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Our objective was to determine the effects of temperature and protein concentration on viscosity increase and gelation of liquid micellar casein concentrate (MCC) at protein concentrations from 6 to 20% during refrigerated storage. Skim milk (~350 kg) was pasteurized (72°C for 16 s) and filtered through a ceramic microfiltration system to make MCC and replicated 3 times. The liquid MCC was immediately concentrated via a plate ultrafiltration system to 18% protein (wt/wt). The MCC was then diluted to various protein concentrations (6–18%, wt/wt). The highest protein concentrations of MCC formed gels almost immediately on cooling to 4°C, whereas lower concentrations of MCC were viscous liquids. Apparent viscosity (AV) determination using a rotational viscometer, gel strength using a compression test, and protein analysis of supernatants from ultracentrifugation by the Kjeldahl method were performed. The AV data were collected from MCC (6.54, 8.75, 10.66, and 13.21% protein) at 4, 20, and 37°C, and compression force test data were collected for MCC (15.6, 17.9, and 20.3% protein) over a period of 2-wk storage at 4°C. The maximum compressive load was compared at each time point to determine the changes in gel strength over time. Supernatants from MCC of 6.96 and 11.61% protein were collected after ultracentrifugation (100,605 × g for 2 h at 4, 20, and 37°C) and the nitrogen distributions (total, noncasein, casein, and nonprotein nitrogen) were determined. The protein and casein as a percent of true protein concentration in the liquid phase around casein micelles in MCC increased with increasing total MCC protein concentration and with decreasing temperature. Casein as a percent of true protein at 4°C in the liquid phase around casein micelles increased from about 16% for skim milk to about 78% for an MCC containing 11.6% protein. This increase was larger than expected, and this may promote increased viscosity. The AV of MCC solutions in the range of 6 to 13% casein increased with increasing casein concentration and decreasing temperature. We observed a temperature by protein concentration interaction, with AV increasing more rapidly with decreasing temperature at high protein concentration. The increase in AV with decreasing temperature may be due to the increase in protein concentration in the aqueous phase around the casein micelles. The MCC containing about 16 and 18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels increased in strength over 10 d of storage at 4°C, likely due either to the migration of casein (CN) out of the micelles and interaction of the nonmicellar CN to form a network that further strengthened the random loose jamming gel structure or to a gradual increase in voluminosity of the casein micelles during storage at 4°C.

Our objectives were to determine if milk casein as a percentage of true protein (CN%TP) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is equivalent to CN%TP estimated by Kjeldahl, and to determine the proportion of casein (CN), casein proteolysis products (CNPP), and serum protein (SP) from milk true protein (TP) that goes into the Kjeldahl noncasein nitrogen (NCN) filtrate and the proportion that stays in the NCN precipitate using SDS-PAGE. Raw milk samples were collected from 16 mid-lactation Holstein cows twice a week for 2 wk. These milks were analyzed for Kjeldahl total nitrogen, nonprotein nitrogen, and NCN content in duplicate, and by SDS-PAGE. The CN%TP determined by Kjeldahl was compared with the CN%TP estimated by SDS-PAGE calculated in 2 ways: as a percentage of only intact caseins divided by TP and as a percentage of both intact caseins and CNPP divided by TP. Three milks varying in fat, lactose, TP, CN, and SP content were formulated. These milks were analyzed in duplicate for Kjeldahl total nitrogen, nonprotein nitrogen, and NCN content, and each of the NCN filtrate and NCN precipitate were analyzed in duplicate by SDS-PAGE for relative quantity (%) of CN, CNPP, and SP. We found that the estimate of CN%TP by Kjeldahl was higher than the estimate of CN%TP by SDS-PAGE that was calculated as only intact CN divided by the total of all protein bands. However, no difference was detected in the estimate of CN%TP by Kjeldahl compared with CN%TP by SDS-PAGE when CNPP were included as CN in the calculation of SDS-PAGE results. Based on SDS-PAGE results, we found that a majority (89%) of the CNPP from the milk (approximately 10.13 out of 11.41% TP) were retained in the Kjeldahl NCN precipitate. Thus, CN%TP measured by Kjeldahl underestimates the amount of proteolytic damage that has been done to CN in milk. It is important for the dairy industry to correctly and rapidly measure the extent of proteolytic damage to milk protein to correctly value milk from a product quality and yield point of view. A rapid and quantitative measure of proteolytic damage to milk protein is needed.

Our research objective was to measure percent removal of whey protein from separated sweet whey using 0.1-µm uniform transmembrane pressure ceramic microfiltration (MF) membranes in a sequential batch 3-stage, 3× process at 50°C. Cheddar cheese whey was centrifugally separated to remove fat at 72°C and pasteurized (72°C for 15 s), cooled to 4°C, and held overnight. Separated whey (375 kg) was heated to 50°C with a plate heat exchanger and microfiltered using a pilot-scale ceramic 0.1-µm uniform transmembrane pressure MF system in bleed-and-feed mode at 50°C in a sequential batch 3-stage (2 diafiltration stages) process to produce a 3× MF retentate and MF permeate. Feed, retentate, and permeate samples were analyzed for total nitrogen, noncasein nitrogen, and nonprotein nitrogen using the Kjeldahl method. Sodium dodecyl sulfate-PAGE analysis was also performed on the whey feeds, retentates, and permeates from each stage. A flux of 54 kg/m2 per hour was achieved with 0.1-µm ceramic uniform transmembrane pressure microfiltration membranes at 50°C. About 85% of the total nitrogen in the whey feed passed though the membrane into the permeate. No passage of lactoferrin from the sweet whey feed of the MF into the MF permeate was detected. There was some passage of IgG, bovine serum albumen, glycomacropeptide, and casein proteolysis products into the permeate. β-Lactoglobulin was in higher concentration in the retentate than the permeate, indicating that it was partially blocked from passage through the ceramic MF membrane.

