Luận văn β d fructofuranosidase production and application to the manufacture of frutooligosaccharides

Preface Oligosaccharides, especially fructooligosaccharides (FOS) are relatively new functional food ingredients that have great potential to improve the quality of many foods In addition to providing[.]

Preface

Oligosaccharides, especially fructooligosaccharides (FOS) are relatively new functional food ingredients that have great potential to improve the quality of many foods In addition to providing useful modifications to food favors and physiochemical characteristics, many of these sugar possess properties that ar beneficial to the heath of consumers These include non-cariogenicity, a low calorific value and the ability to stimulate the growth of beneficial bacteria in the colon Both the production and the applications of food-grade oligosaccharides are increasing rapidly Major uses are in beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy desserts Research continues into the development of new oligosaccharides with a range

of physiological properties and applications in the food industry

FOS has been attracted attention of many researchers with its prebiotical property recently In industrial scale, immobilized fungal β-fructofuranosidase or immobilized cells are used for the manufacture of FOS To improve the yield of FOS, so many studies have been done For this reason, this report will represent some new methods for the production of β-fructofuranosidase and the combination of immobilized fungal β-fructofuranosidase with innovated operations to improve the FOS obtained

1 Introduction (15%)

1.1 β – D – fructofuranosidase

D-fructofuranosidase (FFase, EC 3.2.1.26) is a glycoenzyme that hydrolyses D-fructofuranoside such as sucrose, raffinose, stachyose, ( α-D-Fructofuranosides and β -D-fructopyranosides are not hydrolysed ), also named invertase FFase catalyses the hydrolysis of sucrose into fructose and glucose In addition to releasing D-glucose and D-frucose from sucrose, some microbial β-D-fructofuranosidase may catalyse the synthesis

β-of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to sucrose by different glycosidic bonds depend on the enzyme source (Sangeetha

et al., 2005) This enzyme has been used in food industry to produce inverted sugar and mostly used for the preparation of jams, candies and soft-centered chocolates (Aranda C,

2006) FFase has been found in many different plants and microorganisms FFase from

different sources differs in optinum pH of activity (which may be neutral, acid or alkaline) (Winter H, 2000), optinum temperature of activity,

1.1.1 Catalytic mechanism

There have been many researches on the amino acid residues that present at the active site of FFase However, amino acid involved at the active site of enzyme from different source may various According to the study of Reddy and Maley (1996), the

active site of FFase from Saccharomyces cerevisiae consists of imidazole, carboxylic

and thiol groups Reddy and Maley also indicated that carboxylic groups from Asp-23 and Glu-204 play an important role in the catalytic process Nevertheless, amino acid that participate in the catalytic process of FFase from Arabidopsis thaliane’cell wall (Arabidopsis thaliane is a small flowering plant native to Europe, Asia, and northwestern Africa) are Asp-23 and Glu-203 (M Verhaest, 2006)

The catalytic sucrose process of FFase is divided into three steeps:

- First, FFase links with sucrose to form enzyme-substrate complex at the

Glu-204 by hydrogen bond

- Second, fructosyl residue on sucrose molecule combines with Asp-23 of FFase by valent bond to break the glycosidic bond between glucose and fructose After that, α-glucose receives proton from Glu-204 and releases from enzyme active site

Finally, fructose residue combines with free water in media and separate from Asp-203

-

1.1.2 Soluble β – D – fructofuranosidase

Commercial FFase is often powdered in shape and slight yellow in colour Soluble FFase may be produced from many sources but it mainly produced from

Saccharomyces cerevisiae, As.niger, As.japonicus Soluble enzymes have a high activity

but sensity to temperature, pH, During use, the activity of soluble FFase decreases due

to the change in pH, temperature, conformational changes as a result of friction, amostic pressure imposed by the environs of their use Furthermore, since it is soluble, its cover from a mixture of subtrate and product for use is not economically practical Thus, the advance of immobilized enzyme technology has led to increasing efforts to replace conventional enzymatic process with the preparation as immobilization

