Vol 62, No 2 May 2009 International Journal of Dairy Technology

ORIGINAL RESEARCH

*Author for correspondence. E-mail: prapullasg@yahoo.co.in

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doi: 10.1111/j.1471-0307.2009.00480.x

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Blackwell Publishing LtdOxford, UKIDTInternational Journal of Dairy Technology1364-727X1364-0307Society of Dairy Technology 2009XXX ORIGINAL RESEARCHORIGINAL RESEARCH

Comparative survival and evaluation of functional probiotic properties of spray-dried lactic acid bacteria

KANCHI BHASKER PRAVEEN KUMAR REDDY, ARENAHALLI NINGEGOWDA MADHU and SIDDALINGAIYA GURUDUTT PRAPULLA*Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore 570 020, India

The effect of spray drying on the viability and retention of key probiotic properties like acid and biletolerance and cholesterol assimilation of three probiotic lactic acid bacteria (LAB) (Lactobacillusplantarum CFR 2191, Lactobacillus salivarius CFR 2158 and Pediococcus acidilactici CFR 2193) hasbeen studied. More than 97% survival was exhibited by the three LAB (1% cell suspension) spray driedwith maltodextrin and nonfat skimmed (NFSM) as carriers. LAB cultures spray dried with maltodextrinshowed significantly greater (P ≤ 0.05) retention of functional properties than those with NFSM. Theresults highlight a cost-effective way of producing large quantities of selected probiotic cultures withfunctional properties for neutraceutical application.*Author for correspondence. E-mail: prapullasg@yahoo.co.in

Keywords Probiotics, Lactic acid bacteria (LAB), Lactobacillus plantarum, Lactobacillus salivarius, Pediococcus acidilactici, Spray drying.

I N T RO D U C T I O N

Probiotic lactic acid bacteria (LAB) are of strategicimportance to the dairy and neutraceuticalindustries because of their role in human andanimal health and welfare. They also play a keyrole in the production of lactic-fermented foods,which represent about 20% of the total economicvalue of fermented foods produced throughout theworld (McKay and Baldwin 1990). There has beena considerable interest in incorporating theseprobiotics in functional and pharmaceuticalproducts. It has been recommended that foodscontaining probiotic LAB (probiotic functionalfoods) should contain at least 107 colony-formingunits (cfu) of live microorganisms per gram orper millilitre (Ishibashi and Shimamura 1993) atthe time of consumption, in order to benefit theconsumer. Therefore, in addition to the requirementsof safety and functional characteristics, probioticcultures should also be able to withstand the foodprocessing and storage conditions encounteredduring industrial manufacture (Knorr 1998). Thus,from a commercial point of view, an inexpensivemethod for the large-scale production of shelfstable products containing high levels of viablecells becomes a necessity.

Spray drying, freezing and freeze drying are themost common downstream processes used for thepreparation of dried stable probiotic cultures (Toand Etzel 1997). The importance of starters in the

dairy industry is recognized from the industrialpoint of view, but preparation of starters is timeconsuming (Tamime and Robinson 2007). Adisadvantage in using stock cultures is the need forthe production of large volumes of cultures. Theabove downstream processes acquire importancein this context. The development of concentratedstarter cultures via spray drying for inoculating theproduct directly eliminates much of the time,drudgery and danger customarily involved in thepreparation and maintenance of starter cultures inthe dairy plant (Tamime 2002). Spray drying oflarge quantities of bacterial cultures as a method toreplace the usual liquid bulk starter in the productionof fermented food products has been extensivelyinvestigated (Fu and Etzel 1995; O’Riordan et al.2001; Tamime 2002; Gardiner et al. 2002; Tamimeand Robinson 2007). Despite its low cost in compar-ison to other technical solutions (e.g. freeze drying),spray drying has not been developed commercially,mainly because of low survival rates during drying,and difficulties in rehydrating the product (Porubeanand Sellars 1975). However, spray drying is stillone of the predominant processing tools used in thedairy industry, and is used to produce large amountsof dairy ingredients relatively inexpensively. It hasbeen estimated that the cost of spray drying issix times lower per kilogram of water removedthan the cost of freeze drying (Knorr 1998). Thereare obvious challenges in spray drying of viablecultures, including the requirement that the