The objective of our work was to develop and evaluate the performance of a rapid method for measuring fat, protein, moisture, and salt content of Cheddar cheese using a combination mid-infrared (MIR) transmittance analysis and an in-line conductivity sensor in an MIR milk analyzer. Cheddar cheese was blended with a dissolving solution containing pentasodium triphosphate and disodium metasilicate to achieve a uniform, particle-free dispersion of cheese, which had a fat and protein content similar to milk and could be analyzed using a MIR transmittance milk analyzer. Annatto-colored Cheddar cheese samples (34) from one cheese factory were analyzed using reference chemistry methods for fat (Mojonnier ether extraction), crude protein (Kjeldahl), moisture (oven-drying total solids), and salt (Volhard silver nitrate titration). The same 34 cheese samples were also dissolved using the cheese dissolver solution, and then run through the MIR and used for calibration. The reference testing for fat and crude protein was done on the cheese after dispersion in the dissolver solution. Validation was done using a total of 36 annatto-colored Cheddar cheese samples from 4 cheese factories. The 36 validation cheese samples were also analyzed using near-infrared spectroscopy for fat, moisture, and the coulometric method for salt in each factory where they were produced. The validation cheeses were also tested using the same chemical reference methods that were used for analysis of the calibration samples. Standard error of prediction (SEP) values for moisture and fat on the near-infrared spectroscopy were 0.30 and 0.45, respectively, whereas the MIR produced SEP values of 0.28 and 0.23 for moisture (mean 36.82%) and fat (mean 34.0%), respectively. The MIR also out-performed the coulometric method for salt determination with SEP values of 0.036 and 0.139 at a mean level of salt of 1.8%, respectively. The MIR had an SEP value of 0.19 for estimation at a mean level of 24.0% crude protein, which suggests that MIR could be an easy and effective way for cheese producers to measure protein to determine protein recovery in cheese making.

Nitrogen concentrations in feeds, feces, milk, and urine samples were measured using 2 analytical methods following different drying procedures. Ten samples of corn silage, alfalfa silage, and concentrates collected from 2017 to 2018 at Krauss Dairy Research Center, The Ohio State University (Wooster), were used. A 4-d total collection digestion trial provided fecal samples from 10 cows (1 sample/cow), and another 10 cows were used to collect milk samples (1 sample/cow) and spot urine samples (1 sample/cow). Spot urine samples were acidified immediately to pH

Mammalian milks may differ greatly in composition from cow milk, and these differences may affect the performance of analytical methods. High-fat, high-protein milks with a preponderance of oligosaccharides, such as those produced by many marine mammals, present a particular challenge. We compared the performance of several methods against reference procedures using Weddell seal (Leptonychotes weddellii) milk of highly varied composition (by reference methods: 27–63% water, 24–62% fat, 8–12% crude protein, 0.5–1.8% sugar). A microdrying step preparatory to carbon-hydrogen-nitrogen (CHN) gas analysis slightly underestimated water content and had a higher repeatability relative standard deviation (RSDr) than did reference oven drying at 100°C. Compared with a reference macro-Kjeldahl protein procedure, the CHN (or Dumas) combustion method had a somewhat higher RSDr (1.56 vs. 0.60%) but correlation between methods was high (0.992), means were not different (CHN: 17.2±0.46% dry matter basis; Kjeldahl 17.3±0.49% dry matter basis), there were no significant proportional or constant errors, and predictive performance was high. A carbon stoichiometric procedure based on CHN analysis failed to adequately predict fat (reference: Röse-Gottlieb method) or total sugar (reference: phenol-sulfuric acid method). Gross energy content, calculated from energetic factors and results from reference methods for fat, protein, and total sugar, accurately predicted gross energy as measured by bomb calorimetry. We conclude that the CHN (Dumas) combustion method and calculation of gross energy are acceptable analytical approaches for marine mammal milk, but fat and sugar require separate analysis by appropriate analytic methods and cannot be adequately estimated by carbon stoichiometry. Some other alternative methods—low-temperature drying for water determination; Bradford, Lowry, and biuret methods for protein; the Folch and the Bligh and Dyer methods for fat; and enzymatic and reducing sugar methods for total sugar—appear likely to produce substantial error in marine mammal milks. It is important that alternative analytical methods be properly validated against a reference method before being used, especially for mammalian milks that differ greatly from cow milk in analyte characteristics and concentrations.

Previous research has suggested that the standard noncasein nitrogen (NCN) measurement method for milk overestimates the NCN content of microfiltration (MF) retentate. The objective of this study was to develop a modified method to more accurately measure the NCN content of ultrafiltration and MF retentate products. The standard method is based on precipitation of casein micelles at their isoelectric point (4.6) with acetic acid. In the standard method, a 10-mL milk sample and 75 mL of 38°C water are placed in a 100-mL volumetric flask. One milliliter of 10% acetic acid solution is added and the flask is incubated at 38°C for 10 min. Subsequently, 1 mL of 1N sodium acetate solution is added and mixed. After cooling the contents to 20°C, the flask is made up to 100 mL with water, mixed, and then filtered (Whatman No. 1 filter paper). The N content of the filtrate is then determined by Kjeldahl analysis and referred to as NCN. A method was developed that used a 50-mL centrifugal tube instead of a volumetric flask. This modification facilitated measurement of the pH after addition of acetic acid. Subsequently, the sample was centrifuged (800 × g at 25°C) for 10 min to facilitate filtration with a smaller pore size filter paper (Whatman no. 6). In this study, we evaluated the effect of pH after addition of 1% acetic acid and pH of the final filtrate on NCN analysis. Four pH levels after acetic acid addition (4.0, 4.2, 4.4, and 4.6) and 2 pH levels after sodium acetate addition (4.6 and 4.8) were evaluated. As the pH after acetic acid addition was increased from 4.0 to 4.6, the NCN content significantly decreased. Sodium dodecyl sulfate PAGE results also indicated that the casein fractions present in the filtrate were significantly decreased when the pH was increased from 4.0 to 4.6. The NCN content slightly decreased but the difference was not significant when the final pH of the filtrate was increased from 4.6 to 4.8. Subsequently, the NCN contents of several ultrafiltration and MF samples were determined using the standard method and modified method. The modified method gave significantly lower NCN values for most samples as compared with the standard method.

