evaluation of the effect of malt, wheat and barley extracts on the viability of potentially...

Evaluation of the effect of malt, wheat and barley extracts

on the viability of potentially probiotic lactic acid

bacteria under acidic conditions

D. Charalampopoulos, S.S. Pandiella *, C. Webb

Satake Centre for Grain Process Engineering, Department of Chemical Engineering, UMIST, PO Box 88, Manchester M60 1 QD, UK

Received 21 September 2001; received in revised form 19 April 2002; accepted 18 June 2002

Abstract

In this work, the effect of cereal extracts, used as delivery vehicles for potentially probiotic lactic acid bacteria (LAB), on the

acid tolerance of the cells was evaluated under conditions that simulate the gastric tract. More specifically, the effect of malt,

barley and wheat extracts on the viability of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri

during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. In the absence of cereal extracts all strains

demonstrated a significant reduction in their cell population, particularly L. plantarum. The viability of L. plantarum was

improved by approximately 4 log10 cycles in the presence of malt and 3 log10 cycles in the presence of wheat and barley. The

survival of L. acidophilus and L. reuteri was increased by more than 1.5 and 0.7 log10 cycle, respectively, upon addition of

cereal extracts. In order to evaluate the contribution of the cereal constituents on cell survival, the individual effect of glucose,

maltose and free amino nitrogen (FAN), which were added at concentrations that correlated to the reducing sugar and FAN

content of the cereal extracts, was examined. The viability of L. plantarum was progressively improved as the maltose or

glucose concentration increased; an increase by approximately 2 log10 cycles was observed in the presence of 8.33 g/l sugar.

The survival of L. acidophilus increased by more than 1 log10 cycle, even at very low concentrations of maltose and glucose

(e.g., 0.67 g/l), while L. reuteri stability was enhanced in the presence of maltose but no appreciable effect was demonstrated in

the presence of glucose. Sugar analysis indicated that glycolysis was inhibited in all cases. Addition of tryptone and yeast

extract, used as sources of FAN, enhanced L. acidophilus acid tolerance, but did not affect L. reuteri and L. plantarum. The

results presented in this study indicate that malt, wheat and barley extracts exhibit a significant protective effect on the viability

of L. plantarum, L. acidophilus and L. reuteri under acidic conditions, which could be mainly attributed to the amount of sugar

present in the cereal extracts.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Lactobacillus; Cell viability; Cereals; Acidic conditions

1. Introduction

The inclusion of potentially probiotic microorgan-

isms in the diet has been established in the global food

market leading to the development and commercializa-

tion of numerous probiotic products. In the dairy

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0168 -1605 (02 )00248 -9

* Corresponding author. Tel.: +44-161-200-4429; fax: +44-161-

200-4399.

E-mail address: [email protected] (S.S. Pandiella).

www.elsevier.com/locate/ijfoodmicro

International Journal of Food Microbiology 82 (2003) 133–141

industry, a large variety of milk formulations have been

used as delivery vehicles of probiotic lactic acid bac-

teria (Scheinbach, 1998). Cereals are also suitable

substrates for lactic acid bacteria (LAB) growth, which

has led to the commercialization of cereal-based pro-

biotic products (Salovaara, 1996). Strains of the Lac-

tobacillus genus, such as Lactobacillus acidophilus,

Lactobacillus reuteri, Lactobacillus plantarum, Lacto-

bacillus casei, Lactobacillus rhamnosus and Lactoba-

cillus gasseri constitute a significant proportion of

cultures used in probiotic products (Shortt, 1999).

An important criterion when selecting a probiotic

strain is its ability to survive the acidic conditions of

the gastric tract. The stomach has a pH that may fall to

as low as 1.5 and a salt content not less than 0.5% w/v

(Hill, 1990; Kailasapathy and Chin, 2000). The intrin-

sic tolerance of lactobacilli to low pH values, ranging

between 1 and 4, has been examined in vitro using

solutions such as acidified broths, buffers, complex

media consisting of pancreatic enzymes, or fresh

human gastric fluids (Conway et al., 1987; Charteris

et al., 1998; Gardiner et al., 1999; Vinderola et al.,

2000). Lactobacillus species are considered intrinsi-

cally resistant to acid although differences exist

between species and specific strains, however they

generally demonstrate increased sensitivity at pH

values below 3 (Hood and Zottola, 1988; Jin et al.,

1998; Kailasapathy and Chin, 2000). The acid toler-

ance of lactobacilli is attributed to their ability to

maintain a constant pH gradient between the pH of the

medium and their cytoplasmic pH. When the internal

pH reaches a threshold value cellular functions are

inhibited and the cell dies (Kashket, 1987).

