Lactose Hydrolysis Research Articles

Biotechnological application of Aspergillus oryzae β-galactosidase immobilized on glutaraldehyde modified zinc oxide nanoparticles

The current research demonstrates the synthesis of zinc oxide nanoparticles (ZnO-NPs) via green nanotechnology approach (Azatirachta indica leaves). The size of the synthesized ZnO-NPs was confirmed as 27 nm by TEM. Glutaraldehyde was used to modify the surface of the developed ZnO-NPs in order to promote covalent binding of Aspergillus oryzae β-galactosidase. Enzyme activity was achieved as 93% on glutaraldehyde modified ZnO-NPs. The immobilized enzyme exhibited significant enhancement in activity under extreme temperature and pH variations, as compared to the soluble β-galactosidase (SβG). It was further observed that the immobilized enzyme retained 58% activity at 5% galactose concentration. However, under similar experimental conditions, SβG showed 27% activity. Reusability of immobilized enzyme revealed that it retained 89% activity even after fifth repeated use, and hence could be recovered easily by centrifugation for repeated use in biotechnological applications. Batch reactor experiment indicates that the immobilized enzyme displayed 81% and 70% lactose hydrolysis at 50 °C and 60 °C, respectively as compared to 70% and 58% lactose hydrolysis by soluble enzyme under identical conditions after 9 h.

Stabilization of β-Galactosidase Encapsulated in Pectin-Alginate Hydrogel and Hydrolysis of Whey Lactose and Whole Milk

The operational conditions of β-galactosidase encapsulated in pectin-alginate hydrogel were investigated as well as its catalytic activity in the hydrolysis of lactose from whey and whole milk. β-Galactosidase encapsulated in pectin-alginate hydrogel showed the best results to yields and diffusional effect and pH range. Thermostability showed considerable income gain for half-life (1.7-fold), activation energy of denaturation (31.6-fold), activation of denaturation (217.3-fold at 40 ºC; 266.8-fold at 50 ºC; 345.6-fold at 60 ºC), and entropy of activation of denaturation (−42.0 J mol-1 K-1). Reusability of encapsulated β-galactosidase was observed in 8-cycles for the milk whey lactose hydrolysis (51.9%) and 7-cycles for whole milk (55.6%), and the lactose hydrolysis was observed in 13% in milk whey and 10.3% in whole milk after 10 cycles. These results revealed an industrial application potential of β-galactosidase encapsulated in pectin-alginate hydrogel for lactose hydrolysis of milk whey and whole milk processes.

Development of lactose hydrolyzed milk using micro fluidization assisted crudeβ-galactosidase enzyme of Lactobacillus acidophilus

The objectives of this study were to screen maximumβ-galactosidase enzyme producing Lactobacillus strain out of seven commercial lactobacilli and development of lactose hydrolyzed milk using micro fluidization assisted crude β-galactosidase enzyme from selected maximum β-galactosidaseenzyme producing Lactobacillus strain. From all the screening methods L. acidophilus ATCC 4356 culture was found to possess the maximum β-galactosidase enzyme, so this culture was selected for further studies. Crude β-galactosidase enzyme extract (CEE)was obtained from L. acidophilus ATCC 4356 using microfluidization as cell disruption method. A total of 37.41 (μmol/ mL/min) enzyme activity was obtained from crude extract. Lactose hydrolysis in milk was done using different concentrations (0.5,1 and 1.5%) CEE of L. acidophilus ATCC 4356. CEE@1.5%showed highest 39.96% hydrolysis of lactose when compared with other CEE added milk after 8 h. There was significant difference found when sensory scores were recorded for lactose hydrolyzed milk obtained by using crude enzyme extracts concentrations @1.5% CEE of L. acidophilus ATCC 4356 and1% commercial enzyme. Lactose hydrolyzed milk was successfully developed using crude enzyme extract and being an economical, innovative and therapeutic product, large scale production of the product can be taken up by large players of the field in future.

Design of supported organocatalysts from a biomass-derived difuran compound and catalytic assessment for lactose hydrolysis.

The engineered structures and active sites of enzyme catalysts give rise to high catalytic activity and selectivity toward desired reactions. We have employed a biomass-derived difuran compound to append N-substituted maleimides with amino acid (glutamic acid) substitution by Diels-Alder reaction to mimic the chemical functional groups that comprise the active site channels in enzyme catalysts. The difunctionality of the biomass-derived difuran allows production of Diels-Alder adducts by appending two amino acid moieties to form a difunctional organocatalyst. The catalytic activity of the organocatalyst can be improved by immobilizing the organocatalyst on solid supporting materials. Accordingly, the structures of these immobilized organocatalysts can be engineered to mimic enzymatic active sites and to control the interaction between reactants, products, and transition states of catalytic reactions. Lactose hydrolysis was carried out to provide an example of industrial application of this approach to design and fabricate new supported organocatalysts as artificial enzymes.

