Configuration of a bioreactor for milk lactose hydrolysis

Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications. Immobilization and process recycling can compensate for the low specific activity of this preparation. For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity. We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen. A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention. The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol. The unused biocatalysts stored in milk did not lose activity after 50 d. However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity. A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst. The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor. The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.     

Quantification of lactose using ion-pair RP-HPLC during enzymatic lactose hydrolysis of skim milk

The correct labelling of dairy foods as “lactose-free” requires a suitably sensitive and valid analytical method for the quantification of lactose in complex food matrices. Thus, an ion-pair RP-HPLC method for the simultaneous determination of lactose, glucose and galactose in original skim milk was investigated. The samples derived from an enzymatic lactose hydrolysis approach (0.5 L) using the commercial β-galactosidase Godo-YNL2. After derivatisation with p-aminobenzoic acid and sodium cyanoborohydride, the samples were injected on a RP-C₁₈ column. Tetrabutylammonium hydrogen sulphate was used as the ion-pair reagent in the eluent system. The sugars were quantified using photometric- (UV; 303 nm) and fluorescence-detection (λ_ex 313 nm, λ_em 358 nm). The overall run time was 27 min. The limits of detection (LOD) were estimated at 2 mg L⁻¹ (UV detection) and at 0.13 mg L⁻¹ (fluorescence detection). The limits of quantification were 6 mg L⁻¹ (UV detection) and 0.45 mg L⁻¹ (fluorescence detection). Thus, this analytical method is suitable for sensitive lactose quantification in milk systems of less than 10 mg L⁻¹.     

一种高温乳糖酶的酶学性质及对牛乳中乳糖的水解

对重组大肠杆菌产生的β-半乳糖苷酶学性质的研究表明，该酶最适反应温度为55℃，最适PH值为7．0，在50℃时具有良好的热稳定性，K^ ,Mg^2 ,Mn^2 等对酶有一定的激活作用，而重金属盐如Pb^2 ,Sn^2 ,Zn^2 ,Cu^2 等对酶活有一定抑制作用，对乳糖分解试验研究表明，该酶水解乳糖的能力较强，牛奶中添加2NLU／mL时，55℃保温水解2h可达到55％以上的水解率。     

耐热β-半乳糖苷酶重组菌WB600/pMA5-bgaB发酵条件的研究

耐热β-半乳糖苷酶相比酵母来源的中温乳糖酶用于低乳糖牛奶的生产具有潜在的优越性。在已将来源于嗜热脂肪芽孢杆菌的耐热β-半乳糖苷酶基因(bgaB)克隆入枯草芽孢杆菌得到重组菌WB600/pMA5-bgaB后,对此重组菌的发酵条件进行了研究。重组菌WB600/pMA5-bgaB在发酵过程中具有良好的遗传稳定性,可溶性淀粉和胰蛋白胨是重组菌生长和酶活表达的适合碳源和氮源。WB600/pMA5-bgaB适宜的摇瓶培养条件为接种量3%～5%,装液量30～50/250 mL(三角瓶),起始pH7.0,摇床转速220r/min。在7L反应器中进行分批发酵,研究表明,pH调控和溶氧控制对提高工程菌发酵产酶具有明显帮助,pH控制在7.0,溶氧控制在50%可提高发酵酶活;补料培养后菌体密度OD_(600)可达到47,酶活达到37.5U/mL,比在初始条件下提高了10倍。     

Identification of lactose ureide, a urea derivative of lactose, in milk and milk products

