A.R. Madureira
a, C.I. Pereira
a, K. Truszkowska
b, A.M. Gomes
a,
M.E. Pintado
a, F.X. Malcata
a,aEscola Superior de Biotecnologia, Universidade Cato´lica Portuguesa, R. Dr. Anto´nio Bernardino de Almeida, P-4200-072 Porto, Portugal bWarsaw Agricultural University, Ul. Nowoursynowska 166, 02-787 Warszawa, Poland
Abstract
Various foods may be used to deliver probiotic bacteria into the gastrointestinal tract; one such example is Requeija˜o, a Portuguese whey cheese. Survival and stability of Bifidobacterium animalis strains BLC-1, Bb-12, and Bo, Lactobacillus acidophilus strains LAC-1 and Ki, L. paracasei ssp. paracasei strain LCS-1 and L. brevis strain LMG 6906 inoculated into Requeija˜o, when exposed to simulated gastrointestinal tract conditions, were assessed. Homogenates of inoculated whey cheese in 0.85% (w/v) sterile
saline water were exposed to a solution of hydrochloric acid (pH 2.5–3.0) and pepsin (1000 units mL–1) at 37 1C, and then to 0.3%
(w/v) bile salts after 60 or 120 min of acid exposure. All bacterial strains retained their initial viable cell numbers. Bifidobacterium animalis strains Bb-12 and Bo, and L. brevis strain LMG 6906 exhibited the highest viable cell numbers when exposed to bile salts, whereas the other strains had variable death rates.
Keywords: Gastric acid; Bile salts; Dairy products; Lactobacilli; Bifidobacteria
1. Introduction
An increasing number of dairy foods that carry a variety of health claims are appearing on the world food market. Some of these health claims pertain to probiotic properties when the foods contain microorganisms that have therapeutic properties—including antimicrobial activity, hypocholesterolaemic activity, improved lac-tose utilisation, maintenance of gastrointestinal balance and anticarcinogenic activity (Gomes & Malcata, 1999). The bacterial strains that are claimed to be probiotics typically belong to the genera Lactobacillus and Bifidobacterium.
Ice cream(Hekmat & McMahon, 1992), yoghurt (Reuter, 1990; Laroia & Martin, 1991) and cheese
(Dinakar & Mistry, 1994;Gobbetti, Corsetti, Smacchi, Zocchetti, & de Angelkis, 1998; Gomes & Malcata, 1998;O’Riordan & Fitzgerald, 1998) have been found to be carriers of probiotic organisms; another potential food vector is whey chees—on which, unfortunately, very little (if any) research has been performed to date. Whey is the aqueous portion of milk that is obtained following acid- or rennet-driven coagulation (i.e. pre-cipitation of caseins) in cheesemaking, and is still disposed of in significant overall volumes, especially by small dairy industries, to public sewage systems ( Pinta-do, MacePinta-do, & Malcata, 2001). Requeija˜o, a Portuguese whey cheese, is a dairy product that uses whey proteins. The manufacturing process consists basically of heat processing, aimed at denaturing the whey proteins; this is an economical alternative for small dairy industries, for which the implementation of membrane- or chro-matography-based protein separation and recovery technologies is extremely difficult and expensive.
Corresponding author. Tel.: +351 22 5580004; fax: +351 22 5090351.
Madureira et al. (2003)recently evaluated the viability of probiotic bacterial strains of the Lactobacillus and Bifidobacterium genera in whey cheese matrices; in all cases, viability (at approximately 107cfu g–1) was main-tained for 28 days of refrigerated storage, i.e. well above the threshold that is necessary for a probiotic benefit upon ingestion (Ishibashi & Shimamura, 1993). Never-theless, to ensure that such a benefit will be experienced by the human host, the strains need to survive passage through the gastrointestinal tract—and should also be able to adhere to and colonise the intestine (Havenaar, Brink, & Huis in’t Veld, 1992). This requirement is a major challenge for the bacterial strains, because they are exposed to gastric juice, digestive enzymes and bile salts in the gastrointestinal tract.