Survival of the probiotic strains during gastric

transit is also influenced by the physicochemical

properties of the food carrier used for delivery. The

buffering capacity and the pH of the carrier medium

are significant factors, since food formulations with

pH ranging from 3.5 to 4.5 and high buffering

capacity, such as yoghurt, cheese, and skim milk,

would increase the pH of the gastric tract and thus

enhance the stability of the probiotic strain (Gardiner

et al., 1998; Kailasapathy and Chin, 2000; Zarate et

al., 2000). In this regard, Charteris et al. (1998)

reported improved gastric tolerance of several pro-

biotic lactobacillus species upon addition of milk

proteins. However, Gardiner et al. (1999) suggested

that besides the buffering capacity, there are other

factors, possibly including properties such as the

presence of protective extracellular polysaccharides,

the fat content and the dense matrix of cheese, which

may enhance the survival of the probiotic strain.

In the present study, the effect of malt, wheat and

barley extracts and several dietary constituents indi-

vidually (reducing sugars, free amino nitrogen) on the

viability of potentially probiotic L. plantarum, L.

reuteri and L. acidophilus strains during exposure

for 4 h in a phosphate buffer acidified at pH 2.5

was investigated. The overall aim was to identify the

physicochemical factors that can generally improve

the viability of a probiotic strain and select the

appropriate cereal substrate as probiotic carrier, in

relation to its composition. The strains were selected

as representatives of the major LAB species present in

the human gut (Collins et al., 1998). In addition, they

are intrinsically stable to pH 1 (data not shown), are

able to tolerate bile concentrations ranging from 2% to

4% (data not shown) and grow well in cereal sub-

strates (Charalampopoulos et al., 2002). The media

were prepared by adding the cereal suspensions to the

phosphate buffer and subsequently adjusting the pH to

2.5, before adding the cell culture. Using the above

preparation procedure, the effect of buffering capacity

on cell viability was minimized.

2. Materials and methods

2.1. Microorganisms and culture conditions

The microorganisms used in this study were the

following: L. reuteri NCIMB 11951 (National Col-

lection of Industrial and Marine Bacteria, Aberdeen,

Scotland, UK), isolated from human intestine; L.

acidophilus NCIMB 8821, isolated from human saliva

and Lactobacillus plantarum NCIMB 8826, isolated

from human saliva. The strains were maintained at 4

jC and subcultured monthly on slants prepared from

MRS agar (Oxoid, Basingstoke, Hampshire, UK).

2.2. Preparation of acidified phosphate-buffered

media

A stock solution of phosphate-buffered saline was

prepared by dissolving NaCl (9 g/l), Na2HPO4� 2H2O

(9 g/l) and KH2PO4 (1.5 g/l). Malt, wheat and barley

D. Charalampopoulos et al. / International Journal of Food Microbiology 82 (2003) 133–141134

extracts were prepared by grounding the grains in a

laboratory Falling Number hammer mill (Perten Instru-

ments, Sweden), comprising a sieve of size 0.5 mm. A

100-g portion of the flour obtained was mixed with 400

ml of tap water and the resulting slurry was centrifuged

(6000� g) for 30 min at room temperature. Twenty

milliliters of malt, wheat and barley extracts super-

natant was then collected and added singly into 20 ml

of the stock solution. The pH was then adjusted around

2.40 with 5 N HCl and the acidified media were

sterilized for 15 min at 121 jC. The pH of the media

ranged between 2.30 and 2.40 after sterilization. The

individual effects of glucose, maltose (both from

Sigma, Basingstoke, Hampshire, UK), yeast extract

(Oxoid) and tryptone (Oxoid) were examined by fol-

lowing the same procedure as described above. Equal

volumes of a series of glucose (2–25 g/l), maltose (2–

25 g/l), yeast extract (0.6–3 g/l), tryptone (0.2–1.2 g/l)

solutions were mixed with the stock buffered solutions

and subsequently acidified approximately to 2.35 and

sterilized. The pH of the media dropped to approxi-

mately to 2.20 after sterilization.

