Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk

A recombinant thermostable β-galactosidase from Bacillus stearothermophilus was immobilized onto chitosan using Tris(hydroxymethyl)phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized β-galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized β-galactosidase showed greater relative activity in the presence of Ca²⁺ than the free enzyme and was stable during the storage at 4°C for 6 wk, whereas the free enzyme lost 31% of the initial activity under the same storage conditions. More than 80% of lactose hydrolysis in milk was achieved after 2 h of operation in the reactor. Therefore, THP-immobilized recombinant thermostable β-galactosidase from Bacillus stearothermophilus has the potential for application in the production of lactose-hydrolyzed milk.     

Diversity analysis and quantification of lactic acid bacteria in traditionally fermented yaks’ milk products from Tibet

Fermented yaks’ milk is a characteristic fermented dairy product with a rich microflora (especially lactic acid bacteria [LAB]), that has been produced by Tibetan people in China throughout history. Systematic analysis of the LAB composition of fermented yaks’ milk from central Tibet is essential. In the present study, 38 samples of fermented yaks’ milk were collected from three regions in central Tibet. From these, a total of 211 isolates of LAB were generated using traditional pure culture methods. To accurately analyze their biodiversity, 16S rRNA gene sequence analysis and denaturing gradient gel electrophoresis (DGGE) were used. The isolates were identified as belonging to six genera and 22 species/subspecies. Among them, Lactobacillus species predominated (117 isolates) accounting for 55.45% of all isolates. Overall, L. delbrueckii subsp. bulgaricus (60 isolates; 28.43% of the total) was the most commonly isolated species of LAB from fermented yaks’ milk from central Tibet. Seven Lactobacillus species and the Bifidobacterium genus were confirmed by quantitative PCR (q-PCR). The q-PCR results showed that the abundance of these groups in fermented yaks’ milk from central Tibet ranged from 1.12 ± 0.36 log cfu/ml (for L. sakei from Ningzhong) to 7.77 ± 0.84 log cfu/ml (for L. helvetivus from Ningzhong). Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus, L. fermentum, L. helveticus varied in concentration depending on the region. The results indicated that traditional fermented yaks’ milk from different regions of central Tibet have complex compositions of LAB species. The diversity of LAB might be related to geographical origin.     

Engineering the optimum pH of β-galactosidase from Aspergillus oryzae for efficient hydrolysis of lactose

β-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (pKa) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase.     

Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade β-galactosidase

The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade β-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55-525?g/L), enzyme concentration (0.05-1.575?g/L), temperature (30-50°C) and pH (5.0-6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A?maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525?g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on Michaelis-Menten model, four-step reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7(?). Excellent agreements were observed between simulated and experimental results.     

A novel thermostable β-galactosidase from Bacillus coagulans with excellent hydrolysis ability for lactose in whey

β-Galactosidase is one of the most important enzymes used in dairy industry. Here, a novel thermostable β-galactosidase was cloned and overexpressed from Bacillus coagulans NL01 in Escherichia coli. The phylogenetic trees were constructed using neighbor-joining methods. Phylogeny and amino acid analysis indicated that this enzyme belonged to family 42 of glycoside hydrolases. The optimal pH and temperature were, respectively, 6.0 and 55 to 60°C. The purified enzyme had a 3.5-h half-life at 60°C. Enzyme activity was enhanced by Mn²⁺. Compared with other β-galactosidases from glycoside hydrolase family 42, B. coagulans β-galactosidase exhibited excellent hydrolysis activity. The Michaelis constant (K_m) and maximum rate of enzymatic reaction (V_max) values for p-nitrophenyl-β-d-galactopyranoside and o-nitrophenyl-β-d-galactopyranoside were 1.06 mM, 19,383.60 U/mg, and 2.73 mM, 5,978.00 U/mg, respectively. More importantly, the enzyme showed lactose hydrolysis ability superior to that of the commercial enzyme. The specific enzyme activity for lactose was 27.18 U/mg. A total of 104.02 g/L lactose in whey was completely hydrolyzed in 3 h with addition of 2.38 mg of pure enzyme per gram of lactose. In view of the high price of commercial β-galactosidase, B. coagulans β-galactosidase could be a promising prototype for development of commercial enzymes aimed at lactose treatment in the dairy industry.     