The production of serum protein (SP) and micellar casein from skim milk can be accomplished using microfiltration (MF). Potential commercial applications exist for both SP and micellar casein. Our research objective was to determine the total SP removal and SP removal for each stage, and the composition of retentates and permeates, for a 3×, continuous bleed-and-feed, 3-stage, uniform transmembrane pressure (UTP) system with 0.1-μm ceramic membranes, when processing pasteurized skim milk at 50°C with 2 stages of water diafiltration. For each of 4 replicates, about 1,100 kg of skim milk was pasteurized (72°C, 16 s) and processed at 3× through the UTP MF system. Retentate from stage 1 was cooled to

The objective of our research was to modify the current indirect casein method for bovine milk to enable it to be applied to bovine milk, bovine milk concentrates, and milks of other species that contain a protein concentration up to 9% (wt/wt). Our work used a series of bovine milk concentrates from about 3 to 9% protein with the same casein as a percentage of true protein to determine the amount of buffer required and pH of the noncasein nitrogen (NCN) filtrate to achieve consistent estimates of casein and casein as percent of true protein. As the concentration of protein in milk increased (either in bovine milk concentrates or in milks of other species), the amount of buffer needed for the NCN sample preparation method to achieve a filtrate pH of 4.6 increased. In the first part of the study using a series of bovine milk concentrates, it was demonstrated that the method gave more consistent predictions of casein as a percentage of true protein when the final NCN filtrate pH was between 4.5 and 4.6 at 38°C. When the amount of buffer added to the sample was not sufficient (i.e., the filtrate pH was too high), the filtrates were not clear. A polynomial equation was developed for prediction of the amount of acetic acid or sodium acetate buffer required to achieve pH 4.5 to 4.6 for milk protein concentrations from 3 to 9% protein using bovine milk and milk concentrates. When the equation developed using cow milk was applied to goat, sheep, and water buffalo milks, it correctly predicted the volume of reagents needed to achieve a final NCN filtrate pH of 4.6 at 38°C. We also verified as part of this work that the ability to measure NPN content of milk was not influenced by protein content of milk in the range from 3 to 9% protein. The results of this study will be used as the basis for proposed changes in the official methods for measurement of the casein content of milk to expand the scope of the method so it can be used to achieve accurate results for milk concentrates and milks of other species.

WOS: 000236686700006, PubMed ID: 16606712, There is a need for rapid and simple techniques that can be used to predict the quality of cheese. The aim of this research was to develop a simple and rapid screening tool for monitoring Swiss cheese composition by using Fourier transform infrared spectroscopy. Twenty Swiss cheese samples from different manufacturers and degree of maturity were evaluated. Direct measurements of Swiss cheese slices (similar to 0.5g) were made using a MIRacle 3-reflection diamond attenuated total reflectance (ATR) accessory. Reference methods for moisture (vacuum oven), protein content ( Kjeldahl), and fat (Babcock) were used. Calibration models were developed based on a cross-validated (leave- one- out approach) partial least squares regression. The information-rich infrared spectral range for Swiss cheese samples was from 3,000 to 2,800 cm(-1) and 1,800 to 900 cm(-1). The performance statistics for cross-validated models gave estimates for standard error of cross-validation of 0.45, 0.25, and 0.21% for moisture, protein, and fat respectively, and correlation coefficients r > 0.96. Furthermore, the ATR infrared protocol allowed for the classification of cheeses according to manufacturer and aging based on unique spectral information, especially of carbonyl groups, probably due to their distinctive lipid composition. Attenuated total reflectance infrared spectroscopy allowed for the rapid (similar to 3- min analysis time) and accurate analysis of the composition of Swiss cheese. This technique could contribute to the development of simple and rapid protocols for monitoring complex biochemical changes, and predicting the final quality of the cheese.

The yield of microbial crude protein (CP) from carbohydrate fermentations was examined using trichloroacetic acid (TCA) precipitation of batch cultures. The medium contained ammonium bicarbonate, casein acid hydrolysate, and cysteine hydrochloride as nitrogen sources. Isolated bermudagrass neutral detergent fiber (iNDF) and 60:40 blends of iNDF and sucrose (Suc), citrus pectin (Pec), or corn starch (Sta) (approximately 375 mg of substrate organic matter/vial) were fermented in vitro in two separate fermentation runs with mixed ruminal microbes. Three fermentation tubes for each substrate were destructively sampled at 0, 4, 8, 12, 16, 20, and 24 h. Fermented samples were precipitated at a concentration of 19.4% TCA, and filtered to collect unfermented iNDF and precipitate. Collected residues were analyzed for CP as Kjeldahl N x 6.25. Microbial CP (TCACP) was estimated as TCA-precipitated CP corrected for the TCA-precipitated CP content of substrates at 0 h, and the mean of fermentation blanks from each hour. Medium pH did not decline below 6.49 in any fermentation tube. Comparisons of maximal yields based on the hour in which the measured mean yield was greatest for each substrate in each fermentation indicated that Sta > Suc = Pec > iNDF (P < 0.05). All substrates showed increases in TCACP to their maxima, followed by declines in TCACP. This likely reflects the relative dominance of production or degradation of microbes about the point of substrate limitation. Unlike other substrates, Suc had no detectable lag, and presented a more persistent TCACP yield curve than the other non-NDF carbohydrates (NFC). Regression analysis of TCACP yield over time for iNDF versus other substrates, Pec + Sta versus Suc, and Pec versus Sta indicated that the compared curves were not parallel (P < 0.05). The patterns of TCACP yield over time were cubic for iNDF and Suc, and quartic for Pec and Sta. The maximal yields of TCACP predicted from the regressions were Sta: 34.0 mg at 15.6 h, Pec: 29.9 mg at 13.5 h, Suc: 25.5 mg at 12.6 h, and iNDF: 13.6 mg at 19.3 h. The NDF and NFC carbohydrates examined differed in both maximal yields and temporal patterns of yield of TCACP.

Development of cost-effective amendments for treating dairy slurry has become a critical problem as the number of cows on farms continues to grow and the acreage available for manure spreading continues to shrink. To determine effectiveness and optimal rates of addition of either alum or zeolite to dairy slurry, we measured ammonia emissions and resulting chemical changes in the slurry in response to the addition of amendments at 0.4, 1.0, 2.5, and 6.25% by weight. Ammonia volatilization over 96 h was measured with six small wind tunnels with gas scrubbing bottles at the inlets and outlets. Manure samples from the start and end of trials were analyzed for total nitrogen and phosphorus, and were extracted with 0.01 M CaCl2, 1.0 M KCl, and water with the extracts analyzed for ammonium nitrogen, phosphorous, aluminum, and pH. The addition of 6.25% zeolite or 2.5% alum to dairy slurry reduced ammonia emissions by nearly 50 and 60%, respectively. Alum treatment retained ammonia by reducing the slurry pH to 5 or less. In contrast, zeolite, being a cation exchange medium, adsorbed ammonium and reduced dissolved ammonia gas. In addition, alum essentially eliminated soluble phosphorous. Zeolite also reduced soluble phosphorous by over half, but the mechanism for this reduction is unclear. Alum must be carefully added to slurry to avoid effervescence and excess additions, which can increase soluble aluminum in the slurry. The use of alum or zeolites as on-farm amendment to dairy slurry offers the potential for reducing ammonia emissions and soluble phosphorus in dairy slurry.

