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Fructooligosaccharides (FOS) are prebiotic substances found in many vegetables and natural foods. FOS present many unique properties, such as low caloric values, non-cariogenic, decreasing phospholipids, triglycerides, and cholesterol, helping gut absorption of calcium and magnesium, and using as prebiotics to stimulate the bifidobacterial growth in the human colon. FOS are fructose oligomers that can produce from sucrose by microbial enzymes having transfructosylating activity (Î²-fructofuranosidase, EC 22.214.171.124). Therefore, the interest for applications of FOS in food and pharmaceutical products has been increased. Then, this seminar will summarize an alternative to improve the FOS production yield by immobilized Aspergillus japonicus on lignocellulosic materials, and the cultivation of A. japonicus on solid-state fermentation using agro-industrial residues as support and nutrient source. In addition, the use of lignocellulosic materials and agro-industrial residues could be mostly of interest, because they are inexpensive, natural, renewable, biodegradable and readily available.
TABLE OF CONTENTS
TABLE OF CONTENTS i
LIST OF TABLES ii
LIST OF FIGURES iii
Increase in the fructooligosaccharides yield and productivity 4
Fructooligosaccharides and Î²-fructofuranosidase production by Aspergillus 4
japonicus immobilized on lignocellulosic materials
Increase in the fructooligosaccharides yield and productivity by solid-state 8
Fermentation with Aspergillus japonicus using agro-industrial residues as
support and nutrient source
LITERATURE CITED 13
LIST OF TABLES
1 Fermentative parameters of FOS production by Aspergillus japonicus immobilized in 7
different lignocellulosic materials, and using free whole cells.
2 Fermentative parameters of FOS production by Aspergillus japonicus during the 11
solid-state fermentation using different materials as solid support
LIST OF FIGURES
1 Chemical structure of fructooligosaccharides 2
2 FFase activity during the sucrose fermentation by A. japonicus immobilized or not in 5
different lignocellulosic materials
3 Transfructosylating (Ut) activity during the sucrose fermentation by A. japonicas 5
immobilized or not in different lignocellulosic materials
4 Sucrose consumption during the sucrose fermentation by A. japonicus immobilized 6
or not in different lignocellulosic materials
5 FOS production during the sucrose fermentation by A. japonicus immobilized or not 7
in different lignocellulosic materials
6 The chromatogram profile obtained for the assay of FOS production by A. japonicas 7
immobilized in corn cobs, after 21 h fermentation
7 FOS production by A. japonicus under solid-state fermentation conditions using 9
different materials as solid support that obtained for the assays supplemented with
8 FOS production by A. japonicus under solid-state fermentation conditions using 10
different materials as solid support that obtained for the assays where no nutrients were
add to the media
9 Î²-Fructofuranosidase (FFase) activity during the FOS production by A. japonicus 10
under solid-state fermentation conditions using different materials as solid support.
Assays not supplemented with nutrients.
PROCESS DEVELOPMENT FOR THE ENZYMATIC PRODUCTION
Fructooligosaccharides (FOS) are non-digestible carbohydrates that represent one of the major classes of bifidogenic oligosaccharides. They are compounds of a vegetable origin and are found in varying concentrations in many foods such as asparagus, onions, artichokes, garlic, wheat, bananas, tomatoes and honey. However, because production yield of FOS by using these plant-originated enzymes is very low and mass production of enzyme is also quite limited by seasonal conditions, FOS is produced commercially through the enzymatic synthesis from sucrose by microbial enzymes with transfructosylating activity (Î²-fructofuranosidases, EC.126.96.36.199, also designed as fructosyltransferases EC.188.8.131.52). Most of these enzymes have been found in fungi such as Aspergillus, Aureobasidum, and Penicillium. Fructooligosaccharides (FOS) are specifically defined as mixed chains of fructosyl with a glucose terminal unit; they have a maximum of 5 units and are derived from sugar through natural fermentation processes, producing 1-kestose (GF2), nystose (GF 3) and 1-fructofuranosylnystose (GF4) in which the fructosyl units (F) are linked at the Î² (2"1) position of sucrose (Figure 1).
