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AIR1-CT92-0001
Valorisation of Sugar Beet Pulp by Solid State Fermentation - Proteins and Probiotics |
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Proposal No: | AIR1-CT92-0001 |
| Date Prepared: | September 1999 | |
| Source: |
Final technical report 1997 Progress Report Summary Update to Progress Report Summary |
Summary
This project is a continuation of the ECLAIR project (AGRE-0038), in which solid state fermentation using sugar beet pulp and other substrate such as wheat bran was investigated for the production of hemicellulolytic enzymes and feed proteins.
Enzymes The production of xylanase from Aspergillus niger and Trichoderma reesei, beta-glucanase from Aspergillus niger and phytase from Aspergillus niger and Aspergillus ficuum were studied in detail. The basic conditions for the production of arabinofuranosidase were also investigated. The objective was to produce industrial enzymes for use in the food and animal feed sectors.
The first part of the research was the use of mutagenesis in the case of hemicellulases and the use of genetic engineering for phytase in order to increase the productivity of the microorganisms. This was followed by laboratory, small pilot and semi-industrial fermentation studies. Extraction of enzymes from the biomass, purification and concentration were also investigated.
The most important applications in food and feed studied was an investigation of the influence of enzyme supplementation in wheat and barley based feed diets for monogastric animals (pigs and chickens). The results obtained are generally in line with existing published results. The effectiveness of the enzymes produced in this project was comparable to that of enzymes from external sources.
Production costs were reduced during the project. In one case the production cost is already significantly lower than the present market price. This result confirms that solid state fermentation can be competitive with classical submerged or surface fermentation processes.
Protein enriched beet pulp Fermentation of sugar beet pulp by a selected strain of A. niger was used for the production of protein enriched beet pulp. It was shown that fermented pulp has better nutritional characteristics than non-fermented pulp for chickens and pigs. Some fermented pulp can be added to chickens diets without observing the detrimental effect caused by the addition of non-fermented pulp. Fermented pulp can be added to pigs diets up to a maximum level of around 40% with good results.
Other studies The direct addition, in a barley-based diet, of enzyme containing biomass from the fermenter without extraction of the enzymes was also investigated. This showed that, for equivalent enzyme activities, no significant difference in efficiency was observed. This could be an interesting way to combine the effect of enzymes with protein enrichment and therefore could increase the competitivity of the solid state fermentation process.
Studies on the storability and thermal stability of the enzymes were also carried out, as were toxicology tests, conducted for feed applications according to European Union regulations. No toxic effect was detected.
This programme was started in January 1993 and is a continuation of the research programme ECLAIR AGRE 0038. Solid state fermentation using sugar beet pulp as main substrate and also maize grits and wheat bran, was used for the production of enzymes for food and feed uses and for the production of protein enriched sugar beet pulp. Enzyme extraction and purification from the biomass was studied. In parallel, food and feed application tests were carried out in order to determine the efficiency of the products. Other subjects were also studied, such as stability under industrial use conditions, storability and potential toxicity. Three main areas of activity were:
In addition, animal feeding studies using hemicellulolytic enzymes and protein enriched fermented beet pulp, were conducted by Ebro-Agricolas and IRTA.
As far as enzymes are concerned (hemicellulases or phytase), the objective of the programme was to evaluate the potential industrial competitivity of Lyven's solid state fermentation process compared to other existing fermentation processes such as liquid state or surface fermentation. Phytase is used to improve the nutritional quality of phytic acid rich feed components (mainly wheat bran for pigs). The supplementation with phytase decreases the need for calcium phosphate addition and has a positive effect on environment (less manure to be spread in fields).
The objective for Solvay was to develop a highly phytase producing strain of Aspergillus (using a gene amplification procedure).
For protein enriched beet pulp, the objective was to determinate its economical value as animal feed ingredient in order to evaluate the feasibility of an industrial production. Sugar beet pulp is presently used as a feed for polygastric animals. Stock farming in the European Union is a stable or decreasing activity. Furthermore, sugar beet pulp competes with fodder cereals the price of which will decrease sharply in the years to come. This is why there is an interest in developing new outlets for pulp. Protein enriched fermented beet pulp can allow the use of some beet pulp for monogastric animals and can also improve the nutritional efficiency of beet pulp for polygastric animals.
