Table 2.
Batch‐to‐batch analysis of the NF
| Parameters | Batches | Method of analysis | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| #1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | #9 | #10 | ||
| Composition | |||||||||||
| 3’‐SL sodium salt (% w/w DM) | 93.9 | 94.1 | 93.8 | 91.4 | 92.1 | 91.3 | 88.1 | 96.8 | 92.1 | 90.1 |
HPAEC‐PAD (validated internal method) 1 |
| d‐Lactose (% w/w DM) | 0.3 | 0.3 | 0.4 | 0.4 | 0.3 | < LOQ | 0.5 | < LOQ | < LOQ | < LOQ | |
| Sialic acid (% w/w DM) | 0.2 | 0.3 | 0.3 | 0.4 | 0.2 | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | |
| N‐acetyl‐d‐glucosamine (% w/w DM) | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | |
| 3’‐Sialyllactulose (% w/w DM) | – | – | – | – | – | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | |
| Sum of other carbohydrates (% w/w DM) | 1.8 | 1.1 | 2.0 | 4.8 | 1.2 | 0.1 | 3.1 | 0.0 | 0.3 | 0.0 | Calculation 2 |
| Protein (%) | 0.001 | 0.002 | 0.001 | 0.002 | 0.002 | 0.001 | 0.002 | 0.002 | 0.002 | 0.002 | Nanoquant (modified Bradford) |
| Ash (%) | 3.7 | 4.1 | 3.4 | 2.9 | 6.1 | 7.1 | 6.9 | 3.3 | 6.1 | 8.4 | ASU L 06.00‐4 |
| Water (%) | 8.3 | 8.8 | 8.5 | 8.7 | 6.0 | 6.3 | 6.9 | 6.3 | 6.0 | 6.4 | Karl Fischer titration |
| Sodium (%) | 3.0 | 3.1 | 3.0 | 3.2 | 2.8 | 3.1 | 3.1 | 3.4 | 2.8 | 3.0 | ICP‐MS (ASU L 00.00‐13) |
| Contaminants | |||||||||||
| Arsenic 3 (mg/kg) | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | ASU L 00.00‐135: 2011‐01 – ICP‐MS |
| Cadmium 3 (mg/kg) | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | |
| Lead 3 (mg/kg) | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | 0.011 | |
| Mercury 3 (mg/kg) | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | < LOQ | |
| Aflatoxin M1 (µg/kg) | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | < 0.025 | DIN EN ISO 14501: 2008‐01 – IAC‐HPLC‐FD |
| Microbial parameters | |||||||||||
| Standard plate count (CFU/g) | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | ISO 4833‐2 |
| Yeast and mould (CFU/g) | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | ISO 21527‐2: 2008‐07 |
| Enterobacteriaceae (CFU/g) | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | < 10 | ISO 21528‐2: 2019‐05 |
| Salmonella (in 25 g) | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | DIN EN ISO 6579‐1: 2017‐07 |
| Cronobacter spp. (in 10 g) | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ISO/TS 22964: 2017‐04 |
| Listeria monocytogenes (in 25 g) | ND | – | – | ND | ND | – | – | – | – | – | DIN EN ISO 11290‐1: 2017‐09 |
| Bacillus cereus (in 5 g) | ND | – | – | ND | ND | – | – | – | – | – | ASU L 00.00‐108: 2007‐04 |
| Endotoxins (EU/mg) | 0.018 | 0.010 | 0.011 | 0.026 | < 0.005 | < 0.005 | < 0.005 | < 0.005 | < 0.005 | 0.289 | Ph. Eur. 2.6.14 |
‘–: Not reported; 3’‐SL: 3’‐Sialyllactose; ASU: Official collection of analysis methods according to § 64 of the German Food and Feed Code (LFGB); CFU: Colony forming unit; DIN: German Institute for Standardisation e. V.; DM: Dry matter; EN: European norm; EU: Endotoxin unit; HPAEC‐PAD: High‐performance anion‐exchange chromatography – pulsed amperometric detection; IAC‐HPLC‐FD: Immunoaffinity chromatography – high‐performance liquid chromatography – fluorescence detector; ICP‐MS: Inductively coupled plasma – mass spectrometry; ISO: International Organisation for Standardisation; LOQ: Limit of quantification; ND: Not detected; Ph. Eur.: European Pharmacopeia; TS: Technical specification.
LOQs: d‐Lactose = 0.14% w/w DM; Sialic acid = 0.14% w/w DM; N‐acetyl‐d‐glucosamine = 0.14% w/w DM; 3’‐Sialyllactulose = 1.13% w/w DM.
Sum of other carbohydrates = 100 (% w/w DM) – 3’‐SL sodium salt (% w/w DM) – Quantified carbohydrates (% w/w DM) – Ash (% w/w DM). For those batches of the NF where the levels of any carbohydrate by‐product were below the respective limit of quantification (LOQ), the concentration of the corresponding compound has been considered to be equal to the respective LOQ value for the purpose of calculating the sum of other carbohydrates in the corresponding batch.
LOQs: Arsenic = 0.05 mg/kg; Cadmium = 0.010 mg/kg; Lead = 0.010 mg/kg; Mercury = 0.005 mg/kg.