1.1.3 Immobilized β – D – fructofuranosidase

The immobilization of invertase broadens the field of its application, since it prevents the crystallization of sugar in food products and the assimilation of alcohol in fortified wines (D N Klimovskii, 1967) and provides the possibility of regulating the composition of the volatile components of wine, brandy, and aqueous liqueurs (S Kh Abdurazakova, 1978) Characteristics of enzymes important for their practical use are their dependences on the pH, the temperature and substrate concentration

The effect of pH

-

In general, immobilized enzyme are more stable with the effect of pH than free enzyme As we can see from Fig.1 below that FFase was immobilized to polyamide was more stable than free FFase (D T Mirzarakhmetova, 1998) The activity of free FFase reached the maximum level at pH around 4 and fell down quickly after that On the other hand, immobilized FFase had pH optimum in the 4.5-5.0 region and a narrower symmetrical profile According to D T Mirzarakhmetova, the shift of the pH optimum into the neutral region is probably due to a change in the local concentration of hydrogen ions in the microenviroment of the enzyme through the introduction of amino groups during the modification of the support The observed narrowing of the pH profile of the immobilized FFase may be a consequence of the selective binding of the more neutral forms of the enzyme with the modified support in the immobilization process

Fig.1 Dependence of rate of hydrolysis of immobilized (1) and free FFase

from yeast on the pH of the medium

- The effect of temperature

The same as the effect of temperature, immobilized enzyme are more stable with the effect of temperature than free enzyme The optimal pH of FFase from

Fig.2 Determination of the pH optima for immobilized (1) and free (2) FFase

from yeast The thermostability cureves are shown in Fig.3 As can be seen, the free enzyme was inactivated completely at 60-700C for 0.5-1h In contrary, immobilized enzyme was not inactivated at 700C, event after 3h

Fig.3 Thermostabilitis of immobilized (1) and free (2) FFase: A (300C), B

(500C), C (550C), D (600C), E (700C)

The effect of substrate and product concentration

-

It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum After this point, increases in substrate concentration will not increase the velocity (Worthington, Biochemical corporation, 1972) This is represented graphically in Fig.4

Fig.4 Effect of substrate concentration on the reaction velocity of enzyme

Acid invertases from plants are also inhibited by their reaction products, with Glc acting as a non-competitive inhibitor and Fru as a competitive inhibitor Figure 5 shows the dependence of the concentration of reaction products on the time for the immobilized and native enzymes The activity of the immobilized enzyme was stable for 1 h, while the native enzyme was inactivated after 15 min

Fig.5 Kinetics of the formation of the products of the enzymatic hydrolysis of

sucrose: 1)immobilized enzyme; 2) native enzyme

1.2 Fructooligosacharides (FOS)

In response to an increasing demand from the customer for healthier and controlled foods, a number of so-called alternative sweeteners such as palatinose and various oligosaccharides including isomaltooligosaccharides, soybean oligosaccharides and especially, fructooligosaccharides have emerged since the 1980s They are important primarily because of their functional properties rather than sweetness All of the new products introduced so far, microbial fructooligosaccharides (FOS) from sucrose have attracted special attention and are attributed to the expansion of the sugar market by several factors First, mass production is not complicated Second, the sweet taste is very similar to that of sucrose, a traditional sweetener

calorie-Various fructans of higher molecular weight have been produced by the action of transfructosylation activity from many plants and microorganisms Depending on the enzyme sources, they have difference linkages; for instance, fructosyltransferase from

fungi such as Aureobasidium pullulans and Aspergillus niger produce only the lF-type

FOS while Claviceps purpurea enzymes and asparagus enzymes produce both lF- and 6G

type oligofructosides It is an accepted opinion that fructooligosaccharides is a common name only for fructose oligomers that are mainly composed of 1-kestose (GF), nystose (GF), and lF-fructofuranosyl nystose (GF) in which fructosyl units (F) are bound at the β-2,1 position of sucrose (GF), respectively, which should be distinguished from other kinds of fructose oligomers (Hidaka H Eida, 1986 and Hayash, 1989)

