Milk, freeze-drying

To increase the activity and capability of reproduction of Saccharomyces cerevisiae (SC) Kabatov et al. [3.32] proposed the addition of 10 % skimmed milk, which has been saturated with Ar or N2. The freezing down to -25 °C was done under pressure and continued down to -55 °C. The freeze dried suspension did not change its quality during storage at +4 °C. 

In the Penzyme test, freeze-dried Streptomyces oo-carboxypeptidase is placed in sealed vials to which the milk sample is added. A preliminary incubation is carried out for 5 min at 47 C to cause some inactivation of the enzyme molecules. The degree of inactivation is dependent on the amount of -lactams presumed to be present in the milk sample. Subsequent addition of a reagent tablet containing synthetic o-alanine oligopeptide and o-amino acid oxidase followed by reincubation at 47 C for 15 min results in the release of o-alanine, its amount... 

In the Accusphere test a freeze-dried sphere containing the test organism Streptococcus thermophilus and bromocresol purple as indicator disperses into the test milk sample. The acidification test is very similar the milk sample is heated to be further inoculated with a Streptococcus thermophilus culture containing yeast extract, bromocresol purple indicator, and trimethoprim. It is then incubated for 2.5 h at 45 C (33). In the presence of inhibitory substances, the organism growth is suppressed, acid production is reduced or eliminated, and the color of the indicator remains unchanged. Addition of penicillinase to a positive milk sample results in change of the color of the indicator from purple to yellow when only -lactams are present. 

The Aria test is a routine assay in which a heat-treated milk sample is added to a well of a microtiter plate containing a freeze-dried tablet containing Bacillus subtilis ATCC 6633, nutrients, and triphenyltetrazoliumchloride as redox indicator (33). Following incubation, the normal growth of the organism is inhibited if antibacterials are present, and the uncolored indicator is not reduced into its red form. Detection of sulfonamides requires prior addition of trimethoprim in the milk samples analyzed. 

In contrast to tire previous multiresidue tests, the Valio TlOl test uses a freeze-dried Streptococcus thermophilus TIO l-strain dispersed into the milk. During incubation, the test organisms grow and produce acid, which causes the pH indicator to change color from blue to yellow. In the presence of any inhibitory substances, the indicator remains blue or turns to a greenish color depending on the concentration of inhibitors. 

Before the extraction procedure may commence, the sample must be prepared in such a way that it is in a condition for extraction of the analyte(s). For analyzing sulfonamide residues in liquid samples such as milk, a pretreatment dilution step with water prior to direct fluorometric detection may be required (207). Dilution of milk with aqueous buffer (208) or sodium chloride solution (209) prior to sample cleanup has also been reported. For the analysis of honey a simple dissolution of the sample in water (210, 211) or aqueous buffer (212) is generally required. Semisolid samples such as muscle, kidney, and liver, require, however, more intensive sample pretreatment. The analyte(s) must be exposed to extracting solvents to ensure maximum extraction. The most popular approach for tissue break-up is through use of a mincing and/or homogenizing apparatus. Lyophilization (freeze-drying) of swine kidney has been carried out prior to supercritical-fluid extraction of trimethoprim residues (213). 

When a lactose solution is dried rapidly, its viscosity increases so quickly that crystallization cannot take place. The dry lactose is essentially in the same condition as it was in solution, except for removal of the water. This is spoken of as a concentrated syrup or an amorphous (noncrystalline) glass. Various workers have shown conclusively that lactose in milk powder (spray, roller, or freeze-dried) is noncrystalline and exists in the same equilibrium mixture of a- and /3-lactose as existed in the milk prior to drying (Zadow 1984). 

Blocking buffer. 5% (w/v) freeze-dried nonfat milk (e.g., Marvel, Premier Beverages, Stafford, UK) in TST. Make up fresh as required. The quality of the dried milk is important, since some brands contain significant amounts of fat (see Note 4). 

Freeze-dried nonfat milk (e.g., Marvel, Premier Beverages, Stafford, UK). 

