![[BioMatNet Database - FAIR Program]](../images/Fair.gif) |
FAIR-CT95-1048
Valorising of Lactose by Enzymatic Conversions to Compounds with Application in the Food and Non-Food Industries |
 |
Contract No: | FAIR-CT95-1048 |
| Date Prepared: | November 1999, October 1998 | |
| Source: | Final Report
Abstract Second Annual Progress Report |
Introduction
It has been widely appreciated that the efficient utilisation of renewable resources for the production of food and chemicals will be a hallmark of industrial development in the new millennium. Lactose, the major sugar component of milk and most milk products, is available abundantly and in a chemically defined form. Therefore, it is quite useful for food and pharmaceutical applications as such, and provides an interesting starting material for the synthesis of value-added carbohydrates or carbohydrate derivatives. In spite of this remarkable potential for industrial exploitation, lactose accumulates in estimated quantities of 1.2 million tons per year as a by-product of cheese manufacture by the dairy industries but only a small percentage of that amount is utilised further. The rest, if disposed of in the waste water, arguably can create environmental problems. Taking all this into account, it is not surprising that the idea of utilising lactose as raw material for chemical or biological transformation has been put forward already decades ago. However, a number of problems stemming from difficult process technologies and unfavourable economical situations have hampered so far a broad-spectrum utilisation of lactose in many parts of Europe and worldwide.
This project has been an European effort to push ahead lactose utilisation by pursuing in a multidisciplinary approach a number of routes with the aim of adding value to lactose. To this end, modem enzyme and chemical technologies have been used to achieve the conversion of lactose into products, and it has been tested the utility and functionality of the thus obtained lactose derivatives in view of applications as health-related human food ingredients and chemical commodities. The project results have stimulated industrial activities and led to actions in practice, reflected by the development of a demonstration plant in Austria. In other areas of the project significant progress has been made in scientific/technological matters of the key objectives, and in spite of the fact that the process realisation has not been achieved in all cases, a sound knowledge base has been obtained which defines the requirement of future activities in research and therefore enables targeted process development.
Objectives
The objective of the project is to explore strategies that will add significant value to lactose, which throughout the processing of milk accumulates in very large amounts of estimated 1.2 million tons worldwide annually from the dairy by-product, cheese whey., in which lactose represents approximately 70-75% of the solid components. The ultimate goal of the project is to improve current processes of lactose utilisation, with enzyme technology in its broadest sense being employed, and to identify and to establish promising, biocatalytic approaches towards the synthesis of known and novel products using lactose or compounds derived thereof as starting materials. In addition, the important aspect of applying lactose derivatives with particular reference to the growth promotion of probiotic organisms, which are proven to exhibit beneficial effects on human health, will be fully covered. The long-term objective is to improve the competitiveness of the EU in the versatile area of lactose valorisation, especially by means of enzyme technology, which will help the EU to take the lead in launching novel lactose-based products on the world market. The efficient conversion of the low-value compound lactose into (much) more valuable products could indeed serve as the basis for new industry in the future. In addition, for two economic reasons, the project and the processes to be developed therein will add to novel green technologies. First, with better options to utilise lactose being available, the large biological oxygen consumption of today's effluents from the dairy industries will certainly be reduced. Second, lactose-derivatives will be biodegradable and could - at least with regard to the surfactant and tenside industry - be more environmentally compatible than current products on the market. The project will be divided into three highly interactive parts whose objectives are defined as follows:
Results
To summarise the results of the project, it is best to discuss the research and development from the process- oriented viewpoint.
Lactose hydrolysis To overcome the serious problem of microbial infection during continuously operated processes of the enzymatic hydrolysis of lactose at ambient temperatures, it was decided to focus on thermostable enzymes and the use of these protein biocatalysts at temperatures of 70 ·C or greater. At this high temperature, the growth of most micro-organisms is prevented efficiently, thus enabling the processing of the substrate without the risk of contamination. The enzymes, so-called -glycosidases from the hyperthermophilic Archea Sulfolobus solfataricus and Pyrococcus furiosus were produced in high yield by the over-expression of the coding genes in mesophilic host organisms, and downstream processing was accomplished by simply denaturing all less thermostable proteins at 80 ·C so as to obtain a technical-grade preparation of the biocatalysts.
The enzymes were characterised in terms of:
A major point addressed in the project was the enzymatic formation of oligosaccharides during lactose conversion by principally hydrolytic enzymes, and it has been shown that under optimal reaction conditions in which among others the substrate concentration, the temperature and the pH are major factors to be considered, the production of the oligosaccharides can be avoided completely, or maximised to obtain quite high yields of these valuable products. Mutant enzymes- have been prepared by protein engineering, and some of the mutants are better suited for the production of oligosaccharides than the wild-type enzymes.
