Research Article
Effects of sodium fertilizers and supplements on milk production and mammary gland health
- CLIVE J. C. PHILLIPS, PAUL C. CHIY, DAVID R. ARNEY, OLAV KÄRT
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- 01 February 2000, pp. 1-12
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A series of experiments was conducted to investigate the effects of sodium fertilizers and supplements on the milk production and mammary health of dairy cows. In Expt 1, where sodium fertilizer was applied to productive pastures consisting mainly of the natrophile perennial rye-grass, the herbage sodium content and the milk yield of cows was increased and milk somatic cell count (SCC) reduced. In Expt 2, which used pastures containing less productive, natrophobic grasses and broad-leaved plants in Estonia, sodium fertilizer did not increase herbage sodium content and did not affect milk production or composition. In Expt 3 the sodium content of the diet of individually tethered cows was increased from 1 to either 6 or 11 g/kg dry matter (DM) by adding salt to their restricted feed allowance, and the cows' milk yield was increased by the high level of sodium supplement and milk SCC were reduced by both levels of sodium supplement. The calcium and magnesium status of cows was improved by the sodium supplement. In Expt 4 a low level of supplementary salt was included in the ration of tethered cows to increase the sodium content of the diet from 2 to 3·6 g/kg DM. No effects on milk yield or SCC were found, but the sodium supplement reduced Staphylococcus aureus contamination of the milk, but not the proportion of milk samples infected with Escherichia coli. It was concluded that the optimum dietary sodium concentration for maximum milk yield was greater than the published requirements, and that substantial increases in sodium intake above current requirements also reduced milk SCC.
Effects of morphine and naloxone on the release of oxytocin and on milk ejection in dairy cows
- VLADIMIR TANČIN, WOLF-DIETER KRAETZL, DIETER SCHAMS
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- 01 February 2000, pp. 13-20
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The aim of this study was to investigate the action of opioids (the μ receptor agonist morphine) and the antagonist naloxone on inhibition of oxytocin release and milk let-down in response to milking in dairy cows. In the first experiment, cows were injected with 0, 21, 70 and 210 mg morphine 10 min before milking on four successive days. Plasma oxytocin levels after 1 min manual stimulation of the udder were reduced by 70 and 210 mg morphine, and milk let-down was inhibited at the latter dose. In the second experiment, cows were injected after a control milking with 210 mg morphine (or 350 mg at 10 min before milking the following day if not effective) to inhibit milk flow. On the following day the inhibiting dose of morphine was given with 210 mg naloxone. Naloxone injection given before morphine had no effect on plasma oxytocin concentrations, but abolished the inhibition of oxytocin release by morphine and potentiated oxytocin release in response to milking. Naloxone alone injected the day after control milking increased oxytocin levels during milking, suggesting involvement of the opioid system in milking. A model has been developed for the control of opioid effects during milking. Morphine suppressed oxytocin release during milking in a dose-dependent manner and the effect was reversible by naloxone.
Heterogeneity of cationic amino acid transport systems in mouse mammary gland and their regulation by lactogenic hormones
- REKHA SHARMA, VINOD K. KANSAL
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- 01 February 2000, pp. 21-30
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The mechanism of cationic amino acid transport in lactating mouse mammary gland was investigated. Two Na+-independent systems of arginine transport were discriminated on the basis of their sensitivity to leucine. The leucine-sensitive uptake of arginine (Km 0·4 mM) was through a broad specificity system that interacted with both cationic and neutral amino acids, and was inhibited by preloading mammary tissue with neutral amino acids. The leucine-insensitive uptake was identified as the y+ system (Km 0·76 mM). Preloading mammary tissue with cationic amino acids increased the uptake of arginine by the y+ system. Decreasing the pH of the external medium to 6·0 suppressed the y+ system-mediated uptake by ∼25%, whereas the broad specificity system remained unaffected. Lactogenic hormones upregulated the y+ system-mediated uptake of arginine in pregnant mouse mammary tissue cultured in vitro, although the broad specificity system remained unaffected. The y+ system-mediated uptake increased 2-fold with insulin alone and 4-fold with the combination of insulin, cortisol and prolactin.
