in vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy...

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International Journal of Food Microbiology 91 (2004) 253–260

In vitro assessment of the upper gastrointestinal tolerance of

potential probiotic dairy propionibacteria

Yang Huang, Michelle C. Adams*

School of Applied Sciences, The University of Newcastle, P.O. Box 127, Ourimbah, NSW 2258, Australia

Received 27 February 2003; received in revised form 17 July 2003; accepted 24 July 2003

Abstract

This study aimed to assess the transit tolerance of potential probiotic dairy propionibacteria strains in human upper

gastrointestinal tract in vitro, and to evaluate the effect of food addition on viability of these strains in simulated pH 2.0 gastric

juices. The transit tolerance of 13 dairy propionibacteria strains was determined at 37 jC by exposing washed cell suspensions

to simulated gastric juices at pH values at 2.0, 3.0, and 4.0, and simulated small intestinal juices (pH 8.0) with or without

0.3% bile salts. The viability of dairy propionibacteria in pH 2.0 simulated gastric juice with So-Goodk original soymilk or

Up & GoR liquid breakfast was also determined. The simulated gastric transit tolerance of dairy propionibacteria was strain-

dependent and pH-dependent. All tested strains were tolerant to simulated small intestinal transit. The addition of So-Goodkoriginal soymilk or Up & GoR liquid breakfast greatly enhanced the survival of dairy propionibacteria strains in pH 2.0

simulated gastric juices. Dairy propionibacteria strains demonstrate high tolerance to simulated human upper gastrointestinal

tract conditions and offer a relatively overlooked, yet alternative source for novel probiotics besides Lactobacillus and

Bifidobacterium.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Dairy propionibacteria; Probiotics; Gastric and small intestinal tolerance

1. Introduction (Reinbold, 1985; Grant and Salminen, 1998). Dairy

When selecting a new microbial strain for appli-

cation in probiotic food products, the first constraint is

that it must be a strain that is Generally Recognized

As Safe (GRAS) (Havenaar et al., 1992). Like Lac-

tobacillus species, dairy propionibacteria have been

used as starter cultures in the dairy industry for a long

time, and are considered safe for human consumption

0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2003.07.001

* Corresponding author. Tel.: +61-2-4348-4135; fax: +61-2-

4348-4145.

E-mail address: [email protected]

(M.C. Adams).

propionibacteria, which include Propionibacterium

freudenreichii, P. jensenii, P. acidopropionici and P.

thoenii, have recently shown potential probiotic

effects, such as the production of propionic acid,

bacteriocins, vitamin B12 (Holo et al., 2002; Hugen-

holtz et al., 2002), synthesis of h-galactosidase en-

zyme (Zarate et al., 2000), growth stimulation of

bifidobacteria (Kaneko et al., 1994), and favourable

affects on lipid metabolism and the immune system of

hosts (Perez-Chaia et al., 1995).

Probiotic bacteria that are delivered through food

systems have to firstly survive during the transit

through the upper gastrointestinal tract, and then persist

Y. Huang, M.C. Adams / International Journal of Food Microbiology 91 (2004) 253–260254

in the gut to provide beneficial effects for the host

(Chou and Weimer, 1999). In order to be used as

potential probiotics, dairy propionibacteria strains need

to be screened for their capacity of transit tolerance to

the upper gastrointestinal tract conditions.

The low pH of the stomach and the antimicrobial

action of pepsin are known to provide an effective

barrier against entry of bacteria into the intestinal

tract (Holzapfel et al., 1998). The pH of the stomach

could be as low as pH 1.5 (Lankaputhra and Shah,

1995), or as high as pH 6 or above after food intake

(Johnson, 1977), but generally ranges from pH 2.5 to

pH 3.5 (Holzapfel et al., 1998). The nature of food in

the stomach affects the transit time through the

stomach. Normally, food remains in the stomach

between 2 and 4 h (Smith, 1995), however, liquids

empty from the stomach faster than solids, and only

take about 20 min to pass through the stomach

(GastroNet Australia, 2001). There are no agreed

rules for the screening of acid tolerance of potential

probiotic strains. A range of pH values, from pH 1 to

pH 5, has been used to screen in vitro the acid

tolerance of Lactobacillus, Bifidobacterium and some

dairy propionibacteria strains (Conway et al., 1987;

Lankaputhra and Shah, 1995; Charteris et al., 1998;

Chou and Weimer, 1999; Chung et al., 1999; Zarate

et al., 2000).

