Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate

Bioresource Technology 65 (1998) 139-143 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524198 $19.00 ELSEVIER

PII:SO960-8524(98)00005-4

PRODUCTION OF FRUCTOOLIGOSACCHARIDES BY THE MYCELIA OF ASPERGILLUS JAPONICUS IMMOBILIZED IN CALCIUM ALGINATE Rubens Cruz,” Vinicius, D. Cruz, Marcia Z. Belini, Juliana G. Belote & Claudia R. Vieira Depatiamento de Ci&nciasBiol@cas -

fiC,L de Assis, Universidade Estadual Paulista CP 355, CEP 19800-000, Assis-SfZ Brazil

UNESt? Av. Dom Ant&Go. 2.100.

(Received 20 October 1997; revised version received 26 November 1997; accepted 6 December 1997)

market were represented by lipases, proteases and carbohydrases, aimed exclusively at the degradation of their respective substrates. In recent years, some carbohydrases have also started to acquire importance because of their synthesis ability, especially in the food industry. Among these, perhaps the most important is /Gfructofuranosidase (EC 3.2.1.26) with fructosyltransferase activity that catalyzes fructooligosaccharides (FOS) synthesis from sucrose (Hidaka et al., 1988; Park & Almeida, 1991; Su et al., 1991; Yun & Song, 1993). The FOS are composed of sucrose attached by a p(2-+l)linkage to one to three fructose units and are called, respectively, 1-kestose, nystose and fructosyl nystose (Spiegel, 1994; Toshiaki, 1995). galactooligosaccharides, lactosucrose, Like isomaltooligosaccharides and glucosylsucrose, FOS have attracted attention because of their special physiological effect in promoting the growth of Bifidobacteria in the intestinal tract and in decreasing the content of putrefactive substances (Toshiaki, 1995). Besides this, FOS are non-cariogenic and non-caloric but have a sweet taste, with sweetening power from 40 to 60% that of sugar (Spiegel, 1994; Cdndido & Campos, 1995; Toshiaki, 1995). Some microorganisms of the Aspergillus, Fusarium and Aureobasidium genera have been described as good producers of that enzyme, with potential for industrial purposes (Hidaka et al., 1988; Yun & Song, 1993; Pate1 et al., 1994). After extensive screening a strain identified as Aspergillus japonicus showed the highest ability to produce the enzyme intracellularly. To study the possibility of employing this enzyme in industrial production of FOS, the mycelia were immobilized in calcium alginate, inoculated into highly concentrated sucrose reaction parameters were solution and some established.

Abstract This work describes fructose oligosaccharide (FOS) production by the immobilized mycelia (IM) of a strain of Aspergillus japonicus, isolated from soil. The microorganism was inoculated into 50 ml of medium composed of sugar cane molasses (5.0% of total sugars); yeast powdel; 2*0%, K2Hp04, 0.5%; NaN03, 0.2%; MgSO,. 7H,O, 0.05%; KCl, O-OS%,final pH 5.0, and the flasks were agitated in an orbital shaker at 200 rpm for 60 h, at 30°C. The P-fructofiranosidase activity (Uf), transfictosylating activity (Ut), hydrolyzing activity (Uh), and FOS production were analyzed by high performance liquid chromatography. FOS production was performed in a batch process in a 2-l jar fermenter by IM in calcium alginate beads. The optimum pH and temperature were 5.0-5.6 and 55”C, respectively. No loss of activity was observed when the mycelium was maintained at 60°C for 60 min. Maximum production was obtained using 5.75% (cellular weightlvolume) of mycelia (122.4 Ut g-‘) and 65% sucrose solution (w:v) for 4 h of reaction, when the final product reached 61.2870 of total FOS containing GF, (30.5670), GF, (26*4570), GF, (4,27%), sucrose (9.6%) and glucose (29.10%). In the assay conditions, 23 batches were performed without loss of activity of the IM, showing that the microorganism and the process utilized have potential for industrial applications. 0 1998 Elsevier Science Ltd. All rights reserved Key words: Fructooligosaccharides, fl-fructofuranosidase, fructosyltransferase, Aspergillus japonicus.

INTRODUCTION Until recently, industrial application of enzymes has been restricted to only their hydrolytic action. So, the greatest number of biocatalysts available in the *Author to whom correspondence should be addressed. 139

140

R.Cruzet al.