This study investigated the changes in chemical and microbiological properties of fresh goat milk cheese stored in an open deck refrigerated display cabinet (6 ± 2°C) or in a dark cold room (4 ± 1°C). The effects of partial-vacuum packaging and fluorescent lighting were studied during the cheese shelf life (45 d) and 15 d after. Storage conditions did not affect the pH values (4.3), whereas a slight decrease in moisture (ca. 1%) and in water activity (

In the early 1960s, France started to analyze routinely protein for the DHIA program using the amido black method, and, since 1969, milk producers have been paid on the basis of fat and protein. Progressively, infrared analyzers have replaced the dye binding method. Because of the rather large variability of NPN content and its role on the accuracy of both amido black and infrared methods, analysis in 1974 was changed from CP to true protein for both economical (NPN has little value) and analytical reasons (better accuracy and centralized calibration). Examples are given to illustrate the seasonal and between-herd variability of the proportion of NPN in total N for which urea is the most important and variable NPN compound. Since 1976, the fat:protein price ratio of additional grams to the basic price changed from 74:26 to 34:66. Several studies have shown that the calibration of infrared instruments in true protein instead of CP provides better accuracy in protein testing with a high correlation (r = –.80) between errors and the percentage of NPN in total N. However, a recent study has shown than urea interferes significantly with the infrared signal. Because protein now has a much higher value than fat, a better definition of protein is extremely important for the dairy industry. After 15 yr of experience, both dairy industry and farmers are quite satisfied, however, using a different reference, yet the problem of the comparison of protein results between France and the other countries remains, especially for breeding programs.

Accurate and rapid measurement of the protein content of milk is important from both a product quality and an economic standpoint. The Sprint rapid protein analyzer (CEM Corporation, Matthews, NC) is a commercial system based on a dye-binding technique and can be used for rapid measurement of protein in foods. The objective of the present study was to compare the Sprint method with the reference method (Kjeldahl method). Milk and cream samples were analyzed in duplicate for true protein and crude protein (CP) using the reference method as well as the rapid method. Method comparison statistics (regression analysis, graphical representation, standard deviation of residuals, repeatability, and so on) were used to evaluate the agreement between the 2 methods. Regression coefficients and the intercepts were not significantly different from 1 and zero for CP measurement in milk and cream, respectively. The average coefficient of variance between the duplicate CP measurements for the Sprint method was found to be 0.40, 0.49, and 0.76 for milk, light cream, and heavy cream, respectively. True protein measurement in milk and cream also followed a similar trend. Overall, there exists a sufficient level of agreement between the Sprint rapid protein analyzer and Kjeldahl method for true protein and CP measurement of milk and cream samples.

The use of reversed-phase HPLC for the determination of whey protein denaturation was investigated. Denatured whey proteins and caseins were isolated from undenatured whey proteins by isoelectric precipitation at pH 4.6. Whey protein denaturation was determined by comparing reversed-phase HPLC protein profiles of isolates far heat-treated and unheated NDM from pooled herd, Holstein cows. In general, protein profiles for heat-treated skim milk indicated that whey protein denaturation began at about 40°C, became more rapid at 70°C, and was 95% complete at 85°C. Undenatured whey protein was also quantified as whey protein nitrogen based on their absorbance and nitrogen content compared with known whey protein standards or by augmenting the same samples with a known amount of lysozyme. Whey protein nitrogen values obtained by the reversed-phase HPLC method were in good agreement with those obtained by the modified Kjeldahl nitrogen procedure for three NDM powders prepared from skim milk preheated to 63, 74, and 85°C before spray drying, and by commercial low and high heat NDM standards. Isolation of undenatured whey proteins and their separation by reversed-phrase HPLC takes approximately 1.5h.

This trial was to determine the effect of dietary rapeseed screenings on milk production, milk composition, and feed intake of lactating dairy cows. Pelleted rapeseed screenings were fed at 0, 7, and 14% of diet DM. Assignment of 36 multiparous Holstein cows was based upon parity and previous lactation performance. The experimental period was from parturition through wk 16 of lactation. The control ration had 3.1% ether extract, whereas the ration with 14% rapeseed screenings had 5.4% ether extract. The rapeseed screenings contained 52.2% of C18:1 and 24.2% of C18:2 as percentage of total fatty acids. There were no significant effects of dietary treatment on milk yield or feed intake. There were significant increases in milk fatty acids C18:1 and C18:2 for cows consuming the diets with rapeseed screenings. Treatment did not significantly affect milk protein percentage as measured by Kjeldahl N or by the infrared method. Increased unsaturated fatty acids in the milk of cows fed rapeseed screenings had no apparent effect on infrared fat analysis. There was no interaction of treatment and milk fat analysis method (infrared versus Babcock). Dietary treatment did not affect blood serum nonesterified fatty acid concentrations over the 16-wk period. In conclusion, rapeseed screenings, fed at levels up to 14% of the diet, did not affect milk yield, milk protein, or milk fat percentage but did change milk fatty acid composition.

Cheddar cheese was made in beakers (by the process for aged cheese) to compare milkclotting enzymes for their effect on proteolysis during coagulation, cooking, and cheddaring. All enzymes released more N into whey during cooking and cheddaring than calf rennet except the equal mixture of calf rennet and swine pepsin during cheddaring. Levels of true protein were the same in whey during cooking and during cheddaring with all enzymes, except those from Bacillus polymyxa showed proteolysis of whey proteins. Hydrolysis of casein occurred after cooking and during cheddaring with all enzymes including calf rennet. Data were discussed with respect to protein recoveries in cheese from ultrafiltered milk. Probable reductions in yield were estimated using a factor of 27.06 for converting estimated casein reductions in milk to percentage of yield losses. This factor assumes: the prices of milk fat and cheese are equal; cheddar cheese of the same composition is made of .37 moisture and .017 salt; whey contained .065 fat-free, curd-free solids; milk contained .036 fat and .ON64 casein or was standardid to the same casein:fat ratio, considering casein losses; and yield of cheese was 9.834 kg100kg milk. Yield losses (± least significant differences), compared with losses of calf rennet, were estimated as: bovine pepsin, –.14 (± .08); equal mixture of calf rennet and swine pepsin, –.07 (± .08); Mucor pusillus , –.49 (± .11); M. miehei (preparation I), –.63 (± .11); M. miehei , (preparation II), –.68 (± .12); Endothia parasitica , –1.24 (± .32); B. polymyxa , 4.79% (± .32%). These would be economically important losses of concern to cheese makers. Special attention was given to replications and statistical analyses to reduce least significant differences: quadruplicate Kjeldahl analyses, 6 to 12 replicate trials per enzyme, with comparison among enzymes within days. Comparison of levels of N in whey during simulated cheese making in beakers is suggested for comparing milk-clotting enzymes for their effect on yield of different varieties of cheese. This could be considered a basic loss of yield; losses of curd and fat in whey would be additional losses.

