J. Milk Sci. Biotechnol. Vol. 34. No. 2, pp. 117~135 (2016) 1, 2 Development and Research on a Functional Hydrolyzed Whey Protein Powder Product with

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1 J. Milk Sci. Biotechnol. Vol. 34. No. 2, pp. 117~135 (2016) 1, 2 Development and Research on a Functional Hydrolyzed Whey Protein Powder Product with Sialic Acid as a Marker Compound - II. Repeated 90-day Oral Administration Toxicity Test using Rats Administered Whey Protein Powder containing Highly Concentrated Sialic Acid (23%) produced by Enzyme Separation and Solvent Enrichment Method - Hye-Ji Noh 1, Hyang-Hyun Cho 1, Hee-Kyong Kim 1* and Hong-Bum Koh 2 1 HANILBIOMED Co., Gwangju 67024, Korea 2 College of Veterinary Medicine, Chonnam National University, Gwangju , Korea Abstract The present study was performed to develop a functional raw food material from hydrolyzed whey protein powder (23%-GNANA) medication containing sialic acid as a marker compound that is naturally occurring at 7% concentration in GMP (glycomacropeptide). GMP is used worldwide in foodstuffs for babies and infants and is obtained from the milk protein as safe food. While the purpose of our detailed evaluation was aimed to assess preliminary NOAEL values for and above 2,000 mg/kg/day, a clinical dose allowance for 23%-GNANA (as per characteristic of a functional health product, a highly refined test substance of 23% (v/v) sialic acid combined in GMP), at the same time we also wanted to assess the safety of GMP hydrolyzate lacking sialic acid but with identical properties as GMP. Animal safety evaluation was conducted using 23%-GNANA as the test substance, produced from hydrolyzed whey protein powder (product name: HELICOBACTROL-23; provided by Medinutrol Inc. [Korea]; composed of 23% sialic acid and GMP protein) after isolating the sialic acid using enzymes approved as food additives, with GMP as a raw material, and subsequently increasing the content of xx up to 23% through 80% (v/v) ethanol soaking and concentrating, in accordance with GLP Guideline. The animal safety evaluation mentioned above was made on the basis of toxicity in SPF Sprague-Dawley female and male rats dosed with 10 ml of the test substance diluted to 0, 1,250, 2,500, and 5,000 mg/kg directly into their stomachs for 90 d. This was determined * Corresponding author: Hee-Kyong Kim, College of Veterinary Medicine, Chonnam National University, Gwangju , Korea. Tel: , Fax: , in terms of the general symptoms and animal viability, weight and amount of feed intake, eye examination, uracrasia tests, hematological and blood biochemical disorder tests, 117

2 118 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) blood coagulation test, abnormal intestine weight, abnormalities during postmortem and histopathological examinations. Statistical significance was set at P<0.05. Based on the toxicity determination, a certain minor effect associated with the test substance was observed in male rats with no major effects of the tested substance, in comparison with the control group dosed with sterilized water. Nevertheless, the NOAEL value, evaluated as per toxicity criteria, was verified as 5,000 mg/kg/day (P<0.05). Similarly, for female rats, a certain minor effect associated with the test substance was observed in 5,000 mg/kg/day dosed group, with no major effect, yet the NOAEL value (as assessed as per toxicity criteria) was determined to be 5,000 mg/kg/day (P<0.05), which was the same as for male rats. Accordingly, the NOAEL values of the test substances for all female and male rats were finally verified as 5,000 mg/kg/day (P<0.05). In conclusion, it was determined that the 23%-GNANA test substance exceeds 2,000 mg/kg/day, the clinical allowance characteristic for functional health food, and was finally evaluated to cause no safety concerns when used as a raw material in functional health food production, which was the ultimate goal of the present study. Keywords: Glycomacroptide (GMP), sialic acid evaluation on animal safety and development of raw material for functional health food per the GLP guideline Glycomacropeptide( GMP) rennet (Chymosin) 105 Phe 106 Me para-κ-casein GMP. GMP C-terminal casein-peptide, sialic acid 6~7%. 6.8~8.0 KDa, (Yoon et al., 2000; Brody, 2000). GMP, (Brody, 2000). GMP 1.2~1.5 g/l, 10~25%, (Brody, 2000). GMP Vibrio cholera E. coli enterotoxins, Bifidobacterium, (Moon et al., 2005). GMP N-acetylneuraminic acid(sialic acid), galactose(gal) N-acetylgalactosamine (GalNAc) Thr Ser, sialic acid neuraminidase (Fig. 1)(Wang and Brand- Miller, 2003). Neuraminidase Vibrio cholera Clostridium perfrigens (Springer-Verlag, 1984). Silaic acid nine-carbon sugar family, glucose (Wang et al., 2001). Sialic acid,, ganglioside (Wang et al., 2001; Wang et al., 2007; Wang, 2009), Fig. 1. Sialic acid production from GMP by a Alcalase 2.4FL (Neuraminidase type)