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micro-organisms should survive the relatively hightemperatures (Daemen and Van der stege 1982;Stanton et al. 2003). A number of studies concerningthe survival of various lactic cultures after spraydrying have been reported by various investigators(Kim and Bhowmilk 1990; Teixeira et al. 1995;Mauriello et al. 1999; Gardiner et al. 2000; Corcoranet al. 2004). In addition to the viability of probioticcultures, retention of probiotic properties is highlyrelevant. There are reports that various healthproducts and pharmaceutical preparations con-taining dried cells of LAB are used in the treatmentof gastrointestinal disturbances (Gomes and Malcata1999). Although freeze dried probiotic cultures areavailable commercially, published data related tospray drying of commercially available LAB arelimited at present. The literature reports availableon spray drying of various lactic cultures focusonly on the viability after spray drying. Not mucheffort has been made to examine probioticproperties after spray drying. This aspect thusforms the principal novelty of our study.

Ayurveda is the traditional medical sciencepractised in India and can be traced to the pre-historic period. ‘Sandhana Kalpana’ is the termused for the process of fermentation using herbs,with medicinal properties. Kanjika (Indianfunctional probiotic food, also abbreviated as kanji)is a lactic-fermented product where lactic acidfermentation is the terminal step in food processing,and is prepared from raw material of plant originand devoid of dairy product (Misra 1993). The aimof the present study was to investigate spray dryingas a method of producing concentrated probioticLAB isolated from kanjika, as an ingredient for theneutraceutical and pharmaceutical industry. Foodgrade maltodextrin (MDX) and nonfat skimmedmilk (NFSM) were used as carriers for spray dryingof probiotic LAB. The effect of spray drying on cellsurvival, with an emphasis on retention of probioticproperties like pH tolerance, bile tolerance andcholesterol assimilation has been detailed. Thesurvival of probiotic LAB during storage has alsobeen investigated.

M AT E R I A L S A N D M E T H O D S

MaterialsMRS broth/agar, cholesterol and bile salts wereprocured from HiMedia Laboratories Pvt Ltd,Mumbai, India. X-gal (5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside) and IPTG (iso-propyl-thio-β-D-galactopyranoside) were obtained fromSigma-Aldrich, Missouri, USA. Maltodextrin wasa product from Lobo chemicals, Mumbai, India.Nonfat skimmed milk was a commercial productfrom Karnataka Milk Federation Ltd, Bangalore,India. All the chemicals used were of analyticalgrade.

LAB strainsOut of 17 LAB isolated during the preparation ofkanjika, three strains exhibiting potent probioticproperties like tolerance to low pH, high bile saltconcentration up to 1%, antimicrobial activityagainst food-borne pathogens, β-galactosidaseactivity, antibiotic susceptibility and cholesterol-lowering capacity were selected for studying theeffect of spray drying (Reddy et al. 2007). Theyhave been identified by partial sequencing ofthe 16S rRNA gene (ribotyping) as Lactobacillusplantarum CFR 2191, Pediococcus acidilacticiCFR 2193 and Lactobacillus salivarius CFR 2158.All LAB strains were preserved at –80°C with 20%glycerol (2 mL cryovials).

Preparation of feed solutions for spray-drying applicationFor spray-drying studies, two types of feed solu-tions were prepared. In the first type, overnightcultures of each probiotic LAB were inoculatedinto MRS broth (1% v/v) and incubated at 37°Cuntil the stationary phase was reached. Aftercentrifugation at 9000 × g for 15 min at 4°C, thecells [1, 3 and 5% (w/v) on a wet weight basis]were re-suspended in 10% NFSM solution. Thistype of feed solution was termed nonfat skimmedmilk. In the second type, overnight cultures of eachprobiotic LAB strain [1, 3 and 5% (w/v) on a wetbasis] were re-suspended in 10% MDX solution.This type of feed solution was termed maltodex-trin. The pH of the feed solution varied from 4.0 to4.5. These feed solutions were directly spray dried.Each trial was conducted in duplicate for each ofthe three probiotic strains.

