HOi NGHI KHOA HQC C O N G N G H E SINH HQC T O A N Q U O C 2013
IDENTIFICATION OF A POTENTIAL PROBIOTIC STRAIN. LACTOBACILLUS GASSERIHA4, FROM NATURALLY FERMENTED FOODS
Phu-Ha Ho'-^ and Michelle Catherine VVllls^ „ - .
^Sdiod of Biotechnology and Food Techndogy, Hanoi Univerdty of Sdence and Techndogy
^Schod of Environmental and Life Sciences, University of Neif/castie, Australia
S U M M A R Y
Naturally fermented foods have been studied as sources of lactic acid producing bacteria; however there is limited knowledge on these foods as sources of probiotic bacteija. In das study, we have isolated and identified a lactobacillus strain based on its probiotic functionality. Fermented food was suspended of De Man- Rogosa- Shatpe (MRS) broth, exposed to acidic MRS broth (pH 2.5) and MRS brodi contaming 1% bile salts consecutively. After exposure, the samples were plated onto MRS agar. A potential lactobacilli isolate was then tested for survival In simulated gastric and small intestinal juices. This strain was biochemically identified using its carbohydrate fermentation patterns, The identificalion was verified h y molecular methods using partial I6S rRNA gene sequence, genus specific and species specific PCR assays. The strain was examined for adhesiveness onto human colon cancer cell lines Caco- 2, and then it was mvestigated for health promoting ability. The new selected potential probiotic strain Ladobadllus gasseri HA4 was found to produce a proteinaceous antibacterial compound against Listeria monocytogenes and to competitively reduce adhesion of this food bom patbogen onto Caco-2 cell lines. Its potential application as a probiotic demonstrates naturally fermented foods are a useful source of probiotic microorganisms, and may be more efficacious than strains isolated from a human or animal source.
Keywords: baleriocin-like compound, fermented food, Lactobacillus, probiotic
INTRODUCTION
The sourclng of potential commercial probiotics has been controversial. In recent decades there has been a focus on the isolation of these organisms from the animal host of intended use, with considerable success IdenlHied by the number of these products that have reached commerda! reality. Naturally fermented foods are uncommon In most western cultures, having been replaced wilh products developed using specific commercial strains of starter cultures, primarily selected on their Industry suitability and affect on organoleptic properties, rather than their ability to exert any particular health benefit on the consumer. In East -Asia, however, naturally fermented" marine, cereal,, meat, and vegetable based foods have been used for thousands yearS) and are- still readily available. These foods. Including : sikhae and kimchi from Korea;
miso and narezushi from Japan; pla-ra, kungchao, and nham from Thailand; nem-chua and'dua-muoi from Vietnam;
tempeh from Indonesia; and lempoyak from Malaysia, are common dishes In daily meals. The bacteria from these fermented foods have been studied, but primarily fi^m the view of food technological characteristics and food preservation qualities, such as lactic acid or bacteriocin pn>duction. Recent findings that bacteria isolated from Asian fermented foods demonstrate certain probiotic traits has lead to renewed interest in foods as a source of these organisms (Hur ef aL, 2004).
This study Identifies a potentially novel probiotic lactobacilli strain isolated from Vietnamese fermented vegetables, examines its probiotic qualities, and compares certain traits of the bacterium to a commercial lactobacilli strain isolated fr^)m. human feeces. Significantly this research identifies naturally fenmented food as a polendally rich, yet underestimated source, of novel new probiotic organisms.
MATERIAL AND METHOD
Isolation and screening bacteria from fermented foods
Probiotic screening was undertaken using Vietnamese naturally fennented radish leaves. The bacterial isolation method has been described previously (Pennacchia ef ai.. 2004). Colonies were characterized morphologically and purified on MRS agar. Gram positive and catalase negabve bacilli were maintained in MRS broth with 10% glycerol and stored at - 80°C. _ - ' Sunrtval test of Isolates from simulated gastric and small intestinal Juices . v j y i , . In vitro gastrointestinal survival was tested according to the method described by Huang et at (2004). Centrifuged pellets from 1 ml of 24h culture of each bacterial isolate in MRS broth were washed three times with Phosphate Buffered Saline (PBS) and exposed to gastric juice or small intestinal juice in final count of ca, 10^ cfumf'', and then Incubated at37°C.
