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Survival of bifidobacteria after spray-drying
International Journal of Food Microbiology 74 (2002) 79 – 86 www.elsevier.com/locate/ijfoodmicro
Survival of bifidobacteria after spray-drying Wen-Chian Lian, Hung-Chi Hsiao, Cheng-Chun Chou* Graduate Institute of Food Science & Technology, National Taiwan University, 59, lane 144, Keelung Road, Sec. 4, Taipei, Taiwan Received 2 April 2001; received in revised form 5 October 2001; accepted 14 October 2001
Abstract To investigate the survival of bifidobacteria after spray-drying, Bifidobacterium infantis CCRC 14633, B. infantis CCRC 14661, B. longum ATCC 15708, B. longum CCRC 14634 and B. longum B6 were first spray-dried with different carrier media including 10% (w/w) gelatin, gum arabic and soluble starch. B. infantis CCRC 14633 and B. longum were also determined in skim milk. It was found that survival of bifidobacteria after spray-drying varied with strains and is highly dependent on the carriers used. Among the test organisms, B. longum B6 exhibited the least sensitivity to spray-drying and showed the highest survival of ca. 82.6% after drying with skim milk. Comparisons of the effect of carrier concentrations revealed that spray-drying at 10% (w/w) gelatin, gum arabic or soluble starch resulted in the highest survival of bifidobacteria. In addition, among the various outlet-air temperatures tested, bifidobacteria showed the highest survival after drying at 50 °C. Elevation of outlet-air temperature caused increased inactivation of bifidobacteria. However, the inactivation caused by increased outlet-air temperature varied with the carrier used, with the greatest reduction observed using soluble starch and the least with skim milk. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Survival; Bifidobacteria; Spray-drying
1. Introduction Bifidobacteria are gram-positive, nonmotile bacteria that naturally inhabit the guts of warm-blooded animals and man (Scardovi, 1986). Since their first discovery in 1898 by Tisser, extensive research on the properties and applications of these organisms has been conducted. It has been reported that these organisms are able to exert beneficial effects including improvement of intestinal microflora by preventing colonization of pathogens,
*
Corresponding author. Tel.: +886-2-2363-0231x2717; fax: +886-2-2362-0849. E-mail address: [email protected] (C.-C. Chou).
amelioration of diarrhea or constipation, activation of the immune system and increasing protein digestion (Ishibashi and Shimamura, 1993). Owing to these properties, bifidobacteria are now frequently used to prepare probiotic dietary adjuncts. Incorporation of bifidobacteria in food products such as cheese (Dinakar and Mistry, 1994; Blanchette and Roy, 1995; Roy et al., 1995), yoghurt (Holcomb and Frank, 1991; Hughes and Dallas, 1991) and other milk products (Ishibashi and Shimamura, 1993; Gomes and Malcata, 1999) has become an increasingly popular trend. In addition, various health products and pharmaceutical preparations containing dried cells of bifidobacteria are used in the treatment of gastrointestinal disturbances (Gomes and Malcata, 1999).
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 3 3 - 4
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Spray-drying, which has high production rate and low operation cost, is a well-known technology in the food industry. It is one of the common methods used to prepare food adjuncts which are dry, stable and occupy small volume (Potter, 1980). In addition, spray-drying is used for the preservation and concentration of microorganisms (Fu and Etzel, 1995; Teixeira et al., 1995; To and Etzel, 1997a,b). Furthermore, the use of spray-drying has been reported by various investigators to prepare starter cultures which are used to prepare lactic-fermented products or used as adjuncts to enhance the flavor of cheese (Johnson and Etzel, 1993; Johnson et al., 1995; To and Etzel, 1997a,b). However, microorganisms are subjected to heat and dehydration damage during spray-drying. Therefore, the survival of microorganisms deserves much attention if spray-drying is employed for the preparation of microbial cultures. To date, numerous studies concerning the survival of various lactic cultures affected by spray-drying have been reported by various investigators (Espina and Packard, 1979; Kim and Bhowmik, 1990; Fu and Etzel, 1995; Johnson and Etzel, 1995; Teixeira et al., 1995; To and Etzel, 1997a). In addition, To and Etzel (1997b), Mary et al. (1993) and LiChari and Potter (1970), respectively, described the survival of Bradyrhizobium japonicum, Brevibacterium linens and Salmonella after spray-drying. In this study, bifidobacteria were subjected to spray-drying with various carrier media, and the survival of various bifidobacteria after spray-drying was compared. In addition, the effects of carrier concentration and outlet-air temperature on the survival of bifidobacteria after spray-drying were investigated.
2. Materials and methods 2.1. Microorganisms and cultivation Three strains of B. longum and two strains of B. infantis obtained from various sources (Table 1) were used as the test organisms. After two successive transfers of the test organism in Lactobacilli MRS broth (Difco, Detroit, MI, USA) supplemented with 0.05% cysteine (Sigma, St. Louis, MO, USA) (MRSC broth) at 37 °C for 12– 24 h, as shown in Table 1, the activated culture was again inoculated into MRSC broth at 37 °C for 12–
Table 1 Source of the bifidobacteria and cultivation time required to reach its stationary phase when grown in MRSC broth at 37 °C Strain B. B. B. B. B.
infantis infantis longum longum longum
CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
Cultivation time (h)
Source
12 24 12 15 12
A B C A C
(A) Professor C.P. Chiu, Department of Nutrition and Food Sciences, Fu Jen Catholic University, Taipei, Taiwan, ROC. (B) Professor H.Y. Lin, Department of Food Science, National Chung Hsing University, Taichung, Taiwan, ROC. (C) Food Industry Research & Development Institute, Hsinchu, Taiwan, ROC.
