Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Growth of a Mixed Species Lactic Starter in a Continuous "pH-Stat" Fermentor

Optimum growth conditions for mixed species starter FDs 0172 in skim milk, whey medium, and tryptone medium at 25 C in a “pH-stat” continuous fermentor were investigated. Specific growth rate and productivity were found to be influenced both by the medium and the pH value. Highest productivity was found in skim milk medium between pH 5.5 and 5.9. Lactic acid-producing activity of harvested cells decreased by continuous cultivation at pH values lower than 5.9 to 6.1, especially in tryptone and whey medium. Bacterial balance, CO₂ production, and aroma formation were comparable to the control in skim milk and whey medium at any pH, but differed significantly for cells grown in tryptone medium.     

Propionic acid fermentation of ultra-high-temperature sterilized whey using mono-and mixed-cultures

Summary Unlike sterilization by autoclave (Anderson et al. 1986) high concentrations of cheese whey sterilized by ultra high temperature (UHT) resulted in a medium conducive to microbial growth and propionic acid production. Propionibacterium freudenreichii ss. shermanii, grown with pH control in 12% whey solids and 1% yeast extract sterilized by UHT, produced about 1.9% propionic acid within 70 h; more than 50% of the lactose was not used. Under similar conditions, mixed cultures of P. shermanii and Lactobacillus casei produced more than 3.0% propionic acid. Acclimating the mixed culture to the whey medium resulted in 4.5% propionic acid. The amount of propionic acid produced was further increased to about 6.5% by raising the concentration of whey solids to about 18%. Using the mixed culture, all the lactose was consumed and lactic acid did not accumulate.     

In Vivo-Like Antigenic Surface Properties of Staphylococcus aureus from Bovine Mastitis Induced upon Growth in Milk Whey

Antigenic surface properties of Staphylococcus aureus strains grown in milk whey were compared with TSB-grown bacteria using immuno-gold electron microscopy. It is shown that colloidal gold (CG) particles coated with polyclonal antibody raised against Staphylococcus aureus surface antigen expressed in vivo bound to the surface of S. aureus strain F1440 grown in milk whey, but not to homologous bacteria grown in TSB. S. aureus strains grown in milk whey agglutinated in the presence of the polyclonal antibody, whereas the corresponding bacteria grown in TSB did not agglutinate. Immuno-gold particles did not bind to milk whey-grown bacteria treated with periodate. Periodate-treated milk whey-grown bacteria did not agglutinate in the presence of the polyclonal antibody, whereas periodate treatment had no effect on TSB-grown bacteria.     

Studies on batch production of bacterial concentrates from mixed species lactic starters

Optimum growth conditions for mixed species starter FDs 0172 at constant pH in skim milk, whey, and tryptone medium were investigated. Growth rate and maximum population were optimal at 30 C. pH values between 5.5 and 7.0 did not influence the growth rate and maximum population obtainable. Lactic acid-producing activity declined rapidly after reaching the end of the exponential growth phase. The bacterial balance was found to be influenced by the growth parameters: media, pH, temperature, and neutralizer. Skim milk or whey medium at 25 C, pH 6.5, and neutralized with 20% (vol/vol) NH₄OH kept the bacterial balance almost constant throughout the cultivation. Grown in tryptone medium at constant pH, the changes in bacterial balance and other metabolic activities were striking compared to the other two media tested. The effect of lactate as an inhibitor was found to be complex, changing with the growth conditions. Concentrates made from mixed species starters FDs 0172, FD 0570, CH 0170, CHs 0170, and T 27 were comparable to controls when cultivated at the optimum conditions found and thereafter centrifuged. Aroma production, proteolytic activity, and CO₂ production did not change significantly compared to controls when cultivated at optimum conditions in skim milk or whey medium.     

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola. Received: 29 July 1998 / Received revision: 5 October 1998 / Accepted: 11 October 1998     

Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus

Summary Investigations have been carried out on lactic acid production by Lactobacillus helveticus CNRZ 303 in whey ultrafiltrate. Addition of beet molasses was investigated with good results, although yeast extract proved to be more effective. The size of inoculum and the preculture medium also played a significant role in determining the amount of lactic acid produced during the fermentation process. High lactose consumption (94.09%), together with good lactic acid production (26.09 g/l) and yield (0.90%), were obtained in whey ultrafiltrate supplemented with 1% (w/v) beet molasses (WUM), with a 10% (w/v) inoculum and peptonized milk as preculture medium. Although these results were similar to those obtained when yeast extract was used as supplement, the maximum volumetric productivities proved to be quite different, and were definitely higher with yeast extract. Offprint requests to: L. Chiarini     

Influence of milk whey, nitrogen and phosphorus concentration on oxalic acid production by Aspergillus niger

A factorial design at two levels was used to determine the effect of milk whey concentration and the addition of nitrogen (as NH₄NO₃) and phosphorus (as KH₂PO₄) on the oxalic acid production by Aspergillus niger. The results of the experiments indicated that milk whey contains enough nutrients for fungus growth, therefore medium supplementing with N and P is not necessary. The optimum milk whey concentration was 100 kg/m³ reaching a final oxalic acid concentration of 37 kg/m³ and a maximum production rate of 3.4 kg/m³ · d. The yield of oxalic acid was 0.4, a very high value compared to previous works.     