Figure 1 Chemical structure of fructooligosaccharides
Source: Sabater-Molina et al. (2009)
FOS are sweetener as 0.3-0.6 times that of sucrose, depending on the chemical structure and the degree of polymerization of the oligosaccharide. FOS are highly hygroscopic and their water holding capacity is greater than that of sucrose. The viscosity of a FOS solution is higher than that of sucrose at the same concentration because the greater molecular weight of FOS. The enhanced viscosity of the gastrointestinal content may delay the rate of gastric emptying and the digestion and absorption of nutrients. Their thermal stability also is greater than of sucrose and is highly stable in the normal range of food pH (4.0-7.0).
FOS can substitute sucrose as regards many of its properties, including solubility, freezing and fusion point and crystalline properties. It has been estimated that the caloric value of FOS ranges from 1.5 to 2.0 kcal/g, which represents 40-50% of that of digestible carbohydrates such as sucrose.
Fructooligosaccharides have interesting properties:
â€¢ Low sweetness intensity: this property makes them useful for various kinds of foods where the use of sucrose is restricted due to its high sweetness.
â€¢ Calorie free: the human body lacks the necessary enzymes to hydrolyze the beta bonds, so that they are not hydrolyzed by the digestive enzymes. Thus, since these substances can not be used as an energy source in the body, they are safe for diabetics and people on slimming diets.
â€¢ Non-cariogenic: FOS are not used by Streptococcus mutans to form the acids and insoluble Î²-glucans that are the main causes of dental caries.
FOS behave as soluble food fibre from a physiological point of view. They are non-digestible carbohydrates of a vegetable origin that reach to the large intestine, where they can be fermented by the colonic flora to promote the growth of bifidobacteria and prevent the growth of potentially pathogenic microorganisms. The bacterial degradation of FOS occurs in two stages: in the first stage, the monomers are hydrolyzed by bacterial beta-oxidases. In the second, the monomers released ferment anaerobically to produce volatile fat acids (SCFA) such as acetate, propionate and butyrate, and gases (H2, CO2, CH4).
These properties, together with their other beneficial physiological effects (low carcinogenicity, prebiotic effect, improved mineral absorption, decreased serum cholesterol, phospholipid and triacylglycerol levels) defend the addition of FOS to foods as functional food and their industrial applications are continuously increasing. Major uses focus in beverages (fruit drinks, coffee, cocoa, tea, soda, health drinks and alcoholic beverages), probiotic yogurts. Other current applications of FOS in the food industry include desserts such as jellies, puddings and sherbets; confectionary products such as candy, cookies, biscuits, breakfast cereals; chocolate and sweets; breads and pastries ; table spreads and spreads such as jams and marmalades and meat products such as fish paste and tofu. Some non-food applications have also been proposed for oligosaccharides including drug delivery, cosmetics and mouth washes.
Increase in the fructooligosaccharides yield and productivity
The main drawback of the commercial FOS production by transfructosylation is that the yields are normally low (55-60%). Therefore, and due to the increased demand for using these ingredients in food and pharmaceutical products, there is a great interest in the development of a suitable and economically viable biotechnological process for industrial production of FOS that allow obtaining higher yields and productivities.
Fructooligosaccharides and Î²-fructofuranosidase production by Aspergillus japonicus
immobilized on lignocellulosic materials
Mussatto et al. (2009) researched the the fructooligosaccharides (FOS) and Î²-fructo-furanosidase (FFase) production from sucrose by Aspergillus japonicus ATCC 20236 immobilized on different lignocellulosic materials including brewer's spent grain, wheat straw, corn cobs, coffee husks, cork oak, and loofa sponge.
The inoculum was prepared by adding 1 ml of spore suspension containing around 1.8Ã-107 spores/ml to 500-ml Erlenmeyer flasks containing 1 g of carrier and 100 ml of culture medium with composition as follows (w/v) : sucrose 20% ; yeast extract 2.75% ; NaNO3 0.2% ; MgSO4Â·7H2O r0.05%, K2HPO4 0.05% and KCl 0.05%. All media were sterilized by autoclaving at 121Â°C for 15 min. The flasks were incubated in a rotary shaker at 28 â-¦C and 160 rpm for 48 h. The pH of medium was not controlled during experiments. Assays under the same fermentation conditions described above but without addition of carrier particles (free cells assays ) were also performed for comparison. The samples were collected at regular intervals, enzyme activity and immobilized cells concentration were measured. Sugars (sucrose, glucose, fructose, 1-kestose nystose and 1-fructofuranosylnystose) were determined and quantified during cultivation using HPLC.
