Continuous fermentation of a supplemented whey permeate medium with immobilized Streptococcus salivarius subsp. thermophilus

Int. Dais'Journal 1 (1991) 1-15

Continuous Fermentation of a Supplemented Whey Permeate Medium with Immobilized Streptococcus salivarius subsp, thermophilus

P. Audet, C. Lacroix & C. Paquin Centre de Recherche en Sciences et Technologie du Lait, Facult6 des Sciences de l'Agriculture et de I'Alimentation, Universit6 Laval, Sainte-Foy, Qurbec, Canada G I K 7P4 (Received 12 February 1991; revised version accepted 27 April 1991)

ABSTRACT salivarius subsp, t h e r m o p h i l u s was immobilized in x-carrageenan/locust bean gum mixed gel beads (0.5-2.0 mm diameter), using a dispersion process in a two-phase system. Inoculated beads were used to ferment a supplemented whey permeate medium continuously in a O. 75-1iter bioreactor equipped with external p H control and mechanical stirring. The pH-stat fermentations were conducted at optimal p H of 5.8. inoculation level of 20% (v/v) and various dilution rates (D = 0"5, 1"0. 1.5, 2.0 and 3.0 h-l). Cell production, carbohydrate utilization and acid production were measured at a steady state. High cell release rates from the gel beads into the broth medium, and growth of free cells in the bioreactor allowed for a steady and efficient inoculation and fermentation of the feed, with cell counts in the outflow varying from 1 × 109 to 3.0 X 108 CFU/ml for D from O"5 to 3.0 h-l. Lactic acid concentration in the effluent was 17.3 and 4.3 g/literfor D = O.5 and 3.0 h -t, respectively. Biomass yield showed a slight but not significant tendency to increase with increasing dilution rates. Gel bead integrity was only slightly affected afier 72 h continuous fermentation, particularly for bead diameters in the range 1.3-1.7 ram. even though the selected level of broth supplementation with KCI was deliberately suboptimal for the mechanical properties of the gel beads. This process could be usedfor efficient continuous inoculation and fermentation of dairy fluids, or for continuous single or mixed strain starter production. Streptococcus

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Int. Dairy Journal 0958-6946/92/$03.50 © 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland

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13 Audet. C. LacroL~c. C. Paquin

NOTATION Contribution of entrapped cells to overall cell production rate (%) Reactor dilution rate (h -~ ) Cellular dilution rate (h -t) Medium flow rate (liters/h) Ft PA Lactic acid production rate (Jliter h) Cell production rate (CFU/liter h) Pc PEC Production rate of entrapped cells (CFU/liter h) Production rate of free cells (CFU/liter h) PFc PI Lactic acid content in the sterile medium (g/liter) Lactic acid content in the outflow (g/liter) P2 Cell release rate (CFU/cm 2 h) Rc Average radius of gel beads (mm) SGB Gel bead area in the reactor (cm 2) Lactose content in sterile medium (g/liter) $1 Lactose content in the outflow (g/liter) $2 Batch fermentation time (h) t Vc Bead volume in the reactor (liters) VL Volume of the medium in the reactor (liters) lit Total volume in the reactor (liters) X Free cell population in batch fermentation (CFU/ml) Maximal cell population in batch fermentation (CFU/ml) Xmax Initial cell population in batch fermentation (CFU/ml) x0 Cell content in the outflow (CFU/liter) X2 Yx/s Biomass yield (CFU/g) Initial specific growth rate in batch fermentation (h -1) Specific growth rate (h -~ )

CEc

D Dc

INTRODUCTION The manufacture of fermented dairy products and lactic starters always involves an inoculation step with lactic acid bacteria, followed by fermentation or maturation stages. These stages are traditionally carried out batchwise. Continuous fermentation processes can provide several advantages over batch fermentation, such as increased stability and control, increased volumetric productivity, and reduced capital and maintenance costs (Ohleyer et al., 1985). However, particular attention must be paid to reactor contamination, to washout of cells and in the case of mixed