3 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 119 (Wang et al., 2007; Gorog and Kovacs, 1978; Iijima et al., 2004)., (Ishikawa and Koizumi, 2010; Zimmermann et al., 2007; Organization for Economic Co-operation and Development(OECD), 2008). Sialic acid E. coli, E. coli N-acetyl-D-glucosamine, (Ishikawa and Koizumi, 2010; Zimmermann et al., 2007; OECD, 2008; United States of Food and Drug Administration (USFDA), 2005). Sialic acid, sialic acid. Sialic acid,. Sialic acid,. 23%-GNANA, ( )(Ministry of Food and Drug Safety(KFDA), 2014) OECD test guideline(oecd, 2008) GMP sialic acid 7% NatraPep GMP(Murry Goulburn Co-Operative Co., Australia)., 7% (w/v) GMP Alcalase 2.4FL(Novozymes Co., Denmark) 0.24%(w/v) 7 (57 ) (Fig. 1) HELICOBACTROL (23%-GNANA) ( ) (Fig. 2). 2. Sialic acid Sialic acid Fig. 2. Content evaluation results of sialic acid in hydrolyzed whey protein (23%-GNANA), a test substance, applying the HPLC analysis method compared to standard sialic acid. A: Standard sialic acid (10 ppm), B: Sialic acid content in 23%-GNANA (10 ppm), 10 mm sodium acetate buffer, 8 ml 2 ml, 30 Sonication(25 ) (3,000 rpm 20 min, 25 )., 0.22 μm membrane HPLC System(Agilent 1260, USA) (Table 1). sialic acid Sigma-Aldrich (A2090) 0.1 ppm(w/w), 1 ppm 10 ppm. sialic acid 90~110%. 3. 1) IACUC

4 120 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 1. HPLC operation conditions for analysis of sialic acid HPLC system (Agilent 1260) Pump: G1311C Auto sampler: G1329B Column: G1316A UV detector:g1314f 2. Analysis conditions Wavelength: 205 nm Column: Aminex HPX-87H Ion Exclusion Column ( mm, 9 μm, Bio-Rad Co.) Mobile phase: 10 mm H 2 SO 4 Running time: 20 min Flow rate: 0.5 ml/min Injection volume: 10 μl Temperature: 40 Standard sol.(w/w): Silaic acid 0.1 ppm, 1 ppm, 10 ppm OECD(2008), US FDA(2005) ( )(KFDA, 2014), ( 11737, 2013, ) IACUC(Institutional Animal Care and Use Committee). 2) Taconic (specific pathogen-free, SPF) Sprague-Dawley, 5 48 ( ) ±3, 50±20%, 10~20 /, 12 (08:00 ~20:00 ), 150~300 Lux, 60 db, 20 ppm., Polysulfone (260 W 420 L 150 Hmm) 2~3, 1, (121, 20 min). (Zeigler Bros. Inc., USA), 3) 2, 5, (dose-range finding, DRF) NOEL( ) 5,000 mg/kg/day. 5,000 mg/kg/day( ), 2,500( ) 1,250( ) mg/kg/day. 7%-GNANA. 4) 1 6, 2,,. 5), 1,... 6) ) 5. 3~4 1 ml (CliniTek Advantus TM, SIEMENS, USA). 24. (GLU), (BIL), (KET), (SG), (OB), ph, (PRO), (URO), (NIT) (LEU). 4. 1,500 rpm 5 (MF80, Hanil, Korea), S.M(Sternheimer-Malbin) RBC(red blood cell), WBC(white blood cell), Epithelial cell Casts. 1)., 5 (, Lot No.: 13C07K) (Genesis,