Another barrier probiotic bacteria must survive is

the small intestine. The adverse conditions of the small

intestine include the presence of bile salts and pancre-

atin (Floch et al., 1972; Le Vay, 1988). The transit time

of food through the small intestine is generally between

1 and 4 h (Smith, 1995). The pH of the small intestine is

around pH 8.0 (Keele and Neil, 1965). Bile salt-

resistant lactic acid bacteria can be selected by testing

their survivability in the presence of bile salt and their

growth in selective medium containing various levels

of bile (Gilliland et al., 1984; Ibrahim and Bezkoro-

vainy, 1993; Clark and Martin, 1994; Chung et al.,

1999). A concentration of 0.15–0.3% of bile salt has

been recommended as a suitable concentration for

selecting probiotic bacteria for human use (Goldin

and Gorbach, 1992).

Food is the common delivery system for probiotic

bacteria. Food and food ingredients have been shown to

protect probiotic bacteria from acid conditions and

enhance gastric survival. Milk has been reported to

increase the viability of acid-sensitive Lactobacillus

and Bifidobacterium strains during simulated gastric

tract transit (Conway et al., 1987; Charteris et al.,

1998). The protective effect may be due to the increase

of gut pH after milk addition (Conway et al., 1987).

Amylose maize starch granules at pH 3.5 have also

been found to increase the viability of the more acid-

sensitive Bifidobacterium strains (Wang et al., 1999).

Currently, orally ingested probiotic bacteria for humans

are mainly prepared in conjunction with dairy products

(Goldin and Gorbach, 1992).

The vegetarian consumer is especially conscious

about the kinds of foods they consume, and the

concept of a probiotic product that meets health needs

of vegetarians has market appeal. A frozen vegetarian

soy dessert has been found to be a suitable product for

supporting the viability of Lactobacillus and Bifido-

bacterium probiotic strains (Heenan, 2001). However,

in general, there has been very little study on the

effects of vegetarian foods, such as soy and cereal

beverage, on the gastric transit tolerance of probiotic

bacteria.

In this paper, we isolated six dairy propionibac-

teria strains from raw milk and cheese products. The

upper gastrointestinal transit tolerance of these six

strains were assessed along with seven reference

stains of dairy propionibacteria, by testing (1) the

viability in simulated gastric transit conditions (pH

2.0, pH 3.0 and pH 4.0 gastric juices) and (2) the

viability in simulated small intestinal transit condi-

tions (pH 8.0, with or without 0.3% bile salts). In

addition, the effect of two vegetarian foods, So-

Goodk original soymilk (So-Good) (Sanitarium,

Australia) and Up & GoR liquid breakfast (Sanitar-

ium, Australia), on pH 2.0 simulated gastric transit

tolerance of all 13 dairy propionibacteria strains was

also determined.

2. Materials and methods

2.1. Isolation and identification of dairy propionibac-

teria from raw milk and cheese products

The following seven reference strains were used:

P. acidopropionici ATCC25562, P. acidopropionici

341, P. freudenreichii CSCC2200, P. freudenreichii

CSCC2201, P. freudenreichii CSCC2206, P. freuden-

reichii CSCC2207, and P. freudenreichii CSCC2216.