The production yield of FOS using enzymes originated from plants is low and mass production of enzyme is quite limited by seasonal conditions; therefore, industrial

production depends chiefly on fungal enzymes from either Aureobasidium sp (Yun, J W Jung, 1992) or A niger (Hidaka H Eida, 1986) In 1984, Meiji Seika Co in Japan first succeeded in the commercial production of FOSS (commercial name is Neosugar) by A niger enzyme

1.2.1 Occurrence

Plants

The fructooligosaccharides are found in several kinds of plants, such as onion, wheat, asparagus root, (Shiomi N, 1976) Allen and Bacon, 1956 found transfructosylation activity derived from the leaves of suger beet and were led to the conclusion that in the presence of sucrose, the products of transfer are mainly 1-kestose (

lFfructosylsucrose) with some neokestose (6G-β-fructosylsucrose) An enzyme which transfers the terminal fructosyl residue from the trisaccharide to sucrose to reform a donor molecule was discovered in the Jerusalem artichoke (Edelman, 1966) Onion and asparagus are also important sources of fructosyltransferase (Edelman, 1980) Shiomi et extensively studied the fructosyltransferase extracted from asparagus roots They isolated eleven components of FOS Asparagus oligosaccharides are produced by cooperative enzymatic reactions with at least three kinds of fructosyltransferase: sucrose 1-fructosyltransferase, 6”- fructosyltransferase, and lF-fructosyltransferase They further purified and characterized the individual fructosyltransferases It was found that the general properties resembled those of the Jerusalem artichoke, but its substrate specificity differed Satyanarayana, 1976 described the biosynthesis of oligosaccharides and fructans from agave He isolated various oligosaccharides, (DP 3-15), synthesized them in vitro,

and proposed a reaction mechanism Unlike most enzymes, this agave enzyme is capable

of synthesizing inulotriose from inulobiose The naturally occurring oligosaccharides in agave consists of l-kestose, neokestose, 6-kestose, and their derivatives These oligosaccharides arise not only by transfructosylation reactions but by the stepwise hydrolysis of the higher oligosaccharides and fructans catalyzed by the inherent hydrolytic activity of the enzyme Table 1 show the fructooligosaccharide-synthetic enzymes from plants that were discover by some workers in the past

Table 1: Fructooligosaccharide-synthetic enzymes from plants(1)

Microoganisms

On the other hand, industrial fructooligosaccharides are mainly produced from sucrose by fungal enzyme During the cultivation of several fungi in the sucrose medium, the synthesis of FOSs was observed When the the concentration of sucrose supply in the medium was inadequate, FOSs were ultilized as energy source (Arcamone, 1970) Pazur,

1952 studied transfructosidation of an enzyme of A.oryzae He found two β-2,1 linked tri-

and tetrasaccharides named provisonly 1-inulobiosyl-Dglucose and glucose, respectively (they seem to be 1-kestose and nystose, according to Jong Won

1-inulotriosyl-D-Yun, 1996) The action of C.purpurea enzyme on sucrose also gives rise to a number of oligofructosides including 1-kestose and neokestose (Dickerson, 1972) Fusarium oxysporum is another important enzyme source functioning transfructosylation activity,

which has been studied by many workers Maruyama and Onodera, 1969 isolated two kinds of enzyme showing transfructosylation activity That is everything sciencetists did prior to 1980s

Enzymes with the potential for achiving a high yield of FOS production were

found in the late 1980s and early 1990s Hidaka et al, 1988 studied A niger enzymes;

they fully characterized this enzyme and virtually developed it into the industrial

production of FOS syrup By using A niger enzyme, the maximum FOS conversion

reached 55 - 60% (w/w) based on total sugars Van Balken et, 1991 reported another

fructosyltransferase showing high activity from Aspergillus phoenicis; they produced

FOSS at a 60% yield Furthermore, Takeda et al, 1994 reported a new fungal strain,

Scopulariopsis brevicaulis This strain has the ability of selective production of