A number of different enzyme reactors have been developed for lactose hydrolysis in continuous mode of operation, and their performances optimised with respect to productivity at a reasonably complete degree of lactose conversion, 80%, for example. The possible reactor configurations include the continuously stirred tank reactor equipped with an external ultrafiltration module which is required to retain the enzyme; the immobilised enzyme reactor; the diffusional hollow-fibre reactor. Process strategies have been devised by using these different reactors for the hydrolysis at high temperature of lactose in milk or whey, and of pure lactose.
The hollow-fibre reactor has been scaled up in collaboration with industry in Austria, and the start up of the demonstration plant with a capacity of several hundred annual tons has been successful regarding both the quality of the product, lactose-reduced milk powder, and the stability of the reactor during long-term operation.
Redox processes for lactose valorisation An extensive screening of micro-organisms has been carried out in search for the occurrence of enzyme activities capable of reducing lactose or oxidising the corresponding alcohol, lactitol. These activities seem to be lacking in nature, therefore, the utilisation of advanced protein engineering of existing enzymes, preferably the enzymes whose coding genes have been identified, sequenced and over-expressed in the project, appears to be required to obtain biocatalysts with the desired specificities.
During the screening a polyol dehydrogenase from the bacterium Burkholderia cepacia has been identified which oxidises with good activity and selectivity galactitol into D-tagatose, a low- calorie sweetener. A novel process has been developed towards the production of D-tagatose by using enzyme technology. Lactose serves as the starting material, and the galactose derived via the complete hydrolysis of lactose is used for further biotransforrnation. The process is based on a two-step conversion. In the first step, D-galactose is reduced into galactitol by using NADH- dependent aldose reductase from yeast as the key catalyst. The NADH required for activity is continuously regenerated by using NAD-dependent oxidation of D-glucose, catalysed by glucose dehydrogenase or formate, catalysed by formate dehydrogenase. In the second step, the specific oxidation at C-2 of galactitol is catalysed by the polyol dehydrogenase from B. cepacia.
To improve the yield and the end-concentration of D-tagatose, the oxidation must be coupled to a second reaction so as to achieve efficient regeneration of NAD, and to drive the thermodynamically unfavourable ketose formation. By using the enzymatic reduction of dioxygen (into hydrogen peroxide) catalysed by NADH oxidase, the conversion of polyol into D-tagatose was possible in over 95% yield and good productivity. The reaction conditions for the second step of the process have been optimised, taking into account especially the need of oxygen supply during the reaction.
Lactobionic acid This is a compound that has a number of uses in medicine, food manufacture and chemical industries. As an example, lactobionic acid is used in the 'Wisconsin transplantation solution', because of its excellent metal-chelating properties that reduces oxidative damage to tissue during storage and preservation of organs caused by certain metal ions. Lactobionic acid can be used as a biodegradable cobuilder in washing powder, which can contain up to 40% lactobionic acid. In food technology, lactobionic acid could have possible applications because of its sweet- sour, mildly acidic taste. Further applications are found for its mineral salt complexes, which are used to fortify functional drinks with essential minerals, or its addition to functional food because of its presumed prebiotic effect.
A novel and efficient enzymatic bioprocess has been developed in which lactose is completely and efficiently converted (productivity of 50 g/l/hr), specific productivity of 25 g/l/hr/kU into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the enzyme cellobiose dehydrogenase. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H2O-producing, copper-containing oxidase. Both enzymes are very stable and excreted abundantly by fungal organisms.
Physiological effects of products The successful commercialisation of some lactose derivatives such as lactose-derived oligosaccharides or D-tagatose is expected to be facilitated when specific beneficial effects can be demonstrated that are associated with the consumption of these products. To this end, the capability of the lactose derivatives has been tested to promote the growth of the intestinal bacteria and to evaluate the prebiotic potential of these compounds. After selection of suitable model organisms, a series of tests has been carried out to determine:
Finally, clinical studies were carried out with a strain of Lactobacillus rhamnosus. More research is needed to obtain clear proof of the beneficial effects of lactose derivatives on the colonisation of the strain in vivo for which positive indications have been obtained by using lactulose.