Y. Huang, M.C. Adams / International Journal of Food Microbiology 91 (2004) 253–260 255

Four fresh raw milk, and six cheese samples

(Parmesan, Swiss cheese, Gouda, Grana Papano and

Jarlsberg) were analysed by spreading 0.1 ml decimal

dilutions of milk or homogenized solid samples on the

surface of yeast extract lactate agar (YELA) (3%

tryptone, 3% yeast extract, 1.2% sodium lactate,

1.5% agar, pH 7.0) (Harrigan, 1998). Colonies were

picked after 10-day incubation at 30 jC in anaerobic

conditions. From each sample, typical dairy propio-

nibacteria colonies were isolated and purified by

streaking on sodium lactate agar (SLA) (1% tryptone,

1% yeast extract, 1% sodium lactate, 0.025%

K2HPO4, 0.05% MnSO4, 1.5% agar, pH 7.0) and

incubated for 7 days at 30 jC in anaerobic conditions.

Dairy propionibacteria were identified to species

based on Gram-staining, catalase activity test, maltose

and sucrose fermentation, h-haemolysis test, nitrate

reduction test, and gelatinase activity (Cummins and

Johnson, 1986).

2.2. Preparation of washed bacterial cell suspensions

Prior to simulated upper gastrointestinal transit

assays, dairy propionibacteria strains were serially

transferred twice in sodium lactate broth (SLB) (1%

tryptone, 1% yeast extract, 1% sodium lactate, 0.025%

K2HPO4, 0.05% MnSO4, pH 7.0) and incubated an-

aerobically at 30 jC for 48 h.

Cells in an aliquot (1 ml) of the 48-h culture of each

strain were collected by centrifugation (2500� g, 5

min) and washed three times in PBS buffer (0.02%

KCl, 0.144% Na2HPO4, 0.8% NaCl, 0.024% KH2PO4,

pH 7.0). The total viable count of the washed bacterial

cell suspension was determined prior to assay of transit

tolerance.

2.3. Preparation of simulated gastric and small

intestinal juices

Simulated gastric and small intestinal juices were

prepared fresh daily.

Simulated gastric juices were prepared by sus-

pending pepsin (1:10000, ICN) in sterile saline

(0.5% w/v) to a final concentration of 3 g l� 1

and adjusting the pH to 2.0, 3.0, and 4.0 with

concentrated HCl or sterile 0.1 mol l� 1 NaOH

using a pH meter (Model 8417N, Hanna Instrument,

Singapore).

Simulated small intestinal juices were prepared by

suspending pancreatin USP (P-1500, Sigma) in the

sterile saline to a final concentration of 1 g l� 1, with

or without 0.45% bile salts (Oxoid), and adjusting the

pH to 8.0 with sterile 0.1 mol l� 1 NaOH using the pH

meter.

2.4. Upper gastrointestinal transit tolerance assay

The tolerance of washed cell suspensions of dairy

propionibacteria strains to simulated gastric and small

intestinal transit was determined following the method

of Charteris et al. (1998). An aliquot (0.2 ml) of each

washed cell suspension was transferred to a 2.0-ml

capacity screw-cap eppendorf tube, and then mixed

with 0.3 ml of NaCl (0.5% w/v), and 1.0 ml of

simulated gastric (pH 2.0, pH 3.0, or pH 4.0) or small

intestinal juices (pH 8.0). The mixture was then

vortexed at maximum setting for 10 s and incubated

at 37 jC. When screening gastric transit tolerance,

aliquots of 0.1 ml were removed after 1, 60, 90, and

180 min for determination of total viable count. For

screening small intestinal transit tolerance, aliquots of

0.1 ml were removed after 1 and 240 min for the

determination of total viable count. The viable counts

at 0 min were determined as (viable counts of bacte-

rial suspension� 0.2)/1.5.

2.5. Determination of pH 2.0 gastric transit tolerance

in the presence of So-Goodk original soymilk or Up

& GoR liquid breakfast

The tolerance of washed cell suspensions of dairy

propionibacteria strains to simulated pH 2.0 gastric

juice was determined as described in Section 2.4, with

the exception that 0.3 ml of So-Goodk original

soymilk or Up & GoR liquid breakfast replaced the

sterile saline addition.

