Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Use of novel immobilized beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk

beta-galactosidase from Aspergillus oryzae immobilized in an axial-annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30 degrees C, 40 degrees C, and 50 degrees C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three-parameter rate expression which includes the separate competitive inhibition effects of alpha- and beta-galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the beta anomer of galactose (E(A) = 10.3 kcal/mol). At 40 degrees C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial-annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.     

A new innovative process to produce lactose-reduced skim milk

The research field for applications for lactose hydrolysis has been investigated for some decades. Lactose intolerance, improvement for technical processing of solutions containing lactose and utilisation of lactose in whey are main topics in development of biotechnological processes. In this article, the establishment of a hollow fiber membrane reactor process for enzymatic lactose hydrolysis is reported. Mesophilic beta-galactosidases were circulated abluminally during luminal flow of skim milk. The main problem, microorganisms growth in the enzyme solution, was minimised by sterile filtration and UV irradiation. In order to characterise the process parameters, such as skim milk concentration, enzyme activity and flow rates were varied. In comparison to a batch process, enzyme activity could be used longer and enzyme rest into the product should not occur. Furthermore, the three-dimensional separation of the substrate from the enzyme solution minimise blocking and washing out effects, which restrict processes with immobilised enzymes. A conversion rate of 78.11% was achieved at a skim milk flow rate of 9.9l h(-1), enzyme activity of 120 Uml(-1) and a temperature of 23+/-2 degrees C in a hollow fiber reactor with a membrane area of 4.9 m2.     

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).     

植物乳杆菌ST-Ⅲ发酵特性的研究

在添加5%(v/v)番茄汁的改良配方乳中对植物乳杆菌ST-Ⅲ单菌或与嗜热链球菌及保加利亚乳杆菌混合发酵的生长特性、发酵乳风味进行了研究.结果表明,在脱脂乳中添加5%(v/v)番茄汁可以明显缩短植物乳杆菌ST-Ⅲ单菌发酵时的凝乳时间,提高其活菌数.在改良的配方乳中,三菌混合发酵制备的发酵乳具有良好的酸奶风味、无涩味.但与...     

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

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.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Production of alkaline protease from a newly isolated Exiguobacterium profundum BK-P23 evaluated using the response surface methodology

Protease producing bacteria were isolated from soil in South Korea. These bacteria were screened in skim milk agar medium using skim milk as the substrate. The highest clear zone producing bacterial strain (BK-P23) was selected for further optimization studies. The strain was identified as Exiguobacterium profundum BK-P23 based on morphological, biochemical and molecular characterizations. The results of the 16S rRNA analysis showed that this strain was highly similar to E. profundum. The strain was able to grow under alkaline conditions at pH 8.5 and a temperature of 30°C. In the preliminary optimization experiments, five different parameters, i.e. carbon source (lactose), nitrogen source (corn steep solid), pH, temperature and incubation period were varied with the goal of optimizing enzyme production in low cost medium using the Box-Behnken design combined with response surface methodology. The optimal conditions were determined to be pH 9.0, a temperature of 30°C, lactose (1.0%) as the carbon source and corn steep solid (1.0%) as the cheap additional nitrogen source. In addition, 24 h of incubation was shown to produce the highest protease yield. Overall, the amount of enzyme produced was significantly higher in the optimized medium when compared with the original medium.     

Hydrolysis of lactose in skim milk by immobilized beta-galactosidase in a spiral flow reactor

beta-galactosidase from Aspergillus Oryzae immobilized in a spiral flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Residence time distribution measurements were used to assess the amount of longitudinal dispersion occurring as a consequence of the spiral flow pattern and the semiporous nature of the polymeric material used to construct the spiral. It was possible to model the flow conditions as tubular flow with a Peclet number that was a linear function of the reactor space time. Nonlinear regression methods were used to determine the kinetic parameters of three proposed enzymatic rate expressions. The best fit of the data was obtained using a rate expression containing separate terms for competitive inhibition of the reaction by both the a and beta anomers of galactose. This kinetic model also incorporates the kinetics of the mutarotation between these forms. At 30 degrees C and a space time of 7 minutes, 80% of the lactose present in skim milk can be converted to glucose and galactose.     

