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Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley
International Journal of Food Microbiology 91 (2004) 305 – 313 www.elsevier.com/locate/ijfoodmicro
Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley Merete H. Helland *, Trude Wicklund, Judith A. Narvhus Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432 Aas, Norway Received 21 October 2002; received in revised form 27 June 2003; accepted 16 July 2003
Abstract A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g 1 in the porridge at 0 h. The porridge was fermented for 24 h at 37 jC, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g 1. Most strains reached maximum cell count after 12-h fermentation (7.2 – 8.2 log cfu g 1), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg 1. Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg 1) and acetoin (27 mg kg 1) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose. D 2003 Elsevier B.V. All rights reserved. Keywords: Probiotic bacteria; Maize; Metabolism; Fermentation
1. Introduction Maize is the principal source of food for millions of people, particularly in Latin America and Africa (Yousif and El Tinay, 2000). It is non-allergenic and mild in taste, and is therefore suitable for use as a
* Corresponding author. Tel.: +47-64-94-85-88; fax: +47-6494-37-89. E-mail address: [email protected] (M.H. Helland). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.07.007
basis for weaning food. A weaning food is a semisolid food to be used in addition to breast milk (Milla, 1986). Probiotics (meaning ‘‘for life’’) are live microbial feed or food supplements, which beneficially affect the host by improving its intestinal microbial balance (Fuller, 1989). The first recorded probiotics were fermented milks produced for human consumption (Fuller, 1994). Since then, probiotics have been increasingly included in both fermented and unfermented products (Salovaara, 1998; Saarela et al., 2000).
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This has again lead to an increased focus on the documentation of health effects and safety (O’Brien et al., 1999; Hammes and Hertel, 2002). For functionality of probiotics, it is thought that at least 108 – 109 live bacteria should reach the small intestine daily (Sanders and Huis in’t Veld, 1999). Lactic acid bacteria, the bacterial group to which most probiotic cultures belong, convert fermentable sugars into lactic acid, ethanol and other metabolites, thus lowering the pH and creating unfavourable conditions for the growth of potentially pathogenic microorganisms. Diarrhoeal diseases transmitted by weaning foods are believed to contribute to the high prevalence of diarrhoea in developing countries. The addition of probiotic strains to locally made fermented food would increase the shelf-life, palatability, safety (Motarjemi and Nout, 1996; Hammes and Hertel, 2002) and nutritional value of cereals (Chavan and Kadam, 1989) and, in addition, their probiotic properties would hopefully reduce the incidence of foodrelated diarrhoea. This article investigates the growth and metabolism of probiotics in maize weaning porridge with high energy density, low viscosity and good taste. In addition, the effect of using two levels of inoculum of probiotic cultures was assessed in relation to the cell count, pH and metabolites produced during fermentation.
2. Materials and methods 2.1. Probiotic strains The following probiotic strains were selected: Lactobacillus reuteri SD 2112 from BioGaia Biologics, Stockholm, Sweden, Lb. acidophilus LA5 from Christian Hansen, Denmark, Lb. acidophilus NCDO 1748 from the National Collection of Food Bacteria, Reading, England and Lb. rhamnosus GG (ATCC 53103) from Valio, Helsinki, Finland.
37 jC for 24 h. For the concentrate preparation, the cultures were grown in 1000 ml MRS broth and the cell mass was harvested by centrifugation at 14,000 g for 10 min (Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont Instruments). After centrifugation, the cell pellets were washed once in 0.05 M potassium phosphate buffer (pH 7.0), before being centrifuged once more. The pellets were then resuspended in 100 ml UHT milk (1.5% fat, TINE Norwegian Dairies, Oslo, Norway), distributed into sterile tubes, and stored at 80 jC until required. The bacterial counts in the frozen cultures were estimated by plating on MRS-agar (Merck), after incubating anaerobically for 3 days at 37 jC (GasPakPlus System, Dickinson, Cockeysville, MD, US). The necessary inoculum to give approx. 6 and 7 log cfu g 1 in the maize porridge after inoculation was calculated. 2.3. Porridge preparation Porridge was prepared from 18.5% (dm) maize flour (Champagne maize flour, France, obtained from Cerealia, Norway) and 1.5% (dm) malted barley (Ringnes, Norway), giving a dry matter content of 20% (w/v) (Helland et al., 2002). The barley was ground using a laboratory mill (Falling number AB, No. 68488, type 120, 2800 rpm, Koneteollisuus Oy Helsinki, Finland) with a screen size of 0.5 mm. Flour was weighed and mixed in glass jars, before the addition of distilled water to a final weight of 300 g. The flour and water were mixed and kept for a respite period of 1 h at 40 jC, to allow the amylase to break down starch, before boiling in a steam bath for another hour in order to gelatinise the remaining starch. During the respite period and the boiling step, the samples were stirred every 15 min. After cooking, the porridge was autoclaved at 121 jC for 15 min. The porridge was then cooled down and inoculated with calculated amounts of suspensions of probiotic strains (to give approx. 6 or 7 log cfu g 1 in the porridge after inoculation).
2.2. Preparation of probiotic starter culture inocula 2.4. Analyses Concentrated frozen cultures were prepared in the following way: The cells were routinely propagated on 2 successive days, by 1% inoculation in MRSbroth (Merck, Darmstadt, Germany) and incubated at
Samples were taken every 4 h, until 24 h of fermentation (four strains and two different inoculation amounts). All results are an average of three
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replicates. Statistical analyses were carried out using SAS (SAS Institute, Cary, USA). Tukey’s Studentized Range Test was used to further examine any significant differences ( P < 0.05) between the results.
