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.     

Some low homogenization pressures improve certain probiotic characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K

Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus salivarius ssp. thermophilus, and Lactobacillus acidophilus are dairy cultures widely used in the manufacture of cultured dairy products. Commonly used homogenization pressures in the dairy industry are 13.80 MPa or less. It is not known whether low homogenization pressures can stimulate bacteria to improve their probiotic characteristics. Objectives were to determine the effect of homogenization at 0, 3.45, 6.90, 10.34, and 13.80 MPa on acid tolerance, bile tolerance, protease activity, and growth of L. delbrueckii ssp. bulgaricus LB-12, S. salivarius ssp. thermophilus ST-M5, and L. acidophilus LA-K. The cultures were individually inoculated in cool autoclaved skim milk (4°C) and homogenized for 5 continuous passes. Growth and bile tolerance of samples were determined hourly for 10 h of incubation. Acid tolerance was determined every 20 min for 120 min of incubation. Protease activity was determined at 0, 12, and 24 h of incubation. All homogenization pressures studied improved acid tolerance of L. delbrueckii ssp. bulgaricus LB-12 but had no beneficial effect on protease activity and had negative effects on growth and bile tolerance. A pressure of 6.90 MPa improved acid tolerance, bile tolerance, and protease activity of S. salivarius ssp. thermophilus ST-M5, but none of the homogenization pressures studied had an effect on its growth. Homogenization pressures of 13.80 and 6.90 MPa improved acid tolerance and bile tolerance, respectively, of L. acidophilus LA-K but had no effect on protease activity and its growth. Some low homogenization pressures positively influenced some characteristics of yogurt culture bacteria and L. acidophilus LA-K. Culture pretreatment with some low homogenization pressures can be recommended for improvement of certain probiotic characteristics.     

Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr⁸⁰¹ and Pro⁸⁰² around the active sites of BgaQ with His⁸⁰¹ and Gly⁸⁰², the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.     

Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

A selective medium for the enumeration and differentiation of Lactobacillus delbrueckii ssp. bulgaricus

Modified reinforced clostridial medium (mRCM) was developed and evaluated for the differential enumeration of Lactobacillus delbrueckii ssp. bulgaricus. Lactobacillus bulgaricus, an important species of lactic acid bacteria with health benefits, is used in the production of yogurt and other fermented foods. Our results showed that supplementing reinforced clostridial medium with 0.025% CaCl₂, 0.01% uracil, and 0.2% Tween 80 (mRCM) significantly enhanced the growth rate of L. bulgaricus RR and ATCC 11842 strains as measured by the optical densities of these strains after 12 h of incubation at 42°C. The bacterial populations (plate count) of the RR and ATCC 11842 strains were 0.76 and 0.77 log cfu/g higher in mRCM than in de Man, Rogosa, and Sharpe and reinforced clostridial medium media, respectively. Conversely, the population counts for other bacterial species (Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri) were significantly inhibited in the mRCM medium. The addition of aniline blue dye to mRCM (mRCM-blue) improved the selectivity of L. bulgaricus in mixed lactic bacterial cultures compared with de Man, Rogosa, and Sharpe medium and lactic agar with regard to colony appearance and morphology. The mRCM-blue performed better than the conventional medium in culturing, enumerating, and differentiating L. bulgaricus. Therefore, mRCM-blue could be used as a selective medium to enhance the growth and differentiation of L. bulgaricus in order to meet the increasing demand for this beneficial species of bacteria.     

