Food Microbiology 28 (2011) 1041e1047
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Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population Clemencia Chaves-López a, *, Annalisa Serio a, Maria Martuscelli a, Antonello Paparella a, Esteban Osorio-Cadavid b, Giovanna Suzzi a a b
Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Stazione (TE), Italy Departamento de Biología, Universidad del Valle, Calle 13 N 100-00, Cali, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2010 Received in revised form 7 February 2011 Accepted 21 February 2011 Available online 21 March 2011
Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main objective of this research was to studied the enterococcal population, present in 13 kumis samples traditionally manufactured, for their role as beneficial organisms or opportunistic pathogens. The molecular identification of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in enterococci, were detected in 30 isolates, while 42 were free of virulence determinants. Skim milk media were fermented by all the different isolates and further tested for proteolysis (free NH3 groups), Angiotensin-I Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE inhibitory activity, with E. faecalis KE09 as the best producer (IC50 ¼ 14.25 mg ml1). Moreover, the strains showed a very low tyrosine decarboxylase activity and did not produce histamine during 48 h fermentation in milk. This study underlines the that Colombian kumis is a good source of not virulent enterococci able to produce fermented milks with ACE-inhibitory activity. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Enterococcus Angiotensin converting Enzyme Biogenic amines Fermented milk Kumis
1. Introduction Colombian kumis is a fermented cow milk, widely consumed in rural and urban areas in South West Colombia. Traditionally, kumis is a home-made beverage produced by spontaneous fermentation of raw whole milk for 2 or 3 days depending on room temperature and milk producers. The product of this fermentation is a low alcoholic (1e2%), creamy and sparkling beverage, with a slight degree of sourness. It is stored at about 4e10 C and consumed within 3 days, adding sugar cane and cinnamon before serving. The remaining beverage is used as an inoculum for the following day. In traditional fermented milk products like Colombian kumis, fermentation has a symbiotic origin and depends on the action of two distinct microbial groups: 1) lactobacilli that are reported to play a major fermentative role affecting aroma, texture and acidity of the product, as well as being of some benefit to human health (Montanari et al., 1996) and 2) yeasts, whose presence is crucial for the desirable properties of carbon dioxide and ethanol (Narvhus and Gadaga, 2003). Moreover, also enterococci (Obodai and Dodd, 2006; * Corresponding author. Tel.: þ39 0 861 266913; fax: þ39 0 861 266915. E-mail address:
[email protected] (C. Chaves-López). 0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.02.006
Rahman et al., 2009) and Enterobacteriaceae (Gadaga et al., 1999; Kebede et al., 2007) are often found as contaminating bacteria. In general, research on the microbiota of fermented milk products is addressed to dominant populations as yeasts and lactobacilli, while studies on Enterococcus population are limited. Enterococcus species are often found in fermented milk products and may be present in relevant numbers (Giraffa, 2003), indicating that they can influence sensory characteristics as well as the safety of the product. In fact, enterococci seem to play an important role in the development of the sensory profile of milk products, due to their metabolic activity on citrate and proteins. Moreover, they can exhibit probiotic activity (Foulquié Moreno et al., 2006) and some strains have been used to produce fermented milk with hypotensive and/or Angiotensin-I-converting enzyme (ACE) inhibitory characteristics (Muguerza et al., 2006; Quiros et al., 2007; Regazzo et al., 2010). On the other hand, enterococci can also be opportunistic pathogens in humans, possessing some virulence factors (Mundy et al., 2000) allowing adherence to host tissue, invasion, and resistance to host defence mechanisms (Giraffa, 2003). In addition, enterococci can produce amino acid decarboxylases, and therefore are able to form biogenic amines (BAs) (Bover-Cid et al., 2001; Suzzi and Gardini, 2003). The production of biogenic