Fermentation of a supplemented wh O' permeate medium

3

culture to washout of strains with growth rates lower than the dilution rate and possible dominance of other strains. Driessen et al. ( 1977a, b) proposed a process for manufacturing yoghurt with a mixed culture of Streptococcus salivarius subsp, thermophilus and Lactobacillus delbrueckii subsp, bulgaricus, based on the continuous inoculation of milk at different pH followed by a continuous coagulation stage. The maximum dilution rate was limited by specific growth rate of L. delbrueckii subsp, bulgaricus (1-89 h -~ at pH 5-7). No change in the bacterial balance was observed during steady state at each pH tested (Driessen et al., 1977a). Driessen et al. (1977a, b) and MacBean et al. (1979) confirmed that a mixed culture ofS. salivarius subsp, thermophilus and L. delbrueckii subsp, bulgaricus was stable at pH 5.5-5-7 in a continuous prefermentation process of milk. However, Reichart (1979) carried out continuous prefermentation of milk at different dilution rates, and found that bacterial balance was unstable and maximal dilution rate was limited by the maximum specific growth rate of L. delbrueckii subsp. bulgaricus. Immobilization ofbiocatalysts consists of confining or fixing bacteria, enzymes, animal or plant cells, or yeasts in or on a structure in a particular region of a bioreactor. Cell immobilization brings potential advantages (Kolot, 1984; Nasri et aL, 1987; Scott, 1987; Audet & Lacroix, 1989; Huang et aL, 1991), such as: higher cell density when compared to free cell cultures; improved stability and control during continuous fermentations, with pure or mixed strain cultures: increased reaction rates and higher volumetric productivity; reduced susceptibility of immobilized cells to contamination: and - - e n h a n c e d plasmid stability. Prrvost et al. (1985) have studied the continuous prefermentation of milk using S. salivarius subsp, thermophilus and L. delbrueckii subsp. bulgaricus co-entrapped or separately immobilized in Ca-alginate gel beads. They obtained a final product with constant characteristics because resident time, acidity and continuous inoculation of milk with a constant bacterial balance could be controlled at a desired pH. Coimmobilization of the two strains in the same beads resulted in the domination of S. salivarius subsp, thermophilus against L. delbrueckii subsp, bulgaricus in the gel beads. Immobilization in separate beads was thus preferred to co-immobilization, since bacterial balance could be controlled by varying the initial bead volume for each strain (Prevost et al., 1985; Prrvost & Divies, 1987). In the present study, x-carrageenan and locust bean gum were selected

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P .4udet. C. Lacroix. C. Paquin

for cell entrapment. These biopolymers demonstrated synergistic interaction by improving the theological properties of the resulting entrapment gel during lactic fermentations (Arnaud et al., 1989a, b; Lacroix et al., 1990). When inoculated beads are submerged in a broth medium, rapid bacterial growth takes place preferentially near the surface of the gel beads because of nutrient availability (Bailey & Ollis, 1986; Audet et al., 1988: Arnaud & Lacroix, 1991). Cells released from the biofilm formed near the surface allow very efficient and stable inoculation of the broth medium in batch (Audet et al., 1988) or continuous fermentations (Prrvost & Divies, 1988a, b). This research examined the effect of dilution rate on fermentation characteristics and mechanical stability of gel beads with immobilized Streptococcus salivarius subsp, thermophilus during continuous fermentation of a supplemented whey ultrafiltration permeate. MATERIALS AND METHODS

Materials x-Carrageenan (Satiagel MR 150) and locust bean gum were obtained from Satia, CECA, France. The soybean oil used in the dispersion procedure for immobilization was a commercial grade product. The whey permeate supplemented medium (WPSM) was prepared by ultrafiltration of sweet Cheddar cheese whey as described in Audet et al. (1990) and supplemented with 0-5% (w/v) yeast extract and 0-1 M KCI. It was kept frozen at - 3 0 ° C until use. Lactose content of the medium (St) was 43.5 and 49-6 g/liter and lactic acid content (Pt) was 1.82 and 1.28 g/liter for the respective replicate experiments.

Microorganisms and immobilization procedure Streptococcus salivarius subsp, thermophilus was isolated from Delisle yoghurt (Delisle Inc., Boucherville, Canada). Cultures were propagated in WPSM at 42 °C for 4 h with an inoculum of 10% (v/v). Stationary phase cells were immobilized using a two-phase dispersion process as described by Audet and Lacroix (1989) and Audet et al. (1990). Inoculated gel beads with diameters in the range 0.5-2-0 mm (mean radius F = 0.5 mm) were selected for the experiments by sieving.

