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Symptoms of lactose intolerance are unlikely to occur under usual dietary conditions. Yet, self-described "lactose-intolerant" individuals often restrict dairy and calcium intake. A new study suggests that such individuals have reduced peak bone mass and increased incidence of osteopenia, and are at greater risk of osteoporosis and bone fractures.
Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C. The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.
The trypsin-kallikrein inhibitor aprotinin was modified with lactose. The influence of reactant concentrations, temperature, reaction time and sodium borohydride on the carbohydrate residue content and the inhibiting activity of glycated aprotinin were studied. Glycation of aprotinin neither shifts the pH optimum of the inhibitor-trypsin association reaction nor does it alter the apparent dissociation constant Ki of the complex measured at pH optimum. Glycation by lactose stabilizes aprotinin against denaturation by increased temperature. The distribution of native and modified aprotinin in rat organs after endocardiac injection was studied. Fixation of glycated aprotinin increases 2.5- to 3-fold in liver and decreases 2-fold in kidneys during the observation time (5 min-2 h) compared to native aprotinin.
A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.
Triacylglycerides constitute the bulk of fat in human milk. By centrifuging the milk a layer of fat is obtained, the cream, the fraction of which correlates very well with the total triacylglyceride concentration. It is determined in analogy to the haematocrit and is called galactocrit). Protein is determined with the biuret method in defatted milk. The interference due to lactose in this assay is on average 2 g/l. Lactose is determined with an enzyme kit. The amount of macronutrients in milk determines the energy value. A protocol is proposed, which makes it possible to measure the quantity of human milk drunk by the infant and to calculate the daily energy intake.
The sugar absorption test is the usual test for measurement of intestinal permeability. After intestinal absorption of probe sugars the subsequently excreted sugars are measured in urine. We have developed four enzymatic methods for the measurement of the urinary concentration of the probe sugars mannitol, raffinose, lactose and sucrose. Mannitol, lactose and sucrose are directly measured on Hitachi 917 using mannitol dehydrogenase, beta-galactosidase and invertase, respectively, as enzyme reagents. Raffinose measurement needs a three hours preincubation with alpha-galactosidase, after which the liberated sucrose is measured. The analytical performances such as within- and between-run precision, linearity, lowest detection limit, interference of other sugars and comparison with a gas chromatographic method are described for the four methods. These methods are accurate an can easily be performed in any clinical laboratory.
The response of prolactin receptor and lactose synthetase to suppression of plasma concentrations of prolactin was examined in normal and occluded (teat-sealed) mammary glands of Sprague-Dawley rats, Rats, with mammary glands unilaterally occluded, were given bromocriptine (2.5 mg/kg per 12 h) between 5 and 8 post partum. Bromocriptine reduced plasma prolactin concentrations from 460.4 +/- 120.8 (mean +/- s.e.m.) to 2.56 +/- 0.89 ng/ml within 12h whilst concentrations in control rats were 553.4 +/- 110.25 ng/ml. Lactose synthetase activity declined rapidly, within 24h, in occluded glands of both groups but was maintained for 24 h in normal glands of bromocriptine-treated rats and decreased thereafter. Prolactin receptors also declined significantly within 24h in occluded glands. Desaturation of the prolactin receptor by bromocriptine treatment in vivo was compared with desaturation by exposure of membranes to MgCl2 in vitro. Both treatments enhanced prolactin binding but the increase after treatment with MgCl2 may have been partly artefactual since there was a selective loss of protein from the membranes. These results indicate that the prolactin receptor in rat mammary gland may be maintained after acute suppression of prolactin secretion.
We investigated a possible source of contamination of expired-air carbon monoxide (CO) readings on CO monitors from raised expired-air hydrogen (H2) levels, namely lactose intolerance, the norm in certain ethnic groups. A significant correlation between expired-air CO and H2 readings was observed in four non-smoking lactose maldigesters.
S-Lac lectins are a family of soluble lactose-binding animal lectins, some of which have been implicated in modulating cell-cell and cell-matrix interactions through specific carbohydrate-mediated recognition. We report here the x-ray crystal structure of a representative member of this family, the human dimeric S-Lac lectin, L-14-II, in complex with lactose, at 2.9-A resolution. The two-fold symmetric dimer is made up of two extended anti-parallel beta-sheets, which associate in a beta-sandwich motif. Remarkably, the L-14-II monomer shares not only the same topology, but a very similar beta-sheet structure with that of the leguminous plant lectins, suggesting a conserved structure-function relationship. Carbohydrate binding by L-14-II was found to involve protein residues that are very highly conserved among all S-Lac lectins. These residues map to a single DNA exon, suggesting a carbohydrate binding cassette common to all S-Lac lectins.
