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Uncharacteristic of most whole foods, the major component of tree nuts is lipid; surprisingly, information on the lipid constituents in tree nuts has been sporadic and, for the most part, not well reported. Most published papers focus on only one nut type, or those that report a cultivar lack a quality control program, thus making data comparisons difficult. The present study was designed to quantify the healthful lipid constituents of 10 different types of commercially important tree nuts (i.e., almonds, black walnuts, Brazil nuts, cashews, English walnuts, hazelnuts, macadamias, pecans, pine nuts, and pistachios) according to standardized, validated methods. The total lipid content of each nut type ranged from 44.4 ± 1.9% for cashews to 77.1 ± 1.7% for macadamias. As expected, the major fatty acids present in the tree nuts were unsaturated: oleic (18:1 ω9) and linoleic (18:2 ω6) acids. A majority of the lipid extracts contained <10% saturated fatty acids with the exceptions of Brazil nuts (24.5%), cashews (20.9%), macadamias (17.1%), and pistachios (13.3%). The total tocopherol (T) content ranged from 1.60 ± 1.27 mg/100 g nutmeat in macadamias to 32.99 ± 0.78 in black walnuts. The predominant T isomers in the nut types were α- and γ-T. Tocotrienols were also detected, but only in 6 of the 10 nut types (i.e., Brazil nut, cashews, English walnuts, macadamias, pine nuts, and pistachios). In most cases, total phytosterol contents were greater in the present study than reported in peer-reviewed journal papers and the USDA National Nutrient Database for Standard Reference, which is attributed to total lipid extraction and the inclusion of steryl glucosides in the analysis; the levels were highest for pistachios (301.8 ± 15.4 mg/100 g nutmeat) and pine nuts (271.7 ± 9.1 mg/100 g nutmeat). Minor sterols were also quantified and identified using GC-FID and GC-MS techniques.

The trienzyme digestion including protease, α-amylase, and conjugase (γ-glutamyl hydrolase) for the extraction of total folate from cereal grain products used in AOAC Official Method 2004.05 was optimized using response surface methodology. Digestion times of α-amylase (p < 0.05) and conjugase (p < 0.01) showed significant effects on the response (total folate). Digestion time for Pronase digestion was not significant (p > 0.1). Ridge analysis showed that total times required for the trienzyme digestions ranged from 6.1 h for buckwheat to 8.7 h for CRM 121 (wholemeal flour). The predicted maximal value for CRM 121 (51 μg/100 g) was close to the certified value (50 ± 7 μg/100 g). The optimized extractions for the four cereals suggest that a generalized trienzyme extraction for cereals of 1, 2.5, and 6 h for Pronase, α-amylase, and conjugase, respectively, could be used to approximate total folate in cereals and cereal products. These conditions provide data comparable to AOAC Official Method 2004.05 for the cereals included in this study and would be suitable to approximate total folate in most cereals and cereal products. Compared to the official method, the shortened digestion provides cost savings and minimizes the folate loss possible with prolonged digestions.

Phytosterols in commercially grown Runner, Virginia, and Spanish peanuts (n = 221) from 2005 and 2006 were quantified by a combination of acid hydrolysis and alkaline saponification steps followed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivatives. Δ(5)-Avenasterol, which partially degrades during acid hydrolysis, was quantified after alkaline saponification plus direct analysis of the steryl glucosides isolated by solid-phase extraction. β-Sitosterol, Δ(5)-avenasterol, campesterol, and stigmasterol were identified in peanut lipid extracts as the dominant sterols by retention time mapping and mass spectra with recoveries ∼99%. Clerosterol, Δ(5,24(25))-stigmastadienol, Δ(7)-sitosterol + cycloartenol, and one unidentified sterol were also present but at low levels. Free and esterified phytosterols accounted for ∼80% of the total sterols determined; the remainder was attributed to steryl glucosides. The total sterol level in Spanish market type peanuts (144.1 ± 5.3 mg/100 g) was significantly greater than both Runners (127.5 ± 6.3 mg/100 g) and Virginias (129.3 ± 6.9 mg/100 g) (P < 0.05). Tamspan 90 (146.9 mg/100 g) followed by OLIN (138.5 mg/100 g) showed the highest total sterol content among the cultivars examined. Cultivar effects were strongly significant (P < 0.001) for all phytosterols, whereas production year effects were strongly significant (P < 0.001) for Δ(5)-avenasterol, Δ(5,24(25))-stigmastadienol, and the combined quantities of Δ(7)-sitosterol + cycloartenol, which coeluted. Cultivar × year interactions were strongly significant (P < 0.001) in all sterols except for Δ(7)-sitosterol + cycloartenol (P < 0.01). Total phytosterol contents were markedly higher than those reported in the existing literature for Runner and Virginia type peanuts, partially attributed to the inclusion of steryl glucosides in the analysis.

