Myeloid-derived suppressor cell-like fibrocytes are increased and associated with preserved lung function in chronic obstructive pulmonary disease.

BACKGROUND: The role of fibrocytes in chronic obstructive pulmonary disease (COPD) is unknown. We sought to enumerate blood and tissue fibrocytes in COPD and determine the association of blood fibrocytes with clinical features of disease. METHODS: Utilizing flow cytometry to identify circulating, collagen type 1+ cells, we found two populations: (i) CD45+ CD34+ (fibrocytes) and (ii) CD45+ CD34- [myeloid-derived suppressor cell (MDSC)-like fibrocytes] cells in stable COPD (n = 41) and control (n = 29) subjects. Lung resection material from a separate group of subjects with (n = 11) or without (n = 11) COPD was collected for tissue fibrocyte detection. We examined circulating fibrocyte populations for correlations with clinical parameters including quantitative computed tomography (qCT) and determined pathways of association between correlated variables using a path analysis model. RESULTS: Blood and tissue fibrocytes were not increased compared to control subjects nor were blood fibrocytes associated with lung function or qCT, but were increased in eosinophilic COPD. Myeloid-derived suppressor cell-like fibrocytes were increased in COPD compared to controls [2.3 (1.1-4.9), P = 0.038]. Our path analysis model showed that collagen type 1 intensity for MDSC-like fibrocytes was positively associated with lung function through associations with air trapping, predominately in the upper lobes. CONCLUSION: We have demonstrated that two circulating populations of fibrocyte exist in COPD, with distinct clinical associations, but are not prevalent in proximal or small airway tissue. Blood MDSC-like fibrocytes, however, are increased and associated with preserved lung function through a small airway-dependent mechanism in COPD.

Profiling of sputum inflammatory mediators in asthma and chronic obstructive pulmonary disease.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) display features of overlap in airway physiology and airway inflammation. Whether inflammatory phenotypes in airway disease describe similar mediator expression is unknown. OBJECTIVES: To explore the relationship of airway inflammation and cytokine and chemokine expression in asthma and COPD. METHODS: Subjects with asthma and COPD (n = 54 and n = 49) were studied. Clinical characteristics and sputum were collected at entry into the study. A 2-step sputum processing method was performed for supernatant and cytospin preparation. Meso Scale Discovery and Luminex platforms were used to measure cytokines, chemokines and matrix metalloproteinase levels. RESULTS: Analytes sensitive to dithiothreitol (DTT) that had increased recovery in the 2-step sputum process were IL-1ß, 4, 5, 10, 13, IFN-γ, TNFRI, GM-CSF, CCL2, 3, 4, 5, 13 and 17. There was a differential expression in IL-8, TNFRI and TNFRII between asthma and COPD [mean fold difference (95% CI): IL-8, 2.6 (1.3-5.4), p = 0.01; TNFRI, 2.1 (1.3-5.4), p = 0.03; TNFRII, 2.6 (1.2-5.6), p = 0.02]. In neutrophilic and eosinophilic airway inflammation, TNFα, TNFRI, TNFRII, IL-6, IL-8 and IL-5 could differentiate between these phenotypes. However, these phenotypes were unrelated to the diagnosis of asthma or COPD. CONCLUSION: Recovery of sputum mediators sensitive to DTT can be improved using the described sputum processing technique. Within airway inflammatory sub-phenotypes there is a differential pattern of mediator expression that is independent of disease. Whether these inflammatory phenotypes in asthma and COPD confer distinct pathogeneses, therapeutic responses and clinical phenotypes needs to be further evaluated.

Methadone toxicity in infants: a report of two fatalities.

Fatalities in infants resulting from methadone toxicity are rare within the United Kingdom. We report two cases of fatality attributed to methadone toxicity in infants aged 3(1/2) and 15 months of age, respectively. One of the two cases was also associated with diazepam ingestion. We discuss the difficulties with the interpretation of paediatric forensic toxicology and review the current literature related to methadone and diazepam toxicity in infants and older children.

First-trimester increase in oxidative stress and risk of small-for-gestational-age fetus.

