Food biotechnology

Food biotechnology

PLEASE SCROLL DOWN FOR ARTICLE

On: 17 March 2011

Access details: Access Details: [subscription number 907690007]

Publisher Taylor & Francis

Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,

37-41 Mortimer Street, London W1T 3JH, UK

Food Biotechnology

Publication details, including instructions for authors and subscription information:

http://www.informaworld.com/smpp/title~content=t713597251

Rheological properties of pullulan fermentation broth in a stirred tank fermentor

T Roukas a

a Department of Food Science and Technology, Aristotle University of Thessaloniki, Thessaloniki, Greece

Online publication date: 09 December 2009

To cite this Article Roukas, T.(1999) 'Rheological properties of pullulan fermentation broth in a stirred tank fermentor', Food Biotechnology, 13: 3, 255 — 266

To link to this Article: DOI: 10.1080/08905439909549976

URL: http://dx.doi.org/10.1080/08905439909549976

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes Any substantial or

systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or

distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly

or indirectly in connection with or arising out of the use of this material.

RHEOLOGICAL PROPERTIES OF PULLULAN FERMENTATION

BROTH IN A STIRRED TANK FERMENTOR

T Roukas Department of Food Science and Technology, Aristotle University of

Thessaloniki, Box 250, 54006 Thessaloniki, Greece

ABSTRACT

The rheological properties of the fermentation broth during pullulan

production from synthetic medium by Aureobasidium pullulans P56 in a

stirred tank fermentor were investigated The mycelial forms and the production of extracellular polysaccharide were responsible for the non-newtonian flow behaviour of the fermentation broth The rheological behaviour can be characterized by a power law type of equation The relationship shear rate/shear stress and shear rate/apparent viscosity showed a non-newtonian behaviour of the fermentation broth The power law index was found to be a sensitive factor for the indication of the pseudoplastic behaviour of the broth

255

Copyright © 2000 by Marcel Dekker, Inc www.dekker.com

INTRODUCTION Pullulan is an extracellular water-soluble microbial polysaccharide

produced by strains of Aureobasidium pullulans It is a linear mixed

linkage a-D-glucan consisting mainly of maltotriose units interconnected via a-(l—>6) linkages A number of potential applications has been reported for this biopolymer as a result of its good film-forming properties Pullulan can form thin films which are transparent, oil resistant and impermeable to oxygen Pullulan may be used as a coating and packaging material, as a sizing agent for paper, as a starch replacer in low-calorie food formulations, in cosmetic emulsions, and in other industrial and medicinal applications (Deshpande et al 1992)

During fermentation, the broth consists of the liquid medium, the biomass and any product which is produced by the microorganism Thus, the rheology of the fermentation broth is affected by the composition of the original medium and its modification by the growing culture, the concentration and morphology of the biomass and the concentration of microbial products The rheological properties of fermentation broth in polysaccharide production have a strong effect on the transport phenomena in bioreactors relating to problems of mass transfer, heat transport, and energy consumption An intrinsic feature of extracellular polysaccharide production is the variation of the broth from newtonian to non-newtonian behaviour with the progress of fermentation The rheological behaviour of the fermentation broth is also of great importance for the design, scale-up and operation of bioreactors

The aim of this investigation was to describe the rheological

properties of the fermentation broth during pullulan production from

synthetic medium by A pullulans in a stirred tank fermentor.

MATERIALS AND METHODS Microorganism and culture conditions

Aureobasidium pullulans P56, a strain deficient in melanin

production, was kindly supplied by Prof A Mersmann of the Technical University of Munich The microorganism was maintained on potato

dextrose agar plates at 4°C and subcultured every three weeks Cells for

inoculation of the culture medium were obtained from cultures grown on potato dextrose agar plates at 28°C for 48 h From the petri dish, two

loops of A pullulans cells were transferred to 500 ml conical flasks

containing 150 ml of culture medium (pH 5.5) of the following composition (gH): sucrose 30.0, (NH4)2S04 0.6, yeast extract 0.4, K2HPO4 5.0, MgSO4-7H2O 0.2, and NaCl 1.0 The flasks were incubated at 28°C for

48 h in a rotary shaker incubator (Lab Line Orbit - Environ Shaker, Lab-Line Instr., Inc., Melrose Park, IL) at 200 rpm These cultures were used

to inoculate the production medium at a level of 5% (v/v).The production medium (pH 7.5) had the same composition as described above, but instead of 3% sucrose it contained 5%