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amines and in particular histamine and tyramine is an undesirable property for selection of starter cultures (Buchenhüskes, 1993; Chamba and Jamet. 2008), due to the toxicological effect deriving from their vasoactive and psychoactive properties (Suzzi and Gardini, 2003). To the best of our knowledge, studies on the Enterococcus spp. involved during Colombian kumis production have never been performed so far. Thus, the aim of the present study was to identify and characterise the Enterococcus population in samples of traditional Colombian kumis collected from different areas of South West Colombia. In particular we investigated safety and positive aspects, like the production of peptides with Angiotensin I-converting enzyme (ACE) inhibitory activity, as well as for potential negative traits, such as the presence of several putative virulence factors, vancomycin resistance and the production of BAs. 2. Materials and methods 2.1. Samples collection Samples of traditional kumis fermented naturally, were collected from 13 different production sites located in the Valle del Cauca (South West Colombia). All samples were collected at the end of fermentation. Each sample (approximately 150 ml) was aseptically transferred to a 250 ml sterile screw-capped bottle and kept at 4 C until a maximum of 12 h before the analyses. 2.2. Characterization of Colombian kumis Ten ml of each sample were mixed with 90 ml of 0.85% (w/v) sterile physiological saline, and homogenised in a Stomacher Labblender 400 (Seward, UK) for 2 min. Appropriate serial dilutions in the same diluent were prepared. Lactic acid bacteria were enumerated on MRS agar (Oxoid Ltd, Cambridge, UK) and enterococci on Kanamycin Azide Agar (KAA), both incubated anaerobically by means of anaerobic jars and BBL GasPak anaerobic system envelopes (Becton Dickinson, Cockeysville, USA) at 37 C for 48 h. Potato Dextrose Agar (PDA) added with Chloramphenicol (Oxoid), incubated at 25 C for 72 h, was used for the enumeration of yeasts, and Violet Red Bile Glucose Agar (VRBGA) (Oxoid), incubated at 37 C for 24 h, was used for Enterobacteriaceae. Colonies grown on different substrates were randomly selected or all sampled if the plates contained less than 10 colonies. The purity of the isolates was checked by streaking again and subculturing on appropriate media. .For long term maintenance of isolates, stock cultures were stored at 80 C in 25% (v/v) of glycerol. The pH of each sample was measured by Mettler Toledo MP 220 pHmeter (Mettler Toledo, Spain). The occurrence of BAs in kumis samples was determined as described afterwards. 2.3. Identification and characterisation of presumptive enterococci from kumis Representative colonies on KAA were subcultured onto M17 agar media and tested for their morphology, Gram reaction, catalase, oxidase activity and growth at 10 and 45 C by standard methods. The DNA was extracted by means of Chelex 5% (Sigma Chemicals, St. Louis, Mo.) as previously described (Serio et al., 2007) Specific-PCR reactions with E. faecalis and E. faecium species-specific primers, as previously described (Dutka Malen et al., 1995), were performed. As reference strains, E. faecalis ATCC 19433, E. faecium ATCC 19434, E. durans ATCC 19432 and E. italicus DSM 15952 were employed. In order to confirm the obtained results and to identify the isolates which did not belong to the species E. faecalis and
E. faecium, the gene 16S rDNA of the isolates KE 03, KE 12, KE 68 was amplified as previously described (Marchesi et al., 1998) and then sequenced (BMR Genomics, Padua, Italy). For species attribution, the obtained sequences were matched with those present in the databank of NCBI (National Centre for Biotechnology Information), by means of BLAST (Basic Local Allignment Search Tool) program. Identification and presence of virulence factors as aggregation substance (asa1), gelatinase (gel E), cytolysin (cyl), enterococcal surface protein (esp) and hyaluronidase (hyl), and VanA or VanB genetic determinants for vancomycin resistance were performed as previously described (Serio et al., 2007). Frequency percentage analysis. In order to evidence the most frequent Enterococcus species in the samples, the method previously reported (Osorio-Cadavid et al., 2008) was performed. Briefly, colonies randomly collected from plates at the highest dilution give a high probability to pick up strains belonging to the dominant species (Pulvierenti et al., 2004). In order to study distribution species in our samples the method proposed by Solieri et al. (2006) has been performed. It was considered how many times each species was detected in the samples, without considering the strain number belonging to the species. In this way it was obtained the number of positive samples to each species and the corresponding frequency, defined as the number of sample positive to a species divided by the total number of samples expressed in percentage. Screening of skim milk proteolysis by Enterococcus strains. To evaluate the capability of the 72 strains to hydrolyse milk protein and produce peptides with ACE inhibitory activity, purified colonies were transferred into 4 ml of reconstituted skim milk (Biolife, Milan, Italy) and incubated overnight. Each pre-culture sample, prepared with a single bacterial strain, was inoculated (5% v/v) into triplicate 100 ml of pasteurised skim milk. Samples were analysed to determine pH, proteolysis (measured as free-NH3 groups by OPA method), ACE-inhibitory activity at 48 h of incubation at 28 C. Bacterial growth was determined by plating on M17 agar. The fermentation process was stopped by pasteurisation of fermented milk at 75 C for 1 min.