Lactose hydrolysis by immobilized β-galactosidase on nylon-6: A novel spin-basket reactor

Summary -D-galactosidase from Kluyveromyces lactis immobilized on nylon-6 microbeads was used to hydrolyze lactose in skim milk (containing 28.6% total solids), using a novel spin-basket reactor. More than 75% of lactose was hydrolyzed at 34°C within a short space-time (<7 min) without experiencing any plugging such as typically seen in packed columns.     

大熊猫乳汁蛋白组成

测定了2只大熊猫（ailuropoda melanoleuca)乳汁中蛋白含量,乳糖含量,乳蛋白组成,并与成都麻羊（Capra hircus)初乳和中国荷斯坦牛（Bos taurus)乳进行了比较,大熊猫乳蛋白含量平均为41．52g/L,与中国荷斯坦牛乳接近;乳糖平均含量为15．41g/L,极显著低于牛乳和成都麻羊初乳,乳蛋白SDS－PAGE分析表明,大熊猫乳中酪蛋白（CN）含量明显低于羊乳和牛乳;乳中存在3条蛋白含量的区带,而在成都麻羊和中国荷斯坦牛乳电泳图谱的相应位置未见明显的区带,乳中上皮粘蛋白MUC1仅存在1条分子量约为196kDa的区带,未发现多态现象。     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Optimization of galacto-oligosaccharide production by Bifidobacterium infantis RW-8120 using response surface methodology

Oligosaccharide (OS) production, cell concentration (2×10⁹ colony-forming unit/ml), lactose concentration (25% wt/vol), reaction time (6 h), and temperature (50°C) were chosen as the central condition of the central composite design (CCD) for optimizing the production process using Bifidobacterium infantis RW-8120 in skim milk. Statistical analysis (P<0.01) revealed that the most relevant variable concerning OS production and yield was the lactose concentration. The coefficient of determination (R ²) is good for the second-order OS production model (0.92) and fairly good for the second-order nonlinear OS yield model (0.816). An increase of lactose concentration and temperature resulted in a higher OS production. The optimal values for OS production appear to be near the area associated with the central points of the modeling design except for the lactose concentration, which was 40% (wt/vol) of the final volume. Received 29 March 2002/ Accepted in revised form 09 August 2002     

Enzymatic method to determine biomass in skim milk whey permeate medium

Summary In this study, a new technique for biomass determination was developed. Proteases were used to separate cells from the culture broth in skim milk whey permeate medium and the solubilization of casein was achieved 99% by elastase, 95% by pepsin and 90% by trypsin. Elastase was found to be efficient and reproducible for determining biomass in skim milk whey permeate medium.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Isolation and characterization of two protease-producing mutants from Staphylococcus aureus

Two mutants with increased protease production were isolated after nitrosoguanidine treatment of Staphylococcus aureus 8325N. The wild type produces low amounts of extracellular proteolytic activity. The enzyme was inducible and could only be detected if casein or preferably skim milk powder was used as inducer. The optimal pH, salt concentration, and media for enzyme production were determined. The mutants differed from the wild type in several phenotypic characters. The pattern of extracellular deoxyribonuclease and alkaline phosphatase differed between the mutants and the wild type. Several carbohydrates such as lactose, galactose, and mannitol were not utilized by the mutants, probably owing to a block in the uptake. Glucose could, however, be utilized by the mutants. Reversion frequency to wild type with regard to carbohydrate utilization was spontaneously high, and all revertants regained the parental pattern irrespective of the carbohydrate used for selection. The results suggest that a single locus may control the excretion of extracellular enzymes and carbohydrate uptake in S. aureus.     

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.