a specific detection of Casein, Egg and Lysozyme Validation Report
Different egg and milk products are added to wines as clarification agents, for fine tuning of wine flavour (i.e. selective tannin adsorption) or for conservation. In many countries allergens in wine will have to be labelled according to the legislation. At present, no threshold values are set. This means if e.g. fining agents are used and detected in the wine they will have to be declared on the label of the wine bottle (e.g. Produced with egg. Traces may remain ). Fig. 1 The picture shows a typical allergen RIDASCREEN FAST test kit, containing ready-to-use reagents like microtiter plate, standards, conjugate, substrate/chromogen, stop solution and buffers. RIDASCREEN ELISA test kits for wine analysis The ELISA test kits for allergen analysis from R-Biopharm are well suited to detect allergen residues in wines. The most important parameters are ovalbumin and lysozyme from egg as well as casein from milk. Table 1 gives an overview on the Limit of Detection (LOD), Limit of Quantification (LOQ) as well as the extraction procedure for sample preparation. The sample preparation is straight forward: 1 ml of wine is diluted with 19 ml preheated (60 C) Allergen Extraction Buffer (AEB), which is provided in the test kits. The extract is then heated for 10 min at 60 C and centrifuged for 10 min at 200 g. After filtration, 100 µl of the extract is used in the sandwich ELISA. The extraction procedure is identical for casein, lysozyme and ovalbumin, therefore only one extraction for all three parameters is necessary. Table 1 Overview of allergen test kits for wine and their characteristics Limit of detection Limit of quantification RIDASCREEN FAST Ei/Egg Protein R6402 0.101 mg/kg whole egg powder (0.027 mg/kg egg white protein) 0. mg/kg whole egg powder RIDASCREEN FAST Casein R4612 0.24 mg/kg casein 0. mg/kg casein RIDASCREEN FAST Lysozym R642 0.02 mg/kg lysozyme 0.0 mg/kg lysozyme Extraction AEB* AEB* AEB* Extraction conditions 10 min at 60 C 10 min at 60 C 10 min at 60 C *RIDASCREEN Allergen Extraction Buffer 2
Determination of egg proteins in wine Different spiking materials (extracted egg white, whole egg powder (NIST RM844) and an industrial produced egg white powder) were used in red wine as can be seen in table 2. The recoveries were between 76-110 %. Spiking with cooked egg white 1 gram of milled cooked egg white was extracted with 20 ml preheated allergen extraction buffer (AEB) for 10 min at 60 C. The extract was cooled to room temperature, centrifuged 10 min at 200 g, and filtered. The protein content was measured by the BCA (Pierce) method and resulted in a concentration of 2.8 mg/ml. Theoretically, the extraction of 1 g cooked egg white would result in a protein concentration of. mg/ml (1 g egg white contains 110 mg protein; Souci et al. 2000). Therefore, approximately 0 % of heat-denatured egg white proteins could be solubilized by using the described extraction procedure. Spiking with egg white powder / whole egg powder The spiking with egg white powder or whole egg powder (RM844) is described in the R-Biopharm document Instructions. In this case e.g. a powder with known protein content was used as it allows to calculate the result as egg white protein. The egg powder was dissolved in buffer and then added to the wine at different spiking levels. Direct spiking of wine with egg powder is not possible due to the fact that the powder is not soluble in wine and precipitates. Table 2 of different spiking materials in red wine using RIDASCREEN FAST Ei/ Egg Protein. The RIDASCREEN FAST Ei/Egg Protein has been standardized to whole egg powder from NIST (RM844). When spiking with egg white, the spiking amount must be converted to whole egg powder (multiplication with 4.2). Thus, a spike with 2.0 mg/kg egg white corresponds to a spike with 8.4 mg/kg whole egg powder. material (mg/kg) Target (mg/kg whole egg powder) (mg/kg whole egg powder) Egg white, cooked (extract) 2.0 6.0 18.0 8.4 2.2 7.6 7.4 19.2 61.8 88 76 82 Whole egg powder (RM844) 1. 4. 13. 1. 4. 13. 1.3 4.9 14.1 87 109 104 Egg white powder 1.0 3.0 9.0 4.2 12. 37.4 4. 13.8 36. 107 110 98 3
