Pullulan is a polysaccharide produced by Aureobasidium pullulan. In this study, the effect of pH on the molecular weight of pullulan was investigated. High concentration of pullulan was obtained when initial pH was 6. Pullulan having molecular weight of 500,000–600,000 was produced at initial pH of 3.0, while Pullulan with molecular weight of 200,000–300,000 was produced at pH above 4.5. To obtain high molecular weight pullulan with high concentration, pH was initially controlled at pH 6, followed by pH shift from pH 6 to pH 3. Transition of pH at 2 days of fermentation was observed to be optimum. Higher molecular weight pullulan was also obtained when sucrose concentration was 50 g/l compared to the result obtained at initial sucrose concentration of 20 g/l. Sucrose concentration and pH of the fermentation broth seem to be important parameters in obtaining high molecular weight of pullulan.
PuUulan is an exopolysaccharide produced by Aureobasidium pullulans having a structure of a – l , 6 polymer of maitotriose. The effects of many operating parameters on the biosynthesis of Pullulan have been studied. The effects of pH on imllulan biosynthesis were also r~m~ted(Catiey, 1971; Herald and Kristiansen, 1985; Kato, 1975; Shin, 1988) including optimum pH for high pullulan concentration. Since molecular weight of a polysaccharide is a very important parameter characterizing the physical properties of the polysaccharide, the effects of operating parameters, especially the effects of pH and sucrose on the moleodar weight of pullulan were investigated.
MATER I A L S AND M E T H O D S
Cultivation of Microorganism.
Aureobasidium pullulan IFO 4464 was u.~d in this study. The basic fermentation medium contained sucrose 5%(w/v), “.~HP(h 0.5%, NaCI 0.1 % , MgSO4 0.02°/6 and yeast extract 0.16%. The fermentations were carried out at 28℃;
in 2-1 j a r fermentor(New Brunswick, Bioflo) with the aeration rate of 1 vvm.
Determination of crude Pullulan .
Crude pullulan solution was harvested by adding ethanol into the supernatant obtained by precipitating the cells from the fermentation broth(Catiey,1970). The precipitated crude Pullulan was collected and dried to a constant weight in a vaccum oven at 80-90℃ .
Determination of Average Molecular Weight .
Molecular weight of pullulan was estimated by a combination of Mark-Houwink-Sakuradaequation(Wiley et al., 1987) for pulhilan and the calibration curve drawn by using PEO samples as polymer standards.
RESULTS AND DISCUSSION
When crude Pullulan sample was injected into GPC, high molecular weight peak was detected along with low molecular weight peak. The crude pullulan sample was dried to a constant weight in a drying oven and redissolved into 0.1 M NaC1 solution. As a result black powder which was insoluble in water was found and the remainder was mostly water soluble. When the water soluble fraction was injected into GPC, the high molecular weight peak was not detected, but the low molecular weight peak was detected as shown in Fig.1.
The low molecular weight peak had the same retention time as the standard pullulan . Low molecular weight material was also identified as pullulan through F T – I R analysis(Lee,1991).
Fig. 1 Gel permeation chromatogrmn of pullulan fermentation broth. Samples were obtained at 7 days of fermentation. (a) ethanol precipitate from fermentation broth, (b) water soluble fraction(pullulan) of polysaccharides. Numerals indicateinitial pH.
Fig. 2(a) shows time courses of pullulan concentrations at various initial pH conditions. The high concentration of pullulan(22g/l) was obtained at pH 6 compared to the result at pH 3(13 g/l). Fig. 2(b) shows time courses of molecular weight of pullulan at various initial pH. Pullulan having molecular weight of 500,000-600,000 was produced at initial pH of 3.0, while pullulan with molecular weight of 200,000-300,000 was produced at pH above 4.5. In the range of initial pH 4.5 – 7.5, the molecular weight of pullulan decreased during fermentation from higher molecular weight to 200,000-300,000.
Fig. 2 Time courses of pullulan production at v a r i o u s initial pHs. (a) pullulan concentration o : pH 3, • : pH 4, z~: pH 5, A: pH 6, n: pH 7, (b)molecular weight o : pH 3, $ : . p H 4 . 5 , A: pH 6, • : pH 7.5
Since the molecular weight of pullulan was decreased during the fermentation, separate experiments were performed u s i n g standard pullulan. Pullulan was observed to be simply hydrolyzed by water at pH 4 . 5 – 7 . 5 a n d to be stable only
when pH was 3 as shownin Fig. 3. Polyhydroxybutyrate, a biodegradable polymer,was also reported to be also hydrolyzed in water(Doietal., 1990).It is important to obtain high molecular weight pullulan at a high concentration. For this purpose pH was initially controlled at pH 6,then pH was
shifted at an appropriate time from pH 6 to pH 3 and sucrose (50g/l) was added to make-up the carbon source for pullulan biosynthesis. By following this strategy, 22g/l pullulan with the molecular weight of 500,000-600,000 was obtained. In this case, optimal pH transition time w a s to be decided. It was found from experiments that high molecular weight was obtained in a short time by shifting the pH at 2 days of fermentation, as shown in Fig.4.