Objectives
The objectives of the present project are threefold:
Activities
In the area of lactose hydrolysis (Task 1), hyperthermophilic Archea were screened for new thermostable glycosyl hydrolases, and a beta-glucosidase from Pyrococcus horikoshii was selected for further characterisation. By protein engineering, the specificity of the beta-glucosidase from Pyrococcus furiosus (CelB) was increased toward phosphorylated sugars as a first step of altering and tailoring the substrate spectrum of this enzyme. Furthermore, aspects of the reaction mechanism and molecular principles of the thermostability of the P-glucosidase from Sulfolobus solfataricus (Ss beta-gly) were investigated by site-directed mutagenesis methods. To enable the production of recombinant CelB and Ss beta-gly in amounts required for pilot-scale experiments in lactose hydrolysis, new expression vectors have been constricted.
Furthermore, the expression of CelB in the food-grade microorganism Lactococcus lactis is in progress. Both CelB and Ss beta-gly were compared for suitability for use in a hollow fibre reactor for the hydrolysis of lactose in skimmed milk. A suitable hollow fibre system has been selected and operational parameters for this system have been optimised. The immobilisation of CelB and Ss beta-gly was studied by various methods. Use of a plug flow-type immobilised enzyme reactor for the hydrolysis of lactose was evaluated.
In the conversions of lactose by oxido-reduction (Task 2), recombinant E. coli strains overproducing aldose reductase (ALR) from Candida tenuis and xylitol dehydrogenase (XDH) from Candida mastotermitis have been constructed and made available. Screening methods have been established to start the alteration of the properties of these two enzymes by protein engineering.
Studies on the coenzyme-dependent oxido-reduction of galactose to tagatose via galactitol have been started. The cloning of cellobiose dehydrogenase from Sclerotium rolfsii is in progress, and the wild-type enzyme has been successfully employed for the transformation of lactose into lactobionic acid on a kg scale.
For the production of novel products from lactose (Task 3), several promising routes and reactions have been selected and followed up. Various amides of lactobionic acid (hexadecyl, benzyl, allyl amide) were efficiently produced. These will now be made available in larger quantities to evaluate their performance as surfactants.
Lactose derivatives could be used for the synthesis of polymers that can be used for electrophoretic analysis. Future work will focus on the performances of these novel sugar-based polymers for electrophoretic separation of glycoproteins and sugar derivatives.
CelB and Ss beta-gly have been employed for the synthesis of novel glycosides (alkyl and allyl-beta-D-glucopyranosides) and oligosaccharides (mainly tri- and tetra- galacto-oligosaccharides) from lactose.
Based on the establishment of extensive bacterial collections with strains of probiotic function and of intestinal importance in two laboratories, and on the establishment of a collection of basic mono- and oligo-saccharides and prebiotic sugar preparations (mainly produced from lactose), the behaviour of bacteria in media containing varying carbohydrates was examined (Task 4). Results indicate that there is a strongly strain-dependent behaviour regarding the bacterial growth on different saccharides. Furthermore, the specific adherence properties of the bacteria which might affect their local behaviour in the intestine has to be taken into consideration.
Lactobacillus rhamnosus VTT E-97800, isolated from human faecal material, has been found to have a good capacity to utilize both lactulose and lactitol. Furthermore, it binds very efficiently to human colonic tumour cell lines. Based on these properties, the strain has been selected for further in vitro and clinical trials.
L. rhamnosus VTT E-97800 in combination with lactulose as a prebiotic caused a marked increase in the concentration of lactic acid and a decrease in ammonia. Furthermore, efficient colonisation was observed in preliminary clinical trials. These results form an excellent basis for further clinical trials.
Tagatose will be included in these experiments as soon as larger amounts of this compound become available.
The results of a detailed market analysis (Task 5) suggest that from the industrial point of view, three products are currently of special interest:
Progress
The state of progress of the overall project is matched to the time table of planned activities laid down in the work programme.
Results and Future Actions
Efficient expression systems for CelB, Ss beta-gly, ALR and XDH have been developed, and the downstream processing of the enzymes has been improved. These enzymes are therefore now available in sufficient quantities and the required purity, allowing their use in conversion studies on pre-pilot to pilot scale. Biochemical and mechanistic studies on enzymes employed in the project have reached a point where the information thus obtained is of immediate importance for reaction engineering and process development.
The hydrolysis of lactose has been studied and optimised in several different reactor systems, employing both free and immobilised beta-glucosidases. Lactobionic acid was produced enzymatically and isolated. An enzymatic process for D-tagatose is under development.
Work on the production of new compounds from lactose has been well advanced. Based on the current achievements, conversion studies in line with process development and optimisation will be continued. The screening for novel thermostable enzymes will be continued also, as will be the characterisation of known enzymes, by using protein engineering and other biochemical methods.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
- biomatnet@biomatnet.org
 |
 |
 |
News |
 |
Events |
|
|