2.3. Determination of acid tolerance

Isolated colonies from MRS agar plates were pre-

cultured twice inMRS broth (Oxoid) for approximately

16 h at 37 jC. The 16-h pre-cultured cells were then

centrifuged (5000� g, 10 min, 4 jC), washed twice

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

suspended in Ringer’s solution. A 20-ml portion of the

washed cell suspensions was immediately transferred

aseptically to 40 ml of the sterile acidified phosphate-

bufferedmedium, raising its pH to approximately 2.5 in

all cases, and incubated at 37 jC. Samples of 5 ml were

collected at 0, 60, 120, 180 and 240 min and were

analysed, regarding their viable cell population.

2.4. Bacterial enumeration

The drop method was used for counting the viable

bacterial populations (Collins et al., 1989). The sam-

ples obtained were decimally diluted in sterile quarter-

strength Ringer’s solution, and six 0.12-ml aliquot

dilutions were plated on MRS agar using a pre-

calibrated pipette and incubated at 37 jC for 48 h.

Plates that gave 10–30 colonies per drop were

selected. Colony forming units (cfu/ml) were counted

and the results were expressed as their log10 values.

2.5. Chemical analyses

The soluble free amino nitrogen (FAN) content was

determined by the ninhydrin colorimetric method

accepted by the European Brewery Convention

(1987), using a glycine solution (2 mg/l) as control.

Total soluble sugar concentrations were estimated by

the phenol–sulphuric acid method (Dubois et al.,

1956). The analysis of the samples was conducted

based on a calibration curve (R2 = 0.996, S.E. = 0.001,

with 95% confidence) that was obtained by replicating

three times the assay using an array of maltose stand-

ard solutions (550 mg/l). Reducing sugar concentra-

tions were determined by the 3,5-dinitrosalicylic acid

method (Miller, 1959). Two calibration curves were

obtained, one for glucose (R2 = 0.999, S.E. = 0.01),

and the other for maltose (R2 = 0.999, S.E. = 0.007)

using an array of standard sugar solutions (0.2–1 g/l).

The buffering capacity of each cereal extract was

determined by titrating 100 ml of the medium with

Table 1

Chemical composition of the autoclaved buffered media immediately after addition of the washed cell suspension

Malt medium Wheat medium Barley medium

pH 2.45F 0.06 2.52F 0.04 2.43F 0.05

Total sugars (g/l) 10.83F 0.96 4.60F 0.41 4.21F 0.32

Reducing sugars (g/l)a 8.40F 0.28 4.11F 0.14 3.81F 0.13

Free amino nitrogen (mg/l) 54.80F 8.09 18.92F 0.96 17.55F 1.09

Starch (g/l) NDb ND ND

Buffering capacityc (mmol/pH�l) 18.67 7.33 13.33

Values are means of five determinations (n= 5) with the standard error (F S.E.) calculated with 95% confidence.a Reducing sugar concentration was calculated using the calibration curve of maltose.b ND, Not detected.c The buffering capacity was estimated by titrating 100 ml of the cereal extracts, after sterilization for 15 min at 121 jC, with 1 N HCI.

D. Charalampopoulos et al. / International Journal of Food Microbiology 82 (2003) 133–141 135

HCl (1 N). The values were expressed as the amount

of HCl (mmol) required to drop 1 pH unit per unit

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

2.6. Statistical analysis

The imprecision and inaccuracy of the viable cell

counting method, which consists of a series of

dilutions followed by incubation on solid MRS agar,

was evaluated by repeating the method 20 times for

a randomly selected sample (6 drops from each

diluted sample on the MRS plate). The data were

then analysed by one-way analysis of variance

(ANOVA) test.

The significance of the initial microbial population

on cell viability after 4 h (log10 N0 h� log10 N4 h) was

assessed by conducting a block design experiment for

each strain. The data obtained from 15 experiments

with the control (five sets of three replicates), and 15

experiments with 5 g/l maltose addition (five sets of

three replicates) for each strain were analysed by

ANOVA test.

3. Results

3.1. Chemical composition of media

Table 1 shows the results of the compositional

analyses of the cereal media, prepared after mixing

equal volumes of the washed cell suspension, the

stock buffer solution and the cereal extract. Higher

concentrations of total sugar than reducing sugar were

detected in all samples. Increased amounts of reducing

sugar and free amino nitrogen were observed in the

malt medium. Residual starch was not detected in the

samples, since starch was completely removed during

the extraction procedure.

