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Supercritical fluid processing of materials from aqueous solutions: the application of SEDS to lactose as a model substance.

PURPOSE: The objective of the work was to study the factors influencing the crystallisation of alpha-lactose monohydrate from aqueous solution using the Solution Enhanced Dispersion by Supercritical Fluids (SEDS) technique. METHODS: An aqueous solution of alpha-lactose monohydrate is dispersed with a homogeneous mixture of carbon dioxide-ethanol/methanol using a co-axial nozzle. Crystallised lactose particles were analysed for water content by Karl-Fisher analysis, anomeric composition by Differential Scanning Calorimetry (DSC) and characterised for crystallinity by powder X-ray diffraction and morphology by scanning electron microscopy. RESULTS: Water content in the lactose recrystallised with ethanol was higher compared to the product obtained with methanol as cosolvent. Rate of crystallisation could be altered by varying the CO2 flow thereby modifying the water content in the lactose. At low flow rates of CO2, the crystallisation occurred in a cosolvent rich antisolvent phase causing rapid crystallisation whereas high flow rates of CO2 favoured a much slower crystallisation mechanism in the water rich phase. As a consequence, the morphology changed from thin long bands to large agglomerated chunks with mean particle size between 5 and 31 microns. CONCLUSIONS: The SEDS process is an efficient method for forming micron sized particles of water-soluble compounds with controlled physico-chemical properties.

Calorimetry, Differential Scanning↗

Effects of basolateral amygdala lesions on taste aversions produced by lactose and lithium chloride in the rat.

In Experiment 1, intact rats were given either lactose or sucrose solutions. Although on first exposure they readily consumed lactose, its ingestion produced a conditioned taste avoidance which was partly extinguished by repeated sucrose exposure after lactose conditioning. In Experiment 2, rats with large bilateral electrolytic lesions of the basolateral amygdala and those with either sham or no operations were given two pairings of saline with LiCl injections (upper gastrointestinal tract discomfort) and in a separate condition access to high levels of lactose (lower gastrointestinal tract discomfort). Conditioned taste avoidances were measured both by two-bottle tests and by video recordings of the rats' orofacial and somatic responses. The lesions attenuated LiCl-induced taste aversion but not lactose-induced taste avoidance, results demonstrating that taste avoidance can occur without the basolateral amygdala. The results suggested that aversions based on distaste can be distinguished from avoidances based on danger, not only in terms of orofacial responses but also in terms of their neuroanatomical substrate.

Amygdala↗

Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase.

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.

Animals↗

The lactose and neuraminlactose content of rat milk and mammary tissue.

Specific analyses of rat milk sugar revealed lactose and N-acetylneuraminyl-lactose. The latter showed multiple, interconvertible forms in certain paper-chromatographic systems. Mean lactose concentrations rose from 33.1mumol/ml at day 0 to 100mumol/ml at day 20 of lactation. Mean N-acetylneuraminyl-lactose concentrations rose from 7.1mumol/ml at day 0 to 25.9mumol/ml at day 4 and thereafter declined, decreasing to zero by day 20 of lactation. Similar data are given on the concentrations of these sugars in mammary tissue of rats. Neuraminyl-lactose was also detected in mouse mammary tissue.

Animals↗

Diurnal variation and response to food withdrawal of lactose synthesis in lactating rats.

1. The incorporation of radiolabelled plasma glucose into mammary lactose was used to measure the rate of lactose synthesis in lightly anaesthetized lactating rats. 2. Lactose synthesis showed a diurnal variation with a minimum at 18:00h 3. Food withdrawal for 6h did not affect lactose synthesis in the early morning but greatly decreased it in the afternoon or evening. 4. Plasma glucose, milk sugars and total galactosyltransferase activity (EC 2.4.1.22) did not show the above changes. 5. Measurements of plasma insulin, which varies diurnally, and experiments with injected insulin suggested that variations of insulin within the physiological range do not account for the changes in lactose synthesis described.

Animals↗

Magnesium balance in adult cats fed a dry food rich in lactose.

The question addressed was whether lactose consumption would stimulate apparent magnesium absorption in adult cats. In a cross-over study, eight cats were fed dry diets without or with 10% lactose, which was added at the expense of the glucose component. Urine and faeces were quantitatively collected and the balance of magnesium, calcium and phosphorus was determined. Lactose feeding did not affect mineral absorption. The dry matter content of faeces was unchanged after lactose consumption. Urinary excretion of galactose represented 0.2% of the intake. It is concluded that lactose ingestion at a level of 1.2 g/kg body weight has no effect on mineral absorption and on the consistency of faeces in adult cats.