2.6. Determination of total viable counts

Total viable counts of dairy propionibacteria strains

were determined by a pour plate method using SLA

agar after serial 10-fold dilution in maximum recovery

diluent (Oxoid). SLA plates were incubated anaero-

bically at 30 jC for 6 days, and colonies on SLA

plates were counted using a colony counter (Stuart

Scientific, UK).

rnal of Food Microbiology 91 (2004) 253–260

2.7. Statistical analysis

Results are expressed as the mean and stan-

dard deviation of two determinations. P values

less than 0.05 were regarded as significant differ-

ence between means using a two-tailed Student’s

t-test.

Y. Huang, M.C. Adams / International Jou256

Table 1

Effect of simulated gastric juices (pH 2.0, pH 3.0, pH 4.0) on the viability

Strains pH of simulated Viable count (log

gastric juices0 min 1 m

P. freudenreichii CSCC2200 pH 2.0 8.5 (0.4) 8.2

pH 3.0 8.5 (0.4) 8.3

pH 4.0 8.5 (0.4) 8.3


pH 3.0 8.6 (0.4) 8.9

pH 4.0 8.6 (0.4) 8.7


pH 3.0 7.7 (0.1) 7.7

pH 4.0 7.7 (0.1) 7.6


pH 3.0 8.6 (0.1) 8.9

pH 4.0 8.6 (0.1) 8.9


pH 3.0 8.6 (0.0) 8.7

pH 4.0 8.6 (0.0) 8.6

P. freudenreichii 201a1 pH 2.0 8.4 (0.0) 8.4

pH 3.0 8.4 (0.0) 8.3

pH 4.0 8.4 (0.0) 8.3

P. freudenreichii 201b pH 2.0 8.5 (0.1) 8.3

pH 3.0 8.5 (0.1) 8.3

pH 4.0 8.5 (0.1) 8.2

P. jensenii 702 pH 2.0 8.2 (0.1) 8.3

pH 3.0 8.6 (0.1) 8.6

pH 4.0 8.6 (0.1) 8.6

P. freudenreichii 801 pH 2.0 8.8 (0.1) 8.5

pH 3.0 8.8 (0.1) 8.8

pH 4.0 8.8 (0.1) 8.8


pH 3.0 8.8 (0.0) 8.8

pH 4.0 8.8 (0.0) 8.8


pH 3.0 8.6 (0.0) 8.6

pH 4.0 8.6 (0.0) 8.6

P. acidopropionici 341 pH 2.0 8.3 (0.0) 6.0

pH 3.0 8.3 (0.0) 8.2

pH 4.0 8.3 (0.0) 8.1

P. acidopropionici ATCC25562 pH 2.0 9.1 (0.0) 6.5

pH 3.0 9.1 (0.0) 9.1

pH 4.0 9.1 (0.0) 9.1

Results are shown as mean (S.D.), n= 2.

Viable counts (log cfu/ml) of each strain at 1, 60, 90, and 180 min wer

(Student’s t-test, two tailed).

3. Results

3.1. Isolation and identification of isolated dairy

propionibacteria strains

Six dairy propionibacteria strains were isolated, of

which four were from milk and two from a Swiss-

of 13 dairy propionibacteria strains during 180 min of gastric transit

cfu/ml) during simulated gastric transit tolerance

in 60 min 90 min 180 min

(0.0) 4.2 (0.1)** 0.8 (0.1)*** < 1

(0.2) 8.3 (0.1) 8.3 (0.2) 8.3 (0.3)

(0.2) 8.4 (0.2) 8.4 (0.2) 8.4 (0.2)

(0.1) 4.0 (0.1)* 3.7 (0.2)* 2.0 (0.0)**

(0.1) 8.9 (0.0) 8.9 (0.0) 8.9 (0.0)

(0.0) 8.9 (0.0) 8.8 (0.0) 8.8 (0.0)

(0.0)*** 2.7 (0.0)*** < 1 < 1

(0.0) 6.8 (0.3)* 6.8 (0.3)* 6.8 (0.2)*

(0.1) 7.8 (0.0) 7.9 (0.0) 7.9 (0.1)