The Harland-Ashworth test for measuring undenatured whey protein N in NDM was modified to measure undenatured whey protein N in dried whey. Dried whey was reconstituted in phosphate buffer (pH 6.7) then saturated with NaCl and incubated for 30min at 37°C. Samples were cooled to room temperature and filtered before total absorbance was measured at 280nm. Absorbance measurements were taken at 280nm to prevent difficulties caused by turbidimetric measurements in Harland-Ashworth filtrates. Nonprotein absorbance was measured on TCA deproteinized filtrates and subtracted from total absorbance values to find absorbance of undenatured whey protein. The modified assay produced reproducible estimates of undenatured whey protein in NDM as well as in dried whey. The correlation between absorbance of undenatured whey protein N and the corresponding Kjeldahl results were not influenced by season, heat treatment of samples, solids concentrations, residual milk-clotting enzymes, or pH of whey during heating.

Milk proteins are grouped in three rather arbitrary categories, which are separated by acid precipitations. Each is determined by reference Kjeldahl (N × 6.38) measurement. Quality payment schemes to dairy farmers, which involve a protein payment, are currently almost universally based on total protein. The alternative use of true protein (caseins plus whey proteins and proteosepeptones) has been under debate for some years, and, in France, NPN is discounted in DHI and quality payment testing. Arguments favoring discounting NPN in payments are weighed against the commercial problems that such action creates. The major reason for change is to improve analytical accuracy, but the commercial price to be paid for such a change seems unjustified, and I argue for retention of recording protein as total protein.

Decomposition of livestock manure produces gaseous ammonia. Dietary manipulation is one means to reduce N in manure and ammonia volatilization. The effects of dietary crude protein concentration on N intake, N and urinary urea-N excretion, and ammonia volatilization were measured. Eight Holstein heifers (body weight = 260 to 488 kg) were fed a total mixed ration containing either 9.6 or 11.0% crude protein in a crossover design. Oatlage and concentrate were fed at 77:23 (dry matter basis), and soybean meal was used to alter total dietary crude protein. Seven-day adjustment periods preceded 5-d collection periods. Indwelling urinary catheters were inserted 2 d prior to the collection periods. Daily feces and acidified urine were collected, stirred, and subsampled for total Kjeldahl N, urinary urea N, dry matter, P, K, and ash. Urine collection tubes were split during period 2 to allow for collection of unacidified samples for urea N and total N determinations. Unacidified urine and fecal samples were combined (1:1.3) for collection of volatilized ammonia. Remaining slurries were extracted for total and urea N. Increased dietary crude protein concentration increased N intake, N excretion, urea-N excretion, and N excreted in the urine by the heifers. Dietary manipulation of N intake by reduction of 14.0% (dry matter basis) resulted in a 28.1% decrease in ammonia emission and decreases in the urea N, total N, and percentage N excreted in the urine of 29.6, 19.8, and 7.4%, respectively. Ammonia volatilization was dependent on N quantity and form in the urine.

A longitudinal study was carried out on samples of bulk tank milk collected from 200 farms for 12 mo to evaluate SCC, plasmin and plasminogen activities, psychrotrophic bacteria count, and protein quality in milk and to examine the proteolytic effects of plasmin on milk proteins. Herds were selected randomly from client lists of two dairies to create four groups based on milk SCC of the month before the study; herds were reassigned monthly to one of four groups based on SCC for that month. Overall means were 3.73% fat, 3.13% protein by infrared analysis, 3.16% protein by Kjeldahl analysis, 2.42% casein percentage, 4.65% lactose, 5.43 cells/ml of log SCC, 1.13 U of plasmin, 45.6 U of plasminogen, and log 2.86 cfu/ml of psychrotrophic bacteria. The ANOVA showed a significant effect of month on all factors except SCC, which was fixed by the experimental design. Plasmin and plasminogen activities were high from December to May. Plasmin activities and psychrotroph counts were significantly higher for the high SCC group. Casein percentage and number were significantly higher for the low SCC group.

Total and phosphate buffer insoluble amino acids were determined for corn grain and silage, dried brewer's grain, timothy hay, and soybean meal. In addition, amino acid compositions of the acid detergent residue and neutral detergent residue were determined, and soluble amino acids were estimated for each feedstuff. Variation of amino acid concentrations among protein fractions as percent of total amino acid was measured in the buffer insoluble, soluble, neutral detergent fiber, and acid detergent fiber fractions within a feed as well as among feedstuffs. Insoluble noncell-wall, neutral detergent, and acid detergent amino acids as a percentage of total individual amino acid (neutral detergent fiber or acid detergent fiber amino acid/total individual amino acid × 100) for soybean meal, brewer's grain, corn grain, timothy, and corn silage were 73.2, 8.5, 5.6; 31.2, 54.5, 16.3; 72.1, 16.2, 4.6; 18.0, 55.6, 8.2; 14.6, 20.8, and 4.8. Recovery of amino acid nitrogen as percent of total Kjeldahl nitrogen varied among and within fractions studied.

Digestibilities, nitrogen balances, and voluntary intakes of six silage-hay diets fed to sheep were used to contrast different methods of determination of dry matter. All wet samples of feed, feed refusals, and feces from 24 wethers were analyzed in triplicate for dry matter by: forced air drying at 90C for 24h; vacuum oven drying at 70C under pressure of less than 75mm mercury for 24h; toluene distillation (uncorrected); toluene distillation (acid corrected); and by a chemical saponification method. There were significant differences in dry matter between methods. Dry matter digestibility of hay was overestimated by 6.8% when forced air drying was used rather than the chemical method whereas that of a formaldehyde-treated silage was underestimated by 5.5%; the digestible energy intake of the formaldehyde-treated silage was underestimated by 15.1%, and that of the hay overestimated by 6.9%. Nitrogen balance was 75.9 and 7.4 g/head per day for hay and untreated silage when forced air dry matter and Kjeldahl nitrogen on the dried sample were combined while values were 25.6 and −42.1 g/head per day when data by chemical method were used with Kjeldahl nitrogen on the wet sample. Differences in estimates of dry matter may explain some of the anomalous dietary comparisons for silage and hay.