Effects of glutathione on acid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Streptococcus thermophilus SDMCC18 contains a novel bi-functional glutathione synthetase gene gshF, and it can produce glutathione (GSH). In the present study, we demonstrated that the produced GSH by S. thermophilus SDMCC18 could protect cells against acid challenge and be secreted into the medium. Moreover, using a gshF-defective mutant strain as the control, we found that the GSH conferred a protective role against acid stress to Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. In addition, L. bulgaricus ATCC11842 could import the exogenous GSH into the cytoplasm, leading to an improved growth of strain ATCC11842. Taken together, our study reported a novel proto-cooperation relationship between S. thermophilus and L. bulgaricus in yoghurt fermentation.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Comparison of Associative Growth and Proteolytic Activity of Yogurt Starters in Whole Milk from Camels and Cows

Growth and proteolytic activities were studied using yogurt starter cultures incubated in pasteurized whole milk from camels and cows at 42°C as single and mixed cultures. In general, the growth of four strains of Streptococcus thermophilus and three strains of Lactobacillus delbrueckii ssp. bulgaricus was higher in cow milk than in camel milk. However, proteolysis was higher in camel milk than in cow milk. Lactobacillus delbrueckii ssp. bulgaricus LB12 in combination with streptococci had lowered pH more than did the other lactobacilli. Mixed cultures released the same amount of free amino groups as the corresponding single cultures, except for L. delbrueckii ssp. bulgaricus LB12.     

Inactivation of Escherichia coli K-12 in Apple Juice Using Combination of High-Pressure Homogenization and Chitosan

ABSTRACT:  Apple juice and apple cider were inoculated with Escherichia coli K-12 and processed using a high-pressure homogenizer to study bacterial inactivation. Seven levels of pressure ranging from 50 to 350 MPa were used in the high-pressure homogenizer. Two types of chitosan (regular and water soluble) with 2 levels of concentration 0.01% and 0.1% were investigated for synergistic effect with high-pressure homogenization for the bacterial inactivation. E. coli K-12 inactivation was evaluated as a function of homogenizing pressure at different concentration of 2 types of chitosan in apple juice and cider. High-pressure homogenization (HPH) induced significant inactivation in the range of 100 to 200 MPa, while thermal inactivation was the primary factor for the bacterial inactivation above 250 MPa. Significant ( P < 0.05) 2-way interactions involving pressure and type of substrate or pressure and chitosan concentration were observed during the study. The homogenization pressure and the incremental quantity of chitosan (both types) acted synergistically with the pressure to give higher inactivation. Significantly ( P < 0.05) higher inactivation was observed in apple juice than apple cider at same homogenizing pressure. No effect of type of chitosan was observed on the bacterial inactivation.     

Effect of High-Pressure Homogenization on the Structure of Cassava Starch

Cassava starch suspension was homogenized at different pressures (0, 20, 40, 60, 80, and 100 MPa) with a high-pressure homogenizer. To investigate the effect of high-pressure homogenization on the structure of cassava starch, the samples were characterized using microscopy, laser scattering, and X-ray diffraction techniques, with native and heat gelatinized cassava starches as controlled samples. The temperature of starch suspension increased linearly with applied pressure at a rate of 0.187°C/MPa. Microscopy studies showed that cassava starch was partly gelatinized after high-pressure homogenization, and the degree of gelatinization increased with homogenizing pressure. Results of laser scattering measurements suggested a considerable increase in particle size after homogenization at 100 MPa as a result of granule swelling. The X-ray diffraction pattern showed that there was no evident change after homogenization suggesting that the crystalline structure of starch granules was resistant to high-pressure homogenization.     

Effect of Ultra-high-pressure Homogenization on Structure and on Rheological Properties of Soy Protein-stabilized Emulsions

ABSTRACT: An ultra high-pressure homogenizer (20 to 350 MPa) was used to realize fine food emulsions stabilized by soy proteins. The first aim of the work was to understand how dynamic high-pressure processing affects soybean globulin conformation. Then, the effect of homogenizing pressure on the emulsions structure and rheology was investigated. High-pressure homogenization caused denaturation of proteins due to strong mechanical forces and high temperatures encountered in the valve. Droplet sizes of emulsions were greatly reduced with high-pressure homogenization and Newtonian liquid emulsions were converted into shear-thinning emulsion gels by homogenization at pressures above 250 MPa. Hydrophobic interactions between proteins were supposed to cause the gel-like network structure of emulsions.     