Spray dryingA bench top scale dryer (JISL, Bombay, India) wasused. The inlet air, heated to 140 ± 2°C by anelectrical heater, flowed concurrently with the sprayinto a 12.5-L drying chamber with an outlet temper-ature of 40 ± 2°C. Feed solution was deliveredby a peristaltic pump into two fluid stainless steelatomizers. The spray-dried powder was collected atthe bottom of a cyclone. Spray drying was carriedout in duplicate, and the properties of duplicatesamples from each trial of spray drying were studied.The moisture content of the spray-dried powderswas determined by drying in a hot air oven at 102°C.The weight loss was expressed as a percentage ofthe powder weight (Corcoran et al. 2004).

Cell survivalThe residual viability of spray-dried samples wasdetermined by the standard plate count method.The spray-dried powder (1 g) was rehydrated with10 mL of sterile distilled water to about the samesolid content as that of the feed solution. Therehydrated samples were kept on a shaker for

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30 min to get a homogeneous suspension. Suitablydiluted feed solution samples and those of re-hydrated samples (100 μL each) were platedusing the spread plate method. Colony-formingunits were determined after incubation for 48 h at37°C. The percentage survival of the spray-driedsample was calculated as

% survival = 100 × Nr/Nf, (1)

where Nr was log cfu/mL of rehydrated sample andNf was the log cfu/mL of feed solution.

Acid toleranceThe pH tolerance was tested according to Parket al. (2002). The enumerated spray-dried LABcultures were further grown in MRS broth forthree generations. Cells enumerated for threegenerations with an optical density of 0.280 (50-fold dilution) at 600 nm were inoculated into 9 mLof sterile MRS broth of pH 2 and 2.5 (adjusted with0.1 N HCl). The initial bacterial concentration was107–108 cfu/mL and was maintained throughoutthe experiments. Samples were incubated at 37°Cfor 4 h. One millilitre of sample was serially diluted(6-fold) with sterile saline to neutralize the medium’sacidity and incubated for 24 h at 37°C. The viablecell population was determined by the plate countmethod. The percentage survival was calculatedas the percentage of colonies grown on MRS agarcompared to the initial bacterial concentration.

(2)

Bile salt toleranceBile tolerance of the LAB was determined asdescribed by Park et al. (2002). One millilitre ofinoculum, as mentioned in the acid tolerancemethod, was inoculated into 9 mL of sterile MRSbroth containing 0.3% (w/v) bile salt. Growth wasmonitored by enumerating the viable cells on MRSagar medium after 12 h incubation, using the platecount method. The percentage survival of testsamples was calculated according to equation 2.

Cholesterol loweringA cholesterol-lowering test was carried outaccording to the method described by Searchy andBergquist (1960). LAB cultures were cultivated inMRS broth supplemented either with cholesterolor cholesterol with 0.3% bile salt. About 500 μL ofethanol containing 10 mg of cholesterol was addedto 100 mL of MRS broth with or without bile salt.The cultures were grown for 12–24 h at 37°C.Cells were harvested by centrifugation at 9000 × gfor 10 min at 4°C (Remi cooling centrifuge, C-30,Mumbai, India). Spent broth was collected andused for the cholesterol assay. The uninoculated

broth was used as control (zero percentage assimi-lation). The concentration of cholesterol wasdetermined using a cholesterol calibration graph.The percentage assimilation of cholesterol wascalculated using the formula:

(3)

Scanning electron microscopy of spray-dried powderSpray-dried powder was attached to brass stubsand coated with gold using a scanning electronmicroscopy coating system (Polaron Sputter coatsystem, Model 5001, Polaron Equipment Ltd.,Watfort, UK). Samples were examined with a Leoelectron microscope (Model Leo-435 VP, LeoElectron Microscopy Ltd., Carlzeiss SMT, Cam-bridge, UK) using an accelerating voltage of 20 kV.Micrographs were taken at different magnifications.