Activities
The solid state fermentation process developed by Lyven has significant advantages for the growth of aerobic microorganisms such as moulds and yeasts. The duration of fermentation batches is shorter than for conventional fermentation processes (24 to 72 hours instead of about 5 days). As a consequence of the rapid growth of the microorganisms, Lyven's fermentation process is also much less susceptible to infection than existing industrial processes. This process is already in industrial use by Lyven for the production of pectinases.
Solvay, a Belgian chemical company is active in the enzyme industry worldwide through the Enzymes Department. Solvay has a microbiology research department which provided its laboratory equipments and research teams for microbiology and genetical engineering in order to evaluate the feasibility of phytase production by solid state fermentation in comparison with submerged fermentation.
INRA Dijon, a French public research center, coordinated the work of an engineer doctor thesis for the laboratory scale evaluation of solid state fermentation conditions and for the evaluation of new hemicellulytic enzymes production (arabino-furanosidase). INRA Dijon has a long-term experience of solid state basic and applied fermentation research.
Ebro-Agricolas, a Spanish sugar company, associated with IRTA Reus, a Spanish public research center has industrial interests in the feed industry. It carried out with IRTA research on the potential feed applications of the products developed by the other partners. IRTA, at its Reus research center, has complete facilities for animal feed nutrition tests. Pigs and poultry were used for the present programme to evaluate the efficiency of enzyme supplementation in cereal-based feed diets and the nutritional quality of fermented sugar beet pulp.
Results
Endo III xylanase production Starting from an Aspergillus niger strain developed during the previous ECLAIR contract, the production of endo III (AXA) xylanase was developed as follows. A new fermentation concept was developed in order to be able to use efficiently a wheat bran rich substrate. A new automated fermenter was designed and built. The most important productivity factors were the quality of the inoculum, the quality of the wheat bran substrate and the control of humidity during fermentation. Temperature control and stirring during fermentation were also optimized. As far as extraction and purification was cpncerned, it was found that the high pressure system used for existing industrial production from beet pulp fermentation was found unsuitable for fermented wheat bran. A new multi-layer system was installed and operated satisfactorily. A new microfiltration system was also installed. At the end the overall productivity of the different steps of the process, from inoculum to concentrated and purified enzyme now reaches 60% of the maximum theoretical laboratory productivity. The first efficiency test conducted to compare the AXA xylanase with existing commercial enzymes showed significantly better results for Lyven's enzyme at equivalent AXA concentrations. This could reflect the presence of some secondary enzymatic activity. At the end of the project samples of preparations based on this enzyme were being tested by potential industrial users.
Aspergillus niger beta-glucanase for animal feed applications The productivity of the A. niger strain developed during the previous ECLAIR programme was improved by mutagenesis. A wheat bran rich fermentation substrate was selected for fermentation studies. The new multilayered fermenter was used. Fermentation, extraction and purification developments were similar to those developed for AXA xylanase but less advanced. Animal feed experiments were conducted at IRTA on barley-based feed for broiler chickens, weaning and fattening pigs. Preparations containing commercially available beta-glucanase were compared to Lyven's enzyme. The efficiency of beta-glucanase on the digestibility of barley based diets for broiler chickens was influenced by the quality of barley but significant positive results were always observed. For pigs, the effects of enzyme supplementation on feed performance was much smaller than for chickens. This suggests that pigs utilize barley very efficiently and that non-starch polysaccharides do not negatively affect performance.
Trichoderma.endo I (AXC) xylanase for animal feed applications The strain developed during the previous ECLAIR contract was used for pilot development. Studies were similar to those conducted with A. niger enzymes. with results intermediate between those obtained for AXA and glucanase strains. AXC xylanase was tested at IRTA on wheat based diets for broiler chickens and pigs. Lyven's xylanase was compared with commercially available preparations. It appeared that the high AXC content of Lyven's xylanase is a favourable factor for enzyme efficacy. Significant positive effects of enzyme supplementation were observed for chickens with results depending on the quality of wheat. The influence of the quality of wheat was also observed for pigs. A significant positive effect of AXC xylanase supplementation on growing pigs (+5% for final carcass weight) was observed; probably for the first time since such an effect has not been reported in existing publications to the best of the knowledge of the consortium.
