Table 2.
Batch‐to‐batch analysis of the NF
| Parameters | NF (produced with soy peptone in the fermentation media) | NF (produced without soy peptone in the fermentation media) | Method of analysis | ||||||
|---|---|---|---|---|---|---|---|---|---|
| #1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | ||
| Composition | |||||||||
| 6′‐SL sodium salt (% w/w DM) | 87.0 | 92.0 | 90.0 | 92.0 | 92.0 | 91.0 | 93.0 | 95.0 | HPLC‐CAD (validated internal method) |
| Sialic acid (% w/w DM) | 5.1 | 3.5 | 4.9 | 4.3 | 5.4 | 1.8 | 0.1 | 0.2 | HPLC‐CAD (a) (validated internal method) |
| d‐Glucose (% w/w DM) | < 0.02 | < 0.02 | < 0.02 | < 0.02 | < 0.02 | < 0.05 | < 0.05 | < 0.05 | HPLC‐PAD (b) (validated internal method) |
| d‐Lactose (% w/w DM) | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | < 0.05 | < 0.05 | < 0.05 | HPLC‐PAD (b) (validated internal method) |
| Sum of 6′‐sialyllactulose and 3′‐SL sodium salts (% w/w DM) | 0.4 | 0.4 | 0.5 | 0.5 | 0.4 | 1.7 | 0.5 | 0.3 | HPLC‐CAD (a) , (c) (validated internal method) |
| Sum of other carbohydrates (% w/w DM) | 6.6 | 3.3 | 3.8 | 2.5 | 1.4 | 5.0 | 5.8 | 3.8 | Calculation (d) |
| Water (% w/w) | 5.3 | 5.0 | 5.4 | 5.6 | 5.0 | 7.9 | 8.4 | 8.6 | JP 2.48 (e) – Karl Fischer titration (volumetric/coulometric titration) |
| Ash (% w/w DM) | – | – | 11.6 | – | 11.7 | 11.3 | 11.1 | 11.2 |
JP 2.44 (e) (residue on ignition, gravimetry) |
| Protein (% w/w) | – | – | ≤ 0.01 | – | ≤ 0.01 | ≤ 0.01 | ≤ 0.01 | ≤ 0.01 | Bradford assay (f) (spectrophotometry) |
| Sodium (% w/w DM) | 3.8 | 3.8 | 3.8 | 3.7 | 3.8 | 3.5 | 3.5 | 3.6 |
USP 233 (g) (ICP‐MS or ICP‐OES) |
| pH (5% solution, 25°C) | 6.4 | 6.5 | 6.5 | 6.5 | 6.5 | 6.1 | 5.9 | 5.8 | JP 2.54 (e) (potentiometry) |
| Contaminants | |||||||||
| Arsenic (mg/kg) | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | < 0.01 (i) | < 0.01 (i) | < 0.03 (i) |
AOAC (2019) 999.10 and 2011.14 (i) (AAS and ICP‐OES) |
| Cadmium (mg/kg) | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | < 0.01 (j) | < 0.01 (j) | < 0.01 (j) |
AOAC (2019) 999.10 and 2011.14 (j) (AAS and ICP‐OES) |
| Lead (mg/kg) | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | < 0.02 (k) | 0.03 (k) | < 0.02 (k) |
AOAC (2019) 999.10 and 2011.14 (k) (AAS and ICP‐OES) |
| Mercury (mg/kg) | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | < 0.004 (l) | < 0.004 (l) | < 0.004 (l) |
US EPA, February 2007, Method 7473 (l) (AAS) |
| Aflatoxin M1 (μg/kg) | – | < 0.02 | < 0.02 | < 0.02 | < 0.02 | < 0.02 | < 0.02 | < 0.02 |
AOAC 2000.08 (m) (HPLC) |
| Microbial parameters | |||||||||
| Total plate count (CFU/g) | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | ISO 4833‐1:2013 (n) (colony count) |
| Yeasts and moulds (CFU/g) | < 100 | < 100 | < 100 | < 100 | < 100 | < 10 (o) | < 10 (o) | < 10 (o) | ISO 21527‐2:2008 (p) (colony count) |
| Enterobacteriaceae (in 10 g) | ND | ND | ND | ND | ND | ND | ND | ND | ISO 21528‐1:2017 (detection or qualitative method) |
| Salmonella spp. (in 25 g) | – | – | ND | – | – | ND | ND | ND | ISO 6579‐1:2017 (detection or qualitative method) |
| Cronobacter spp. (in 10 g) | ND | ND | ND | ND | ND | ND | ND | ND | ISO 22964:2017 (detection or qualitative method) |
