growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

9
Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates D. Charalampopoulos, S.S. Pandiella and C. Webb Satake Centre for Grain Process Engineering, UMIST, Department of Chemical Engineering, Manchester, UK 2001/21: received 19 March 2001, revised 31 July 2001 and accepted 16 November 2001 D. CHARALAMPOPOULOS, S.S. PANDIELLA AND C. WEBB. 2002. Aims: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. Methods and Results: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8 10–10 11 log 10 cfu ml )1 , depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l )1 total fermentable sugars) and free amino nitrogen (approx. 80 mg l )1 ). The consumption of sugars during the exponential phase (10–12 h) resulted in the accumulation of lactic acid (1 06–1 99 g l )1 ) and acetic acid (0 29–0 59 g l )1 ), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17–43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3 40 and 3 77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7 20–9 43 log 10 cfu ml )1 ) than in the malt medium. This could be attributed to the low sugar content (3–4 g l )1 total fermentable sugar for each medium) and the low free amino nitrogen concentration (15 3–26 6 mg l )1 ). In all fermentations, the microbial growth ceased at pH values (3 73–4 88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. Conclusions: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. Significance and Impact of the Study: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria. Correspondence to: S.S. Pandiella, Satake Centre for Grain Process Engineering, UMIST, Department of Chemical Engineering, PO Box 88, Manchester M60 1QD, UK (e-mail: [email protected]). ª 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 92, 851–859

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Page 1: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

Growth studies of potentially probiotic lactic acid bacteriain cereal-based substrates

D. Charalampopoulos, S.S. Pandiella and C. WebbSatake Centre for Grain Process Engineering, UMIST, Department of Chemical Engineering, Manchester, UK

2001/21: received 19 March 2001, revised 31 July 2001 and accepted 16 November 2001

D. CHARALAMPOPOULOS, S .S . PANDIELLA AND C. WEBB. 2002.

Aims: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum,

Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media

were investigated. The objectives were to identify the main factors influencing the growth and

metabolic activity of each strain in association with the cereal substrate.

Methods and Results: All fermentations were performed without pH control. A logistic-type

equation, which included a growth inhibition term, was used to describe the experimental data.

In the malt medium, all strains attained high maximum cell populations (8Æ10–10Æ11

log10 cfu ml)1, depending on the strain), probably due to the availability of maltose, sucrose,

glucose, fructose (approx. 15 g l)1 total fermentable sugars) and free amino nitrogen (approx.

80 mg l)1). The consumption of sugars during the exponential phase (10–12 h) resulted in the

accumulation of lactic acid (1Æ06–1Æ99 g l)1) and acetic acid (0Æ29–0Æ59 g l)1), which

progressively decreased the pH of the medium. Each strain demonstrated a specific preference

for one or more sugars. Since small amounts of sugars were consumed by the end of the

exponential phase (17–43%), the decisive growth-limiting factor was probably the pH, which at

that time ranged between 3Æ40 and 3Æ77 for all of the strains. Analysis of the metabolic products

confirmed the heterofermentative or homofermentative nature of the strains used, except in the

case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway.

All strains produced acetic acid during the exponential phase, which could be attributed to the

presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to

consume the remaining sugars and accumulate metabolic products in the medium, probably

due to energy requirements for cell viability, while Lact. acidophilus entered directly into the

decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact.

reuteri, attained lower maximum cell populations (7Æ20–9Æ43 log10 cfu ml)1) than in the malt

medium. This could be attributed to the low sugar content (3–4 g l)1 total fermentable sugar

for each medium) and the low free amino nitrogen concentration (15Æ3–26Æ6 mg l)1). In all

fermentations, the microbial growth ceased at pH values (3Æ73–4Æ88, depending on the strain)

lower than those observed for malt fermentations, which suggests that substrate deficiency in

sugars and free amino nitrogen contributed to growth limitation.

Conclusions: The malt medium supported the growth of all strains more than barley and

wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum

appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and

Lact. reuteri.

Significance and Impact of the Study: Cereals are suitable substrates for the growth of

potentially probiotic lactic acid bacteria.

Correspondence to: S.S. Pandiella, Satake Centre for Grain Process Engineering, UMIST, Department of Chemical Engineering, PO Box 88, Manchester M60 1QD,

UK (e-mail: [email protected]).

ª 2002 The Society for Applied Microbiology

Journal of Applied Microbiology 2002, 92, 851–859

Page 2: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

INTRODUCTION

Many lactic acid bacteria strains, characterized as probiotics,

have been proven to beneficially affect humans or animals by

improving the properties of their indigenous gut flora

(Fuller 1989; Havenaar and Huis in’t Veld 1992). The

incorporation of potentially probiotic lactic acid bacteria of

human origin in traditional fermented foods has been

established in the dairy industry, leading to the production

of different types of fermented milks and yoghurts (Gomes

and Malcata 1999). Strains of the Lactobacillus genus, such

as Lactobacillus acidophilus, Lact. casei, Lact. reuteri, Lact.gasseri and Lact. paracasei, constitute a significant proportion

of the lactic acid bacteria used in commercial probiotic milk-

based products (Shortt 1999). New applications of probiotic

micro-organisms in foods have been introduced into the

market or are still in the development phase, such as frozen

yoghurt, soy yoghurt, dairy desserts, cheese, ice-cream,

bread and chocolate (De Vuyst 2000).