EFFECTS OF LACTOSE HYDROLYSIS AND MILK TYPE ON THE QUALITY OF LACTOSE-FREE YOGHURT

The purpose of the work was to investigate the influence of different lactose hydrolysis processes, the contribution of the enzyme and the milk type on the characteristics of the obtained lactose-free yogurt. The analysis was performed on non-hydrolyzed yogurt (control sample), the pre-hydrolyzed yogurt (that was hydrolyzed before fermentation), and the co-hydrolyzed yogurt (concurrent addition of β-galactosidase and starter culture). According to the obtained results, at the end of the fermentation time, an advanced hydrolysis degree was reached (over 80%) both for yogurt samples obtained from pre-hydrolyzed milk and obtained by co-hydrolysis. The optimal method from the economic point of view is to obtain yogurt by co-hydrolysis. The sensory quality of the yogurt samples obtained from hydrolyzed milk by co-hydrolysis was characterized by a better flavor than the control sample, for yogurt from both types of milk. This may be due to the availability of a greater amount of glucose for the production of aromatic compounds, a sweeter taste than natural yogurt, with a light caramel flavor, a firm coagulum, a porcelain appearance, without whey removal. Cow’s milk yogurt showed higher viscosity values to goat’s milk yogurt for both pre-hydrolyzed and co-hydrolyzed milk. Lactose hydrolysis determined the reduction of the syneresis index of the yogurt compared to the control samples. The studies led to the development of lactose-free yogurt with improved sensory and rheological properties recommended for people with lactose intolerance.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Kluyveromyces lactis strain GD-YNL.

The food enzyme β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) is produced with the non-genetically modified Kluyveromyces lactis strain GD-YNL by Godo Shusei Co., Ltd. The food enzyme is intended to be used for the hydrolysis of lactose in milk processing, production of fermented milk products and whey processing. The food enzyme is also intended for lactose hydrolysis in milk products at home. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 54mg TOS/kg body weight per day in European populations. As the production strain qualifies for the qualified presumption of safety approach of safety assessment and as no issue of concern raised from the production process, no toxicological studies other than assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Study of UF-retentate concentration degree in low- and lactose-free products’ development

The article provides a comparative evaluation of some organoleptic characteristics of low- and lactose-free milk samples produced on the basis of ultrafiltration concentrates of skim milk with different mass fraction of solids. Using the ranking method, it was found that the sample produced on the basis of UV concentrate with a mass fraction of solids of 27 % followed by enzymatic hydrolysis of lactose has the best indices of taste and sweetness. The results obtained were necessary for the development of the recipe and technology for the production of low- and lactose-free milk.

Исследование особенностей культивирования молочнокислых бактерий в подсырной сыворотке и уфпермеате для получения β-галактозидаз

Lactic acid microorganisms are potential sources of the β-galactosidase enzyme, which can be used for the hydrolysis of lactose in the production of low- and lactose-free food products, as well as a catalyst in the biosynthesis of lactulose, galactooligosaccharides, and other valuable food additives. The need of lactic acid bacteria for the presence in the cultivation medium of additional sources of carbon, nitrogen, and other components significantly increases the cost of bacterial β-galactosidases. Lactose-containing raw materials, in particular whey and its UV permeate, are a promising and fairly cheap analogue of expensive complex media for the cultivation of lactic acid microorganisms. In this work, the features of cultivation of various types of lactic acid bacteria (Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus LGG, Lactococcus lactis ssp.) in different types of secondary milk raw materials (whey and UV permeate) were studied.. It has been shown that cheese whey is a more favorable medium for their growth than UV permeates. The highest concentration of lactic acid was obtained in experiments with L. acidophilus. To accelerate the growth of lactic acid bacteria in the permeate; it was proposed to use the addition of 2% peptone.

Determination of lactose in milk by high resolution ultrasonic spectroscopy

The application of high-resolution ultrasonic spectroscopy for determination of the amount of lactose in milk and milk-based drinks is described. The methodology is based on high precision real time ultrasonic monitoring of hydrolysis of lactose in milk by the enzyme β-galactosidase, allowing for quantitation of lactose present at reaction completion. The method can also be used to control in real-time the hydrolysis of lactose for the precise preparation of milks with preferred lactose concentrations. The elimination of sample preparation steps and the minimal need for user interaction represent significant simplification and time savings over existing methods. Comparison of the ultrasonic data with results from HPLC-RI showed excellent agreement in the determination of lactose in normal, skimmed and chocolate milks.