High-pressure treatment of milk: effects on casein micelle structure and on enzymic coagulation
- ERIC C. NEEDS, ROBERT A. STENNING, ALISON L. GILL, VICTORIA FERRAGUT, GILLIAN T. RICH
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- 01 February 2000, pp. 31-42
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High isostatic pressures up to 600 MPa were applied to samples of skim milk before addition of rennet and preparation of cheese curds. Electron microscopy revealed the structure of rennet gels produced from pressure-treated milks. These contained dense networks of fine strands, which were continuous over much bigger distances than in gels produced from untreated milk, where the strands were coarser with large interstitial spaces. Alterations in gel network structure gave rise to differences in rheology with much higher values for the storage moduli in the pressure-treated milk gels. The rate of gel formation and the water retention within the gel matrix were also affected by the processing of the milk. Casein micelles were disrupted by pressure and disruption appeared to be complete at treatments of 400 MPa and above. Whey proteins, particularly β-lactoglobulin, were progressively denatured as increasing pressure was applied, and the denatured β-lactoglobulin was incorporated into the rennet gels. Pressure-treated micelles were coagulated rapidly by rennet, but the presence of denatured β-lactoglobulin interfered with the secondary aggregation phase and reduced the overall rate of coagulation. Syneresis from the curds was significantly reduced following treatment of the milk at 600 MPa, probably owing to the effects of a finer gel network and increased inclusion of whey protein. Levels of syneresis were more similar to control samples when the milk was treated at 400 MPa or less.
Purification of goat β-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor
- FRÉDÉRIC SANNIER, STÉPHANIE BORDENAVE, JEAN-MARIE PIOT
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- 01 February 2000, pp. 43-51
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This paper presents a novel contribution to the purification of goat β-lactoglobulin by using an ultrafiltration membrane enzymic reactor. The basis of the purification process was the enzymic hydrolysis of contaminating proteins, α-lactalbumin and traces of serum albumin, by pepsin at 40 °C and pH 2, conditions under which β-lactoglobulin is resistant to peptic digestion. Simultaneously, β-lactoglobulin and peptides were separated by ultrafiltration. β-Lactoglobulin was retained in the reactor while peptides generated by hydrolysis from α-lactalbumin and serum albumin permeated through the membrane. The process was made continuous by the addition of fresh whey to replace the lost permeate. Three mineral membranes with 10, 30 and 50 kDa molecular mass cut-off were tested and the 30 kDa membrane was selected for the continuous process. The simultaneous purification and concentration of β-lactoglobulin from clarified goats' whey was achieved in a single step. The ultrafiltration membrane enzymic reactor could treat eight reactor volumes of clarified whey. The recovery of β-lactoglobulin was 74%, its purity was 84% and its concentration 6·6-fold that in the initial clarified whey.
Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides
- ANNE PIHLANTO-LEPPÄLÄ, PÄIVI KOSKINEN, KATI PIILOLA, TUOMO TUPASELA, HANNU KORHONEN
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- 01 February 2000, pp. 53-64
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The aim of this study was to identify whey-derived peptides with angiotensin I-converting enzyme (ACE) inhibitory activity. The bovine whey proteins α-lactalbumin and β-lactoglobulin were hydrolysed with pepsin, trypsin, chymotrypsin, pancreatin, elastase or carboxypeptidase alone and in combination. The total hydrolysates were fractionated in a two step ultrafiltration process, first with a 30 kDa membrane and then with a 1 kDa membrane. Inhibition of ACE was analysed spectrophotometrically. The peptides were isolated by chromatography and identified by mass and sequencing analysis. The most potent inhibitory peptides were synthesized by the 9-fluorenylmethoxycarbonyl solid phase method. Inhibition of ACE was observed after hydrolysis with trypsin alone, and with an enzyme combination containing pepsin, trypsin and chymotrypsin. Whey protein digests gave a 50 % inhibition (IC50) of ACE activity at concentration ranges within 345–1733 μg/ml. The IC50 values for the 1–30 kDa fractions ranged from 485 to 1134 μg/ml and for the <1 kDa fraction from 109 to 837 mg/ml. Several ACE-inhibitory peptides were isolated from the hydrolysates by reversed-phase chromatography, and the potencies of the purified peptide fractions had IC50 values of 77–1062 μM. The ACE-inhibitory peptides identified were α-lactalbumin fractions (50–52), (99–108) and (104–108) and β-lactoglobulin fractions (22–25), (32–40), (81–83), (94–100), (106–111) and (142–146).
