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Page 1: Production of potentially probiotic beverages using single and mixed cereal substrates fermented with lactic acid bacteria cultures

at SciVerse ScienceDirect

Food Microbiology 30 (2012) 239e244

Contents lists available

Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Production of potentially probiotic beverages using single and mixed cerealsubstrates fermented with lactic acid bacteria cultures

Sorbhi Rathore, Ivan Salmerón, Severino S. Pandiella*

School of Chemical Engineering and Analytical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK

a r t i c l e i n f o

Article history:Received 29 April 2010Received in revised form27 August 2011Accepted 3 September 2011Available online 24 September 2011

Keywords:ProbioticLactic acid bacteriaCerealsFermentationMixed cereal substratesMixed cultures

* Corresponding author. Tel.: þ44 161 306 4429; faE-mail address: [email protected] (S.S

0740-0020/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.fm.2011.09.001

a b s t r a c t

In the present work, single and mixed cereal substrates were fermented with lactic acid bacteria to studyand compare the effect of the media formulation on fermentation parameters. Three cereal flours namelymalt, barley and barley mixed with malt (barleyemalt) were selected and fermented with two probioticstrains: Lactobacillus plantarum (NCIMB 8826) and Lactobacillus acidophilus (NCIMB 8821). The effect ofthe single and mixed cereal flour suspensions on the fermentation of these two strains of lactic acidbacteria (LAB) was studied at an incubation temperature of 30 �C for 28 h. It was found that the LABgrowth was enhanced in media containing malt and significant amounts of lactic acid were produced(0.5e3.5 g/L). A cell concentration between 7.9 and 8.5 Log10 CFU/mL and a pH below 4.0 was achievedwithin 6 h of fermentation. Though the cell populations in the mixed culture fermentations of mixedsubstrates were similar to the ones obtained with single cereal flours, significant differences in theproduction of lactic acid were observed. These results suggest that the functional and organolepticproperties of these cereal-based probiotic drinks could be considerably modified through changes in thesubstrate or inocula composition.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Increasing consumer awareness towards healthy diets and chang-ing eating habits due to urbanization has created a huge marketdemand for new functional foods with a beneficial effect on health.Probiotic foods are fermented formulates containing sufficient num-bers of selected live microorganisms (6e7 Log10 CFU/mL based ona100 mLdailydose) thatbeneficiallymodify the intestinalmicrobiotaof the host (Fuller, 1989; Havenaar and Huis in’t Veld, 1992; Mattila-Sandholm et al., 1999). Today most probiotics available are dairybased, but cereals can prove to be a healthier option for developingnew non-diary probiotic foods since they can overcome some of thedisadvantages associated with fermented dairy products like lactoseintolerance, allergy and the impact in cholesterol levels (Prado et al.,2008). Different cereals have complex nutrient composition andwhenmixed in a certainproportion they can considerablymodify theproperties of the food. Two substrates may change the nutrientavailability for fermentation and affect the microorganism growthand metabolism. On the other hand, mixed culture fermentationsprovidecomplexgrowthpatterns that canalsoconsiderablyaffect theorganoleptic and functional properties of the food.

x: þ44 161 306 4399.. Pandiella).

All rights reserved.

There is a considerable amount of work done on cereal-containing foods fermented with complex indigenous cultures(Blandino et al., 2003). The studies using fermented cereals for theproduction of probiotic foods in most cases utilize single cerealsubstrates and cultures as delivery vehicles for potentially probioticlactic acid bacteria (Charalampopoulos et al., 2003; Kedia et al.,2008; Angelov et al., 2006; Muyanja et al., 2003; Helland et al.,2004). Kedia et al. (2007) have also used mixed cultures for thefermentation of single cereals and cereal fractions, but no work hasbeen reported to study the influence of the cereal composition andinocula on the fermentation parameters.

The success of new probiotic formulations does not only rely onthe ability to provide enough probiotic cells that may survive thehuman gastrointestinal tract. The organoleptic properties of theseproducts must also be acceptable by the consumer, which in manycases is related to the organic acid content (fermented productsthat are too sour or acidic might not be acceptable by consumers).An appropriate selection of substrate composition and strains isnecessary to efficiently control the distribution of the metabolicend-products (Lönner and Åkesson, 1988; De Vuyst, 2000).