After fermenting for 24 h, porridge inoculated with Lb. rhamnosus GG or Lb. reuteri contained significantly higher viable counts than porridge inoculated with Lb. acidophilus LA5 or Lb. acidophilus 1748.
2.4.1. Bacteriological Samples were diluted in peptone – saline water (0.9%, w/v saline; 0.1%, w/v peptone). Porridge (0.1 g) was weighed into 9.9 ml peptone –saline water and diluted until proper dilution. Bacterial counts were determined (pour plate) on MRS-agar (Merck), after incubating anaerobically for 3 days at 37 jC (GasPakPlus System).
3.2. pH
2.4.2. Chemical The concentrations of organic acids, volatile organic compounds (voc.) and sugars (maltose, glucose and fructose) in the samples, were determined according to methods previously described by Narvhus et al. (1998) and Gadaga et al. (2001). PH was measured during fermentation using a Radiometer pH meter, (PHM 92, Lab pH Meter, Copenhagen) with a combined glass electrode and temperature probe, which was calibrated using buffers of pH 4.01 and 7.00 (Merck).
3. Results The results are presented according to changes in viable counts, pH, organic acids, voc. and sugars during a 24-h fermentation period. Unless otherwise stated, significant results refer to P < 0.05. 3.1. Viable counts All strains investigated showed good growth at both inoculation levels in maize porridge at 37 jC, reaching maximum populations of 7.2 –8.2 log cfu g 1 in 12 h (Fig. 1a). For porridge inoculated with Lb. acidophilus LA5 or Lb. rhamnosus GG, no significant difference was observed in the cell count after 12h fermentation, despite differences in the inoculation rate. After 20 h, it was not possible to detect any significant difference in the viable counts of Lb. acidophilus 1748. For Lb. reuteri, significant differences in the viable counts, due to the inoculation rate, were observed throughout the fermentation period.
The pH dropped from 5.8 to 3.1– 3.7 during the fermentation period, (Fig. 1b). After 12 h, the pH was reduced below 4 for all strains, except Lb. acidophilus 1748 inoculated to give approx. 6 log cfu g 1 at inoculation (pH 4.4). During the first 12 h, reduction of pH was significantly greater for most samples inoculated at approx. 7 log cfu g 1, but no differences were observed between high inoculation rate of Lb. acidophilus LA5 and low inoculation rate of Lb. rhamnosus GG, or between high inoculation rate of Lb. acidophilus 1748 and low inoculation rate of Lb. rhamnosus GG and Lb. reuteri. The fastest and greatest reduction was seen for Lb. rhamnosus GG ( P < 0.05). 3.3. Organic acids For most organic acids analysed, small differences in production/reduction of organic acids were seen and could be attributed both due to the different strains used and to differences in viable counts at inoculation. The different strains produced lactic acid in amounts ranging from 1360 to 4000 mg kg 1. The largest production was seen as a result of growth of Lb. rhamnosus GG (Fig. 2a). During the first 12 –16 h of fermentation, significantly less lactic acid was produced in porridge inoculated with approx. 6 log cfu g 1, compared to porridge inoculated with approx. 7 log cfu g 1. All strains were able to metabolise citric acid when growing in maize porridge (Fig. 2b). Lb. reuteri was shown to be very active, being able to break down all citric acid during the first 4 –8 h. No citric acid was detected after 12 –16 h in porridge inoculated with Lb. acidophilus LA5 or Lb. acidophilus 1748. Growth of Lb. rhamnosus GG in maize porridge was characterised by only partial utilisation of citric acid, which was reduced from 35 to 17 –20 mg kg 1. The concentration of succinic acid and pyruvate were also monitored during fermentation (results not
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Fig. 1. Viable counts (a) and changes in pH (b) in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( – x – ), Lb. reuteri, LR (- -.- -), ( – . – ) and Lb. acidophilus 1748 (- -E- -), ( – E – ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
shown). For most strains, only small changes were detected in the concentrations of succinic acid during the fermentation period. Only Lb. reuteri utilised most of the succinic acid present in the maize porridge, with a reduction of approx. 560 mg kg 1. Lb. rhamnosus GG utilised approx. 250 mg kg 1, while Lb. acidophilus LA5 and Lb. acidophilus 1748, utilised approx. 140 mg kg 1 and 60 mg kg 1 succinic acid, respectively. In maize porridge inoculated with Lb. reuteri or Lb. rhamnosus GG, pyruvic acid concentration did not change.