2.4. Dynamics of peptides with ACE inhibitory activity production and BA accumulation in vitro Among 72 Enterococcus strains, 3 E faecalis and 2 E. faecium were selected to evaluate the dynamics of ACE inhibitory activity production and BA accumulation. These strains were chosen due to their ability to produce fermented milk with high ACE inhibitory activity (> 63%), in absence of virulence factors and vancomycin resistence. Samples (250 ml of skim milk) were prepared as above described, and aliquots of 4 ml were periodically collected (0, 6, 10, 18, 24, 30, 36, and 48 h) for the following analyses: cell counts, pH, peptide content, ACE inhibitory activity and BAs accumulation. Assay for ACE inhibitory activity and peptides quantification. The measurement of ACE inhibitory activity was carried out spectrophotometrically using the pH 4.6 soluble fraction obtained by centrifugation (10.000 g 10 min at 4 C) as previously reported (Pan et al., 2005). Briefly the supernatant was filtered by ultrafiltration and then freeze-dried. The residual was dissolved in 1.0 ml of 0.1 M borate buffer containing 0.3 M NaCl (pH 8.3), and centrifuged. The supernatant was filtered using 0.45 mm PVDF filters. Then, 200 ml of HHL buffer (5 mM Hip-His-Leu in 0.1 M borate buffer containing 0.3 M NaCl, pH 8.3) were mixed with 80 ml of sample solution and pre-incubated for 3 min at 37 C. After addition of 20 ml of ACE (Rabbit lung ACE, dissolved in distilled water, 0.1 units/ml), the mixture was incubated for 30 min at 37 C. The reaction was stopped by adding 250 ml of 1.0 N HCl and mixed with 1.7 ml of ethyl acetate. The hippuric acid formed was extracted with
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ethyl acetate and the absorbance was measured at 228 nm. Unfermented skim milk was used as reference. ACE inhibitory activity index of the samples was calculated according to the formula:
ACE inhibitor activity index ¼ ½ðB AÞ=ðB CÞ 100%; where A is the absorbance in the presence of ACE and in presence of the ACE-inhibitory component, B is the absorbance with ACE and without the ACE-inhibitory component, C is the absorbance without ACE or ACE inhibitor component.The protein content of the samples was determined using DC protein assay (Bio-Rad Laboratories, CA, USA) with bovine serum albumin as standard. Peptide content was measured using o-phthaldialdehyde method as previously described by Church et al. (1983), briefly as follow: Fifty microlitres of the sample were added with 1 ml of OPA reagent (25 ml of 100 mM borax, 2.5 ml of 20% (w/w) SDS, 40 mg of ophthaldialdehyde solution -dissolved in 1 ml of methanol- and 100 ml of b-mercaptoethanol and then adjusted to 50 ml with deionised water). The reaction mixture was incubated for 2 min at ambient temperature, and the absorbance was measured spectrophotometrically at 340 nm. The peptide content was quantified using hydrolized casein as standard. Results were expressed as mg of hydrolysed casein peptone per ml of sample. Unfermented milk was used as blank and was subtracted from each sample value. Hydrolysis of the peptides by pepsin and pancreatin. To study both the peptides stability and the formation of new ACE-inhibitory peptides, aliquots of 5 ml of fermented skim milk, prepared as described for the screening of skim milk proteolysis, were subjected to hydrolysis. The samples were first hydrolysed with pepsin (EC 3.4.4.1; 1:60,000, 3400 U mg1) (Sigma Aldrich) at the following conditions: 20 mg pepsin g1 of protein, 37 C, pH 3.5, 4 h. The reaction was stopped by boiling water for 10 min and neutralised to pH 7.0 adding of NaOH solution (2N). The remained neutralised suspension was further digested with 40 mg g1 of pancreatin (EC 232-468-9; 800-2500 units/mg protein) at 37 C for 4 h, then the enzyme was inactivated by boiling for 10 min followed by cooling to room temperature and centrifuging (10.000 g x, 30 min). The supernatant was used for ACE inhibitory activity determination calculated as follows:
ACE inhibitor activity ¼ ðB AÞ=ðB CÞ 100%; where A is the absorbance in the presence of ACE and in presence of the ACE-inhibitory component, B is the absorbance with ACE and without the ACE-inhibitory component, C is the absorbance without ACE or ACE inhibitor component. This activity was also calculated as IC50. The IC50 value was defined as the concentration
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of peptide (mg/ml) required to reduce 50% of absorbance peak height of the hippuric acid, which was determined by regression analysis of ACE inhibition (%) versus protein concentration. Biogenic amines determination. The enterococci were screened for the capacity to produce BAs using the decarboxylase medium as previously described by Bover-Cid and Holzapfel, 1999). To quantify the BAs produced during the skim milk fermentation, aliquots (1.6 ml) of inoculated skim milk were analysed by HPLC procedure, after acid extraction and derivatization of samples as previously described (Martuscelli et al., 2005). For the determination of BAs in kumis, samples were homogenised (ratio 4:1) with 0.1 M HCl added with 100 mg 1 of internal standard (1,7-diaminoheptane, Sigma), and centrifuged at 1400 g at 4 C for 15 min. The supernatant was recovered and the extraction and derivatization were carried out as described above.