This work is part of a project studying the development of

a novel food fermentation process, based on the use of

cereals as substrates for potentially probiotic lactic acid

bacteria. Cereals, such as wheat and rye, are used for

sourdough production, a fermentation process that now-

adays is performed by using defined mixed starter cultures,

consisting of lactic acid bacteria strains isolated from natural

environments and yeasts (Lonner and Akesson 1988; Ganzle

et al. 1998). Other cereal-based fermented foods are pro-

duced indigenously in Asia and Africa by natural lactic acid

fermentation under uncontrolled conditions (Oyewole

1997). However, as natural fermentations rely on microbial

populations present in the raw material, these products

exhibit substantial variations in flavour and quality (Meraz

et al. 1992; Giraud et al. 1998). The good adaptation of

lactic acid bacteria in cereals suggests that the utilization of a

potentially probiotic strain as starter culture in a cereal

substrate would produce a fermented food with defined and

consistent characteristics and possibly health-promoting

properties. However, several technological aspects have to

be considered in the design of such a novel food fermen-

tation process, such as the composition and processing of the

raw material, the growth capacity and productivity of the

starter culture and the stability of the final product during

storage (De Vuyst 2000).

Probiotic products are usually standardized, based on the

presumption that culture viability is a reasonable measure of

probiotic activity, thus the ability of the strain to attain a

high cell population is of primary importance. A concen-

tration of approx. 107 cells ml)1 at the time of consumption

is considered functional (Gomes and Malcata 1999; Shortt

1999). High cell growth and acidification rates would also

result in the reduction of fermentation times and enhance

the viability of the specific strain by preventing growth of

undesirable micro-organisms present in the raw material

(Marklinder and Lonner 1992). Lactobacilli are very

fastidious micro-organisms that require fermentable carbo-

hydrates, amino acids, vitamins of the B-complex, nucleic

acids and minerals to grow, regardless of the specific

nutrient requirements of the strain (Gomes and Malcata

1999). Thus, the substrate composition and nutritional

requirements of the strain considerably affect the overall

performance of the fermentation. Microbial growth also

depends on environmental factors, such as pH, temperature

and accumulation of metabolic end-products. Several

investigators have demonstrated the effects of these para-

meters on the growth of lactic acid bacteria based on

experiments and have used empirical mathematical models

to describe them (Mercier et al. 1992; Ganzle et al. 1998;

Lejeune et al. 1998).

The distribution of the metabolic end-products is another

important parameter when evaluating a microbial food

process. Lactic acid-fermented foods are usually character-

ized by a sour taste, which is mainly attributed to lactic and

acetic acids produced via the homo- or heterofermentative

metabolic pathways. Consequently, an appropriate selection

of the strain is necessary to efficiently control the distribu-

tion of the metabolic end-products (Lonner and Akesson

1988; De Vuyst 2000).

In this work, the overall growth kinetics of the potentially

probiotic Lact. fermentum, Lact. reuteri, Lact. acidophilus and

Lact. plantarum strains in malt, barley and wheat media have

been studied. The main objectives were to identify the key

factors influencing the growth and metabolic activity of each

strain in association with the cereal substrate in order to

evaluate the production process of a novel cereal-based

probiotic food. Subsequent research will aim to develop a

mathematical model that could predict the growth and

acidifying properties of specific probiotic lactic acid bacteria

in different cereal substrates, taking into account their

different sugar compositions.

MATERIALS AND METHODS

Micro-organisms and culture conditions

The micro-organisms used in this study were as follows:

Lact. reuteri NCIMB 11951 (National Collection of Indus-

trial and Marine Bacteria, Aberdeen, Scotland, UK),

isolated from human intestine; Lact. acidophilus NCIMB

8821, isolated from human saliva; Lact. fermentum NCIMB

12116, isolated from ogi (fermented corn gruel indigenous to

South Nigeria) and Lact. plantarum NCIMB 8826, isolated

from human saliva. The strains were maintained at 4�C and

subcultured monthly on slants prepared from MRS agar

(Oxoid, Basingstoke, Hampshire, UK). Colonies isolated

from MRS agar plates were precultured twice in MRS broth

852 D. CHARALAMPOPOULOS ET AL.

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 851–859

Page 3: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

(Oxoid) for approx. 12 h at 37�C. The 12-h precultured cells

were then centrifuged (5000 g, 10 min, 4�C), washed twice

with sterile quarter-strength Ringer’s solution and resus-

pended in Ringer’s solution. The bacterial suspensions were

then used to inoculate the fermentation media at 2Æ5% (v/v)

for Lact. fermentum, 3% (v/v) for Lact. reuteri, 7Æ5% (v/v)

for Lact. acidophilus and 2Æ5% (v/v) for Lact. plantarum. In

all cases, the initial microbial concentration was approxi-

mately 107 cfu ml)1.