Samples were taken at 1 min, 90 min and 180 min and plate counted on MRS agar in duplicate. Sunfl'val bacteria after go min or 180 min assay were expressed relative to the log of bacteria count at 1 miri.
Identification of a potential probloHe bacterium
Based upon in vitro gastrointestinal tolerance a single (.aclodaciffi/s strain was selected for fiirther analysis. Identification of this Lactobadllus strain was Initially undertaken using the API 50CHL kit (Blo-Merieux). -Confirmation of identification was then undertaken using molecular analysis based on partial 16S rRNA gene sequencing, and genus- and species- cnecific PCR assays. Approximately 430 bps of bacterial 163 rRNA gene was amplified using universal primers 968f and 1401 r in PCR condition as described in the study by Nubel et a/. (1996). PCR product was purified using QIA Quick Spin Kit (Qiagen) and then size verified by 1% w/v agarose gel electrophoresis. The DNA sequencing'was undertaken using an MegaBACE DNA sequencer (Amersham) based on the Sanger method. Inquiry for DNA similarity was peri'ormed
HOI NGHI KHOA HQC C O N G NGHe.SiNH HOC T O A N Q U 6 C 2013
using Genomic BLAST from Gen-Bank database fhttD://www ncbi nim nih gov/BLAST) identifying strains L johnsonii NCC 533 (accession NC_005362) and L gasseri ATCC 33323 (accession NC_008530) as having the highest homologies. The genus identification was confimied as Lactobacillus by genus specific PCR assay as described in the study by Dubemet et a l . (2002) using primers LbLMAI-rev and R16-1 wilh expected band o f 250 bps. The species was verified using species specific PCR assays. The method for Lactobadllus acidophilus and L gasseri was described by Ktjfon et al. (2004) using pnmers IDL04F and IDL22R with an expected band of 606 bps, and primers IDL42R and IDL04F with an expected band of 272 bps, respectively. For L johnsonii the meBiod descnljed by Ventura and Zink (2002) was undertaken, using primer set L I I , 338r and P6 with two bands of 1471 and 1192 bps identifying L. johsonll speciesandthepresenceof one band of 1192 bps indicative of other species.
A d h e s i o n onto human epithelial cell lines Caco-2
The human gut epithelial cell line, Caco-2, was grown in RPMI medium (Gibco) supplemented witti 2 0 % Fetal Bovine serum (Gibco). An ovemighl broth culture of the selected bacterium was centrifuged at 20000 x g for 5 min, washed three times witti PBS bufler, and tiien resuspended in PBS to acquire i t f cfu m\'\ F w tiie adhesion assay, monolayers of Caoo-2 was prepared by inoculating 1x10* cells per well tn 24-wetI tissue culture plate until post-confluent for seven days. The adhesion assay was conducted as described by Huang et al. (2004). The adhered bacteria were viewed using scanning electronic microscopy (Riilips XL-30).
Antimicrobial assay
The Isolated lacbsbadlli strain was cultijred in MRS media (Oxoid) at 37° C under anaerobic conditions. Indicator bactena used In this stijdy were.' Usteria monocytogenes ATCC 7644, Pseudomonas aeroginosa A T C C 27853, Yersinia enlerocditica ATCC 9650, EschericNa co/(0157'H7 VT-N NTCC 12900; Staphylococcus aureus ACM 4988, Salmonella typhimurium ATCC 14028, and Enterococd 306R. Antimicrobial activity of Uie lactobacilli w a s screened using the spot lest metiiod as described by Fernandez et al. (2003). The plates were incubated at 37° C aerobicaily for 24 h. Inhibition zones were observed and measured.