24 h. It was then serially diluted and served as the inoculum. To prepare the cell paste, 1.0 ml of the inoculum was inoculated into 400 ml of MRSC broth in a 500-ml screw-cap Erlenmeyer flask, and incubated at 37 °C for a period of 12– 24 h. When growth of test organism became stationary, as indicated in Table 1, cells were harvested by centrifugation (8200 g for 15 min at 4 °C) and were washed twice with 0.01-M sodium phosphate buffer (pH 7.0) (PB). The cell paste was kept at 4 °C and was used the same day. 2.2. Preparation of feed suspensions Feed solutions were prepared essentially following the procedures described by Fu and Etzel (1995) and Kim and Bhowmik (1990) with minor modification. Various solutions containing different carrier agents including 30% (w/w) gelatin (Genfont, Taipei, Taiwan), 35% (w/w) soluble starch (Genfont), 35% (w/w) gum arabic (Panbiochem, Taipei, Taiwan) and 15% (w/w) skim milk (Anchor, Wellington, New Zealand) were first prepared and autoclaved at 121 °C for 15 min. Fresh cell paste was dispersed in PB and was vigorously shaken with a vortex mixer (Genie 2, Scientific Indnipment, Bohemia, NY, USA) until it becomes homogeneous. The cell suspension was then mixed with carrier solution. With the addition of more PB, the final feed solution was adjusted to contain 6% (w/w) cell paste and various amounts of carrier solid (%, w/w) as stated in Results and discussion below. The feed solution contained ca. 1010 –1011 cfu/g dry weight bifidobacteria.
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2.3. Spray-drying A mini spray-dryer Buchi B-191 (Buchi, Flawil, Switzerland) was used. Inlet-air was filtered and heated electrically to 100 F 1 °C after passing through a blower. A peristaltic pump delivered the feed solutions to a two-fluid stainless steel atomizer, with a liquid jet having inside diameter of 0.7 mm. Outlet-air temperatures of 50– 60 °C were controlled by adjusting the flow rate of the feed solution. Dried powder samples were collected from the base of cyclone and mixed thoroughly with a spatula. The samples were stored in tightly sealed sterile bottles at 4 °C.
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ulation with the initial population, which corresponds to 100% survival. Moisture content of samples was determined according to AOAC method (AOAC, 1984) 2.6. Statistical analysis The mean values and the standard deviation were calculated from the data obtained with triplicate trials. These data were then compared by the Duncan’s multiple range method (SAS, 1989).
3. Results and discussion 2.4. Scanning electron microscopy (SEM) The samples of spray-dried powder were analyzed in an Akashi scanning electron microscope (Model ABT55, Akashi, Tokyo, Japan) in order to examine the external appearance of the particles. Microparticles were placed on a carbon adhesive paper and were coated with ˚ gold metal with an ion sputtering coater (Hitachi 200-A E101, Tokyo, Japan). Images of the specimen were viewed with an accelerating voltage of 30 kV. 2.5. Enumeration of bifidobacteria and determination of moisture content To enumerate bifidobacteria, samples were serially diluted with PB and pour plated on MRSC agar. Colonies were counted after incubation at 37 °C for 72 h. However, samples of dried powder were first suspended in PB and homogenized with a stomacher (Lab-Blender 400, Seward, England) for 1 min before further dilution with PB. This treatment ensured the complete release of the entrapped bifidobacteria from inside the dried particles. Percentage of survival after drying was calculated by dividing the surviving pop-
To develop Bifidus products as dietary adjunct and to exhibit probiotic effect on human beings, the Bifidobacterium strains selected must be of human-origin species (Ishibashi and Shimamura, 1993). In this study, three strains of B. longum and two strains of B. infantis, which are the most common species in human infants (Modler et al., 1990), were used as the test organisms. It was reported that stage of growth affects the heat resistance of microorganisms, which are least sensitive to heat at their stationary phase (Hurst and Collins, 1974; Teixeira et al., 1994). In addition, Teixeira et al. (1995) subjected Lactobacillus bulgaricus to spraydrying and recommended drying the cells in their stationary growth phase to obtain a high number of viable cells. Thus, in the present study, cells of bifidobacteria in their stationary phase were collected and spray-dried. As shown in Table 1, grown in MRSC broth, the cultivation time required by bifidobacteria to enter their stationary phase varied with strains. B. infantis CCRC 14661 required 24 h of cultivation, the longest period among the organisms tested, before reaching its stationary phase. On the other hand, B. infantis CCRC 14633, B. longum ATCC 15708 and B.
Table 2 Yield, cell density and water content of bifidobacteria cell pastes Organism B. B. B. B. B.
infantis infantis longum longum longum 1
Cell paste (g/100-ml broth) CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
1
0.82 F 0.02ab 0.79 F 0.01b 0.62 F 0.02d 0.84 F 0.01a 0.69 F 0.02c
Cell density (cfu/g) 11
2.2 F 0.027 10 2.2 F 0.026 1011 1.9 F 0.071 1011 1.7 F 0.070 1011 2.1 F 0.081 1011
Water content (%) 74.8 F 1.00 75.6 F 1.45 75.0 F 1.47 75.5 F 0.96 74.6 F 1.01
Values in the same column with same letter were not significantly different ( p > 0.05) according to Duncan’s multiple range test.
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Table 3 Survival of bifidobacteria after spray-drying1 with various carriers2 Survival (%)3
Strain
B. B. B. B. B.
infantis infantis longum longum longum
CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
Gum arabic (10%)
Gelatin (10%)
Soluble starch (10%)
Skim milk (10%)
B2.15 F 0.37d A3.47 F 0.69d B26.19 F 1.16c B30.95 F 4.57b C41.16 F 0.29a
C1.30 F 0.15d B0.15 F 0.04e A54.33 F 3.08b A39.40 F 2.80c B63.74 F 4.56a
D0.92 F 0.07d C0.08 F 0.04e C16.56 F 1.86b C10.79 F 0.54c D29.06 F 0.91a
A15.99 F 0.23b –4 – – A82.59 F 0.52a
1
Outlet temperature of spray-drier was 50 °C. Means in the same row with different letters A, B, C or D and the same column with different letters a, b, c, d or e differ significantly ( p < 0.05) according to Duncan’s multiple range test. 3 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) by the initial population in cell suspension (cfu/g dry weight) before spray-drying. 4 Not determined. 2
longum B6 entered their stationary phase after only 12 h of cultivation in MRSC broth. The maximum population of bifidobacterium was found to be ca. 9.0 log cfu/ ml at stationary phase, regardless of strain. Table 2 shows the water content and yield of cell paste of various Bifidobacterium strains obtained at their stationary phase. Yields of cell paste ranged between 0.62 and 0.84 g per 100 ml of MRSC broth. The cell pastes contained ca. 11.0 log cfu/g with water contents ranging between 74.6% and 75.6%.