Cereal-based fermentation media

Malt, wheat and barley grains were used to prepare the

fermentation media following the same procedure. The

grains were ground in a laboratory Falling Number hammer

mill (Perten Instruments, Huddinge, Sweden) with a sieve

of size 0Æ5 mm. A sample (50 g) of the flour obtained was

mixed with 450 ml tapwater and the resulting slurry

centrifuged (6000 g) for 30 min at room temperature. The

starch-free supernatant fluid was collected and immediately

sterilized at 121�C for 45 min. Sedimentation of solids

(possibly protein mixtures) was observed after sterilization

and approx. 4–5% (w/w) of solids were present in the final

fermentation media. The extraction and sterilization pro-

cedures were repeated four times. The sugar content, pH,

free amino nitrogen (FAN) and buffering capacity (mmol

HCl per pH unit per unit volume) of the three media were

analysed. The small differences observed in the initial

composition of the media were probably due to reactions

between sugars and amino acids during sterilization. The

malt medium had a pH value between 5Æ12 and 5Æ21 and

contained maltose (4Æ82–5Æ43 g l)1), sucrose (6Æ36–

6Æ98 g l)1), glucose (2Æ00–2Æ05 g l)1) and fructose (0Æ95–

1Æ12 g l)1); the FAN concentration was between 75Æ2and 82Æ1 mg l)1 and the buffering capacity was

8Æ45 mmol pH)1 l)1. The barley medium had a pH value

between 5Æ69 and 5Æ83 and contained maltose (0Æ91–

1Æ19 g l)1), sucrose (1Æ56–1Æ78 g l)1), glucose (0Æ17–

0Æ20 g l)1) and fructose (0Æ16–0Æ18 g l)1); the FAN

concentration was between 15Æ3 and 15Æ9 mg l)1 and the

buffering capacity was 6Æ54 mmol pH)1 l)1. The wheat -

medium had a pH value between 6Æ23 and 6Æ38 and contained

maltose (0Æ82–1Æ09 g l)1), sucrose (1Æ88–2Æ12 g l)1), glucose

(0Æ15–0Æ18 g l)1) and fructose (0Æ14–0Æ16 g l)1); the FAN

concentration was between 20Æ1 and 26Æ6 mg l)1 and the

buffering capacity was 3Æ30 mmol pH)1 l)1. All fermenta-

tions were performed under no pH control in 500-ml screw-

capped bottles, without agitation. The temperature was set

at 37�C, which is usually the optimum for lactic acid

bacteria of human origin. Samples were collected approx.

every 2 h for the first 12 h and then at intervals of 8–12 h

during the next 36 h. All fermentations were performed in

duplicate.

Analytical methods and statistics

Bacterial enumeration. Fermentation samples were deci-

mally diluted in sterile quarter-strength Ringer’s solution

and four 0Æ15-ml aliquot dilutions were plated on MRS agar

using a precalibrated pipette and incubated at 37�C for 48 h.

Colony-forming units were counted (cfu ml)1) and the

results expressed as their log10 values. Throughout this

work, cell values are given as mean values of eight replicate

measurements and the S.E. of the mean was calculated with

95% confidence.

Buffering capacity. The buffering capacity of each cereal

medium was determined by titrating 100 ml of the medium

with HCl (1 mol l)1). The values were expressed as the

amount of HCl (mmoles) required to drop 1 pH unit per

unit volume (l) (Pai et al. 2001).

Determination of ethanol and acetic acid. Ethanol and

acetic acid were determined in sample supernatant fluids by

gas chromatography using a gas chromatograph (5890;

Hewlett-Packard, Palo Alto, CA, USA) equipped with a

flame ionization detector, set at 250�C, a ChemStation

Integrator (Hewlett-Packard) and a capillary column

(30 m · 0Æ25 mm; HP-5; Hewlett-Packard). The tempera-

ture was initially held at 90�C for 2Æ5 min and then

increased at 8�C min)1 to 138�C; the total time of analysis

was 10 min. The retention times were approximately 1Æ1and 3Æ8 min for ethanol and acetic acid, respectively.

The data presented are mean values of three replicate

measurements.

Determination of free amino nitrogen, sugars and lacticacid. The FAN content was determined by the ninhy-

drin method (Magne and Larher 1992). Glucose, fruc-

tose, sucrose, maltose, D- and L-lactic acid were

determined using enzymatic methods (Boehringer

Mannheim). The total lactic acid concentration was

calculated by adding the values for D- and L-lactic acids.