This study evaluated the survival of strains of Lactobacillus acidophilus, Lactobacillus paracasei, Bifi-dobacterium animalis and Lactobacillus brevis in a whey cheese model that was exposed to conditions that are typically encountered in the gastrointestinal tract, viz. simulated gastric juice (with a low pH and containing pepsin), followed by bile salts (normally secreted into the duodenum).
The microorganisms used in this study (and their sources) are listed in Table 1. To prepare a suitable inoculumfor incorporation into Requeija˜o cheese, the bacteria were grown in MRS broth (Merck, Darmstadt, Germany) and then cultured twice in skim milk incubated at 37 1C (or at 30 1C for L. brevis), for 48 h in both cases. The media used to grow the B. animalis and L. acidophilus strains were supplemented with 0.05% (w/v) cysteine-HCl (Merck) to lower the redox potential (which, although not inevitable, enhances growth substantially).
Bovine sweet whey (obtained as a by-product of the manufacture of a full-fat cheese) and milk used in the production of experimental Requeija˜o cheese were provided by Lacticı´nios das Marinhas (Esposende, Portugal); upon arrival, both feedstocks were immedi-ately refrigerated and were stored at 7 1C.
Seven batches of whey cheese were manufactured in parallel. Production followed the traditional procedure: bovine whey and raw milk (4:1, v/v) were added to a pan, and the mixture was gradually heated to 90 1C with gentle manual stirring, and kept at that temperature for 20 min; the resulting, floating curd was collected from the surface with a sterile skimmer, poured into sterile plastic moulds—which were covered immediately (to guarantee near aseptic conditions), and left to drain under a constant force (approximately 20 N) for 20 min at roomtemperature.
The whey cheeses were aseptically inoculated with bacterial inoculumprepared in skimmilk, as described above, at 10% (v/w) and were subsequently homo-genised in sterile bags using a Stomacher Lab Blender 400 for 5 min at 260 rev min–1 (Seward Medical, London, UK).
The inoculated whey cheeses at 10% (w/v), using sterile 0.85% (w/v) saline water as the dilution medium (Merck), were homogenised for 5 min at 260 rev min–1in the Stomacher; the resulting homogenates were trans-ferred to sterile flasks. Eight homogenates were prepared for each probiotic bacterial strain; two were used as controls, with no pH adjustment and no addition of pepsin or bile salts. The pH of the remaining six homogenates was adjusted to approximately 2.5–3.0 with sterile, 1M hydrochloric acid. Pepsin solution
was filter sterilised using a 0.22 mm membrane filter (Millipore, Billerica, MA, USA) and was added to the six homogenates to a final concentration of 1000 units mL–1. These homogenates were incubated as follows: two were incubated for 120 min (to simulate exposure to gastric juice); two were incubated for 60 min under gastric conditions, then a solution of filter-sterilised 0.3% (w/v) bile salts (Becton Dickinson, Sparks, MD, USA) was added and the homogenates were incubated for a further 120 min (to simulate conditions prevailing in the stomach and the duode-num); two were incubated for 120 min under gastric conditions, then the bile salts were added and the homogenates were incubated for a further 120 min (to simulate a slower gastric transit followed by a typical duodenumtransit). The homogenates were incubated at 371C. All assays were performed in duplicate for each bacterial strain.
Table 1
Strains selected for the study and their source
Strain Source B. animalis BLC-1 DELVO-PROs DSM (Moorebank, Australia) L. acidophilus LAC-1 L. paracasei ssp. paracasei LCS-1
B. animalis Bo CSK—frozen concentrates (Leeuwarden, The Netherlands) L. acidophilus Ki
L. brevis LMG 6906 Laboratoriumvoor Microbiologie en Microbiele Genetica—Rijksuniversiteit (Gent, Belgium)
B. animalis Bb-12 Christian Hansen (Hoersholm, Denmark)
Manufacture of whey cheeses
Microorganisms Materials and methods
Viable cell counts were determined by preparing serial decimal dilutions in 0.1% (w/v) peptone water (Sigma, St. Louis, MO, USA), which were subsequently plated (in duplicate) on MRS agar (Merck), using theMiles & Misra Method (1938), and incubated at 371C for 48 h. For the B. animalis and L. acidophilus strains, the MRS agar (Merck) was supplemented with 0.05% (w/v) cysteine-HCl and was incubated under anaerobic con-ditions (Gas-Pak Plus system, from Becton Dickinson, Maryland, MA, USA) for 48 h at 37 1C (30 1C for L. brevis). Thus, the total number of replicates of viable counts for each experimental condition was four. The pH of each homogenate was measured after 0, 60 and 120 min of incubation, and after 240 min for the control samples.