The main aim of this work is to study the effect of mediaformulation and inoculum on the fermentation parameters of novelcereal-based drinks produced with probiotic lactic acid bacteria.Single and mixed cereals substrates made from barley and malt

Page 2: Production of potentially probiotic beverages using single and mixed cereal substrates fermented with lactic acid bacteria cultures

Table 1Cell growth observed at 24 h of fermentation of cereal substrates with single andmixed lactic acid bacteria cultures.a

Media L. plantarum L. acidophilus Mixed culture

Malt 8.59� 0.03 8.69� 0.09Barley 7.91� 0.08 8.55� 0.04BarleyeMalt 8.53� 0.04 8.43� 0.05 8.2� 0.4

a Cell growth observed at 24 h of fermentation of cereal substrates with singleand mixed lactic acid bacteria cultures.

S. Rathore et al. / Food Microbiology 30 (2012) 239e244240

were used in single and mixed culture fermentations with Lacto-bacillus plantarum and Lactobacillus acidophilus. Cell growth, FAN,TRS and organic acids weremonitored during fermentation of cerealsubstrates without the addition of supplements or pH control.

2. Materials and methods

2.1. Cereal fermentation substrates

The barley grains (Brewing Research International, UK) weremilled using a Falling Number hammer mill (Perten Instruments,Huddinge, Sweden) fitted with a 0.5 mm aperture sieve. The maltwas prepared from the same barley grains through steeping,germination and drying (steeping at 20 �C for 36 h; germination at10 �C for 24 h and 18 �C for 72 h; and kilning at 45 �C for 20 h and95 �C for 3 h). The germinated barley was then milled into flour inthe same way. The single cereal fermentation media were preparedby dissolving 65 g of the barley or malt flours in 1.3 L of distilledwater (5% w/w). The mixed cereal media were prepared by mixingequal quantities of the single cereal flours.

Starch in the fermentation media was first gelatinised andpartially hydrolysed by keeping the suspensions in a water bath at95� 5 �C for 1.5 h. The media were then transferred to a bath at50� 5 �C for 30 min and then to a cold water bath. The media werethen filtered using Whatman filter paper (grade 1) and finallyautoclaved at 121 �C at 15 psi for 15 min.

2.2. Microorganisms and inoculation

The microorganisms used in study were L. acidophilus NCIMB8821 (National Collection of Industrial and Marine Bacteria, Aber-deen, Scotland, UK) and L. plantarumNCIMB8826, both isolated fromhuman intestine. In vitro and in vivo studies supporting the probioticcharacteristics of L. plantarum NCIMB 8826 have been previouslyreported by Vesa et al. (2000) and Borchers et al. (2009). Black et al.(1989) and Marteau et al. (1990) have also proved the probioticcharacteristics of L. acidophilus strains similar to the one used in thiswork. Thus L. acidophilus NCIMB 8821 could be considered a poten-tially probiotic strain though further in vitro and in vivo studiesshould be performed to confirm its probiotic properties.

The strainswerefirst sub-cultured three times for 12 h at 37 �C inMan Rogosa Sharp broth (MRS, Merck) from the frozen stock con-taining MRS broth and 10% glycerol before using it as an inoculumand stored at�30 �C. The starter culture was obtained by overnightincubation at 37 �C in MRS broth. The culture was centrifuged at4000 rpm for 7 min (Sigma 6K15, Sigma-Zentrigugen, Germany),washed in 0.1% sterile NaCl water (Sigma Aldrich, UK) and re-suspended in 10 mL 0.1% sterile NaCl.

2.3. Batch fermentation

The cereal suspensions were inoculated with 1% (v/v) pure ormixed culture (ratio 1:1). All fermentations were carried out at30 �C in a shaker incubator (New Brunswick Scientific CO. INC, NJ,USA) set at 150 rpm till 28 h under no pH control. Sampling wasdone every 2 h at the beginning of fermentation, which was carriedout for 28 h. The samples collected were centrifuged at 4000 rpmfor 7 min and the supernatant was stored at �30 �C for chemicalanalysis.

2.4. Cell growth

For cell counting the Miles and Misra drop method (Hedges,2002) was used. Suspensions of fermented media were decimallydiluted in 0.1% sterile saline (NaCl) solution and 10 mL of the diluted

fermentation sample was dropped onto the MRS nutrient agarplates (Fluka, 69964 biochemika, UK) and incubated for 48 h at37 �C.

2.5. Analytical methods

2.5.1. pHThe pH of the samples was monitored during the fermentation

using a digital pHmetre (Eutech instruments, Cyberscan) calibratedwith buffers at pH 4.0 and 7.0 (Fisher Scientific, UK).