5 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 121 Gowa Co. Ltd., Japan),..,. 2)., EDTA-2K CBC bottle (Vacutainer 3 ml, BD, USA) (ADVIA 2120, SIEMENS, USA). (WBC), (RBC), (HGB), (HCT), (MCV), (MCH), (MCHC), (PLT), (RDW), (HDW), (RET), (NEU), LYM(Lymphocyte), MONO(Monocyte), EOS(Eosinophil) BASO(Basophil) LUC(Large unstained cells). 3) clot activator vacutainer tube(bd, USA), 15~20. 3,000 rpm 10 (MF80, Hanil, Korea) (KONELAB 20XT, Thermo, USA). aspartate aminotransferase(ast), alanine aminotransferase(alt), alkaline phosphatase(alp), blood urea nitrogen (BUN), creatinine(cre), glucose(glu), total cholesterol(cho), total protein(pro), creatine phosphokinase(cpk), albumin(alb), total bilirubin(bil), albumin/globulin ratio(a/g), triglyceride(tg), inorganic phosphorus(ip), Ca 2+. Na +, K +, Cl (744 Na + /K + /Cl Analyzer, SIEMENS, USA). 4) 1.8 ml 3.2% sodium citrate 0.2 ml vacutainer tube(vacutainer 1.8 ml, BD, UK), 3,000 rpm 10 (MF80, Hanil, Korea) PT(prothrombin time), APTT(activated partial thromboplastin time). : K-98(ACL 7000 Instrumentation Laboratory, USA) Nephelometric Analysis (sec). 5) Isofluran 6) (CP-64, Sartorius, Ger).. (ovary), (adrenal gland), (pituitary), (thymus), (prostate), (testis), (epididymis), (spleen), (kidney), (heart), (lung), (brain) (liver). 7) 10%, Davidson, Bouin.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ( ).,. Hematoxylin & Eosin(H & E). (5,000 mg/kg/day). 5. (parametric multiple comparison procedures) (non parametric multiple comparison procedures), SPSS 14.0.,,,, one-way ANOVA test. Levene test Duncan multiple range test, Dunnett s T-test. (severity) (Table 3). 6. weight-based classification (Park and Cho, 2011) (Table 2).

6 122 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 2. Definitions of LOAEL, NOAEL and NOEL by three steps of weight-based classification in toxicity test Weight-based classification Important compoundrelated changes Minor compoundrelated changes Noncompoundrelated changes Definitions of criteria The lowest exposure level at which there are statistically or biologically significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate control. The highest exposure level at which there are no statistically or biologically significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate control. Some effects may be produced at this level, but they are not considered to be adverse or precursors to adverse effects. The highest exposure level at which there are no effects (adverse or nonadverse) observed in the exposed population, when compared with its appropriate control. Criteria of toxicity Criteria of toxicity Non-adverse effect No effect Evaluation endpoints LOAEL NOAEL NOEL Table 3. Table of severity values for statistical analysis by item of urinalysis signs during the 90 days oral administration of 23%-GNANA, a test substance to the rats Severity GLU - +/ BIL - +/ KET - +/ SG (Specific gravity) PRO - +/ ph >=9.0 URO (EU/dL) >=8 NIT - + OB (μlmol/l) - +/ LEU - +/ RBC 0 +/ WBC 0 +/ Epithelial cell 0 +/ Cast 0 +/ GLU (glucose), BIL (bilirubin), KET (ketone body), LEU (leukocyte), OB (occult blood), PRO (protein), URO (urobilinogen), NIT (nitrite), SG (specific gravity) Weight-based Classification (important compound-related changes), (minor compound-related changes) (noncompound-related changes) 3. (noncompound-related changes), NOEL(No Observed Effect Level), NOAEL(No Observed Adverse Effect Level) LOAEL (Lowest Observed Adverse Effect Level). - adverse - non-adverse effect. NOAEL (adverse effect), non-adverse effect, LOAEL adverse effect. NOEL (USFDA, 2005). NOAEL NOEL. 1. Sialic acid Fig. 3 23%-NANA sialic acid. 23%-GNANA sialic acid, GMP sialic acid 100%, 23%(v/v) sialic acid GMP 84% 3%.,. 2. 1) Table 1, %-GNANA 90,,. 2)

7 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 123 Table 5, , (weight gains) (p<0.05), -. 3) Table 6, %-GNANA, (p<0.05), -. Fig. 3. Manufacturing process diagram for 23%-GNANA, a test substance containing 23% sialic acid, through the enzyme (Alcalase 2.4 FL) separation mechanism of sialic acid, the marker compound having the glycomacropeptide (GMP) as a substrate, and the ethanol refining process. 4) Table 7, %-GNANA,. Table 4. Investigation results of clinical signs through the autopsy after the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) a) Days Signs Groups (mg/kg/day) b) 0 No finding 0 / 10 a)* 0 / 10 a)* 0 / 10 a) 0 / 10 a) 0 / 10 a) 0 / 10 a) 0 / 10 a) 0 / 10 a) 1~7 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 8~14 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 15~21 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 22~28 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 29~35 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 36~42 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 43~49 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 50~56 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 57~63 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 64~70 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 71~77 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 78~84 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 85~91 No finding 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / 10 0 / Terminal sacrifice 10 / / / / / / / / 10 Number of animals with the sign / Number of animals examined. * : Represents a significant difference at p<0.05 level compared with the vehicle control %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day)