The nature of the active antimicratilal substance produced by the lactobacilli was determined b y t h s well difiijsion method. 50 pi of prepared ladobadtli supematant was placed into each well, with 50 ii\ of MRS broth used as negative conlrol. A solution o f 0.25 mg m f ' nisin from Streptococcus lactis (Sigma) was used as positive conlrol. Clear rings of more Ihan 1 mm were recorded as positive. The dear ring o f neub'alized and catalase treated supematant was recorded as supernatant containing bacteriodrv-like substance. Protease sensitivity of bacteriocin-like substance was investigated as described by Bromberg ef al. (2005). Ceil-ti^e supematant of a 24h lactobacilli culhjre was neutralized, heated In boiling water for five minutes, cooled to the room temperatijre and subjected to tiie ti-eatment with proteolytic enzymes.
The test enzymes were bypsin (Sigma) in 40 mM Tris-HCl, pH 8.2; a-chymotrypsin (Sigma), in 20 m M Tris-HCI, pH 8.0;
pronase E (Sigma) in 20 mM Tris-HCI. pH 8.0; and pepsin (ICN) in 0.002 M HCI in tiie concentration of 0.2g ml"'. The mter-stenle enzymes solutions were added Io cell-free supematant at pH 7.0 a l ratio of 1/1 (v/v). T h e controls i n d u d e d a set of tested enzyme solutions alone and the cell-free supematant in 0.1 M sodium phosphate buffer. All samples and cdribDis. were Incubated at 37° C for 2 h and tiien were healed In boiling water for 5mln to inactivate tiie enzymes.
Subsequentiy, they were tested for antibacterial actiirfty using well diffusion metiiod as described previously witii the indicator bacterium L. monocytogenes ATCC 7644. The original neuti"alized cell-free supematant was set as positive - contiol for tiie antibacterial assay.
Determlttatlon and c o m p a r i s o n o f the competitive adhesion potential o f the i s o l a t e d lactobacilli s t r a i n a n d a commercial human originated probiotic strain w f t h L. monocytogenes ATCC 7644
A commerdally available probiotic strain of L^tobadllus fermentum, previously isolated fi^m a human source, was selected as a comparison to the ladobadlli isolated in this study. This commercial sti-ain was described in company literature -as demonsti3ting "unrivalled pattiogen fighting ability". The adhesion assay w a s conducted using tiie metiiodology described by Hirano e t a/ (2003) witii modifications. Post-confluent Caco-2 cell monolayers were prepared and washed ttiree limes with PBS buffer (pH7.4). Overnight culhires of botti lactobacilli and L. monocytogenes cultures were centrifuged at 1700 x g for 15 min at 4° C, then washed three times wUh PBS (pH7.4). These bactenal pellets were resuspend in PBS to obtain a final concentration between 1x10* and 1x10^ cfu ml"'. Then 1ml of each baderial suspension was added into the prepared tissue culture wells in duplicate in following anangements: lactobacilli was added first incubated for one hour, then L monocytogenes was added; lactobacilli and L monocytogenes added at tiie same time; L monocytogenes was added, incubgted for one hour, then added lactobadlli. L monocytogenes was inoculated into Uie tissue culture wells independentiy as the conti-ol. After total Incubatron time of three hours Uie tissue culture monolayers were washed three times with PBS, and then were detached from the culture plate by adding 1 ml of trypsm/EDTA solution (Gibco). The detached layers were serially diluted and plated onto Listerial selective agar (Oxold) to detennine the number of adhering L monocytogenes.
Statistical analysis
An^ysis of variance was measured using a paired t-tesl, tw tails wiUi Uie level o f significant measured at a P level of 0.05 (Microsoft Office Excel 2003).