Optimisation of immobilisation conditions for chick pea β-galactosidase (CpGAL) to alkylamine glass using response surface methodology and its applications in lactose hydrolysis

Response surface methodology was advantageously used to optimally immobilise a β-galactosidase from chick pea onto alkylamine glass using Box–Behnken experimental design, resulting in an overall 91% immobilisation efficiency. Analysis of variance was performed to determine the adequacy and significance of the quadratic model. Immobilised enzyme showed a shift in the optimum pH; however, optimum temperature remained unaffected. Thermal denaturation kinetics demonstrated significant improvement in thermal stability of the enzyme after immobilisation. Galactose competitively inhibits the enzyme in both soluble and immobilised conditions. Lactose in milk whey was hydrolysed at comparatively higher rate than that of milk. Immobilised enzyme showed excellent reusability with retention of more than 82% enzymatic activity after 15 uses. The immobilised enzyme was found to be fairly stable in both dry and wet conditions for three months with retention of more than 80% residual activity.     

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

Six strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows’ milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilot-scale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 - S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product.     

Estrone and 17β-estradiol concentrations in pasteurized-homogenized milk and commercial dairy products

Some individuals fear that estrogens in dairy products may stimulate growth of estrogen-sensitive cancers in humans. The presence of estrone (E₁) and 17β-estradiol (E₂) in raw whole cow's milk has been demonstrated. The objectives of this study were to determine if pasteurization-homogenization affects E₂ concentration in milk and to quantify E₁ and E₂ concentrations in commercially available dairy products. The effects of pasteurization-homogenization were tested by collecting fresh raw milk, followed by pasteurization and homogenization at 1 of 2 homogenization pressures. All treated milks were tested for milk fat globule size, percentages of milk fat and solids, and E₂ concentrations. Estrone and E₂ were quantified from organic or conventional skim, 1%, 2%, and whole milks, as well as half-and-half, cream, and butter samples. Estrone and E₂ were quantified by RIA after organic solvent extractions and chromatography. Pasteurization-homogenization reduced fat globule size, but did not significantly affect E₂, milk fat, or milk solids concentrations. Estrone concentrations averaged 2.9, 4.2, 5.7, 7.9, 20.4, 54.1 pg/mL, and 118.9 pg/g in skim, 1%, 2%, and whole milks, half-and-half, cream, and butter samples, respectively. 17β-Estradiol concentrations averaged 0.4, 0.6, 0.9, 1.1, 1.9, 6.0 pg/mL, and 15.8 pg/g in skim, 1%, 2%, whole milks, half-and-half, cream, and butter samples, respectively. The amount of fat in milk significantly affected E₁ and E₂ concentrations in milk. Organic and conventional dairy products did not have substantially different concentrations of E₁ and E₂. Compared with information cited in the literature, concentrations of E₁ and E₂ in bovine milk are small relative to endogenous production rates of E₁ and E₂ in humans.     

Kinetics of lactose hydrolysis and galactooligosaccharides formation in beverages based on goat’s milk and its permeate

Food Science and Biotechnology - The aim of this work was the analysis of galactooligosaccharides (GOS) formation in a model mixture of goat’s milk and its permeate from microfiltration and...     