8 124 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 5. Weekly investigation results of the effects on the body weight changes during the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Weeks ** Groups (mg/kg/day) b) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 8.72 * ± ± ± ± ± ± ± ± 8.52 * ± ± ± ± ± ± ± ± 9.62 ** ± ± ± ± ± ± ± ±11.89 * ± ± ± ± ± ± ± ±10.94 * ± ± ± ± ± ± ± ±11.08 * ± ± ± ± ± ± ± ± 8.92 * ± ± ± ± ± ± ± ± 7.02 * ± ± ± ± ± ± ± ±12.14 * ± ± ± ± ± ± ± ±12.33 * ± ± ± ± ± ± ± ±10.15 * ± ± ± ± ± ± ± ± 8.06 * ± ± ± ± ± ± ± ±10.31 * Gains a) 142.1± ± ± ± ± ± ± ±10.48 * * : Represents a significant difference at p<0.05 level compared with the vehicle control ** : Weight gains are body weight difference between week 13 and week 0 %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) Table 6. An assessment of the food consumption changes affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Weeks Groups (mg/kg/day) a) ± ± ± ± ± ± ± ±0.98* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.64 Average 23.0± ±1.01 * 23.7± ± ± ± ± ±0.37 * : Represents a significant difference at p<0.05 level compared with the vehicle control %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day)

9 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 125 Table 7. A clinical investigation of the ophthalmic findings affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Sites a Findings Groups (mg/kg/day) b) Left eye Normal 0 / 5 * 0 / 5 * 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 Right eye Normal 0 / 5 * 0 / 5 * 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 / 5 a : Number of animals with the finding / Number of animals examined * : Represents a significant difference at p<0.05 level compared with the vehicle control. %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) 5) Table 14, 15, %-GNANA (absolute weight) (relative weight). ( ) (p<0.05), -. (, ) (% body weight) (p<0.05)., non-adverse effect -. ( ) (% body weight) (p<0.05), -. (, ) (% body weight) (p<0.05). non-adverse effect -. 6) Table 8, 10, %-GNANA. SG (specific gravity, ) (p<0.05), PRO (p<0.05).., -. PRO Table 8. An assessment of the urinalysis changes affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests ** Result Severity Male Groups (mg/kg/day) Female G1 % G2 G3 G4 G1 G2 G3 G4 0 1,250 2,500 5, ,250 2,500 5,000 GLU BIL KET SG / * 1 * 1 * 1 4 * 3 * * 2 * 2 * 1 * 1 *

10 126 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 8. Continued Tests ** Result Severity ph PRO Male Groups (mg/kg/day) Female G1 % G2 G3 G4 G1 G2 G3 G4 0 1,250 2,500 5, ,250 2,500 5, * / * * 1 1 * 3 * * * 1 * URO a) NIT OB LEU / / No. of animals a) The unit of urobilinogen is ehrlich unit/dl, * : Represents a significant difference at p<0.05 level compared with the vehicle control, ** : GLU: Glucose, BIL: Bilirubin, KET: Ketone body, SG: Specific gravity, PRO: Protein, URO: Urobilinogen, NIT: Nitrite, OB: Occult blood, LEU: Leukocyte, %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High -dose group (G4: 5,000 mg/10 ml/day) -. PRO LEU (p<0.05), (historical data) Table 9, %-GNANA.,. 1) Table 11, %-GNANA. RBC (p<0.05), HDW (p<0.05) -. 2) Table 12, %-GNANA. ALB AST

11 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 127 Table 9. An assessment of the changes in urine sediments affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests Result Severity RBC Male Groups (mg/kg/day) a) Female G1 % G2 G3 G4 G1 G2 G3 G4 0 1,250 2,500 5, ,250 2,500 5, * * / 1 1 * 1 1 WBC * * Epithelial cell Casts +/ * * * * / 1 3 * * 1 No. of animals " "= no finding * : Represents a significant difference at p<0.05 level compared with the vehicle control. %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) Grade RBC (mean/field) WBC (mean/field) Epithelial cell Casts (mean/field) / 20 fields 0 +/ 4 5 Few / 20 fields ~8 6~20 Around 1 / few field 2~5 Table 10. An assessment of the changes in urine volume and color affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests Groups (mg/kg/day) a) Volume (ml) 12.8±1.89 * 9.0±2.24 * 11.4± ± ± ± ± ±3.98 Color-yellow 5 * 5 * No. of animals 5 * 5 * * : Represents a significant difference at p<0.05 level compared with the vehicle control. %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) (p<0.05), -. ALB (p<0.05), (historical data) -. 3) Table 13, %-GNANA, PT (p<0.05), -. 4) Table 16, %-GNANA. 1 (redness), -