In porridge inoculated with Lb. acidophilus LA5, a small increase (approx. 10 mg kg 1) in the amount of pyruvic acid was observed after 8 to 12 h (depending on the inoculation amount), followed by a decrease (approx. 11 mg kg 1). Lb. acidophilus 1748 produced the highest amounts of pyruvic acid and significant differences attributable to the viable counts at time zero were observed after fermenting for 8 –16 h and after 24-h fermentation. After fermenting for 8 –16 h, the concentration of pyruvic acid detected in maize porridge inoculated with approx. 7
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Fig. 2. Changes in the levels of (a) lactic acid and (b) citric acid in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( x ), Lb. reuteri, LR (- -.- -), ( . ) and Lb. acidophilus 1748 (- -E- -), ( E ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
log cfu g 1 of Lb. acidophilus 1748 was significantly higher than in porridge inoculated with the respective low inoculation rate. 3.4. Volatile compounds Less than 2 mg kg 1 acetaldehyde was present in the maize porridge. This amount constantly increased in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, but decreased in porridge inoculated with Lb. reuteri and Lb. rhamnosus GG (Fig. 3a). For Lb. acidophilus LA5 and Lb. acid-
ophilus 1748, significantly higher concentrations of acetaldehyde were seen in porridge with the highest rate of inoculation. In porridge inoculated with approx. 7 log cfu g 1 of Lb. acidophilus LA5, about 7.5 mg kg 1 acetaldehyde was detected, while porridge inoculated with approx. 6 log cfu g 1 of Lb. acidophilus LA5 contained about 5.7 mg kg 1 acetaldehyde after 24-h fermentation. The concentration of ethanol, diacetyl and acetoin was also monitored during fermentation (results not shown). Lb. reuteri produced large amounts of ethanol and the production was independent of the inoculation
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rate after fermenting for 20 and 24 h ( P>0.05), but Lb. reuteri inoculated at a high rate showed a significantly higher production than Lb. reuteri inoculated at a low rate, between 4 and 16 h. The mean concentrations of ethanol in porridge inoculated with high and low inoculation rate of Lb. reuteri were 1160 and 1040 mg kg 1, respectively, after 24-h fermentation. Porridge inoculated with Lb. rhamnosus GG contained 8 –9 mg kg 1 ethanol after 24-h fermentation, with no significant difference detected between high and low inoculation rate of Lb. rhamnosus GG. Only Lb. rhamnosus GG produced high concentrations of diacetyl in the maize porridge, producing approx. 6.3 mg kg 1 during 12-h fermentation. The concentration of diacetyl in porridge inoculated with Lb. acidophilus LA5 was about 0.2 mg kg 1. Diacetyl concentration in porridge inoculated with Lb. acidophilus 1748 was dependent upon the rate of inoculation, producing 1 – 1.5 mg kg 1 diacetyl, with significantly higher concentrations of diacetyl after 12 – 20-h fermentation in porridge inoculated with approx. 7 log cfu g 1, but with no significant difference after 24-h fermentation. For Lb. rhamnosus GG, the concentrations of diacetyl were independent of the viable counts at inoculation ( P>0.05), except for concentrations detected after fermenting for 4 h ( P < 0.05). Lb. rhamnosus GG was also the only strain producing acetoin in maize porridge, with levels ranging from 23 to 28 mg kg 1 after 12-h fermentation, depending on the inoculation level. No significant differences were found between the inoculation levels, except for concentrations detected after 8 h fermentation. 3.5. Maltose, fructose and glucose (RI) The concentration of maltose in maize porridge was between 11,000 and 13,000 mg kg 1 (results not shown). Lb. reuteri was the only strain able to metabolise maltose, and levels were reduced by about 4800 and 2600 mg kg 1 for high and low inoculation rate, respectively.
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Glucose was reduced by all four cultures, with Lb. rhamnosus GG being the most active (Fig. 3b). In porridge inoculated with Lb. rhamnosus GG, glucose was reduced by approx. 2700 mg kg 1 after 24 h, from a mean initial concentration of 3000 mg kg 1. Lb. acidophilus 1748 was the least active strain. Depending on the inoculation level, glucose was reduced by 570 and 810 mg kg 1, during the fermentation period. Only small reductions in the initial fructose concentration (600 – 1000 mg kg 1) were observed (Fig. 3c). Lb. reuteri did not utilise fructose, and the largest reduction (360 mg kg 1) was seen in porridge inoculated with Lb. acidophilus LA5. Changes in levels of fructose were independent upon the inoculation level ( P>0.05), until after 12-h fermentation ( P < 0.05).
4. Discussion Several criteria, such as viability, technological suitability, competitiveness, safety, and functionality are important when selecting appropriate probiotic strains for a fermented product (Klaenhammer and Kullen, 1999). Microorganisms alter the taste of food positively or negatively by producing metabolic byproducts such as lactic acid, alcohol, acetic acid, CO2, and diacetyl. In this study, the viable count on inoculation of the starter culture appeared to be of great importance during the first hours of the fermentation period, since a delayed production of most metabolites was observed for all strains in porridge inoculated with approx. 6 log cfu g 1. All strains examined showed good growth in maize porridge with added barley malt. High viable counts are necessary to get the desired acid production and reduction in pH, which again will affect the product shelf-life. After 12 h of fermentation, the viable cell count was well above the suggested minimum limit of 6 log cfu g 1 for efficacy of a probiotic product (Vinderola et al., 2000).