2.5.2. Free amino nitrogen (FAN) and total reducing sugars (TRS)The concentration of FAN was estimated by the Ninhydrin

colorimetric method (European Brewery Convention, 1987). Theabsorbance of the samples was measured at 570 nm in a spectro-photometer (Shimadzu UV mini 2140-VIS). To measure TRS thedinitrosalicylic acid method (DNS) was used (Bernfeld, 1951). Theabsorbance of the samples wasmeasured at 540 nm using the samespectrophotometer.

2.5.3. Determination of organic acidsHigh-performance liquid chromatography (HPLC) was per-

formed to separate, identify, and quantify organic acids by usinga Varian ProStarModel 220 HPLC equipment (Varian Inc., UK), fittedwith a variable wavelength detector based upon ultravioletevisiblespectrophotometry set at 210 nm. The column used was a 5 mmsilica Thermo Hypersil C18 (column no.4-38273, Thermo FisherScientific Ltd.). The mobile phase used was 0.005 M H2SO4. Allanalyses were performed in triplicate. Lactic acid concentration inthe fermentation samples (g/L) was quantified by UV detector at210 nm.

3. Results

3.1. Growth patterns of single cultures

Results of cell growth observed at 24 h of fermentation in allmedia are presented in Table 1. The cereal media (malt, barley andbarleyemalt) were inoculated with 1% (v/v) starter culturesuspension of L. plantarum and L. acidophilus with the objective ofachieving a cell concentration between 106 and 107 CFU/mL, whichwould be the cell concentration required at the time of consump-tion (based on a 100 ml daily intake of the of fermented product)(Gomes and Malcata, 1999; Rasic and Kurmann, 1983). High viablecounts are necessary to get the desired acid production andreduction in pH, which affect the products organoleptic properties,its shelf-life and prevents product contamination. For L. plantarumfermentations the cell populations increased rapidly at the begin-ning and after 6 h the growth reached the stationary phase. After24 h of fermentation (results not shown) the cell concentrationstarted to decrease. The L. plantarum strain used in the studyshowed good growth in single malt, barley and barleyemalt (mixedcereal) media, which indicates that the fermentation conditionswere appropriate for growth and that the mixed media provides all

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S. Rathore et al. / Food Microbiology 30 (2012) 239e244 241

the required nutrients. After 6 h of fermentation, the viable cellcounts were well above the suggested minimum limit of 6e7 Log10CFU/mL (Mattila-Sandholm et al., 1999) for efficacy of a probioticproduct in all the cereal fermentations with L. plantarum andL. acidophilus. In the barleyemalt mixed media fermented withL. plantarum the lag phase was slightly longer but the cell pop-ulations achieved the same levels as in the single malt media. Thegrowth patterns observed for L. acidophiluswere very similar. WithL. acidophilus fermentations, the single cereal malt media producedthe highest cell concentrations at 24 h as compared to L. plantarum.The barleyemalt (mixed) media reached similar cell populations(approximately 8.5 and 8.4 Log10 CFU/mL at 24 h for L. plantarumand L. acidophilus respectively).

3.2. Evolution of FAN and TRS in single culture fermentations

Results presented in Fig. 1 show differences in TRS and FANinitial concentrations in both L. plantarum and L. acidophilusfermentations. In all cases the barley medium has lower TRS andFAN initial concentrations and decrease through fermentation.Media with malt or barleyemalt mixture have much higher initialTRS and FAN values, and concentrations increase or decreasethrough fermentation depending on the composition of the mediaor the Lactobacillus strain.

3.3. pH and organic acid production in single culture fermentations

The profiles of lactic acid evolve in a different way depending onthe composition of the cereal medium and the lactobacillus strainused for fermentation. Lactic acid and pH profiles obtained in allfermentations between 0 and 28 h are shown in Fig. 2. Lactic acidincreased in all cases through fermentation. Malt showed thehighest lactic acid increments followed by the barleyemalt mixtureand single barley media. In this study more lactic acid wasproduced by L. acidophilus at 24 h in barleyemalt (mixed) media

Fig. 1. TRS and FAN content in L. acidophilus and L. plantarum (pure culture

than by L. plantarum (2.3 g/L and 1.8 g/L respectively). In all cases,pH drops rapidly during the first 4 h and then further decreasesslowly through fermentation. After 24 h, the pH was found to bebelow 3.5 in mixed and single cereal media.