2.5. Statistical analyses All the experiments were carried out in triplicate and all the analyses were carried out in duplicates. The average and standard deviations were calculated for the experimental data, using analysis of variance (ANOVA). One-way ANOVA was used to compare mean values of the data following Tukey Test. Spearman’s rank correlation coefficient was used to evidence the possible correlation between ACE inhibition and free NH3 groups production during skim milk fermentation.
3. Results 3.1. Microbiota and chemical-physical characteristics of Colombian kumis Colombian kumis beverages from 13 different producers were analysed for microbial populations and for some phenotypic traits related to the safety and health of the product, such as ACE inhibitory activity and BA occurrence. Cell counts of microbiota isolated from the samples are shown in Table 1. Significant differences (p < 0.05) were found among the microbial groups determined in the different kumis samples. In particular, LAB and yeasts were the prevalent populations, ranging from 7.05 to 9.53 log CFU ml1 and from 6.26 to 8.65 log CFU ml1, respectively. Enterococci counts markedly varied between 4.29 and 8.30 log CFU ml1, while Enterobacteriaceae were present only in four samples at low numbers (log 2.10e3.70 CFU g1). Moreover, kumis samples had pH values ranging from 3.9 to 4.5.
Table 1 Cell numbers (log CFU g1) of the most important microbial groups founds in the traditional Colombian kumis. Sample
Yeast
Mesophilic LAB
Enterococci
Enterobacteriaceae
1 2 3 4 5 6 7 8 9 10 11 12 13
8.25 0.17aa 8.65 0.21a 7.00 0.14b 8.05 0.21ad 7.24 0.15b 6.26 0.17c 7.03 0.24b 7.85 0.12d 7.95 0.21d 7.74 0.19d 6.69 0.17c 6.79 0.15b 8.31 0.15a
9.31 0.27a 9.09 0.15a 8.60 0.15b 9.01 0.13a 7.42 0.53c 7.05 0.21c 9.59 0.23a 9.46 0.21a 8.56 0.21b 7.59 0.14c 8.80 0.14b 9.53 0.07a 9.37 0.34a
8.30 0.14a 7.95 0.22a 4.91 0.27b 4.29 0.16b 6.46 0.21c 4.47 0.06b 6.59 0.44c 4.29 0.29b 6.34 0.20c 5.28 0.14d 6.51 0.05c 6.60 0.14c 5.00 0.28d
3.21 n.da n.d n.d 2.10 n.d 3.70 n.d n.d n.d n.d n.d 3.40
0.16a
0.21b 0.31c
0.10a
pH 4.4 0.2a 4.1 0.1b 4.3 0.1a 3.9 0.2b 4.5 0.1a 4.1 0.1b 4.3 0.2a 4.2 0.1 ab 4.3 0.3 ab 4.3 0.2 ab 4.1 0.2a 4.2 0.1 ab 4.4 0.2a
n.d: not detected in 10 ml of sample. a Mean valuesstandard deviations for three batches of each sample of kumis analysed in duplicate. Different letters in the same column mean significant differences (p < 0.05) among the samples.