OBJECTIVE: Investigation of increased oxidative stress in early pregnancy and association with an increased risk of small-for-gestational-age (SGA) fetus. DESIGN: Longitudinal case-control study. SETTING: University Hospitals of Leicester NHS Trust, Leicester, UK. POPULATION: Low-risk pregnant women with no current or pre-existing medical illness were recruited at a large teaching hospital from 2004 to 2006. METHODS: Recruitment performed at the time of the dating ultrasound scan (12+/-2 weeks of gestation). Spot urine samples collected at 12+/-2 and 28+/-2 weeks of gestation were analysed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by liquid chromatography with tandem mass spectrometry). SGA was defined as birthweight <10th centile based on customised centile calculator (www.gestation.net). This identified the cases (n=55), whereas controls (n=55) were mothers whose babies were appropriate for gestational age (AGA, birthweight 10th-90th centile). Statistical analysis was performed using GraphPad Prism v.5. The relationship between maternal urinary 8-oxodG at different gestations and customised SGA was investigated by nonparametric tests. MAIN OUTCOME MEASURES: Customised SGA and AGA pregnancies. RESULTS: Urinary 8-oxodG concentrations were significantly increased in pregnancies with subsequent SGA compared with concentrations in normal pregnancies; 12 weeks: 2.8 (interquartile range [IQR] 1.96-3.67) versus 2.2 (IQR 1.26-3.28) pmol 8-oxodG/micromol creatinine (P=0.0007); 28 weeks: 2.21 (IQR 1.67-3.14) versus 1.68 (IQR 1.16-2.82) pmol 8-oxodG/micromol creatinine (P<0.0002). Concentrations decreased significantly between week 12 and 28 (P=0.04 and P=0.02 for controls and cases). CONCLUSIONS: In this study, urinary 8-oxodG at 12 and 28 weeks were elevated in SGA compared with AGA pregnancies. This may reflect early placental changes predating clinical features of SGA.

Granulocyte-macrophage colony-stimulating factor expression in induced sputum and bronchial mucosa in asthma and COPD.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of GM-CSF and its receptor in airway samples in asthma and COPD across disease severity needs to be further defined. METHODS: Sputum GM-CSF was measured in 18 control subjects, 45 subjects with asthma and 47 subjects with COPD. Enumeration of GM-CSF+ cells in the bronchial submucosa and airway smooth muscle bundle was performed in 29 control subjects, 36 subjects with asthma and 10 subjects with COPD. RESULTS: The proportion of subjects with measurable GM-CSF in the sputum was raised in those with moderate (7/14) and severe (11/18) asthma, and in those with COPD GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage II (7/16), III (8/17) and IV (7/14) compared with controls (1/18) and those with mild asthma (0/13); p = 0.001. The sputum GM-CSF concentration was correlated with the sputum eosinophilia in subjects with moderate to severe asthma (r(s) = 0.41; p = 0.018). The median (interquartile range) GM-CSF+ and GM-CSFR+ cells/mm(2) of submucosa was increased in severe asthma (1.4 (3.0) and 2.1 (8.4)) compared with those with mild to moderate asthma (0 (2.5) and 1.1 (5)) and healthy controls (0 (0.5) and 0 (1.6)), (p = 0.004 and p = 0.02, respectively). CONCLUSIONS: The findings support a potential role for GM-CSF in asthma and COPD and suggest that overexpression of GM-CSF in sputum and the bronchial mucosa is a particular feature of severe asthma.

Induced sputum and bronchial mucosal expression of interleukin-13 is not increased in chronic obstructive pulmonary disease.

BACKGROUND: The Th2 cytokine interleukin-13 (IL-13) has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). We sought to examine IL-13 expression in COPD subjects in induced sputum and bronchus specimens. We hypothesized that inflammatory cells expressing IL-13 localize to the airway smooth muscle bundle and bronchial glands. METHODS: Interleukin-13 was measured in sputum samples from subjects with COPD (n = 34) across a range of severity (Global initiative for chronic Obstructive Lung Disease 2-4) and controls (n = 14) using ELISA. IL-13+ cells and inflammatory cells were enumerated within surgically resected proximal airway using immunohistochemical techniques from subjects with COPD (n = 10), smoking (n = 10) and nonsmoking controls (n = 8). RESULTS: Sputum IL-13 was measurable in only 6/34 subjects with COPD and was not found in the smoking or nonsmoking control subjects. In subjects with COPD and controls there was a paucity of IL-13+ cells. The distribution of inflammatory cells within different airway compartments was similar in COPD and controls except for an increase in CD3(+) lymphocytes within bronchial glands in COPD (P = 0.04). CONCLUSIONS: Our findings do not support a role for IL-13 in COPD. However, the tissue localization of inflammatory cells to airway compartments, particularly the increase of T cells in glands in COPD may be important in disease.

Reduced fat process cheese made from young reduced fat Cheddar cheese manufactured with exopolysaccharide-producing cultures.