1-kestose, a major component of FOS Table 2shows microorganisms that were discovered

as the fructooligosaccharide-producing sources

Table 2: Fructooligosaccharide-producing microorganisms

Moreover, using imobillized cell and enzyme for the production of FOS has been developing Production of FOS from sucrose catalyzed by β-D-fructofuranosidase was

achieved by Chien and Lee (2001) with the use of mycelia of Aspergillus japonicus

immobilized in gluten One gram of mycelia-immobilized particials having a cell content

of 20% (w/w) was incubated with 100ml sucrose solution with an initial content of 400g.L-1 After a reaction period of 5h, the FOS yield was 61% of the total sugars When

Aspergillus oryzae was used, the cultural conditions and reaction parameters have been

standardized to get FOS yield of 58% (Sangeetha, Ramesh and Prapulla, 2002)

Besides fungi, bacterial strain have been reported to produce FOS A transfructosylating enzyme, which produces FOS from sucrose, have been isolated from

Bacillus macerans FG-6 which, unlike other FTases, produced selectively GF5 and GF6

fructooligosaccharides The final yield of FOS was reported to be 33% when 50%

sucrose was used as substrate (Park and Oh, 2001) Lactobacillus reutri strain 121 has

been reported to produce 10g.L-1 (95% kestose and 5% nystose) in the supernatants when grown on sucrose containing medium (Van Hijum, Van Geel-Schutten, 2002)

More recently, high content FOS is produced by removing the liberated glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS This aspect will be represented later

1.2.2 Chemical structure

FOS are easily understood as inulin-type oligosaccharides of D-fructose attached

by β-(2 ->1) linkages that carry a D-glucosyl residue at the end of the chain They constitute a series of homologous oligosaccharides derived from sucrose usually represented by formula GFnas depicted in Figure 6

Fig 6 Chemical structure of fructooligosaccharides

A research group of Meiji Seika Co, the first commercial producer of FOS,

introduced the chemical structure of FOS produced from A niger fructosyltransferase

Until now, FOSs are widely known that oligosaccharides contain 1-kestose, nystose and

1f - fructofuranosyl nystose However, this definition is not completely true FOSs are not

only contain these sugar but also others sugar with higher polymerization Aspergillus sydowi produce six different FOSs showing a high degree of polymerization (DP 3-13)

(Muramasu, 1988) Structure analysis is important in the study of FOSsbecause, as the mentioned above, the degree of polymerization and linkages of FOSs vary with the enzyme sources

1.2.3 Enzyme mechanisms

The reaction mechanism of the fructosyltransferase to form FOSs depends on the source of the enzyme In plants and some microorganisms, a series of enzymes act together whereas a single enzyme works in most other microorganisms For instance, fructosan metabolism in Jerusalem artichoke is established by the combination of two enzyme: sucrose:sucrose l-fructosyltransferase (SST) and β (2->1) fructan:β(2->1) fructan l-fructosyltransferase (FFT) In the first instance, SST converts sucrose into glucose and an oligosaccharide but unable to promote polymerization above the trisaccharide level; further higher polymers are consecutively synthesized by FFT The overall reaction mechanism was expressed as follows:

where GF is a sucrosyl group and n is the number of extrasucrosyl fructose residues Agave enzyme catalyzed a stepwise transfructosylation reaction to give rise to higher FOS formation where synthesis of FOSs from sucrose takes place as follows:

GF + fructosyltransferase -> F-fructosyltransferase +G F-fructosyltransferase + GF -> GF2 + frucotsyltransferase

Here, it is notable that glucose, not fructose, acts as the acceptor of the fructose molecule from sucrose GF2, GF3, and GF4, cannot act as donors of the fructosyl moiety for the synthesis of higher oligosaccharides but act as acceptors of fructose from sucrose only for the synthesis of higher oligosaccharides This mechanism is identical with that of chicory enzyme reported by Singh and Bhatia, 1971 Gupta and Bhatia, 1980 proposed a

model for the fructosyltransferase in F oqsporum They suggested that fructose is

transferred from the donor site to the fructosylated nucleotide bridge and this, in turn, transfers the fructose moiety to the sucrose at the acceptor site to form GF2, GF4 was the highest glucofructosan, suggesting that the acceptor site is perhaps just big enough to accommodate up to GF4 This seems a similar result with the cases of fructosyltransferase

from A niger (Hirayama, 1989 and Hidaka, 1988) and A pullulan (Yun, J W, 1992 and