Inability of dairy propionibacteria to grow in milk from low inocula
- PASCAL PIVETEAU, SÉAMUS CONDON, TIMOTHY M. COGAN
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- 01 February 2000, pp. 65-71
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Growth of propionibacteria in complex media was independent of the initial number of cells; in contrast, growth of propionibacteria in milk and whey did not occur if the initial level of cells was <106 cfu/ml. Addition of vitamins, minerals or complex nitrogen sources to the milk or whey, or incubation under anaerobic conditions had no effect on the lack of growth. Addition of freeze-dried whey, prepared from skim milk reconstituted from powder, to a complex medium prevented growth from low inocula in the complex medium, demonstrating the presence of an inhibitor or inhibitors in the whey. The inhibitor(s) was heat stable, had a low molecular mass and retained its activity for at least 4 weeks at 20 °C. Pregrowth of some lactic acid bacteria, used as starter cultures in Swiss-type cheese manufacture, in milk for 2 weeks at 20 °C removed the inhibition, which explains how propionibacteria develop in Swiss-type cheese from low numbers even though they are inhibited in milk.
Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet
- ELSAYED I. ELAGAMY
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- 01 February 2000, pp. 73-81
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Camel calf rennet (CCR) and buffalo calf rennet (BCR) were prepared from dried abomasa to study their physicochemical properties and electrophoretic behaviour and to carry out an immunological characterization of the rennet proteins. CCR was more thermostable than BCR. The milk clotting activity of both rennets increased as pH decreased. The optimum temperatures for CCR and BCR were 50 and 45 °C respectively. CCR was more sensitive to increased CaCl2 in milk than BCR. Addition of NaCl to milk in the range 0–100 g/l resulted in a marked decrease in the clotting activity of both rennets. When the rennets were treated with acetone, the activity of BCR was completely destroyed, but that of CCR was unaffected. The proteolytic activity of CCR was higher than that of BCR and pepsin towards both camel and cows' milk caseins at pH 6·0. SDS-PAGE electrophoretic patterns of CCR and BCR proteins gave two major bands with molecular masses estimated as 52 and 39 kDa for CCR and 50 and 35 kDa for BCR. Immunodiffusion and immunoelectrophoresis using anti-CCR serum demonstrated immunological cross reactivity between CCR and BCR.
Method of quantifying the loss of acidification activity of lactic acid starters during freezing and frozen storage
- FERNANDA FONSECA, CATHERINE BÉAL, GEORGES CORRIEU
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- 01 February 2000, pp. 83-90
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We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (Δtm) between the tm values of the concentrated cell suspension before and after freezing. During storage at −20 °C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9–14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.