In a previous study, exopolysaccharide (EPS)-producing cultures improved textural and functional properties of reduced fat Cheddar cheese. Because base cheese has an impact on the characteristics of process cheese, we hypothesized that the use of EPS-producing cultures in making base reduced fat Cheddar cheese (BRFCC) would allow utilization of more young cheeses in making reduced fat process cheese. The objective of this study was to evaluate characteristics of reduced fat process cheese made from young BRFCC containing EPS as compared with those in cheese made from a 50/50 blend of young and aged EPS-negative cheeses. Reduced fat process cheeses were manufactured using young (2 d) or 1-mo-old EPS-positive or negative BRFCC. Moisture and fat of reduced fat process cheese were standardized to 49 and 21%, respectively. Enzyme modified cheese was incorporated to provide flavor of aged cheese. Exopolysaccharide-positive reduced fat process cheese was softer, less chewy and gummy, and exhibited lower viscoelastic moduli than the EPS-negative cheeses. The hardness, chewiness, and viscoelastic moduli were lower in reduced fat process cheeses made from 1-mo-old BRFCC than in the corresponding cheeses made from 2-d-old BRFCC. This could be because of more extensive proteolysis and lower pH in the former cheeses. Sensory scores for texture of EPS-positive reduced fat process cheeses were higher than those of the EPS-negative cheeses. Panelists did not detect differences in flavor between cheeses made with enzyme modified cheese and aged cheese. No correlations were found between the physical and melting properties of base cheese and process cheese.

Effect of vacuum-condensed or ultrafiltered milk on pasteurized process cheese.

Milk was concentrated by ultrafiltration (UF) or vacuum condensing (CM) and milks with 2 levels of protein: 4.5% (UF1 and CM1) and 6.0% (UF2 and CM2) for concentrates and a control with 3.2% protein were used for manufacturing 6 replicates of Cheddar cheese. For manufacturing pasteurized process cheese, a 1:1 blend of shredded 18- and 30-wk Cheddar cheese, butter oil, and disodium phosphate (3%) was heated and pasteurized at 74 degrees C for 2 min with direct steam injection. The moisture content of the resulting process cheeses was 39.4 (control), 39.3 (UF1), 39.4 (UF2), 39.4 (CM1), and 40.2% (CM2). Fat and protein contents were influenced by level and method of concentration of cheese milk. Fat content was the highest in control (35.0%) and the lowest in UF2 (31.6%), whereas protein content was the lowest in control (19.6%) and the highest in UF2 (22.46%). Ash content increased with increase in level of concentration of cheese milk with no effect of method of concentration. Meltability of process cheeses decreased with increase in level of concentration and was higher in control than in the cheeses made with concentrated milk. Hardness was highest in UF cheeses (8.45 and 9.90 kg for UF1 and UF2) followed by CM cheeses (6.27 and 9.13 kg, for CM1 and CM2) and controls (3.94 kg). Apparent viscosity of molten cheese at 80 degrees C was higher in the 6.0% protein treatments (1043 and 1208 cp, UF2 and CM2) than in 4.5% protein treatments (855 and 867 cp, UF1 and CM1) and in control (557 cp). Free oil in process cheeses was influenced by both level and method of concentration with control (14.3%) being the lowest and CM2 (18.9%) the highest. Overall flavor, body and texture, and acceptability were higher for process cheeses made with the concentrates compared with control. This study demonstrated that the application of concentrated milks (UF or CM) for Cheddar cheese making has an impact on pasteurized process cheese characteristics.

Bioavailability of vitamin D from fortified process cheese and effects on vitamin D status in the elderly.

We conducted 2 studies to determine the effect of vitamin D-fortified cheese on vitamin D status and the bioavailability of vitamin D in cheese. The first study was designed to determine the effect of 2 mo of daily consumption of vitamin D3-fortified (600 IU/d) process cheese on serum 25-hydroxyvitamin D (25-OHD), parathyroid hormone (PTH), and osteocalcin (OC) concentrations among 100 older (> or =60 yr) men and women. Participants were randomized to receive vitamin D-fortified cheese, nonfortified cheese, or no cheese. Serum levels of 25-OHD, PTH, and OC were measured at the beginning and end of the study. There were no differences in 25-OHD, PTH, or OC after 2 mo of fortified cheese intake. The vitamin D-fortified cheese group had a greater decrease in 25-OHD than other groups, due to higher baseline 25-OHD. A second study was conducted to determine whether the bioavailability of vitamin D2 in cheese (delivering 5880 IU of vitamin D2/56.7-g serving) and water (delivering 32,750 IU/250 mL) is similar and whether absorption differs between younger and older adults. The second study was a crossover trial involving 2 groups of 4 participants each (younger and older group) that received single acute feedings of either vitamin D2-fortified cheese or water. Serial blood measurements were taken over 24 h following the acute feeding. Peak serum vitamin D and area under the curve were similar between younger (23 to 50 yr) and older (72 to 84 yr) adults, and vitamin D2 was absorbed more efficiently from cheese than from water. These studies demonstrated that vitamin D in fortified process cheese is bioavailable, and that young and older adults have similar absorption. Among older individuals, consuming 600 IU of vitamin D3 daily from cheese for 2 mo was insufficient to increase serum 25-OHD during limited sunlight exposure.