Fig. 3. Changes in the levels of (a) acetaldehyde (b) glucose and (c) fructose in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( x ), Lb. reuteri, LR (- -.- -), ( . ) and Lb. acidophilus 1748 (- -E- -), ( E ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
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A pH below 3.5 has been reported to inhibit Enterobacteriaceae and other Gram-negative bacteria (Granum, 1999). It has also been shown that acid adaptation and increased resistance to subsequent acid stress can increase the survival time in acidic environment (as low as pH 3.0). This has been shown for food-borne pathogens such as Listeria (Hill et al., 1995) and Escherichia coli O157:H7 (Garren et al., 1997; Cheng et al., 2003). Although, in the lactic fermented milk product Yakult (pH 3.6), reduced survival of the acid-adapted E. coli O157:H7 was observed (Cheng and Chou, 2001). Extents of increased acid tolerance will also vary with the strain and the types of organic acids produced (Cheng et al., 2003). With inoculation amounts of approx. 7 log cfu g 1, all four strains reached a pH below 4.2 within 8 h of fermentation. After 12 h, the pH was found to be below 4.4 in porridges inoculated with approx. 6 log cfu g 1. After 20-h fermentation, differences in pH between different inoculation rates were not significant for strains Lb. reuteri, Lb. acidophilus LA5 and Lb. rhamnosus GG. This shows that if the objective is to reduce the pH below 4.4, the inoculation rate is of minor importance with respect to pH, provided that the porridge is fermented for 12 h or more. Fermentable carbohydrates are transformed into organic acids, mainly acetic and lactic acid and/or ethanol. Lb. reuteri was the only strain that utilised maltose as a carbohydrate source, but all strains reduced glucose and, to a certain extent, also fructose. It appears that Lb. rhamnosus GG preferentially utilised glucose as an easily metabolised carbohydrate source, shown by a reduction of 2470 – 3060 mg kg 1, depending on the inoculation rate. All strains produced lactic acid as a result of the metabolism of glucose, and the greatest production was seen for Lb. rhamnosus GG. Depending on inoculation rate, Lb. rhamnosus GG produced 3600– 4000 mg kg 1 lactic acid, which corresponds well with the amount of glucose and fructose reduced. Lb. rhamnosus GG metabolised almost all the glucose and also some of the fructose present in the porridge. Fructose reduction by high and low inoculation rate of Lb. rhamnosus GG was 402 and 217 mg kg 1, respectively. The high concentration of lactic acid produced resulted in these porridges reaching a pH as low as 3.1 after 20-h fermentation.
Pyruvate was only detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748 and may have originated from the fermentation of both glucose and citrate. Diacetyl is an important aromatic compound in several fermented products and is normally dependent upon the rate of citrate or altered pyruvate metabolism (Axelsson, 1998). Lb. rhamnosus GG produced 6 mg kg 1 diacetyl, which is high considering that 1 –2 mg kg 1 is usual in fermented milk and the reported taste threshold value for diacetyl is 0.03 mg kg 1 (Imhof et al., 1994). Some of the diacetyl produced was probably also reduced to acetoin, since detected concentrations of acetoin and diacetyl followed similar patterns throughout the fermentation period. Small amounts of diacetyl, but no acetoin, were detected in porridge inoculated with Lb. acidophilus 1748 and Lb. acidophilus LA5. The pathway for acetoin production, via a-acetolactate, could be inhibited in Lb. acidophilus in the presence of pyruvate. (Benito de Ca´rdenas et al., 1990). Succinate may be an end product when heterofermentative lactobacilli metabolise glucose and citrate (Axelsson, 1998). However, in this study, Lb. reuteri metabolised almost all succinic acid present in the porridge (560 mg kg 1) and no production was detected. Ethanol, but no acetaldehyde, was detected in porridge inoculated with Lb. rhamnosus GG and in porridge inoculated with Lb. reuteri, which might imply that these two strains possess active alcohol dehydrogenase. Heterofermentative Lb. reuteri produced large amounts of ethanol, which may be above the taste threshold for ethanol of 100 – 800 mg kg 1 (Imhof et al., 1994). Most ethanol is probably a result of the breakdown of glucose with the resultant production of lactate, ethanol and CO2 (Axelsson, 1998). Ethanol can also be produced from pyruvate by pyruvate formate lyase (Hugenholtz, 1993). Acetaldehyde is expected to accumulate in porridge inoculated with Lb. acidophilus, since its alcohol dehydrogenase activity is rather low (Imhof and Bosset, 1994). Lb. acidophilus can also produce acetaldehyde directly from pyruvate or some amino acids (Gonzalez et al., 1994). This agrees with our results, where both strains of Lb. acidophilus produced diacetyl and acetaldehyde. These results have shown the importance of the inoculation rate. Low inoculation rates could be
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favourable from an economical point of view, but depending on the strain, it may then be necessary to ferment the samples for a longer time period (at least 12 h). Inoculation rate will be even more important if the purpose is to compare the metabolites of strains in a certain time-interval, since the detected metabolites followed a defined profile depending on the rate of inoculation. Acknowledgements The authors gratefully acknowledge the expert technical help of Kari Olsen for the HSGC and HPLC analyses, and the gift of cultures from Ivan Casas, Biogaia Biologics (Lb. reuteri), Valio (Lb. rhamnosus GG), The National Collection of Food bacteria (Lb. acidophilus 1748). References Axelsson, L.T., 1998. Lactic acid bacteria; classification and physiology. In: Salminen, S., von Wright, A. (Eds.), Lactic Acid Bacteria. Marcel Dekker, New York, pp. 1 – 72. Benito de Ca´rdenas, I.L., Cerutti De Guglielmone, G., Lesdema, O., Oliver, G., 1990. Diacetyl and acetoin production from pyruvate by cell suspensions of lactobacilli. Milchwissenschaft 45, 775 – 777. Chavan, J.K., Kadam, S.S., 1989. Nutritional improvement of cereals by fermentation. Crit. Rev. Food Sci. Nutr. 28, 349 – 401. Cheng, H.-Y., Chou, C.-C., 2001. Acid adaptation and temperature effect on the survival of E. coli O157:H7 in acidic fruit juice and lactic fermented milk product. Int. J. Food Microbiol. 70, 189 – 195. Cheng, H.-Y., Yu, R.-C., Chou, C.-C., 2003. Increased acid tolerance of Escherichia coli O157:H7 as affected by acid adaptation time and conditions of acid challenge. Food Res. Int. 36, 49 – 56. Fuller, R., 1989. A review: probiotics in man and animals. J. Appl. Bacteriol. 66, 365 – 378. Fuller, R., 1994. Probiotics: an overview. In: Gibson, S.A.W. (Ed.), Human Health: The Contribution of Microorganisms. Springer, London, pp. 63 – 73. Gadaga, T.H., Mutukumira, A.N., Narvhus, J.A., 2001. Growth characteristics of Candida kefyr and two strains of Lactococcus lactis subsp. lactis isolated from Zimbabwean fermented milk. Int. J. Food Microbiol. 70, 11 – 19. Garren, D.M., Harrison, M.A., Russel, S.M., 1997. Retention of acid tolerance and acid schock responses of Escherichia coli O157:H7 and non-O157:H7 isolates. J. Food Prot. 60, 1478 – 1482.