Fermentation procedure Duplicate fermentations were carried out continuously for at least 72 h in a 750 ml vertical reactor (Bioflo model C-30, New Brunswick Sci. Co.,

Fermentation of a supplemented whe3"permeate medium

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Edison, N J). Mixing was provided by two marine impellers set at I00 rpm. Temperature was maintained at 42°C. The reactor was flushed with pure sterile nitrogen in order to maintain anaerobic conditions. Once the desired pH value of 5.8 had been reached, pH was maintained by a Radiometer p H M 84 titrator (Radiometer, Copenhagen), coupled with a magnetic valve dispensing 6 N NH4OH. Preincubated beads (20% by volume) were placed in the fermentor for a total fermentation volume of 500 ml. Bead volume (100 ml) was measured by displacement in a graduated cylinder. Two peristaltic pumps were used for continuous feeding of the reactor (Fig. !). A screen of 0.5 m m mesh size was installed at the outlet pipe in order to prevent gel bead washout. Five dilution rates (D = 0-5, 1.0, 1-5, 2.0 and 3.0 h -~) were selected and randomized in the experiments. Cell production, carbohydrate utilization and acid production were measured at steady state (obtained after a m i n i m u m of six residence times) during 3 h for each dilution rate before changing to another dilution rate. Bead volume was determined at the end of the 72 h continuous fermentation by volume displacement. Two replicates of the entire experiment were carried out.

Analytical methods and cell enumeration Sugars and organic acids were determined in duplicate by HPLC and cell enumeration was carried out in duplicate by a pour-plate technique as described by Audet et al. (1988).

N 2 inlet

_um;

6 N NH 4OH

e i ( 0 . 5 mm mesh size)

Fresh sterile

X1 Sl P1 F1

= 0 CFUIL (g/L) (g/L) (L/h)

pH = 5.8 T = 42'~C M i x i n g S p e e d = 100 rpm T o t a l V o l u ~ (Vt) = 500 mL Bead Volume {Vg) = 100 mL Bead diameter range = 0.5 - 2.0 m m h.1 Dilution rates (D) = 0.5,1.0, 1.5, 2.0, 3.0

r , u Tut,,ow

X2 $2 P2 F2

(CFU/L) (g/L) {g/t) = F1 (IJh)

Fig. 1. Diagram of the continuous fermentation procedure.

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P. Audet, C. LacroLr. C. Paquin

Bead diameter distribution analysis Diameter analysis was performed using an image analysis system (Quantinet 520, Cambridge Instrument, Cambridge, UK). Beads were sampled before and after each continuous fermentation experiment in order to determine possible changes of the bead diameter distribution. Samples of beads were drained and stained with pure India ink for at least three days. About 150-300 beads were taken for image analysis measurement, which was repeated four times. Results were expressed in diameter and volume, considering each particle as a sphere.

Statistical analysis and calculations Analysis of variance was carded out using the General Linear Model Procedure (Proc GLM, SAS, 1985). Fermentations were performed in duplicate and treatments (dilution rates) randomized according to splitblock design (Little & Hills, 1978). Orthogonal contrasts for dilution rates were calculated to test for polynomial effects (linear, quadratic, cubic and quartic). Variance homogeneity was tested by a Burr-Foster Q-test (pa > 0.01). A logarithmic transformation was used for certain variables (X2, Rc and Yx/s) to ensure variance homogeneity. The following general formulae were used for calculating or estimating several parameters of the continuous fermentation. Reactor dilution rate: D - Fi

VT

(h-')

(I)

(h-')

(2)

(g/liter h)

(3)

(CFU/liter h)

(4)

(CFU/liter h)

(5)

Cellular dilution rate: Dc _ FI

VL

Lactic acid production rate: PA

= D(P2 - G )

Cell production rate: Pc = DX2

Production rate of free cells: Pvc -

VL

VT

Fermentation of a supplemented wh~, permeate .tedium

7

Production rate of entrapped cells: PEc = Pc - Pvc

(CFU/liter h)

(6)

(CFU/cm'- h)

(7)

(cm 2)

(8)

Cell release rate: Rc = X2(FI - ~ VL)

SOB

Gel bead area in the bioreactor: S ~ _- 3Vo g X 104

Contribution of entrapped cells on overall cell production rate: (%)

(PEc)

CEc= 1 0 0 \ p c )

(9)

Biomass yield: X,

Yx/s - ($1 --$2)

(CFU/g)

(10)

Calculation of fermentation parameters such as Pro PEO Rc and CEC requires values for specific growth rates,/~, of the bacterial strain used in the experiment. It should be mentioned that the production rate of entrapped cells (PEc) and cell release rate (Rc) are two expressions of the same result since PEC = Rc X (SGB/VT). Data from pH controlled (pH 5-8) batch fermentations with free cells of the same S. salivarius subsp, thermophilus in a similar medium (Audet et al., 1988) were fitted with the following equation which describes cell growth in batch fermentation with product inhibition (Weiss & Ollis, 1980; YOndem et al., 1989): In(X) = l n ( X 0 ) + p t - l n [ 1 -

