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Protein enrichment : The scale up of fermentation from laboratory to pilot will be carried out in order to:

- Test and optimize the fermentation process which has been developed at laboratory scale.

- Produce sufficient quantities of fermented pulp in order to conduct nutritional tests on animals (mainly pigs and poultry).

- Evaluate the economy of the process. It will be necessary, starting from the pilot fermenter operating conditions, to evaluate the potential industrial costs (investment, raw materials and nutrients, energy, manpower, etc.). The production costs will be compared to the potential valorization for animal feed uses.

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:

- Genetic engineering:
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.

- 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.

- Nutrition test:
The product of the previous points, i.e. phytase samples, will be assessed in vitro and in vivo.
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.


- Enzymes. The production of xylanase from Aspergillus niger and Trichoderma reesei, glucanase from Aspergillus niger and phytase from A. niger and A. ficuum were studied in detail. The basic conditions for the production of arabinofuranosidase were also investigated. The first step of the research to increase, by mutagenesis for hemicellulases and by genetic engineering for phytase, the productivity of the microorganisms. This was followed by laboratory, small pilot and semi industrial fermentation studies. Methods for extraction, purification and concentration of enzymes from the biomass were also studied. Food and feed uses were also studied, particularly the influence of enzyme supplementation on wheat and barley based feed diets for monogastrics (pigs and chickens). The results obtained are generally in line with published results with the efficiency of the enzymes from SSF comparable to those on the market. Production costs were significantly reduced, confirming that solid state fermentation can be competitive with classical submerged or surface fermentation processes. Patent and technical problems caused an interruption of the research on phytase.

- 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, which in trials was found to have better nutritional characteristics than non fermented pulp for chickens and with good results. for igs (up to a maximum level of around 40%).

- Other studies. The direct addition, in a barley based diet, of enzyme containing biomass from the fermenter without extraction of the enzymes gave results equivalent to those for separated enzymes. This could be an interesting way to combine the effect of enzymes with protein enrichment. Studies on the storability and thermal stability of the enzymes were also conducted, as were toxicology tests for feed applications according to European Union regulations. No toxic effect was detected.


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.
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.


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.


This project is coordinated by Generale Sucriere (France) with Ebro Agricolas (Spain) and Solvay (Belgium) {contractors} and INRA, Dijon (France), IRTA (Spain) and LWEN (France) {sub-contractors}.

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Générale Sucrière
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Balmes 103
08008 Barcelona
Rue De Ransbeek 310
1120 Bruxelles