(0.1) 4.9 (0.1)** 2.9 (0.2)** 1.4 (0.0)***

(0.1) 8.9 (0.1) 8.9 (0.1) 8.9 (0.0)

(0.0) 8.8 (0.0) 8.9 (0.1) 8.8 (0.1)

(0.0) 2.0 (0.0)*** 0.7 (0.1)*** < 1

(0.1) 8.7 (0.1) 8.7 (0.1) 8.7 (0.1)

(0.0) 8.6 (0.1) 8.7 (0.0) 8.7 (0.0)

(0.0) 5.0 (0.1)*** 3.9 (0.0)*** 3.5 (0.2)***

(0.0) 8.3 (0.1) 7.8 (0.7) 8.3 (0.0)

(0.0) 8.3 (0.0) 8.4 (0.2) 8.5 (0.1)

(0.1) 5.6 (0.2)*** 5.2 (0.1)*** 4.9 (0.0)***

(0.1) 8.3 (0.0) 8.4 (0.0) 8.4 (0.2)

(0.1) 8.4 (0.1) 8.4 (0.0) 8.4 (0.0)

(0.2) 6.0 (0.0)*** 4.0 (0.1)*** 2.6 (0.0)***

(0.0) 8.5 (0.0) 8.4 (0.0) 8.5 (0.0)

(0.0) 8.8 (0.1) 8.3 (0.3) 8.5 (0.2)

(0.0) 7.8 (0.0)*** 5.9 (0.2)*** 3.5 (0.2)***

(0.0) 8.8 (0.0) 8.8 (0.1) 8.9 (0.0)

(0.0) 8.8 (0.1) 8.9 (0.0) 8.9 (0.1)

(0.1) 7.6 (0.2)** 6.8 (0.0)*** 3.9 (0.2)***

(0.0) 8.8 (0.0) 8.9 (0.0) 8.9 (0.0)

(0.0) 9.0 (0.0) 8.9 (0.0) 8.9 (0.0)

(0.0) 7.3 (0.1)*** 4.7 (0.1)*** 3.1 (0.3)***

(0.0) 8.6 (0.0) 8.5 (0.0) 8.7 (0.0)

(0.0) 8.8 (0.0) 8.7 (0.0) 8.8 (0.0)

(0.0)** 4.9 (0.2)*** 4.64 (0.23)*** 3.0 (0.0)***

(0.0) 8.0 (0.1)* 8.0 (0.0)** 7.5 (0.4)***

(0.0) 8.2 (0.1) 8.3 (0.1) 8.2 (0.1)

(0.3)** 5.5 (0.1)*** 5.4 (0.2)*** 4.9 (0.0)***

(0.0) 9.0 (0.0)* 8.5 (0.0)*** 8.0 (0.0)***

(0.0) 9.2 (0.0) 9.2 (0.0) 9.2 (0.0)

e compared with that at 0 min, *p< 0.05, **p< 0.01, ***p< 0.001


cheese sample. These six isolates were Gram-positive,

catalase positive, non-spore forming, and irregular

short rods. Strains 201a1, 201b, 801, 901, and 1001

were identified as P. freudenreichii, and strain 702 was

identified as P. jensenii.

3.2. Effect of simulated gastric juices with different

pH on viability

The effect of different pH of simulated gastric

juices on viability of 13 dairy propionibacteria strains

is presented in Table 1. The average final pH of the

simulated transit mixture was 2.3, 3.8, and 6.0 for the

pH 2.0, pH 3.0, and pH 4.0 gastric juices, respectively.

In general, each strain showed lower viability in

simulated gastric juice at pH 2.0 than in simulated

gastric juices with pH 3.0 or pH 4.0.

When the simulated gastric juice was at pH 2.0, all

the strains showed progressive reduction in viability

during 180 min of simulated gastric transit, especially

P. freudenreichii CSCC2200, P. freudenreichii

CSCC2206 and P. freudenreichii CSCC2216, which

lost total viability after 180 min of simulated gastric

transit.