X0 (1 - e " ' ) ]

Xmax

(11)

Specific growth rates in batch fermentation 0i) were estimated from the first derivative of eqn (11) as follows: d(ln(X)) /~

-

dt

[ -

/J -

X0pe"' Xm~,

-z-X-~o(1 -

] e"')

(12)

Value// is the slope of the cell growth curve at a specific fermentation time (t) corresponding to a given lactic acid concentration for batch fermentation ofS. salivarius subsp, thermophilus. It was assumed that the specific growth rate/./for free cells was the same at a given lactic acid concentration in batch or in continuous fermentation.

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P. Audet. C. Lacrog~. C. Paquin

Coefficients of eqn (l 1) (p, Xo, Xmax) were obtained using the nonlinear regression procedure (Proc NLIN. Marquardt computational method. SAS. 1985). The estimates for/,1. X0 and Xm~x from the three replicates of the free cell batch fermentation (Audet et al., 1988) were 1.98 +_ 0-16 h -~, 2.0 x 106 + 0.5 x 106 CFU/ml and 8"6 X 10 9 + 2"5 X 109 CFU/ml, respectively. RESULTS A N D DISCUSSION Reproducible results were obtained with both replicates of the fermentation experiments. Table 1 presents basic data (X_,, Pz and $2) required for estimating other calculated results reported in Table 1 and in Figs 2 and 3. Cell counts in the outflow (X2) decreased significantly with increasing reactor dilution rate D. A high and stable level of inoculation was maintained, even for cellular dilution rates Dc (calculated on the basis of the extraparticulate volume) exceeding specific growth rate (forD and D c in the range of 1.57-2.93 h -~ and 1.96-3-66h -~, respectively). The amount of lactic acid (P2) and residual lactose ($2) in the outflow respectively decreased and increased significantly with increasing D (Table 1). Figure 2 shows cell production rate for continuous fermentation as influenced by dilution rates. Cell prodution rate appeared to increase with D up to a m a x i m u m between 1.5 and 2.0 h -~, and then remained stable with a slight tendency to decrease. However. only the linear effect was statistically significant (pc, < 0.01). Figure 3 shows that lactic acid production rate behaved similarly, with a m a x i m u m at D between 1.5 and 2-0 h -I. Outflowing cells could originate from either free cell growth in the reactor or from growth of entrapped cells with subsequent release from gel beads. The respective contribution of free cell growth and entrapped cell growth to the total biomass production is a function of the dilution rate. Entrapped cell growth takes place preferentially in micro-colonies near the gel bead surface because of nutrient availability, and cells are released from the bead surface into the medium (Audet et al., 1988; Arnaud & Lacroix, 1991). This results in a dense cell layer near the bead surface and a less colonized inner core, due to lack of nutrients caused by diffusional limitations. Cell release rates from the gel beads expressed per hour and cm 2 area of gel bead increased significantly with D (Table 1). The contribution of immobilized cells to overall cell production rate in the continuous fermentations also increased with D from 7% for D --- 1-57 h-~ up to 49% forD = 2-93 h-~ (Table 1). At dilution rates higher than 1-5 h -~, immobilized cells contributed to a substantial part of the global cell production rate. W h e n the specific growth rate (/,i), which is a

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Fig. 2. Mean cell production rate in continuous fermentation at various dilution rates. (Significant linear effect of D on logl0(Pc), p , < 0,01), 15 _= E

.£ "6A "lD

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Fig. 3. Mean lactic acid production rate in continuous fermentation at various dilution rates. (Significant linear, quadratic (Pa < 0,001 ) and cubic effect (Pa < 0.05) of D on PA).

function of the steady-state lactic acid concentration at the selected cellular dilution rate, exceeds this dilution rate (,fi > Dc), cell release rate (Rc) and contribution of immobilized cells to overall cell production rate (CEc) cannot be calculated since free cell growth is sufficient for inoculating the medium under these conditions. Conversely, when Dc >/~ (i.e. for Dc = 1-96, 2.49 and 3.71 h-~), free cell growth cannot provide a steady inoculation of the medium since the reactor is then operating under washout conditions. Entrapped cell growth is then solicited more, providing a massive contribution of cells released from gel beads into the medium, and ensuring a steady inoculation at higherD. Biomass yield (Yzas) shown in Table 1, expressed as the n u m b e r of cells produced per gram of lactose consumed, displayed a slight but not