Fermentation conditions

The fermentation was carried out in a 9 I stirred tank fermentor with a working volume of 6 I The fermentor was built in our department

It consisted of a glass vessel with stainless-steel endplates and three equally spaced vertical baffles Agitation was provided by a six-flat-blade

impeller (diameter 6 cm) located 4 cm above the bottom of the vessel The fermentor was sterilized at 121°C for 15 min After cooling, 6 1 of production medium was added to the fermentor The medium was inoculated with 300 ml of inoculum The fermentor was incubated at 28°C in a control temperature chamber The impeller speed was 650 rpm and the sterile air flow 3 1/min

Analytical Techniques

At specific time intervals, fermentation broth was removed from the fermentor and analysed Total biomass (mycelial and yeast cells) dry weight was determined by centrifugation of the broth at 10,000xg for 20 min, washing the sediment with distilled water and drying at 105°C overnight The first supernatant was combined with the washings, and the polysaccharide was precipitated with 2 volumes of ethanol at 4°C for 1 h The precipitate was filtered through a pre-weighed whatman GF/A filter and dried at 105°C overnight Residual sugars as glucose were determined in the filtrate according to Dubois et al (1956) The shear stress (T) and the apparent viscosity (na) of the fermentation broth were measured with a Brookfield vjscometer DV-II fitted with a small samples adaptor SC4-18/13R Determinations were made at 25 °C and shear rates (y) of 7.9,15.8, 39.6 and 79.2 s"1 The consistency index (k) and the power law index (n) were evaluated from a log-log plot of equation: na =kvtvl

(Berovic et al 1991) Polysaccharide yield and sugar utilization were expressed as grams polysaccharide/100 g sugar utilized and grams sugar utilized/100 g initial sugar, respectively Microscopic observations were made on a haemocytometer using a standard 14 Zeiss microscope,

equipped with phase contrast All experiments were done twice and reported data are averages

RESULTS AND DISCUSSION Pullulan production in a stirred tank f ermentor

The production of pullulan from synthetic medium by A pullulans

P56 in batch culture is shown in Fig 1 Pullulan concentration increased steadily during the entire time of fermentation up to a level of 34 gF at the 168 h of incubation and then decreased A glucoamylase activity,

named glucoamylase B, was recently detected in fermentation broth of A pullulans at the late stages of fermentation {West and Stronfus, 1996); this

enzyme is capable of degrading not only starch but also pullulan McNeil and Kristiansen (1987) and Gibbs and Seviour (1992) found maximum polysaccharide levels of 20 and 6 gr1 respectively, when different strains

of A pullulans were grown in synthetic medium in a stirred tank fermentor.With a mixed culture of A pullulans and Kluyveromyces fragilis,

Shin et al (1989) obtained pullulan concentrations of 17.5 and 15.5 gh1

using inulin and Jerusalem artichoke extracts as carbon sources, respectively Roukas and Biliaderis (1995) have reported a 6 gr1 pullulan concentration from carob pod extract in shake flask culture, whereas in similar experiments by LeDuy and Boa (1983), maximum polysaccharide levels of 12-14 gr1 were found for various strains of A pullulans grown in

peat hydrolyzates There are several reasons for such variability in pullulan production, including the strain of microorganism, the chemical composition of the substrate, the fermentation system, and generally the

, 70

60

50

o

CQ

-o-Bfotrass

-o-RAian

- * - Sugars

- • - M^o*im (% of total bfamassj

0 24 48 72 9 5 1 2 0 144 1 6 8 1 9 2 216

FIGURE 1

Fermentation kinetics of A pullulans P56 during pullulan production from

synthetic medium in a stirred tank fermentor

conditions employed during fermentation The biomass concentration increased rapidly during the first 48 h of fermentation, then rose slightly

to its maximum value, and finally remained constant until the end of the fermentation A maximum biomass concentration (6.8 gl1) was obtained after 168 h of incubation (Fig 1) The concentration of residual sugars decreased during fermentation coinciding with the increase in biomass and pullulan production (Fig 1) At the maximum concentration of pullulan obtained, the pullulan yield and the sugar utilization were 68.5% and 99%, respectively

|

20

18

16

14

S 12

•S

T îo

S

•s 8

to 6

4

2

0

-°-24h

-t>-48h

-*-72h -o-96h -»-12Gh

- » - 1 4 * -*-168h -*-192h -*-216h

-—o "

