Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk.

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500-4,000 Da; 4-20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy.

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and 1H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.

[Tests of Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. I. Verification of its bacteriostatic and bacteriocidal effects and its interference effects]. / Testování nového konzervacního prípravku vzorku mléka Milkofix pro úcely infracervené analýzy základního slození mléka. I. Overení bakteriostatických a baktericidních vlastností a interferencního vlivu.

Hygienic, ecological and health problems of sample preservation for an analysis of basic milk components make us continually to develop a safer chemical preservative substance which will preserve the original sample composition for the time required and which will not influence the analyses. Trzický (1990) proposed Milkofix (M), a preservative substance on the basis of silver compound. The author reports on minimum risks of the use of this preparation, in comparison with traditional preservatives. Preservative efficiency of Milkofix was compared with other preservatives: K2Cr2O7 (C), NaN3 (A) and bronopol (B). The following concentrations were used: A--0.0085 g NaN3 and 0.0630 g NaCl, B--0.0050 g bronopol and 0.0500 g NaCl, C--0.0330 g K2Cr2O7 and 0.0670 g KCl in tablet, M--0.1250 g of the mixture, all amounts are per 25 ml milk. The observed antibacterial efficiency of M could be seen in a slower decrease in actual acidity, and/or in an increase in titratable acidity in M-treated samples unlike untreated ones (N). From the starting value pH 6.3 (Fig. 1), the value of N treatment dropped to 3.8 after two days, the values of M and A treatments dropped to 4.9 after nine days and to 5.7 after twelve days, respectively. As for SH, the values increased within the same interval from 6.5 (2.5 mmol/l) to 28.6 in N, and to 22.3 in M and 9.4 in A (Fig. 3). There was a similar trend when the milk samples were stored at a temperature of 4 degrees C, but the differences between the preservation methods were not so clear in comparison with storage at a temperature of 20 degrees C (Figs. 1 and 3). The standardized SH value of 9.0 (2.5 mmol) for infraanalyzer measurements was exceeded after 24 hours in N samples, after four days in M samples and after 12 days in A samples at a temperature of 20 degrees C. The observation of the growth of microorganism counts (CPM) showed that this growth was slower in M than in N, but faster in the samples of C treatment (Fig. 5). The generative time of CPM in N made 1.6 hours, in M 2.4 hours and in C 7.9 hours. The lag phase of these mixed cultures was 24 hours in M, 60 hours in C and in N treatment the lag phase was zero.(ABSTRACT TRUNCATED AT 400 WORDS)

[Testing Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. II. Verification of its preservative effects in relation to infrared analysis]. / Testování nového konzervacního prípravku vzorku mléka Milkofix pro úcely infracervené analýzy základního slození mléka. II. Overení konzervacního úcinku ve vztahu k infracervené analýze.

For the purposes of the infrared analysis of the basic milk composition, Milkofix, an ecologically friendly preparation used for milk sample preservation (Trzický, 1990), was compared with untreated samples (N) and with samples preserved with sodium azide (A), bronopol (B) and potassium dichromate (C) at a storage temperature of 20 degrees C (I) and 4 degrees C (II) in samples kept for 14 and 18 days. Pursuant to the recommendations cited in literature, the preservatives had these concentrations: A = 0.0085 g NaN3 and 0.0630 g NaCl; B = 0.0050 g bronopol and 0.0500 g NaCl; C = 0.0330 g K2Cr2O7 and 0.0670 g KCl; M = 0.1250 g, all amounts are per 25 ml milk. Three bulk milk samples were used which were analyzed on an automatic Milko-Scan 133 B infraanalyzer (Foss Electric, Denmark) every day. On the basis of a graphical evaluation of the results by IDF recommendations (1985) the times within which the applicable results could be obtained were determined for the various methods of milk sample treatment (Figs. 1 to 6): N I--0 days; A I--9; B I--10; C I--13; M I--4; N II--10; A II--5; B II--11; C II--15; M II--10 (Tab. I). The results recorded for Milkofix are in agreement with the conclusions drawn in the previous study, where the intervals of four and nine days were determined. The days to milk sample coagulation were as follows: N I--1 day, M I--10 days. The coagulation in A I, B I, C I and N II samples was not observed even after 13-day storage and in A II, B II, C II and M II samples not even after 17 days of storage. The results for particular components (fat, proteins, lactose) of milk samples differently treated in time are presented in Tabs. II, III and IV. A system of evaluating criteria (Tab. V) was used to determine the order beginning from the most convenient method of milk sample treatment for the given purpose: 1. C II, 2. C I, 3. B II and A I, 4. B I, 5. M II, 6. N II, 7. A II, 8. M I and 9. N I.(ABSTRACT TRUNCATED AT 400 WORDS)

[The effect of aging of milk samples on the accuracy of infrared analysis of milk composition]. / Vliv stárnutí vzorku na presnost infracervené analýzy základního slození mléka.