Application of capillary electrophoresis to the characterization of processed cheeses
- BEATRIZ MIRALLES, MERCEDES RAMOS, LOURDES AMIGO
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- 01 February 2000, pp. 91-100
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Capillary electrophoresis using a hydrophilically coated capillary and a low pH buffer containing urea has been used to characterize processed cheeses. Different electrophoretic patterns were obtained depending on the ingredients used in the blend such as acid casein, rennet casein, sodium and calcium caseinates and skim milk powder. Isoelectric casein, and sodium and calcium caseinates were shown to contain intact non-glycosylated κ-casein (κ-CN), while rennet casein contained only trace amounts of κ-CN and mainly para-κ-CN. Therefore, the addition of casein or caseinate to processed cheeses has been detected by analysing the intact non- glycosylated κ-CN. Quantitation of intact non-glycosylated κ-CN in processed cheeses of known and unknown composition was carried out using a regression curve from standard mixtures of 150–550 g isoelectric casein/kg total rennet casein. This capillary electrophoresis method successfully confirmed the addition of isoelectric casein or caseinate to processed cheeses of known composition. The quantitative determination range was 0·605–3·688 mg κ-CN/ml. This method cannot be used for measuring additions of rennet casein or any caseinates that have been exposed to chymosin.
SHORT COMMUNICATION
Ultrasound technique for measuring mammary cistern size of dairy ewes
- ANNA NUDDA, GIUSEPPE PULINA, RITA VALLEBELLA, ROBERTA BENCINI, GIUSEPPE ENNE
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- 01 February 2000, pp. 101-106
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Sarda ewes, ∼4·5 million animals producing 500 000 tonnes milk annually, are one of the most important breeds of dairy sheep in the Mediterranean area. Several studies (Casu & Labussière, 1972; Labussière et al. 1981; Gallego et al. 1983; Rebello de Andrade et al. 1989; Bencini, 1993) have shown that milk production is influenced by mammary gland size and cistern dimension. The size of the mammary cistern affects both milk secretion rate and milk emission kinetics during milking.
Milk secretion rate is controlled at the mammary gland level mainly by a protein feedback inhibitor of lactation (FIL), which is produced by mammary epithelial cells and secreted together with milk into the alveoli (Wilde & Peaker, 1990). As the alveoli are the site of action of the FIL (Henderson & Peaker, 1984), the FIL affects the rate of secretion when the milk is stored in the secretory tissue, whereas it is inactive in the milk stored in the cistern. As a consequence, the action of the FIL should be less in animals with a greater cistern volume, because a large proportion of milk is stored in the mammary cistern and the time during which the milk is in contact with the alveoli is reduced. This hypothesis is supported by the finding that the milk production of cows (Dewhurst & Knight, 1992; Knight & Dewhurst, 1992, 1994) and sheep (Karam et al. 1971; Enne et al. 1972) with large cistern storage capacities was almost unaffected by changes in the frequency of milking.
Cistern volume also affects milk emission kinetics and the proportion of stripped milk obtained at milking (Labussière, 1988). Cisternal milk is immediately available for removal, whereas alveolar milk is available only after operation of the ejection reflex, necessary in dairy ewes for complete udder emptying (Bruckmaier et al. 1997). Therefore, in animals readily able to expel alveolar milk into the cistern before the whole cisternal milk fraction is removed (Pazzona et al. 1978; Bruckmaier et al. 1997), a larger cistern volume enables milking time to be reduced by eliminating or restricting the need for stripping.
On the basis of the above considerations, the volume of the mammary gland cistern could be proposed as a selection objective to improve milk production and milking ability of dairy ewes. For this purpose, a quick, accurate and economic method for measuring it is needed. The ultrasound technique allows the internal structure of the mammary gland cistern to be observed clearly in sheep (Ruberte et al. 1994; Pulina et al. 1996; Bruckmaier et al. 1997), cows (Bruckmaier et al. 1994b) and goats (Bruckmaier et al. 1994a). Cistern size has been measured by ultrasound in dairy cows (Bruckmaier et al. 1994b) and sheep (Pulina & Nudda, 1996), where a positive correlation between milk yield and cistern area calculated from the ultrasound images of mammary glands was found. However, area estimation requires the use of expensive ultrasound equipment or of a digitizing tablet. In both cases, area measurement is difficult owing to the irregular shape of the cistern.