With the widespread consumption of milk, the complete characterization of the constituents of milk and milk products is important in terms of functionality and safety. In this study, a novel nonreducing carbohydrate was separated from powdered skim milk and was identified using electron spray ionization-mass spectrometry (m/z 385.1[M + H⁺]), ¹H, ¹³C, ¹H¹H-correlation spectroscopy, and heteronuclear single quantum-nuclear magnetic resonance spectra. The carbohydrate was identified as a lactose derivative of urea, N-carbamoyl-o-β-d-galactopyranosyl-(1–4)-d-glucopyranosylamine (lactose ureide, LU). For the HPLC analysis of LU in milk and milk products, benzoylated LU, hepta-o-benzoyl lactose ureide (melting point 137–139 °C; m/z 1,113 [M + H⁺]; wavelength of maximum absorption, λ_max, 229 nm; molar extinction coefficient, ε, 8.1037 × 10⁷), was used as a standard. The crude nonreducing carbohydrate fraction from raw milk, thermally processed milk, and milk products such as powdered milks were directly benzoylated and subjected to HPLC analysis using an octadecylsilyl column to determine the quantity of LU. The content of LU in 10% solutions of powdered skim milk and powdered infant formula (5.0 ± 1.1 and 4.9 ± 1.5 mg/L, respectively) were almost 3-fold higher than that of UHT milk (1.6 ± 0.5 mg/L) and higher than that of low-temperature, long-time-processed (pasteurized at 65 °C for 30 min) milk (1.2 ± 0.3 mg/L) and the fresh raw milk sample (0.3 ± 0.1 mg/L). A time-course of the LU content in raw milk during heating at 110 °C revealed that LU increased with time. From these results, it is likely that LU is formed by the Maillard-type reaction between the lactose and urea in milk and milk products. Because the concentration of LU in milk increased with the degree of processing heat treatment, it could serve as an indicator of the thermal deterioration of milk. Although it is known that the human intestine is unable to digest LU, the gastrointestinal bacteria in human subjects are able to digest and utilize urea nitrogen in formation of essential amino acids that are available to the host human. These findings suggest that LU in milk might have a functional role in human health.     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

Feeding lactose to increase ruminal butyrate and the metabolic status of transition dairy cows

Twenty-four multiparous Holstein cows (775 ± 24 kg body weight; 3.4 ± 0.11 body condition score) were used in a randomized complete block design experiment to determine the impact of increased ruminal butyrate from the fermentation of lactose on metabolism and lactation. Dietary treatments were either a corn-based control diet (CON) or a diet containing lactose at 15.7% of diet dry matter (LAC). Experimental diets were fed from 21 d before expected calving through 21 d in milk (DIM). Blood was sampled at −21, −14, −7, −2, 2, 7, 14, and 21 DIM, rumen fluid at −21, −7, and 7 DIM, and liver tissue via biopsy at 7 and 14 DIM. Pre- and postpartum dry matter intake (DMI) through 28 DIM averaged 12.8 and 17.7 kg/d, respectively, and did not differ between treatments; however, cows fed LAC did not exhibit a prepartum decrease in DMI. Milk yield was unaffected by treatments and averaged 45.7 kg/d during the first 70 DIM. Plasma glucose, insulin, and non-esterified fatty acids were not affected by dietary treatments. Feeding LAC increased the ruminal proportion of butyrate both pre- (11.3 vs. 9.2 ± 0.45%) and postpartum (13.0 vs. 10.3 ± 0.67%). Likewise, circulating plasma β-hydroxybutyrate was increased both pre- (6.1 vs. 4.2 ± 0.31 mg/dL) and postpartum (14.6 vs. 8.34 ± 1.7 mg/dL) when feeding LAC compared with CON. Liver lipid content was decreased (8.6. vs. 14.7 ± 1.5% of wet weight) in cows fed LAC relative to those fed CON, whereas liver glycogen was not affected by dietary treatments. Feeding lactose to transition dairy cows increased the proportion of butyrate in the rumen and β-hydroxybutyrate in plasma and decreased liver lipid but did not affect lactation performance.     

Production,purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1

Food Science and Biotechnology - Bacillus methanolicus LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized...     

耐高温β-半乳糖苷酶的分离纯化与酶学性质分析

为从嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)XG24 发酵液中纯化到β- 半乳糖苷酶,并对酶学性质进行研究,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow 阴离子交换层析和Sephadex G-75 分子筛凝胶过滤层析等方法进行分离纯化。结果表明：经过系列步骤纯化后,酶纯度提高了54.5 倍,回收率20.4%,酶比活力达32.7U/mg。以邻- 硝基酚-D- 半乳糖吡喃糖苷(ONPG)为底物,研究β- 半乳糖苷酶的酶学性质。最适pH6.5,最适作用温度65℃。此菌株产β- 半乳糖苷酶在70℃以下和pH4.0～8.0 范围内具有较好的稳定性;Mg2+、Mn2+、Fe2+和Co2+ 对此酶有明显激活作用,而Cu2+、Ag+、Hg2+ 几乎完全抑制酶活性。以ONPG 为底物酶的Km 值为4.32mmol/L。SDS-PAGE 和凝胶过滤层析测得酶蛋白为单肽链蛋白,表观分子质量64kD。因此,嗜热脂肪芽孢杆菌XG24 β- 半乳糖苷酶在乳制品工业中具有潜在的应用价值。     

Construction of an enzymatic route using a food-grade recombinant Bacillus subtilis for the production and purification of epilactose from lactose

Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure.     