Table 3 shows the survival of various Bifidobacterium strains after spray-drying with 10% of different carriers. The inlet- and outlet-air temperature was constant at 100 and 50 °C, respectively. Generally, it was found that the percentage of survival of B. infantis strains was lower than B. longum strains after drying with same carrier. Of these Bifidobacterium strains tested, survival after spray-drying was greatest for B. longum B6 regardless of the carrier used. Reduction in cell viability after spray-drying is mainly due to heat
Fig. 1. SEM micrographs of spray-dried microparticles containing B. longum B6. Spray-drying was conducted with 10% of gum arabic (A), gelatin (B), soluble starch (C) and skim milk (D).
W.-C. Lian et al. / International Journal of Food Microbiology 74 (2002) 79–86
inactivation (To and Etzel, 1997a,b). Therefore, it is believed that the better survival of B. longum B6 may be attributed to the less sensitivity of this organism than other bifidobacteria tested to heat. In all instances, it was found that spray-drying resulted in the reduction of viable bifidobacteria, with a population reduction of ca. 1.0– 2.0 log/g dry weight, under the test conditions. Regardless of the Bifidobacterium strains and carriers, the powder obtained after spray-drying contained bifidobacteria with a population of ca. 109 – 1010 cfu/g dry weight, meeting the number required for use as a probiotic Bifidus product (Ishibashi and Shimamura, 1993). Fig. 1 shows the scanning electron micrographs of the microparticles obtained after drying with various carriers. Regardless of carriers used for drying, these microparticles were spherical and varied in size. Similar to the observation of Charpentier et al. (1998), the gum arabic, gelatin and soluble starch microparticles showed various extents of flat ball effect on their surfaces (Fig. 1A, B and C), as if they had been dehydrated. On the other hand, the surface of skim milk microparticles appeared grainy with visible cracks (Fig. 1D). These cracks may facilitate the escape of heat from inside the particle after drying causing less heat injury to the entrapped microorganisms. Thus, this may be one of the reasons that contributed to the higher survival of bifidobacteria after spray-drying with skim milk than other carriers (Table 3). Skim milk contains a mixture of protein, carbohydrate, etc. Gum arabic and soluble starch are carbohydrates, while gelatin is a denatured protein. In addition to difference in chemical characteristics, these carriers possess different physical properties such as thermal conductivity, thermal diffusivity, etc. Therefore, it is reasonal to expect that these carriers tested in the present study may exert different degree of protective effect on the entrapped cells of test organism when subjected to heat inactivation during spray-drying and, thus, lead to a different extent of the survival of bifidobacteria. Comparing the effect of gum arabic, gelatin and soluble starch on the survival of bifidobacteria after drying, it was noted that B. infantis strains survived better with gum arabic followed by gelatin and soluble starch. On the other hand, B longum strains survived better with gelatin as a carrier compared to gum arabic and soluble starch. Further testing with B. infantis CCRC 14633 and B. longum B6 showed that the
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percentage of survival increased to 16.0% and 82.6%, respectively, when dried with 10% skim milk. These results show that the survival of bifidobacteria was highly dependent on carriers and it varies with strains. Espina and Packard (1979) reported that survival of L. acidophilus after spray-drying was higher with 25% milk – solid – nonfat (MSNF) than 40% MSNF. We compared the effect of feed concentration on survival of B. longum B6 and B. infantis CCRC 14633 by suspending bifidobacterium cells in feed solutions containing various concentrations of carrier before they were subjected to spray-drying (Table 4). Percentage of survival of B. longum B6 with 10% gum arabic or soluble starch was found to be 41.2% and 29.1%, respectively, the highest among the various concentrations of carrier tested. As the concentration of gum arabic or soluble starch increased, the percentage of survival of B. longum B6 decreased. When gelatin was used as the feed solution, increasing gelatin concentration from 2.0% to 10.0% resulted in an increase in the percentage of survival of B. longum B6 from 58.85% to 63.74%. However, further increasing the gelatin concentration from 10.0% caused the reduction of survival after drying.
Table 4 Survival of bifidobacteria after spray-drying1 with different concentrations of carriers2 Concentrations Survival (%)3 (%) Gelatin
Gum arabic
Soluble starch
B. longum B6 2 58.85 F 0.05a 10 A63.74 F 4.56a 20 B2.07 F 0.03b 30 –
–4 B41.16 F 0.29a A6.51 F 0.13b A3.16 F 0.51c
– C29.06 F 0.91a C1.56 F 0.13b B0.24 F 0.01c
B. infantis CCRC 14633 2 1.03 F 0.10a – 10 B1.30 F 0.15a A2.15 F 0.37a 20 A0.52 F 0.04b A0.65 F 0.10b 30 – A0.04 F 0.01c 1
– C0.92 F 0.0524a B0.09 F 0.0061b B0.01 F 0.0001c
Outlet temperature of spray drying was 50 °C. Means in the same row with different letters A, B or C and the same column for each test organism with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 3 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) with initial population in cell suspension (cfu/g dry weight) before spray-drying. 4 Not determined. 2
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Table 5 Effect of outlet temperature on the survival of bifidobacteria after spray-drying with various carriers1 Temperature (°C)
B. longum B6 50 55 60 B. infantis CCRC 14633 50 55 60
Survival (%)2 Gelatin (10%)
Gum arabic (10%)
Skim milk (10%)
Soluble starch (10%)
B63.74 F 4.56a C9.64 F 0.20b C8.20 F 0.02b
C41.16 F 0.29a B32.79 F 1.89b B23.38 F 2.30c
A82.59 F 0.52a A71.19 F 3.45b A63.21 F 2.38c
D29.06 F 0.91a D0.32 F 0.04b D0.08 F 0.00c
C1.30 F 0.15a C0.41 F 0.04b C0.01 F 0.00c
B2.15 F 0.37a B0.65 F 0.08b B0.06 F 0.09c
A15.99 F 0.23a A8.81 F 0.16b A1.64 F 0.30c
D0.92 F 0.07a D0.03 F 0.01b C0.01 F 0.00c
1
Means in the same row with different letters A, B, C or D and the same column for each strain with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 2 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) by the initial population in cell suspension (cfu/g dry weight) before spray-drying.