The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media

Mixed starters containing Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus strains were produced on commercial starter media (MB Complete, Thermolac, Marlac), as well as on milk. With the exception of Marlac, the starters were cultured under pH control. The effect of media and incubation temperature (22 or 32°C) on population ratios, on specific acidifying activities (SAA) of the cultures as well as on their ability to produce aroma compounds in milk was studied. The starters had higher contents in lactobacilli when they were produced at 32°C, whereas a tendency to obtain higher Leuconostoc populations was observed at 22°C. With respect to the lactococci, there was a significant interaction between temperature and growth medium for both strains. Thus, Le. cremoris T2 reached higher populations at 32°C if grown in MB complete and Thermolac, whereas in Marlac and skim milk, viable counts were higher at 22°C. The lactococci represented 50% of the total population of the culture at the beginning of the incubation, but they composed between 80% and 99% of the total population following fermentation. The best medium for growth of Leuconostoc was milk, but populations of only 10⁸ cfu/ml were reached. The lactobacilli did not grow well in MB Complete, and their development was best in the low-phosphate Marlac medium. The cultures grown on Marlac had the highest SAA values, whereas those grown on MB complete had the lowest. Overall, more ethanol and diacetyl were detected in the fermented milks when the starters used to inoculate them were produced at 22°C. Journal of Industrial Microbiology & Biotechnology (2000) 25, 288–297. Received 23 June 2000/ Accepted in revised form 22 September 2000     

Antibacterial Effect of Bovine Lactoferrin Against Udder Pathogens

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coli isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

The Effect of UV-C Pasteurization on Bacteriostatic Properties and Immunological Proteins of Donor Human Milk

Background

Human milk possesses bacteriostatic properties, largely due to the presence of immunological proteins. Heat treatments such as Holder pasteurization reduce the concentration of immunological proteins in human milk and consequently increase the bacterial growth rate. This study investigated the bacterial growth rate and the immunological protein concentration of ultraviolet (UV-C) irradiated, Holder pasteurized and untreated human milk.

Methods

Samples (n=10) of untreated, Holder pasteurized and UV-C irradiated human milk were inoculated with E. coli and S. aureus and the growth rate over 2 hours incubation time at 37°C was observed. Additionally, the concentration of sIgA, lactoferrin and lysozyme of untreated and treated human milk was analyzed.

Results

The bacterial growth rate of untreated and UV-C irradiated human milk was not significantly different. The bacterial growth rate of Holder pasteurized human milk was double compared to untreated human milk (p<0.001). The retention of sIgA, lactoferrin and lysozyme after UV-C irradiation was 89%, 87%, and 75% respectively, which were higher than Holder treated with 49%, 9%, and 41% respectively.

Conclusion

UV-C irradiation of human milk preserves significantly higher levels of immunological proteins than Holder pasteurization, resulting in bacteriostatic properties similar to those of untreated human milk.     

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium

AIMS: To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk-based medium. METHODS AND RESULTS: The influence of temperature and pH was studied through fermentation and modelling. Fermentations under non-pH controlled conditions with S. thermophilus ST 111 indicated that the EPS production was low in milk medium, even if additional nitrogen sources were supplemented. Under pH-controlled conditions, addition of whey protein hydrolysate to the milk medium resulted in a fivefold increase of the EPS production. This medium did not contain polysaccharides interfering with EPS isolation. Primary and secondary modelling of different fermentations revealed an optimum temperature and pH of 40 degrees C and constant pH 6.2, respectively, for growth in milk medium supplemented with whey protein hydrolysate. Maximum EPS production was observed in the range of 32-42 degrees C and constant pH 5.5-6.6. Whereas growth and maximum EPS production were clearly influenced by temperature and pH, the specific EPS production was only affected by stress conditions (T = 49 degrees C). CONCLUSIONS: Addition of whey protein hydrolysate to milk medium resulted in an increased growth and EPS production of S. thermophilus ST 111 under pH-controlled conditions. A modelling approach allowed studying the influence of temperature and pH on the kinetics of both growth and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an appropriate milk-based medium and a combined model of temperature and pH can be of practical importance for the production of yoghurt or other fermented milks as well as for process optimization of the large-scale production of starter strains to be used for their EPS production.     