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Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley Merete H. Helland *, Trude Wicklund, Judith A. Narvhus Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432 Aas, Norway Received 21 October 2002; received in revised form 27 June 2003; accepted 16 July 2003
Abstract A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g 1 in the porridge at 0 h. The porridge was fermented for 24 h at 37 jC, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g 1. Most strains reached maximum cell count after 12-h fermentation (7.2 – 8.2 log cfu g 1), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg 1. Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg 1) and acetoin (27 mg kg 1) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose. D 2003 Elsevier B.V. All rights reserved. Keywords: Probiotic bacteria; Maize; Metabolism; Fermentation
1. Introduction Maize is the principal source of food for millions of people, particularly in Latin America and Africa (Yousif and El Tinay, 2000). It is non-allergenic and mild in taste, and is therefore suitable for use as a
* Corresponding author. Tel.: +47-64-94-85-88; fax: +47-6494-37-89. E-mail address: [email protected] (M.H. Helland). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.07.007
basis for weaning food. A weaning food is a semisolid food to be used in addition to breast milk (Milla, 1986). Probiotics (meaning ‘‘for life’’) are live microbial feed or food supplements, which beneficially affect the host by improving its intestinal microbial balance (Fuller, 1989). The first recorded probiotics were fermented milks produced for human consumption (Fuller, 1994). Since then, probiotics have been increasingly included in both fermented and unfermented products (Salovaara, 1998; Saarela et al., 2000).
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This has again lead to an increased focus on the documentation of health effects and safety (O’Brien et al., 1999; Hammes and Hertel, 2002). For functionality of probiotics, it is thought that at least 108 – 109 live bacteria should reach the small intestine daily (Sanders and Huis in’t Veld, 1999). Lactic acid bacteria, the bacterial group to which most probiotic cultures belong, convert fermentable sugars into lactic acid, ethanol and other metabolites, thus lowering the pH and creating unfavourable conditions for the growth of potentially pathogenic microorganisms. Diarrhoeal diseases transmitted by weaning foods are believed to contribute to the high prevalence of diarrhoea in developing countries. The addition of probiotic strains to locally made fermented food would increase the shelf-life, palatability, safety (Motarjemi and Nout, 1996; Hammes and Hertel, 2002) and nutritional value of cereals (Chavan and Kadam, 1989) and, in addition, their probiotic properties would hopefully reduce the incidence of foodrelated diarrhoea. This article investigates the growth and metabolism of probiotics in maize weaning porridge with high energy density, low viscosity and good taste. In addition, the effect of using two levels of inoculum of probiotic cultures was assessed in relation to the cell count, pH and metabolites produced during fermentation.
2. Materials and methods 2.1. Probiotic strains The following probiotic strains were selected: Lactobacillus reuteri SD 2112 from BioGaia Biologics, Stockholm, Sweden, Lb. acidophilus LA5 from Christian Hansen, Denmark, Lb. acidophilus NCDO 1748 from the National Collection of Food Bacteria, Reading, England and Lb. rhamnosus GG (ATCC 53103) from Valio, Helsinki, Finland.
37 jC for 24 h. For the concentrate preparation, the cultures were grown in 1000 ml MRS broth and the cell mass was harvested by centrifugation at 14,000 g for 10 min (Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont Instruments). After centrifugation, the cell pellets were washed once in 0.05 M potassium phosphate buffer (pH 7.0), before being centrifuged once more. The pellets were then resuspended in 100 ml UHT milk (1.5% fat, TINE Norwegian Dairies, Oslo, Norway), distributed into sterile tubes, and stored at 80 jC until required. The bacterial counts in the frozen cultures were estimated by plating on MRS-agar (Merck), after incubating anaerobically for 3 days at 37 jC (GasPakPlus System, Dickinson, Cockeysville, MD, US). The necessary inoculum to give approx. 6 and 7 log cfu g 1 in the maize porridge after inoculation was calculated. 2.3. Porridge preparation Porridge was prepared from 18.5% (dm) maize flour (Champagne maize flour, France, obtained from Cerealia, Norway) and 1.5% (dm) malted barley (Ringnes, Norway), giving a dry matter content of 20% (w/v) (Helland et al., 2002). The barley was ground using a laboratory mill (Falling number AB, No. 68488, type 120, 2800 rpm, Koneteollisuus Oy Helsinki, Finland) with a screen size of 0.5 mm. Flour was weighed and mixed in glass jars, before the addition of distilled water to a final weight of 300 g. The flour and water were mixed and kept for a respite period of 1 h at 40 jC, to allow the amylase to break down starch, before boiling in a steam bath for another hour in order to gelatinise the remaining starch. During the respite period and the boiling step, the samples were stirred every 15 min. After cooking, the porridge was autoclaved at 121 jC for 15 min. The porridge was then cooled down and inoculated with calculated amounts of suspensions of probiotic strains (to give approx. 6 or 7 log cfu g 1 in the porridge after inoculation).