12 128 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 11. An assessment of the changes in hematological values affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests ** Units Groups (mg/kg/day) WBC 10 3 /μl 3.87±1.392 # 1.57± ± ± ± ± ± ±0.871 RBC 10 6 /μl 8.94± ± ±0.253 * 7.97± ± ± ± ±0.305 HGB g/dl 15.55± ± ± ± ± ± ± ±0.442 HCT % 47.07± ± ± ± ± ± ± ±1.215 MCV fl 52.70± ± ± ± ± ± ± ±1.988 MCH pg 17.44± ± ± ± ± ± ± ±0.854 MCHC g/dl 33.08± ± ± ± ± ± ± ±0.762 RDW % 12.45± ± ± ± ± ± ± ±0.382 HDW g/dl 2.67± ± ± ± ± ±0.098 * 2.66± ±0.090 PLT 10 3 /μl 1,052±109 1,097±161 1,037±118 1,051±111 1,069.40±113 1,122±131 1,032±98 1,1300±159 MPV fl 8.25± ± ± ± ± ± ± ±0.508 RET % 1.46± ± ± ± ± ± ± ±0.382 NEU LYM % 18.60± ± ± ± ± ± ± ± /μl 0.73± ± ± ± ± ± ± ±0.095 % 76.18± ± ± ± ± ± ± ± /μl 2.95± ± ± ± ± ± ± ±0.780 MONO % 2.54± ± ± ± ± ± ± ±0.795 EOS % 2.08± ± ± ± ± ± ± ±1.397 BASO % 0.11± ± ± ± ± ± ± ±0.057 LUC % 0.51± ± ± ± ± ± ± ±0.057 No. of animals * : Represents a significant difference at p<0.05 level compared with the vehicle control, ** : WBC (White blood cell count), RBC (Red blood cell count), HGB (Hemoglobin conc.), HCT (Hematocrit), MCV (Mean corpuscular volume), MCH (Mean corpuscular hemoglobin), MCHC (Mean corpuscular Hb conc.), RDW (Red cell distribution width), HDW (Hb conc. distribution width), PLT (Platelet), MPV (Mean platelet volume), RET (Reticulocyte), # : The data shown represent means±s.d. (n=10), %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day). 2 (retention of clear fluid),, -. 1, 1 -, ) Table 17, %-GNANA.. minimal( ), slight( ), moderate( ), severe( ) 4. (pituitary gland) 1 (pars distalis) (cyst) (slight). 1 (atropy) (degeneration) severe (prostate) 1 (Inflammatory cells infiltration) minimal, 2 minimal slight -. (kidney) 2 protein cast minimal

13 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 129 Table 12. An assessment of the changes in serum biochemical values affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests ** Units Groups (mg/kg/day) ALB g/dl 3.23±0.073 # 3.36± ±0.064 * 3.26± ±0.057 * 3.25± ± ±0.112 * ALP U/L 505.5± ± ± ± ± ± ± ±19.96 ALT U/L 31.1± ± ± ± ± ± ± ±2.50 AST U/L 94.3± ± ± ± ± ± ± ±9.02 BIL mg/dl 0.06± ± ± ± ± ± ± ±0.014 BUN mg/dl 12.78± ± ± ± ± ± ± ±3.202 CHO mg/dl 64.9± ± ± ± ± ± ± ±10.69 CPK U/L 339.0± ± ± ± ± ± ± ± CRE mg/dl 0.39± ± ± ± ± ± ± ±0.116 GLU mg/dl 157.4± ± ± ± ± ± ± ±19.84 IP mg/dl 5.59± ± ± ± ± ± ± ±0.689 PRO g/dl 6.0± ± ± ± ± ± ± ±0.28 TG mg/dl 66.6± ± ± ± ± ± ± ±8.008 A/G ratio 1.15± ± ± ± ± ± ± ±0.085 Ca 2+ mg/dl 9.94± ± ± ± ± ± ± ±0.278 Na + mg/dl ± ± ± ± ± ± ± ±1.350 K + mmol/l 4.17± ± ± ± ± ± ± ±0.279 Cl mmol/l ± ± ± ± ± ± ± ±1.65 No. of animals * : Represents a significant difference at p<0.05 level compared with the vehicle control ** : AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), ALP (Alkaline phosphatase), BUN (Blood urea nitrogen), CRE (Creatinine), GLU (Glucose), CHO (Total cholesterol), PRO (Total protein), CPK (Creatine phosphokinase), ALB (Albumin), BIL (Total bilirubin), TG (Triglyceride), IP (Inorganic phosphorus), A/Gratio (Albumin/Globulin ratio), Ca 2+ (Calcium ion), Na + (Sodium ion), K + (Potassium ion), Cl (Chloride ion) %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) # : The data shown represent means±s.d. (n=10) Table 13. An assessment of the changes in plasma coagulation values affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Tests Groups (mg/kg/day) a) PT(sec) 10.30±0.461 # 9.11±0.425 * 9.52±0.546 * 8.99± ± ± ± ±0.394 APTT(sec) 18.10± ±0.703 * 18.36± ± ± ± ± ±1.365 No. of animals * : Represents a significant difference at p<0.05 level compared with the vehicle control %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) # : The data shown represent means±s.d. (n=10)