3.4. Mixed culture fermentation of mixed substrates

The evolution of cell populations, pH, TRS, FAN, lactic acid andacetic acid concentrations during fermentation of a mixed bar-leyemaltmediawithmixedculturesof L. acidophilusandL. plantarumare shown in Fig. 3. The cell populations in mixed culture fermen-tations of barleyemalt (mixed) substrates were similar to fermen-tations carried out with single cultures of single and mixed cereals(see Table 1). However, in this case there was a slight increase in thecell concentration after 24 h with decrease in pH, which was notobserved in single culture fermentations (data not shown). The pHdecreased and reached values close to 3.0 at the end of fermentation.TRS and FAN fluctuate at the beginning of fermentation and ingeneral decrease through fermentation. Therewas a rapid increase oflactic and acetic acid between 4 and 24 h but the maximumconcentrations observed (0.79 and 0.25 g/L respectively) were lowerthan the ones observed in single culture fermentations.

4. Discussion

Our results show that growth of L. plantarum and L. acidophilus issignificantlyenhanced inmedia containingmalt. At 6 h theviable cellconcentration in the fermented products was above the minimumdose recommended for a probiotic product to confer a therapeuticeffect (6 Log10 CFU/mL based on a 100 mL daily dose) (Sanders andHuis in’t Veld, 1999). The cell concentrations observed are in agree-ment with similar studies conducted by previous authors (Hellandet al., 2004; Angelov et al., 2006; Mattila-Sandholm et al., 1999;Kedia et al., 2008). It was found that fermentation of barleyemaltmixed media with L. plantarum started at a lower cell population of

s) fermentations of Malt (B), Barley (C) and BarleyeMalt (;) media.

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Fig. 2. Change in pH and Lactic acid production observed in L. acidophilus and L. plantarum (pure cultures) fermentations of Malt (B), Barley (C) and BarleyeMalt (;) media.

S. Rathore et al. / Food Microbiology 30 (2012) 239e244242

around 6 Log10 CFU/mL but reached the same level as that of singlemalt media (8.6 Log10 CFU/mL). This could be attributed to thesimultaneous presence of considerable amounts ofmonosaccharides(glucoseand fructose) anddisaccharides (maltose andsucrose) in themalt medium (approximately 3 and 12 g/L respectively) as reportedby Charalampopoulos et al. (2002).

In this study the initial fluctuations in the TRS concentration inL. plantarum fermentations could be due to further breakdown ofsugars in malt. The overall decrease in TRS concentration could beattributed to the consumption of total reducing sugars during theexponential phase of growth. It has been shown by Samuel et al.(1980) and Gobbetti et al. (1994) that L. plantarum exhibits speci-fic preference towards glucose.

The increase in the FAN concentration observed at the end ofL. acidophilus fermentation of malt media could be due to the actionof proteases secreted by the bacteria as reported by Kedia et al.(2007). The FAN concentration in most fermentations decreases(with the exception ofmalt fermentedwith L. acidophilus) toprovidemaintenance energy for cells (Charalampopoulos et al., 2002). InL. acidophilus fermentations as the cell population reached statio-nary phase FAN and TRS concentration also became constant. Thisdemonstrated that only small quantities of reducing sugars arerequired by L. acidophilus during this phase. On the other hand,initial increase inTRS concentration in the barleyemaltmedia couldbe a result of the hydrolysis of flour components as compared to therate of free sugars uptake by L. acidophilus. As demonstrated byCharalampopoulos et al. (2002) presence of 36 and 47 mg/L FANincreased the viability of L. acidophilus bymore than 0.6 Log10 cycle ,which suggested that the FAN content of malt and barley couldcontribute to the protective effect of these cereal extracts. Since cellswith added malt extract displayed higher viability than barley inthat work, sugar appears more important for stabilization whichagreeswithMichida et al. (2006). Therefore, the higher TRS and FANcontent of malt basedmediamay be due to the breakdown of starchand proteins during themalting process and could also be one of the

factors contributing to the increased cell viability observed in thisstudy.