A similar trend in the effect of feed concentration on the survival was also noted for B. infantis CCRC 14633. With 10% gelatin, gum arabic and soluble starch, respectively, B. infantis CCRC 14633 exhibited the highest percentage of survival of 1.30%, 2.15% and 0.92%, respectively, after spray-drying. Increasing the concentration (w/w) of gelatin, gum arabic or soluble starch from 10% to 20% or more caused reduced survival of the test organism after drying and was in accordance with the observation of Espina and Packard (1979) on L. acidophilus. It is reported that several factors for and against survival of microorganisms are interrelated during the spray-drying process. First, as water activity decreases on the surface of the particle, wet bulb temperatures are ex-
ceeded. It is at this point that bacteria may be subjected to killing temperatures (Elizondo and Labuza, 1974). However, it is also reported that bacteria are less sensitive to effect of heat in the intermediate moisture range (Karel, 1995). On the other hand, higher solid content in the feed solution would result in larger particles, which are subjected to greater heat damage than smaller ones. Microorganisms entrapped in the particles would also be subjected to more heat damage (Espina and Packard, 1979). This may contribute to the decreased survival of test organisms as observed in the present study. During spray-drying, lethal thermal injury is the main reason for reduced cell viability (To and Etzel, 1997a). Various investigators have reported that in-
Table 6 Effect of outlet temperature on the moisture content of powder after spray-drying with various carrier1 Temperature (°C)
Moisture (%)2 Gelatin
Gum arabic
Skim milk
Soluble starch
B. longum B6 50 55 60
C7.76 F 0.09a C6.13 F 0.04b C5.72 F 0.07b
A10.20 F 0.23a A9.99 F 0.17b A8.64 F 0.35c
B8.93 F 0.06a B7.63 F 0.57b B6.93 F 0.26c
D6.41 F 0.03a D5.03 F 0.05b D4.22 F 0.03c
B. infantis CCRC 14633 50 55 60
A9.96 F 0.03a B8.54 F 0.06b B7.72 F 0.07c
A10.31 F 0.26a A9.97 F 0.13b A8.61 F 0.36c
B8.70 F 0.14a B7.91 F 0.59b C6.99 F 0.21c
C7.91 F 0.07a C6.24 F 0.04b D5.91 F 0.08c
1
Spray-drying was conducted with 10% of carrier. Means in the same row with different letters A, B, C or D and the same column for each strain with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 2
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creasing outlet-air temperature reduces the survival of microorganisms after spray-drying (LiChari and Potter, 1970; Labuza et al., 1972; Espina and Packard, 1979; Kim and Bhowmik, 1990; To and Etzel, 1997a,b). Among these, Espina and Packard (1979) spray-dried L. acidophilus with reconstituted nonfat dry milk and observed a sharp decrease in numbers of survivors as the outlet-air temperature was raised. They indicated that the number of viable L. acidophilus decreased from 7.0 108 to 2.6 107 cfu/g of solids at 75 °C and 3.6 106 and 1.8 106 cfu/g solids at 80 and 85 °C, respectively. To and Etzel (1997a,b) spray-dried B. linens with condensed skim milk and indicated that the percentage of survival of B. linens was halved for every 5 °C increase in the outlet-air temperature using a 4.5-l drying chamber. The data in Table 5 show the survival of bifidobacteria after spray-drying at outlet-air temperatures of 50, 60 and 70 °C, and Table 6 shows the moisture content of the collected dried powder. As expected, a decrease in the percentage of survival of test organisms (Table 5) and in water content (Table 6) was found as the outletair temperature was raised regardless of carriers (Table 5). For example, drying with 10% gelatin, the percentage of survival of B. longum B6 decreased sharply from 63.74% to 8.20% as the outlet-air temperature was increased from 50 to 60 °C (Table 5), while the water content of the dried powder decreased from 7.76% to 5.72% (Table 6). However, the magnitude in the reduction of survival caused by increased temperature varied with the kind of carriers (Table 5). In general, the effect of outlet-air temperature was most pronounced when using soluble starch as the carrier. For example, increasing the outlet-air temperature from 50 to 55 °C, approximately 90.8- and 30.7-fold reduction in survival were noted for B. longum B6 and B. infantis CCRC 14633, respectively, after drying with 10% soluble starch. On the other hand, when drying with other carriers, elevation of outlet-air temperature caused a lesser magnitude in the reduction of survival.
4. Conclusion The results of this study demonstrate that survival of bifidobacteria after spray-drying varies with strains and is highly dependent on the kinds of carrier, as well as outlet-air temperatures employed during spray-drying.
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Among the test organisms, B. longum B6 was the least susceptible to spray-drying under the test conditions. Survival was the lowest with soluble starch as the carrier for drying. Increasing the outlet-air temperature resulted in reduced survival of bifidobacteria, while the magnitude of reduced survival varied with the carriers used. Generally, elevation of outlet-air temperature caused the largest reduction in the survival of bifidobacteria after spray-drying with soluble starch while the magnitude of survival reduction caused by this temperature effect was the least when spray-drying with skim milk.
Acknowledgements This work was supported by the National Science Council (NSC 89-2312-B-002-017) (NSC 89-2316-B002-029), Taiwan, ROC.
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during storage of spray-dried Bradyrhizobium japonicum cell concentrates. J. Appl. Bacteriol. 74, 340 – 344. Modler, H.W., McKellar, R.C., Yaguchi, M., 1990. Bifidobacteria and bifidogenic factors. Inst. Food Sci. Technol. J. 23, 29 – 41. Potter, N.N., 1980. Food Science, 3rd edn. The AVI Publishing, Westport, CT, pp. 283 – 289. Roy, D., Desjardins, M.L., Mondou, F., 1995. Selection of bifidobacteria for use under cheese making conditions. Milchwissenschaft 50, 139 – 142. SAS, 1989. SAS User’s Guide: Statistics, Version 6 edn. SAS Institute, Gary, NC. Scardovi, V., 1986. The genus Bifidobacterium. In: Holt, J.G. (Ed.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams and Wilkins, Baltimore, MD, pp. 1418 – 1434. Teixeira, P., Castro, H., Kirby, R., 1994. Inducible thermotolerance in Lactobacillus bulgaricus. Lett. Appl. Microbiol. 18, 218 – 221. Teixeira, P., Castro, H., Kirby, R., 1995. Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus. J. Appl. Bacteriol. 78, 456 – 462. To, B.C.S., Etzel, M.R., 1997a. Spray drying, freeze drying, or freezing of three different lactic acid bacteria species. J. Food Sci. 62, 576 – 578. To, B.C.S., Etzel, M.R., 1997b. Survival of Brevibacterium linens ATCC 9174 after spray drying, freeze drying, or freezing. J. Food Sci. 62, 167 – 170.
