Download miễn phí Luận văn Β-D-fructofuranosidase production and application to the manufacture of frutooligosaccharides
Contents
Preface
1.Introduction . page 1
1.1 β– D – fructofuranosidase . page 1
1.1.1 Catalytic mechanism . page 1
1.1.2 Soluble β– D – fructofuranosidase. page 1
1.1.3 Immobilized β– D – fructofuranosidase . page 2
1.2 Fructooligosacharides (FOS) . page 5
1.2.1 Occurrence . page 5
1.2.2 Chemical structure . page 7
1.2.3 Enzyme mechanisms. page 9
1.2.4 Physicochemical properties . page 10
2. β-D-Fructofuranosidase production . page 11
2.1 Material . page 12
2.2 Production line . page 13
2.2.1 Process discription . page 13
2.2.2 Factors effecting fermentation . page 16
2.2.2.1 Time . page 16
2.2.2.2 pH. page 17
2.2.2.3 others factors. page 19
3. Fructooligosaccharides production . page 21
3.1 Process. page 21
3.1.1 Enzyme production. page 22
3.1.2 Enzyme extraction . page 22
3.1.3 Substrates . page 22
3.1.4 Cell immobilization . page 23
3.1.5 Enzyme immobilization . page 24
3.1.6 Fructooligosaccharides syntheisis. page 26
3.1.7 Fructooligosaccharide purification . page 28
3.1.8 Concentration . page 28
3.1.9 Sterilization . page 28
3.2 Equipment diagram . page 29
3.2.1 Laboratorial scale . page 29
3.2.2 Industrial scale . page 30
4. Application . page 30
4.1 β-frucofuranosidase . page 30
4.2 FOS . page 31
4.2.1 Apllication. page 31
4.2.2 Market trend . page 31
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Although many articles on FOSs have been published so far, the extensive data on
the physicochemical properties are scarcely available. Gross, 1962 reported chemical
properties of ome kestosides such as 1-kestose, 6-kestose, and neokestose. The specific
rotation ([α]D20 ) and melting temperature of 1-kestose are 28.5 and 199-200°C
respectively. It forms fine white crystals fairly rapidly. The relative sweetness of 1-
kestose, nystose, and 1F-fructofuranosyl nystose to 10% sucrose solution are 31, 22, and
16%, respectively. Indeed, FOSs have a nice, clean sweet taste typically 0.3–0.6 times as
sweet as sucrose depending on the chain length – sweetness decreases with increasing
chain length.
FOSs are highly hygroscopic; it is difficult to keep the lyophilized products stable
under atmospheric conditions for prolonged periods. The solubility of FOSs is rarely
higher (up to 80% in water at room temperature copared to inulin, just 35%, Douwina
Bosscher, 2008). The viscosity of an FOS solution is relatively higher than that of sucrose
when at the same concentration, and the thermal stability is also higher than that of
sucrose (Neosugar User’s guide, Meiji Seika Co., Kawasaki- shi, Japan, 1982).
FOSs are highly stable in the normal pH range for food (5.0-10.0) and at
refrigerated temperatures over one year. Nevertheless, as a whole, when the pH falls
below 4.0 and treatment temperature is high, they can be hydrolyzed.
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There have been few published studies comparing the physicochemical properties
of FOSs from sucrose, there is a strong indication that FOSs resemble sucrose in many
properties such as solubility, freezing and boiling points, crystal data, etc.
FOSs can also be used to alter the freezing temperature of frozen food to control
the intensity of browing due to the Maillard reaction in heat-processed food. They also
provide high moisture retaining activity preventing excessive drying (Mussato and
Mancilha, 2007).
The caloric value of value of purified fructooligosaccharides (%Sc-FOS > 95%)
has been estimated to be 1.5–2.0 kCal/g. This is approximately 40–50% the caloric value
of digestible carbohydrates such as sucrose.
2. β-D-Fructofuranosidase production
Yeast FFase have been widely studied in Saccharomyces cerevisiae (Taussig and
Carlson, 1983; Reddy and Maley, 1990,1996), Schwanniomyces occidentailis (Miguel
Alvoro-Benito, 2007), Aspergillus niger (Ashok Kuman Balasub ramaniem, 2001),
Aspergillus japonicus (S.I Mussatto, 2009), Aspergillus aculeatus (Iraj Ghazi,
2005),…Among of them, S.cerevisiae is considered as the organism of choice for FFase
production because of its hight sucrose fermentability (Rouwen horst et at., 1991). A
mutant with improved FFase production and the provision of appropriate fermentation
conditions are required for better yield of enzyme (Gomez et al., 2000; ShaWq et al.,
2004). Furthermore, immobilized cells are also a good choice for the production of high
yield of FFase due to they promote an increase in fermentor cell density that
consequently contribute to increased productivity.
The production level of FFase depends to a great extent on the microorganism,
basal substrate and microbial production process. Moreover, the fermentation operation
mode also influences the efficency of the process. Submerged fermentation has been
preferred over the solid-state for FFase production as it is inviromentally friendly,
requires less manpower and give higher yields (Koo et at., 1988). For this reason, most
researches these days concentrate on submerged fermentation for FFase production.