3.2. Statistical evaluation of cell enumeration method

and cell viability

The ANOVA test that was applied to the data

obtained from the randomly selected sample showed

that variations between the 20 sample means were not

significant (F20,99 = 0.86 < Ftable, P < 0.05), which

suggests that they were probably due to random

imprecision and not to inaccuracy. Therefore, cell

values displayed in this study are mean values of six

measurements, while the standard error (S.E.) of the

mean was calculated with 95% confidence.

Fig. 1. Evolution of L. plantarum (a), L. acidophilus (b) and L. reuteri

(c) cell population in the presence of cereal extracts during exposure

at pH 2.5 for 4 h. Experimental data symbols: (.) control, (y) malt,

(n) wheat, (E) barley. Cell values are means of six determinations

(n= 6), with standard error (F S.E.) calculated with 95% confidence.


The ANOVA test that was applied to the data from

the block design experiments did not indicate signifi-

cant differences between the mean values of cell

viability (L. plantarum: F4,10 = 2.35 < Ftable in the

control, F4,10 = 2.88 < Ftable after addition of 5 g/l

maltose; L. acidophilus: F4,10 = 2.14 <Ftable in the

control, F4,10 = 2.01 < Ftable after addition of 5 g/l

maltose; L. reuteri: F4,10 = 2.98 <Ftable in the control,

F4,10 = 2.69 <Ftable after addition of 5 g/l maltose).

Therefore, it was concluded that the initial cell pop-

ulation did not affect the cell viability, which con-

sequently could be estimated by the expression log10N0 h� log10 N4 h for all the experimental data. The

standard deviation (S.D.) was calculated by the

expression

S:D: ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðS:D:Þ2N0

þ ðS:D:Þ2N4 h

q;

and the S.E. (S:E: ¼ S:D:=ffiffiffin

p) was estimated with

95% confidence.

3.3. Effect of malt, wheat and barley on cell viability

Fig. 1a, b, c demonstrates the evolution of L.

plantarum, L. acidophilus and L. reuteri cell popula-

tion during exposure for 4 h in the acidified buffered

medium, in the presence and absence of malt, wheat

and barley extracts. In the control experiments, with-

out addition of cereal extracts, L. plantarum cell

population decreased 4.82F 0.12 log10 cycles, while

L. reuteri and L. acidophilus showed a 1.25F 0.17

and 1.95F 0.10 log10 cycle reduction in their cell

population, respectively.

The addition of cereal extracts affected the acid

tolerance of all strains, improving generally their via-

bility. The cell population of L. plantarum decreased

0.61F 0.10, 1.68F 0.12 and 1.59F 0.14 log10 cycle

in the presence of malt, wheat and barley, indicating a

considerable improvement in cell viability by approx-

imately 4 log10 cycles in the presence of malt, and 3

log10 cycles in the presence of wheat and barley.

Similarly, the cell population of L. acidophilus de-

creased 0.12F 0.16, 0.32F 0.12 and 0.37F 0.11

log10 cycle in the presence of malt, wheat and barley

demonstrating an improvement in cell viability by

more than 1.5 log10 cycle in all cases. The cell counts

of L. reuteri declined 0.40F 0.10, 0.65F 0.08 and

0.47F 0.11 log10 cycle in the presence of malt, wheat

and barley, respectively, which indicates an improve-

ment in cell viability by approximately 0.8 log10 cycle.

3.4. Effect of yeast extract and tryptone on cell

viability

Table 2 demonstrates the effect of yeast extract and

tryptone, added singly at different concentrations into

the buffer solution, on cell viability during exposure at

Table 2

Effect of tryptone and yeast extract addition on the viability of L. plantarum and L. acidophilus during 4 h exposure at pH 2.5

Additive FANa (mg/l) L. plantarum (N log10 cfu/ml) L. acidophilus (log10 cfu/ml)

N (0 h) N (2 h) N (4 h) D(N0 h�N4 h) N (0 h) N (2 h) N (4 h) D(N0 h�N4 h)

Tryptone (g/l)