Comparison of effect of vacuum-condensed and ultrafiltered milk on cheddar cheese.

The objective of this study was to compare the effects of vacuum-condensed (CM) and ultrafiltered (UF) milk on some compositional and functional properties of Cheddar cheese. Five treatments were designed to have 2 levels of concentration (4.5 and 6.0% protein) from vacuum-condensed milk (CM1 and CM2) and ultrafiltered milk (UF1 and UF2) along with a 3.2% protein control. The samples were analyzed for fat, protein, ash, calcium, and salt contents at 1 wk. Moisture content, soluble protein, meltability, sodium dodecyl sulfate-PAGE, and counts of lactic acid bacteria and nonstarter lactic acid bacteria were performed on samples at 1, 18, and 30 wk. At 1 wk, the moisture content ranged from 39.2 (control) to 36.5% (UF2). Fat content ranged from 31.5 to 32.4% with no significant differences among treatments, and salt content ranged from 1.38 to 1.83% with significant differences. Calcium content was higher in UF cheeses than in CM cheeses followed by control, and it increased with protein content in cheese milk. Ultrafiltered milk produced cheese with higher protein content than CM milk. The soluble protein content of all cheeses increased during 30 wk of ripening. Condensed milk cheeses exhibited a higher level of proteolysis than UF cheeses. Sodium dodecyl sulfate-PAGE showed retarded proteolysis with increase in level of concentration. The breakdown of alphas1- casein and alphas1-I-casein fractions was highest in the control and decreased with increase in protein content of cheese milk, with UF2 being the lowest. There was no significant degradation of beta-casein. Overall increase in proteolytic products was the highest in control, and it decreased with increase in protein content of cheese milk. No significant differences in the counts of lactic starters or nonstarter lactic acid bacteria were observed. Extent as well as method of concentration influenced the melting characteristics of the cheeses. Melting was greatest in the control cheeses and least in cheese made from condensed milk and decreased with increasing level of milk protein concentration. Vacuum condensing and ultrafiltration resulted in Cheddar cheeses of distinctly different quality. Although both methods have their advantages and disadvantages, the selection of the right method would depend upon the objective of the manufacturer and intended use of the cheese.

Reduction of salt (NaCl) losses during the manufacture of cheddar cheese.

The objective of this study was to evaluate the effects of cheese-making technologies, including homogenization of cream, ultrafiltration, and vacuum condensing of milk, on the retention of salt in Cheddar cheese. One part of pasteurized, separated milk (0.58% fat) was ultrafiltered (55 degrees C, 16.0% protein), another vacuum condensed (12.5% protein), and the third was not concentrated. Cheddar cheese was manufactured using 6 treatments by standardizing unconcentrated milk to a casein-to-fat ratio of 0.74 with unhomogenized 35% fat cream (C), homogenized (6.9 MPa/3.5 MPa) 35% fat cream (CH), ultrafiltered milk and unhomogenized cream (UF), ultrafiltered milk and homogenized cream (UFH), condensed milk and unhomogenized cream (CM), and condensed milk and homogenized cream (CMH). Treatments C and CH had 3.7% fat and 3.5% protein, and the respective values for the remaining treatments were 4.9 and 4.6. The milled curd was dry salted at 2.7% by weight. The salt content of the cheeses receiving homogenization treatment was higher at 1.83 and 1.70% for CH and UFH, respectively, compared with their corresponding controls at 1.33%. The salt content in cheeses from CMH was 1.64% and was not affected by homogenization. Salt retention in C increased from 41.7 to 59.2% in CH, and in UF it increased from 42.5 to 54.5% in UFH. There was a corresponding decrease in the salt content of whey from these cheeses.

Estimation and fortification of vitamin D3 in pasteurized process cheese.