Moreover, Compared with the traditional batch operation, repeated batch, fed-batch, or
continuous operating modes often improve the efficiency of the fermentation process
(Liu Y, Liu D, 2004). Repeated batch cultivation is a well-known method for enhancing
the productivity of microbial cultures because it skips the turnaround time and the lag
phase, thus increasing the process productivity (Radmann EM, 2007; Huang W-C, 2008).
In addition, cell immobilization is particularly feasible for repeated batch fermentation
because the process is characterized by its easy operation, convenient separation of cells
from the broth, and high density of cells (Liu Y, Liu D, 2004). Furthermore, fermentation
with immobilized cells is a convenient manner to reduce the fermentation time during
repeated batch fermentation due to the elimination of the time needed for cell growth
(Yang X, 2005). S.I.Mussatto, 2009 also studied a system by using A.japonicus
immobilized in vegetable fibe as a feasible operation strategy to increase the process
yield. With these recent achievement, this report will represent the production of FFase
by using submerged fermentation with some new methods to increase the yield of
product.
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2.1 Material
Material for the production of FFas are microorganism and nutrient.
Saccharomyces cerevisiae: is oftens isolated from different soil samples and fruits
such as plum, peach, banana, mango,… S.cerevisiae best grows in yeast peptone sugar
agar (YPSA) medium containing (g/l): yeast extract 3.0, peptone 5.0, sucrose 20.0 and
sugar 20.0 at pH 6.0 and room temperature (Ikram ul – Haq, 2006). In order to increase
the yield of obtained, some workers mutated S.cerevisiae by different methods such as
UV irradiation or chemical mutagenesis,… (Ginka et at, 2004; Ikram ul – Haq, 2006).
Subsequently, mutant S.cerevisiae is cultured in YPSA medium, harvested during the
exponential phase growth (about 1.6.106 cells/ml), wash with distilled water and plated
on suitable medium before fermentation. Medium for the production of FFase by
S.cerevisiae was improved by many authors. In general, medium have to contain sucrose
or raffinose which known as the best carbon sourse to get the highest yield of FFase. For
instance, S.cerevisiae which mutated by UV irradiation was inoculated in sterilized
medium containing (mg/ml): yeast extract 3.0, peptone 5.0, raffinose 20.0, agar 20.0 and
2-deoxy-D-glucose 0.02-0.10 (Ikram ul – Haq, 2006).
Aspergillus japonicus is also considered as a potential source for FFase
production. There have been so many research on the production of FFase by
As.japonicus such as Wen Chang chen, 1997; Ching-shan chien, 2001; S.I Mussatto,
2009;… As.japonicus can produce both intra- and extracellular FFase. As.japonicus is
maintained on potato dextro agar (PDA) medium at 40C and spores are maintained by
mixing with glycerine solution in ultrafreeze at -800C. Spores are produced by growing
the strain on PDA medium at 300C for 7-8 days. The best culture for the fermentation
containing (mg/ml): sucrose 20.0, yeast extract 2.75, NaNO3 0.2, K2HPO4 0.5,
MgSO4.7H2O 0.05 and KCl 0.05 (S.I. Mussatto, 2009). Before use, the medium is
sterilized at 1210C for 20min. Spore suspension used in the fermentation contains around
1.8.107 spore/ml.
Besides S.cerevisiae and As.japonicus, some other microorganism such as
Schwaniomyces occidentialis (Miguel Alvaro Benito, 2007); Bifidobacterium lactis
(Carolina Janer, 2004); Aspergillus aculaeatus (Iraj Ghazi, 2005);… were disicribed as a
good souce for FFas production. Table 3 shows the microorganism and the medium for
the production of FFase which have been researched recently.
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Table 3: Microorganism and medium for the production of FFase
Microorganism Medium Author
Sch.occidentalis YEPD (1%, w/v, yeast extract, 2%, w/v, peptone,
2%, w/v, glucose) or Lactose Medium (0.3%,
w/v, yeast extract from Difco, 0.35%, w/v,
bactopeptone, 0.5%, w/v, KH2PO4, 0.1%, w/v,
MgSO4·7H2O, 0.1%, w/v, (NH4)SO2, 2%, w/v,
lactose).
Miguel A´ lvaro-
Benito, 2007
S.cerevisiae (mg/ml) yeast extract 3.0, peptone 5.0, raYnose
20.0, agar 20.0 and 2-deoxy-D-glucose 0.02–0.10
Ikram ul-Haq,
2007
(% w/v) sucrose 20.0, yeast extract 2.75, NaNO3
0.2, K2HPO4 0.5, MgSO4. 7H2O 0.05, and KCl
0.05
S. I. Mussatto,
2009
As. japonicus
20% sucrose, 2% yeast extract (Difco), 2%
NaNO3, 0.05% MgSO4-7H20, and 0.5% K2HPO4.
Wen-Chang Chen,
2001
As. niger (g/l): (NH4)2SO4-45; KH2PO4-23; FeSO4-0.1;
MgSO4 · 7H2O-7; sucrose-50; urea-11 and yeast
extract-5, initial pH 5.
Ashok Kumar
Balasubramaniem,
2001
2.2 Production line
2.2.1 Process discription
Microorganism preparation was described above. After inoculating, the
fermentation experiment is carried out in a fermentor. In laboratory scale operation,
microorganism is cultivated in flasks. The flasks are cultivated in a rotary shaking
inoculator at 30°C for 48 h. The agitation rate is often kept at 200 revolutions per minute.
On the other hand, in large scale operation, the fermentation proce...