Death rates were calculated using linear regression of log (viable numbers) versus time data, at the two distinct phases of the experimental set-up, viz. exposure to artificial gastric juice alone (using all data encompassing the 60- and 120-min periods) and exposure to artificial gastric juice plus bile salts (using all data encompassing the subsequent periods, after correcting for the time before adding the bile salts).
One-way analysis of variance (ANOVA) was carried out using SPSS v. 11.5 (SPSS, Chicago, IL, USA) to quantify the effects of the parameters bacterial strain and incubation time on the viable counts, when exposed to artificial gastric juice and to artificial gastric juice plus bile salts. The least significant difference (LSD t-test), at the 5% significance level, was applied to all experimental data, to assess statistically significant differences among all possible combinations of parameters.
The survival of each probiotic bacterial strain in the gastrointestinal tract model systems, and the death rates of cells exposed to gastric juice and to gastric juice plus bile salts are shown inFig. 1andTable 2, respectively. The bacterial strains exhibited different viability profiles upon exposure to the simulated stomach and duodenum contents. The results of the ANOVA are tabulated in Tables 3 and 4. As shown in Fig. 1, the probiotic bacterial strains in the control samples maintained their viable cell numbers throughout incubation.
Inspection ofTable 2suggests that all bacteria tested, except L. paracasei ssp. paracasei LCS-1 and B. animalis Bb-12 in descending order of significance, were resistant to the action of artificial gastric juice and essentially maintained their initial viable cell numbers—with negligible death rates after both 60 and 120 min of exposure. Of the resistant strains, B. animalis BLC-1 was the most resistant to artificial gastric juice, at every time of incubation up to 120 min (Fig. 1 and Table 2). Survival was strain-dependent; in particular, signifi-cantly different profiles were observed for B. animalis BLC-1, B. animalis Bb-12 and B. animalis Bo, and for L. acidophilus LAC-1 and L. acidophilus Ki. This observa-tion was confirmed by the high F-value associated with the strain effect (F ¼ 87:20; Po0:0001) and, for the Bifidobacterium species, by the different death rates (Tables 2 and 3). Lactobacillus brevis LMG 6906 and L. paracasei ssp. paracasei LCS-1 also exhibited different profiles fromthe remaining strains, as well as between themselves (Po0:05); in particular, L. paracasei ssp. paracasei LCS-1 had a reduction in viable cell numbers of approximately 2 log cycles and the highest death rate, as mentioned above (Table 2).
Table 4 indicates that the type of strain had a significant effect on the viability profiles (F ¼ 83:04; Po0:0001). The strains that were least affected were B. animalis Bo and L. brevis LMG 6906—which main-tained their viable cell counts when exposed to artificial gastric juice plus bile salts, with no statistically significant differences between them(P40:05); B. animalis Bb-12 behaved in a similar manner (Table 2). Lactobacillus paracasei ssp. paracasei and B. animalis BLC-1 showed similar trends upon addition of bile salts, viz. their viability was completely lost within 30 min of incubation—which was illustrated by the highest death rates among the bacterial strains investigated (see Fig. 1(iii) and (iv), and Table 2). Furthermore, it should be noted that these bacterial strains suffered a similar decrease in viability for cells pre-incubated in artificial gastric juice for 60 min and for 120 min. These results were confirmed by the ANOVA; the time of exposure significantly affected the viability profiles (F ¼ 9:35; Po0:0001).