When the simulated gastric juice was at pH 3.0, 10

out of 13 tested strains retained a similar level of

viability during simulated gastric tract transit for up to

Table 2

Effect of simulated small intestinal juices on the viability of 13 dairy pro

Strains Viable count (log cfu/ml) during

Absence of bile salts

0 min 1 min

P. freudenreichii CSCC2200 8.2 (0.0) 8.2 (0.0)





P. freudenreichii 201a1 8.6 (0.0) 8.4 (0.0)

P. freudenreichii 201b 8.2 (0.1) 8.2 (0.1)

P. freudenreichii 801 8.8 (0.1) 8.6 (0.0)



P. jensenii 702 8.6 (0.1) 8.6 (0.0)

P. acidopropionici 341 8.1 (0.1) 8.2 (0.0)

P. acidopropionici ATCC25562 8.0 (0.1) 8.1 (0.1)


Viable counts (log cfu/ml) of each strain at 1 and 240 min were compa

tailed).

180 min. Only three strains, P. freudenreichii CSC-

C2206, P. acidopropionici 341, and P. acidopropio-

nici ATCC25562, showed a 1-log reduction of viabil-

ity after 180 min of simulated gastric tract transit.

When the simulated gastric juice was at pH 4.0, all

of the tested 13 strains retained the same level of via-

bility during 180 min of simulated gastric tract transit.

3.3. Effect of simulated small intestinal transit on

viability

All 13 tested strains retained the same viability

during 240 min of simulated small intestinal transit in

the absence of bile salt (Table 2). However, in the

presence of 0.3% of bile salts, two strains (P. freu-

denreichii CSCC2207 and P. acidopropionici 341)

showed a slight reduction of viable counts, 0.2-log

and 1.0-log, respectively (Table 2).

3.4. Effects of soy milk and cereal liquid breakfast

addition on viability during simulated gastric tract

transit with pH 2.0 simulated gastric juice

The effect of two kinds of liquid vegetarian foods,

So-Goodk original soymilk (So-Good) and Up &

GoR liquid breakfast (Up & Go), on viability during

simulated gastric transit with pH 2.0 simulated gastric

pionibacteria strains during 240 min of small intestine transit

simulated small intestinal transit tolerance

In the presence of 0.3% bile salts

240 min 0 min 1 min 240 min

8.3 (0.0) 8.4 (0.0) 8.5 (0.0) 8.5 (0.0)

8.9 (0.1) 8.8 (0.0) 8.8 (0.1) 8.8 (0.0)

7.8 (0.1) 7.9 (0.0) 8.0 (0.1) 7.9 (0.0)

8.8 (0.0) 8.7 (0.0) 8.7 (0.1) 8.5 (0.0)**

8.5 (0.0) 8.6 (0.0) 8.7 (0.0) 8.6 (0.0)

8.5 (0.1) 7.6 (0.0) 7.5 (0.1) 7.6 (0.1)

8.5 (0.1) 7.6 (0.0) 7.6 (0.1) 7.7 (0.1)

8.9 (0.0) 7.8 (0.1) 7.7 (0.1) 7.7 (0.1)

8.9 (0.0) 7.6 (0.1) 7.7 (0.0) 7.7 (0.1)

8.8 (0.0) 7.7 (0.0) 7.7 (0.0) 7.7 (0.1)

8.5 (0.1) 8.6 (0.1) 9.0 (0.0) 8.7 (0.3)

8.3 (0.2) 7.8 (0.1) 6.5 (0.1)** 6.8 (0.3)*

8.6 (0.1) 7.7 (0.0) 7.7 (0.14) 7.8 (0.1)

red with that at 0 min, *p< 0.05, **p< 0.01 (Student’s t-test, two


juice is present in Table 3. The pH of the gastric

transit test mixtures was pH 2.3, pH 5.2, and pH 5.3

for the samples of control, So-Good and Up & Go,

respectively.