14 130 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 14. An assessment of the changes in absolute organ weights affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Organ weight (g)** Groups (mg/kg/day) a) Adrenal(L) ± # ± ± ± ± ± ± ± Adrenal(R) ± ± ± ± ± ± ± ± Pituitary ± ± ± ± ± ± ± ± Thymus ± ± ± ± ± ± ± ± Ovary(L) ± ± ± ± Ovary(R) ± ± ± ± Prostate ± ± ± ± Testis(L) ± ± ± * ± Testis(R) ± ± ± ± Epididymis(L) ± ± ± ± Epididymis(R) ± ± ± ± Spleen ± ± ± ± ± ± ± ± Kidney(L) ± ± ± ± ± ± ± * ± * Kidney(R) ± ± ± ± ± ± ± * ± * Heart ± ± ± ± ± ± ± ± Lung ± ± ± ± ± ± ± ± Brain ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± No. of animals * : Represents a significant difference at p<0.05 level compared with the vehicle control. ** : Weight (g): unit of absolute weight of organ %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) # : The data shown represent means ± S.D. (n=10)., 4 slight. 3 minimal, 1 slight. 2 minimal. 5 minimal. protein cast, -. (kidney) protein cast 1 minimal, 1 slight. 2 minimal, 2 slight. 3 minimal, 1 slight -. (kidney) mineralization 1 minimal. 3 minimal, 1 moderate, 2 severe, 6 minimal, 1 slight. 1 minimal, -. (liver) (vacuolation) 1 minimal, Bile duct hyperplasia minimal, -. (lung) (Osseous metaplasia) 1 minimal, 1 -. (heart), 1 (myocarditis) minimal, 2 minima - (Table 12). (stomach) (ulcer of the glandular stomach) 1, (Result summary and Toxicity evaluation) Table %-GNANA 90

15 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 131 Table 15. An assessment of the changes in relative organ weights (% of body weight) affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Organ weight (g) ** Groups (mg/kg/day) a) Adrenal(L) ± *# ± * ± ± ± ± ± ± Adrenal(R) ± ± ± ± ± ± ± ± Pituitary ± ± ± ± ± ± ± ± Thymus ± ± ± ± ± ± ± ± Ovary(L) ± ± ± ± Ovary(R) ± ± ± ± Prostate ± ± ± ± Testis(L) ± ± ± ± Testis(R) ± ± * ± ± Epididymis(L) ± ± ± ± Epididymis(R) ± ± ± ± Spleen ± ± ± ± ± ± ± ± Kidney(L) ± ± ± ± ± ± ± * ± Kidney(R) ± ± ± ± ± ± ± * ± Heart ± ± ± ± ± ± ± ± Lung ± ± ± ± ± ± ± ± Brain ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± No. of animals * : Represents a significant difference at p<0.05 level compared with the vehicle control. ** : Ratio (%): Relative ratio of organ weight against body weight # : The data shown represent means±s.d. (n=10). %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) Table 16. An assessment of the changes in necropsy findings affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Organs Observed signs Groups (mg/kg/day) a) Thymus Redness Testis(R) Aatropy Uterus Retention of clear fluid Adrenal(R) Aatropy No. of animals %: Control group (G1), Low-dose group (G2: 1,250 mg/10 ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) ,250, 2,500, 5,000 mg/kg/day. sialic acid NOAEL(no observed adverse effect level) 5,000 mg/kg/day. -, - 5,000 mg/kg/day.