The inaccuracy and imprecision of each of the enzymatic

methods was determined by conducting a block design

experiment, consisting of three sets of six replicate

measurements. The one-way AnoVa test applied to the

data showed that variations between block means were, in

all cases, not significant (P > 0Æ05), which suggests that

they were probably due to imprecision and not inaccur-

acy. Thus, the S.D. of each method was calculated after

treating all of the measurements from the three blocks as

a single set. The S.D. values obtained were 0Æ10 for lactic

acid, 0Æ12 for maltose, 0Æ14 for sucrose, 0Æ08 for glucose

and 0Æ09 for fructose. Each value was regarded as

expressing the S.E. of all individual measurements with

95% confidence.

GROWTH OF LACTIC ACID BACTERIA IN CEREAL-BASED SUBSTRATES 853

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 851–859

Page 4: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

RESULTS

Growth kinetics of lactic acid bacteria in malt,wheat and barley media

A common model used for the simulation of microbial

growth is the logistic equation (Mercier et al. 1992; Ozen

and Ozilgen 1992; Lejeune et al. 1998):

dX

dt¼ lX ¼ lmaxXð1 � X=XmaxÞ ð1Þ

where l is the specific growth rate, lmax is the maximum

specific growth rate predicted by the model, t is time, X is

the cell concentration and Xmax the maximum attainable cell

concentration. This equation represents both the exponen-

tial (X < < Xmax) and the stationary phase (X ¼ Xmax) of

growth. The term (1 ) X/Xmax) implies that micro-organ-

isms inhibit their own growth either by competing for the

same carbon and nitrogen sources present in the fermenta-

tion medium or by accumulating inhibitory compounds.

Integration of eqn (1) yields the following expression for cell

concentration:

X ¼ X0Xmax expðlmaxtÞXmax � X0 þ X0 expðlmaxtÞ ð2Þ

The kinetic parameters of eqn (2) (see Table 1) were

estimated from the experimental data by selecting the set of

parameters that minimize the sum of the square deviation

between experimental and computed values. To measure the

precision of the model an AnoVa was performed using as

null hypothesis that the residuals from the non-linear

regression are non-zero only because of uniform imprecision

in the measurement of the response variable (cell number)

and not due to the lack of fit. The ratio F was in all cases less

than Ftable (Draper and Smith 1981).

The experimental data and simulations of Lact. fermentum,

Lact. reuteri, Lact. plantarum and Lact. acidophilus in malt,

wheat and barley media are presented in Fig. 1. An

exponential growth phase of 6–8 h was observed for Lact.fermentum and Lact. acidophilus, while Lact. reuteri and Lact.plantarum grew exponentially until 10–12 h of fermentation.

The viable cell densities of Lact. fermentum, Lact. plantarumand Lact. reuteri declined slightly during the stationary

phase (12–48 h); however, the cell population of Lact.acidophilus significantly declined after the end of the

exponential phase. Since all fermentations were performed

under no pH control, the organic acids formed via the

metabolic pathways decreased the pH of the media.

In malt medium Lact. fermentum exhibited a higher

maximum specific growth rate and population density than

Lact. reuteri (see Table 1). Lactobacillus plantarum reached

the highest population densities (10Æ11 ± 0Æ18 log10

cfu ml)1). At the end of the exponential phase pH values

dropped from 5Æ20 ± 0Æ09 to 3Æ77 ± 0Æ09, 3Æ72 ± 0Æ09,

3Æ73 ± 0Æ09 and 3Æ40 ± 0Æ09 for Lact. fermentum, Lact.reuteri, Lact. acidophilus and Lact. plantarum, respectively.

Lactobacillus fermentum and Lact. plantarum attained high

cell populations growing in barley medium (see Table 1).

However, Lact. reuteri and Lact. acidophilus did not grow

well. Based on the model parameters, the increase in their

cell population was 1Æ16 ± 0Æ08 and 0Æ73 ± 0Æ09 log10

cfu ml)1, respectively. At the end of the exponential phase

pH values decreased from 6Æ20 ± 0Æ09 to 4Æ61 ± 0Æ09,

4Æ88 ± 0Æ09, 3Æ93 ± 0Æ09 and 3Æ92 ± 0Æ09 for Lact. fermen-tum, Lact. reuteri, Lact. acidophilus and Lact. plantarum,

respectively.

In the wheat medium the four lactic acid bacteria strains

displayed similar growth patterns to those observed in barley

medium (see Table 1). The growth of Lact. reuteri and Lact.acidophilus was again inhibited, leading to an increase in cell

population of only 1Æ03 ± 0Æ07 and 0Æ70 ± 0Æ08 log10

cfu ml)1. At the end of the exponential phase pH values

dropped from 5Æ85 ± 0Æ09 to 4Æ50 ± 0Æ09, 4Æ40 ± 0Æ09,

3Æ73 ± 0Æ09 and 3Æ83 ± 0Æ09 for Lact. fermentum, Lact.reuteri, Lact. acidophilus and Lact. plantarum, respectively.