Storage stability of spray dried probiotic culturesFrom the duplicate samples of the spray dryingtrials, two sets each were stored at 30°C ± 2 and4°C ± 2 for 60 days, respectively. Storage stabilityin terms of viability of the three spray-dried probioticcultures was assessed over a period of 60 days.

Statistical analysisSpray-drying experiments were carried out induplicates and the samples were then evaluated forprobiotic properties in duplicates from each set ofspray drying. The mean and standard deviationwere calculated for n = 4. One-way analysis ofvariance (anova) at P ≤ 0.05 was used to expressthe statistical differences between the treatedand the active cells. anova was done using Origin6 statistical software (Originlab Corporation,Massachusetts, USA).

R E S U LT S

Effect of cell concentration on viability of the spray-dried probiotic LABThe effects of cell concentration (1, 3 and 5%) onthe viability of LAB during spray drying are shownin Figure 1. As can be seen from the results, allthree probiotic LAB exhibited more than 97%survival, when a 1% cell suspension was spraydried either with MDX or NFSM (10%). This isprobably due to the fact that the higher ratio of thecarriers to the concentration of cells provided aprotective effect on the cells (Figure 2a). Thisresults in less damage to the cells during spraydrying (Figure 2b). However, it was found that thepercentage of viability decreased significantly (P ≤0.05) with an increase in the cell concentration in

% survival

log cfu of viablecells survived

log cfu of initial viablecells inoculated

100= ×

% assimilation Conc. of cholesterol in control Conc. of cholesterol in test sample

Conc. of cholesterol in control 100

=−

×

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the case of P. acidilactici. These are gram-positivetetrad-forming cocci and do not have the lipidbi-layer and hence are more susceptible to heatdamage. Scanning electron micrographs (Figure 2a)clearly indicate the oozing out of the cytoplasmfrom the cells damaged during spray drying. In thecase of L. plantarum and L. salivarius, there was nosignificant decrease (P ≥ 0.05) in viability at all thethree concentrations of cells used. This indicatesthat bacilli are less susceptible to heat damageduring spray drying. Contrary to this, there was nosignificant difference (P ≥ 0.05) in the percentagesurvival of all the three probiotic LAB studiedwhen NFSM was used as the carrier. Similarly,varying concentration of cells also did not have anypronounced effect on the percentage survival.

Acid tolerance of spray-dried probiotic LABL. salivarius was found to retain only 60% survivalat pH 2.0 in the initial experiments before spraydrying, and hence acid tolerance studies of spray-dried L. salivarius at pH 2.0 were not conductedafter spray drying. The percentage survival of theother two studied probiotic LAB at pH 2.0 ispresented in Figure 3. As can be seen from theresults, the active cells of P. acidilactici exhibited atolerance of 85% at pH 2.0. There was a significantdecrease (P ≤ 0.05) in the percentage survival ofP. acidilactici after spray drying with MDX. Thedecrease was more pronounced with the increasein percentage cell suspension. The percentagesurvival of spray-dried P. acidilactici (1% cellsuspension) was reduced to 70%, corresponding toa 17.6% loss in comparison with that of active cells(85%). The percentage survival decreased from70 to 58% with an increase in percentage cellsuspension from 1 to 5%.

The acid tolerance (pH 2.0) of L. plantarum wasfound to be retained to a significant level (P ≤ 0.05)even after spray drying either with MDX or withNFSM. The varying concentration of cell suspensiondid not affect the acid tolerance. Based on theseresults, it can be said that a stable spray-dried prepa-ration of the studied L. plantarum (1% cell suspension)can be prepared using MDX as the carrier.

Acid tolerance of the spray-dried LAB was alsostudied at pH 2.5 and the results are presentedin Figure 4. P. acidilactici and L. plantarum werefound to retain their acid-tolerant property (up to95%) in all the cell suspensions (1–5%) spray driedwith MDX. A significant loss (P ≤ 0.05) in viabilitywas observed in the case of P. acidilactici, spraydried with NFSM at all the concentration of cellsstudied. Loss in viability (22%) was observed inthe case of L. salivarius spray dried with MDX.The decrease was more pronounced (P ≤ 0.05)when NFSM was used as the carrier. The loss inpercentage survival was around 55% in comparisonwith that of active cells (90%).