Tocopherols in commercially grown normal, mid- and high-oleic Runner peanuts from 2005 and 2006 were quantified to give accurate vitamin E contents. Tocopherols were extracted from raw peanuts by a direct solvent extraction procedure using 10% ethyl acetate in hexanes that provided percent recoveries of 105.4, 101.2, 103.9, and 102.8 for alpha-tocopherol (T), beta-T, gamma-T, and delta-T, respectively. No significant (P > 0.05) differences were noted in total tocopherol levels in normal- (22.4 mg/100 g), mid- (23.9 mg/100 g), and high-oleic (22.4 mg/100 g) Runner peanuts. alpha-T levels did vary significantly among the Runner cultivars classified by their oleic acid content (mid, 11.7 mg/100 g; normal, 10.9 mg/100 g; high, 9.8 mg/100 g). Cultivar effects were highly significant (P < 0.001) for alpha-, beta-, gamma-, and delta-T and total tocopherol contents, whereas production year effects were highly significant for alpha- and beta-T levels. Year x cultivar interactions were not significant (P > 0.05). Cluster analysis segregated the cultivars into two major groups represented by lower alpha-T and higher gamma-T levels (cluster A) and high alpha-T and low gamma-T levels (cluster B) (P < 0.05). The mean alpha-T level in Runner peanuts (151 samples) was 10.5 +/- 1.5 mg/100 g, which is 26.7% greater than the imputed value for peanuts, all types (NBD 16087) provided by the USDA National Nutrient Database for Standard Reference.

Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.

An interlaboratory verified, liquid chromatographic (LC) method is presented for the analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in soy-based infant formula. The extraction procedure uses sample dehydration with magnesium sulfate followed by extraction with isopropanol, hexane-ethyl acetate (85 + 15, v/v). After evaporation and filtration, the sample extract is injected directly onto a normal-phase LC system with fluorescence detection. All-rac-alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing 0.50% (v/v) and 0.125% (v/v) isopropanol, respectively. A zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" is the minimum level of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in 21 Code of Federal Regulations 107.100. The following recoveries and RSD values represent an average (n = 25) of the 5 levels for each analyte: all-rac-alpha-tocopheryl acetate, 100% (RSD = 3.5%); retinyl palmitate, 97.2% (RSD = 2.1%). Two additional laboratories analyzed the fortified ZRM samples. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 99.0% (RSD = 4.0%), and retinyl palmitate, 96.2% (RSD = 1.4%) at the second laboratory. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 102% (RSD = 1.4%) and retinyl palmitate, 95.7% (RSD = 2.0%) at the third laboratory. In addition, 6 replicates of the same commercial soy-based infant formula powder were run by the 3 laboratories.

An interlaboratory-verified, liquid chromatographic (LC) method is presented for determination of all-racemic alpha-tocopheryl acetate and retinyl palmitate in infant formula. The extraction procedure uses matrix solid-phase dispersion. A sample is mixed with C18, and the mixture is packed into a reservoir and eluted with selective solvents to extract the analytes. After evaporation and filtration, the sample extract is injected directly into a normal-phase LC system with fluorescence detection. All-racemic alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing isopropanol at 0.2% (v/v) and 0.125% (v/v), respectively. A nonfortified zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" represents the minimum levels of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in Title 21 of the Code of Federal Regulations, part 107.100. Recoveries of retinyl palmitate ranged from 83.8 to 107%, and those of all-racemic alpha-tocopheryl acetate ranged from 87.7 to 108%. Two additional laboratories analyzed the ZRM samples at 4 spiking levels with 6 replicates. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 92.2 to 104% and from 91.7 to 101%, respectively, in the second laboratory. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 85.3 to 97.0% and from 86.6 to 110%, respectively, in the third laboratory. Relative standard deviations for all 3 laboratories ranged from 0.2 to 7.5% with an average of 2.9%. In addition, each laboratory analyzed a commercial milk- and commercial soy-based infant formula. Excellent agreement in results was obtained between the 3 laboratories for vitamins A and E in all matrixes.

Retention of immunoglobulin A (IgA) and IgA specific for enteropathogenic Escherichia coli (EPEC) was determined in human milk that was processed and stored under refrigeration or frozen conditions. Human milk was subjected to three different processing conditions: (a) deaerated, vacuum packaged in metalized polyester bags and pasteurized (64°C for 6 min, 10 s); (b) vacuum packaged and pasteurized; (c) vacuum packaged. Samples were stored at 4°C for 7 d and at -20°C for 28 d. An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to EPEC was developed and used to monitor these antibody levels, while radial immunodiffusion (RID) plates were used to provide information on total IgA of the milk samples. In addition, the dissolved oxygen content of the samples was monitored. It was found that pasteurization and deaeration did not adversely affect the IgA levels, and levels of total IgA and IgA specific for EPEC remained stable or increased slightly during storage in most samples regardless of the storage temperatures.