Fermentation of a supplemented wh~" permeate medium

I1

significant tendency towards a maximum at D = 2.0 h-I for both replicates. Biomass and lactic acid productivities per day could be taken into consideration for comparing continuous and batch fermentations. In continuous fermentations, these productivities were calculated from X~ and P2 respectively by multiplying each of them by flow rate (F~) and time (24 h). In batch fermentations (Audet et al., 1988), biomass and lactic acid productivities per day were calculated by multiplying the cellular and lactic acid concentration at stationary phase by the medium volume in the reactor (500 ml), considering a single batch fermentation per day. Biomass and lactic acid productivities per day in continuous fermentation increased from 4.1 × 10~2 to 1.0 × 10~3CFU/day and from 72-6 to 154-5 g/day respectively with increasing D from 0.35 to 2.93 h -I. Lactic acid production per day reached a maximum of 159.5g/day at D = 1.99 h-~. In batch fermentations with free cells ofS. salivarius subsp. thermophilus (Audet et al., 1988), biomass and lactic acid productivities per day were 3-1 × 1012CFU/day and 9.57 g/day, respectively. Continuous fermentation increased biomass and lactic acid daily productivities by factors varying from 1-3 to 4-6 and from 7-6 to 16.7, respectively. These increased productivities demonstrated one of the advantages of continuous fermentations for producing biomass or metabolites. Higher daily productivities could be attained since the outflowing inoculated medium was not exhausted and could be further fermented in a second stage. The effect of the 72 h continuous fermentation on the mechanical stability of the gel beads was determined for the two replicates by measuring gel bead volume and size distribution at the beginning and end of the fermentations using an image analysis system. It should be emphasized that in this study, KCI was added to the WPSM at a suboptimal concentration (0.1 M instead of 0.3 M) in order to decrease bead resistance, thus amplifying the phenomenon of bead disruption in a short time period. Lacroix et al. (1990) have shown that 0-3 M KCI supplementation in the broth medium is required for best rheological properties of the beads during Lactobacillus casei entrapped cell fermentation. In recent work with the same organism, complete mechanical stability of the gel beads was observed during a 50 h continuous fermentation, at a mixing rate as high as 150 rpm (unpublished data). Moreover, Huang et al. (1991) showed total stability of the gel beads entrapping Leuconostoc mesenteroides during 10 days' continuous fermentation in Lactobacilli MRS broth medium (Difco Laboratories, Detroit, USA) supplemented with 0-1 M KCI. KCI supplementation is not required for mechanical integrity of the gel beads during fermentation in milk based media, as synergistic interactions between the gel matrix

12

P Audet. C. LacroL~. C. Paquin

and the medium result in a large gel strengthening effect (unpublished data). Bead volume in the reactor decreased by about 4-5% during 72 h continuous fermentation for both replicates. Bead samples were taken before and after continuous fermentation in order to determine which bead diameter range was most affected. We observed that the bead volume distribution was changed after fermentation (Fig. 4). Figure 4(a) shows the evolution of bead volume distribution during 72 h continuous fermentation for the first replicate. Beads in the range 0.9-1.3 mm and over 1.7 mm appeared to be the most affected. Beads of intermediate size were less susceptible, and an optimal diameter class could be selected between 1.3 and 1.7 mm. Increased % volume for diameters lower than 0.9 m m could be explained by particles originating from disrupted larger beads. Figure 4(b) shows the evolution of bead volume distribution for another experiment conducted under adverse conditions for bead integrity, i.e. using two flat blade impellers. A large bead volume loss was observed (>20%), resulting from higher shear stresses and collisions in the reactor. Two p h e n o m e n a could explain these results: (1) large beads were more sensitive to shear forces and collisions between beads, and with the impellers and the probes inside the reactor (mixing effect); (2) small beads were more damaged by cell growth within the gel, resulting in a weakening of the theological properties of the gel. This latter effect could be explained by the colonization of the entire gel bead volume for small beads. For large beads, cell growth does not occur in the inner core due to diffusional limitations, resulting in better mechanical properties of the biocatalyst (Arnaud & Lacroix, 1991).

