Stabilization of cetyltrimethylammonium bromide permeabilized yeast whole cell lactase

Summary Cetyltrimethylammonium bromide-permeabilized cells ofK. fragilis loose -galactosidase activity due to leaking of the enzyme into the medium. This leakage of the enzyme can be prevented by storing the permeabilized cells either in buffer containing 50% glycerol or by treating the permeabilized cells with 0.2% glutaraldehyde at 4°C for 10 min. In repeated batch hydrolysis of lactose in milk, glutaraldehyde treated cells could be repeatedly used very efficiently.     

Growth of cucumber cells in media with lactose or milk whey as carbon source

Lactose utilisation by cucumber cell suspension cultures starts only after a long lag phase and is accompanied by an increase of an extracellular lactosespecific -galactosidase activity. Supplementing the lactose medium with sucrose shortens the lag phase.Milk whey permeate seems to contain a factor(s) which inhibits lactose utilisation. After supplementing the medium with sucrose or its hydrolysis products, growth and substrate utilisation is as efficient as in Murashige and Skoog medium. Galactose also induces growth, but growth and substrate utilisation are slower. In whey medium, supplemented with sucrose, the extracellular -galactosidase activity again accompanies growth induction.Abbreviations MS Murashige and Skoog medium - MW milk whey medium - NAD nicotinamide-adenine dinucleotide - ONPG o-nitrophenyl--D-galactopyranoside - PNPG p-nitrophenyl--D-galactopyranoside     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

Semicontinuous lactic fermentation of whey byLactobacillus bulgaricus I. Experimental results

Summary A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum in semicontinuous fermentation of whey byLactobacillus bulgaricus. The volume of the inoculum varied from 10% to 80% of the reactor working volume.Nomenclature N number of fermentation cycles after the first fermentation - P lactic acid productivity - S_w average total sugars concentration of the whey as lactose (the standard deviation is indicated) - T average fermentation time (the standard deviation is indicated) - V_a average total consumption of NH₄OH solution (the standard deviation is indicated) - V_i volume of recently fermented medium used as inoculum of the next fermentation cycle - V_w volume of whey added to the reactor at the beginning of each fer mentation cycle - volume fraction of inoculum=V_i/(V_i+V_w)     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

Ethanol production from deproteinized whey byβ-galactosidase coimmobilized cells ofSaccharomyces cerevisiae

Summary The performance of -galactosidase coimmobilized cells ofSaccharomyces cerevisiae was evaluated during shake flask fermentation of deproteinized cheese whey lactose to ethanol. The performance of the coimmobilized enzyme treatment was compared to that of a treatment using acid prehydrolyzed whey lactose (a readily available substrate). Enzyme coimmobilization resulted in a slower rate and a lower extent of substrate utilization, thus giving a lower maximum ethanol concentration (13.5 versus 16.7 g/l). It did result, however, in a better ethanol yield (95% vs. 89% theoretical). It appears that, compared to acid prehydrolysis of whey lactose, through -galactosidase coimmobilization we could succeed in obtaining substantial process simplifications, thus saving in equipment and operating cost, while gaining in ethanol yield at the cost of some reasonable loss in the rate and the extent of lactose utilization.     

Production of ethanol and biomass from various carbohydrates byKluyveromyces fragilis

Summary Growth ofKluyveromyces fragilis NRC 2475 and the production of ethanol by the yeast were studied in the media containing one of the following sugars: glucose, lactose, galactose, or a glucose-galactose (50% 50%) mixture as a carbon source.The largest biomass yield and the lowest yield of ethanol were obtained in the medium containing glucose. The medium containing galactose gave the lowest yield of biomass and the largest yield of ethanol. When lactose was used for the growth and production of ethanol the obtained results for both biomass and ethanol were between those obtained with glucose and galactose.The ethanol productivities, expressed in terms of ethanol produced either per unit of cells, or per unit of cells and time, were the highest in the system with galactose and the lowest in that with glucose.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Use ofClostridium acetobutylicum P262 for production of solvents from whey permeate

Summary The production of solvents from whey permeate in batch fermentation usingClostridium acetobutylicum P262 was examined. An overall reactor productivity of 0.24 g/l.h was observed, representing a marked improvement over reports using other strains of clostridia. Using a semi-synthetic medium galactose was shown to be as effective a substrate as glucose. When whey permeate was used in which the lactose was hydrolysed prior to fermentation, preferential uptake of glucose over galactose was observed, and such hydrolysis provided no advantage to the fermentation process.     

Detecting concentrations of milk components by an iterative optical technique

This paper introduces a theoretical and practical model for reconstructing the scattering properties of a participating media. Our theory is based on a robust generalization of the Gerchberg–Saxton (G–S) algorithm. At the end of this algorithm the reduced scattering coefficient μ′_s of a given substance, can be estimated from the standard deviation (STD) of the retrieved phase of the remitted light. We use the theory to compute the phase's STD that directly correlated to the optical properties for different types of milk components, and we derive a novel appearance model for milk parameterized by the lactose and protein contents. Our results show that we are able to detect the possibility of lactose and milk proteins' quantitative signature by the G–S optical tool, en route to the design of a novel milk‐content‐monitoring tool.

Sketch of the experimental setup for light intensity measurements and reduced scattering coefficient reconstruction. The samples were prepared from various milk components: whey protein, sodium casienate and lactose, at different concentrations.     

Oligosaccharide formation byTrichoderma harzianum in lactose containing medium

Summary A soil isolate ofTrichoderma harzianum produced oligosaccharides in lactose containing medium. The yield of trisaccharides was 32.1 per cent based on total saccharides. The main product formed was 6-galactosyllactose.     