2.2. Preparation of probiotic starter culture inocula 2.4. Analyses Concentrated frozen cultures were prepared in the following way: The cells were routinely propagated on 2 successive days, by 1% inoculation in MRSbroth (Merck, Darmstadt, Germany) and incubated at
Samples were taken every 4 h, until 24 h of fermentation (four strains and two different inoculation amounts). All results are an average of three
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replicates. Statistical analyses were carried out using SAS (SAS Institute, Cary, USA). Tukey’s Studentized Range Test was used to further examine any significant differences ( P < 0.05) between the results.
After fermenting for 24 h, porridge inoculated with Lb. rhamnosus GG or Lb. reuteri contained significantly higher viable counts than porridge inoculated with Lb. acidophilus LA5 or Lb. acidophilus 1748.
2.4.1. Bacteriological Samples were diluted in peptone – saline water (0.9%, w/v saline; 0.1%, w/v peptone). Porridge (0.1 g) was weighed into 9.9 ml peptone –saline water and diluted until proper dilution. Bacterial counts were determined (pour plate) on MRS-agar (Merck), after incubating anaerobically for 3 days at 37 jC (GasPakPlus System).
3.2. pH
2.4.2. Chemical The concentrations of organic acids, volatile organic compounds (voc.) and sugars (maltose, glucose and fructose) in the samples, were determined according to methods previously described by Narvhus et al. (1998) and Gadaga et al. (2001). PH was measured during fermentation using a Radiometer pH meter, (PHM 92, Lab pH Meter, Copenhagen) with a combined glass electrode and temperature probe, which was calibrated using buffers of pH 4.01 and 7.00 (Merck).
3. Results The results are presented according to changes in viable counts, pH, organic acids, voc. and sugars during a 24-h fermentation period. Unless otherwise stated, significant results refer to P < 0.05. 3.1. Viable counts All strains investigated showed good growth at both inoculation levels in maize porridge at 37 jC, reaching maximum populations of 7.2 –8.2 log cfu g 1 in 12 h (Fig. 1a). For porridge inoculated with Lb. acidophilus LA5 or Lb. rhamnosus GG, no significant difference was observed in the cell count after 12h fermentation, despite differences in the inoculation rate. After 20 h, it was not possible to detect any significant difference in the viable counts of Lb. acidophilus 1748. For Lb. reuteri, significant differences in the viable counts, due to the inoculation rate, were observed throughout the fermentation period.
The pH dropped from 5.8 to 3.1– 3.7 during the fermentation period, (Fig. 1b). After 12 h, the pH was reduced below 4 for all strains, except Lb. acidophilus 1748 inoculated to give approx. 6 log cfu g 1 at inoculation (pH 4.4). During the first 12 h, reduction of pH was significantly greater for most samples inoculated at approx. 7 log cfu g 1, but no differences were observed between high inoculation rate of Lb. acidophilus LA5 and low inoculation rate of Lb. rhamnosus GG, or between high inoculation rate of Lb. acidophilus 1748 and low inoculation rate of Lb. rhamnosus GG and Lb. reuteri. The fastest and greatest reduction was seen for Lb. rhamnosus GG ( P < 0.05). 3.3. Organic acids For most organic acids analysed, small differences in production/reduction of organic acids were seen and could be attributed both due to the different strains used and to differences in viable counts at inoculation. The different strains produced lactic acid in amounts ranging from 1360 to 4000 mg kg 1. The largest production was seen as a result of growth of Lb. rhamnosus GG (Fig. 2a). During the first 12 –16 h of fermentation, significantly less lactic acid was produced in porridge inoculated with approx. 6 log cfu g 1, compared to porridge inoculated with approx. 7 log cfu g 1. All strains were able to metabolise citric acid when growing in maize porridge (Fig. 2b). Lb. reuteri was shown to be very active, being able to break down all citric acid during the first 4 –8 h. No citric acid was detected after 12 –16 h in porridge inoculated with Lb. acidophilus LA5 or Lb. acidophilus 1748. Growth of Lb. rhamnosus GG in maize porridge was characterised by only partial utilisation of citric acid, which was reduced from 35 to 17 –20 mg kg 1. The concentration of succinic acid and pyruvate were also monitored during fermentation (results not
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Fig. 1. Viable counts (a) and changes in pH (b) in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( – x – ), Lb. reuteri, LR (- -.- -), ( – . – ) and Lb. acidophilus 1748 (- -E- -), ( – E – ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
shown). For most strains, only small changes were detected in the concentrations of succinic acid during the fermentation period. Only Lb. reuteri utilised most of the succinic acid present in the maize porridge, with a reduction of approx. 560 mg kg 1. Lb. rhamnosus GG utilised approx. 250 mg kg 1, while Lb. acidophilus LA5 and Lb. acidophilus 1748, utilised approx. 140 mg kg 1 and 60 mg kg 1 succinic acid, respectively. In maize porridge inoculated with Lb. reuteri or Lb. rhamnosus GG, pyruvic acid concentration did not change.