The effect of a decrease (and/or fermentation) in the lactose content during milk storage under different conditions was investigated on the accuracy of the results obtained on a Milko-Scan apparatus to contribute to the present knowledge of this problem. The results were in agreement with some results cited in the literature. These wavelengths are used for infrared spectrophotometry on the above apparatus: for fat 3.48 microns, for proteins 6.46 microns and for lactose 9.60 microns. Bulk milk samples used for the tests were untreated or treated with potassium dichromate, bronopol, sodium azide and Milkofix at the temperatures of storage in darkness 20 degrees C and 4 degrees C. The differences against the reference values (measured on the first day) were determined and evaluated in milk composition and characteristics as arising during milk storage. These differences were used in form of either cumulative means of differences (Figs. 1 to 5) or individual differences (Fig. 8). In the first part significant correlation coefficients (P less than 0.001) were calculated for the relationship between the variations of lactose content and the fat and protein contents: r = -0.59 and/or -0.73 (Figs. 6 and 7). This suggests that the decrease in the lactose content by 0.10% recorded by the infrared analysis and caused by lactose decomposition is accompanied by a "seeming" increase in the fat and protein content by about 0.04%. In the second part the correlation coefficients for the fat and protein contents r = -0.96 and -0.96 (P less than 0.001; Figs. 9 and 10; Tab. II) were calculated on the basis of an observation of the lactose decrease in an untreated milk sample (20 degrees C for 28 hours). These coefficients are somewhat different from the preceding ones; this is due to the lower homogeneity of the first set where the milk samples were treated in a different way, but the coefficients confirm the same conclusions. The values of the correlation coefficients for the dependence between the development of the acquired titratable acidity (SH) and the variations of fat (F), protein (P) and lactose (L) contents were as follows: r = 0.95; 0.95; -0.99 (P less than 0.001; Figs. 12, 13; Tab. II). Thus the above-mentioned "seeming" increase in the F and P contents can be explained to the extent of 92.2% from the decrease in the L content, which also causes the increase in titratable acidity to the extent of 98.0%.(ABSTRACT TRUNCATED AT 400 WORDS)

[Comparison of the effectiveness of Milkofix, a preservative preparation, with traditional preservative agents in the determination of somatic cell count in milk samples using a fluoro-optic-electronic method]. / Srovnání úcinnosti konzervacního prípravku Milkofix s tradicními konzervacními prostredky pro úcely stanovení poctu somatických bunek ve vzorcích mléka fluoro-opto-elektronickou metodou.

Milkofix (M), a health friendly preservative substance, to be used for milk sample preservation (Trzický, 1990), was compared with other preservatives. Untreated milk samples (N) were tested against samples treated with sodium azide (A; 0.0085 g NaN3 and 0.0630 g NaCl), bronopol (B; 0.0100 g bronopol and 0.090 g NaCl), potassium dichromate (C; 0.0330 g K2Cr2O7 and 0.0670 g KCl) and Milkofix (M; 0.1250 g). The doses of the preservatives A, B, C and M are per 25 ml milk. The somatic cell counts (SB) were determined on a FOSSOMATIC 90 apparatus (FOSS ELECTRIC, DENMARK). In the treated milk samples taken from individual cows the values of SB counts were significantly higher than in N samples if determined within eight hours after sampling (Tab. I): in A higher by 18.6%, B by 26.3%, C by 26.4% and M by 24.3% (P less than 0.05). The significantly higher values of SB counts were recorded in bulk milk samples treated with preservatives in comparison with N samples immediately after sampling: in A by 6.0%, in B, C and M by 12.9% (P less than 0.01; Tab. II). After one-day storage of N samples at a temperature of 4 degrees C these differences are insignificant (P greater than 0.05), thus the results of N, A, B, C and M samples obtained after one-day storage at 4 degrees C can be taken as actual and mutually comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relation of lactose levels in milk and indicators of mammary gland health in the first third of lactation]. / Vztah obsahu laktózy v mléce k ukazatelum zdravotního stavu mlécné zlázy v první tretine laktace.

A total of 662 bucket milk samples from cows of two breed groups were examined: red and white breed = Bohemian Pied cattle with different genetic proportions of Ayrshire and Red Holstein improvement breeds; black and white breed = Black and White Lowland breed and different degree of absorptive crossing with a genetic proportion of the Holstein breed. Samples of daily milk yields were taken in the first three months of lactation once a month within a year. A possibility of using lactose content as an auxiliary indicator for detection of the mammary gland secretion disorders in the initial lactation stage was evaluated. The average values of the different indicators and their variability are summarized in Tab. I showing also the significance in a statistical model of included effects. Lactose content (L) was 4.88 +/- 0.20%, chloride content (Cl-) 113.7 +/- 22.4 mg/100 ml, somatic cell (SC) count 474 +/- 805 thousand/ml, SC count log corresponds to the geometrical mean of 234 thousand/ml, titratable acidity (SH) 7.34 +/- 0.83 x 2.5 mmol/l, chloride-lactose ratio (ClL) 2.27 +/- 0.51, conductivity (gamma) 442.4 +/- 34.5 mS/m and mastitis test (MT-NK) 0.72 +/- 1.18. The efficiency of the used statistical model was highest for Cl- content (Tab. I, R2 = 0.41), and it was lowest for SC counts (R2 = 0.07), while it increased to the twofold value (R2 = 0.15) after logarithmic transformation of SC counts. The breed group exerted a significant effect on Cl-, SC, log SC, SH, ClL, gamma and MT-NK (Tab. I). The breed group of red and white cows (Tab. II) had higher component contents and better indicators of the udder health state (Cl-, SC, log SC, ClL, gamma and MT-NK). The month of lactation influenced significantly SC, log SC, SH and gamma (Tab. I). A decrease in SC counts with the accruing month of lactation was observed (Tab. II), the trend of gamma and SH was opposite. The effect of lactation number was found to be significant for L, SC, log SC, SH, ClL, gamma and MT-NK (Tab. I). A tendency of a gradual decrease with the lactation number was observed in these indicators: L, SH, proteins and solids-non-fat (Fig. 1), while Cl-, gamma and ClL showed an opposite tendency. The year season influenced significantly L, log SC, SH, ClL, gamma and MT-NK (Tab. I, Fig. 2).(ABSTRACT TRUNCATED AT 400 WORDS)