Medium Micro-organism lmax (h)1) X0 (log10 cfu ml)1) Xmax (log10 cfu ml)1)

Malt Lact. fermentum 0Æ62 ± 0Æ04 6Æ85 ± 0Æ08 9Æ68 ± 0Æ03

Lact. plantarum 0Æ41 ± 0Æ03 6Æ90 ± 0Æ10 10Æ11 ± 0Æ18

Lact. reuteri 0Æ38 ± 0Æ02 6Æ20 ± 0Æ07 8Æ86 ± 0Æ06

Lact. acidophilus 0Æ19 ± 0Æ02 6Æ89 ± 0Æ06 8Æ10 ± 0Æ06

Barley Lact. fermentum 0Æ43 ± 0Æ05 6Æ90 ± 0Æ11 9Æ12 ± 0Æ05

Lact. plantarum 0Æ20 ± 0Æ02 6Æ71 ± 0Æ13 9Æ43 ± 0Æ10

Lact. reuteri 0Æ13 ± 0Æ01 6Æ14 ± 0Æ04 7Æ28 ± 0Æ05

Lact. acidophilus 0Æ18 ± 0Æ03 7Æ02 ± 0Æ04 7Æ73 ± 0Æ03

Wheat Lact. fermentum 0Æ53 ± 0Æ05 6Æ93 ± 0Æ09 9Æ28 ± 0Æ04

Lact. plantarum 0Æ23 ± 0Æ02 7Æ21 ± 0Æ05 9Æ29 ± 0Æ06

Lact. reuteri 0Æ13 ± 0Æ01 6Æ22 ± 0Æ03 7Æ20 ± 0Æ04

Lact. acidophilus 0Æ15 ± 0Æ01 7Æ02 ± 0Æ02 7Æ71 ± 0Æ03

Table 1 Numerical values of estimated

microbial growth parameters in malt, barley

and wheat media based on eqn (2)

854 D. CHARALAMPOPOULOS ET AL.

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 851–859

Page 5: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

Substrates and products

In order to investigate the ability of each strain to utilize the

available carbohydrates and to study the distribution of the

primary products of lactic acid metabolism, samples from all

fermentations were taken after the end of the exponential

phase (12 h) and at the end of the fermentation (48 h). They

were analysed for their content of sugars, organic acids and

ethanol. The results from malt fermentations are shown in

Tables 2 and 3.

Lactobacillus fermentum and Lact. reuteri produced mainly

lactic and acetic acids and ethanol (see Table 2). The molar

ratios of lactic acid produced to ethanol plus acetic acid at

the end of the exponential phase were 0Æ72 ± 0Æ10 and

0Æ62 ± 0Æ11, respectively. During the stationary phase

(12–48 h) the lactic acid and ethanol concentrations

increased, while the acetic acid concentration remained

constant for both microbes, giving an overall molar ratio

throughout the fermentation of 0Æ90 ± 0Æ11 and 0Æ95 ± 0Æ10,

respectively. Analysis of the samples taken at the end of the

exponential phase of Lact. fermentum indicated a steady

decrease in all sugars, but mainly maltose, while Lact. reutericonsumed primarily glucose and fructose (see Table 3).

Lactobacillus plantarum demonstrated a homofermentative

metabolic pattern producing mainly lactic acid and small

amounts of acetic acid. Sugar analysis indicated a significant

depletion of glucose and a similar consumption of fructose,

maltose and sucrose (see Table 3). The growth of Lact.acidophilus was associated with the production of lactic acid

and comparably significant amounts of acetic acid. The low

concentrations of metabolites coincided with a relatively low

consumption of the available carbohydrates, mainly glucose

(see Table 3). In contrast to the other microbes, sugar

consumption was not observed between 12 and 48 h.

DISCUSSION

The aim of this study was to monitor and compare the

growth characteristics of potentially probiotic strains of

Lact. fermentum, Lact. reuteri, Lact. plantarum and Lact.acidophilus species in natural cereal substrates formulated

without the addition of supplements. Important technolo-

gical points in the design and evaluation of this type of food

fermentation process are the composition of the raw

material, specific growth rate of the starter culture applied,

final cell population, acidification rate and distribution of the

primary metabolic products.

The empirical logistic-type model (eqn (2)), that was used

to simulate the growth of each of the four microbes in malt,

barley and wheat media, described well the clear exponential

phase (l ¼ lmax, X < < Xmax), the deceleration stage of the

exponential phase (l < lmax, X < Xmax) and the constant

stationary phase (l ¼ 0 h)1, X ¼ Xmax). This could be due

to the fact that the model can describe both the effect of a

product inhibition and the depletion of an essential nutrient

on microbial growth (Lejeune et al. 1998). This model

cannot, however, describe the declining stationary phase.

