Screening of lactases suitable for the preparation of low-lactose prebiotic liquid milk and optimisation of their combination

Lactose intolerance is an important factor restricting the consumption of dairy products. Lactase is used to hydrolyze lactose in milk while generating galactooligosaccharides (GOS), thereby reducing the incidence of lactose intolerance. We used cow milk as raw materials, and selected enzyme preparations with high lactose hydrolysis rate and strong GOS generation ability from 14 commercially available lactase enzymes. The lactose hydrolysis rate is 5.85%-81.38%, and the GOS content is 0.03 g/L- 13.10 g/L. The mixing experiment design determined the two lactase enzymes (E10 and E11) ratio and the optimal enzymatic hydrolysis process of low-lactose prebiotic milk: compound lactases (E10:E11=0.756:0.244) addition 0.11%, 55℃for 5h, lactose hydrolysis rate and GOS content were 98.02% and 19.69g/L, respectively, and the remaining lactose content was about 0.97 g/L.

Lactose quantification in UHT milk by high-performance liquid chromatography and cryoscopy (freezing point depression)

Due to the large number of people with lactose maldigestion, the dairy industries have increased production and diversity of low lactose and lactose-free foods. Consequently, the need to control the lactose hydrolysis process has also risen. This study aimed to correlate freezing point depression (cryoscopy) and lactose concentration, quantified by high-performance liquid chromatography (HPLC), in UHT milk. To accomplish this, UHT milk samples were subjected to seven lactose hydrolysis treatments, using lactase enzyme, resulting in different lactose concentrations. All samples were subjected to HPLC analysis and freezing point measurement, using a cryoscope. The results were plotted on a graph and a linear regression was performed. There was a strong correlation between lactose concentration and freezing point (R = 0,9973) and the coefficient of determination (R2) was 0,9946, which means that 99,46% of the variability of the response data is explained by the linear regression model. Therefore, the results point to the feasibility of estimating the lactose concentration in milk during the hydrolysis process for the production of low lactose milk, by cryoscopy, a quick analysis, with lower cost compared to HPLC and that is already among the analyses commonly performed in dairy industries.

Multitasking in the gut: the X-ray structure of the multidomain BbgIII from Bifidobacterium bifidum offers possible explanations for its alternative functions

β-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some β-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain β-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain β-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective β-galactosidase modification for industrial purposes in the future.

Valorisation of whey for lactose recycling products production = Sūkalu valorizācija laktozes pārstrādes produktu ieguvei

The doctoral thesis “Valorisation of whey for lactose recycling products production” was developed from 2016 to 2021. Experiments were carried out in the research laboratories of the Faculty of Food Technology, Latvia University of Life Sciences and Technologies; Dairy Innovation Institute, California Polytechnic State University (USA); Institute of Microbiology and Biotechnology, Latvia University; Faculty of Chemistry, Latvia University; Institute of Solid State Physics, Latvia University and J.S. Hamilton Baltic Ltd. The aim of the doctoral thesis was to improve the lactose hydrolysis process for obtaining glucose-galactose and oligosaccharide syrups. The hypothesis of the doctoral thesis – the two-stage fermentation increases the sweetness of glucose-galactose syrup. The hypothesis of the doctoral thesis has been confirmed by the defended thesis: 1. The presence of cations affects the β-galactosidase activity in the sweet and acid whey permeate. 2. The chemical composition and quality of whey affect the physical properties of lactose. 3. Enzymatic reactions affect the functional and sensory properties of syrups. The research objects – sweet and acid whey permeates, glucose isomerase, commercial β-galactosidases and glucose-galactose syrup. The following tasks were set to achieve the aim of the doctoral thesis: 1. To evaluate the effect of cation concentration to ensure the β-galactosidase activity in substrate. 2. To investigate the physical properties of whey lactose in order to better understand its behaviour. 3. To study the changes of monosaccharide concentration in the lactose hydrolysis, varying with the solids concentration of the substrates and enzyme units. 4. To assess the possibilities of glucose isomerase to increase the sweetness of glucose-galactose syrup. 5. To evaluate the sensory properties of the developed syrups. The novelty of the doctoral thesis: 1. The study of the relationship of lactose hydrolysis process in the formation of galacto-oligosaccharides and lactulose. 2. The combination of β-galactosidase and glucose isomerase increases the sweetness of glucose-galactose syrup. The economic significance of the doctoral thesis: 1. The studies have shown the possibility to obtain syrup that can be used as sugar and sweeteners replacer in the food industry and to produce functional products. 2. A technological solution for hydrolysis of lactose is proposed, comprehensively evaluating the physical properties of lactose, fermentation parameters and whey composition. The doctoral thesis consists of three chapters: Chapter 1 describes the composition of whey and the possibilities of using it. An overview of the chemical and physical properties of lactose, lactose hydrolysis methods, the application of β-galactosidases and the properties of glucose-galactose syrup are provided. Chapter 2 summarises the materials and methods used in the thesis. Chapter 3 provides a summary of the results obtained in the study, the properties of commercial enzymes in different cation concentrations, the stability of enzymes in the gastrointestinal tract model, methods for the determination of lactose, the properties of dehydrated permeates are evaluated. The influence of factors on the hydrolysis of permeates and the profile of the obtained sugars was analysed. Possibilities for lactulose synthesis are considered. Sensory analysis of glucose-galactose syrups and syrups obtained in the two-stage fermentation are given. During the PhD studies the author had an internship at the Dairy Innovation Institute California Polytechnic State University (USA), where the experimental work was done. Internship was provided by the Baltic – American Freedom Foundation (BAFF) and the Council on International Education Exchange (CIEE). The study was partly financed by the LLU programme “Strengthening Research Capacity at the Latvia University of Agriculture” grant (Contract No. 3.2.-10/2017/LLU/27) “The optimization of bioprocesses for lactose recycling products”. The study was partly financed by the doctoral studies grant “Transition to the new doctoral funding model at the Latvia University of Life Sciences and Technologies” (Contract No. 3.2.-10/90). The thesis is written in English, it consists of 111 pages, 32 tables, 41 figures, 3 appendixes, and 233 bibliographic sources.

Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

Background Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous β-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L−1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L−1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L−1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and β-lactose and GlcNAc feeding strategy. Conclusions Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products.

Whole conversion of agro-industrial wastes rich in galactose-based carbohydrates into lipid using oleaginous yeast Aureobasidium namibiae

Background Raw materials composed of easily assimilated monosaccharides have been employed as carbon source for production of microbial lipids. Nevertheless, agro-industrial wastes rich in galactose-based carbohydrates have not been introduced as feedstocks for oleaginous yeasts. Results In this study, Aureobasidium namibiae A12 was found to efficiently accumulate lipid from soy molasses and whey powder containing galactose-based carbohydrates, with lipid productions of 5.30 g/L and 5.23 g/L, respectively. Over 80% of the fatty acids was C16:0, C18:0, C18:1, and C18:2. All kinds of single sugar components in the two byproducts were readily converted into lipids, with yields ranging between 0.116 g/g and 0.138 g/g. Three α-galactosidases and five β-galactosidases in the strain were cloned and analyzed. Changes of transcriptional levels indicated GalB and GalC were key α-galactosidases, and GalG was key β-galactosidase. In 10 L fermentor, lipid production from SM and WP achieved 6.45 g/L and 6.13 g/L, respectively. β-galactosidase was responsible for lactose hydrolysis; sucrase and α-galactosidase both contributed to the efficient hydrolysis of raffinose and stachyose in a cooperation manner. Conclusions This is a new way to produce lipids from raw materials containing galactose-based carbohydrates. This finding revealed the significance of sucrase in the direct hydrolysis of galactose-based carbohydrates in raw materials for the first time and facilitated the understanding of the efficient utilization of galactose-based carbohydrates to manufacture lipid or other chemicals in bioprocess. Graphic abstract

A Comprehensive Bioprocessing Approach to Foster Cheese Whey Valorization: On-Site β-Galactosidase Secretion for Lactose Hydrolysis and Sequential Bacterial Cellulose Production

Cheese whey (CW) constitutes a dairy industry by-product, with considerable polluting impact, related mostly with lactose. Numerous bioprocessing approaches have been suggested for lactose utilization, however, full exploitation is hindered by strain specificity for lactose consumption, entailing a confined range of end-products. Thus, we developed a CW valorization process generating high added-value products (crude enzymes, nutrient supplements, biopolymers). First, the ability of Aspergillus awamori to secrete β-galactosidase was studied under several conditions during solid-state fermentation (SSF). Maximum enzyme activity (148 U/g) was obtained at 70% initial moisture content after three days. Crude enzymatic extracts were further implemented to hydrolyze CW lactose, assessing the effect of hydrolysis time, temperature and initial enzymatic activity. Complete lactose hydrolysis was obtained after 36 h, using 15 U/mL initial enzymatic activity. Subsequently, submerged fermentations were performed with the produced hydrolysates as onset feedstocks to produce bacterial cellulose (5.6–7 g/L). Our findings indicate a novel approach to valorize CW via the production of crude enzymes and lactose hydrolysis, aiming to unfold the output potential of intermediate product formation and end-product applications. Likewise, this study generated a bio-based material to be further introduced in novel food formulations, elaborating and conforming with the basic pillars of circular economy.

The Potency of Indigenous Lactobacillus farciminis LIPI12-2-LAB033 Isolated from Non-Dairy Product of Indonesian Fermented Food as a New Source of β-galactosidase Enzyme

The β-galactosidase is an enzyme that plays an essential role in the lactose hydrolysis into glucose and galactose. This study examines the potential of β-galactosidase from several lactic acid bacteria (LAB) isolated from non-dairy products Indonesian fermented foods and purifies them to increase their specific activity. The enzyme was extracted using ultrasonication, purified with ammonium sulfate, and dialyzed with a cellulose membrane (11 kDa). The result of isolates tests showed that Lactobacillus farciminis LIPI12-2-LAB033 had the highest specific activity of 13.9 U/mg protein. Precipitation using 40% ammonium sulfate increased the specific activity up to 19.6 U/mg protein. This enzyme works optimally at a temperature of 40 °C and pH of 7. The specific activity of this enzyme increases to 75.6 U/mg protein after dialysis. The dialysis process purifies the enzyme 5.44 times with a yield of 26.7%. These findings indicate that Lactobacillus farciminis LIPI12-2-LAB033 can be considered as a source of β-galactosidase enzyme production.