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ACE inhibitory activity of the peptides produced during kumis fermentation was evidenced in all the samples, with values ranging from 32.21% to 65.72% (data not shown), whereas the total BA content ranged from not detectable values to 15.31 mg l1. In particular, ethylamine, putrescine, cadaverine and spermidine were detected (Fig. 1). 3.2. Identification and characterisation of the enterococci isolates Among the different microbial groups, the study focused on enterococci to evaluate their role in kumis. Seventy-two isolates of presumptive enterococci obtained from KAA were phenotypically characterised. All the isolates showed the typical characteristics commonly used to identify enterococci. By means of speciesspecific primers, 57 isolates were identified as E. faecalis (79.0%) and 12 as E. faecium (17.0%). By sequencing the gene 16S rRNA, the other isolates were identified as 2 E. serioliocida (synonimous of Lactococcus garviae; access numbers: KE03: HM573319; KE12: HM573320) (3.0%), 1 Vagococcus penaei (Access number KE68: HM573318) (1.0%). All strains were analysed for the presence of several known virulence determinants, by using PCR. The major part of the isolates (42 of 75) were free of virulence factors, while 33 isolates harboured at least one gene (Fig. 2). In particular, all the E. faecium strains from kumis did not possess the virulence genes searched, with the exception of KE13, positive for cyl A gene. The most frequent gene was esp (8 isolates), encoding for an extracellular protein. In addition, 14 isolates harboured two genes, the most widespread combination being asa1 and cyl, present in 8 E. faecalis. On the other hand, only 5 strains possessed the gene gelE (alone or in combination), encoding for gelatinase, while none of the enterococci from kumis had the hyl gene. It is noteworthy that enterococci with code from KE50 and KE62, isolated from kumis samples of the same region and showing a high homology degree (data not shown), possessed one or more virulence genes, in particular the association asa1 and cylA. Among enterococci from kumis, the vancomycin resistance detected by VanA- and VanB- specific PCR was very limited. In fact, only E. faecium KE49 possessed the gene Van A, encoding for the resistance to vancomycin and teicoplanin, while KE02 and KE 13, showed the gene VanB, determining resistance to vancomycin but not to teicoplanin.
16
14
BAs content (mg.liter -1 )
12
8 7 positive strains (number)
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6 5 4 3 2 1 0 as
a1
cy
lA
es
p
lE ge as
a1
+
cy
lA
as
a1
+
p es as
a1
+g
E el
l cy as
+e
a1
sp
+
cy
lA
+
es
p
Va
nA
Va
nB
Virulence genes Fig. 2. Occurrence of virulence determinants among enterococcal isolates from , Enterococcus spp . Colombian kumis. E. faecalis , E. faecium , E. seriolicida
3.3. Fermentation of skim milk by the Enterococcus species All the enterococcal strains were tested in skim milk to determine their fermentative capacity and to evaluate the production of BAs and of peptides with ACE inhibitory activity. The pH decreased during fermentation, with intra and interspecies variations. In particular, the pH values of skim milk ranged from 4.25 to 5.20 in the samples fermented by E. faecalis, from 4.49 to 4.96 by E. faecium, and from 4.81 to 4.96 by E. seriolicida. The cell counts at the end of fermentation ranged from 7.5 Log CFU ml1 to 8.3 log CFU ml1 (data not shown), confirming that all the strains carried out fermentation. Table 2 shows the pH values, the peptide content as OPA index, and the ACE-inhibitory activity index (of the eleven strains that were shown to possess ACE-inhibitory activity after 48 h fermentation at 28 C). In particular, 9 strains of E. faecalis reduced between 39.67% and 82.01% the activity of ACE and hydrolysed skim milk proteins producing from 1.20 mg ml1 to 2.45 mg ml1 peptides. Unfortunately, 6 of these strains were positive for one or two virulence factors, as reported in Table 2. On the other hand, the production of peptides with ACE-inhibitory activity in the samples fermented by E. faecium was limited to the strains KE01 and KE73, with values of 84.35 and 65.83% respectively, and peptide content of 2.40 and 1.24 mg ml1 respectively. The two E. seriolicida strains did not show any ACE-inhibitory activity. Statistical analysis did not show any correlation between free NH3 groups and ACE-inhibitory activity.