Two death phases were apparent when L. acidophilus LAC-1 was pre-incubated in artificial gastric juice for 60 min: during the first 60 min of contact with bile salts, the viable cell counts decreased by approximately 2 log cycles, followed by an essentially complete loss of viability during the next 30 min of incubation (Fig. 1). When pre-incubated in artificial gastric juice for 120 min, the viable cell numbers initially decreased by
Sampling and microbiological analyses Resistance to artificial gastric juice
Mathematical analyses
Statistical analyses
Resistance to artificial gastric juice plus bile salts
approximately 3 log cycles within 90 min, and then lost the remaining viability within the subsequent 30 min of contact with bile salts (seeFig. 1(i)).
For L. acidophilus Ki, better results were obtained when the cells were pre-incubated in artificial gastric juice for only 60 min compared with 120 min. In the former case, the viable cell counts decreased by approximately 4 log cycles within the first 30 min of exposure to bile salts; this level was then retained until the end of the 120-min incubation period (seeFig. 1(ii)). In contrast, cultures that had been pre-incubated in artificial gastric juice for 120 min had no detectable viable cells within 60 min of incubation.
Bifidobacterium animalis Bo did not experience further significant changes when exposed to bile salts after 60 min of incubation in artificial gastric juice. However, cells that had been exposed to artificial gastric juice for 120 min decreased by approximately 1 log cycle within the first 30 min after addition of bile salts; the viable cell numbers then remained essentially constant until the end of the incubation period (see Fig. 1(v)). This bacterial strain was the most resistant to the addition of bile salts to the artificial gastric juice, with a death rate of only 0.0061 log(cfu g–1) m in–1.
Bifidobacterium animalis Bb-12 inoculated into whey cheese underwent a decrease of approximately 1.5 log
0 1 2 3 4 5 6 7 8 9 0 210 240 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 0 1 2 3 4 5 6 7 8 9 BS BS 30 60 90 120 150 180 Time (min) Time (min) 0 30 60 90 120 150 180 210 240 Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers Log (cfu mL –1 ) Viable numbers 0 30 60 90 120 150 180 210 240 Time (min) 0 30 60 90 120 150 180 210 240 Time (min) (i) (iii) (iv) (v) (ii) Time (min) 0 30 60 90 120 150 180 210 240 Time (min) 0 30 60 90 120 150 180 210 240 Time (min) 0 30 60 90 120 150 180 210 240 (vi) (vii)
Fig. 1. Average (7standard deviation) viable cell numbers of (i) L. acidophilus strain LAC-1, (ii) L. acidophilus strain Ki, (iii) L. paracasei ssp. paracasei strain LCS-1, (iv) B. animalis strain BLC-1, (v) B. animalis strain Bo, (vi) B. animalis strain Bb-12 and (vii) L. brevis strain LMG 6906, when exposed to artificial gastric juice for 60 min and to 0.3% bile salts (BS) for 120 min (J), and to artificial gastric juice for 120 min and to 0.3% BS for 120 min (K). Control samples, held for 240 min, are also shown (&).
cycles within 60 min when pre-incubated for 60 min in artificial gastric juice; the viable cell numbers were maintained thereafter. However, when the cells were pre-incubated in artificial gastric juice for 120 min, there was a decrease of approximately 1.5 log cycles within the
first 30 min after addition of bile salts, followed by an increase of approximately 1 log cycle in the next 30 min; finally, the viable cell numbers decreased a further 0.5 log cycle over the remaining 60-min period (seeFig. 1(vi)).
As mentioned above, L. brevis LMG 6906 did not undergo a significant change when exposed to bile salts after pre-incubation for 60 or 120 min in artificial gastric juice; there was a two-fold greater effect in the latter case than the former, with an approximately 2 log cycles decrease in viable cell numbers (see Fig. 1(vii)); B. animalis Bo showed good resistance to bile salts, with a death rate of 0.0061 log(cfu g–1) m in–1(seeTable 2).
An important factor that affects the survival of probiotic bacterial strains in food is pH; survival is constrained at low pH values. Hence, whey cheese should be a good food carrier for probiotic strains because it has a pH in the range 6.0–6.5, and never lower than 4.5 even after 28 days of storage; good viability profiles of some of the strains investigated in this work have been shown previously (Madureira et al., 2003).
Before reaching the intestine, probiotic bacteria must first survive the deleterious action of gastric juice during passage through the stomach. In general, the acid tolerance of lactic acid bacteria depends on the pH profile of H+-ATPase and on the composition of the cytoplasmic membrane—which is largely influenced by the type of bacterium, the type of growth medium and the incubation conditions (Hood & Zotolla, 1988).