δ13C values and phytanic acid diastereomer ratios: combined evaluation of two markers suggested for authentication of organic milk and dairy products

Authentication of organic milk by suitable markers is currently attracting more and more interests in food control. In this study, we aimed to compare the efficiency of the markers stable carbon isotope ratio (δ¹³C value) with the SRR/RRR phytanic acid diastereomer ratio (SRR/RRR) of milk fat for distinguishing different feeding systems. For stable carbon isotope ratio analysis by elemental analysis–isotope ratio mass spectrometry (EA-IRMS), we first developed a simple sample preparation method based on milk fat extracts allowed to evaporate the solvent in tin capsules for liquid samples. The δ¹³C values and reproducibility measured with this alternative sample pre-treatment method excellently matched those obtained with the current standard method. Applied to milk samples, the results of the EA-IRMS analysis were linked to the SRR/RRR, and both markers allowed to distinguish milk from cows fed with hay (δ¹³C value > ?28.0 ‰, SRR/RRR <1.5) from feed used in conventional milk production which contained maize silage (C₄-plants) (p < 0.001). Milk fat of organic retail cheese samples was also highly depleted in ¹³C (δ¹³C value ?30.0 ‰ ± 1.1), and the SRR/RRR was low (<1.5). However, seven cheese samples showed inconsistent δ¹³C values and SRR/RRR, most likely due to the feeding of grass silage. Both parameters (δ¹³C values and SRR/RRR), together with the phytanic acid content of milk fat, also allowed distinguishing between the feeding of high amounts of pasture, hay, and/or grass silage.     

Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842

Three different neutralizers (NaOH, KOH, NH₄OH) were employed for pH maintenance during the growth of Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, used as a source of β-galactosidase extracts. The crude enzymatic extract (CEE) was obtained by bead milling of the cell paste, collected from the cultivation of the source microorganism in skim milk at 43 °C and constant pH. Lactose hydrolysis kinetics in skim milk and proteolytic activity during the hydrolysis were evaluated. The use of NH₄OH as a neutralizer resulted in significantly (P<0.05) higher enzyme activity of the CEE than that obtained using NaOH or KOH. The kinetic parameters, k_cat and K_m, of the Michaelis–Menten model were determined for lactose hydrolysis in skim milk using 1% (v/v) addition of a CEE. There was no significant (P>0.05) difference in k_cat among the different extracts, with a clear temperature dependence following Arrhenius kinetics. The rate of lactose hydrolysis was dependent on the initial enzyme activity and temperature. The highest initial rate was observed at 65 °C; however, the enzyme deactivation occurred within 1–1.5 h. The proteolytic activity determined by HPLC peptide mapping was significantly (P<0.05) higher in the moderate temperature range (20 and 37 °C) than at 7 or 55 °C. Industrial relevance: Since lactose intolerance affects a large proportion of the world's population, an economically feasible and effective process with a cheap source of β-galactosidase may have a substantial potential. The use of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842 appears to be a promising approach for development of a technologically feasible process of lactose hydrolysis for food or non-food uses.     

Prediction of blood β-hydroxybutyrate content and occurrence of hyperketonemia in early-lactation,pasture-grazed dairy cows using milk infrared spectra

The objective of this study was to evaluate the ability of milk infrared spectra to predict blood β-hydroxybutyrate (BHB) concentration for use as a management tool for cow metabolic health on pasture-grazed dairy farms and for large-scale phenotyping for genetic evaluation purposes. The study involved 542 cows (Holstein-Friesian and Holstein-Friesian × Jersey crossbreds), from 2 farms located in the Waikato and Taranaki regions of New Zealand that operated under a seasonal-calving, pasture-based dairy system. Milk infrared spectra were collected once a week during the first 5 wk of lactation. A blood “prick” sample was taken from the ventral labial vein of each cow 3 times a week for the first 5 wk of lactation. The content of BHB in blood was measured immediately using a handheld device. After outlier elimination, 1,910 spectra records and corresponding BHB measures were used for prediction model development. Partial least square regression and partial least squares discriminant analysis were used to develop prediction models for quantitative determination of blood BHB content and for identifying cows with hyperketonemia (HYK). Both quantitative and discriminant predictions were developed using the phenotypes and infrared spectra from two-thirds of the cows (randomly assigned to the calibration set) and tested using the remaining one-third (validation set). A moderate accuracy was obtained for prediction of blood BHB. The coefficient of determination (R²) of the prediction model in calibration was 0.56, with a root mean squared error of prediction of 0.28 mmol/L and a ratio of performance to deviation, calculated as the ratio of the standard deviation of the partial least squares model calibration set to the standard error of prediction, of 1.50. In the validation set, the R² was 0.50, with root mean squared error of prediction values of 0.32 mmol/L, which resulted in a ratio of performance to deviation of 1.39. When the reference test for HYK was defined as blood concentration of BHB ≥1.2 mmol/L, discriminant models indicated that milk infrared spectra correctly classified 76% of the HYK-positive cows and 82% of the HYK-negative cows. The quantitative models were not able to provide accurate estimates, but they could differentiate between high and low BHB concentrations. Furthermore, the discriminant models allowed the classification of cows with reasonable accuracy. This study indicates that the prediction of blood BHB content or occurrence of HYK from milk spectra is possible with moderate accuracy in pasture-grazed cows and could be used during routine milk testing. Applicability of infrared spectroscopy is not likely suited for obtaining accurate BHB measurements at an individual cow level, but discriminant models might be used in the future as herd-level management tools for classification of cows that are at risk of HYK, whereas quantitative models might provide large-scale phenotypes to be used as an indicator trait for breeding cows with improved metabolic health.     