Conventional analyses and high pressure liquid chromatography were used to detect sweet-type cheese whey replacement for nonfat milk solids in ice milk mixes at substitutions below and above the allowable 25%. Sweet whey replacement was monitored by noting abnormally high lactic acid and lactose, lower protein (using Kjeldahl nitrogen and dye binding), high lactose to protein ratios, and small but distinctive amounts of glycomacropeptide as determined by its solubility characteristics (soluble in 12% trichloroacetic acid but precipitated by phosphotungstic acid) and sialic acid content. The following compositional characteristics of ice milk would be suspect in sweet whey adulteration above 25% replacement: lactic acid (lactate) above .05%, lactose to protein ratio above 2.1, and glycomacropeptide precipitate, (per 100g mix) having a dry weight above 55mg, and sialic acid content above 5.2 μ moles.

The suitability of California raw milks for the production of cheese was evaluated during 1983 by measuring the distribution of nitrogen in 1417 samples of farm milk from four major dairy regions of California. Both Kjeldahl (431 samples) and the Udy dye-binding procedures (986 samples) were used. True protein concentrations measured by Kjeldahl varied from 2.66 to 4.41% and averaged 3.32%. For samples measured by Udy, true protein concentrations ranged from 2.56 to 3.93% and averaged 3.15%. Casein as a percent of true protein varied from 69.1 to 80.4% and averaged 76.4% for samples analyzed by the Kjeldahl procedure. For samples analyzed by the Udy procedure, casein as percent of true protein ranged from 72.8 to 80.7% and averaged 77.0%. There was close agreement between values obtained by the Udy and Kjeldahl procedures, which suggests that milk can be chosen for cheese production on the basis of its casein content using the Udy procedure.

A nephelometric procedure for determination of protein and fat content of milk has been developed. Protein is aggregated into a colloidal suspension by the addition of an anhydrous reagent consisting of 15.75% acetic anhydride, 83.75% glacial acetic acid, and .5% p-toluenesulfonic acid. Protein then is redissolved, and the fat is aggregated into an emulsion by addition of an aqueous surfactant solution consisting of 5% IGEPAL DM-970 in water. Correlation of the nephelometric method with the standard Kjeldahl protein determination gives a coefficient of .977; that of the nephelometric and Babcock fat determination gives .985.

Fast protein liquid chromatography was used to determine the extent of whey protein denaturation in various heat-treated milk samples: Sordi-indirect UHT (145°C/3 s), Dasi-direct UHT (142°C/3 s), HTST (80°C/30 s), and batch (63°C/30 min). Results were compared with other published methods (differential scanning calorimetry, whey protein nitrogen index, and Kjeldahl nitrogen on salt fractions). Results of the differential scanning calorimetry method were too erratic to be used to quantify whey protein denaturation. The remaining methods (fast protein liquid chromatography, Kjeldahl nitrogen, and whey protein nitrogen index) gave reproducible results and the extent of denaturation (highest to lowest) was consistently predicted as Sordi > Dasi > HTST > batch. There was no difference between fast protein liquid chromatography and Kjeldahl nitrogen, but there was a significant difference between fast protein liquid chromatography and whey protein nitrogen index and between Kjeldahl nitrogen and whey protein nitrogen index. Fast protein liquid chromatography appears to be an effective method to determine whey protein denaturation in heat-treated milks.

Ability to hydrolyze proteins in skim milk and whey was determined for four commercial neutral pH enzyme-based cleaners for ultrafiltration systems. The four enzyme-based cleaners (A, B, C, and D) had a use strength pH of 7.0 to 8.4. Pasteurized skim milk and pasteurized sweet whey (adjusted to pH 8.0) were heated to 43°C, and 1 L of each was placed in Erlenmeyer flasks (A, B, C, D, and control). Flasks were placed in a waterbath at 43°C. Enzyme-based cleaners were added to give use strength concentrations and distilled water used to adjust for differences in amounts of cleaner added. Samples were taken over 24 h. Percentage of NPN by Kjeldahl and percentage of protein by dye binding were determined. Samples were taken after 8 h for gel electrophoresis. Samples containing enzymes A and D had increased percentages of NPN and decreased percentages of protein as measured by dye binding both in skim milk and whey. Enzyme A hydrolyzed whey proteins more effectively than enzyme D. Enzyme D, however, hydrolyzed casein more effectively than enzyme A. No changes in NPN or protein concentration were observed in skim milk or whey using enzyme B or C when compared with controls. Using SDS-PAGE to quantify the activity of four enzyme cleaners in skim milk and whey confirmed the action of enzymes A and D on whey proteins and casein and indicated no activity by enzymes B and C. Some commercial enzyme-based cleaners were ineffective in hydrolyzing casein or whey proteins. Other enzyme-based cleaners required extended times to hydrolyze whey proteins or casein to NPN.

Recovery of ammonia from ammonium sulfate solutions. (.01 M to .0005 M ) by a modified Conway microdiffusion procedure did not differ significantly from recovery by micro-Kjeldahl distillation. Total and whey-protein nitrogen concentrations of milk were determined by microdiffusion and microdistillation of Kjeldahl digests. Protein values by microdiffusion and microdistillation for 38 samples of milk from individual cows (3.38 and 3.42%) and 40 samples of acid-prepared whey (.87 and .88%) did not differ significantly although results by microdiffusion were consistently lower (1 to 3%) than microdistillation values. The correlation coefficient for the two methods was .99. Recovery of nitrogen by the microdiffusion procedure is influenced by the digestion catalyst. The microdiffussion procedure provides a means of reducing work time while maintaining the quantitative accuracy of the micro-Kjeldahl distillation.

Summary 1.A semimicro-Kjeldahl method and apparatus is described. 2.Mercuric oxide is recommended as a catalyst with boric acid as the ammonia receiving agent and methyl red-methylene blue as the indicator. 3.The semimicro method was found to check very closely with the Official Method. 4.The semimicro method is useful for determining small amounts of nitrogen, it is time saving, well adapted to routine analysis, and reduces the cost of reagents per determination.