In porridge inoculated with Lb. acidophilus LA5, a small increase (approx. 10 mg kg 1) in the amount of pyruvic acid was observed after 8 to 12 h (depending on the inoculation amount), followed by a decrease (approx. 11 mg kg 1). Lb. acidophilus 1748 produced the highest amounts of pyruvic acid and significant differences attributable to the viable counts at time zero were observed after fermenting for 8 –16 h and after 24-h fermentation. After fermenting for 8 –16 h, the concentration of pyruvic acid detected in maize porridge inoculated with approx. 7
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Fig. 2. Changes in the levels of (a) lactic acid and (b) citric acid in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( x ), Lb. reuteri, LR (- -.- -), ( . ) and Lb. acidophilus 1748 (- -E- -), ( E ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
log cfu g 1 of Lb. acidophilus 1748 was significantly higher than in porridge inoculated with the respective low inoculation rate. 3.4. Volatile compounds Less than 2 mg kg 1 acetaldehyde was present in the maize porridge. This amount constantly increased in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, but decreased in porridge inoculated with Lb. reuteri and Lb. rhamnosus GG (Fig. 3a). For Lb. acidophilus LA5 and Lb. acid-
ophilus 1748, significantly higher concentrations of acetaldehyde were seen in porridge with the highest rate of inoculation. In porridge inoculated with approx. 7 log cfu g 1 of Lb. acidophilus LA5, about 7.5 mg kg 1 acetaldehyde was detected, while porridge inoculated with approx. 6 log cfu g 1 of Lb. acidophilus LA5 contained about 5.7 mg kg 1 acetaldehyde after 24-h fermentation. The concentration of ethanol, diacetyl and acetoin was also monitored during fermentation (results not shown). Lb. reuteri produced large amounts of ethanol and the production was independent of the inoculation
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rate after fermenting for 20 and 24 h ( P>0.05), but Lb. reuteri inoculated at a high rate showed a significantly higher production than Lb. reuteri inoculated at a low rate, between 4 and 16 h. The mean concentrations of ethanol in porridge inoculated with high and low inoculation rate of Lb. reuteri were 1160 and 1040 mg kg 1, respectively, after 24-h fermentation. Porridge inoculated with Lb. rhamnosus GG contained 8 –9 mg kg 1 ethanol after 24-h fermentation, with no significant difference detected between high and low inoculation rate of Lb. rhamnosus GG. Only Lb. rhamnosus GG produced high concentrations of diacetyl in the maize porridge, producing approx. 6.3 mg kg 1 during 12-h fermentation. The concentration of diacetyl in porridge inoculated with Lb. acidophilus LA5 was about 0.2 mg kg 1. Diacetyl concentration in porridge inoculated with Lb. acidophilus 1748 was dependent upon the rate of inoculation, producing 1 – 1.5 mg kg 1 diacetyl, with significantly higher concentrations of diacetyl after 12 – 20-h fermentation in porridge inoculated with approx. 7 log cfu g 1, but with no significant difference after 24-h fermentation. For Lb. rhamnosus GG, the concentrations of diacetyl were independent of the viable counts at inoculation ( P>0.05), except for concentrations detected after fermenting for 4 h ( P < 0.05). Lb. rhamnosus GG was also the only strain producing acetoin in maize porridge, with levels ranging from 23 to 28 mg kg 1 after 12-h fermentation, depending on the inoculation level. No significant differences were found between the inoculation levels, except for concentrations detected after 8 h fermentation. 3.5. Maltose, fructose and glucose (RI) The concentration of maltose in maize porridge was between 11,000 and 13,000 mg kg 1 (results not shown). Lb. reuteri was the only strain able to metabolise maltose, and levels were reduced by about 4800 and 2600 mg kg 1 for high and low inoculation rate, respectively.
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Glucose was reduced by all four cultures, with Lb. rhamnosus GG being the most active (Fig. 3b). In porridge inoculated with Lb. rhamnosus GG, glucose was reduced by approx. 2700 mg kg 1 after 24 h, from a mean initial concentration of 3000 mg kg 1. Lb. acidophilus 1748 was the least active strain. Depending on the inoculation level, glucose was reduced by 570 and 810 mg kg 1, during the fermentation period. Only small reductions in the initial fructose concentration (600 – 1000 mg kg 1) were observed (Fig. 3c). Lb. reuteri did not utilise fructose, and the largest reduction (360 mg kg 1) was seen in porridge inoculated with Lb. acidophilus LA5. Changes in levels of fructose were independent upon the inoculation level ( P>0.05), until after 12-h fermentation ( P < 0.05).
4. Discussion Several criteria, such as viability, technological suitability, competitiveness, safety, and functionality are important when selecting appropriate probiotic strains for a fermented product (Klaenhammer and Kullen, 1999). Microorganisms alter the taste of food positively or negatively by producing metabolic byproducts such as lactic acid, alcohol, acetic acid, CO2, and diacetyl. In this study, the viable count on inoculation of the starter culture appeared to be of great importance during the first hours of the fermentation period, since a delayed production of most metabolites was observed for all strains in porridge inoculated with approx. 6 log cfu g 1. All strains examined showed good growth in maize porridge with added barley malt. High viable counts are necessary to get the desired acid production and reduction in pH, which again will affect the product shelf-life. After 12 h of fermentation, the viable cell count was well above the suggested minimum limit of 6 log cfu g 1 for efficacy of a probiotic product (Vinderola et al., 2000).