log 10

cfu

ml–1

log 10

cfu

ml–1

log 10

cfu

ml–1

Fig. 1 Growth of lactic acid bacteria in (a) malt, (b) barley and (c)

wheat media. The solid lines represent the growth curves predicted

with eqn (2). Experimental data symbols: j, Lactobacillus plantarum;

d, Lact. fermentum; m, Lact. reuteri and e, Lact. acidophilus

GROWTH OF LACTIC ACID BACTERIA IN CEREAL-BASED SUBSTRATES 855

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 851–859

Page 6: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

The malt medium supported well the growth of Lact.plantarum, Lact. fermentum and Lact. reuteri, which showed

increases in their cell populations of 3Æ21 ± 0Æ28, 2Æ83 ± 0Æ16

and 2Æ66 ± 0Æ13 log10 cfu ml)1 at the end of the exponential

phase (12 h), respectively. This could be attributed to the

simultaneous presence of considerable amounts of monosac-

charides (glucose and fructose) and disaccharides (maltose and

sucrose) in the malt medium (approximately 3 and 12 g l)1,

respectively). Our results showed that each microbe demon-

strated a specific preference for one or more sugars during the

exponential phase (see Table 3). The preference of Lact.plantarum towards glucose has also been suggested by Samuel

et al. (1980) and Gobbetti et al. (1994). The preferential

utilization of maltose by the sourdough-originated Lact.fermentum strain and glucose and fructose by the intestinal

Lact. reuteri strain could be due to the origins of these strains,

since sourdough strains of both species have been shown to

effectively metabolize maltose (Stolz et al. 1995). Regarding

Lact. acidophilus, the small increase in cell populations

(1Æ21 ± 0Æ12 log10 cfu ml)1) could be explained by the

possible absence of specific nutrients in the malt medium,

such as free amino acids, B-vitamins or minerals. This species

has complex growth requirements (Gomes and Malcata 1999),

usually exhibiting poor growth in synthetic media without the

addition of large amounts of supplements, such as yeast

extract and peptone (Taillandier et al. 1996; Elli et al. 1999).

The small amounts of the available sugars consumed

during the exponential phase by all strains (19% for Lact.fermentum, 17% for Lact. reuteri, 43% for Lact. plantarumand 16% for Lact. acidophilus) indicated that the sugar

content of the malt medium is not the decisive growth-

limiting factor. In agreement with the above results, Passos

et al. (1994) demonstrated a 45% reduction in sugar during

the Lact. plantarum exponential phase (10 h) in cucumber

juice; the pH was 3Æ53 when growth ceased. In addition,

Venkatesh et al. 1993) reported an incomplete consumption

of the available sugars (17%) and a fast cessation of Lact.bulgaricus growth (approx. 10 h) in fermentations performed

in synthetic media without pH control, while at constant pH

(5Æ6) a 90% reduction in sugar and a longer exponential phase

(approx. 18 h) were observed. Since all experiments were

performed under uncontrolled conditions, the accumulation

of lactic and acetic acids produced via the metabolic pathways

progressively decreased the pH of the medium. These

organic acids can inhibit microbial growth in their undisso-

ciated form, dissociated form or indirectly by the protons

(H+) that are released in the medium (Passos et al. 1993).

The relatively low concentrations of total lactic acid at the

end of the exponential phase (from 0Æ81 to 1Æ82 g l)1) are not

considered inhibitory to cell growth (Giraud et al. 1991;

Passos et al. 1993; Ganzle et al. 1998). Therefore, the most

significant factor in growth limitation was probably pH

Table 3 Sugar content of the malt medium during fermentation with Lactobacillus fermentum, Lact. reuteri, Lact. plantarum and Lact. acidophilus

Concentration (g l)1) at different times (h)

Lact. fermentum Lact. reuteri Lact. plantarum Lact. acidophilus

Substrates 0 12 48 0 12 48 0 12 48 0 12 48

Maltose 5Æ27 3Æ49 2Æ10 5Æ53 5Æ17 1Æ86 4Æ78 4Æ06 2Æ25 4Æ82 4Æ67 4Æ80

Sucrose 7Æ33 6Æ88 3Æ85 6Æ98 6Æ40 2Æ08 6Æ36 3Æ67 2Æ30 6Æ88 6Æ38 6Æ42

Glucose 2Æ10 1Æ64 0Æ03 2Æ14 1Æ14 0Æ08 2Æ11 0Æ28 0Æ17 2Æ16 1Æ03 1Æ00

Fructose 1Æ14 0Æ73 0Æ08 1Æ08 0Æ32 0Æ09 0Æ98 0Æ16 0Æ05 0Æ95 0Æ28 0Æ09

Table 2 Primary metabolites produced during growth of Lactobacillus fermentum, Lact. reuteri, Lact. plantarum and Lact. acidophilus in the malt

medium. The prefermented malt medium contained 0Æ05 g l)1 lactic acid (La); acetic acid (Ac) and ethanol (Et) were not detected