0.0 0.0 9.78F 0.08 8.22F 0.12 4.90F 0.15 4.89F 0.14 8.65F 0.02 8.22F 0.12 6.59F 0.07 2.06F 0.06

0.24 13.06F 0.28 9.94F 0.08 8.06F 0.08 4.29F 0.21 5.65F 0.20 8.51F 0.06 8.07F 0.10 6.92F 0.15 1.59F 0.13

0.60 28.40F 0.76 9.75F 0.06 7.87F 0.12 4.06F 0.21 5.69F 0.19 8.54F 0.19 7.99F 0.10 6.86F 0.15 1.69F 0.21

0.80 35.68F 0.96 9.80F 0.09 8.16F 0.11 4.30F 0.22 5.51F 0.21 8.54F 0.19 8.16F 0.11 7.13F 0.07 1.41F 0.17

1.20 46.97F 3.60 9.51F 0.04 8.23F 0.10 4.29F 0.23 5.14F 0.20 8.51F 0.06 8.23F 0.10 7.54F 0.10 0.97F 0.08

Yeast extract (g/l)

0.0 0.0 9.91F 0.07 8.12F 0.17 4.54F 0.27 5.38F 0.24 8.48F 0.14 8.13F 0.10 6.50F 0.15 1.98F 0.18

0.60 13.58F 0.21 9.82F 0.08 7.70F 0.20 4.15F 0.16 5.67F 0.16 8.51F 0.05 8.06F 0.09 6.33F 0.18 2.18F 0.16

1.50 28.01F 0.57 9.91F 0.08 7.57F 0.16 4.16F 0.20 5.65F 0.18 8.57F 0.08 7.92F 0.14 6.57F 0.24 2.00F 0.22

2.00 36.11F 0.85 9.77F 0.06 7.66F 0.19 4.10F 0.20 5.67F 0.18 8.52F 0.11 8.25F 0.07 7.15F 0.04 1.36F 0.10

3.00 47.00F 2.21 9.82F 0.07 7.31F 0.10 3.87F 0.18 5.95F 0.17 8.54F 0.10 8.26F 0.12 7.33F 0.15 1.21F 0.16

Cell values are means of six determinations (n= 6), with the standard error (F S.E.) calculated with 95% confidence.a The FAN content was calculated using a glycine solution as control (2 mg/l). The values are means of five determinations (n= 5).


pH 2.5. The evaluation of yeast extract and tryptone

additions were based on the soluble free amino nitrogen

(FAN) content of these nitrogen sources, ranging from

13 to 47 mg/l. In general, no differences were observed

between yeast extract and tryptone on the viability of

each strain. L. reuteri was unaffected by the FAN

addition at all concentrations tested (data not shown).

In the case of L. acidophilus, an improvement in cell

viability by more than 0.5 log10 cycle compared to the

control was observed when the FAN concentration was

approximately 36 and 47 mg/l. Regarding L. planta-

rum, a slight negative effect of free amino nitrogen on

cell population was observed during exposure for 4 h.

3.5. Effect of maltose and glucose on cell viability

Table 3 demonstrates the effect of glucose and

maltose additions (ranging from 0.67 to 8.33 g/l) on

the cell viability of each strain. L. reuteri was not

appreciably affected by glucose; however, in the

presence of maltose cell viability was gradually

improved and showed a 0.8 log10 cycle increase in

the presence of 8.33 g/l maltose. L. acidophilus

exhibited a rapid improvement in cell viability even

after addition of 0.67 g/l glucose and maltose (approx-

imately by 1 log10 cycle), which was further enhanced

by increasing the glucose and maltose concentrations.

The tolerance of L. plantarum was considerably

enhanced by progressively increasing glucose and

maltose concentrations. An improvement by more

than 3 log10 cycles was observed upon addition of

8.3 g/l of each sugar. Sugar analysis did not indicate

any consumption of sugars after exposure for 4 h at

pH 2.5 (results not shown).

4. Discussion

The primary barrier to the survival of probiotic

microorganisms in the stomach is mainly the low pH,

which is related to the high hydrochloric acid con-

centration of the secreted gastric acid (Hood and

Zottola, 1988). Selection of intrinsically resistant

strains of potentially probiotic LAB is performed

using synthetic solutions that simulate the gastric

juice, such as broths and buffers acidified to a pH

ranging between 1 and 4 (Conway et al., 1987; Hood

and Zottola, 1988; Charteris et al., 1998). It has been

suggested that besides the intrinsic acid tolerance, the

nature of the food carrier affects the stability of the

probiotic microorganisms (Gardiner et al., 1998;

Zarate et al., 2000).