Production of phytase Recombinant strains of A. niger and A. ficuum were developed satisfactorily by Solvay, but it was decided that they could not be used because of a competitor's patent. Therefore another strain of A. niger developed by classical mutagenesis was studied.
Fermentation tests on beet pulp and wheat bran were conducted and compared to submerged fermentation. Productivity by solid state fermentation appeared to give better results. But, due to the slow growth of the strain, a fully aseptic fermentation must be used. As no sufficiently aseptic pilot fermenter was available, it was decided to postpone further research on phytase.
Protein enriched beet pulp Lyven developed the production of protein enriched fermented beet pulp by cultivation of a selected strain of A. niger. At pilot plant scale, enriched pulp contained about 17 % proteins after 48 hours and 23 % after 72 hours. Fermented sugar beet pulp (FSBP) was compared with non fermented pulp (NFSBP) and with control diets without addition of pulp. The experiences with broiler chickens showed that, although the apparent metabolizable energy of beet pulp is significantly improved after fermentation, it remains too low for allowing its blending in high proportions with standard chicken diets. On the contrary, the apparent digestible energy (ADE) of NFSBP is already much higher for pigs than for chickens. After fermentation, the ADE of A. niger FSBP is equivalent to that of a gluten feed at a level of addition of 25 % of the base feed. At the highest level of addition, an significnat decrease of ADE was observed, which is probably due to the limited capacity for pigs to ferment fibres. In both cases (chickens and pigs), the nutritional results for A. niger FSBP were significantly better than for Fusarium oxysporum FSBP tested during the previous ECLAIR programme.
Discussion
This project is a continuation of the previous project under the ECLAIR programme - AGRE-0038, valorization of sugar beet pulp by solid state fermentation, conducted from 1st November 1990 to 31st December 1991. The above mentioned programme led to promising potential for the production and commercial utilization of proteins and probiotics. The first topic, protein enrichment of sugar beet pulp by solid state fermentation with filamentous fungi was treated by Südzucker. The second topic, development of a microbial feed additive with probiotic characteristics was treated by the subcontractor Enzymatix Ltd, Cambridge, UK. The two parallel studies conducted during the ECLAIR contract up to the end of 1991 will be carried on. It has examined the use of solid state fermentation with sugar beet pulp as the main substrate, as well as evaluating maize grits and wheat bran. The objective is the production and assessment of fungal enzymes. The work includes enzyme extraction and purification, as well as parallel studies covering applications in baking and animal nutrition. A novel solid state fermentation system has been developed, from which improved xylanases have been obtained. Note: during 1995, studies were carried out up to the end of June by Generale Sucriere associated with Lyven and Ebro Agricolas associated with IRTA. No study was carried out by Solvay during this period.
INTRODUCTION
This project aims to improve systems previously developed under the ECLAIR programme in which enzymes were grown in solid state fermentation using agricultural by-products (sugar beet pulp and wheat bran) as substrates. This includes studies of both the fermentation process and on the end products, with a focus on plant cell wall degrading enzymes and xylanases in particular. These enzymes have been tested in animal feed and bread making applications.
OBJECTIVES
Protein enrichment : The scale up of fermentation from laboratory to pilot will be carried out in order to:
Probiotics : The six previously selected strains will be tested for their resistance to gastrointestinal conditions (resistance to antibiotics and bile acids), then through animal feed trials. A first trial with piglets will be carried out at the University of Stuttgart-Hohenheim. A comparison will be made between a control diet (with no additive), a diet containing a classical antibiotic (tylosin) and a diet containing a mixture of the 6 selected probiotics. Further trials will be carried out at IRTA Reus. The potential industrial production of the selected strains will be evaluated. SSF can be used only for 2 of the 6 selected microorganisms.
Production of hemicellulases : The basic purpose of the proposed project is to apply SSF, using sugar beet pulp as the main substrate, for the manufacture of a range of hemicellulases. The hemicellulolytic activities selected presently are endo-xylanase, endo-beta-glucanase, arabanase and arabunofuranosidase. Other hemicellulolytic activities could be also studied if necessary. The improvement of feed digestibility (fodder cereals, cellulose containing feeds) will be the main subject of application studies. Other applications will be considered such as fodder silage and composing.