The aim of this study was to test the use of linear measurements taken directly from ultrasound images to estimate cistern size in dairy ewes.
Expression polymorphism of κ-casein gene in Holstein cows
- GILLES ROBITAILLE, DENIS PETITCLERC
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- 01 February 2000, pp. 107-111
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Several studies have reported associations between κ-casein (κ-CN) genetic variants A and B and milk composition and properties (for review, see Ng-Kwai- Hang, 1998). For instance, genetic variant κ-CN B was associated with higher protein, casein and κ-CN contents. The objective of the present study was to demonstrate a differential expression of κ-CN genetic variants A and B at the mRNA level in mammary epithelial cells during lactation.
Distribution of nitrogen in goats' milk and use of capillary electrophoresis to determine casein fractions
- ANTONIA GARCÍA-RUIZ, ROSINA LÓPEZ-FANDIÑO, LUCIDIA LOZADA, JAVIER FONTECHA, MARÍA J. FRAGA, MANUELA JUÁREZ
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- 01 February 2000, pp. 113-117
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The last few years have seen a number of advances in the understanding of genetic polymorphisms of caprine caseins, and especially in molecular characterization of the allelic variants and analysis of their frequencies in Spanish and other breeds (Grosclaude et al. 1994; Jordana et al. 1996). Although more is being discovered about the influence of these polymorphisms on the yield and characteristics of cheeses (Remeuf, 1993; Pirisi et al. 1994), little work has been done on the quantitation of different casein fractions of goats' milk throughout lactation. Measurements have been made using SDS-PAGE (Quiles et al. 1990) and cation- exchange FPLC (Brown et al. 1995), and Recio et al. (1997a) have demonstrated the potential of capillary electrophoresis (CE) for the analysis and quantitation of milk proteins. Use of CE has resulted in the development of rapid automated analysis with very high resolution, requiring very small sample and buffer volumes and with a significantly reduced amount of solvent waste.
The aim of the present study was to determine the influence of herd and milking period on the contents of the various nitrogen and casein fractions, the latter being determined by CE, in milk from goats of the Murciana-Granadina breed.
Microbial resistance of caseinate films crosslinked by gamma irradiation
- MOHAMED RESSOUANY, CAROLINE VACHON, MONIQUE LACROIX
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- 01 February 2000, pp. 119-124
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The need to improve the shelf life and quality of foods, combined with the urgency to reduce waste generated by synthetic non-biodegradable packaging, have resulted in an increasing interest in biodegradable or edible materials (Chen, 1995). The challenge in the development of such materials lies in achieving controlled lifetime together with compatibility with foods and the environment. Natural polymers or polymers derived from natural monomers such as food proteins offer the greatest opportunities, since their biodegradability and environmental compatibility are assured (Krochta & de Mulder-Johnston, 1997).
Milk proteins such as caseins have been formulated into coatings and films for improving food quality (Baker et al. 1994). Unfortunately, the highly hydrophilic nature of these proteins limits the possibility of producing films with the required properties. Gamma irradiation, which induces the formation of crosslinks in the protein structure, is known to improve the mechanical properties and water resistance of such materials (Brault et al. 1997; Mezgheni et al. 1998a; Ressouany et al. 1998), and can also sterilize materials for possible biomedical applications (Kaetsu, 1995).
Gamma irradiation is slowly becoming accepted in the food industry as a means of safely improving the shelf life of various fruits and vegetables and eliminating bacterial contamination in meats (Pszczola, 1997). All these features (sterility, increased mechanical strength, controlled life time) make gamma irradiation a promising technique for the production of commercial biodegradable films.