The objective of this study was to develop methods for the estimation and fortification of vitamin D3 in pasteurized Process cheese. Vitamin D3 was estimated using alkaline saponification at 70 degrees C for 30 min, followed by extraction with petroleum ether:diethyl ether (90:10 vol/vol) and HPLC. The retention time for vitamin D3 was approximately 9 min. A standard curve with a correlation coefficient of 0.972 was prepared for quantification of vitamin D3 in unknown samples. In the second phase of the study, pasteurized Process cheeses fortified with commercial water- or fat-dispersible forms of vitamin D3 at a level of 100 IU per serving (28 g) were manufactured. There was no loss of vitamin D3 during Process cheese manufacture, and the vitamin was uniformly distributed. No losses of the vitamin occurred during storage of the fortified cheeses over a 9-mo period at 21 to 29 degrees C and 4 to 6 degrees C. There was an approximately 25 to 30% loss of the vitamin when cheeses were heated for 5 min in an oven maintained at 232 degrees C. Added vitamin D3 did not impart any off flavors to the Process cheeses as determined by sensory analysis. There were no differences between the water- and fat-dispersible forms of the vitamin in the parameters measured in fortified cheeses.

Nitric oxide from enteric nerves acts by a different mechanism from myogenic nitric oxide in canine lower esophageal sphincter.

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (NO) synthase (NOS) in plasma membrane limits tone by opening large conductance Ca(2+)-dependent K(+) channels (BK(Ca) channels) and hyperpolarizing the membrane. We examined whether K(V) channels were involved and whether NO from enteric nerves and from NO donors used the same mechanisms. With nerves inactive, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone but less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammonium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone increase from any combination of these agents. More than iberiotoxin or 4-AP, TEA decreased relaxations in response to sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (Sin-1) by approximately 50%. In whole-cell patch-clamp recordings, TEA and 4-AP reduced outward K(+) currents additively by >90% at depolarization of +90 mV. Thus, K(+) channels in addition to BK(Ca) channels are opened by myogenic NO, and exogenous NO had relaxing effects both related and unrelated to K(+) channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerves. 4-AP relaxed tone, an effect that was abolished and reversed by L-NOARG. 4-AP apparently released NO and acetylcholine from nerves. The putative Cl(-) channel blocker niflumic acid (NFA; 30-100 microM) dose dependently reduced tone, but tone, restored by 10(-6) M carbachol or 20 mM TEA, was still relaxed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) at 500 to 1000 microM did not inhibit relaxation to EFS or SNP. The addition of TEA (20 mM) to DIDS (1000 microM) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relaxations to SNP like TEA alone. Reduction in extracellular ¿Cl(-) by isethionate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular ¿Cl(-) and TEA did not abolish relaxation to EFS until DIDS was added. Thus, multiple K(+) channels are opened by myogenic NO, and openings of these channels, as well as DIDS-sensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.

Influence of salt on the quality of reduced fat cheddar cheese.

Cream was homogenized in a two-stage homogenizer (17.25 MPa in the first stage and 3.43 MPa in the second stage); blended with skim milk to produce milk containing 1.25% fat, which was pasteurized (63 degrees C for 30 min); and then manufactured into reduced fat Cheddar cheese. After milling, the curd was divided into three equal portions of 13 kg each. Three salting rates, 2.3, 3.8, and 5%, yielded cheeses with 1.3, 1.7, and 2.0% salt and 2.7, 3.7, and 4.5% salt in the moisture phase. Cheese moisture contents ranged from 45% (2.0% salt) to 47.7% (1.3% salt), and fat contents ranged from 14.6 to 15.1%. In the texture profile analysis, the hardness and fracturability of the cheeses increased as the salt content increased. Both parameters decreased during ripening, but cheeses with 4.5% salt in the moisture phase remained the hardest. Cheeses with the most salt had the least desirable body characteristics, but there were no differences in flavor. Intensity of bitterness was lowered as the amount of salt in cheese increased. During ripening, the number of lactic acid bacteria decreased more slowly in cheese with 2.7% salt in the moisture phase than in those with 3.7 or 4.5% salt in the moisture phase. As the salt content increased, proteolysis and the general rate of ripening decreased. Degradation of alpha s-casein was reduced by higher percentages of salt, but no differences were found in the degradation of beta-casein.

Growth characteristics of bifidobacteria in infant formulas.