RESULTS AND DISCUSSION
IdentificaHon of a potential probiotic Lactobacillus strain
Bacteria rf different morphological types were obtained from Uie VieUiamese food after exposure to MRS with tow pH ' and bile salts (results not shown). For this shjdy. one Lacfodaciffus sfrain » - - -
HOI NGH! KHOA HQC C O N G N G H E SINH HQC TOAN QU6C 2013
klenfafied as I-IA4, was identified as a Ladooadlli sp, based o p o i r i t i j uolonymorphotogy-on-MRS-agarr-Sram-and—
catalase reaction, its selection for further analysis was determined by survivability under In vitro simulated gastrointestinal conditions (Rgure 1).
No significant difference in viability of HtA4 was found after 90 min o r 180 min Ueatment in gastiic juice at pH 3.0 (P >
0.05). Viability at pH 2 and 2.5 was significantiy affected compared to pH 3 (P < 0.05), however I-1A4 maintained at c a . 4 log cfu/ml after 180 min exposure a l pH 2.0. The isolate was affected by presence of bile salt In simulated small intestinal
\\iKe. In the jUKe without bile salts, tiiere was no significant redudion in bacterial count after 180 min (P > 0.05). HA4 viability decreased In the presence of bile salts, however, r e f i n e d nearly 6 log cfii/ml after 180 min of treatment (Rgure
Figure 1. Survive of HA4 In simulated gastric Juice and small Intestinal Juice.
A B, C, gastric juices pH 2.0,2.5.3.0 respectively D, E: small intestinai juices with and without 0.3% bile salts respecbvsly.
Results are ^ o w n as means. Enor bar is standani enor of means.
n=3.
HA4 was affected b y low pH (Figure 1), however, survivabinty at low pH can also be enfianced through the use of a food matrix that buffers that bacteria through the stomach (Michida et al., 2006). Regardless, HA4 did retein a high level o f viability under the test conditions, and tor this reason was fUrUiOT analyzed for its potential probiotic application.
Strain HA4 adhered o n t o Caco-2 cell lines
It is known that probiotics need to colonize Uie gastrdnteslinal t r a d to exert a positive health benefit, and Uie first stage of this is adherence. HA4 demonsfa^ted adherence onto human Caco-2 epithelial cell lines (Rgure 2). Jacobsen ef al.
(1999) previously reported thai survival and reisolation of lactobadlli in human Gl h a d was strongly con-elated to their in vitio pH 2.5_tolerance and Caco-2 adhesion.
Identification o f potential probiotic L a c t o n e / / / / s p . HA4
l i A 4 was Mentified as Lactobadllus addophilus accoroing to Ihe API 5DCHL (Bio-M4rieux) biochemical fermenteUon profile, virith a good identification (97.1%). Using genus spedfic primers, a 250 bp PCR p r o d u d (Figure 3) confirmed the genus as Ladobadllus. however, Is insufficient to discriminate homology groups. Bacteria, previously known as L addof^ilus, have been reported as a highly heterogeneous group by DNA homology and other p h e n o l i c tests. This Acidophilus group now is dassifled Into six subgroups known as L addophilus, L amylovonis, L. crispatus. L galllnanim. L. johnsonii and L gasseri (Fujisawa etal.. 1992). "In Uiis stody. using 16s rRNA gene prifners, a PCR "
produd of 430 bp was amplified and subsequentiy sequenced. The obtained nucleotide sequence was demonsti'ated as ha\^ng a 9 8 % (404/411 identities) similarity to Lactobadllus jc^nsonil NCC 533 and 98% (403/411 identities) similarity t o Ladobadllus gasseri ATCC 33323 fhtto://www.ncbi.nlm.nih.qov/BLASTl. There are two species; L johnsonS and L ga^eri, in llteratore are reported very similar in genotype (Venhrra ef al., 2002). Considering tiiis complexity, in our shJdy, PCR assays were conducted with sets o f spedes-spedfic primers for L. addophilus, L. johnsonii, and L gassel species as described above.Ttie resulting PCR products from stiain HA4 did not show any spedfic band wiUi L acidophilus specific primers (date not shown), only one band of 1200 bps wilh L. johnsonii specific primers and a specific 270 bp band witii L. gasseri spedfic primers (Figure 3). TTiis result suggested that tiie isolated sti^in belonged to L.
gasseri spedes. Confirmation by anoUier reliable molecular metiiod should not be disregarded. NeverUieless, tiie fact HA4 belonged to ttie genus Lacft>bac///us supported its appropriateness in application as a probiotic sti^in.