Effect of genetic variation on the tryptic hydrolysis of bovine beta-lactoglobulin A, B, and C

The structure, stability, and hydrolysis characteristics of beta-lactoglobulin (LG) A are different from those of either beta-LG B or beta-LG C. Purified samples of these proteins were hydrolyzed with trypsin and the rates of loss of native monomeric beta-LG structure were measured using sodium dodecyl sulfate PAGE. At the same time, the appearance of many individual peptides were identified and followed in time by HPLC, measuring their concentration as a function of solution pH, temperature, protein concentration, and added urea or palmitate. The identity of the peptides was confirmed by liquid chromatography-mass spectrometry. This semiquantitative exploration showed that the rate of hydrolysis was in the order beta-LG A > beta-LG B > beta-LG C under most circumstances, and that 12 of the 18 trypsin-susceptible bonds were cleaved at very similar rates that were governed by the variant type. Consequently, the rate of hydrolysis of the intact protein was related to the overall structural stability of the individual proteins and the accessibility of certain peptide bonds to the enzyme. The hydrolysis of mixtures of 2 or more variants or of denatured beta-LG gave more heterogeneous peptide mixtures.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Hydrolysis of lactose in whey permeate for subsequent fermentation to ethanol

Fermentation of lactose in whey permeate directly into ethanol has had only limited commercial success, as the yields and alcohol tolerances of the organisms capable of directly fermenting lactose are low. This study proposes an alternative strategy: treat the permeate with acid to liberate monomeric sugars that are readily fermented into ethanol. We identified optimum hydrolysis conditions that yield mostly monomeric sugars and limit formation of fermentation inhibitors such as hydroxymethyl furfural by caramelization reactions. Both lactose solutions and commercial whey permeates were hydrolyzed using inorganic acids and carbonic acid. In all cases, more glucose was consumed by secondary reactions than galactose. Galactose was recovered in approximately stoichiometric proportions. Whey permeate has substantial buffering capacity-even at high partial pressures (>5500 kPa[g]), carbon dioxide had little effect on the pH in whey permeate solutions. The elevated temperatures required for hydrolysis with CO2-generated inhibitory compounds through caramelization reactions. For these reasons, carbon dioxide was not a feasible acidulant. With mineral acids reversion reactions dominated, resulting in a stable amount of glucose released. However, the Maillard browning reactions also appeared to be involved. By applying Hammet's acidity function, kinetic data from all experiments were described by a single line. With concentrated inorganic acids, low reaction temperatures allowed lactose hydrolysis with minimal by-product formation and generated a hexose-rich solution amenable to fermentation.     

Enhancement of lactase activity in milk by reactive sulfhydryl groups induced by heat treatment

The effects of heat treatments of milk and whey prior to lactose hydrolysis with Kluyveromyces lactis beta-galactosidase were studied. It was observed that heat treatment of milk significantly increases lactase activity, with a maximum activity increase found when milk was heated at 55 degrees C. In whey from 55 up to 75 degrees C, beta-galactosidase activity decreased slightly. Nevertheless, heating whey at 85 degrees C for 30 min raised the rate of hydrolysis significantly. Electrophoretic patterns and UV spectra proved that the activity change correlated with milk protein denaturation, particularly that of beta-lactoglobulin. Heating whey permeate did not increase the enzyme activity as heating whole whey; but heating whey prior to ultrafiltration also resulted in enzyme activation. Measurement of free sulfhydryl (SH) groups in both whey and heated whey permeate showed that the liberation of free SH is highly correlated to the change of the activity. Furthermore, this activation can be reversed by oxidizing the reactive sulfhydryl groups, proving that the observed effect may be related to the release of free SH to the medium, rather than to the denaturation of a thermolabile protein inhibitor.     

Optimization of a dual-functional biocatalytic system for continuous hydrolysis of lactose in milk

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Nucleotide sequencing,purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.     

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.     

葵花籽仁酶解体系中绿原酸的分离纯化技术

葵花籽仁富含绿原酸,以水酶法提取葵花籽油后的酶解液为原料,采用乙醇沉淀法去除酶解液中的多糖,滤液超滤浓缩后得到绿原酸粗品。然后,分别采用乙酸乙酯法、正丁醇法、石-硫沉淀法、大孔树脂法纯化绿原酸,以紫外分光光度法测定提取物中绿原酸含量,结果表明,乙酸乙酯法纯化效果最好,绿原酸纯度达89.88%。