Copper inhibits postacidification of yogurt and affects its flavor: A study based on the Cop operon

Yogurt and its related products are popular worldwide. During transportation and storage, Lactobacillus delbrueckii ssp. bulgaricus in yogurt continues to metabolize to form lactic acid, the postacidification phenomenon of yogurt. Postacidification of yogurt is a widespread phenomenon in the dairy industry. Many scholars have done research on controlling the postacidification process, but few report on the molecular mechanisms involved. In this study, we used a molecular-assisted approach to screen food additives that can inhibit postacidification and analyzed its effects on yogurt quality as well as its regulatory mechanism from multi-omics perspectives in combination. The copper ion was found to upregulate the expression of the LDB_RS05285 gene, and the copper transporter-related genes were regulated by copper. Based on the metabolic-level analysis, copper was found to promote lactose hydrolysis, accumulate a large amount of glucose and galactose, inhibit the conversion of glucose to lactic acid, and reduce the production of lactic acid. The significantly greater abundance of l-isoleucine and l-phenylalanine increased the abundance of 3-methylbutyraldehyde (∼1.2 times) and benzaldehyde (∼7.9 times) to different degrees, which contributed to the formation of the overall flavor of yogurt. Copper not only stabilizes the acidity of yogurt, but also it improves the flavor of yogurt. Through this established method involving quantitative and correlation analyses at the transcriptional and metabolic levels, this study provides guidance for the research and development of food additives that inhibit postacidification of yogurt and provide a reference for studying the changes of metabolites during storage of yogurt.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Aspergillus sp. strain GD-FAL.

The food enzyme β-galactosidase (EC 3.2.1.23) is produced with the non-genetically modified Aspergillus sp. strain GD-FAL by Godo Shusei Co., Ltd. The food enzyme is intended to be used in milk processing for the hydrolysis of lactose. The absence of viable cells of the production organism in the food enzyme was not demonstrated. Based on the assumption that all milk/dairy products are enzymatically treated, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.301mg TOS/kg body weight per day in European populations. The data provided did not allow concerns of genotoxicity of the food enzyme to be excluded. The systemic toxicity could not be assessed in the absence of an appropriate repeated dose 90-day oral toxicity study. Consequently, a margin of exposure was not calculated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the remaining concerns on genotoxicity, the inadequacies of the 90-day repeated dose oral toxicity study in rats and the missing data regarding the absence of viable cells of the production strain in the food enzyme, the Panelcould not conclude on the safety of this food enzyme.

Estimation of selectivities in transglycosylation systems with multiple transglycosylation products

Transglycosylation can be used to produce oligosaccharides that act as prebiotics. The maximum yield of the oligosaccharides depends on the selectivities for transglycosylation, not only over primary hydrolysis of the original donor molecule, but also over secondary hydrolysis of the oligosaccharides. A recent approach to determining these selectivities only considered systems in which there is a single transglycosylation product. The current work extends this approach, determining selectivities for cases involving multiple transglycosylation products. A model is used to analyze literature data for the production of galactooligosaccharides through the transgalactosylation of lactose, catalyzed by β-galactosidases of Bacillus circulans and Aspergillus oryzae. In the case studies, the selectivity for using oligosaccharides rather than lactose as the galactosyl acceptor ranged from 1.0 (i.e. not selective) to 2.4. Selectivities for transgalactosylating lactose with lactose over primary hydrolysis of lactose ranged from 113 to 919. The selectivities for producing oligosaccharides over secondary hydrolysis of the oligosaccharides decreased with increase in the initial lactose concentration; the values obtained ranged from 6 to 159. Knowledge of these selectivities is useful for guiding the improvement of enzymes for transglycosylation processes.

Immobilisation of β-galactosidase onto double layered hydrophilic polymer coated magnetic nanoparticles: Preparation, characterisation and lactose hydrolysis

The surface of polydopamine (PDA) coated magnetic particles was grafted spontaneously with diaminopolyethylene glycol (DAPEG) as a second polymer layer by conjugation of PDA with DAPEG without using any activating agent. The amount of immobilised β-galactosidase was found to be 49.7 mg g−1 support. The optimum pH for the free and immobilised enzyme was observed at 5.5 and 6.0, respectively. The optimal temperature for the immobilised β-galactosidase was 15 °C higher compared with the free counterpart. The apparent Km values for the free and immobilised enzymes were found to be 3.69 and 4.35 mmol L−1, with Vmax values of 7.72 and 5.54 U mg−1, respectively. Thermal stability of β-galactosidase was increased upon immobilisation. Finally, the operational stability of the immobilised β-galactosidase was studied for lactose hydrolysis in a continuous reactor. After 96 h continuous operation, the remaining activity of the immobilised enzyme was 94%.