Evaluation of quality changes in udder quarter milk from cows with low-to-moderate somatic cell counts

Much emphasis has been put on evaluating alterations in milk composition caused by clinical and subclinical mastitis. However, little is known about changes in milk composition during subclinical mastitis in individual udder quarters with a low-to-moderate increase in milk somatic cell count (SCC). This information is needed to decide whether milk from individual udder quarters with a moderate-to-high increase in milk SCC should be separated or not. The aim of this study was to determine how milk composition in separate udder quarters is affected when cow composite milk has low or moderately increased SCC levels. Udder quarter and cow composite milk samples were collected from 17 cows on one occasion. Milk yield was registered and samples were analyzed for SCC, fat, total protein, whey proteins, lactose, citric acid, non-protein nitrogen (NPN), lactoferrin, protein profile, free fatty acids (FFAs), lactate dehydrogenase (LDH), proteolysis, sodium and potassium. Bacteriological samples were collected twice from all four quarters of all cows. The cows were divided into three groups depending on their SCC at udder quarter level. The first group comprised healthy cows with four udder quarters with low SCC, <50 000 cells/ml; composition was equal when opposite rear and front quarters were compared. In the second and the third groups, cows had one udder quarter with 101 000 cells/ml < SCC < 600 000 cells/ml and SCC > 700 000 cells/ml, respectively. The remaining udder quarters of these cows had low SCC (<100 000 cells/ml). Despite the relatively low average cow composite SCC = 100 000 cells/ml of Group 2, milk from affected udder quarters exhibited lower casein number, content of lactose and β-casein (β-CN), while the content of whey protein, sodium, LDH and α-lactoalbumin (α-la) were higher compared to healthy opposite quarters. In addition to these changes, milk from affected udder quarters in Group 3 also exhibited lower values of potassium and αs1-casein (αs1-CN) and higher values of lactoferrin when compared to milk from opposite healthy quarters. This indicates that even when the SCC in cow composite milk is low, there might exist individual quarters for which milk composition is changed and milk quality impaired.     

Biovalorization of saccharides derived from industrial wastes such as whey: a review

Whey is a liquid waste issued from the transformation of milk into cheese. Whey is a major environmental problem for the dairy industry due to its high organic load, linked to its high content of lactose. It can be valorized by biological processes based on lactose fermentation into different products such as (1) lactic acid (as food additive), (2) 2,3-butanediol (as feedstock to get products such as methyl-ethyl-ketone or 2-butene for the pharmaceutical and chemical industries), (3) biogas (to obtain energy). The production of 2,3-butanediol from saccharides, such as glucose, has been actively studied over previous decades using several types of microorganisms such as Enterobacter aerogenes, Paenibacillus polymyxa, Klebsiella sp., Serratia marcescens and Escherichia coli. Some of these have even been genetically modified to improve the 2,3-butanediol production. The potential whey fermentation process into 2,3-butanediol depends on several operating conditions such as microorganisms, composition of the culture medium, temperature, pH and aeration. This review first presents a summary of the situation of milk and cheese production in Canada and around the world. It also describes the different kinds of whey and their treatment techniques. Finally, this paper describes the production of 2,3-butanediol from saccharides by various microorganisms under different operating conditions.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Hydrolysis of lactose solutions and wheys by whole cells of Kluyveromyces bulgaricus

Summary Some factors affecting the hydrolysis of lactose solution and whey by whole cells of Kluyveromyces bulgaricus were studied. The K_m values were 59 mM and 32 mM for whole cells and cell-free extracts respectively. Optimum hydrolysis activity was observed at 48°C. At this temperature, 80% hydrolysis was obtained in lactose solution and whey after 3.5 and 9 min respectively by yeast cells (15 mg/ml) with an activity of 1.13 U/mg. Protein concentration in whey did not have an inhibitory effect. In whey permeate, cells were reused eight times with a hydrolysis degree of more than 80% but in lactose phosphate buffer, the hydrolysing capacity was lost quickly.     

Permeabilization ofKluyveromyces lactis cells for milk whey saccharification: A comparison of different treatments

Summary In this paper we compare the effectiveness of various lactose permeabilization treatments forKluyveromyces lactis. Best yields are obtained with 4:96 toluene: ethanol and with 70% ethanol in mild conditions, expression of intracellular ?-galactosidase activity being increased 40-fold with respect to untreated cells. Permeabilized cells hydrolyse lactose from milk whey more rapidly than control cells and do not further metabolize monosaccharides. This is a desired characteristic in terms of their potential commercial application.     

Properties and potential advantages of β-galactosidase from Bacteroides polypragmatus

Summary A -galactosidase (EC 3.2.1.23) from the mesophilic obligate anaerobe, Bacteroides polypragmatus, was purified 172 fold by p-aminophenyl--D-thiogalactopyranoside agarose affinity chromatography followed by Bio-Gel P300 chromatography. The presence of Mg²⁺ and a reducing agent such as dithiothreitol (DTT) or mercaptoethanol was required for enzyme activity. The optimum pH and temperature, as determined from hydrolysis of the substrate analogue o-nitrophenyl--D-galactopyranoside (ONPG), for enzyme activity were 6.8 and 45°C, respectively. There was negligible activity loss during incubation at 35°C for up to 13 h. The K_m values obtained with ONPG and lactose as substrates were 0.43 mM and 9.09 mM respectively. The enzyme obtained by affinity chromatography was shown to hydrolyze the lactose component of cheese whey; the amount of lactose hydrolyzed was 32% of that expected with pure lactose as the substrate in buffer containing Mg²⁺ and DTT.NRCC Publication Number 24295     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

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.