The Mark II Pro-Milk Tester was evaluated for its accuracy, repeatability, reliability, and operational limitations. During 1 yr the instrument calibration was checked continually with preanalyzed calibration milks prepared by combining individual cow milks at specific fat contents. Protein ranged from 2.96 to 4.24% by a semi-micro Kjeldahl method. Plant milk samples (833) were analyzed with the instrument and protein of these samples was from 2.75 to 3.65% by the Kjeldahl method. From regression analysis calibration maintained with calibration milks was unsuitable for use with the plant milks. For the 1 yr the standard deviation of difference between Kjeldahl and Pro-Milk results for calibration milks was .031 whereas for the plant milks it was .074. Repeatability of the Pro-Milk test (±.0110) was about the same as with the Kjeldahl (± .0114), and mean difference between Kjeldahl and Pro-Milk were small both for calibration milks (.0007) and plant milks (.0049). The larger standard deviation of difference with plant milks, therefore, was the result of a different requirement for calibration slope. Six adjustments of calibration were required during the year, three with changing from one dye lot to another and three with the same dye lot. As there was no noticeable drift in instrument read-out at any time, changes in calibration could be required because of differences in purity of dye or because of seasonal changes in mean dye binding capacity of milk proteins. Calibration of the Pro-Milk Tester needs to be monitored continually and its results adjusted to conform to those of an official method for the type of milk to be analyzed routinely with the instrument.

Summary A rapid and simple method for determining the undenatured serum protein in milk, based on measurement of the volume of precipitated serum protein, is described. It involves the acid precipitation of the casein and the denatured serum protein and their removal by filtration. The soluble undenatured whey proteins in 7 ml. of filtrate are precipitated with 3 ml. of 10% phosphotungstic acid. Upon controlled centrifugation, the volume of the precipitate is obtained and converted to milligrams of whey protein nitrogen per gram of nonfat milk solids by means of a standard curve or the volume of precipitate may be used directly as the unit of measurement. Thus, the suitability of milk for a specific purpose is indicated. The relation between milliliters of precipitate and milligrams of undenatured whey protein nitrogen as determined by Kjeldahl analysis has a correlation coefficient of 0.97. A standard curve is given which can be used routinely with the indicated equipment.

Summary Hippuric acid was identified as a constituent of the nonprotein nitrogen fraction of milk. A method of measuring the hippuric acid content of milk was developed. This method is based on continuous ether extraction of tryptic digested, acidified skimmilk and measurement of extracted nitrogen by the Kjeldahl procedure. The amounts of hippuric acid in 14 samples of skimmilk, gathered during the period July to January, were determined. These values ranged from 3.1 to 6.4mg./100ml. of skimmilk and averaged 5.1mg./100ml.

The time required for the determination of nitrogen has been materially reduced since Kjeldahl (1) first proposed his original method. This reduction has been made possible by introducing into the reaction mixture potassium sulphate and catalysts such as mercury and copper. Just recently, the use of selenium has been advocated as a catalyst. Although Lauro (2) was the first to investigate the use of selenium, this substance has been combined with other catalysts by several investigators (3 to 11 inc.) Of these workers, several (7, 8, 10) have found this element to reduce materially the amount of nitroge n obtained in the analysis. Sandstedt (11) also has found with selenium some loss of nitrogen on long heating. Nearly all of the investigations of the determination of nitrogen in which selenium has been used have been with cereal products. In this communication, there are reported the results obtained when selenium is used with other catalysts in the determination of n~trogen in dairy products. No experimental data are available on the efficiency of selenium when combined with copper, mercury, and strong hydrogen peroxide.

Pasteurized homogenized milk was analyzed for 2 yr for variations in the noncasein proteins. Milk was chemically fractionated into the principal noncasein fractions, and the concentration of each fraction was determined by Kjeldahl nitrogen and correlated with season. Data were analyzed statistically, employing (as the regression model) the first two terms of a trigonometric series. Results indicate that all of the fractions but one varied with season and the variation was cyclic in character. The total albumin fraction did not vary. Also, with the exception of β -lactoglobulin, all of the fractions were highest in concentration during the summer and lowest in winter months. β -Lactoglobulin concentration followed a reverse variation. The equation for the regression model and appropriate constants for each fraction are included. Also included are the predicted maximum and minimum concentration values for each fraction plus the date these values are expected.

Summary A rapid method for estimating the degree of proteolysis in ripening cheese was studied by measuring absorption of ultraviolet light by a clear, sodium citrate-hydrochloric acid extract of cheese at pH 4.4±0.05; these measurements were closely correlated with the per cent of soluble nitrogen in the cheese extract. Measurements of the optical density of the cheese from 40 lots of varying ages revealed peaks of all absorption spectra between 274 and 275 mµ. The coeffieient of correlation between optical density of the cheese extract and percentage of soluble nitrogen measured in the cheese extract by Kjeldahl was 0.958. Concentrations of soluble tyrosine and soluble tryptophane were measured spectrophotometrically at optical densities of 270 and 290 mµ. Equations were developed by Vierordt's method of analysis of two component systems for this pair of wave lengths. Relationships between these compounds and percentage of soluble nitrogen in the cheese were linear and highly significant; the coefficient of correlation between soluble tyrosine and percentage of soluble nitrogen was 0.964; whereas, that between soluble tryptophane and percentage of soluble nitrogen was 0.886. Flavor intensity estimated in the 40 lots of cheese by professional judges showed a linear relationship to soluble tyrosine.

Summary A commercial spectrophotofluorimeter was used to determine the relationship between the intensity of the fluorescence of milk in the ultraviolet region and its protein content. The effect of pH, temperature, milk contaminants, and milk protein aggregation on the fluorescence was studied. By dissociating the milk micelles with a diluent containing citrate, phosphate, and urea, stable fluorescing systems were obtained for study. A double-reciprocal plot of the data obtained showed a good correlation between fluorescence intensity and protein content as determined by Kjeldahl methods. In examining the milk of 88 cows of differing breed, age, and period of lactation it was found that the Standard error of the estimate was 0.079% protein. It was concluded that the intensity of fluorescence of milk in the ultraviolet could be used to make accurate and rapid determinations of the protein content of milk, but a number of disadvantages must also be considered before recommending the technique for general use. These include the large dilution of milk samples required, the necessity for rigid temperature control, adverse effects of milk contamination, the relatively small slope of the standard curve, and the fact that commercial fluorimeters suitable for routine use are not available at present.

Summary A highly significant correlation of +0.975 between Orange G dye and Kjeldahl methods of protein determinations demonstrated that for most practical purposes the protein content of milk can be determined as effectively by the use of the rapid Orange G dye binding method as by the time-consuming Kjeldahl method. The correlation of +0.839 between the percentage of total protein determined by formol titration and the Kjeldahl method indicated that the Orange G dye method has an advantage in accuracy compared to formol titration as a rapid method for determination of protein in milk. This is, in part, due to effects from stage of lactation and month of the year on the formol factors for total protein.