METHODS Culture medium and microorganisms A culture collection of 1350 fungi of the Aspergillus genus was utilized: these have been maintained in slants on dextrose-agar-potato (PDA-Difco) in the Biochemistry and Microorganism laboratories of the FCL, UNESP-Assis. For the screening studies and cellular growth of the screened strain, aliquots of 0.5 ml of a spore suspension (1.8 x 10’ cells) were incubated in 250 ml Erlenmeyer flasks containing 50 ml of medium composed of sucrose, 2.5%; peptone, 1.5%; yeast extract, 0.5%; K2HP04, 0.5%; NaN03, 0.2%; MgS04*7H20, 0.05%; KCl, 0*05%, final pH 5.0. The flasks were agitated in an orbital shaker at 180 rpm for 60 h, at 30°C. One of these flasks, cultivated for only 24 h with the selected strain was utilized as seed culture for the studies of mycelia immobilization and FOS production. These utilized a 2-l jar fermenter (Tecnal) containing 1.5 1 of medium in which sucrose was replaced by sugar cane molasses (5.0% total sugars), and peptone and yeast extract by 2.0% of yeast powder, aeration rate of 1.2 wm with culture time 60 h and temperature 30°C. Mycelia were separated from culture media by filtration and the filtrate utilized as extracellular enzyme source. A lot of 1.4 g (dry weight) of mycelia disrupted in a mortar, suspended in 50 ml of 50 mM McIlvaine buffer, pH 5.0, was centrifuged at 3000 rpm for 10 min and the Supernatant utilized as intracellular enzyme source. All experiments on microorganism growth, enzyme properties and FOS production were conducted in triplicate and the relative standard deviation was less than 5%. Fructofuranosidase activity The reaction mixture (12 ml) for determining the enzyme activity was composed of 2.0 ml of enzyme source with 10 ml of 60% (w/v) sucrose solution in 150 mM McIlvaine buffer, pH 5.0, as substrate. The enzyme reaction was carried out at 50°C for 60 min and was stopped by heating in boiling water for 5 min. Products in the reaction mixture were measured by high-performance liquid chromatography (HPLC) in a Shimadzu LC-10A chromatograph equipped with a refractive index detector, model RID-6A, and a Supelcosyl LC-NH2 column of 250 x 4.6 mm, in a room at 20°C. A system composed of acetonitrilewater (80:20) as solvent and a flow rate of 2.0 ml min-’ were utilized. Glucose, fructose, sucrose and a mixture of 1-kestose, nystose and fructosyl nystose from WACO (Japan) were utilized as standards. In all the analyses, fructose traces were counted as glucose. P-Fructofuranosidase activity (Uf) was calculated from the amount of sucrose consumed in the reaction. The transfructosylating activity (Ut) and the hydrolyzing activity (Uh) were determined from the amounts of transferred fructose and released

fructose, respectively, as described by Su et al. (1991). One unit of each enzyme activity was defined as the enzyme amount required to consume, transfer or release 1 PM of the respective saccharide per minute in the assay conditions. Determination of dry mycelia weight Samples of 1 g of mycelia were washed with distilled water, pre-dried by compression among several layers of filter paper and the dry weight was determined after further drying at 105°C. Crude enzyme properties Aliquots of 2.0 ml of the intracellular enzyme solution were incubated with 10 ml of a 60% (w:v) sucrose solution in 50 mM McIlvaine buffer in the pH range 3.5-7.5 for 3 h, to determine the pH effect at 50°C. The same reaction system was utilized to measure the temperature effect on enzyme activity at pH 5.0 in the temperature range 35-75°C. Thermal stability was determined by measuring the remaining activity after maintenance of the enzyme for 60 min in the same buffer system and temperature range. Mycelia immobilization of selected strain For the immobilization procedure, mycelia of the strain codified as 119T were held in 1.5% glutaraldehyde solution at 4°C for 24 h, centrifuged at 3500 rpm for 10 min, washed in deionized water and suspended in 75 mM McIlvaine buffer, pH 5.0, in the ratio 1:lO (dry weight:volume). After light agitation until the whole homogenization, 0.5% (w:v) of collagen plus 2.5% (w:v) of sodium alginate were slowly added to the suspension, and the gel produced poured into a column with a small opening at the bottom. The drops from this falling into a 2.0% CaCl, plus 50% (IO min-‘) sucrose solution under constant and weak agitation, changed into calcium alginate beads. The beads were kept at 4°C for 24 h before use. FOS production by IM The optimal sucrose concentration and reaction time for FOS production were determined in a 2-1 jar fermenter (Tecnal) by incubation of 5.75% (cell weight/v) of IM (122.4 Ut g- ‘) at 5O”C, pH 5.0. The stability of the immobilized mycelia and the optimum conditions for FOS synthesis were investigated in the same reactor by the batch system at the optimum condition of pH, temperature and at the same mycelia: sucrose solution ratio (5.75%). The change of sucrose to FOS was followed by HPLC analysis. RESULTS AND DISCUSSION Microorganisms Table 1 shows the production P-fructofuranosidase (Uf) by the