Animal Feed↗

Isolation of a Xanthomonas campestris strain with elevated beta-galactosidase activity for direct use of lactose in xanthan gum production.

AIMS: To isolate a Xanthomonas campestris strain that can use lactose directly for xanthan gum production. METHODS AND RESULTS: The presence of indigenous beta-galactosidase gene in the wild-type Xc17 was detected by PCR and Southern hybridization. Treatment of Xc17 with nitrous acid resulted in the isolation of Xc17L with a 3.5-fold elevation of beta-galactosidase activity capable of growing in lactose-based medium. Xc17L is stable for at least 100 generations in terms of beta-galactosidase expression. The amounts of xanthan produced by Xc17L in lactose-based medium are comparable to those in glucose-based medium. CONCLUSIONS: Xc17L is potentially useful for xanthan production from whey, a waste containing lactose. SIGNIFICANCE AND IMPACT OF THE STUDY: A lactose-utilizing strain of X. campestris strain can be constructed without incorporation of any exotic DNA or antibiotic resistance gene and therefore concern of a gene-modified organism and fear of a spread of an antibiotic-resistant gene are avoided.

Bacteriological Techniques↗

Microbial flora in the digestive tract and action of lactose on mineral metabolism.

Germ-free (GF) and conventional (CV) adult rats were given for 4 weeks a semi-synthetic diet containing 10 p. 100 lactose (L) or no lactose (LO). The axenic state had an unfavourable effect on sodium and potassium absorption. On the contrary, it increased the absorption and retention of calcium, phosphorus and magnesium. The favourable effect of lactose on magnesium metabolism was not different in GF and CV rats. On the contrary, lactose increased the absorption and retention of phosphorus and calcium more in GF than in CV animals. It augmented the amount of calcium present in a soluble, ultrafiltrable form in the caecum of only the GF rats. These results are discussed and compared to previous data on younger rats. They lead to the hypothesis that the voluminous caecum of the GF rat is the site of a calcium absorption which is accentuated by presence of lactose in the diet.

Animals↗

Production of volatile fatty acids as a result of bacterial interactions in the cecum of gnotobiotic rats and chickens fed a lactose-containing diet.

Volatile fatty acid (VFA) productions from lactose and lactate by a Clostridium butyricum and a Veillonella alcalescens strain, alone or in combination with a Lactobacillus acidophilus strain, were determined both in vitro in culture media and in vivo in the ceca of gnotobiotic animals. Gnotobiotic rats, which possess intestinal lactase, and chickens, which are devoid of it, were used. Both animal species were fed a diet containing 4% lactose. The in vitro results showed that the C. butyricum strain fermented lactose and D-lactic acid to butyric and acetic acids, whereas L-lactic acid was not fermented. The V. alcalescens strain did not ferment lactose and fermented L better than D-lactic acid. The in vivo results showed that high VFA concentrations were obtained in the ceca of chickens either disassociated with V. alcalescens or C. butyricum and Lactobacillus strains or monoassociated with C. butyricum. VFA concentrations in the ceca of rats were low, whatever strain the rats harbored. In addition, an antagonistic effect of the C. butyricum strain against the Lactobacillus strain was evidenced both in rats and chickens. It is suggested that the absence of a host lactase makes the chick a good model for lactose intolerance studies in human newborns.

Anaerobiosis↗

Metabolic effects of dietary lactose in adult female rats.

As an outgrowth of our interest in the potential toxicity of dietary galactose, we investigated the metabolic effects of high lactose diets in Long-Evans female rats. Seventy-five Long-Evans female rats (25-day-old) were randomized to receive one of 3 diets for 7 months: glucose diet (CON); low lactose diet (10.5%, LLD); or a high lactose diet (41.9%, HLD). Necropsy was performed seven months after randomization. HLD animals had significantly lower body weights than controls (P < 0.01). These animals continued to grow, however at a retarded rate compared to the CON group. The HLD group also had significantly lower triglyceride and non-esterified fatty acid levels than the CON group (P < 0.01 and P < 0.05). Serum glucose concentrations were lower in the HLD group compared to CON animals (P < 0.05), while serum insulin levels were lower than both the LLD and CON animals (P < 0.01 and P < 0.05). Leptin exhibited a similar trend. Thyroid studies revealed no difference in TSH between groups. Free T4 was significantly higher in HLD rats compared to LLD and CON rats while free T3 was lower in the HLD group (P < 0.05). This indicates a possible impairment in T4 to T3 conversion. Our data suggests that a long-term high lactose diet is associated with a decrease in insulin and leptin levels, and an increase in the insulin to glucose ratio. However, these changes are seen in the presence of a decreased body mass. A significant effect on thyroid hormone metabolism is also seen, and may be an adaptive mechanism in lactose-fed rats.