Lact. fermentum Lact. reuteri Lact. plantarum Lact. acidophilus

12 h 48 h 12 h 48 h 12 h 48 h 12 h 48 h

Lactic acid (g l)1) 1Æ39 ± 0Æ1 2Æ41 ± 0Æ1 1Æ06 ± 0Æ1 2Æ96 ± 0Æ1 1Æ99 ± 0Æ1 5Æ74 ± 0Æ1 0Æ81 ± 0Æ1 1Æ07 ± 0Æ1Acetic acid (g l)1) 0Æ59 ± 0Æ02 0Æ57 ± 0Æ01 0Æ50 ± 0Æ02 0Æ62 ± 0Æ02 0Æ29 ± 0Æ06 0Æ32 ± 0Æ05 0Æ38 ± 0Æ04 0Æ37 ± 0Æ03

Ethanol (g l)1) 0Æ53 ± 0Æ02 0Æ93 ± 0Æ06 0Æ48 ± 0Æ02 1Æ11 ± 0Æ04 ND ND ND ND

Molar ratio

La : Ac + Et

0Æ72 ± 0Æ10 0Æ90 ± 0Æ11 0Æ62 ± 0Æ11 0Æ95 ± 0Æ10 4Æ56 ± 1Æ17 11Æ95 ± 2Æ07 1Æ42 ± 0Æ32 1Æ92 ± 0Æ33

YP/Sb 0Æ84 ± 0Æ29 0Æ42 ± 0Æ06 0Æ88 ± 0Æ39 0Æ42 ± 0Æ04 0Æ66 ± 0Æ21 0Æ76 ± 0Æ14 0Æ68 ± 0Æ42 0Æ62 ± 0Æ28

ND, Not detected.

YP/Sb, Product yield.

856 D. CHARALAMPOPOULOS ET AL.

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which, at the end of the exponential phase, ranged between

3Æ40 and 3Æ77 for all strains. Thus, the higher cell populations

of Lact. plantarum compared with the other microbes could

be attributed to its ability to continue to grow in malt

medium until the pH value dropped to 3Æ40, similar to the

values reported by Giraud et al. (1991) and Passos et al.(1993). Lactobacillus plantarum maintains a proton (pH) and

charge gradient between the inside and outside of the cells

even in the presence of large amounts of lactate and protons

(Giraud et al. 1998). No information was found in the

literature regarding the growth-limiting pH values of Lact.fermentum and Lact. reuteri, which in this study were

3Æ77 ± 0Æ09 and 3Æ72 ± 0Æ09, respectively. Ganzle et al.(1998) reported a value of 3Æ8 for Lact. sanfranciscensis, a

heterofermentative Lactobacillus, in sourdough fermentation

performed under similar non-nutrient-limiting conditions.

In this study, the limiting pH value for the growth of Lact.acidophilus was 3Æ83 ± 0Æ09, while Lonner and Akesson

(1988) reported pH values between 3Æ4 and 3Æ6, depending on

the type of cereal substrate used. However, in that study

these values did not correspond to the end of the exponential

phase. Our results showed that, during the stationary phase,

all strains except Lact. acidophilus continued to consume

sugars and accumulate organic acids (see Tables 2 and 3),

decreasing the pH of the medium to approx. 3Æ10, probably

due to energy requirements for the preservation of cell

viability (Passos et al. 1994). Lactobacillus acidophilus entered

directly into the decline phase, presumably due to its

inability to withstand the low pH of the medium

(3Æ83 ± 0Æ09) at the end of the exponential phase.

The low pH values, compared with the moderate concen-

trations of lactic and acetic acids detected at the same time

(12 h), could be attributed to the low buffering capacity of

the malt medium used in this study (8Æ45 mmol pH)1 l)1),

confirming the significance of substrate composition on the

overall fermentation process. Therefore, the supplementa-

tion of malt medium with additives that enhance its buffering

capacity would result in reduced acidification rates and

increased fermentation times. In milk and vegetable lactic

acid fermentations, citric and acetic acids are added in order

to enhance the buffering capacity of the substrates (Boquien

et al. 1988; Passos et al. 1994). In the prefermented malt

medium acetic acid was not detected, while the amount of

citric acid present in cereals is usually small (Tamine et al.1997). Other factors contributing to the buffering capacity

could be the protein and ash content of the malt medium

(Marklinder and Johansson 1995).

The similar fermentation patterns of the four strains in

barley and wheat media could be associated with the FAN

content (15Æ3–26Æ6 mg l)1) and sugar composition of the

media, consisting mainly of maltose (0Æ9–1Æ3 g l)1) and

sucrose (1Æ4–2Æ0 g l)1). In accordance with existing reports

(Salovaara and Valjakka 1987; Ganzle et al. 1998; Bvochora

et al. 1999), the concentrations of these constituents were

significantly lower than those in malt medium (75Æ2–

82Æ1 mg l)1 FAN and 14Æ5–16Æ8 g l)1 total available carbo-

hydrates). Interestingly, the microbial growth ceased at

higher pH values than those observed for malt fermenta-

tions, which suggests that the growth-limiting factor was not

only pH but that deficiency in nutrients also contributed to

growth limitation. A deficiency in specific vitamins or

minerals could contribute to growth limitation but barley

and wheat contain significant amounts of these nutrients

(Palmer 1989). Therefore, the poor growth of Lact. reuteri(1Æ16 ± 0Æ08 and 1Æ03 ± 0Æ07 log10 cfu ml)1 increase in