Summary S. lactis organisms caused a rapid increase in both soluble nitrogen and tyrosine and tryptophan during the first 24 hours, followed by a smaller but gradual increase during the rest of the experimental period (72 to 90hours). Marked increases in tyrosine and tryptophan were found when the production of soluble nitrogen as determined by the Kjeldahl procedure still was negligible. Considerably more soluble nitrogen and tyrosine and tryptophan were produced when the reaction was controlled at pH 6.0 to 7.5 than in comparable samples without controlled pH. The cell-free culture medium did not show proteolytic activity.

The cheese industry faces many challenges to optimize cheese yield and quality. A very precise standardization of the cheese milk is needed, which is achieved by a fine control of the process and milk composition. Thorough analysis of protein composition is important to determine the amount of protein that will be retained in the curd or lost in the whey. The fluorescence-based Amaltheys analyzer (Spectralys Innovation, Romainville, France) was developed to assess pH 4.6-soluble heat-sensitive whey proteins (sWP*) in 5 min. These proteins are those that can be denatured upon heat-treatment and further retained in the curd after coagulation. Monitoring of sWP* in milk and subsequent adaptation of the process is a reliable solution to achieve stable cheese yield and quality. Performance of the method was evaluated by an accredited laboratory on a 0 to 7 g/L range. Accuracy compared with the reference Kjeldahl method is also provided with a standard error of 0.25 g/L. Finally, a 4-mo industrial trial in a cheese plant is described, where Amaltheys was used as a process analytical technology to monitor sWP* content in ingredients and final cheese milk. Calibration models over quality parameters of final cheese were also built from near-infrared and fluorescence spectroscopic data. The Amaltheys analyzer was found to be a rapid, compact, and accurate device to help implementation of standardization procedures in the dairy industry.

Concentrations of protein calculated from total Kjeldahl nitrogen and total Kjeldahl nitrogen corrected for the nonprotein nitrogen were determined in samples of herd milk collected from dairy farm tanks in California's San Joaquin Valley (45 samples) and from the area north of San Francisco Bay (55 samples). Means for the San Joaquin Valley and North Bay Area were for protein determined from nitrogen corrected for nonprotein nitrogen 3.15 and 3.61%, for total nitrogen 522 and 598 mg/100 g milk, and for nonprotein nitrogen 28.6 and 32.6 mg/100 g. Nonprotein nitrogen was 5.5% of the total nitrogen in the North Bay Area and 5.5% of total nitrogen in the San Joaquin Valley. Regional differences were statistically significant. Protein content of milk and dairy products should be determined from Kjeldahl total nitrogen concentration corrected for the nonprotein nitrogen, and instrumental procedures, such as infrared and dyebinding, should be calibrated to actual protein content excluding nonprotein nitrogen.

Nonprotein nitrogen in pooled sweet (Cheddar and Dagano) and acid (cottage) wheys was estimated by currently proposed dialysis and chemical precipitation methods. Kjeldahl nitrogen analyses of (a) membrane retentates after water dialysis, and (b) 12% trichloroacetic acid plus .2% phosphotungstic acid filtrates indicated that nonprotein nitrogen values vary significantly with the method of sample preparation. Membrane porosity influence nonprotein nitrogen values for molecular weight cut-offs of 3500, 6000 to 8000 and 12,000 to 14,000. Dialyzable nitrogen values with all membranes were lower than 12% trichloroacetic acid soluble nitrogen for both wheys and higher than the 12% trichloroacetic acid + .2% phosphotungstic acid soluble nitrogen for all but the 3500 molecular weight cut-off membrane. The dialyzable nonprotein nitrogen fraction was heterogeneous but more that 80% was less than 3500 in molecular weight.

The spectrophotometric method of Haugaard and Damm (1) for estimating the protein content of skimmilk at wavelength 277 m μ was modified for raw and pasteurized milk, sterile concentrate, and cream as follows: Add 0.5ml of butylamine to 0.5ml of milk, heat to 65C for 5min, add 25ml of a mixture of 0.3% each of disodium ethylenediamine—tetraacetate (EDTA) and sodium lauryl sulfate (SLS), add five drops of hydrochloric acid and filter. Add one drop of 30% sodium hydroxide solution and read the optical density at 280 m μ . For milk changed by the Maillard-type reaction, add 0.5ml of butylamine to 0.5ml of milk, heat to 65C and add two drops of sodium borohydride. The remainder of the procedure is the same. Results for whole milk, sterile concentrated milk, and cream agreed with the Kjeldahl method.

Protein determinations from Orange-G dye by two spectrophotometers (Bausch and Lomb Type 33-29-40, Beckman Model DB) and nitrogen by micro-Kjeldahl were available on 1,378 monthly test-day samples of milk. These samples were collected for 20 months. The objective was to determine the best procedure for obtaining samples to estimate prediction equations for each month when micro-Kjeldahl determinations are not available on all milk samples. One sampling procedure involved the selection of six samples for each month completely at random, while the other involved dividing the data into high, middle, and low thirds according to their dye-binding readings and selecting two observations from each third for each month. Considerable variability was present in the magnitude of the correlations between the Kjeldahl and dye-binding estimates of per cent protein by both systems of sampling, indicating that six observations for each month were not sufficient to establish the monthly prediction equations.

A method for routine determination of protein, fat, and lactose contents of milk is based on the ability of a scintillation counter to measure coloration or opalescence through attenuation of photons emitted from sealed miniature carbon-14 and hydrogen-3 radioactive standards. A series of simplified and accurate analytical procedures enable full advantage to be taken of the automatic facilities on the modern liquid scintillation counter. The methods provide several advantages over existing procedures. Accuracy of quantification was high as assessed by comparing the results with those derived by recommended Kjeldahl, Gerber, and colorimetric procedures for protein, fat, and lactose determinations, respectively.

Equations for protein in sow's milk by Orange G dye binding procedures were: per cent protein=9.184-4.526 (absorbance) at Sensitivity 1 for samples above 6.5% protein, and per cent protein=7.317-6.061 (absorbance) at Sensitivity 2 for samples containing 3.0 to 6.5% protein. Forty-two samples of sow's milk were analyzed with a Beckman Model B spectrophotometer. Sow's milk had more protein than cow's milk, requiring 1ml milk per 25ml Orange G dye. Protein analyses by Orange G dye binding were 0.95 correlated with Kjeldahl estimates of protein. These equations differed from those developed for estimating protein of cow's milk by the Orange G procedure.