The result of FFase activity was presented in the figure 2, the cell immobilized in corn cobs giving the highest value (44.81 U/ml). Maybe some nutritional component of corn cobs favored the FFase production by the microorganism. Then, corn cobs are material to be used as immobilization carrier aiming to maximize the FOS production.
However, free cells presented FFase activity higher than those observed for immobilized cells (figure 2), but their Ut value was lower than those observed for cells immobilized in corn cobs. By comparing the Ut/Uh ratio for the different assays, the highest value (4.15) was found after 36 h cultivation for cells immobilized in corn cobs, which was 30% higher than that observed for the free cells system. Although, it is known that FFases compsed with hydrolytic (Uh) and transfructosylating (Ut) activities. The Ut/Uh ratio indicates the relative strength of the transfructosylating activity of the produced strain, and for an efficient production of FOS, high Ut/Uh ratio is preferable.
Figure 2 FFase activity during the sucrose fermentation by A. japonicus immobilized or not in different lignocellulosic materials.
Figure 3 Transfructosylating (Ut) activity during the sucrose fermentation by A. japonicus immobilized or not in different lignocellulosic materials.
The time course of sucrose uptake and FOS production was showed in Figure 4 and 5. All experiment presented similar kinetic behaviors with an initial lag phase among 0 and 12 h approximately, followed by an almost complete sucrose uptake between 12 and 24 h. In this period, sucrose was quickly changed into glucose and 1-kestose (GF2), and after completed 24 h fermentation the FOS production obtained the most elevated level in almost all media. Maximum production of FOS was obtained in 21 h fermentation with cells immobilized in corn cobs, a final product containing GF2 (46.83%), GF3 (16.31%), GF4 (2.75%), residual sucrose (10.53%), glucose (20.93%) and fructose (2.65%) was obtained (figure 6). The total of FOS concentration decreased slowly after 24 h fermentation, as result of the sucrose exhaustion and formation of FOS with longer chain (GF3 and GF4) from1-kestose (GF2).
Figure 4 Sucrose consumption during the sucrose fermentation by A. japonicus immobilized or not in different lignocellulosic materials.
Figure 5 FOS production during the sucrose fermentation by A. japonicus immobilized or not in different lignocellulosic materials.
Figure 6 The chromatogram profile obtained for the assay of FOS production by A. japonicus immobilized in corn cobs, after 21 h fermentation.
Table 1 Fermentative parameters of FOS production by Aspergillus japonicus immobilized in different lignocellulosic materials, and using free whole cells.
The table 1 showed the fermentative parameters of FOS production, corn cobs and coffee husks have the major amounts of cells and highest yield of FOS concentrations. Nevertheless, cells immobilized in corn cobs obtained the maximum FOS production in a shorter time (21 h), giving the highest value of FOS productivity (6.61 g/l h). Such result was 23.3% higher than that achieved in the free cells assay.
All estimated media gave FOS yield (YP/S) at least similar to that of free cells system (Table 1), independently of the carrier used for cells immobilization. Such yields were also similar or higher than the maximum theoretical normally found for FOS production by microorganisms (55-60%, w/w). Once more, the highest values were attained from cells immobilized in corn cobs and coffee husks. When considering only the consumed substrate, the YP/S value obtained for cells immobilized in corn cobs was 14% higher than that obtained by free cells.
From the results can be concluded that corn cobs can be successfully used as carrier for immobilization of A. japonicus, for the production of FOS and FFase because high amounts of cells adhered to this material and produced FOS and FFase enzyme with high productivity and Ut/Uh ratio.
Increase in the fructooligosaccharides yield and productivity by solid-state fermenta-
tion with Aspergillus japonicus using agro-industrial residues as support and nutrient source
Mussatto et al. (2010) improved the FOS yield and productivity by used three agro-industrial residues in solid-state fermentation systems for the production of FOS and Î²-fructofuranosidase by A. japonicus.