Production of phytase : The objectives will focus on three major activities:
This improvement of the recombinant clones will be done through the construction of new cloning vectors in which the phytase gene is under the control of different constitutive promoters. Two host strains will be used for comparison purposes, namely Asp. ficuum NRRL3135 and an industrial Aspergillus strain of the niger group. The host strains will be transformed with the new vectors; transformants will be assessed under SSF and liquid fermentation.
The first objective will be to assess, both in SSF and in liquid, the recombinant clones obtained. The second will be to optimize the fermentation conditions with the most productive clone of each strain in order to compare the performances of both processes.
The product of the previous points, i.e. phytase samples, will be assessed in vitro and in vivo.
ACTIVITIES
Fermentation: It was found that the fermenter developed for fermentations using wheat bran based substrates can also be profitably used for sugar beet pulp based substrates. A flexible multi substrate fermenter design is, therefore, available for industrial development. However, for infection susceptible fermentations, the present design does not provide sufficient sterilisation possibilities. A fully sterilisable fermenter is being developed.
Production of hemicellulolytic enzymes: During the first part of 1995, the main objective was the genetic improvement of previously selected strains and the scale-up of fermentation and extraction techniques. Further studies were carried out on the new fermenter designed in 1994 for fermentation media containing a high proportion of wheat bran or maize grits. The main problem was the control of the humidity level of the biomass during fermentation. A humidification system has now been installed. Solid state fermentation on a wheat bran based substrate gave good results. At the end of the research programme, the pilot plant productivity was sufficient to envisage a full size industrial production. Several Aspergillus strains were used to produce xylanase enriched enzyme preparations which could be used to improve the quality of wheat flour for breadmaking (French type bread). Although less advanced than the research on breadmaking xylanase, the production of Trichoderma reesei xylanase and Aspergillus niger ß glucanase has been developed satisfactorily. Further studies are necessary to improve productivity.
Protection of the enzymes against thermal degradation: Some protection was obtained with a coating process using mainly polydextrose. However, the level of protection remains insufficient for industrial applications and needs to be further improved.
Xylanase for breadmaking applications: The main objective of enzyme addition is to increase the volume of the loaf without having a deleterious effect on other aspects of bread quality. Breadmaking tests showed that this enzyme preparation was more effective than existing commercial enzymes. Although the reasons for this improved performance are not yet fully explained, it is suggested that solid state fermentation gives a range of secondary enzymatic activities different from those obtained by the usual submerged fermentation processes. Preliminary tests showed that the same enzyme could be valuable as a bromate replacer. Bromates are presently used mainly in the USA as improvers for breadmaking. Their use should be prohibited in the near future and a number of studies are being carried out to find replacement improvers, which could include enzymes.
Feeding trials and enzyme stability: Studies of the efficacy of xylanase and ß-glucanase supplementation in wheat and barley diets for pigs and chickens have been carried out. These included investigations of enzymes during storage, following pelletisation. Nutrition tests showed that the enzymes are efficient for the improvement of the feed digestibility of barley and wheat based diets. Fermented sugar beet pulp using a strain of Aspergillus niger has been tested for animal nutrition in comparison with non-fermented sugar beet pulp. It has been found to have a nutritional value significantly improved when compared to non-fermented pulp and also to that produced using Fusarium oxysporum developed during the previous ECLAIR programme.
CONCLUSIONS
Significant improvement concerning the industrial feasibility of enzyme production by solid state fermentation have been obtained, whilst nutritional test results confirmed and completed previous results. Poultry feed is the main outlet for such enzymes. Marketing tests are being conducted in coordination with industrial feed producers.
PARTICIPANTS
This project is coordinated by Generale Sucriere (France) with Ebro Agricolas (Spain) and Solvay (Belgium) {contractors} and INRA, Dijon (France), IRTA (Spain) and Lyven (France) {sub-contractors}.
Results
Conclusions
The development of xylanase production was satisfactory. Laboratory application tests showed that its efficiency was greater than that of commercial enzymes. Tests quantities have been sent to industrial users for confirmation of these results. The production costs of this enzyme is significantly below current market price. A. niger glucanase produced by SSF was effective in broiler chicken barley-based diets. Significant improvement of production costs were obtained. Solid state fermentation with A. niger significantly improved the nutritional value of pulp for chickens. However, the benefits were not suffficient to allow for incorporation of high proportions of fermented sugar beet pulp in practical diets.
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