Since all the experiments were performed under uncontrolled pHconditions, the rapid drop in pH in L. plantarum fermentations wasdue to the accumulation of lactic acid produced via the metabolicpathways. L. acidophilus continued to consume sugars and accumu-late organic acids, decreasing the pHof themedium to 3.0e3.3 due tothe energy requirements for the preservation of cell viability (Passoset al.,1994). For all cerealflour fermentations (bothwith L. acidophilusand L. plantarum) the pHwas below 3.5 which demonstrated that 1%(v/v) inoculum was sufficient to achieve the pH value that has beenreported to inhibit Enterobacteriaceae and other Gram-negativebacteria (between 3.5 and 4.0) (Mbugua, 1985; Chavan and Kadam,1989; Nout, 1991; Steinkraus, 1996; Muyanja et al., 2003). More-over, low pH (around 3.7 or below) prevents the growth of coliforms(Muyanja et al., 2003), and food formulations with pH ranging from3.5 to 4.5 help increasing the pH of the gastric tract and thus enhancethe stability of the probiotic strain (Kailasapathy and Chin, 2000;Gardiner et al., 1998; Zárate et al., 2000). The pH and lactic acidformationwere in accordancewith studies conducted by Sulma et al.(1991), Choi et al. (1994) and Dziedzoaze et al. (1996). The lactic acidproduced in this study was much higher than the one reported byHelland et al. (2004) in maize porridge with added malted barley. Asreported by Passos et al. (1993) and Campbell-Platt (1994), theproduction of lactic acid and organic acids inhibits microbial growthin their undissociated form, dissociated form or indirectly byreleasing the protons (Hþ) in the medium.

Some differences in fermentation parameters (cell population,TRS, FAN and lactic concentration) were found in this studybetween mixed media fermented with single and mixed cultures,which could be attributed to the interactions between the strains inthe mixed culture fermentation. L. plantarum has been identified asthe dominant organism at the end of several natural lactic acidfermentations (Nout, 1980; Mbugua, 1984; Brauman et al., 1996),probably due to its acid tolerance (Fleming andMcFeters,1981) and

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Fig. 3. Fermentation of barleyemalt cereal flour media inoculated with a mixture ofL. plantarum and L. acidophilus in a 1:1 ratio showing different fermentation param-eters. (C) CFU, (;) pH; (:) TRS, (-) FAN and (C) Lactic acid, (A) Acetic acid.

S. Rathore et al. / Food Microbiology 30 (2012) 239e244 243

greater ability to utilize the substrates (Oyewole and Odunfa, 1990),including dextrins (Akinrele, 1970). Moreover, low pH could be thegrowth limiting factor of L .acidophilus since L. plantarum is able tomaintain a proton gradient between the inside and outside of thecells even in the presence of large amounts of lactate and protons(Giraud et al., 1998). TRS and FAN are consumed through fermen-tation but are not depleted, which suggest they are not limitinggrowth factors in these fermentations.

The decrease in pH and increase in lactic acid followed the sametrend as reported in other natural fermented foods (Sulma et al.,1991; Choi et al., 1994; Dziedzoaze et al., 1996). Lactate andacetate are important flavour compounds in fermented cereals(Onyango et al., 2000), and the latter has been reported to act asa flavour enhancer, sensitizing consumers towards other aromaticcompounds in products such as sourdough (Gobbetti and Corsetti,1997).

Our study has shown a considerable difference in the productionof lactic acid between the single andmixed culture fermentations ofthebarleyemaltmixedmedia (see Figs. 2 and3). This couldbedue tointeractions between the lactobacilli species during fermentation.L. acidophilus is an obligately homofermentative bacteria thatproduces a single end product (lactic acid) from glycolysis ofcarbohydrates (EM pathway). L. plantarum is a facultatively heter-ofermentative bacteria that produces lactic acid and acetate orsignificant amounts of other end-products besides lactic acid

(Pentose Phosphate Pathway). The two species may influence eachother’smetabolism,which can lead to different profiles of importantorganoleptic compounds. There might also be other complicatedinteractions between metabolites where a compound produced byone organism may be metabolized further by another (Axelsson,1998). Kedia et al. (2007) also reported lower concentrations oflactic acid inmixed cultures of lactic acid bacteria and yeast in an oatfermented media. These results highlight the importance of theselection of the substrate composition and inocula in the develop-ment of the organoleptic properties of these fermented products.

5. Conclusion

The results of this study indicate that in pure culture fermen-tations of single and mixed cereal media, the two selected Lacto-bacillus strains grow above probiotic levels with a considerableproduction of lactic acid. Mixed culture fermentations in mixedcereal substrates produced similar amounts of cell populations, butthe production of organic acids was considerably lower, whichcould have a strong impact on the organoleptic properties of theproducts. Malt proved to be the best substrate (as single and mixedmedia) for cell growth which could be attributed to its chemicalcomposition. It also seems that the low pH could be the mainlimiting factor for microbial growth, though this would also dependon the lactic acid bacteria strain(s) used for fermentation.

Further research is required to study the sensorial properties ofthese fermented products and those formulated with other sup-porting LAB strains to develop a novel probiotic product acceptableby consumers. The non-digestible components in the fermentedcereal media should also be studied for their potential as probiotics.

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