of intracellular six strains that

Production of fructooligosaccharides Table 1. Enzyme activity* and FOS production* pre-screened strains Strain code Uf g-r** Ut g-l** Uh g-l** Ut/Uh 118B 119T*** 1146F 1170Ft 225T 675Ft

253.2 257.9 2595 255.2 254.8 251.5

104.0 122.4 132.8 115.9 126.9 117.0

*Means of three experiments, **Dry weight. ***Selected strain.

53.6 13.9 24.2 54.2 24.0 27.9

1.94 8.81 5.48 2.14 5.28 4.19

by the 100

FOS (%) 51.21 61.01 57.77 53.23 57.37 57.01

SD less than 5%.

exhibited the best performance. It can be seen that the final yield of FOS was not only determined by Uf or Ut activity, but also by the Ut/Uh ratio of the enzyme, a high ratio leading to a high FOS yield. These two conditions for an enzyme with an efficient production of FOS have already been observed by Hidaka et al. (1988) and Su et al. (1991) using Aspergillus niger ATCC 20611 and a strain of Aspergillus japonicus, respectively. The strain coded as 119T did not have a ,!&fructofuranosidase productivity (Uf) significantly superior to the others, but showed a high Ut/Uf ratio, and was selected for the rest of this work. Figure 1 shows the growth profile of the organism and enzyme production expressed as transfructosylating activity. The maximum intracellular Ut was obtained at the end of the exponential phase at 48 h of fermentation, and the extracellular activity 24 h later. Fast microorganism growth and enzyme production are positive factors in considering industrial use of a microorganism. In the past few years, several Aspergillus japonicus strains have been described as potentially adequate for p-fructofurano-

20 3

4

5

I

7

0

PH Fig. 2. Effect of pH on the activity of intracellular /I-fructofuranosidase from Aspetgillus japonicus: v, Ut activity; ?,?Uf activity; A, total FOS.

sidase industrial production (Su et al., 1991; Hayashi et al., 1992a; Duan et al., 1993; Chen, 1996). Characterization

of the crude enzyme

Figure 2 shows the effect of pH on the production of FOS for the intracellular enzyme. Uf activity (consumption of sucrose) presents a well defined peak in the pH range 50-56. The synthesis of FOS, however, developed, parallel to Ut activity, in a broader band of pH, following a model observed by Jung et al. (1989) for fructosyltransferase of Aureobasidium pullulans. Highest transfructosylase activity was found at 55-6O”C, as shown in Fig. 3, similar to the enzyme of another strain of Aspergillus japonicus, described by Hayashi et al. (1992b). When the enzyme was submitted to those temperatures for

100

Fig. 1. Effect of incubation time on cellular growth and ,&fructofuranidase production: +, mycelia; 0, sugar concentration; A, Uf intracellular; V, Uf extracellular.

Fig. 3. Temperature effect on the activity and stability of /I-fructofuranosidase from Aspeqillus japonicus. O, Ut stability; V, Ut stability; o, Uf activity.

142

R. Cruz et al.

60 min, no loss of activity was detected. At 65”C, the enzyme maintained about 95% of Uf and Ut activities. These activities, however, dropped to approximately 30% at 70°C. So, the thermostability of the enzyme was higher than that exhibited by the fructosyltransferases from some strains of Aureobasidium (Jung et al., 1989; Lee et al., 1992), Aspergillus niger (Hirayama et al., 1989) and a strain of Aspergillus juponicus (Hayashi et al., 1992a), already proposed as appropriate for industrial use. FOS production by the immobilized mycelium Sucrose concentration Figure 4 shows that increasing the concentration of sucrose in the reaction system produced a linear increase in the production of FOS, up to an optimum concentration of 65%. The higher synthesis of FOS in concentrated solutions of sucrose was first observed by Hidaka et al. (1988), although they tested concentrations of, at the most, 50%. Park and Almeida (1991) verified a considerable increase in FOS production and a parallel decrease in the content of free fructose in the middle of the reaction when the sucrose concentration was increased from 30 to 60%, which was explained by the competition among the water and the substrates used as acceptors in the reactions catalyzed by the P-fructosyltransferase. Higher sucrose concentrations also produced high 1-kestose concentrations, with a consequent decrease of the oligosaccharides with longer fructose chains such as nystose and fructosyl nystose. In accordance with the model proposed by Jung et al. (1989), the fructooligosaccharide synthesis was sense sequential in the always