16 132 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) Table 17. An assessment of the changes in histopathological findings affected by the 90-day oral administration of 23%-GNANA, a test substance to the male and female rats (n=10) Organs Observed signs Male groups (mg/kg/day) Testis Atropy degeneration - 1 Prostate Inflammatory cells infiltration 2 1 Kidney Protein casts Liver Bile duct hyperplasia 1 - Lung Osseous metaplasia - 1 Heart Focal myocarditis 2 1 Stomach Ulcer of the glandular stomach 1 - " - "=no finding, = no slide Organs No. of animals Observed signs Female groups (mg/kg/day) G1 (0) G2 (1,250) G3 (2,500) G4 (5,000) Pituitary gland Cyst in pars distalis 1 - Kidney Protein casts Mineralization Liver Vacuolization of hepatocytes 2 2 No. of animals %: Control group (G1), Low-dose group (G2: 1,250 mg/10ml/day), Medium-dose group (G3: 2,500 mg/10 ml/day), High-dose group (G4: 5,000 mg/10 ml/day) Table 18. Result of toxicity test by weight-based classification in SD rats (male) administrated orally with 23%-GNANA for 90 days Weight-based classification Important compoundrelated changes Minour compoundrelated changes Noncompoundrelated changes Not detected Finding Dose (mg/kg/day) Urinalysis (SG) 1,250, 2,500, 5,000 Relative weight of organs (kidney) 5,000 Absolute of organs (kidney) 5,000 Histological examination of organ (kidney: protein casts) 0(5/10), 5,000(6/10) Urinalysis (PRO) 1,250 Hematological values (RBC) 1250 Serum biochemical values (ALB) 1,250 Serum biochemical values (AST) 2,500 Plasma coagulation values (PT) 1,250 Relative weight of organs (testis) 1,250 Absolute weight of organs (left testis) 2,500 Necropsy findings (redness of thymus) 0, 1,250 Necropsy findings (right testis, atropy) 5,000 Histological examination of organ (testis) 5,000 Histological examination of organ (inflammatory cells infiltration) 0, 5,000 Histological examination of organ (kidney: bile duct hyperplasia) 0(1/10) Histological examination of organ (lung: osseous metaplasia) 5,000(1/10) Histological examination of organ (heart: myocarditis) 0(2/10), 5,000(1/10) Histological examination of organ (stomach: ulcer of the glandular stomach) 0(1/10)

17 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 133 Table 19. Result of toxicity test by weight-based classification in SD rats (female) administrated orally with 23%-GNANA for 90 days Weight-based classification Important compoundrelated changes Minour compoundrelated changes Noncompoundrelated changes Not detected Finding Dose (mg/kg/day) Absolute weight of organ (kidney) 5,000 Body weight 5,000 Food consumption decrease 5,000 Urinalysis (PRO, LEU) 5,000 Hematological values (HDW) 2,500 Serum biochemical values (ALB) 5,000 Necropsy findings (right adrenal, atropy) 5,000 Necropsy findings (uterus) 5,000 Histological examination of organ (kidney: protein casts) 0(4/10), 1,250(2/10), 2,500(2/10), 5,000(2/10) Histological examination of organ (kidney: mineralization) 0(1/10), 1,250(7/10), 2,500(6/10), 5,000(1/10) Histological examination of organ (kidney: vacuolation) 0(1/10), 5,000(1/10) sialic acid NOAEL (no observed adverse effect level) 5,000 mg/kg/day. 23%-GNANA NOAEL 5,000 mg/kg/day. GMP sialic acid sialic acid 23%, Sprague-Dawley 90., (Table 1, 18). (weight gains), (5,000 mg/kg/day), - (Table 6, 19). (body weight changes), 1~2. (Table 5, 18, 19)., RBC, HDW, (historical data) - (Table 11, 18, 19)., ALB, -. ALB, - (Table 12, 18, 19)., (, ),. -., -. -, (Table 14, 18, 19)., 1.,,. -. 1, (atropy) (degeneration)

18 134 Journal of Milk Science & Biotechnology 제 34 권제 2 호 (2016) (severe), - (Table 16, 18, 19)., (pituitary gland), (pars distalis) (cyst) (slight). (kidney) protein casts,, 2, 4, 6 (minimal) (slight), (minimal) 5. protein cast (minimal) 3, (slight) 1, (minimal) (slight) 1, (minimal) 2, (slight) 2 (Table 17, 18, 19). protein cast F , , 2, 4, 6,, -. 4, 2, 2, 4 -. (kidney) mineralization 1, 1, 5, 7., (KFDA, 2014)., -. (liver) 1 (minimal) (vacuolation), -. 1 (myocarditis) (minimal), 2 (minimal) ,000 mg/kg/day 23% G-NANA NOAEL (no observed adverse effect level, ) 5,000 mg/kg/day (Table 18, 19). GMP 1.2~1.5 g/l 10~25%, (Yoon et al., 2000; Brody, 2000). GMP 6~7% sialic acid,,, ganglioside (Wang et al., 2001; Wang et al., 2007; Wang, 2009), (Wang et al., 2007; Gorog and Kovacs, 1978; Iijima et al., 2004)., (Ishikawa and Koizumi, 2010; Zimmermann et al., 2007)., sialic acid GMP, 23%, 2,000 mg/kg/day., GMP sialic acid. sialic acid 23% (whey protein of hydrolysis). sialic acid 23%(v/v) GMP(glycomacropeptide) 87%(v/v) ( : 23%-GNANA). (KFDA, 2014) OECD(2008)., 0, 1,250, 2,500 5,000 mg/kg/day SPF Sprague-Dawley 90.,,,,,,,,.,.,, (P<0.05).,, SG PRO, - (non-adverse effect) (5,000 mg/kg/day). Weight-based classification( )., NOEL(No Observed Effect Level)