Throughout this study, differences ( < 0.5 log10cycle) in the initial cell populations (log10 N0 h) of

Table 3

Effect of glucose and maltose addition on the viability of L. plantarum, L. acidophilus and L. reuteri during 4 h exposure at pH 2.5

Sugar (g/l) L. plantarum (log10 cfu/ml) L. acidophilus (log10 cfu/ml) L. reuteri (log10 cfu/ml)

N (0 h) N (4 h) D(N0 h�N4 h) N (0 h) N (4 h) D(N0 h�N4 h) N (0 h) N (4 h) D(N0 h�N4 h)

Glucose

0.0 9.76F 0.09 4.93F 0.13 4.82F 0.13 8.53F 0.08 6.49F 0.15 2.04F 0.15 8.57F 0.10 7.45F 0.12 1.13F 0.13

0.67 9.68F 0.07 4.84F 0.21 4.84F 0.19 8.59F 0.06 7.52F 0.10 1.07F 0.10 8.53F 0.09 7.39F 0.07 1.14F 0.10

1.67 9.72F 0.07 5.27F 0.12 4.45F 0.12 8.47F 0.11 7.70F 0.12 0.77F 0.13 8.50F 0.11 7.45F 0.09 1.05F 0.11

3.34 9.75F 0.10 5.76F 0.12 3.98F 0.14 8.48F 0.07 7.90F 0.08 0.58F 0.10 8.48F 0.10 7.44F 0.04 1.04F 0.09

5.00 9.81F 0.09 6.77F 0.18 3.05F 0.17 8.55F 0.09 8.28F 0.07 0.27F 0.09 8.56F 0.09 7.62F 0.06 0.94F 0.09

6.67 9.72F 0.08 7.20F 0.13 2.52F 0.13 8.53F 0.08 8.17F 0.11 0.36F 0.12 8.57F 0.08 7.53F 0.09 1.04F 0.11

8.33 9.73F 0.09 7.78F 0.06 1.96F 0.10 8.55F 0.09 8.22F 0.08 0.33F 0.10 8.60F 0.10 7.60F 0.12 1.00F 0.13

Maltose

0.0 9.48F 0.07 4.49F 0.16 4.99F 0.15 8.52F 0.08 6.52F 0.07 2.00F 0.09 8.61F 0.11 7.35F 0.13 1.26F 0.14

0.67 9.50F 0.06 4.45F 0.21 5.05F 0.19 8.54F 0.07 7.41F 0.07 1.13F 0.09 8.54F 0.09 7.22F 0.11 1.32F 0.11

1.67 9.66F 0.04 4.40F 0.17 5.26F 0.15 8.50F 0.08 7.93F 0.07 0.57F 0.10 8.66F 0.13 7.45F 0.06 1.21F 0.12

3.34 9.57F 0.09 5.50F 0.17 4.07F 0.17 8.45F 0.08 7.88F 0.07 0.57F 0.10 8.58F 0.08 7.54F 0.16 1.04F 0.16

5.00 9.51F 0.08 5.74F 0.06 3.77F 0.09 8.61F 0.06 8.04F 0.03 0.49F 0.07 8.60F 0.11 7.59F 0.07 1.01F 0.10

6.67 9.51F 0.06 7.15F 0.12 2.36F 0.12 8.52F 0.10 8.05F 0.03 0.47F 0.06 8.55F 0.10 7.65F 0.06 0.90F 0.10

8.33 9.47F 0.04 8.07F 0.09 1.40F 0.08 8.50F 0.08 8.10F 0.11 0.42F 0.12 8.52F 0.10 8.00F 0.08 0.52F 0.10

Cell values are means of six determinations (n= 6), with the standard error (F S.E.) calculated with 95% confidence.


each strain were observed, which could be ascribed to

differences in environmental conditions during the

preparation of the inocula. Statistical analysis indi-

cated that the initial cell population did not affect cell

viability, which was estimated as the reduction in cell

population after exposure for 4 h in a phosphate buffer

acidified at pH 2.5.