L

^

i < ' i i

Shear rate (s 1 )

60 70 80

FIGURE 2

Shear stress as a function of the shear rate at different fermentation times

Shear stress as a function of the shear rate

The shear stress as a function of the shear rate is shown in Fig 2

As shown in Fig 2, all the rheograms of the fermentation broth were characterized with non-newtonian behaviour It was interesting that the shear stress for a given shear rate at first increased, reached a maximum, and then decreased with the progress of fermentation This was due to the decrease in polysaccharide concentration at the end of the fermentation (Fig 1) The maximum shear stress (19.0 dynes/cm2) was obtained at shear rate of 79.2 s"1 after 168 h of incubation

50 •

40 •

30 •

20 •

10

•

-O-7.9 5-1 -O-15.8S-1 -*-39.6s-1 -X-79.2S-1

/ \ / \

Time(h)

FIGURE 3

Change of the apparent viscosity during pullulan production by A

pulfuhns P56 at different shear rates.

Chanqe of the apparent viscosity durinq pullulan production

The apparent viscosity increased as fermentation progressed up to

168 h and then decreased (Fig 3) The maximum viscosity corresponded

to the maximum biomass and polysaccharide concentration (Figs 1 and 3) This was due to the increase in biomass and polysaccharide concentration up to 168 h and the decrease in pullulan production

beyond this time (Fig.l) A pullulans is a polymorphic microorganism

which has a complex life-cycle involving yeast-like cells and mycelial forms The yeast-like cells in the polysaccharide fermentation made a minimal contribution to the high culture viscosity Microscopic examination

Shear rate (s ')

FIGURE 4

Apparent viscosity as a function of the shear rate at different fermentation times

of the fermentation broth indicated that increasing the fermentation time the number of yeast-like cells was decreased, while the number of mycelial forms increased (Fig 1) Thus, the apparent viscosity of the fermentation broth was primarily due to the mycelial and polysaccharide formation The maximum apparent viscosity (51 cp) was obtained at shear rate of 7.9 s'1 after 168 h of incubation The above results show that the mycelial forms and the polysaccharide concentration were mainly responsible for the rheological behaviour of the fermentation broth

Apparent viscosity as a function of the shear rate

As shown in Fig 4, the apparent viscosity decreased with the increase of the shear rate from 7.9 to 79.2 s"1 In all fermentation times, the curves

indicated non-newtonian behaviour and pseudoplastic charecteristic of the fermentation broth This also means that the rheological behaviour of the

broth during pullulan production from synthetic medium by A pullulans

can be described by the power law model na=kvn'1

Changes of the consistency index and power law index during pullulan production

The consistency index followed a pattern similar to polysaccharide concentration (Figs 1 and 5) On the other hand, the power law index decreased rapidly during the first 72 h of fermentation, then increased slightly, and decreased after 120 h of incubation (Fig 5).The increase in the consistency index as the fermentation proceeds up to 168 h can be explained by increasing the biomass and pullulan concentration The maximum consistency index (9.5 Pas") and the minimum power law index (0.58) were obtained after 168 and 216 h of fermentation, respectively The power law index is a sensitive indicator of the non-newtonian behaviour of the broth The larger deviations of the power law index from 1.0 at higher fermentation times indicated that the broth has more tendency toward psewdoplastic behaviour

In conclusion, results showed some important aspects on the rheological behaviour of the fermentation broth during pullulan production

from synthetic medium by A pullulans in a stirred tank fermentor The

mycelial forms and the pullulan concentration influenced the rheological properties of the fermentation broth The rheological behaviour of the broth changed from newtonian to non-newtonian; the latter can be characterized by the power law model The changes in the apparent

168 192 216

FIGURE 5

Changes of the consistency index and power law index during pullulan

production by A puffu/ans P56 in a stirred tank fermentor.

viscosity, consistency index, and power law index were correlated with growth of microorganism and the polysaccharide formation

REFERENCES

Berovic, M., Cimerman, A., Steiner, W and Koloini, T 1991 Submerged

citric acid fermentation: rheological properties of Aspergillus niger

broth in a stirred tank reactor Appl Microbiol Biotechnol 34: 579-581

Deshpande, M.S., Rale, V.B and Lynch, J.M 1992 Aureobasidium puffulans in applied microbiology: a status report Enzyme Microb.

Technol 14: 514-527