| Listeria monocytogenes (in 25 g) | ND | ND | ND | ND | ND | ND | ND | ND | ISO 11290‐1:2017 (detection or qualitative method) |
| Presumptive Bacillus cereus (CFU/g) | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 |
ISO 7932:2004 (q) (colony count) |
| Endotoxins (EU/mg) | 0.006 | 0.011 | 0.020 | 0.034 | 0.026 | < 0.0002 (r) | < 0.0002 (r) | < 0.0002 (r) | JP 4.01 (e) (kinetic‐turbidimetric method) |
‘–’: Not reported; 6′‐SL: 6′‐Sialyllactose; 3′‐SL: 3′‐Sialyllactose; AAS: Atomic absorption spectroscopy; AOAC: Association of Official Analytical Collaboration; CFU: Colony forming units; DM: Dry matter; EU: Endotoxin units; HPLC‐CAD: High‐performance liquid chromatography – charged aerosol detection; HPLC‐PAD: High‐performance liquid chromatography – pulsed amperometric detection; ICP‐MS: Inductively coupled plasma – mass spectrometry; ICP‐OES: Inductively coupled plasma – optical emission spectroscopy; ISO: International Organisation for Standardisation; JP: Japanese Pharmacopoeia; LOD: Limit of detection; LOQ: Limit of quantification; MW: Molecular weight; ND: Not detected; US EPA: United States Environmental Protection Agency; USP: United States Pharmacopoeia; w/w: Weight per weight.
For batches #1 to #5, the LOD and LOQ for sialic acid, 6′‐sialyllactulose and 3′‐SL sodium salts are, respectively, 0.03% w/w DM and 0.2% w/w DM (as 6′‐SL sodium salt). For batches #6 to #8, the LOD and LOQ for sialic acid are, respectively, 0.15% w/w DM and 0.45% w/w DM (as sialic acid); the LOD and LOQ for 6′‐sialyllactulose + 3′‐SL sodium salts are, respectively, 0.13% w/w DM and 0.18% w/w DM (as 6′‐SL sodium salt).
For batches #1 to #5, the LOD for d‐glucose and d‐lactose is 0.02% w/w DM, and their LOQ is 0.05% w/w DM (as d‐lactose). For batches #6 to #8, the LOD for d‐glucose and d‐lactose is 0.05% w/w DM, and their LOQ is 0.05% w/w DM (as d‐glucose and d‐lactose, respectively).
6′‐sialyllactulose and 3′‐SL sodium salts peaks on the HPLC‐CAD chromatograms overlap.
Sum of other carbohydrates = 100% w/w DM – 6′‐SL (acid) (% w/w DM) – quantified carbohydrates (i.e., sialic acid, d‐glucose, d‐lactose, 6′‐sialyllactulose (acid) and 3′‐SL (acid); % w/w DM) – sodium (% w/w DM). Concentrations in acid form for the respective carbohydrates have been theoretically calculated.
Consistent with the compendial method specified in the 17th edition of the Japanese Pharmacopoeia (2016).
Evaluated using a limit test at 100 ppm.
Method is consistent with the compendial method specified in the United States Pharmacopoeia 35th revision (2011). Batches #1 to #5 were analysed by ICP‐MS (LOQ = 1.25%). Batches #6 to #8 were analysed by ICP‐OES (LOQ = 0.005%).
LOQ for arsenic, cadmium, lead and mercury is 0.05 mg/kg.
Method based on AOAC (2019) 999.10 and 2011.14. LOD = 0.01 mg/kg. LOQ = 0.03 mg/kg.
Method based on AOAC (2019) 999.10 and 2011.14. LOD = 0.01 mg/kg. LOQ = 0.03 mg/kg.
Method based on AOAC (2019) 999.10 and 2011.14. LOD = 0.02 mg/kg. LOQ = 0.03 mg/kg.
Method based on U.S. EPA, February 2007, Method 7473, Mercury Analyser. LOD = 0.004 mg/kg. LOQ = 0.01 mg/kg.
LOQ = 0.02 μg/kg.
LOD = 10 CFU/g.
LOD = 10 CFU/g (in‐depth plating).
LOD = 100 CFU/g (surface plating).
LOD = 10 CFU/g.
LOQ = 0.0002 EU/mg.