46

60

66

60

65

GF+GF,+GF,+GF, as a consequence of the increasing K,,, values for such products presented by the transfructosylase. Thus, high concentrations of the preceding oligosaccharide are always necessary for the synthesis of its homologue with one more fructose unit. This would also explain why the content of 1-kestose is higher at the beginning of the enzymatic reaction, as seen in Fig. 5. Taking account of the fact that increasing the length of the fructose chain decreases the sweetening power of the FOS (Spiegel, 1994; Toshiaki, 1995), in the industrial process it would be interesting to utilize more concentrated sucrose solutions. However, the FOS mixture composition has not attracted the attention of many researchers. Reaction time Figure 5 shows that, in assay conditions, the total FOS synthesis was time-dependent up to 3 h, when the sucrose concentration in the reaction medium had dropped to 11%. When the reaction time was extended to 4 h, no increase in total FOS was detected, but sucrose concentration decreased to 10%. When the reaction time was extended to over 4 h, despite little change in the total FOS content, there was a linear increase in GF3 and GF, contents with a corresponding loss of 1-kestose. Therefore, if the purpose is to obtain FOS for its sweet taste or as a sweetener product, the ideal reaction time is between 3 and 4 h. This is a short time compared with the results obtained by other authors using free mycelia from other sources as biocatalyst. Hidaka et al. (1988) and Su et al. (1991) reached the maximum sucrose conversion for FOS (about 60%) only after 24 h of reaction. The lower enzyme:sucrose ratio

70

Suavse cmwentmtion(9th) Fig. 4. Sucrose concentration and FOS production by the IM of Aspergillus japonicus. Reaction system: mycelia 575% (cell w/v), pH 5.0, reaction time 4 h, temperature 50°C. +, Glucose; A, sucrose; n, 1-kestose; o, nystose; v, fructosyl nystose; ?? , total FOS.

Fig. 5. Reaction time Aspergillus japonicus. sucrose 60% (w/v), sucrose; ?,?1-kestose;

and FOS production by the IM of Mycelia 5.75% (cell w/v), pH 5.0, temperature 50°C. v, Glucose; o, o, nystose; A, fructosyl nyStOSe; 0,

total FOS.

143

Production of fn*ctooligosaccharides

Table 2. FOS production by the IM from AspergiUus juponicus* in optimal** conditions of pH, temperature, reaction time and sucrose concentration 1-Kestose (%)

Nystose (%)

Fruc. Nyst. (%)

Total FOS (%)

9.30

31.70

25.90

4.10

61.70

10.20 9.40 9.60

30.33 29.64 3056

26.82 26.44 26.45

4.26 4.46 4.21

61.40 60.54 61.28

Run

Glucose (%)

Sucrose (%)

1

28.90

: Mean

29.17 29.24 29.10

*Cell concentration 5.75% (dry weight/v). **pH 5.5, temperature 55”C, reaction time 4 h, sucrose concentration

(5.6-6 Ut g- ‘) and lower sucrose concentration (50%) used by those authors could explain these differences. Optimal conditions As shown in Table 2, at optimal conditions of pH (5*5), temperature (55”Q sucrose concentration (65%) and reaction time (4 h), for a ftxed ratio mycelia:sucrose solution of 5.75% (cell weight/v), it was possible to obtain a final product with 61.3% of total FOS containing GF2 (30*5%), GF, (26*5%), GF, (4.3%) residual sucrose (9.6%) and glucose (29.1%). In general, the total contents of FOS obtained by the use of free enzyme, free cells or immobilized enzyme from other microorganisms have been from 55 to 60% (Park & Almeida, 1991; Su et al., 1991; Toshiaki, 1995) for the utilization of solutions with initial concentrations of 30-50% of sucrose. As the same conversion rates were obtained from more concentrated sucrose solutions and in a shorter reaction time, it can be concluded that the conditions used in the present work produce larger yields and are more appropriate than those previously described. Although studies on the retention time of the mycelial activity are not yet finished, no loss was observed after 23 batches (92 h of effective use), suggesting that the process and microorganism now used present good potential for industrial use.

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