In the absence of cereal extracts, the viable cell

population of L. plantarum decreased 4.82F 0.12

log10 cycles after 4 h, suggesting that this strain is

intrinsically sensitive to acidic conditions. Interest-

ingly, addition of cereal extracts significantly improved

L. plantarum viability by approximately 4 log10 cycles

in the case of malt and 3 log10 cycles in the cases of

wheat and barley. The similar survival patterns

observed in the presence of wheat and barley extracts

could be associated with their chemical composition,

both consisting of similar amounts of total sugars

(4.60F 0.41 and 4.21F 0.32 g/l, respectively), reduc-

ing sugars (4.11F 0.14 and 3.81F 0.13 g/l) and FAN

(18.92F 0.96 and 17.55F 1.09 mg/l). The higher

concentrations of total than reducing sugar could be

attributed to the presence of sucrose or other soluble

oligosaccharides. The higher buffering capacity of the

malt extract compared to that of barley and wheat

would justify the observed enhanced protective effect

of malt, as Gardiner et al. (1999) suggested when

comparing yoghurt and cheese. However, in the present

study, the effect of buffering capacity on cell viability

was minimized. Therefore, the higher total sugar,

reducing sugar, and FAN content of malt than that of

wheat and barley observed due to the breakdown of

starch and proteins during themalting process, could be

the main factors contributing to the increased cell

viability observed in this case.

Since cereals are very complex substrates, the above

observations were evaluated by studying individually

the effect of primary diet constituents, such as sugars

(maltose and glucose) and FAN (tryptone and yeast

extract) on L. plantarum viability. The results demon-

strated that addition of either yeast extract or tryptone,

which resulted in FAN concentrations ranging from 13

to 47 mg/l, did not improve cell viability, suggesting

that the soluble FAN has no obvious positive effect on

L. plantarum stability. The acid tolerance of L. planta-

rum was enhanced upon addition of glucose and

maltose. Cell viability was progressively improved as

sugar concentration increased from 1.5 to 8.33 g/l; in

the presence of 8.33 g/l of maltose or glucose the cell

population decreased approximately 2 log10 cycles less

than the control. These results indicate that glucose and

maltose exert a protective effect on L. plantarum

viability under acidic conditions, which could partly

justify the protective effect of cereals. However, addi-

tion of maltose and glucose at concentrations which

correlated to the reducing sugar content of malt, wheat

and barley resulted in reduced protection than that

observed upon addition of cereal extracts. This could

be attributed to the presence of non-reducing sucrose in

malt, barley and wheat, which could possibly affect the

acid tolerance of L. plantarum in a similar way to

glucose and maltose, while a possible protective effect

due to the presence of soluble oligosaccharides can not

be excluded.

L. acidophilus exhibited higher acid tolerance than

L. plantarum, although a significant decrease of

approximately 2 log10 cycles was observed after 4 h

exposure at pH 2.5. Addition of cereal extracts

enhanced cell viability more than 1.5 log10 cycle in

all cases, which suggests that there are no significant

differences on the protective effect of cereal extracts on

L. acidophilus survival. These results confirmed the

overall protective role of cereal extracts, but also

illustrated some differences in the behaviour of L.

acidophilus compared to L. plantarum, which was

verified in the experiments studying the individual

effect of sugar and FAN. The viability of L. acidophilus

was increased by more than 0.6 log10 cycle in the

presence of 36 and 47 mg/l FAN, which suggests that

the FAN content of malt, wheat and barley could

contribute to the protective effect of these cereal

extracts. Enhanced viability of L. acidophilus upon

addition of milk proteins has also been reported by

Charteris et al. (1998), however, in that study this was

partly attributed to the buffering capacity of the milk

protein solution added to the buffer. In the present

study, maltose and glucose affected significantly the

survival of L. acidophilus even at very low concen-

trations, such as 0.67 and 1.66 g/l. At concentrations

similar to the reducing sugar concentration of malt,

wheat and barley, a similar improvement of more than

1.5 log10 cycle in cell viability was observed. There-

fore, the significant effect of sugars, even at very low

concentrations on L. acidophilus viability could possi-

bly justify the similar level of protection induced by

malt, wheat and barley on L. acidophilus.


L. reuteri cell population decreased approximately

1 log10 cycle after 4 h exposure at pH 2.5, and could

be characterized as the most intrinsically acid tolerant

lactobacillus among the three strains used in this

study. In the presence of cereal extracts, an improve-

ment in cell viability by approximately 0.8 log10 cycle

was observed. FAN did not affect considerably cell

survival (data not shown). Interestingly, addition of

glucose did not improve cell viability, while maltose

addition increased cell survival by approximately 0.7

log10 cells. However, the intrinsic stability of the L.

reuteri strain used was very high and it is therefore

very difficult to evaluate the individual protective

effect of the dietary components of cereal extracts

on cell viability.