10
3.4. ACE-inhibitory activity dynamics in skim milk 8
6
4
2
Median 25%-75% Non-Outlier Range Outliers Extremes
0 eth
put
cad
spd
tot
Fig. 1. Biogenic amines content in 13 traditional Colombian kumis samples at consuming time (after 3 days of fermentation).
The five strains listed in Table 2, characterised by the absence of all the investigated virulence factors and by the production of ACEinhibitory peptides from 63.20%, were evaluated for the ACEinhibitory peptides production dynamics. Subsequent to the inoculum (5.3e5.8 log CFU ml1) an increase in cell counts was observed for all the strains after 6 h of fermentation with the maximum levels (7.2e7.6 log CFU ml1) after 30 h for E. faecalis KE06 and KE09 and for E. faecium KE01. The cell growth of all the strains determined a low reduction of pH during the first 10 h of fermentation. After this period to 24 h, there was a significant (p < 0.05) pH drop (1.0e1.5 units), particularly for E. faecalis KE09, E. faecalis KE06 and E. faecium KE01 (data not shown).
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pH
E. faecalis KE 02 4.54 0.03 KE 06 4.45 0.01 KE 09 4.52 0.02 KE 17 4.38 0.01 KE 36 4.25 0.03 KE 40 4.34 0.01 KE 45 4.52 0.01 KE 59 4.57 0.01 KE 61 4.41 0.02 E. faecium KE01 4.76 0.01 KE73 4.49 0.01
OPA Index (mg ml1)
ACE index (%)
1.38 0.17 1.78 0.22 2.45 0.11 1.79 0.31 1.44 0.25 1.73 0.19 2.00 0.15 1.73 0.27 1.20 0.18
76.09 71.98 82.01 63.20 55.40 59.92 69.73 52.88 39.67
2.40 0.35 1.24 0.21
84.35 4.28 65.83 3.76
4.21 2.47 4.35 2.24 3.03 2.48 3.35 1.87 2.41
Virulence factor VanB n.d n.d n.d gelE asa1 Esp asa1 cylA gelE cylA asa1
2 1,8
-1
Strain
a Peptide content (mg.mliter )
Table 2 Characteristics of skim milk fermented by E. faecalis and E. faecium after 48 h at 28 C and virulence factor of the strains.
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1,6 1,4 1,2 1 0,8 0,6 0,4 0,2
n.d n.d
0 0
10
b
30
40
50
40
50
100 90 80
ACE inhibition (%)
A similar trend in the evolution of free NH3 groups was observed in all the strains with an increase until stationary phase, but at a low rate. Also in this case, the most acidifying strains E. faecium KE01 and E. faecalis KE09 showed higher values of free-NH3 groups during the first 10 h (Fig. 3a). The increase in the peptide content was often accompanied by an increase in ACE-inhibitory activity of the product. In fact, in all the samples the ACE-inhibitory activity grew until the late log phase to stationary phase (30 h) with different production rates; after that, a significant activity decrease was observed (Fig. 3b). The best ACE-inhibitory peptide production, with a significantly higher average content (P < 0.05), was observed in skim milk fermented by the strain E. faecium KE01, whereas the lowest values were determined for E. faecalis KE17. The Spearman’s rank test revealed a significant positive correlation between values of free NH3 groups and ACE-inhibitory activity only for the strains E. faecalis KE09 and E. faecium KE73 (Sr ¼ 0.82; p ¼ 0.041).
20
Fermentation time (hours)
n.d: not detected.