Survival of bifidobacteria after spray-drying Wen-Chian Lian, Hung-Chi Hsiao, Cheng-Chun Chou* Graduate Institute of Food Science & Technology, National Taiwan University, 59, lane 144, Keelung Road, Sec. 4, Taipei, Taiwan Received 2 April 2001; received in revised form 5 October 2001; accepted 14 October 2001
Abstract To investigate the survival of bifidobacteria after spray-drying, Bifidobacterium infantis CCRC 14633, B. infantis CCRC 14661, B. longum ATCC 15708, B. longum CCRC 14634 and B. longum B6 were first spray-dried with different carrier media including 10% (w/w) gelatin, gum arabic and soluble starch. B. infantis CCRC 14633 and B. longum were also determined in skim milk. It was found that survival of bifidobacteria after spray-drying varied with strains and is highly dependent on the carriers used. Among the test organisms, B. longum B6 exhibited the least sensitivity to spray-drying and showed the highest survival of ca. 82.6% after drying with skim milk. Comparisons of the effect of carrier concentrations revealed that spray-drying at 10% (w/w) gelatin, gum arabic or soluble starch resulted in the highest survival of bifidobacteria. In addition, among the various outlet-air temperatures tested, bifidobacteria showed the highest survival after drying at 50 °C. Elevation of outlet-air temperature caused increased inactivation of bifidobacteria. However, the inactivation caused by increased outlet-air temperature varied with the carrier used, with the greatest reduction observed using soluble starch and the least with skim milk. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Survival; Bifidobacteria; Spray-drying
1. Introduction Bifidobacteria are gram-positive, nonmotile bacteria that naturally inhabit the guts of warm-blooded animals and man (Scardovi, 1986). Since their first discovery in 1898 by Tisser, extensive research on the properties and applications of these organisms has been conducted. It has been reported that these organisms are able to exert beneficial effects including improvement of intestinal microflora by preventing colonization of pathogens,
*
Corresponding author. Tel.: +886-2-2363-0231x2717; fax: +886-2-2362-0849. E-mail address: [email protected] (C.-C. Chou).
amelioration of diarrhea or constipation, activation of the immune system and increasing protein digestion (Ishibashi and Shimamura, 1993). Owing to these properties, bifidobacteria are now frequently used to prepare probiotic dietary adjuncts. Incorporation of bifidobacteria in food products such as cheese (Dinakar and Mistry, 1994; Blanchette and Roy, 1995; Roy et al., 1995), yoghurt (Holcomb and Frank, 1991; Hughes and Dallas, 1991) and other milk products (Ishibashi and Shimamura, 1993; Gomes and Malcata, 1999) has become an increasingly popular trend. In addition, various health products and pharmaceutical preparations containing dried cells of bifidobacteria are used in the treatment of gastrointestinal disturbances (Gomes and Malcata, 1999).
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 3 3 - 4
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Spray-drying, which has high production rate and low operation cost, is a well-known technology in the food industry. It is one of the common methods used to prepare food adjuncts which are dry, stable and occupy small volume (Potter, 1980). In addition, spray-drying is used for the preservation and concentration of microorganisms (Fu and Etzel, 1995; Teixeira et al., 1995; To and Etzel, 1997a,b). Furthermore, the use of spray-drying has been reported by various investigators to prepare starter cultures which are used to prepare lactic-fermented products or used as adjuncts to enhance the flavor of cheese (Johnson and Etzel, 1993; Johnson et al., 1995; To and Etzel, 1997a,b). However, microorganisms are subjected to heat and dehydration damage during spray-drying. Therefore, the survival of microorganisms deserves much attention if spray-drying is employed for the preparation of microbial cultures. To date, numerous studies concerning the survival of various lactic cultures affected by spray-drying have been reported by various investigators (Espina and Packard, 1979; Kim and Bhowmik, 1990; Fu and Etzel, 1995; Johnson and Etzel, 1995; Teixeira et al., 1995; To and Etzel, 1997a). In addition, To and Etzel (1997b), Mary et al. (1993) and LiChari and Potter (1970), respectively, described the survival of Bradyrhizobium japonicum, Brevibacterium linens and Salmonella after spray-drying. In this study, bifidobacteria were subjected to spray-drying with various carrier media, and the survival of various bifidobacteria after spray-drying was compared. In addition, the effects of carrier concentration and outlet-air temperature on the survival of bifidobacteria after spray-drying were investigated.
2. Materials and methods 2.1. Microorganisms and cultivation Three strains of B. longum and two strains of B. infantis obtained from various sources (Table 1) were used as the test organisms. After two successive transfers of the test organism in Lactobacilli MRS broth (Difco, Detroit, MI, USA) supplemented with 0.05% cysteine (Sigma, St. Louis, MO, USA) (MRSC broth) at 37 °C for 12– 24 h, as shown in Table 1, the activated culture was again inoculated into MRSC broth at 37 °C for 12–
Table 1 Source of the bifidobacteria and cultivation time required to reach its stationary phase when grown in MRSC broth at 37 °C Strain B. B. B. B. B.
infantis infantis longum longum longum
CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
Cultivation time (h)
Source
12 24 12 15 12
A B C A C
(A) Professor C.P. Chiu, Department of Nutrition and Food Sciences, Fu Jen Catholic University, Taipei, Taiwan, ROC. (B) Professor H.Y. Lin, Department of Food Science, National Chung Hsing University, Taichung, Taiwan, ROC. (C) Food Industry Research & Development Institute, Hsinchu, Taiwan, ROC.