Production of High-Viscosity Whey Broths by a Lactose-Utilizing Xanthomonas campestris Strain

Xanthomonas campestris BB-1L was isolated by enrichment and selection by serial passage in a lactose-minimal medium. When BB-1L was subsequently grown in medium containing only 4% whey and 0.05% yeast extract, the lactose was consumed and broth viscosities greater than 500 cps at a 12 s⁻¹ shear rate were produced. Prolonged maintenance in whey resulted in the loss of the ability of BB-1L to produce viscous broths in whey, indicating a reversion to preferential growth on whey protein, like the parent strain.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

The Influence of Site of Antigen Deposition on the Local Immune Response in the Mammary Gland of the Ewe

Experiments were carried out to compare the local antibody responses in mammary glands of ewes immunized by infusion of antigen (killed Brucella abortus) into the lactiferous sinuses (trans-epithelial presentation of antigen) or injection into the mammary tissue near the supramammary lymph node (interstitial presentation of antigen). Although both methods of antigen presentation resulted in similar antibody levels in blood, infusion of antigen into the lactiferous sinus resulted in significantly higher levels of agglutinating antibody in milk whey than did injection of antigen. When within-animal comparisons were made, infusion of antigen was also significantly superior to injection of antigen in terms of levels of non-agglutinating antibody in milk as determined by Coomb's antiglobulin assays. Evidence from immunoglobulin estimations in milk whey suggested that any elevation in concentrations of immunoglobulins (including IgA) in milk from ewes, which had received injections of antigen into mammary tissue, was associated with chronic inflammatory damage to these glands.     

Indigestion in Young Calves

Increasing the lactose content of different milk replacers or milk diets by approx. 30 % of the dry matter increased the frequency of diarrhoea the first 10–12 days in young calves on all occasions in 5 experiments comprising 120 calves. For all diets taken together, this effect was highly significant. Total daily intakes of lactose amounted to 200–480 g. When lactose was given on top of the milk rations, the growth rate increased significantly, whereas the growth rate was usually insignificantly reduced when lactose replaced other nutrients in milk diets or milk replacers, their levels of protein and fat becoming low. Albumin and total protein in blood plasma were significantly lower when the dietary protein level was low. Milk replacers with 20 or 40 % whey powder, replacing skim milk powder, performed equally well, but gave significantly less growth than the old-fashioned feeding of whole milk-skim milk. Intake of hay and barley and a number of clinical and histological or pathological parameters did not vary consistently with dietary level of whey powder or lactose. Feeding whole milk all the time resulted in low intake of hay and barley and poorly developed forestomachs, but high dressing-out %. Substituting soya for part of the skim milk powder in milk replacers gave abomasal content with no curds. In most cases, pH in the rumen appeared to be nearly up to neutral until the calves ate ground barley, about 1 month old.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

Distribution of lead in human milk fractions

The purpose of this study was to determine the concentration and distribution of lead, calcium, iron, zinc, and copper in major fractions (fat, casein, whey) of mature milk from 38 nursing adult women with low environmental lead exposure. The potential associations between milk lead and maternal blood lead and between milk and blood lead and essential mineral data (nutritional status, dietary intake, and milk concentration) were investigated. Maternal blood lead (geometric mean, 60 μg/L) was negatively associated, although modestly, with dietary calcium intake (r=−0.32, p=0.02). Lead in whole milk (geometric mean, 1.2 μg/L) was positively associated with calcium in whole milk (r=0.56, p=0.005). Distribution of lead in milk fractions was 63%, 28%, and 9%, in whey, fat, and casein, respectively. Milk distribution of essential minerals was 67–76%, 17–18%, and 7–17% in whey, fat, and casein, respectively. Lead in milk whey was positively associated with lead in maternal blood (r=0.49, p=0.02). However, milk lead was not affected by nutritional status, dietary intake, and milk composition of the essential minerals. The high percentage of lead in the milk whey fraction, as seen for the essential minerals, suggests that most lead in human milk is bioavailable to the infant.     

The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus

 Addition of the growth retardant ancymidol to Narcissus shoots and lower inner leaf sections isolated from shoots cultured in liquid medium induced hyperhydric malformations associated with morphogenetic changes. Meristematic centers initiated on the basal proximal ends appeared over the entire surface of the hyperhydric leaf sections after 6 weeks in culture. The meristematic centers which formed clusters on the leaf sections developed later into buds. In leaf sections grown in the liquid medium lacking ancymidol, hyperhydricity was not induced, and regeneration was not observed. Starch and protein levels and ascorbate peroxidase and catalase activities were examined in shoots and isolated leaf sections that were either hyperhydric or non-hyperhydric. In ancymidol-treated, hyperhydric leaf sections, ascorbate peroxidase and catalase activities were lower than in control, untreated leaf sections. The changes in starch and protein levels and in antioxidant enzymatic activities appeared to be related to the onset of meristematic-center initiation and further bud development on Narcissus hyperhydric leaf sections. Received: 6 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000