Formation of nanoemulsions stabilized by model food-grade emulsifiers using high-pressure homogenization: Factors affecting particle size

Nanoemulsions are finding increasing utilization in the food and beverage industries for certain applications because of their unique physicochemical and functional properties: high encapsulation efficiency; low turbidity; high bioavailability; high physical stability. In this study, we examined the impact of system composition and homogenization conditions on the formation of nanoemulsions using a high-pressure homogenizer (microfluidizer). The mean particle diameter decreased with increasing homogenization pressure and number of passes, with a linear log–log relationship between mean particle diameter and homogenization pressure. The minimum droplet diameter that could be produced after 6 passes at 14 kbar depended strongly on emulsifier type and concentration: SDS < Tween 20 < β-lactoglobulin < sodium caseinate. Small-molecule surfactants formed smaller droplets than proteins, which was attributed to their ability to rapidly adsorb to the droplet surfaces during homogenization. The impact of phase viscosity was examined by using different octadecane-to-corn oil ratios in the oil phase and different glycerol-to-water ratios in the aqueous phase. The minimum droplet size achievable decreased as the ratio of disperse phase to continuous phase viscosities (η_D/η_C) decreased for SDS-stabilized emulsions, but was relatively independent of η_D/η_C for β-lactoglobulin-stabilized emulsions. At low viscosity ratios, much smaller mean droplet diameters could be achieved for SDS (d ∼ 60 nm) than for β-lactoglobulin (d ∼ 150 nm). The information reported in this study will facilitate the rational design of food-grade nanoemulsions using high-pressure homogenization methods.     

Proteolytic profiles of yogurt and probiotic bacteria

Nine strains of Streptococcus thermophilus, 6 strains of Lactobacillus delbrueckii ssp. bulgaricus, 14 strains of Lactobacillus acidophilus and 13 strains of Bifidobacterium spp. were screened for proteolytic, amino-, di-, tri- and endopeptidase activity by using the o-pthaldialdehyde-based spectrophotometric assay. Strains showing the highest and lowest proteolytic activity were further studied for their peptidase activities at the extracellular and intracellular levels. The amounts of free amino groups released by S. thermophilus, L. delbrueckii ssp. bulgaricus and L. acidophilus strains were higher than that by Bifidobacterium strains. Aminopeptidase activity was detected for all bacterial strains both at the extracellular and intracellular levels. The specific activity towards the six substrates studied was higher at the intracellular level for all strains. High dipeptidase activity was demonstrated by all bacterial strains for L. delbrueckii ssp. bulgaricus, L. acidophilus, and Bifidobacterium spp. whereas S. thermophilus had greater dipeptidase activity at the extracellular level. All bacterial cultures tested were able to hydrolyse large biologically active peptides, bradykinin, Ala–Ala–Ala–Ala–Ala and the tripeptide substrate Gly–Ala–Tyr at both the extracellular and intracellular levels. However, with the substrates ending with a C-terminal of phenylalanine, the hydrolysis only occurred at the intracellular level.     

Emulsions stabilized by heat-treated collagen fibers

The emulsifying properties of collagen fiber were modified by heat treatment at temperatures ranging from 50 to 85 °C for 20 or 60 min. In addition to heat treatment, the influence of pH (3.5 and 9.2) and the emulsifying process (rotor-stator device and high-pressure homogenizer) were evaluated on oil-in-water emulsions stabilized by collagen fiber through visual analysis (stability), microstructure and rheological measurements. Emulsions homogenized using solely the rotor-stator device showed phase separation and a larger mean droplet size (d₃₂), except for the emulsion composed by non-heated collagen fiber. The alkaline emulsions showed lower kinetic stability, since collagen fibers have a lower net charge (zeta potential) at higher pH values, decreasing the electrostatic stability process. Heat treatment slightly decreased the protein charge and significantly reduced the insoluble protein content, suggesting a decrease in the emulsifying properties of the collagen fiber. The use of high-pressure homogenization (20-100 MPa) made it possible to produce acid emulsions with a reduced droplet size and distribution. At 20 MPa, the emulsions showed a higher d₃₂ value (between 3.17 and 1.18 μm), while at 60 and 100 MPa the emulsions presented lower d₃₂ values (between 0.74 and 0.94 μm) without any significant variation between the different heat-treated collagen fibers, but showing a noticeable decrease in emulsion viscosity and elasticity with increases in the homogenization pressure and heat treatment.     