70 60 50 40 30 20 10 0 0
10
20
30
Fermentation time (hours) Fig. 3. Evolution of peptide contents and ACEI activity in fermented milks. Changes of peptide content (a) and inhibitory activity of angiotensin I-converting enzyme (b) , during skim milk fermentation at 28 C. E. faecium KE01:, E. faecalis KE06 , E. faecalis KE17 , E. faecium KE73 . E. faecalis KE09
3.5. Hydrolysis under simulated gastrointestinal conditions To select strains of enterococci for functional kumis production, the stability of the peptides and of the ability to form new ACEinhibitory peptides after a simulate physiological digestion were determined in all the five strains. With this aim, pepsin and pancreatin digestion of supernatant of fermented skim milk was performed (Fig. 4). Except for E. faecalis KE09, all strains showed a significant increase of ACE-inhibitory activity after hydrolysis with pepsin. After the subsequent incubation with pancreatin, ACEinhibitory activity slightly increased in the samples inoculated with strains E. faecium KE01 and E. faecalis KE09 and KE17, and decreased with strains E. faecalis KE06 and E. faecium KE73. Mainly, under simulated gastrointestinal conditions, the skim milk samples fermented by E. faecium KE01, E. faecalis KE06, KE09 and KE17 possessed an increased ACE-inibitory activity. To evaluate the power of peptide activities after simulated gastrointestinal digestion, IC50 was determined. Although this value can be overestimated in presence of free amino acids interfering with the calculation of the peptide concentration and, more generally, due to the possible breakdown of the large peptides resulting from the ACE activity (Minervini et al., 2003), this value can be considered a useful tool to compare the different fermented milks. Before digestion, the IC50 values for E. faecalis KE06, KE09 and KE17 were 18.12 0.73 mg ml1, 14.25 1.0 mg ml1, and 28.12 0.8 mg ml1 respectively, while those for E. faecium KE01 and KE73 were 15.63 1.11 mg ml1 and 16.44 1.2 mg ml1 respectively. It is interesting to point out that the sample fermented by E. faecalis KE09 showed the highest ACE-inhibitory activity
(82.01 4.35), and the best activity power, as evidenced by the low IC50 values (14.25 mg ml1). 3.6. Kinetics of biogenic amines production A matter of concern with respect to the possible negative treats of enterococci is indeed the production of BA. For this reason, BA production in skim milk during 48 h fermentation by five selected Enterococcus strains was investigated. The preliminary screening for decarboxylase activity (data not shown), carried out in synthetic medium in presence of the BA precursors, gave a positive result for phenylalanine, ornithine, tryptamine and lysine decarboxylation in all the strains. Tyrosine was decarboxylated by all the strains except for E. faecalis KE09 and E. faecium KE73. Finally, histidine decarboxylation test was negative for all the strains. The quantification of BAs produced during skim milk fermentation evidenced the presence of very low quantities of these compounds (lower than 8.43 mg L1) and in particular of spermidine, ethylamine, phenyletylamine and tyramine. Fig. 5 shows the kinetics of production of tyramine, the most abundant BA, by the selected Enterococcus isolates during 48 h of skim milk fermentation. 4. Discussion In this study we report that enterococci constitute together with other LAB and yeasts the main microbial groups of traditional Colombian kumis. In other traditional fermented milks, such as
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ACE inibitory activity (%)
90 80 70 60 50 40 30 20 10 0 E. faecium KE01
E. faecalis KE06
E. faecalis KE09
E. faecalis KE17
E. faecium KE73
Strain
Fig. 4. ACEI activity after “in vitro” digestion of fermented skim milk. Inhibitory activity of angiotensin I-converting enzyme during simulated physiological digestion of fermented skim milk with Enterococcus spp at 28 C by 30 h. Undigested-, with pepsin ; with pepsin þ pancreatine .
Gariss (Abdelgadir et al., 2008), Amasi (Gran et al., 2003), Nyarmie (Obodai and Dodd, 2006) and Koumiss (Hao et al., 2010), Enterococcus spp. are frequently reported, although their counts are not specified. The high levels of this species in the different samples of Colombian kumis suggest that enterococci could play an important role in the characteristics of this product, probably through proteolysis, lipolysis and citrate breakdown, hence contributing to their typical taste and flavour. This is the first report about Enterococcus species associated with Colombian kumis, and according to our findings E. faecalis is the dominant species, followed by E. faecium, as also reported in other dairy fermented products (Giraffa, 2003). During milk fermentation, the main chemical changes are lactic acid production and proteolysis of some milk proteins. With this respect, although Enterococcus species are not generally considered highly proteolytic microorganisms (Macedo et al., 2000; Sarantinopoulos et al., 2001; Serio et al., 2010), in this study the strains from Colombian kumis seemed to possess a somewhat proteolytic activity and the differences of free NH3 group amounts measured during skim milk fermentation might be related to the different proteinases of the strains. In fact, in E. faecalis, proteinases able to hydrolise caseine, bovine seroalbumine, b-lactoglobuline e a-lactoalbumine were distinguished (Hegazi, 1990; García de Fernando et al., 1991; Pritchard and Coolbear, 1993).