Table 3

Effect of soymilk and liquid cereal breakfast addition on the viability of 1

simulated gastric juice at pH 2.0

Strains Conditions Viable count (lo

0 min

P. freudenreichii CSCC2200 Control 8.5 (0.4)

So-gooda 8.8 (0.0)

Up & Gob 8.8 (0.0)


So-good 8.7 (0.0)

Up & Go 8.7 (0.0)


So-good 7.7 (0.0)

Up & Go 7.7 (0.0)


So-good 8.7 (0.0)

Up & Go 8.7 (0.0)


So-good 8.6 (0.0)

Up & Go 8.6 (0.0)

P. freudenreichii 201a1 Control 8.4 (0.0)

So-good 8.4 (0.0)

Up & Go 8.4 (0.0)

P. freudenreichii 201b Control 8.5 (0.1)

So-gooda 8.5 (0.1)

Up & Gob 8.5 (0.1)

P. jensenii 702 Control 8.2 (0.1)

So-good 8.2 (0.1)

Up & Go 8.2 (0.1)

P. freudenreichii 801 Control 8.8 (0.1)

So-good 8.8 (0.1)

Up & Go 8.8 (0.1)


So-good 8.7 (0.1)

Up & Go 8.7 (0.1)


So-good 8.6 (0.0)

Up & Go 8.6 (0.0)

P. acidopropionici 341 Control 8.3 (0.0)

So-good 8.3 (0.0)

Up & Go 8.3 (0.0)

P. acidopropionici ATCC25562 Control 9.1 (0.0)

So-good 9.1 (0.0)

Up & Go 9.1 (0.0)


Viable counts (log cfu/ml) of each strain at 1 and 180 min were compare

t-test, two tailed).a So-good, So-Goodk original soymilk.b Up & Go, Up & GoR liquid breakfast.

In general, the addition of both liquid vegetarian

foods significantly improved the viability of each

strain through the pH 2.0 simulated gastric transit

( p < 0.05) (Table 3).

3 dairy propionibacteria strains during 180-min gastric transit with

g cfu/ml) during simulated gastric transit tolerance

1 min 180 min

8.2 (0.0) < 1

8.5 (0.1)* 8.5 (0.0)*

8.5 (0.1)* 8.5 (0.0)

8.8 (0.1) 2.0 (0.0)**

8.8 (0.0) 8.9 (0.1)

8.8 (0.0) 8.9 (0.0)

4.7 (0.0)*** < 1

7.5 (0.0)*** 7.8 (0.0)

7.6 (0.1) 7.8 (0.0)

8.8 (0.1) 1.4 (0.0)***

8.9 (0.0) 8.8 (0.1)

8.8 (0.1) 8.9 (0.0)

8.5 (0.0) < 1

8.7 (0.1) 8.8 (0.1)

8.5 (0.1) 8.7 (0.2)

8.4 (0.0) 3.5 (0.2)***

8.4 (0.1) 8.4 (0.1)

8.3 (0.1) 8.4 (0.0)

8.3 (0.1) 4.9 (0.0)***

8.4 (0.2) 8.3 (0.0)

8.3 (0.2) 8.4 (0.0)

8.3 (0.2) 2.6 (0.0)***

8.2 (0.1) 8.3 (0.2)

8.2 (0.1) 8.0 (0.1)*

8.5 (0.0) 3.5 (0.2)***

8.6 (0.0) 8.6 (0.0)

8.5 (0.0) 8.4 (0.1)

8.8 (0.1) 3.9 (0.2)***

8.7 (0.0) 8.8 (0.0)

8.7 (0.0) 8.8 (0.0)

8.5 (0.0) 3.1 (0.3)***

8.6 (0.0) 8.7 (0.0)

8.6 (0.0) 8.7 (0.0)

6.0 (0.0)*** 3.0 (0.0)***

8.1 (0.1) 8.3 (0.0)

8.1 (0.1) 8.4 (0.1)

6.5 (0.3)*** 4.9 (0.0)***

9.1 (0.0) 9.3 (0.0)

9.2 (0.0) 9.2 (0.0)

d with that at 0 min, *p< 0.05, **p< 0.01, ***p< 0.001 (Student’s


4. Discussion

This study compared the effect of different pH of

simulated gastric juices on the viability of 13 dairy

propionibacteria strains during 180-min simulated

gastric transit. There was no loss of viability for all

strains at pH 4.0; in contrast, at pH 3.0, 10 strains

retained the same level of viability, while at pH 2.0,

all strains showed reduced viability (Table 1). These

results are comparable to the findings of Zarate et al.