In the present study, we have investigated the microbial resistance of calcium caseinate films irradiated at 4 or 64 kGy. A minimum dose of 4 kGy was required to produce complete sterility in the films without inducing many crosslinks in the protein. A dose of 64 kGy was used to induce maximum crosslinking density (Ressouany et al. 1998). The rate of modification of the films was evaluated using three methods. Firstly, microbiological counts were performed on films incubated with a strain of Pseudomonas aeruginosa. This strain was chosen because it produces a wide range of proteinases and is often implicated in the process of food spoilage. These results were compared with those obtained for soluble N. Since conversion to CO2 is one of the standard test procedures to assess biodegradability (El-Din Sharabi & Bartha, 1993), we also analysed the CO2 produced by the biodegradation of the crosslinked films using a modified version of a standard test procedure (American Society for Testing Materials, 1992). This measures the aerobic degradation of plastic materials inoculated with municipal sewage sludge. We have replaced the sludge by a concentrated bacterial strain (Ps. aeruginosa) and compared these results with the two previous methods. This last method was used to measure the resistance of the crosslinked films to bacterial degradation.
In vitro digestion of caseinophosphopeptide–iron complex
- NABIL AÎT-OUKHATAR, SAÏD BOUHALLAB, FRANÇOIS BUREAU, PIERRE ARHAN, JEAN-LOUIS MAUBOIS, DOMINIQUE L. BOUGLÉ
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- 01 February 2000, pp. 125-129
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Caseins bind strongly to Fe by their phosphoseryl residues (Hegenauer et al. 1979; Brulé & Fauquant, 1982; Bouhallab et al. 1991; Emery, 1992) and keep it soluble at the alkaline pH of the duodenum (Manson & Annan, 1971; Bouhallab et al. 1991). It has been suggested that this strong binding prevents the release of free Fe during digestion and impairs its absorption (West, 1986; Hurrell, 1997), but in human studies hydrolysis by digestive enzymes to give low molecular mass peptides improves Fe absorption (Hurrell et al. 1988, 1989).
β-Casein peptide 1–25 (β-CN(1–25)) is the phosphorylated N-terminal fragment of β-CN. Its molecular mass is 3124 Da and it contains four phosphoseryl residues that bind four Fe atoms and keep them soluble (Bouhallab et al. 1991).
Preliminary results showed that binding Fe to β-CN(1–25) enhances its bioavailability in the rat (Aît-Oukhatar et al. 1997) and its absorption by the duodenal rat loop model (Pérès et al. 1997). We recently showed that the β- CN(1–25)–Fe complex is hydrolysed to a lesser extent than free β-CN(1–25) during duodenal digestion, and that the phosphorylated region of the peptide to which Fe is bound, β-CN(15–24), cannot be detected in the digestive lumen (our unpublished results).
These previous studies did not examine the effect of luminal digestion that could release free inorganic Fe. We considered the possibility that Fe bound to β-CN(1–25) is not released in the digestive lumen, and that it reaches the absorbing surface of the small intestine brush border membrane as a complex. To test this hypothesis we examined in vitro the influence of pH and digestive enzymes on the dialysability of the β-CN(1–25)–Fe complex.
Influence of ions on growth and production of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772
- GERT J. GROBBEN, INGEBORG C. BOELS, JAN SIKKEMA, MARK R. SMITH, JAN A. M. DE BONT
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- 01 February 2000, pp. 131-135
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Several lactic acid bacteria produce exopolysaccharides (EPS), either attached to the cell wall or excreted into the environment as slime material. EPS produced by Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) and Streptococcus thermophilus play an important role in improving the texture and stability of yogurt and preventing syneresis (Cerning, 1990; Nakajima et al. 1990). The amount and composition of the EPS produced by lactic acid bacteria are dependent on a number of factors, such as temperature, initial pH, carbon source and the availability of minerals, vitamins and other medium components.
In previous work it was shown that the production and sugar composition of the EPS from Lb. bulgaricus NCFB2772 are affected by the carbohydrate source (Grobben et al. 1995, 1996). In a simplified defined medium, from which several vitamins and trace elements were omitted, EPS production by Lb. bulgaricus significantly increased, although growth of the strain was reduced (Grobben et al. 1998).