Four experiments evaluated the efficacy of Na or Cl or their combination added to weanling pig diets that contained plasma protein and lactose on pig performance and N digestibility. The four experiments used a total of 563 crossbred pigs weaned at 22+/-1 d of age averaging 6.4 kg body weight. The basal diet in each experiment contained 5.8% plasma protein and 20% lactose and analyzed .20% Na and .23% Cl. In Exp. 1, NaCl was added to treatment diets at 0, .20, .40, or .60%. The trial was conducted for a 21 d period in a randomized complete block (RCB) design in seven replicates. Improved growth rates (P < .01) and gain:feed ratios (P < .01) occurred up to a dietary salt level of .40%. In Exp. 2, we evaluated the interaction of Na and Cl on pig performance. The experiment was a 2 x 2 factorial arrangement in a RCB design conducted in seven replicates. Total dietary Na was .20 or .36%, and Cl was included at .25 or .45%. Although there was a numerical increase in pig gains with added Na, the response was not significant (P > .15), but both gains (P < .01) and gain:feed ratios (P < .01) increased at the higher dietary Cl level. In Exp. 3, we evaluated the effect of five dietary levels of Cl added at .06% increments to a basal diet that analyzed .34% Na and .20% Cl on postweaning pig performance. The experiment was a RCB design conducted in eight replicates. A growth response (P < .01) to the .38% Cl level occurred during the initial 14-d postweaning period and to the .32% Cl level from 14 to 21 d. Gain:feed ratio increased each week with added Cl, but it was significant only for the period from d 0 to 7 d (P < .01). A N digestibility trial, using the diets of Exp. 3, constituted Exp. 4, and groups of three pigs per stainless steel metabolism crate were pair-fed to pigs fed the basal diet. The experiment was a RCB design conducted in three replicates over a 3-wk period. The results demonstrated a weekly decrease in fecal N (P < .01), no effect on urinary N (P < .15), improved N retention (P < .01), and an improved apparent N digestibility (P < .01) to the .38% dietary Cl concentration during the initial 2 wk postweaning. These experiments suggest that although plasma protein contributed Na and Cl to the initial diets of weaned pigs, additional Na and Cl, but particularly Cl, improved pig growth, N retention, and N digestibility. The results suggest a dietary minimum of .38% total Cl level during the initial 2 wk postweaning.
Forty-five neonatal pigs were used to compare the utilization of casein plus L-tryptophan (Try) and L-arginine (Arg) and of isolated soybean protein (ISP) plus DL--methionine (Met), L-lysine (Lys) and Try, with either glucose or lactose used as the carbohydrate source, from 1 to 29 days of age. A reference diet containing dried skim milk and soybean meal proteins was utilized. No interaction (P greater than .05) occurred between protein and carbohydrate sources. The diets containing lactose or glucose produced similar pig perforamnce, nutrient digestibility and serum levels of glucose, protein and urea nitrogen from 1 to 29 days of age. However, pigs fed the diet containing casein had higher (P less than .05) average daily gain and higher gain to feed ratios from 1 to 15 days of age than did pigs fed the diet containing ISP, while both groups were similar in performance from 15 to 29 days of age. Ether extract, crude protein, energy, ash and dry matter digestibilities and serum levels of protein were greater (P less than .05) for pigs fed the diets containing casein than for those fed the diets containing ISP. Nitrogen free extract digestibility and serum glucose and urea nitrogen were similar for pigs fed the diets containing casein or ISP. The diets that the pigs received from 1 to 29 days of age did not influence their subsequent nursery performance from 29 to 64 days of age, when all groups were fed the same diet. These results indicate that casein plus Try and Arg was superior to isolated soybean protein plus Met, Lys and Try as a source of protein for the baby pig from 1 to 15 days of age, while both protein sources produced similar perforamnce from 15 to 29 days of age.