Rates of thermal destruction of Staphylococcus aureus were determined in mature human milk using a continuous flow high-temperature short-time pasteurization system. D and z values for inactiviation of S. aureus were determined from data on survivors capable of forming colonies in an appropriate selective medium. The effects of thermal injury on D and z values were assessed by survivor colony forming units (CFU) on Staphylococcus medium 110 (SMI 10), nutrient agar (NA), Trypticase Soy Agar (TSA), Trypticase Soy Agar with 7.5% NaCi (TSAS) and Baird-Parker medium (BP) (Difco Laboratories, Detroit, MI). D values for inactivation of S. aureus at 52, 58 and 60 and 62°C were used to predict D at 60°C of 15.3 s and 24.3 s when based on survivor CFU in SM 110 and nutrient agar, respectively. The z value was 4.9°C in either medium. D-values for inactivation of S. aureus at 60, 62, 64 and 67°C were used to predict D at 60°C of 41.2 s, 41.0 s and 34.7 s when based on survivor CFU in BP, TSA and TSAS, respectively. The z values were 6.5, 6.5 and 6.4°C, respectively.

A comparative review of the specificities of various B-vitamin analysis methods is presented. Methods of vitamin analysis employing protozoa, viz. Tetrahymena and Ochromonas , are compared to the commonly used methods of vitamin assay, including officially accepted methods. Since 1968 the protozoan techniques have routinely been used to evaluate clinical vitamin status. This is due to their more mammalian-like response to the various forms of the vitamins that occur in natural materials. Protozoa have more developed ingestive and digestive systems than bacteria and yeast, which allow them to respond to conjugated forms of the vitamins (e.g. thiamin pyrophosphate and folic acid polyglutamates). Also, fewer problems are encountered with non-specific stimulation (e.g. fatty acid stimulation of the Lactobacillus casei assay for riboflavin or the sparing of the vitamin B 12 requirement of Lactobacillus leichmannii by deoxyribosides). Application of the protozoan methods to analysis of foods indicate significantly lower levels of biotin and vitamin B 12 than official methods, while significantly higher levels of riboflavin, vitamin B 6 , niacin and pantothenic acid are found in low-acid foods. This review also summarizes extraction methods for the B-vitamins from food samples and indicates the importance of enzymatic hydrolysis in extract preparation.

The bacteriological profiles of human milk samples collected from individual donors under supervised conditions of collection were compared to pooled human milk samples obtained from a commercial human milk bank. Total aerobic counts and total coliform counts of individual donor samples were lower than those of pooled, banked human milk. All of the 200 isolates from ten individual samples were staphylococci with Staphylococcus epidermidis predominating (82%). Only 1% of the isolates was identified as Staphylococcus aureus . Forty-two percent of the 100 isolates from five pooled samples were staphylococci and all of these staphylococci were coagulase-negative. Three percent of the isolates from the pooled samples were Streptococcus faecalis . The remainder (55%) were gram-negative organisms. S. epidermidis was the microorganism that was isolated most frequently from either individual (9 of 10) or pooled (3 of 5) samples.

A continuous flow high-temperature short-time pasteurization system was used to determine kinetic parameters (D- and z-values) for thermal destruction of the bacterial pathogens, Escherichia coli and Klebsiella pneumoniae , in mature human milk. D-and z-alues of each bacterium were determined from data on survivors enumerated on both selective media, Violet Red Bile agar or MacConkey's, and on a non-selective medium, nutrient agar (NA). For E. coli , D-values were determined at 58, 60, 62 and 64°C. The predicted value of D at 60°C is 31.5 s. The z-value for E. coli is 3.2°C. D-values for K. pneumoniae were determined at 52, 56 and 58°C. Based on these data the predicted value of D at 60°C is 1.3 s. The z-value for K. pneumoniae , is 2.8°C. For both E. coli and K. pneumoniae , counts on NA tend to be higher than on selective media. This is undoubtedly due to the inhibitory nature of the selective media. This also suggests that some degree of thermal injury may occur for each organism.

Three composites of human milk samples were subjected to different processing conditions: (1) deaerated, vacuum packaged in metalized polyester bags and pasteurized at 56°C for 8 min; (2) vacuum packaged and pasteurized; (3) vacuum packaged. On days 0, 4, 7, 14, 28, 64, and 96 of storage, each treatment was analyzed for dissolved oxygen content and viable microflora. On days 0, 4, and 14, randomly selected isolates from each treatment were identified to the species level. Heat treatment of the milk samples reduced the number of viable microorganisms and resulted in a shift in the type of bacteria in the milk. Pasteurized samples contained primarily non-pathogenic skin commensals, while non-pasteurized samples were populated by species of Pseudomonas and other Gram negative microorganisms, including potential pathogens. Deaeration did not affect either the number or types of microorganisms surviving the heat treatment.