Figure 1 Effect of cell concentration in spray drying with maltodextrin (MDX) and nonfat skimmed milk on the survivability of Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus salivarius. The results are means based on data from four replicates, and standard deviations are indicated by error bars. The means of different cell suspensions of P. acidilactici with MDX differ significantly (P ≤ 0.05).

Figure 2 Scanning electron micrographs of the spray-dried Pediococcus acidilactici. (a) Damaged cells with cytoplasm oozing out due to improper coating of the cells with carriers. (b) Cell protected with a coated layer of carrier.

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Bile tolerance and cholesterol assimilation of spray-dried probiotic lactic acid bacteriaThe effect of cell concentration and carriers on theretention of bile tolerance (0.3%) and cholesterolassimilation in the presence of bile was studied.The results are depicted in Figure 5a,b & c for L.plantarum, L. salivarius and P. acidilactici, respec-tively. As can be seen from the results, there was nosignificant difference (P ≥ 0.05) in bile tolerance inall the combinations tested. In comparison with theactive cells, there was a decrease in cholesterolassimilation with an increase in cell concentration,when both MDX and NFSM were used as thecarriers. With increased cell concentration, andconsidering the cholesterol assimilation of activecells as 100%, the loss in cholesterol assimilationsignificantly varied (P ≤ 0.05) from 21–32, 0–8and 6–7% for L. plantarum, L. salivarius andP. acidilactici when MDX used as the carriers.However, with the NFSM the loss in cholesterolassimilation significantly varied (P ≤ 0.05)from 18–35, 31–71 and 12–22% for L. plantarum,L. salivarius and P. acidilactici, respectively withincrease in cell concentration from 1–5%.

Survival of spray-dried probiotic cultures during storageThe maximum survival rate during the period of60 days for the three probiotic LAB (L. salivarius,

P. acidilactici and L. plantarum) is presented inFigure 6(a,b,c), respectively. It was found to be60% at 4°C for all the three strains with both thecarriers (MDX and NFSM). However, duringstorage at 30°C for 60 days, survival decreasedto 45% for L. salivarius and 50% for both P. acidi-lactici and L. plantarum when MDX was used as acarrier. When NFSM was used as carrier, survivalwas found to decrease to 38% for L. salivarius,53% for P. acidilactici and 67% for L. plantarum.This indicates that MDX can be used in place ofNFSM for the spray drying of selected probioticLAB.

D I S C U S S I O N

The present investigation details the effect ofdifferent carriers and cell concentrations on thespray drying of probiotic LAB. The advantage ofspray drying is that it allows large quantities ofcultures to be dried at low cost (Mauriello et al.1999), but the disadvantage is extreme cell damageduring the process and hence the reduced survivalof the organism during spray drying and storage(Brennan et al. 1986). Inlet and outlet temperaturecould also be one of the major reasons for celldamage/death during spray drying (Kim andBhowmik 1990; Mauriello et al. 1999). The optimumtemperature for the growth of most LAB is around

Figure 3 Effect of cell concentration and carriers on acid tolerance of spray-dried Pediococcus acidilactici and Lactobacillus plantarum at pH 2: percentage survival in comparison with that of initial cell concentration of the feed solution. The results are means based on data from four replicates and standard deviations are indicated by error bars. The means of cell suspensions of P. acidilactici of both carriers maltodextrin (MDX) and nonfat skimmed milk (NFSM) shows significant difference (P ≤ 0.05) when compared with active cells.

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40°C. Considering this factor, the experimentswere carried out at a constant outlet temperatureof 40°C and corresponding inlet temperature of140°C.

Percentage survival was more in the case ofspray-dried P. acidilactici (3–5% cell suspension)with NFSM in comparison with that of spray-driedP. acidilactici with MDX (Figure 1). Cell damageduring spray drying is mainly due to the denaturingof cell membrane proteins. NFSM could probablyhave a protective effect against cell damage. Therewas no significant loss in the acid-toleranceproperty in the case of L. plantarum at pH 2 and2.5. P. acidilactici exhibited tolerance only atpH 2.5. This could be due to fact the P. acidilacticibeing cocci do not have the protective lipid bi-layer. L. salivarius has been earlier reported to besensitive to heat treatment (Gardiner et al. 2000).