A n t i m i c r o b i a l activity o f HA4 and characteristics o f active c o m p o u n d
HA4 demonstiated a broad spectrum of antimicrobial actiirily toward indicator organisms with diameters of inhibitory rings in tiie range ftom 10 mm {Listeria monocytogenes ATCC 7644) to 30 mm {Yersinia enterocolitica ATCC 9650) w h i d i in most cases w a s likely to be due to a pH effect This was confirmed when tiie neuti-allsed and catalase tieated supematant was relesled against tiie test bacteria. Only L monocytogenes remained sensitive (Figure 4). To determine if ttie antimicrobial action was due to a bacteriocin, ttie supematant was taeated wiUi trypsin, Q-chymoti^)sin, pronase E, and pepsin, and once again retested against ttie L monocytogenes (Figure 5). The lack of inhibition confimned the action to be the result of a baderiocin. The performance of the bacteriocin-iike substance showed that it likely betonged to tiie Class lla bacteriodns, which are descnbed to have quite limited spectrum on Gram positive bacteria (Dnder ef al.. 2006).
- i monocytogenes has also been reported to be Uie mpsl sensitive baderia to bacteriocins of ttiis Class.
HOI NGHI KHOA HQC C6HG N G H ^ SINH HOC T O A N Q U O C 2013
Fleure 3, Agarose gel electrophoresis of PCR products.
Lane 1: PCR produd (250 tq>s) of HA4 genome with Lactobacillus genus specific pdmeis. U n e 2: Sln^e band of PCR produd (1200 bps) of HA4 genome witti Ljohnsot^specdie primers. l.ane 3:
PCR produd (270 bps) of HA4 genome *Wi L gasserf specific primers. Lane M: lOObp DIM ladder.
Figure 4. Inhibition of L. monocytogenes by HA4 catalase treated supemalant (well 1), neutralized supernatant (well 2). and non-treated supematant (well 3). No activity was showi by MRS brolh (well 4) and InhibiSon activily was shown by 25 mg ml' of Nisin (well 5).
Figure 5. Proteinase sensitivity f t-monocytogenes Figure 6. Adhesion of tested lactic acid bacteria and L.
InhlbHory activity of HA4 supernatant monocytogenes.
C l : tiypsin solution, C2: a-chymotrypsin solution, C3: pronase E A' Count of L monocytogenes in exposure to strain L. gasseri sduHon, C4: pepsin solution. 1, 2, 3, 4;'neutralized HA4 HA4 B Count L monoc^ogenes in exposure to the commercial supematant treated with trypsin, a-chymotrypsin, pronase E, and- L fermentum. Results are shown as means. Error bar Is standard pepsin, respectively. 5: neutraSzetf supem^ant treated with 0.1M error of means, n=2.
sodium phosphate buffer, 6 non-trmRsd supematanL
Competitive adhesion o f Lactobacilli strains w i t h L. monocytogenes o n Caco-2 cell lines
Treahnent w t t i HA4 significantiy reduced Uie number of L. monocytogenes tiiat adhered onto Caco-2 cell lines b y more than 1 log cfu ml"' (P < 0.05). There was no significant difference in L. monocytogenes count when o f HA4 was added betore, after or al Uie same time witii L monocytogenes (P>0.05). When Uie commercial L. fermentum was tested, tiie L monocytogenes count was slightiy reduced, however, with no stetistical significance (P>0.05) (Figure 6). Potential for HA4 to inhibit L. monocytogenes identified a specific application for I-1A4 as a probiotic. In addition, the f a d that HA4 actively adhered to human epithelial cells suggested that this strain may have synergistic properties in pathogen inhibition, and a competitive adherence assay was underteken against L. monocytogenes. Addition o f HA4 reduced I . monocytogenes adherence to human epitiielial cells by more than 1 log cfu/ml. Interestingly, under our study conditions • tiie commercial L feimantum, which was reported isolated from the human samples, showed no significant reduction of L. monocytogenes adhesion. What is evident in this study is Ihe food based isolate did perfoim better under t i i e test conditions, even though the dalms made on the commercial strain Indicate that "no other probiotic has demonstrated the capacity to Inhibit such a range of patiiogens". Antimicrobial activity was described In lactic acid baderia isolated from Vietnamese ti^adilionally femiented foods, e g fmm "nem chua'(Phan et al., 2005). However, to tiie best knowledge of tiie autiiors. no data on antilisteria! activity by tiie means of competitive adhesion was reported.