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis

The enzymatic transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis (KlβGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).     

Agar-immobilized basil–lactic acid bacteria bioproducts as goat milk taste-masking agents and natural preservatives for the production of unripened goat cheese

Goat milk cheeses have become popular recently; however, many consumers do not choose these products because they have specific sensory properties that are not acceptable to all consumers and the shelf life of the cheese is short. The concept of this work was to increase overall acceptability and shelf life of unripened goat milk cheese by using Ocimum basilicum and lactic acid bacteria (Lactobacillus plantarum LUHS135, Lactobacillus paracasei LUHS244, Pediococcus pentosaceus LUHS100, Pediococcus acidilactici LUHS29, and Lactobacillus brevis LUHS140) bioproducts (basil-LAB) immobilized in agar. A basil-LAB bioproduct could be a promising multifunctional ingredient for cheese manufacturing because it has a low pH, high LAB count, and high total phenolic compound content (after fermentation pH decreased by 25.4%, LAB count averaged 7.2 log₁₀ cfu/g, and total phenolic compound content increased by 30.9%). Use of different LAB in the preparation of basil-LAB bioproducts had a significant influence on cheese pH and hardness, and compared with cheese samples prepared with nonfermented basil, cheese samples prepared with basil-LAB bioproducts had, on average, higher pH (by 2.6%) and lower hardness (by 36.0%), similar to the control cheese (without basil). Overall acceptability of cheese was significantly influenced by the basil-LAB bioproduct immobilization process; in all cases, cheese samples prepared with fermented and immobilized basil-LAB bioproduct had better acceptability (5 points). After 120 h of storage, cheese samples prepared with basil-LAB bioproducts fermented with LUHS135, LUHS244 and LUHS140, no enterobacteria were found, and we detected strong negative and moderate negative correlations, respectively, of LAB count with enterobacteria count and yeast/mold count (r = ?0.7939 and r = ?0.4495, respectively). Finally, immobilization increased LAB viability in fresh goat milk cheese, which led to a reduction in enterobacteria and mold/yeast contamination during storage and an increase in overall acceptability compared with nonimmobilized basil-LAB. Therefore, basil-LAB bioproducts fermented with LUHS135, LUHS244, and LUHS140 strains can be recommended for preparing fresh goat milk cheese with extended shelf life and high acceptability.     