barley and wheat, respectively) and Lact. acidophilus(0Æ70 ± 0Æ08 and 0Æ73 ± 0Æ09 log10 cfu ml)1 increase in

barley and wheat, respectively) could be attributed to the

low concentrations of sugars (mainly glucose and fructose)

and FAN. These observations are in good agreement with

the literature (Marklinder and Lonner 1992). In the case of

Lact. plantarum and Lact. fermentum, although maltose and

sucrose were not completely depleted during the exponential

phase, their initial low concentrations were probably limit-

ing, resulting in lower final cell counts when compared with

malt fermentations. However, these cell populations, ran-

ging between 9Æ1 and 9Æ5 log10 cfu ml)1, still enhance the

potentially probiotic activities of these strains. The increase

in the cell population of Lact. plantarum (2Æ72 ±

0Æ23 cfu ml)1 in barley and 2Æ08 ± 0Æ11 cfu ml)1 in wheat)

and Lact. fermentum (2Æ22 ± 0Æ16 cfu ml)1 in barley and

2Æ35 ± 0Æ09 cfu ml)1 in wheat) suggests that these strains

are less fastidious than Lact. reuteri and Lact. acidophilusstrains, being able to grow better under nutrient-limiting

conditions.

When designing and evaluating a novel fermentation

process, besides considering the factors influencing micro-

bial growth, the distribution of the end metabolic products is

also very important. The organoleptic properties of cereal-

based and dairy fermented foods produced by lactic acid

fermentation are very much dependent on the amounts of

organic acids produced. For example, in wheat sourdough a

molar ratio of lactic to acetic acid from 2 to 2Æ7 is considered

an optimal value for its sensory quality (Gobbetti et al. 1995)

while, in yoghurt, a 0Æ85–0Æ90% lactic acid content is

considered an optimum value (Oberman and Libudzisz

1998). In general, lactic acid is described as ‘acid-sour’ and

acetic acid as ‘acid-sharp’ (De Vuyst 2000).

Theoretically, under strict anaerobic conditions, hetero-

fermentative lactic acid bacteria produce 1 mole of lactic acid

and 1 mole of ethanol per mole of glucose utilized. However,

in the presence of oxygen or compounds that can serve as

electron acceptors, such as fructose, citric and malic acids,

there is a shift of the metabolic pathway towards acetic acid

production instead of ethanol (Stolz et al. 1995). Lactobacillusfermentum and Lact. reuteri showed a heterofermentative

GROWTH OF LACTIC ACID BACTERIA IN CEREAL-BASED SUBSTRATES 857

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Page 8: Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

pattern by producing approx. equimolar amounts of lactic

acid and ethanol plus acetic acid (see Table 2). Both strains

indicated an overproduction of acetic acid during the

exponential phase, which could be attributed to the presence

of small amounts of oxygen and fructose. Since acetic acid

production persisted in wheat and barley fermentations (data

not shown), where very small amounts of fructose were

present, it is more likely that acetic acid is formed via oxygen

utilization. In addition, Stolz et al. (1995) demonstrated the

inability of a Lact. reuteri strain isolated from sourdough to

utilize fructose as an electron acceptor. After cell growth

ceased, the formation of lactic acid and ethanol continued

following a heterofermentative pattern, and no acetic acid was

produced, which suggests that there is an association between

acetic acid and growth (Gobbetti et al. 1995). However, the

total product yields based on sugar consumption were very

low, less than 50% (see Table 2), probably due to the fact that

substrate consumption was mainly associated with cell

maintenance and survival (Goncalves et al. 1997; Fu and

Mathews 1999). The small amount of acetic acid observed

with the homofermentative Lact. plantarum could be attrib-

uted to the presence of oxygen (Murphy and Condon 1984;

Bobillo and Marshall 1991), while the resulting product yield

of 0Æ76 ± 0Æ14 was similar to those found in other studies

(Saucedo et al. 1990; Fu and Mathews 1999). The high

concentration of acetic acid detected in the case of Lact.acidophilus, shifting the metabolism to an almost heterofer-

mentative pathway, cannot be sufficiently explained.

In this study, the growth characteristics of four potentially

probiotic lactic acid bacteria in cereal substrates were

investigated. A general conclusion is that in a non-pH-

controlled lactic acid fermentation of cereals the main

inhibitors of microbial growth are pH and nutrient limita-

tions, probably sugars or FAN. Further research will aim to

develop a mathematical model that will enable prediction of

the growth of potentially probiotic lactic acid bacteria in

cereal substrates, taking into account the effect of pH and

the substrate composition.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support provided to

D.C. by the Hellenic State Scholarships Foundation. They

are also grateful to Elizabeth Davenport and Janet Wilson

for their help in performing the GC analysis and Konstan-

tinos Stathopoulos for his comments during the preparation

of the manuscript.

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