24 h. It was then serially diluted and served as the inoculum. To prepare the cell paste, 1.0 ml of the inoculum was inoculated into 400 ml of MRSC broth in a 500-ml screw-cap Erlenmeyer flask, and incubated at 37 °C for a period of 12– 24 h. When growth of test organism became stationary, as indicated in Table 1, cells were harvested by centrifugation (8200 g for 15 min at 4 °C) and were washed twice with 0.01-M sodium phosphate buffer (pH 7.0) (PB). The cell paste was kept at 4 °C and was used the same day. 2.2. Preparation of feed suspensions Feed solutions were prepared essentially following the procedures described by Fu and Etzel (1995) and Kim and Bhowmik (1990) with minor modification. Various solutions containing different carrier agents including 30% (w/w) gelatin (Genfont, Taipei, Taiwan), 35% (w/w) soluble starch (Genfont), 35% (w/w) gum arabic (Panbiochem, Taipei, Taiwan) and 15% (w/w) skim milk (Anchor, Wellington, New Zealand) were first prepared and autoclaved at 121 °C for 15 min. Fresh cell paste was dispersed in PB and was vigorously shaken with a vortex mixer (Genie 2, Scientific Indnipment, Bohemia, NY, USA) until it becomes homogeneous. The cell suspension was then mixed with carrier solution. With the addition of more PB, the final feed solution was adjusted to contain 6% (w/w) cell paste and various amounts of carrier solid (%, w/w) as stated in Results and discussion below. The feed solution contained ca. 1010 –1011 cfu/g dry weight bifidobacteria.
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2.3. Spray-drying A mini spray-dryer Buchi B-191 (Buchi, Flawil, Switzerland) was used. Inlet-air was filtered and heated electrically to 100 F 1 °C after passing through a blower. A peristaltic pump delivered the feed solutions to a two-fluid stainless steel atomizer, with a liquid jet having inside diameter of 0.7 mm. Outlet-air temperatures of 50– 60 °C were controlled by adjusting the flow rate of the feed solution. Dried powder samples were collected from the base of cyclone and mixed thoroughly with a spatula. The samples were stored in tightly sealed sterile bottles at 4 °C.
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ulation with the initial population, which corresponds to 100% survival. Moisture content of samples was determined according to AOAC method (AOAC, 1984) 2.6. Statistical analysis The mean values and the standard deviation were calculated from the data obtained with triplicate trials. These data were then compared by the Duncan’s multiple range method (SAS, 1989).
3. Results and discussion 2.4. Scanning electron microscopy (SEM) The samples of spray-dried powder were analyzed in an Akashi scanning electron microscope (Model ABT55, Akashi, Tokyo, Japan) in order to examine the external appearance of the particles. Microparticles were placed on a carbon adhesive paper and were coated with ˚ gold metal with an ion sputtering coater (Hitachi 200-A E101, Tokyo, Japan). Images of the specimen were viewed with an accelerating voltage of 30 kV. 2.5. Enumeration of bifidobacteria and determination of moisture content To enumerate bifidobacteria, samples were serially diluted with PB and pour plated on MRSC agar. Colonies were counted after incubation at 37 °C for 72 h. However, samples of dried powder were first suspended in PB and homogenized with a stomacher (Lab-Blender 400, Seward, England) for 1 min before further dilution with PB. This treatment ensured the complete release of the entrapped bifidobacteria from inside the dried particles. Percentage of survival after drying was calculated by dividing the surviving pop-
To develop Bifidus products as dietary adjunct and to exhibit probiotic effect on human beings, the Bifidobacterium strains selected must be of human-origin species (Ishibashi and Shimamura, 1993). In this study, three strains of B. longum and two strains of B. infantis, which are the most common species in human infants (Modler et al., 1990), were used as the test organisms. It was reported that stage of growth affects the heat resistance of microorganisms, which are least sensitive to heat at their stationary phase (Hurst and Collins, 1974; Teixeira et al., 1994). In addition, Teixeira et al. (1995) subjected Lactobacillus bulgaricus to spraydrying and recommended drying the cells in their stationary growth phase to obtain a high number of viable cells. Thus, in the present study, cells of bifidobacteria in their stationary phase were collected and spray-dried. As shown in Table 1, grown in MRSC broth, the cultivation time required by bifidobacteria to enter their stationary phase varied with strains. B. infantis CCRC 14661 required 24 h of cultivation, the longest period among the organisms tested, before reaching its stationary phase. On the other hand, B. infantis CCRC 14633, B. longum ATCC 15708 and B.
Table 2 Yield, cell density and water content of bifidobacteria cell pastes Organism B. B. B. B. B.
infantis infantis longum longum longum 1
Cell paste (g/100-ml broth) CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
1
0.82 F 0.02ab 0.79 F 0.01b 0.62 F 0.02d 0.84 F 0.01a 0.69 F 0.02c
Cell density (cfu/g) 11
2.2 F 0.027 10 2.2 F 0.026 1011 1.9 F 0.071 1011 1.7 F 0.070 1011 2.1 F 0.081 1011
Water content (%) 74.8 F 1.00 75.6 F 1.45 75.0 F 1.47 75.5 F 0.96 74.6 F 1.01
Values in the same column with same letter were not significantly different ( p > 0.05) according to Duncan’s multiple range test.
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Table 3 Survival of bifidobacteria after spray-drying1 with various carriers2 Survival (%)3
Strain
B. B. B. B. B.
infantis infantis longum longum longum
CCRC 14633 CCRC 14661 ATCC 15708 CCRC 14634 B6
Gum arabic (10%)
Gelatin (10%)
Soluble starch (10%)
Skim milk (10%)
B2.15 F 0.37d A3.47 F 0.69d B26.19 F 1.16c B30.95 F 4.57b C41.16 F 0.29a
C1.30 F 0.15d B0.15 F 0.04e A54.33 F 3.08b A39.40 F 2.80c B63.74 F 4.56a
D0.92 F 0.07d C0.08 F 0.04e C16.56 F 1.86b C10.79 F 0.54c D29.06 F 0.91a
A15.99 F 0.23b –4 – – A82.59 F 0.52a
1
Outlet temperature of spray-drier was 50 °C. Means in the same row with different letters A, B, C or D and the same column with different letters a, b, c, d or e differ significantly ( p < 0.05) according to Duncan’s multiple range test. 3 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) by the initial population in cell suspension (cfu/g dry weight) before spray-drying. 4 Not determined. 2
longum B6 entered their stationary phase after only 12 h of cultivation in MRSC broth. The maximum population of bifidobacterium was found to be ca. 9.0 log cfu/ ml at stationary phase, regardless of strain. Table 2 shows the water content and yield of cell paste of various Bifidobacterium strains obtained at their stationary phase. Yields of cell paste ranged between 0.62 and 0.84 g per 100 ml of MRSC broth. The cell pastes contained ca. 11.0 log cfu/g with water contents ranging between 74.6% and 75.6%.