1. 14C-Sulphamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide; 220 mg/kg of body weight) was given orally or i.v. to lactating dairy cows. Milk collected from 0-48 h after dosing accounted for 2.0% (oral dose) and 1.1% (i.v. dose) of the total 14C-activity administered. 2. Sulphamethazine accounted for 70-79% (oral dose) and 54-75% (i.v. dose) of the total 14C in milk samples collected from 0-48 h after dosing. N4-acetylsulphamethazine accounted for 1-2% (oral dose) and 1-4% (i.v. dose) of the 14C in milk. 3. The major 14C-labelled metabolite in the milk was isolated and identified as the N4-lactose conjugate of sulphamethazine, a unique type of metabolite not previously reported. This metabolite accounted for 10-14% (oral dose) and 9-20% (i.v. dose) of the 14C-activity in the milk collected from 0-48 h after dosing with 14C-sulphamethazine. 4. N4-lactose conjugates of sulphapyridine, sulphamerazine, sulphathiazole, sulphadimethoxine and sulphaquinoxaline were present in the milk from cows orally dosed with these five sulphonamide drugs.
The purpose of this investigation was to study the effects of lubricant concentration, dosator pressure, and dosator piston height on the properties of lactose plugs. An apparatus designed to simulate Macofar 13/2 capsule filling equipment was used, and the relationship between the variables and the responses was established using a Box-Behnken three-factor, three-level experimental design followed by multiple regression. Plug porosity, both under compression and after ejection, was found to be dependent on dosator pressure. Plug weight and length were dependent on dosator piston height, and uniformity of plug weight was independent of all the factors studied. Plug ejection pressure was dependent on dosator pressure and dosator piston height, even when ejection pressure was corrected for changes in plug length. Lubricant concentration had no significant effect on any plug property, and it must be concluded that 0.5% magnesium stearate provides adequate lubrication for lactose plugs. No interaction between variables was noted, and values of the variables raised to the power 2 had no significant effect, permitting a simplified experimental design to be adopted for future work.
A capillary electrophoresis (CE) method for the simultaneous separation of 11 metabolically important organic acids (oxalic, formic, citric, succinic, orotic, uric, acetic, pyruvic, propionic, lactic, and butyric), 10 amino acids (Asp, Glu, Tyr, Gly, Ala, Ser, Leu, Phe, Lys, and Trp), and lactose has been optimized, validated, and tested in dairy products. Repeatability and linearity were calculated for each compound, with detection limit values as low as 0.2 x 10(-2) mM for citric acid and Gly. The method was applied to analyze yogurt and different varieties of commercial cheeses. This method yielded specific CE patterns for different varieties of cheese. Also, it has been shown to be sensitive enough to measure small changes in composition of some of those compounds in fresh cheese stored under accelerated ripening conditions for 2 d at 32 degrees C (e.g., from 1728.3 +/- 45.0 to 1166.7 +/- 4.5 mg/100 g of DM in the case of lactose, or from 23.5 +/- 0.6 to 76.8 +/- 16.7 mg/100 g of DM in the case of acetic acid).
Abilities to accumulate beta-lactoglobulin and ortic acid were compared to lactose in dispersed cell cultures of lactating bovine mammary tissue. The inverse of the amount accumulated of each milk constituent at a given time in the culture medium was a linear function of the inverse of the cell density. The amount of lactose had no effect on its own subsequent accumulation, but added orotic acid and beta-lactoglobulin inhibited their own production. The accumulation of certain milk constituents in the culture medium is a factor in the expression and loss of normal function in the in vitro cultures which may be related to the observed effects of milk accumulation in vivo on the rate of milk synthesis and mammary involution.
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.
The objectives of this work were 1) to examine the responsiveness of SCC, lactose concentration, and NAGase activity in milk to changes in bacteriological status and 2) to develop models for predicting bacteriological status of mammary glands. Data included 550 cows in 10 commercial herds. Natural logarithm NAGase and log cell count were most responsive to changes in bacterial status. The log NAGase was relatively more effective in identifying major from minor pathogen infections, whereas log SCC was better able to differentiate between infected and uninfected classes. Non-transformed NAGase, SCC, and lactose were considerably less responsive to infection status. Logistic regression of bacterial status on herd, lactation number, milk, log SCC, log NAGase, and stage of lactation was performed. The least significant variables were removed in a stepwise process. Final predictors of infection status were herd, log SCC, and log NAGase. The role of log SCC was to discriminate infection from no infection, whereas log NAGase discriminated major from minor pathogens. The log NAGase, alone or in combination with log SCC, added substantially to the detection power of the model. Chi-square goodness of fit tests found no significant differences between observed and predicted infection probabilities. Substitution of herd averages for log SCC and log NAGase for the herd variables resulted in significant differences between predicted and observed herd infection probabilities.
The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.