Fig. 3. Changes in the levels of (a) acetaldehyde (b) glucose and (c) fructose in maize porridge during fermentation at 37 jC with Lb. acidophilus LA5 (- -n- -), ( – n – ), Lb. rhamnosus GG (- -x- -), ( x ), Lb. reuteri, LR (- -.- -), ( . ) and Lb. acidophilus 1748 (- -E- -), ( E ). The porridge was inoculated to give approx. 6 log cfu g 1 (- - -) or approx. 7 log cfu g 1 (—) in the porridge after inoculation. Vertical lines represent standard deviations.
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A pH below 3.5 has been reported to inhibit Enterobacteriaceae and other Gram-negative bacteria (Granum, 1999). It has also been shown that acid adaptation and increased resistance to subsequent acid stress can increase the survival time in acidic environment (as low as pH 3.0). This has been shown for food-borne pathogens such as Listeria (Hill et al., 1995) and Escherichia coli O157:H7 (Garren et al., 1997; Cheng et al., 2003). Although, in the lactic fermented milk product Yakult (pH 3.6), reduced survival of the acid-adapted E. coli O157:H7 was observed (Cheng and Chou, 2001). Extents of increased acid tolerance will also vary with the strain and the types of organic acids produced (Cheng et al., 2003). With inoculation amounts of approx. 7 log cfu g 1, all four strains reached a pH below 4.2 within 8 h of fermentation. After 12 h, the pH was found to be below 4.4 in porridges inoculated with approx. 6 log cfu g 1. After 20-h fermentation, differences in pH between different inoculation rates were not significant for strains Lb. reuteri, Lb. acidophilus LA5 and Lb. rhamnosus GG. This shows that if the objective is to reduce the pH below 4.4, the inoculation rate is of minor importance with respect to pH, provided that the porridge is fermented for 12 h or more. Fermentable carbohydrates are transformed into organic acids, mainly acetic and lactic acid and/or ethanol. Lb. reuteri was the only strain that utilised maltose as a carbohydrate source, but all strains reduced glucose and, to a certain extent, also fructose. It appears that Lb. rhamnosus GG preferentially utilised glucose as an easily metabolised carbohydrate source, shown by a reduction of 2470 – 3060 mg kg 1, depending on the inoculation rate. All strains produced lactic acid as a result of the metabolism of glucose, and the greatest production was seen for Lb. rhamnosus GG. Depending on inoculation rate, Lb. rhamnosus GG produced 3600– 4000 mg kg 1 lactic acid, which corresponds well with the amount of glucose and fructose reduced. Lb. rhamnosus GG metabolised almost all the glucose and also some of the fructose present in the porridge. Fructose reduction by high and low inoculation rate of Lb. rhamnosus GG was 402 and 217 mg kg 1, respectively. The high concentration of lactic acid produced resulted in these porridges reaching a pH as low as 3.1 after 20-h fermentation.
Pyruvate was only detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748 and may have originated from the fermentation of both glucose and citrate. Diacetyl is an important aromatic compound in several fermented products and is normally dependent upon the rate of citrate or altered pyruvate metabolism (Axelsson, 1998). Lb. rhamnosus GG produced 6 mg kg 1 diacetyl, which is high considering that 1 –2 mg kg 1 is usual in fermented milk and the reported taste threshold value for diacetyl is 0.03 mg kg 1 (Imhof et al., 1994). Some of the diacetyl produced was probably also reduced to acetoin, since detected concentrations of acetoin and diacetyl followed similar patterns throughout the fermentation period. Small amounts of diacetyl, but no acetoin, were detected in porridge inoculated with Lb. acidophilus 1748 and Lb. acidophilus LA5. The pathway for acetoin production, via a-acetolactate, could be inhibited in Lb. acidophilus in the presence of pyruvate. (Benito de Ca´rdenas et al., 1990). Succinate may be an end product when heterofermentative lactobacilli metabolise glucose and citrate (Axelsson, 1998). However, in this study, Lb. reuteri metabolised almost all succinic acid present in the porridge (560 mg kg 1) and no production was detected. Ethanol, but no acetaldehyde, was detected in porridge inoculated with Lb. rhamnosus GG and in porridge inoculated with Lb. reuteri, which might imply that these two strains possess active alcohol dehydrogenase. Heterofermentative Lb. reuteri produced large amounts of ethanol, which may be above the taste threshold for ethanol of 100 – 800 mg kg 1 (Imhof et al., 1994). Most ethanol is probably a result of the breakdown of glucose with the resultant production of lactate, ethanol and CO2 (Axelsson, 1998). Ethanol can also be produced from pyruvate by pyruvate formate lyase (Hugenholtz, 1993). Acetaldehyde is expected to accumulate in porridge inoculated with Lb. acidophilus, since its alcohol dehydrogenase activity is rather low (Imhof and Bosset, 1994). Lb. acidophilus can also produce acetaldehyde directly from pyruvate or some amino acids (Gonzalez et al., 1994). This agrees with our results, where both strains of Lb. acidophilus produced diacetyl and acetaldehyde. These results have shown the importance of the inoculation rate. Low inoculation rates could be
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