Exploring the industrial potential of Lactobacillus delbrueckii ssp. bulgaricus by population genomics and genome-wide association study analysis

Lactobacillus delbrueckii ssp. bulgaricus is one of the most commonly used starter cultures for yogurt production. However, how its genetic background affects acid production capacity is unclear. This study investigated the industrial potential of L. delbrueckii ssp. bulgaricus using population genomics and GWAS analysis. To meet our goal, population genetics and functional genomics analyses were performed on 188 newly sequenced L. delbrueckii ssp. bulgaricus strains isolated from naturally fermented dairy products together with 19 genome sequences retrieved from the NCBI database. Four distinct clusters were identified, and they were correlated with the geographical sites where the samples were collected. The GWAS analysis about acidification fermentation results with sucrose-fortified reconstituted skim milk revealed a significant association between l-lactate dehydrogenase (lldD; Ldb2036) and the bacterial acid production rate. Our study has broadened the understanding of the population structure and genetic diversity of L. delbrueckii ssp. bulgaricus by identifying potential targets for further research, development, and use of lactic acid bacteria in the dairy industry.     

β-Carotene nanodispersions: preparation,characterization and stability evaluation

The aim of the present study was to investigate the preparation of β-carotene nanodispersions as potential active ingredients for food formulations. Nanodispersions containing β-carotene were obtained by a process based on an emulsification–evaporation technique. The preparation method consisted of emulsifying an organic solution of β-carotene in an aqueous solution containing emulsifier using two different homogenizers (a conventional homogenizer and a microfluidizer), followed by direct solvent evaporation under reduced pressure. The influence of different homogenizing conditions (pressure and cycle) and two organic/aqueous phase ratios on particle size parameters and content of β-carotene was investigated. In addition, the stability of β-carotene nanodispersions was carried out at a storage temperature of 4 °C. The particle size distribution of β-carotene in nanodispersions was demonstrated with a laser diffraction particle size analyzer and the retention of β-carotene in the prepared nanodispersions was studied by high-pressure liquid chromatography. In general, homogenization pressure and cycle had significant (P < 0.05) effects on various particle size parameters. A volume-weighted mean diameter (D_4,3) of β-carotene nanoparticles, ranging from 60 to 140 nm, was observed in this study.     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

高压均质对大豆β-伴球蛋白结构及功能特性的影响

使用差示扫描量热仪、扫描电镜和傅里叶变换红外光谱仪研究高压均质后大豆β-伴球蛋白的结构和功能特性。结果表明：大豆β-伴球蛋白的变性温度为(74±1)℃,高压均质后大豆β-伴球蛋白粒径明显变小,经20MPa和30MPa均质处理后,大豆β-伴球蛋白结构发生了明显变化。高压均质可有效提高大豆β-伴球蛋白溶解性、乳化活性和乳化稳定性,并降低乳析率。经20MPa和35MPa压力处理的大豆β-伴球蛋白的保水性明显提高,经25MPa和30MPa压力处理的大豆β-伴球蛋白的持油性明显提高。     

Survival of Lactic Acid Bacteria and Their Lactases under Acidic Conditions

Effects of sonication, survival, and β-galactosidase activity of four lactic cultures were investigated in pH 1.5-3.5 range. Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophilus exhibited the highest β-galactosidase activity in skim milk and broth systems, respectively. The β-galactosidases from L. delbruekii subsp. bulgaricus, S. thermophilus, and L. acidophilus showed optimum activity in the neutral pH range and 55°C. Viable count of all four cultures decreased most rapidly at pH 1.5, but L. acidophilus and L. delbruekii subsp. bulgaricus survived better than the other organisms. The decrease of enzyme activity of unsonicated cultures with pH was slight, especially at pH 3.5. However, acidification of sonicated cultures to pH 3.5 or lower resulted in rapid and permanent loss of enzyme activity.