19 유청가수분해단백분말 (Sialic acid 23% 함유 ) 의랫드를이용한 90 일반복경구투여독성시험평가연구 135 5,000 mg/kg/day NOAEL(No Observed Adverse Effect Level) 5,000 mg/kg/day., NOAEL 5,000 mg/kg/day., 1,000 mg/kg/ day 5. ( -,, ). 1. Brody, E. P Biological activities of bovine glycomacropeptide. British Journal of Nutrition. 84:S39-S Boorman, G. A. et al Pathology of the fischer rat. Academic Press, INC., pp Gikins, Mary L. A Clinical laboratory parameters for Crl:CD (SD) rats. Charles River Laboratories. 4. Gorog, P. and Kovacs, I. B Anti-inflammatory effect of sialic acid. Agents and Actions. 8: Iijima, R., Takahashi, H., Namme, R., Ikegami, S. and Yamazaki, M Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Letters 561: Ishikawa, M. and Koizumi, S Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli. Carbohydrate Research 345: Keenan, C., Elmore, S., Francke-Carroll, S., Kemp, R., Kerlin, R., Peddada, S. and Pletcher, J Best practices for use of historical control data of proliferative rodent lesions. Toxicol. Pathol. 37: Lewis, R. W., Billington, R., Debryune, E., Gamer, A., Lang, B. and Carpanini, F Recognition of adverse and nonadverseeffects in toxicity studies. Toxicologic Pathology 30: Ministry of Food and Drug Safety (KFDA) Notice No (Jul. 30, 2014) Guidelines for toxicity tests in drugs, etc. KFDA Notice No (Feb. 12, 2015). 10. Moon, Y. I., Lee, W. J. and Oh, S Glycomacropeptide hydrolysed from bovine κ-casein:. Chromatographic changes of κ-casein macropeptide as related to trichloroacetic acid concentration. Korean J. Food Sci. Ani. Resour. 17(1): Oh, S. J., Kim, S. H., Jeon, W. M., Kim, B. C. and Ki, Y. K Glycomacropeptide hydrolysed from bovin κ- casein: I. The fractionation of glycomacropeptide. Korean J. Food Sci. Ani. Resour. 17(1): Organization for Economic Co-operation and Development (OECD) OECD guidelines 408 for the testing of chemicals: Repeated Dose 90 day oral toxicity study in oodents Organization for Economic Co-operation and Development. Paris, France. pp Park, Y. C. and Cho, M. H A new way in deciding NOAEL based on the findings from GLP-toxicity test. Toxicology Research 27: Springer-Verlag, R. S Sialic acids: Chemistry, metabolism and function. Carbohydrate Research 129: United States of Food and Drug Administration (USFDA) Guidance for industry estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Pharmacology and Toxicology. United States of Food and Drug Administration. Rockville. MD, USA. pp Wang, B Sialic acid is and essential nutrient for brain development and cognition. Annu. Rev. Nutr. 29: Wang, B. and Brand-Miller, J The role and potential of sialic acid in human nutrition. European Journal of Clinical Nutrition. 57: Wang, B., Brand-Miller, J., McVeagh, P. and Petocz, P Concentration and distribution of sialic acid in human milk and infant formulas1-3. Am. J. Clin. Nutr. 74: Wang, B., Yu, B., Karim, M., Hu, H. S., McGreevy, Y., Petocz, H. P., Held, S. and Miller, J. B Dietary sialic acid supplementation improves learning and memory in piglets1-3. Am. J. Clin. Nutr. 85: Yoon, Y. C., Cho, J. K., Song, C. H., Lee, S. and Chung, C. I Purification of the glycomacropeptide from cheese whey. Korean J. Food SCI. ANI. Resour. 20: Zimmermann, V., Hennemann, H. G., Dauβmann, D. and Kragl, U Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-D-glucosamine new strategies for the optimisation of neuraminic acid synthesis. Appl. Microbiol. Biotechnol. 76: Received June 1, 2016 Revised June 19, 2016 Accepted June 22, 2016