A few Bifidobacterium species have been shown to be less acid-resistant than lactobacilli (Dunne et al., 2001). The three B. animalis strains (i.e. BLC-1, Bo and Bb-12) examined in this study were all resistant to artificial gastric juice, because they were able to maintain their initial viable cell numbers throughout the various incubation periods. Nevertheless, different behaviours have also been observed (Berrada, Lemeland, Laroche, Thouvenot, & Piaia, 1991) for a variety of Bifidobacter-ium species; for example, the viable cell counts of one strain decreased by 0.5 log cycle when exposed to stomach-like acidity (i.e. pH 3), whereas those of another strain decreased by 4 log cycles.
The acid resistance of L. acidophilus strains is well documented. Hood and Zotolla (1988) found that L. acidophilus could survive at a pH as low as 4, and that such pH values did not affect the ability of the organismto attach to human intestinal cells. Further-more, L. acidophilus strains plated on MRS agar at pH 2.5 containing pepsin (1000 units mL–1) and incubated for 180 min underwent a decline of about 1 log cycle within 120 min (Oh, Kim, & Worobo, 2000).
Table 3
ANOVA of the effects of various parameters on the viable counts of probiotic bacterial strains when exposed to artificial gastric juice
Source Number of
degrees of freedom
Mean square F-ratio
Bacteria 6 4.71 87.20a Time 4 0.65 12.06a Bacteria * time 24 0.60 11.08a Error 35 0.05 Total 70 Corrected total 69 aSignificant at 0.01%. Table 4
ANOVA of the effects of various parameters on the viable counts of probiotic bacterial strains when exposed to artificial gastric juice (GJ) plus bile salts (BS)
Source Number of
degrees of freedom
Mean square F-ratio
Time 4 18.44 9.35a Conditionc 1 2.32 1.18b Bacteria 6 163.80 83.04a Time * conditionc 4 2.99 1.52b Time * bacteria 24 6.19 3.14a Conditionc* bacteria 6 2.44 1.24b Error 70 1.97 Total 140 Corrected total 139 a Significant at 0.01%. b Not significant. c Condition—exposure to GJ/BS. Table 2
Estimated death rates (7 standard deviation) for the observed declines in viable counts of probiotic bacterial strains exposed to artificial gastric juice (GJ) and artificial gastric juice plus bile salts (GJ/BS) Strain Death rate (log(cfu g–1) m in–1)
GJ GJ/BS L. acidophilus LAC-1 0.001770.0013 0.064270.0908 L. acidophilus Ki 0.001070.0010 0.051470.0741 L. paracasei ssp. paracasei LCS-1 0.029370.0006 0.187370.0351 B. animalis BLC-1 0.003070.0010 0.222170.0004 B. animalis Bo 0.000970.0010 0.006170.0040 B. animalis Bb-12 0.012270.0019 0.011970.0004 L. brevis LMG 6906 0.000370.0027 0.011170.0050 Discussion
Lactobacillus paracasei ssp. paracasei was the only strain that underwent a significant decrease in viable cell numbers (about 2 log cycles relative to the initial concentration) during exposure to artificial gastric juice. Vinderola, Prosello, Ghilberto, and Reinheimer (2000) observed a decrease in viability of about 4 log cycles within 180 min when using homogenates of Argentinian Fresco cheese inoculated with a similar strain, viz. L. casei, and acidified to pH 2; at pH 3, this bacterial strain decreased by only 1 log cycle.Jacobsen et al. (1999)also observed loss of viability in MRS at pH 2 for L. paracasei ssp. paracasei fromtwo sources (a human clinical isolate—strain 19058-4, and dairy strains— CHCC 2166, 3981, 3982 and 3740). Conversely,Morelli et al. (2003)obtained an increase of at least 1 log cycle over the intake period (15 days) for faecal isolates of L. paracasei.