In a field study, two red wines (Pinot noir and St. Laurent) were clarified with egg white proteins. The egg protein concentration was monitored as can be seen in table 3. Before fining, both wines had values below the LOQ of 0. mg/l whole egg powder. After adding liquid egg white the concentrations were 30 and 410 mg/l. These concentrations are close to the calculated target value of 381 mg/l. During stirring of the wine, the concentrations remained at this level. After six and twelve hours of sedimentation the concentration in the wines decreased considerably, due to the fact that most of the egg white is present in the precipitate. The first filtration over diatomaceous earth led to concentrations of 1.1 mg/l for Pinot noir and 9.4 mg/l for St. Laurent. After filtration over a 0.6 µm filter and bottling, the concentrations were below the LOQ for Pinot noir and 1.1 mg/l for St. Laurent (see also Lacorn et. al.: Determination of Residual Egg white Proteins in red Wines during and after Fining. Am. J. Enol. Vitic. 62:3 (2011). Table 3 Measurement of egg protein at different processing stages Concentration of mg whole egg powder/l wine Processing stage Pinot noir St. Laurent Wine, untreated < LOQ* < LOQ* Wine, treated with liquid egg white 410 30 Wine, sedimented 6 hrs 280 6 Wine, sedimented 24 hrs 38 3 Wine, filtered 1.1 9.4 Wine, bottled < LOQ 1.1 *LOQ: Limit of Quantification 0. mg/kg whole egg powder (RM844) Different German and Italian wines from the market were analyzed for egg. In one Italian wine a concentration of 1.7 mg/kg whole egg powder was detected. Table 4 Egg concentration in different commercially available wines Wine Optical density Concentration in mg/kg whole egg powder Italian red wine 1 0.78 1.7 Italian red wine 2 0.120 < LOQ Italian red wine 3 0.202 < LOQ German red wine 0.26 < LOQ 4
Determination of casein in wine Different casein containing spiking materials such as skim milk powder and UHT (Ultra High Temperature) milk were used as can be seen in table. The recoveries were between 78-117 %. Spiking with casein Spiking with casein or skim milk powder is described in the R-Biopharm document Instructions. The spiking with milk (approx. 26 g casein/kg milk) was carried out directly by adding milk to the wine. Casein or skim milk powder (approx. 4 g casein/ kg skim milk powder) were solved in urea-containing PBS-buffer and then added to the wine. If casein or skim milk powder were added directly to the wine, precipitates were observed and consequently low recoveries. Table a of different spiking materials in white wine using RIDASCREEN FAST Casein material (mg/kg) Target (mg/kg casein) (mg/kg casein) Casein 0.6 2.2 4.0 8.2 0.6 2.2 4.0 8.2 0.7 2.0 4.6 117 91 11 109 UHT milk 38.0 38.0 23.1 10 10 0.7 7.8 10.1 0.8 78 101 114 Skim milk powder 2.3 1.4 1.1 7.0 0.9 8.2 82 117 Table b of Casein spike material in red wine using RIDASCREEN FAST Casein material (mg/kg) Target value (mg/l casein) (mg/l casein) Casein ---- 1.9 10.0 ---- 1.9 10.0 < LOD 1.9 10.2 ---- 100 100 102
Determination of lysozyme in wine For the spiking, lysozyme from Sigma (L6876, a lyophilzed powder from chicken egg white) was used. The lysozyme was diluted to the required concentration and then pipetted to the wine. The recoveries were between 100-120 % (see table 6). Table 6 of lysozyme in white and red wine using RIDASCREEN FAST Lysozym Wine (mg/l) (mg/l lysozyme) White wine (Custoza) 0.0 0.1 1.0.0 0.06 0.11 1.0. 120 111 10 111 Red wine (Shiraz) 0.0 0.1 1.0.0 0.0 0.11 1.08.0 100 110 108 101 In a second experiment two different commercial lysozyme preparations were analyzed. LALLZYME Lyso-easy is a ready-to-use solution of lysozyme (22 % solution, no additives, stable at room temperature for 6 months), specific for gram-positive bacteria, such as lactic acid bacteria (LAB). Lyso-easy is used at various winemaking stages such as in grape must to reduce the risk of spoilage LAB and at the end of the alcohol fermentation to delay the onset of the malolactic fermentation. BactiCare from Erbslöh is an enzyme produced from egg white protein. Table 7 of commercial lysozyme preparations for wine processing Commercial preparation used for spiking Lallzyme Lyso-easy TM (Lallemand) White wine (Custoza) BactiCare (Erbslöh) Red wine (Shiraz) Lysozyme (mg/l) *Values in brackets are out of calibration range (mg/l lysozyme).4 4.1.1.6.8 6.0.3 6.3 107 83 103 (113)* 11 119 10 (12)* The recovery in red wine for both products was measured between 83 and 119 % (see table 7). Therefore, it can be concluded that the ELISA RIDASCREEN FAST Lysozym is well suited to detect lysozyme in wine. 6
Conclusion The RIDASCREEN test kits for the determination of casein, egg and lysozyme are robust and reliable methods to determine residual concentrations of fining, clarification and conservation agents in wine. 7