(2000), in which four dairy propionibacteria strains

have been shown to survive well at pH 4 and one

strain slightly lose viability at pH 3 but all tested four

strains lose viability substantially at pH 2. The vari-

ability of dairy propionibacteria strains to survive at

pH 2 and pH 3 suggests that the acid tolerance of

dairy propionibacteria is strain-specific, and pH val-

ues of 2 and 3 could be considered as critical for the

selection of potential probiotic dairy propionibacteria.

Although pH could be used as a suitable direct

measure for selection of probiotic strains, most pro-

biotics are consumed in food products. The presence of

food and food ingredients has been reported to improve

viability of microorganisms during gastric transit (Con-

way et al., 1987; Charteris et al., 1998; Wang et al.,

1999; Zarate et al., 2000). The suggested mechanism

for the beneficial effect of food and food ingredients is

the pH increase of the gastric contents resulting from

the addition of the food (Conway et al., 1987; Charteris

et al., 1998; Wang et al., 1999; Zarate et al., 2000). In

the current study, the viability of all 13 tested dairy

propionibacteria strains during pH 2.0 simulated gas-

tric transit was significantly improved with the addition

of So-Goodk original soymilk and Up & GoR liquid

breakfast (Table 3). One of the factors contributing to

the improvement of the viability of tested dairy pro-

pionibacteria strains is the increase of pH in the reaction

mix after the addition of those foods, since the final pH

of the simulated gastric contents was increased to

above pH 5, which was close to that of the simulated

gastric mixture (pH 6.0) with pH 4.0 gastric juices.

The improved viability of microorganisms during

pH 2.0 simulated gastric transit with the addition of

food indicates that low-acid tolerant strains need not be

excluded from probiotic applications, providing they

can be delivered to the intestine in high numbers, and

preferably as part of a buffered food or encapsulated

delivery system.

Small intestinal transit tolerance (including bile

tolerance) is essential for probiotic strains to colonise

the small intestine. Most of tested 13 strains demon-

strated high levels of small intestinal transit tolerance,

with no loss of viability after exposure to simulated

small intestinal juices for 240 min (Table 2). Only two

strains, P. freudenreichii CSCC2207 and P. acidopro-

pionici 341, showed a small reduction in their viabil-

ity (0.2-log and 1.0-log, respectively) in the presence

of bile salts (Table 2).

In probiotic selection, small intestine tolerance is of

potentially more importance than gastric survival.

With the development of new delivery systems and

the use of specific foods, evidence clearly demon-

strates that acid-sensitive strains can be buffered

through the stomach. However, to exert a positive

effect on the health and well being of a host, probiotics

need to colonise and survive in the small intestine

(Havenaar et al., 1992), and it is the condition of this

environment that may in fact be an essential selection

criteria for future probiotics.

This current study has demonstrated that although

the viability of dairy propionibacteria is affected by

pH 2.0, most of the tested strains survived well at pH

3.0 and pH 4.0. Furthermore, survival of the dairy

propionibacteria in simulated gastric juice at pH 2.0 is

significantly enhanced by the addition of food prod-

ucts. Simulated small intestine conditions have little

effect on the viability of dairy propionibacteria. Over-

all, dairy propionibacteria showed high capacity of

upper gastrointestinal transit tolerance and will pro-

vide an alternative source to Lactobacillus and Bifi-

dobacterium for future probiotic development.

Acknowledgements

The authors acknowledge the support from The

University of Newcastle, Australia.

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