There has been little work on L. brevis.Ro¨nka¨ et al. (2003) showed that strains of L. brevis (GRL1 and GRL62) suffered a decrease in their viable cell numbers when inoculated in MRS acidified to pH 2.0. In the same work, there was no effect on the viability when L. brevis was inoculated in MRS broth supplemented with 0.3% (w/v) bile salts. This was opposite to the current study, in which the cells were more sensitive to the addition of bile salts than to acid. Thus, the reduction in cell numbers when bile salts were added after 120 min of exposure to artificial gastric juice may have been due to weak resistance to acid rather than to the bile salts themselves.
The current study was based on an original ap-proach—that combined the effect of exposure to gastric juice, followed by the effect of exposure to bile salts on the viability of probiotic strains incorporated in a food matrix. After 60 or 120 min of exposure to artificial gastric juice, 0.3% (w/v) bile salts was added to the homogenates and the incubation was extended for a further 2 h. This approach simulated the two situations that prevail during transit through the gastrointestinal tract: passage through the stomach, followed by release of bile salts in the small intestine.
In terms of survival, the best results were obtained for B. animalis Bo, B. animalis Bb-12 and L. brevis; all three strains resisted the action of bile salts well, irrespective of the time of pre-incubation with artificial gastric juice. Artificial gastric juice had an adverse effect on the survival of L. paracasei ssp. paracasei LCS-1, which was intensified by the addition of bile salts. For B. animalis BLC-1, addition of bile salts led to immediate cell death. The protective effect of food as a vector for probiotic strains is of crucial importance.Kos, Suskovic, Goreta, and Matosic (2000) studied the effect of whey protein concentrate (WPC) on the viability of L. acidophilus M92, and found that addition to WPC protected the cells fromthe low pH of simulated gastric juice, and even increased their tolerance to higher concentrations
of bile salts. In typical studies (Jacobsen et al., 1999;Oh et al., 2000), the resistance to bile salts has been investigated independently of the resistance to gastric juice and in the absence of a protection system; in this type of study, almost all L. acidophilus strains resist exposure to 0.3% (w/v) bile salts. In a more recent study, Cebeci and Gu¨rakan (2003) evaluated the two effects simultaneously and concluded that they caused the death of a strain, L. plantarum. In the current study, no conclusions on the protective effect of whey cheese on the strains examined can be drawn, because there was no control comparison in the absence of whey cheese. However, it should be pointed out that the main objective was to screen for the viability of these strains when inoculated into whey cheese and exposed to conditions that prevail in the gastrointestinal tract, because the viability of these bacteria in whey cheese has already been demonstrated (Madureira et al., 2003).
The results obtained in this study suggest that the resistance of probiotic strains to passage through the gastrointestinal tract, when delivered in a whey cheese matrix, is strain-dependent. The best viability profiles throughout the period of exposure to the combination of artificial gastric juice and bile salts were obtained for L. brevis, B. animalis Bo and B. animalis Bb-12. Lactobacillus acidophilus LAC-1 and L acidophilus Ki resisted exposure to artificial gastric juice, but showed different behaviour in the presence of bile salts—the decrease in their viable cell numbers may compromise viability. Lactobacillus paracasei ssp. paracasei LCS-1 and B. animalis BLC-1 were the least resistant to artificial gastric juice plus bile salts.
Several parameters may determine the extent to which probiotic strains survive passage through the upper gastrointestinal tract, viz., the degree of stomach acidity and the period of exposure, as well as the concentration and the period of exposure to bile salts. However, in vivo studies are still necessary to fully validate all previous in vitro studies (including our study), viz., microbiological analyses of faecal samples after feeding of the inoculated products are required, as other factors (e.g. pancreatic juice) also play a role.
Partial funding of this research work was provided via project ‘‘PROCHEESE—probiotic cheese fromwhey with in situ-generated exopolysaccharide’’ (POCTI/ 1999/AGR/36163), administered by Fundac-a˜o para a Cieˆncia e Tecnologia (FCT, Portugal), and project PROBIOSORO, administered by ADI: Ageˆncia de
Conclusions
Inovac-a˜o—POCTI: Progama Operacional de Cieˆncia, Tecnologia e Inovac-a˜o. The authors are indebted to the technical support provided by Ms. Ana Ferreira and Ms. Joana Carvalho.
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