Hayashi, 1991) in that GF4, is the biggest molecule of FOS in both cases The enzyme reaction mechanism (Figure 7) can be expressed as follows:

GFn + GFn -> GFn-1 + GFn+1

Fig 7.Network of the reaction mechanism for the production of

fructooligosaccharides from sucrose catalyzed by

fructosyltransferase derived from A pullans: G, GF, GF2, GF3 and

GF4 means glucose, sucrose, 1-kestose, nystose, and

lFfructofuranosyl nystose, respectively (Redrawn from ref, 1955)

1.2.4 Physicochemical properties

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

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

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

Table 3: Microorganism and medium for the production of FFase

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´ Benito, 2007

lvaro-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,

On the other hand, in large scale operation, the fermentation process is taken place in a dadecated fermentor with contains drive motor, heaters, pumps, gas control, vessel, intrumenstation and sensors These base components combine to perform some important functions such as: maintain a specific temperature, provide adequate mixing and aeration, allow monitoring and/or control of dissolved oxygen, allow feeding of nutrient solutions and reagents,… The production medium is sterilized by heating it to 121ºC at a pressure

of 1.2 Kgf/cm2 and maintaining those conditions for 30 minutes Heat is supplied by circulating steam through the fermenter jacket Air is filtered by passing it through polypropylene filter Cold water is then circulated through the fermenter’s jacket and the broth is cooled to about 30 ºC The production line of FFase production is shown below:

Fig 8: β-D-Fructofuranosidase production line

The process is monitored continuously through periodic measurement of the following parameters: temperature, pH, activity,… When the peak activity is reached, the batch (crude enzyme) is harverted The crude enzyme is purified by different methods such as: ultrafiltration, gel filtration, ion-exchange chromatography,…Ultrafiltration is used to separate the biomass from the culture fluid, which is later used as a source of fructosyltransferase for the production of FOS For commercial FFase, purified FFase is dried by spray drier or freeze drier to obtain powder product

To determine the highest yield of FFas in the process, the activity is examed during the operation Depending on the authors, FFase activity is defined by different ways For instance, one FFase activity unit is defined as the amount of enzyme which released 1.0 mg of sucrose in 5 min at 350C and pH 5.5 (Ikram ul-Haq, 2007) In most experiments, FFase activity is measured by exame the amount of glucose released in the

whole time of the reaction The mount of glucose is measured by determining color intensity by a UV/Vis spectrophoto meter after glucose reacts with DNS reagent

microfilter

Gel filter

waste

Fig 9: FFase production diagram

2.2.2 Factors effecting fermentation

2.2.2.1 Time

In batch fermentation, enzyme submerged culture production begin after a lag

phase of approximately 8-12h and reached a maximum at the onset of stationary phase

Afterwards, enzyme productivity declined sharply possibly due to the decrease in nutrient

avaiblability in the medium or carbon catabolite repression, and the expression of FFase

in yeast is repressed by monosaccharides such as glucose and fructose (Herwig.et.at.,

2001) Therefore, the growth stage of a culture is a critical factor for the optimal enzyme production IKram ul.Hag, 2008 studied mutant

S.cerevisiae to

improved the production of FFase

by submerged fermentation Time course profiles for FFase production by

wildstyle S.cerevisiae

IS-14 and mutan

S.cerevisiae UMF are

shown in fig 10 As the result, maximum FFase production by mutant S.cerevisiae (34.72±2.6U/ml with 17.05±1.2 g/L sugar consumption

and 7.85±1.8 g/L dry cell mass) was observed

48 h after the onset of incubation Therefore the

rate of volumetric productivity was

improved approximately

31-fold over the parental strain Longer incubation

times did not increase FFase production possibly due to the

Fig 10 FFase production in submerged culture by

Saccharomyces cerevisiae IS-14 (top) and mutant UME-2

(bottom), sucrose concentration 30 g/ L, temperature 30 °C,