CONCLUSION Entrapment of S. salivarius subsp, thermophilus or other lactic acid bacteria could be effective in providing a steady inoculum for continuous production of biomass or metabolites. Cell counts in the broth medium and cell production rates in the reactor remained high even at high dilution rates, due to the increase of cell release rate from the gel beads into the fermentation medium, preventing cell washout. Although the experiments were conducted under suboptimal conditions, the optimum bead diameter for mechanical integrity of the biocatalyst during long term continuous fermentation could be close to 1-5 mm, without apparent bead loss in the range 1.3-1.7 mm. Typical losses for 72 h continuous fermentations were about 5% in WPSM with 0-1 M KC1.

Fermentation of a supplemented wh O" permeate medium

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IB Bead volume distr, before fermentation 40

•

Bead volume distr, after fermentation

30

20

o~ 10

M

Diameter class (mm)

(b) Fig. 4. Typical bead volume distributions belbre and after 72 h continuous fermentation. (a) Mixing provided by two marine impellers setat 100 rpm: (b) mixing provided by two flat blade impellers set at 100 r'pm.

However, in other investigations, continuous fermentations with entrapped lactic acid bacteria resulted in total stability of the gel beads for over 10 days. ACKNOWLEDGEMENTS The authors thank the Natural Sciences and Engineering Research Council of Canada, MESS-Action Structurante and FCAR for providing financial support, and Dr Serge Guiot and Miss Sylvie Rochelot of the

14

P Audet. C. LacroL~. C. Paquin

Institut de Recherche en Biotechnologie (6100 Ave. Royalmount, Montrral, Qc, C a n a d a ) for technical assistance in image analyses.

REFERENCES Arnaud, J.-P., Choplin, L. & Lacroix, C. (1989a). Rheological behavior of kappa-carrageenan/locust bean gum mixed gels. J. Text. Stud. 19, 419-30. Arnaud, J.-P. & Lacroix, C. (1991). Diffusion of lactose in x-carrageenan/locust beam gum gel beads with or without entrapped growing lactic acid bacteria. Biotechnol. Bioeng. (in press). Amaud, J.-P., Lacroix, C. & Choplin, L. (1989b). Effect of lactic acid fermentation on the rheological properties of x-carrageenan/locust bean gum mixed gels inoculated with S. thermophilus. BiotechnoL Bioeng., 34, 1403-8. Audet. P. & Lacroix, C. (1989). Two-phase dispersion process for the production ofbiopolymer gel beads: Effect of various parameters on bead size and their distribution. Process Biochem.. 24, 217-26. Audet, P., Paquin, C. & Lacroix, C. (1988). Immobilized growing lactic acid bacteria with x-carrageenan-locust bean gum gel, AppL Microbiol. Biotechnol.. 29, 11-18. Audet, P., Paquin, C. & Lacroix, C. (1990). Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in r-carrageenan locust bean gum gel beads. AppL MicrobioL Biotechnol.. 32, 662-8. Bailey, J. E. & Ollis, D. F. (1986). Biochemical Engineering Fundamentals (2nd edn). McGraw-Hill, New York. Driessen, F. M., Ubbels, J. & Stadhouders, J. (1977a). Continuous manufacture of yogurt I: Optimal conditions and kinetics of the prefermentation process. Biotechnol. Bioeng.. 19, 821-39. Driessen, F. M., Ubbels, J. & Stadhouders, J. (1977b). Continuous manufacture of yogurt II: Procedure and apparatus for continuous coagulation. Biotechnol. Bioeng.. 19, 841-51. Huang, J., Lacroix, C., Daba, H., Pandian, S. & Simard, R. E. (1991). Increased strain stability and bacteriocin prodution in continuous cultures of immobilized Leuconostoc mesenteroides cells.Appl. Env. MicrobioL (submitted for publication). Kolot, F. B. (1984). Immobilized cells for solvent production. Process Biochem.. 19, 7-13. Lacroix, C., Paquin, C. & Arnaud, J.-P. (1990) Batch fermentation with entrapped growing cells ofLactobacillus casei. Optimization of the rheological properties of the entrapment gel matrix. Appl. Microbiol. Biotechnol., 32, 403-8. Little, T. M. & Hills, F. J. (1978). Agricultural Experimentation. Design and Analysis. John Wiley, New York. MacBean, R. D., Hall, R. J. & Linklater, P. M. (1979). Analysis of pH-stat continuous cultivation and the stability of mixed fermentation in continuous yogurt production. Biotechnol. Bioeng., 21, 1517-41. Nasri, M., Sayadi, S., Barbotin, J. N. & Thomas, D. (1987). The use of the immobilization of whole living cells to increase stability of recombinant plasmids in Escherichia coil J. BiotechnoL, 6, 147-57.

Fermentation of a supplemented wh0" permeate medium

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