Animal Feed↗

Hormones, IgG and lactose changes around parturition in plasma, and colostrum or saliva of multiparous sows.

Blood, colostrum and saliva samples were serially taken from 6 multiparous sows from day 109 of gestation until day 3 postpartum. Plasma was assayed for oestradiol-17beta (E2), progesterone (P4), prolactin (PRL), cortisol, immunoglobulin G (IgG) and lactose. Colostrum was assayed for E2, P4, IgG and lactose. Lactoserum, obtained after ultra centrifugation of colostrum, was assayed for PRL. Saliva was assayed for cortisol. Time-related variations in hormone, IgG and lactose concentrations measured in plasma were parallel to those measured in colostrum, lactoserum or saliva. However, the concentrations were higher in colostrum or lactoserum and lower in saliva than in plasma. Ratios of concentrations of cortisol in saliva and PRL in lactoserum over those in plasma did not vary with time and averaged 0.2 and 1.6, respectively. Conversely, the ratios of concentrations of E2 and P4 in colostrum over those in plasma varied with time (P < 0.05) but were quite constant before the end of parturition, averaging 2.7 and 3.6, respectively. The ratios of concentrations of IgG and lactose in colostrum over those in plasma also varied with time (P < 0.05). The concentrations of hormones in plasma on the one hand and in colostrum, lactoserum or saliva on the other hand were significantly correlated but correlations varied with time (PRL across periods: r = 0.31; cortisol across periods: r = 0.60; E2 during parturition: r = 0.83; P4 before parturition: r = 0.82; P4 during parturition: r = 0.67). The present results indicate that around parturition, assays of hormones in colostrum or saliva can be used to study the hormonal status of sows. Furthermore, variations in colostrum and plasma concentrations of IgG and lactose are good indicators of the transition from colostrum to milk synthesis.

Animals↗

Lactose malabsorption and intolerance and peak bone mass.

BACKGROUND & AIMS: Lactose malabsorption per se is not associated with alterations of bone mineral density (BMD) or calcium intake, but when intolerance symptoms are present a lower calcium intake and reduction of BMD values are evident. The purpose of this study was to evaluate whether lactose intolerance interferes with the achievement of an adequate peak bone mass in young adults. METHODS: Of 103 enrolled healthy subjects, 55 proved to be lactose malabsorbers with H(2) breath test after lactose administration, and 29 of them experienced intolerance symptoms (diarrhea, abdominal pain, bloating, flatulence). Lumbar and femoral BMD by dual-energy X-ray absorptiometry was measured, and calcium intake and biochemical indices of bone and mineral metabolism were evaluated. RESULTS: Lumbar and femoral BMD, calcium intake, and mineral metabolism did not differ between malabsorbers and absorbers, although among malabsorbers, intolerant subjects showed significant alterations of all these parameters in comparison with tolerant subjects. A strict correlation was evident between BMD values and both severity of symptoms and calcium intake and between calcium intake and severity of symptoms. CONCLUSIONS: Lactose intolerance prevents the achievement of an adequate peak bone mass and may, therefore, predispose to severe osteoporosis.

Absorptiometry, Photon↗

Recurrent abdominal pain of childhood due to lactose intolerance.

The role of lactose malabsorption was studied prospectively in 80 schoolchildren with recurrent abdominal pain. Malabsorption was documented in 40 per cent (16 of 59 whites, 12 of 16 blacks and four of five Hispanic children) on the basis of elevated levels of hydrogen in their breath. Those with lactose malabsorption, however, were not clinically distinguishable on the basis of past milk ingestion (P greater than 0.05), weekly pain frequency (median, five vs. six times), presence of diarrhea (40 vs. 27 per cent) or symptom response to lactose load. In children with malabsorption who completed a six-week diet trial, 70 per cent reported increased frequency of pain (P less than 0.002) when placed on their usual lactose-containing diet. Lactose malabsorption has a substantial role in the symptoms of children with recurrent abdominal pain, and it should be considered before performing invasive procedures or assuming a psychogenic origin.