The use of HPLC for the analysis of bovine milk proteins was introduced by Diosady et al. (59), who compared SE-HPLC on two SynChropac GPC-100 columns in series and RP-HPLC on a lO-jUm-particle-sizc RP-8 column, both with UV detection, for the separation of dialyzed freeze-dried whey proteins. Column temperature was 40°C and 47°C, respectively. Samples were eluted with Tris-buffer pH 6 for SE-HPLC and a linear gradient of two solvents, i.e., 98% 0.5 M KH2P04, pH 2, and 98% isopropanol, each with 2% 2-methoxyethanol for RP-HPLC. They... 

Figure 22.Two examples of rennet-coagulate milk gel examined after freeze drying of the sample, (x17,000). [From 88].

More recently, pyrraline was found to increase almost linearly with time during the storage of freeze-dried milk at 70 °C. The amount formed increased with moisture content, reaching more than 5000 mg kg 1 protein in 50 h with 9% moisture.354 Values of up to 3100 mg kg 1 protein were found in some samples of milk or whey powder. Remarkably high amounts (200-3700 mg kg 1 protein) were also found in bakery products, indicating that up to 15% of the lysine residues may have been modified. 

The method of sample preparation to be used for a given analysis is governed by the nature and concentration of the analyte, the nature (solid or liquid) and type of matrix, the available sample amount, and also by the instrumental technique employed. Freeze-dried samples will require some form of digestion or dissolution in order to be analyzed by a classic atomic technique (i.e., using nebuliza-tion). Liquids might be analyzed by direct nebulization, but this is not always possible due to matrix interferences. Milk pretreatment may be necessary under such circumstances. 

The main problem in carrying out total multielemental determinations in milk (as in other biological samples) is the nature of the matrix, which may interfere with the analytical technique employed for the measurement. In this sense, pretreatment of the samples becomes necessary so as to minimize matrix effects as much as possible (e.g., by destroying the organic matrix). An alternative to such destructive acid attacks is the direct analysis in milk whey samples by simply diluting the sample previously obtained by centrifugation. The main preparation procedures for milk samples (whole, skimmed, or freeze-dried) can be classified as follows (a) use of diluted solutions in order to minimize matrix and molecular... 

Stapelfeldt, H., Nielsen, K.N., Jensen, S.K., Skibsted, L.H. 1999. Free radical formation in freeze-dried raw milk in relation to its a-tocopherol level. J. Dairy Res. 66, 461 466. 

Influence of Cryoprotectants on Viability and Heterologous Activity of Lyophilized Yeasts in Simulated Gastrointestinal Conditions To evaluate the influence of cryoprotectants on both the survival rate and CA4H activity of WRP45073A1 in simulated gastrointestinal conditions, 1010 viable freeze-dried yeasts and 200 pmol of franx-cinnamic acid were simultaneously introduced into the TIM. Yeast cells were lyophilized in the presence of the milk protein-trehalose mix, trehalose, lactose, or maltose, as previously explained (see above). The freeze-dried samples were introduced into the artificial stomach suspended in 300 mL of yeast culture medium without any storage period. The number of viable cells introduced into the TIM was determined from previously obtained survival rates (cf. Section 5.5.3.1). 

Conclusion Although the impact of freeze drying on both the survival rate and heterologous activity of yeasts in the artificial digestive system was found to be adverse, lyophilization appears to be a convenient technique for the dehydration of recombinant S. cerevisiae. Among the tested cryoprotectants, the association of milk proteins and trehalose was the most efficient to maintain the CA4H activity of... 

Figure 6. Scanning electron micrographs of freeze-dried protein fractions from soy milk (a, b, c) untreated, (d, e, f) 60-sec microwave, (g, h, i) hot water treatment. Note relative size of pores (arrows). (0—0) Unheated (B-B) 60-sec microwave (A—A) hot water.

Probiotics may be encapsulated in protein-based emulsions. Picot and Lacroix (2003) prepared microcapsules by emulsifying milkfat containing micronized skim milk powder (as a surrogate for freeze-dried bacteria) with heat denatured whey proteins and then spray drying. Incorporation rates of up to 58% milk fat and 29% skim milk... 

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