima, R., Takahashi, H., Namme, R., Ikegami, S. and Yamazaki, M. 2004. Novel biological funtion of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Letters 561:163-166. https://doi.org/10.1016/S0014-5793(04)00164-4
  5. Kelleher, S. L., Chatterton, D., Nielsen, K. and Lonnerdal, B. 2003. Glycomacropeptide and ${\alpha}$-lactalbumin supplementation of infant formula affects growth and nutritional status in infant Rhesus monkeys. Am. J. Clin. Nutr. 77: 1261-1268. https://doi.org/10.1093/ajcn/77.5.1261
  6. Kilara, A. and Panyam, D. 2003. Peptides from milk proteins and their properties. Crit. Rev. Food Sci. Nutr. 43:607-633. https://doi.org/10.1080/10408690390251138
  7. Korea Food and Drug Administration (KFDA). 2015. Guidelines for toxicity tests in drugs, etc. KFDA Notice No. 2014-67 (Feb. 12).
  8. Korea Food and Drug Administration (KFDA). 2014. The above study was conducted in accordance with the Ministry of Food and Drug Safety. KFDA Notice No. 2014-136 (Jul. 30.).
  9. Maron, D. M. and Ames, B. N. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215. https://doi.org/10.1016/0165-1161(83)90010-9
  10. Marshall, S. C. 1991. Casein macropeptide from whey. A new product opportunity. Food Res. Quar. 15:86-91.
  11. Moon, Y. I., Lee, W. J. and Oh, S. J. 2005. Glycomacropeptide hydrolysed from bovine ${\kappa}$-casein; II. Chromato-graphic changes of ${\kappa}$-casein macropeptide as related to Trichloroacetic acid concentration. Korean J. Food Sci. Ani. Resour. 17(1):478-482.
  12. Organization for Economic Cooperation and Development. 2008. OECD Guidelines 408 for the testing of chemicals: Repeated Dose 90‐day Oral Toxicity Study in Rodents.
  13. Organization for Economic Cooperation and Development. 1993. OECD guideline for the testing of chemicals, 'Guidelines for the management of non-clinical studies' OECD Test guidelines (No. 473) and GLP Center Standard Operating Procedures.
  14. Schauer, R. and Wien, S. V. 1984. Sialic acids: Chemistry, metabolism and function. Carbohydrate Research 129:5-7. https://doi.org/10.1016/0008-6215(84)85324-0
  15. Takahashi, N., Asakawa, S., Shunichi, D. and Tadashi, I. 1992. Hypoallergenic nutritive composition. Freench Pat. Appl. Fr 2667 850A1.
  16. Wang, B. 2009, Sialic acid is and essential nutrient for brain development and cognition. Annu. Rev. Nutr. 29:177-222. https://doi.org/10.1146/annurev.nutr.28.061807.155515
  17. Wang, B., Brand-Miller, J., McVeagh, P. and Petocz, P. 2001. Concentration and distribution of sialic acid in human milk and infant $formulas^{1-3}$. Am. J. Clin. Nutr. 74:510-515. https://doi.org/10.1093/ajcn/74.4.510
  18. Wang, B. and Brand-Miller, J. 2003. The role and potential of sialic acid in human nutrition. European Journal of Clinical Nutrition 57:1351-1369. https://doi.org/10.1038/sj.ejcn.1601704
  19. Wang, B., Yu, B., Karim, M., Hu, H., Yun, S., McGreevy, P., Petocz, P., Held, S. and Brand-Miller, J. 2007. Dietary sialic acid supplementation improves learning and memory in $piglets^{1-3}$. Am. J. Clin. Nutr. 85:561-569. https://doi.org/10.1093/ajcn/85.2.561
  20. Yoon, Y. C., Cho, J. K., Song, C. H., Lee, S. and Chung, C. I. 2000. Purification of the glycomacropeptide from cheese whey. Korean J. Food Sci. Ani. Resour. 20:159-165.