Table 3 shows the survival of various Bifidobacterium strains after spray-drying with 10% of different carriers. The inlet- and outlet-air temperature was constant at 100 and 50 °C, respectively. Generally, it was found that the percentage of survival of B. infantis strains was lower than B. longum strains after drying with same carrier. Of these Bifidobacterium strains tested, survival after spray-drying was greatest for B. longum B6 regardless of the carrier used. Reduction in cell viability after spray-drying is mainly due to heat
Fig. 1. SEM micrographs of spray-dried microparticles containing B. longum B6. Spray-drying was conducted with 10% of gum arabic (A), gelatin (B), soluble starch (C) and skim milk (D).
W.-C. Lian et al. / International Journal of Food Microbiology 74 (2002) 79–86
inactivation (To and Etzel, 1997a,b). Therefore, it is believed that the better survival of B. longum B6 may be attributed to the less sensitivity of this organism than other bifidobacteria tested to heat. In all instances, it was found that spray-drying resulted in the reduction of viable bifidobacteria, with a population reduction of ca. 1.0– 2.0 log/g dry weight, under the test conditions. Regardless of the Bifidobacterium strains and carriers, the powder obtained after spray-drying contained bifidobacteria with a population of ca. 109 – 1010 cfu/g dry weight, meeting the number required for use as a probiotic Bifidus product (Ishibashi and Shimamura, 1993). Fig. 1 shows the scanning electron micrographs of the microparticles obtained after drying with various carriers. Regardless of carriers used for drying, these microparticles were spherical and varied in size. Similar to the observation of Charpentier et al. (1998), the gum arabic, gelatin and soluble starch microparticles showed various extents of flat ball effect on their surfaces (Fig. 1A, B and C), as if they had been dehydrated. On the other hand, the surface of skim milk microparticles appeared grainy with visible cracks (Fig. 1D). These cracks may facilitate the escape of heat from inside the particle after drying causing less heat injury to the entrapped microorganisms. Thus, this may be one of the reasons that contributed to the higher survival of bifidobacteria after spray-drying with skim milk than other carriers (Table 3). Skim milk contains a mixture of protein, carbohydrate, etc. Gum arabic and soluble starch are carbohydrates, while gelatin is a denatured protein. In addition to difference in chemical characteristics, these carriers possess different physical properties such as thermal conductivity, thermal diffusivity, etc. Therefore, it is reasonal to expect that these carriers tested in the present study may exert different degree of protective effect on the entrapped cells of test organism when subjected to heat inactivation during spray-drying and, thus, lead to a different extent of the survival of bifidobacteria. Comparing the effect of gum arabic, gelatin and soluble starch on the survival of bifidobacteria after drying, it was noted that B. infantis strains survived better with gum arabic followed by gelatin and soluble starch. On the other hand, B longum strains survived better with gelatin as a carrier compared to gum arabic and soluble starch. Further testing with B. infantis CCRC 14633 and B. longum B6 showed that the
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percentage of survival increased to 16.0% and 82.6%, respectively, when dried with 10% skim milk. These results show that the survival of bifidobacteria was highly dependent on carriers and it varies with strains. Espina and Packard (1979) reported that survival of L. acidophilus after spray-drying was higher with 25% milk – solid – nonfat (MSNF) than 40% MSNF. We compared the effect of feed concentration on survival of B. longum B6 and B. infantis CCRC 14633 by suspending bifidobacterium cells in feed solutions containing various concentrations of carrier before they were subjected to spray-drying (Table 4). Percentage of survival of B. longum B6 with 10% gum arabic or soluble starch was found to be 41.2% and 29.1%, respectively, the highest among the various concentrations of carrier tested. As the concentration of gum arabic or soluble starch increased, the percentage of survival of B. longum B6 decreased. When gelatin was used as the feed solution, increasing gelatin concentration from 2.0% to 10.0% resulted in an increase in the percentage of survival of B. longum B6 from 58.85% to 63.74%. However, further increasing the gelatin concentration from 10.0% caused the reduction of survival after drying.
Table 4 Survival of bifidobacteria after spray-drying1 with different concentrations of carriers2 Concentrations Survival (%)3 (%) Gelatin
Gum arabic
Soluble starch
B. longum B6 2 58.85 F 0.05a 10 A63.74 F 4.56a 20 B2.07 F 0.03b 30 –
–4 B41.16 F 0.29a A6.51 F 0.13b A3.16 F 0.51c
– C29.06 F 0.91a C1.56 F 0.13b B0.24 F 0.01c
B. infantis CCRC 14633 2 1.03 F 0.10a – 10 B1.30 F 0.15a A2.15 F 0.37a 20 A0.52 F 0.04b A0.65 F 0.10b 30 – A0.04 F 0.01c 1
– C0.92 F 0.0524a B0.09 F 0.0061b B0.01 F 0.0001c
Outlet temperature of spray drying was 50 °C. Means in the same row with different letters A, B or C and the same column for each test organism with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 3 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) with initial population in cell suspension (cfu/g dry weight) before spray-drying. 4 Not determined. 2
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Table 5 Effect of outlet temperature on the survival of bifidobacteria after spray-drying with various carriers1 Temperature (°C)
B. longum B6 50 55 60 B. infantis CCRC 14633 50 55 60
Survival (%)2 Gelatin (10%)
Gum arabic (10%)
Skim milk (10%)
Soluble starch (10%)
B63.74 F 4.56a C9.64 F 0.20b C8.20 F 0.02b
C41.16 F 0.29a B32.79 F 1.89b B23.38 F 2.30c
A82.59 F 0.52a A71.19 F 3.45b A63.21 F 2.38c
D29.06 F 0.91a D0.32 F 0.04b D0.08 F 0.00c
C1.30 F 0.15a C0.41 F 0.04b C0.01 F 0.00c
B2.15 F 0.37a B0.65 F 0.08b B0.06 F 0.09c
A15.99 F 0.23a A8.81 F 0.16b A1.64 F 0.30c
D0.92 F 0.07a D0.03 F 0.01b C0.01 F 0.00c
1
Means in the same row with different letters A, B, C or D and the same column for each strain with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 2 Percentages were calculated by dividing the viable population in powder (cfu/g dry weight) by the initial population in cell suspension (cfu/g dry weight) before spray-drying.