Bio-conversion of whey lactose using enzymatic hydrolysis with β-galactosidase: an experimental and kinetic study

ABSTRACT Lactose in cheese whey is increasingly challenging to metabolise under normal conditions. The hydrolysis of whey lactose into glucose and galactose using enzymatic methods has been acclaimed to confer benefits like enhanced substrate availability for better degradation in anaerobic digestion. In the present study, whey lactose was subjected to hydrolysis using the enzyme β-galactosidase derived from Aspergillus oryzae fungus to reduce the difficulty of lipid and fat transformation in anaerobic digestion. The individual and combined effects of hydrolysis parameters, pH, enzyme load, reaction time and temperature were studied using Response Surface Methodology by Central Composite Design. The optimum conditions were determined based on variance analyses and surface plots; pH 4.63, temperature 40.47°C, reaction time 25.96 min and enzyme load 0.49%. Results showed a maximum lactose hydrolysis value of 86.21%, while the predicted value was 87.44%. Indeed, enzyme hydrolysis induced a change of soluble chemical oxygen demand around 24.6% and 75.8% reduction in volatile fatty acid concentration. Upon anaerobic digestion, the pre-hydrolysed whey revealed a 3.6-fold higher bio-methane production than that of raw hey, and a visible decrease in volatile fatty acid concentrations. The resultant data agreed with the Gompertz model, and lag phase times were significantly reduced for hydrolysed whey.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Aspergillus oryzae strain GL470.

The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the Aspergillus oryzae strain GL 470 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in five food manufacturing processes; lactose hydrolysis in milk processing, production of fermented milk products, whey processing, manufacture of enzyme‐modified dairy ingredients and in the manufacture of galacto‐oligosaccharides. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.388 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of repeated dose 90‐day oral toxicity studies in rats. The Panel identified a no observed adverse effect level of 7,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 5,043. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel concluded that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided and considering the most recent complete toxicological data set, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Neobacillus sp. strain AE-LT.

The food enzyme β‐galactosidase (EC 3.2.1.23) is produced with the non‐genetically modified Neobacillus sp. strain AE‐LT by Amano Enzyme Inc. The strain is not cytotoxic and does not harbour any known virulence factor or antimicrobial resistance gene. The presence of viable cells of the production strain in the food enzyme could not be excluded, but the likelihood of this being a hazard is considered low. The food enzyme is intended to be used for lactose hydrolysis in milk processing and the manufacture of galacto‐oligosaccharides (GOS). The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 2.971 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,223 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 412. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Aspergillus oryzae strain AE-LA.

The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the non‐genetically modified Aspergillus oryzae strain AE‐LA by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used for lactose hydrolysis in milk processing, production of fermented milk products, whey processing and the manufacture of enzyme‐modified dairy ingredients. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.651 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,656 mg TOS/kg bw per day, the highest dose tested. This results in a margin of exposure of at least 1,003. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Kluyveromyces lactis strain AE-KL.

The food enzyme β‐galactosidase (β‐d‐galactoside galatohydrolase, EC 3.2.1.23) is produced with the non‐genetically modified Kluyveromyces lactis strain AE‐KL by Amano Enzyme Inc. As the production strain meets the requirements for a Qualified Presumption of Safety (QPS) approach to safety assessment and as no other issues of concern were identified, the Panel considered that toxicological tests were not needed for the assessment of this food enzyme. The food enzyme is intended to be used for lactose hydrolysis in milk processing (including infant formulae), production of fermented milk products and manufacture of galacto‐oligosaccharides (GOS). The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 7.933 mg TOS/kg body weight (bw) per day in European populations. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the QPS status of the production strain and the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme β-galactosidase from the genetically modified Kluyveromyces lactis strain KLA.

The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the genetically modified Kluyveromyces lactis strain KLA by DSM Food Specialties B.V. The genetic modifications did not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used for the lactose hydrolysis in milk processing, production of fermented milk products and whey processing. It is also intended for lactose hydrolysis in milk products at home. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 11.876 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme fulfils the requirements for the Qualified Presumption of Safety (QPS) approach to safety assessment. As no concerns arising from its genetic modification or from the manufacturing process have been identified, the Panel considered that toxicological tests are not needed for the assessment of this food enzyme. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Kluyveromyces lactis strain GAL.

The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the non‐genetically modified Kluyveromyces lactis strain GAL by DSM Food Specialties B.V. It is intended to be used for the lactose hydrolysis in milk processing, production of fermented milk products and whey processing. It is also intended to be used for lactose hydrolysis in milk products at home. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 10.78 mg TOS/kg body weight per day in European populations. As the production strain of K. lactis strain GAL qualifies for the Qualified Presumption of Safety (QPS) approach to safety assessment and no issue of concern arose from the production process, no toxicological data are required. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.