Experiments were carried out in petri dishes containing approximately 3.0 g of the sterilized materials. The materials were moistened with a 200 g/l sucrose solution to attain 70% moisture content, inoculated with a concentrated spore suspension to give 2Ã-106 spores/g material. The petri dishes were incubated at 28 â-¦C during 48 h. Cultivations were performed with the sucrose solution supplemented or not with the following nutrients (g/l): yeast extract (27.5), NaNO3 (2.0), K2HPO4 (5.0), MgSO4Â·7H2O (0.5), and KCl (0.5). The samples were collected at regular intervals. Sugars (sucrose, glucose, fructose, 1-kestose nystose and 1-fructofuranosylnystose) were determined by HPLC and enzyme activity was measured.
The result of FOS production was presented in figure 7 and 8, all the material residues used in the experiments can produced FOS but not when using the synthetic fiber (figure 8), as result they performed as nutrient source for the microorganism. Among the three tested materials, it was observed a reduction in the FOS production when corn cobs and cork oak were used as nutrient source, when compared to the results obtained from nutrient supplemented media. Such results suggest that these materials contain nutrients that are metabolized by the microorganism, but probably these nutrients are not found in enough amounts to reproduce the results achieved from supplemented media.
On the other hand, coffee silverskin gave FOS production results similar in both assays, supplemented or not with nutrients, suggesting that this material is nutritionally richer than cork oak or corn cobs, and that is able to be used as nutrient source for FOS production with no requirement of additional nutrient supplementation.
Figure 7 FOS production by A. japonicus under solid-state fermentation conditions using different materials as solid support that obtained for the assays supplemented with nutrients.
Figure 8 FOS production by A. japonicus under solid-state fermentation conditions using different materials as solid support that obtained for the assays where no nutrients were add to the media (the materials were moistened with sucrose solution only).
The result of FFase activity was showed in the figure 9, the highest enzyme activity values were obtained in the medium containing coffee silverskin, which is in agreement with the elevated production of FOS observed in this medium. The lowest FFase results obtained in the media containing corn cobs or cork oak could be related to the presence of a lower nutrient amount in these two residues, when compared to coffee silverskin.
Figure 9 Î²-Fructofuranosidase (FFase) activity during the FOS production by Aspergillus japonicus under solid-state fermentation conditions using different materials as solid support. Assays not supplemented with nutrients.
Table 2 Fermentative parameters of FOS production by Aspergillus japonicus during the solid-state fermentation using different materials as solid support. Assays supplemented (S) or not supplemented (NS) with nutrients.
The fermentative parameter values obtained in these SSF assays are summarized in Table 2. When comparing the FOS production results from supplemented media, Corn cobs gave yielded a 2-fold higher FOS production than that achieved using synthetic fiber. On the other hand, with exception of coffee silverskin, FOS production for the other solid supports was strongly affected in the assays without nutrient addition. The almost null production of FOS when using synthetic fiber clearly shows the microorganism necessity for some nutritional source to produce FOS.
When observing the yield and productivities values showed in Table 2, it can be noted that similar FOS production yields (YP/S) were obtained for the non-supplemented media containing the agro-industrial residues. Such yields, correspondent to 61-70%, were higher than the values usually reported for the commercial FOS production (55-60%), and represent an important improvement for the FOS production at industrial level.
The FOS productivity (QP), coffee silverskin promoted the highest value (8.05 g/l h) of this parameter, which was almost 60% higher than those attained when using corn cobs or cork oak as nutrient source. Such difference was due to the faster FOS production in this medium, which was maximum after only 16 h of fermentation.
Corn cobs, coffee silverskin and cork oak can be used as nutrient source for FOS production by A. japonicus through SSF systems. Among these materials, coffee silverskin is able to provide all the nutritional requirements for the microorganism development since the results obtained when using this material as nutrient source were similar to those achieved when the medium was supplemented with nutrients. The highest enzymatic activity results were also achieved when using coffee silverskin as solid support. Such results are very promising and contribute to the establishment of a strategy for FOS production at industrial level with higher yields and productivities than those commonly achieved, and with lower operational costs due to the use of agro-industrial residues as nutrient source. In fact, SSF with coffee silverskin and A. japonicus can be considered an interesting strategy for synthesizing both products, FOS and FFase, at the industrial level.
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