Under acidic conditions, the anaerobic fermentable

microorganisms regulate their cytoplasmic pH by

several mechanisms, the most important of which is

the translocation of protons from the cytoplasm to the

environment by an ATPase at the expense of ATP

(McDonald et al., 1990; Sanders et al., 1999). Nannen

and Hutkins (1991) have demonstrated that the spe-

cific activity of ATPases from several LAB increases

as the extracellular pH moves from neutral to 5.0.

High ATPase activity was observed for L. plantarum

and L. acidophilus after exposure at pH 3 and 3.5,

respectively (Kullen and Klaenhammer, 1999; Hong

et al., 1999).

In fermentation studies performed at pH values

ranging from 4 to 7, lactic acid bacteria continue to

consume sugars during their stationary phase and

produce additional ATP required for preservation of

cell viability (Venkatesh et al., 1993). In the study of

Giraud et al. (1991), a L. plantarum strain consumed

approximately 40 g/l of sugars 10 h after the end of

the exponential phase at pH 6, while at pH 5 and pH 4

the amount of sugars consumed was decreased. In the

present study, no consumption of glucose or maltose

was detected after exposure of L. plantarum, L. acid-

ophilus and L. reuteri for 4 h at pH 2.5, in all the

concentrations tested. It is important to note that the

sensitivity of the method used for sugar analysis is

0.05 g/l, which suggests that even slight changes

would have been detected, since the amount of sugars

added was not very high (0.67–8.33 g/l). The inhib-

ition of glycolysis was probably due to inactivation of

the enzymes involved in the metabolic processes or

damage to biological regulating systems (Venkatesh et

al., 1993; Hong and Pyun, 2001). Hong et al. (1999)

reported a 50% reduction of glucose (approximately

1.5 g/l) after a 2-h incubation of a L. plantarum strain

isolated from kimchi at pH 2; however the effect on

cell viability was not studied. The differences

observed in the present study regarding L. plantarum

glycolytic activity could be attributed to the intrinsic

properties of the strain and the experimental methods

used to test cell viability (inoculum preparation, media

composition).

Inhibition of glycolysis does not exclude a possibly

increased ATPase activity of L. plantarum and L.

acidophilus in the presence of sugars, which could

explain the fact that cell viability progressively

increased by increasing glucose and maltose concen-

trations. In addition, Glaasker et al. (1998) suggested

that L. plantarum could resist external hyperosmotic

conditions imposed by sugar stress (lactose and glu-

cose) by equilibrating the internal and external con-

centrations after some time (about 30 min) by a

system involving diffusion of the sugar in the cyto-

plasm. The affinity constant of this transport system

was very high (>18 g/l lactose); however, an adapta-

tion of the strain to one stress, such as a mild osmotic

stress, could invoke acid tolerance (Hill et al., 1995;

Sanders et al., 1999; Hong and Pyun, 2001).

Another mechanism for pH homeostasis used by

lactobacilli involves decarboxylation of amino acids,

which results in production of additional ATP, enabling

extrusion of cytoplasmic protons by ATPase (Sanders

et al., 1999; Siegumfeldt et al., 2000). The increased

viability of L. acidophilus that was observed in the

present study upon addition of 36 and 47 mg/l FAN

could be attributed to the above mechanism. Further

studies will investigate the mechanisms of the possible

protective effect of sugars and FAN on L. plantarum

and L. acidophilus viability under acidic conditions.

The results presented in this study suggest that

malt, wheat and barley extracts exert a protective

effect on L. plantarum, L. acidophilus and L. reuteri

viability under acidic conditions, which was associ-

ated with the chemical composition of these cereal

extracts. Supporting experiments, studying the indi-

vidual effect of dietary constituents on cell survival

indicated that these effects could be mainly attributed

to the presence of soluble sugars in the cereal extracts,

and to a less extent to the free amino nitrogen content,

depending on the strain.


Acknowledgements

The authors acknowledge the financial support

provided to Dimitris Charalampopoulos by the

Hellenic State Scholarships Foundation. They are

also grateful to the Satake (Japan) for providing some

of the equipment used in this work.

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