A similar trend in the effect of feed concentration on the survival was also noted for B. infantis CCRC 14633. With 10% gelatin, gum arabic and soluble starch, respectively, B. infantis CCRC 14633 exhibited the highest percentage of survival of 1.30%, 2.15% and 0.92%, respectively, after spray-drying. Increasing the concentration (w/w) of gelatin, gum arabic or soluble starch from 10% to 20% or more caused reduced survival of the test organism after drying and was in accordance with the observation of Espina and Packard (1979) on L. acidophilus. It is reported that several factors for and against survival of microorganisms are interrelated during the spray-drying process. First, as water activity decreases on the surface of the particle, wet bulb temperatures are ex-
ceeded. It is at this point that bacteria may be subjected to killing temperatures (Elizondo and Labuza, 1974). However, it is also reported that bacteria are less sensitive to effect of heat in the intermediate moisture range (Karel, 1995). On the other hand, higher solid content in the feed solution would result in larger particles, which are subjected to greater heat damage than smaller ones. Microorganisms entrapped in the particles would also be subjected to more heat damage (Espina and Packard, 1979). This may contribute to the decreased survival of test organisms as observed in the present study. During spray-drying, lethal thermal injury is the main reason for reduced cell viability (To and Etzel, 1997a). Various investigators have reported that in-
Table 6 Effect of outlet temperature on the moisture content of powder after spray-drying with various carrier1 Temperature (°C)
Moisture (%)2 Gelatin
Gum arabic
Skim milk
Soluble starch
B. longum B6 50 55 60
C7.76 F 0.09a C6.13 F 0.04b C5.72 F 0.07b
A10.20 F 0.23a A9.99 F 0.17b A8.64 F 0.35c
B8.93 F 0.06a B7.63 F 0.57b B6.93 F 0.26c
D6.41 F 0.03a D5.03 F 0.05b D4.22 F 0.03c
B. infantis CCRC 14633 50 55 60
A9.96 F 0.03a B8.54 F 0.06b B7.72 F 0.07c
A10.31 F 0.26a A9.97 F 0.13b A8.61 F 0.36c
B8.70 F 0.14a B7.91 F 0.59b C6.99 F 0.21c
C7.91 F 0.07a C6.24 F 0.04b D5.91 F 0.08c
1
Spray-drying was conducted with 10% of carrier. Means in the same row with different letters A, B, C or D and the same column for each strain with different letters a, b or c differ significantly ( p < 0.05) according to Duncan’s multiple range test. 2
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creasing outlet-air temperature reduces the survival of microorganisms after spray-drying (LiChari and Potter, 1970; Labuza et al., 1972; Espina and Packard, 1979; Kim and Bhowmik, 1990; To and Etzel, 1997a,b). Among these, Espina and Packard (1979) spray-dried L. acidophilus with reconstituted nonfat dry milk and observed a sharp decrease in numbers of survivors as the outlet-air temperature was raised. They indicated that the number of viable L. acidophilus decreased from 7.0 108 to 2.6 107 cfu/g of solids at 75 °C and 3.6 106 and 1.8 106 cfu/g solids at 80 and 85 °C, respectively. To and Etzel (1997a,b) spray-dried B. linens with condensed skim milk and indicated that the percentage of survival of B. linens was halved for every 5 °C increase in the outlet-air temperature using a 4.5-l drying chamber. The data in Table 5 show the survival of bifidobacteria after spray-drying at outlet-air temperatures of 50, 60 and 70 °C, and Table 6 shows the moisture content of the collected dried powder. As expected, a decrease in the percentage of survival of test organisms (Table 5) and in water content (Table 6) was found as the outletair temperature was raised regardless of carriers (Table 5). For example, drying with 10% gelatin, the percentage of survival of B. longum B6 decreased sharply from 63.74% to 8.20% as the outlet-air temperature was increased from 50 to 60 °C (Table 5), while the water content of the dried powder decreased from 7.76% to 5.72% (Table 6). However, the magnitude in the reduction of survival caused by increased temperature varied with the kind of carriers (Table 5). In general, the effect of outlet-air temperature was most pronounced when using soluble starch as the carrier. For example, increasing the outlet-air temperature from 50 to 55 °C, approximately 90.8- and 30.7-fold reduction in survival were noted for B. longum B6 and B. infantis CCRC 14633, respectively, after drying with 10% soluble starch. On the other hand, when drying with other carriers, elevation of outlet-air temperature caused a lesser magnitude in the reduction of survival.
4. Conclusion The results of this study demonstrate that survival of bifidobacteria after spray-drying varies with strains and is highly dependent on the kinds of carrier, as well as outlet-air temperatures employed during spray-drying.
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Among the test organisms, B. longum B6 was the least susceptible to spray-drying under the test conditions. Survival was the lowest with soluble starch as the carrier for drying. Increasing the outlet-air temperature resulted in reduced survival of bifidobacteria, while the magnitude of reduced survival varied with the carriers used. Generally, elevation of outlet-air temperature caused the largest reduction in the survival of bifidobacteria after spray-drying with soluble starch while the magnitude of survival reduction caused by this temperature effect was the least when spray-drying with skim milk.
Acknowledgements This work was supported by the National Science Council (NSC 89-2312-B-002-017) (NSC 89-2316-B002-029), Taiwan, ROC.
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