10 8
-1
Tyramine (mg.liter )
9 7 6 5 4 3 2 1 0 0
6
12
18
24
30
36
42
48
54
Fermentation time (hours) Fig. 5. Tyramine content during skim milk fermentation. Kinetics of tyramine production during skim milk fermentation by E. faecium and E. faecalis isolated from Colombian kumis at 28 C. E. faecium KE01:; E. faecalis KE06 ; E. faecalis KE09 ; E. faecalis KE17 ; E. faecium KE73 .
Proteolysis of milk by hydrolysis or fermentation involves the production of peptides that may exhibit different biological activities, such as antihypertensive, immunomodulating, osteoprotective, antilipemic, opiate, antioxidative and antimicrobial activities (Möller et al., 2008). Among the antihypertensive peptides, ACEinhibitory peptides have been proved to reduce blood pressure (Mundy et al., 2000), for this reason, foods containing these peptides can be regarded as co-adjuvant in the treatment of mild hypertension (Seppo et al., 2003). In this context, some Colombian kumis samples showed values of ACE-inhibitory activity up to 65%, these values are similar to those reported by Chen et al. (2010) in Koumiss (a traditional fermented mare’s milk). It is reported that the type of bacteria used as a starter can be one of the major factors influencing the synthesis of bioactive peptides in dairy products, and the protein substrate seems to be important for ACE-inhibitors production (Gobetti et al., 2002; Leclerc et al., 2002). On the other hand, in this work it was evidenced that milk fermented individually with some strains of E. faecalis (9) and E. faecium (2), showed ACE inhibition activities with values between 40% and 84%, indicating that Enterococcus species could contribute to the values of ACE-inhibitory activity in Colombian kumis. Our results evidenced also an increase of the ACE-inhibitory activity of the samples after simulated gastrointestinal digestion; these skim milks may contain ACE-inhibitor precursors which can lead to the release of ACE-inhibitory peptides during the simulated digestion. Noticeable are the values of IC50 observed in this study after digestion at 30 h of fermentation, in fact they were lower than those reported previously by Muguerza et al. (2006) using different strains of E. faecalis (34e59 mg ml1). As evidenced by the data on the production kinetics of ACE-inhibitory peptides, the fermentation of milk by Enterococcus seems to be prone to a dynamic system where peptides are constantly released; some of them are subsequently hydrolysed and probably utilised for cell growth, while others accumulate over fermentation. In fact, for all the strains a maximum ACE-inhibitory activity was detected after 30 h of fermentation, followed by an activity decrease. A major production of ACE-inhibitors for 3 strains of E. faecalis during the first 24 h, with a significant reduction at 48 h has been previously reported (Quirós et al., 2007). On the other hand, the maximum ACE-inhibitory activity after 3 h of fermentation, and a decrease after 6 h for the strains of L. casei, S. thermophilus and L. debruekii subsp bulgaricus has been reported (Ramachandran and Shah, 2008.). Recently, several strains of enteroccoci with bioactive potential and satisfying stringent technological characteristics as well, have already been evaluated and used as starters of the production of fermented milk with hypotensive and/or angiotensin-I-converting enzyme (ACE)-inhibitory activity (Muguerza et al., 2006; Quirós et al., 2007; Regazzo et al., 2010). However in these studies virulence factors and BAs production were not considered. In our study, 5 strains that did not harbour any of the virulence factors and did not carry VanA or VanB determinants, were able to produce fermented milks showing considerable ACE-inhibitory activity, and produced very low quantities of tyramine in skim milk. Although decarboxylase-positive strains were isolated from kumis samples, only limited quantities of biogenic amines were detected in final products; moreover, in spite of the abundance of enterococci population, histamine, tyramine and phenyethylamine were never detected. Data regarding BAs in fermented milk are scarce (Maga, 1978), however, it should be underlined that in this type of products casein degradation is lower with respect to cheese, thus resulting in a reduced availability of amino acid substrates, generally decreasing BAs formation (Shihata and Shah, 2000). In fact, some enterococci are able to form tyramine in milk only when tyrosine is abundant (Martuscelli et al., 2005). In conclusion, our findings indicate that the E. faecalis and E. faecium strains, with their particular metabolic profiles,
C. Chaves-López et al. / Food Microbiology 28 (2011) 1041e1047
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