Four species of bifidobacteria, Bifidobacterium bifidum (ATCC 15696), Bifidobacterium breve (ATCC 15700), Bifidobacterium infantis (ATCC 15697), and Bifidobacterium longum (ATCC 15708), were grown anaerobically at 37 degrees C in lactobacilli MRS broth with 0.5% cysteine-HC1 and inoculated at 2.5% into three types of infant formula (based on soy, or milk, or casein hydrolysate) and in nonfat milk followed by incubation at 37 degrees C for 24 h. In most cases, the logarithmic phase of growth for all species varied from the first 8 to 12 h postinoculation. Generation times for B. longum and B. breve were similar, and times for B. infantis were shortest, in all of the formulas. Trends for lactic acid production for all species in all the formulas were similar to trends for acetic acid production. Counts for formulas based on soy or milk were similar for all species except B. bifidum, and those for casein-hydrolyzed formula were always lowest for all species except B. bifidum, for which count was maximal with the formula based on soy. Results suggest that growth characteristics of bifidobacteria in infant formula were species specific and formula dependent and that growth was maximal in the formula based on milk.

Effect of bifidogenic factors on growth characteristics of bifidobacteria in infant formulas.

A bifidogenic factor, lactulose or fructooligosaccharides, was added (0.5%) into infant formula based on soy or infant formula with hydrolyzed casein. The infant formulas were then inoculated (2.5%) with Bifidobacterium bifidum (ATCC 15696), Bifidobacterium breve (ATCC 15700), Bifidobacterium infantis (ATCC 15697), or Bifidobacterium longum (ATCC 15708) or their mixture (mixed culture) and incubated at 37 degrees C for 24 h. Lactulose did not influence maximal counts or generation times in either formula for any species except B. infantis, which had lower counts. Trends of developed acidity and pH of the mixed culture in the infant formulas with or without lactulose were similar to those for B. breve. Maximal counts and generation times remained unchanged with or without fructooligosaccharides for all species and the mixed culture, except for B. bifidum in the formula based on soy, for which maximal counts did not occur. Growth in either formula was inhibited for B. infantis with lactulose and B. breve with fructooligosaccharides past 8 h of inoculation.

Growth and viability of Bifidobacterium bifidum in cheddar cheese.

Bifidobacterium bifidum (ATCC 15696) was grown in MRS broth containing cysteine.HCl at 37 degrees C, and the cells were harvested by centrifuging at 1300 x g for 15 min at 4 degrees C. Equal volumes of the cell slurry and a 2.5% solution of kappa-carrageenan were mixed and transferred by drops into a solution of .3 M KCl at 20 degrees C under an atmosphere of nitrogen. The gelled beads were separated, frozen, and lyophilized immediately. This preparation and a commercial powder preparation were added to Cheddar cheese curd at milling as two treatments. Treatments did not affect cheese composition. Soluble protein increased during ripening at 7 degrees C but without differences between treatments; SDS-PAGE patterns of proteolysis were also similar. Lactic acid content of cheeses increased during ripening, but differences between treatments were minor. Acetic acid and ethanol, common metabolites of bifidobacteria, were not detected during ripening. Bifidobacteria remained viable and increased in numbers in cheese during this 24-wk study but did not affect the flavor, flavor intensity, texture, or appearance of the cheese compared with that of the control.

Growth characteristics of bifidobacteria in ultrafiltered milk.

Five replicates of raw skim milk were ultrafiltered at 54 degrees C to total protein concentration ratios of 2:1, 3:1, 4:1, and 5:1. Bifidobacterium bifidum and Bifidobacterium longum were inoculated at 5% into skim milk that was not ultrafiltered (1:1) and into ultrafiltered skim milks followed by incubation at 37 degrees C. The mean maximum bacterial count (colony-forming units per milliliter) range and protein concentration ratio were from 6.25 x 10(8) to 1.69 x 10(9), skim milk; 4.42 x 10(8) to 3.56 x 10(9), 2:1; 2.62 x 10(8) to 3.94 x 10(9), 3:1; 6.67 x 10(8) to 1.88 x 10(9), 4:1; and 2.90 x 10(9) to 3.59 x 10(9), 5:1. The mean developed acidity at maximum B. bifidum population in skim milk was .16%, and pH was 5.55. The 5:1 concentrate had a higher mean developed acidity of .57% at pH 5.35, which was similar to that of the skim milk. Trends were similar for B. longum. Because of the increased buffering capacity of highly concentrated ultrafiltered milks, pH 5.5 or higher was maintained longer, along with high developed acidity. Scanning electron micrographs showed distinct morphological variations between bifidobacteria grown in broth versus those grown in the milks.