Abdomen↗

Familial aquagenic urticaria associated with familial lactose intolerance.

Aquagenic urticaria is a rare disorder characterized by the occurrence of pruritus and wheals after temporary contact with water. The familial occurrence of aquagenic urticaria over 3 generations is reported here in association with familial lactose intolerance, a condition in which the enzyme lactase encoded on chromosome 2, is deficient. In two patients, a young man and his mother, we verified the appearance of pruritic hives 5 to 10 minutes after contact with water of any temperature. Other types of physical urticaria were absent, and mastocytosis was excluded by extensive laboratory investigations; lactose intolerance was confirmed in both patients by H(2)-exhalation test. In these patients the clinical symptoms did not respond to antihistamines or UV-radiation therapy. Four other members of the family had wheals from water contact, two of whom had lactose intolerance. Two other members had lactose intolerance only. Although the association of aquagenic urticaria with lactose intolerance may be coincidental, attention is drawn to the fact that the 2 conditions, known to be familial, may coexist in the same family, possibly based on an association of gene loci.

Adult↗

Unidirectional reconstitution into detergent-destabilized liposomes of the purified lactose transport system of Streptococcus thermophilus.

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl-beta-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (approximately 5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-beta-D-glucoside. For reconstitutions mediated by n-dodecyl-beta-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Amino Acid Sequence↗

Alanine insertion scanning mutagenesis of lactose permease transmembrane helices.

A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of approximately 2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.

Alanine↗

Influence of lactose on phosphate metabolism in rats.

1. Lactose, sucrose or maltose were administered to rats by gavage together with 32PO4, and blood 32P was determined. 2. PO4 uptake into blood was increased only when lactose and PO4 were administered into the gut. 3. Weaned male rats were fed on a control diet or a diet containing 30, 60 or 120 g lactose/kg. After a 5 d period of adaptation, a 6 d P balance was carried out. After a further 24 d blood levels of PO4, and calcium and PO4 uptake by brush-border membrane vesicles prepared from the renal cortex, were determined. 4. The absorption and retention of P were higher in lactose-fed rats. Faecal P excretion decreased with increasing dietary lactose content. Urinary P excretion was lower in the rats fed on 30 and 60 g lactose/kg whereas the 120 g lactose/kg diet increased urinary P. 5. Membrane vesicles isolated from rats fed on the diets containing 30 and 60 g lactose/kg showed a higher uptake of PO4, but with the 120 g lactose/kg diet uptake was lower than with membrane vesicles isolated from rats fed on the control diet. 6. A statistically significant increase in the serum PO4 level was observed in the rats fed on the diet with 120 g lactose/kg. 7. The presence of lactose in the diet caused a stimulatory effect on PO4 absorption and retention.

Animals↗

Effect of the microbial lactase (EC 3.2.1.23) activity in yoghurt on the intestinal absorption of lactose: an in vivo study in lactase-deficient humans.

Breath hydrogen excretion was measured in eight lactase (EC 3.2.1.108)-deficient volunteers ingesting 18 g lactose in the form of milk, yoghurt and heated yoghurt. Total excess hydrogen excretion (area under curve) was significantly lower after yoghurt and heated yoghurt, than after milk: 103 (SE 29), 191 (SE 32), and 439 (SE 69) respectively (P less than 0.001). The oro-caecal transit time of fermentable components from yoghurt and heated yoghurt (mainly lactose) was longer than that from milk: 165 (SE 17), 206 (SE 19), v. 103 (SE 19) min (P less than 0.01). An intestinal perfusion technique was used in the same subjects after ingestion on two consecutive days of 18 g lactose in yoghurt and heated yoghurt. Significantly less lactose was recovered from the terminal ileum after yoghurt than after heated yoghurt meals: 1740 (SE 260) v. 2825 (SE 461) mg (P less than 0.05), and approximately one-fifth of the lactase activity contained in yoghurt reached the terminal ileum. These findings indicate that more than 90% of the lactose in yoghurt is digested in the small intestine of lactase-deficient subjects and suggest that both the lactase activity contained in the viable starter culture and a slow oro-caecal transit time are responsible for this excellent absorption.

Adult↗