In agreement with this earlier study, a considerableloss in acid tolerance at pH 2.5 after spray dryingwas observed. There was no significant change inthe bile tolerance property of all the three probioticLAB after spray drying.

All the three cultures studied were found toretain more than 80% of their cholesterol-assimila-tion properties when a 1% cell suspension wasspray dried with MDX. The cholesterol-assimilationproperty assumes greater importance in the contextof increasing cardiovascular disease in the urbanpopulation, wherein probiotic supplementationis being viewed as an alternate therapeuticapproach. Of the two carriers tested, it was foundthat viability was retained to a significant extentwith NFSM, whereas better retention of the pro-biotic properties with reasonably good viabilitywas observed with MDX. It is generally more

Figure 4 Effect of cell concentration and carriers on acid tolerance of spray-dried Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus salivarius at pH 2.5: Percentage survival in comparison with that of initial cell concentration of the feed solution. The results are means based on data from four replicates, and standard deviations are indicated by error bars. The mean values of cell concentration of L. salivarius show significant difference (P ≤ 0.05).

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Figure 5 Effect of spray drying on bile tolerance and cholesterol assimilation by, Lactobacillus plantarum, Lactobacillus salivarius and Pediococcus acidilactici. The results are mean of four replicates, and standard deviations are indicated by error bars. Mean values of cholesterol assimilation of L. plantarum of both maltodextrin (MDX) and nonfat skimmed milk (NFSM) carriers show significant difference when compared to active cells (P ≤ 0.05). Mean values of cholesterol assimilation of L. salivarius NFSM carriers show significant difference when compared to active cells (P ≤ 0.05).

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common to use NFSM for the preparation ofspray-dried LAB, but the present investigation hasclearly demonstrated the effective use of MDX asa cost-effective, widely available carrier for thepreparation of spray-dried LAB.

Storage stability is another important factorfor the commercialization of LAB preparations.The results have clearly indicated the reasonablestability of three spray-dried probiotic LAB underthe specified conditions of 4°C for 60 days.

C O N C L U S I O N

In the present study, it was found that the con-centration of cell suspension had a significanteffect on the viability of spray-dried probioticLAB. The findings highlight the need to considerthe technological properties of probiotic strains;and provide additional data on the retention offunctional probiotic properties, in addition to theviability of cells, after spray drying. The resultsindicate that MDX was the better carrier for spraydrying than NFSM. The use of NFSM as a carrierresulted in spray-dried preparations of LAB withhigher percentages of survival; however it failed toprotect the functional properties of the probioticLAB. Based on these findings, it can be saidthat among the three cultures studied, percentagesurvival and retention of probiotic propertieswere significantly higher in the case of L.plantarum CFR 2191. It can be concluded thatenough scope exists for the preparation ofspray-dried L. plantarum CFR 2191 for food andpharma applications. However, in-depth studiesare needed to evaluate their performance duringboth pilot scale and ultimately industrial scalespray drying.

AC K N OW L E D G E M E N T S

The authors are grateful to Life Science ResearchBoard, Defence Research and DevelopmentOrganization, for funding the project. PraveenKumar Reddy is thankful to Council of Scientificand Industrial Research for the senior researchfellowship. The authors thank the Director ofCentral Food Technological Research Institute forsupporting the work. S.G. Jayaprakash, technician,food engineering department, is kindly acknow-ledged for his help in spray drying.

Figure 6 Percentage survival of spray-dried Lactobacillus salivarius (a), Pediococcusacidilactici (b) and Lactobacillus plantarum (c) with maltodextrin (MDX) and nonfat skimmed milk (NFSM) as a carrier during storage at 4°C (open triangle and diamond) and 30°C (closed triangle and diamond). The results are mean of four replicates. All mean scores show significant difference (P ≤ 0.05).

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