CONCLUSION
From tills stody. it is wortii considering the limitations of examining only animal hosts as sources of potential probiotics.
This shidy has successfully demonsh^led Uiat Vietnamese fermented vegetables potentially contein probiotic bacteria, ttie properties of which are a l least as useftil as tiiose found inprobiotics from human origin. Witii Uie added benefit that baderia ftom natiJiaily fennented products may be intrinsically suitable for reapplication into foods, these products as a source of probiotics should not be discounted.
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PHAN LAP VI KHUAN T|£M NANG PROBIOTIC. Lactobacillus gasseri HA4, TO"
THU'C PHAM LEN MEN T y NHI^N
H 6 Phii Hk*'' vk Michelle Catherine W i l l s '
V / # n Cdng ngh$ sinh hpc vk Cdng ngh$ tiii/c ph&m, Tnrdng Dal hgc Bich khoa Ha N^i
^Trudng B9I hgc Newcastle. Australia
T6MTAT . _ ^ , _ . , . .
Tbife phim len men t^ nhiSn ti nguon cung cSp vi khuin lactic ddi dao; tiq' ohifti vhi c6n chua c6 nhi^u ngbi6n.ciiu sau sSc vi thifc phim len men vdi vai tro la nguin vi khuan probiotic. Trong nghien ciiu niy, chung tai da phan l$p vi djnh tfin dugc chiiag vi khuan lactic dva trSn m&t s6 dac tinh probiotic. MSu tiivc phim 16n men duyc ho^t h6a tron| m6i tnrdng De Man- Rogosa- Shatpe (MRS) d i ^ ctilnh pH d^n 2,5; sau S6 ho(it h6a trong MRS c6 chiia 1% mu6i m§t Sau khi tiSp xiic v6i hai mdi tmerag nay, vi khuAn dugc * phan l?p trSn mfii trudng MRS rSn. Vi khuln lactic phan Igp dirge xac djnh khd nSng sing s6t qua dich d? diy vi djch ni$t gii l?p.
CMng niy dagc djnh tSn bing phuong phap hoa sinh, sau dfi dugc kiem tra lai bang phircmg phap giii trinh ti.r gen 16S ARN, phan una PCR vdi cjip moi dac hi|u djnh t6n gifing va ten loii. Cbong nay sau do dugc tiiir nghigm khi nSng bim dinh tren ddng tc bio biSu m6 ni$t nguftr Caco-2 v i dinh gii cac d^c tinh c6 Igi cbo site khde. Cluing vi kbuln tiSm nfing probiotic mdi Itfa chgn Lactobacillus gassen HA4 c6khi ning i?o ra hgp chit khing vi-sinh v i i b i n chit protein khang List^a monocytogenes v i cd khi nang cpnh tranh lira giam sg bim dinh cila vi khuln niy tren Caco-2. TiSm ning sir dyng vi khuln mdi phln li_p nhu m^t probiotic cho tlily thirc p l ^ 'en men tif nhifin la nguin probiotic doi dio, v i thim chi cd the hi?u qua hon chiing vi khuan phin l?p tir ngudi hole dSng v^t.
Titkitda: Hgp chit Wong tf bateriocta, thgc phim len men, Lactobadllus, probiodc.
i. Email: ha [email protected]