Improvement of emulsifying properties of Maillard reaction products from β-conglycinin and dextran using controlled enzymatic hydrolysis

A novel emulsifier was prepared by conjugating soy β-conglycinin and dextran (MW 67 kDa) under dry-heated Maillard reaction followed by trypsin hydrolysis with the degree of hydrolysis (DH) at 2.2% and 6.5%. The emulsifying properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 2.2% and DH 6.5%) were investigated using zeta-potential, droplet size and creaming index of the emulsions. The results showed that hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) were capable of forming a fine emulsion (d₄₃ = 0.62 ± 0.04 μm, pH 7.0) which remained stable during 4 weeks of storage. A variety of physicochemical and interfacial properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates were investigated. Hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) had a much higher fraction of protein adsorption (F_ads) and a significantly lower saturation surface load (Γ_sat) compared with β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 6.5%). This might be due to its higher molecular flexibility, which benefited the adsorption and unfolding of peptide molecules at the droplet interface. These might explain its markedly improved emulsifying capability. The conjugation of β-conglycinin and dextran effectively enhanced the hydrophilicity of the oil droplets surfaces and improved the steric repulsion between the oil droplets. Therefore the emulsions were still stable after 4 weeks of storage against pH, ionic strength and thermal treatment. This study demonstrated that controlled enzymatic hydrolysis of protein–polysaccharide conjugates could be an effective method for preparing favourable emulsifiers.     

Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85°C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.     

A novel method for total protein analysis of protein mixtures using enzyme hydrolysis and derivatisation with o-phthaldialdehyde – Application to dairy products

A novel approach to analysis of proteins in dairy products was demonstrated using enzyme hydrolysis followed by derivatisation with o-phthaldialdehyde (OPA) and fluorescence detection. Unlike the response of other dye-binding methods, enzyme hydrolysis significantly eliminated dependence of the fluorescence intensity upon protein primary sequence and, compared with chemical hydrolysis, was achieved under relatively mild conditions. Enzyme hydrolysis and OPA-derivatisation steps were successfully automated using a flow-injection manifold, with a method coefficient of repeatability <6%. The enzyme-OPA (eOPA) method was applied to analysis of total (TP) and serum-soluble proteins (SSP, proteins soluble at pH 4.6) in reference standards spanning two decades of protein concentration. Method accuracy was very good for analysis of TP and acceptable for analysis of SSP. Discrepancies between the eOPA and standard methods for SSP were explained by sample preparation and not analytical factors. The eOPA method may be suitable for automation into a portable or miniaturised device.     

Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase

In this study, lactulose synthesis from whey lactose was investigated in batch and continuous systems using immobilized β-galactosidase. In the batch system, the optimal concentration of fructose for lactulose synthesis was 20%, and the effect of galactose, glucose and fructose on β-galactosidase activity was determined for hydrolysis of whey lactose and the transgalactosylation reaction for lactulose synthesis. Galactose and fructose were competitive inhibitors, and glucose acted as a noncompetitive inhibitor. The inhibitory effects of galactose and glucose were stronger in the transgalactosylation reaction than they were in the hydrolysis reaction. In addition, when immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity was retained to the extent of 52.9% after 10 reuses. Lactulose was synthesized continuously in a packed-bed reactor. We synthesized 19.1 g/l lactulose during the continuous flow reaction at a flow rate of 0.5 ml/min.     

Physical and nutritional conditions for optimized production of bacteriocins by lactic acid bacteria – A review

ABSTRACT

There has been an increasing debate about the use of synthetic chemical compounds and the consequences of their use in food preservation. In this context, the utilization of some natural compounds produced by bacteria, showing an inhibitory effect against microorganisms associated with food contamination, have gained attention as preservation technology. In order to improve the production and yield costs of bacteriocins, detailed studies are necessary to determine the conditions that allow an optimized production and extraction of bacteriocins from lactic acid bacteria (LAB). In this context, this article aims to discuss the information regarding the main factors that influence bacteriocin production by LAB. The biosynthesis of bacteriocins can be influenced by various culture conditions, such as the composition of the medium, pH, temperature and growth kinetics of the microorganisms. One of the limiting factors for the use of bacteriocins on a large scale in food preservation is the economic factor. In order for the production costs of bacteriocins to be reduced, making them attractive, it is necessary to know the optimum parameters of production, thus maximizing productivity and making costs more attractive.     

A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products

A multiplex PCR able to identify cows’, goats’ and sheep's milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments of different length.The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism.The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheep's and cows’ milk in dairy products with a good sensitivity threshold (0.5%).