Fig. 3 Molecular weight of pullulan for various conditions, o : distilled water,
- : 0.1 M NaCI, a : constant pH(pH ffi 6), • : constant pH (pH = 3)
Fig. 4 Molecular weight of pullulan for various pH transition times. @ indicate pH transition time. o : 2 day, • : 2.5 day, ~ : 3.5 day
To clarify the environmental effects on the molecular weight of pullulan, different q~anti~es of sucrose were added just lifter shifting the pH of the fermentation broth. Table 1 shows ~he molecular weights of pullulan when 500 ml of sucrose solution of pH 3 was added after cultivating at pH 6. Higher molecular weight pullulan was obtained when sucrose concentration was 50 g/I compared to the result obtained at sucrose concentration of 20 g/l. Sucrose concentration seems to be an important parameter in regulating the molecular weight of pullulan. Even though the detailed mechanism of molecular weight determination and the function of intracellular enzymes relating to polysaccharide biosynthesis are not clarified, the results from this study can be applied for the production of biopolymers .
Table 1. Average molecular weight of pullulan in the case of pH shift from pH 6 to pH 3 with different concentrations of sucrose.
|sucrose concentration at pH shift|
|fermentation time(day)||S = 20g/l||s = 50g/l|
Catley, B.J. (1970). F.E.B.S. Letters, 10, 190–193.
Catley, B.J. (1971). Appl. Microbiol., 22, 650–654.
Doi, Y., Kanesawa, Y., Kunioka, M. and Saito, T. (1990). Macromolecules, 23, 26–31
Herald, P.J and Kristiansen, B. (1985), Biotechnol. Bioeng., 27, 1516–1519.
Kato, A, Makoto, S. (1975). U.S. Pat., 3,912,591.
Lee, K.Y. (1991). Ph. D. Thesis, Seoul National University, Seoul, Korea.
Shin, Y.C. (1988), Ph.D. Thesis, KAIST, Korea.
Wiley, B.J., Arcidiacono, S., Sousa, S., Mayer, J.M. and Kaplan, D.L. (1987) Report, NATRIC/TR-88/012: Order No. AD-A191040, p29.
Introduction of Pullulan
Pullulan is natural water-soluble polysaccharide, fermented by Auveobasidium pullulan . It consists mainly of maltotriose units linked through 伪-1, 6 – glucosidic bonds. The average molecular weight is 2脳105 Da. Pullulan can be developed into various products. It is an excellent film-former, producing a film which is heat sealable with good oxygen barrier properties. It can be used widely in both the pharmaceutical and food industries, such as encapsulating agents, adhesives, thickening, and extending agent. Pullulan has been used as a food ingredient for over 20 years in Japan. It has Generally Regarded As Safe (GRAS) status in the US for a much wider range of applications.
Tabletting: Pullulan is an excellent tabletting excipient.
Pan coating: Pullulan with its excellent adhesive properties and extremely low viscosity is ideally suited for pan-coating chewing gum, chocolate and candies.
Agglomeration: Pullulan has about double the adhesive strength of food starch. This property combined with its lack of flavor enables it to be used effectively in typical agglomeration processes.
Sauces and dressings: Due to its ‘adhesive properties’ use of granular pullulan in sauces improves their ‘on product’ retention without unduly increasing sauce viscosity.
Innovative soft candy: Incorporation of 2-3% Pullulan, enables innovative soft chewy candies to be made by a cold extrusion process without resorting to conventional boiling. This enables heart sensitive materials to be used in these products.
Coatings: Pullulan solutions which are odorless and flavorless can be used as a coating for a wide range of food products to improve appearance, reduce blemishes and increase shelf life.
Capsules: Pullulan can be formed into capsules for use with pharmaceutical and nutraceutical products. Its non-animal origin ensures there are no safely concerns with consumers and it is suitability for all consumers groups.
Safty of Pullulan
Animal experiments showed that pullulan polysaccharides does not cause any biological toxicity and abnormal state, can be safely used in food and medicine.In Japan are classified as no use restrictions with starch additives.
Advantages of Pullulan
1.Pullulan film sugar isolated from oxygen, but good moisture permeability
2.High adhesion, high dispersion stability
3.Stable aqueous solution, tolerate a wide PH range, won’t produce gel phenomenon
4.Soluble in water, offers an excellent foam stability
5.Edible and biodegradable
The high binding and film-forming properties of pullulan make it a valuable ingredient for coating. Not only can Pullulan help produce strong, smooth and highly glossed surfaces but also increase shelf life.
Dressing and Sauces
Due to its ‘adhesive property’ use of pullulan in sauces improves their ‘on product’ retention without unduly increasing sauce viscosity. It is acknowledged that it is difficult to retain sauces on the surface of some pre-cooked meat and fish products; but this problem can be overcome by incorporating pullulan into the formulation.
Innovative Soft Candy
Incorporation of 2-3% Pullulan enables innovative soft chewy candies to be made by a cold extrusion process without resorting to conventional boiling. This enables heat sensitive materials to be used in these products.
Pullulan can be formed into capsules for use with pharmaceutical and nutraceutical products. Benefits of capsules made from Pullulan:
Its Nature origin ensures it is no GMO
Cost Control- production line still be used to make capsules with Pullulan as the material
Oxygen impermeableeffectively keep ingredients stable, oil in particular
Stable, no cross-linking
Ratify in many countries
Transparent and shining
1. Is sample available before order?
Yes, sample available and usually free charges, but freight is yours.
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Sample in 1-5 days, bag order 5-15 days.
6. How long has your company run?
We have engaged in exporting for more than six years.
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Nanjing Joint Friends Chemical Co.,Ltd
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