Microbiological Characteristics of Kumis, a Traditional Fermented Colombian Milk, with Particular Emphasis on Enterococci Population

Food Microbiology xxx (2011) 1e7

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Food Microbiology

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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 Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Stazione (TE), Italy b Departamento de Biología, Universidad del Valle, Calle 13 N 100-00, Cali, Colombia article info abstract

Article history: Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main Received 8 October 2010 objective of this research was to studied the enterococcal population, present in 13 kumis samples Received in revised form traditionally manufactured, for their role as beneﬁcial organisms or opportunistic pathogens. The 7 February 2011 molecular identiﬁcation of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the Accepted 21 February 2011 dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in entero- Available online xxx cocci, 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 NH groups), Angiotensin-I Keywords: 3 Enterococcus Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two Angiotensin converting Enzyme E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to Biogenic amines 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE 1 Fermented milk inhibitory activity, with E. faecalis KE09 as the best producer (IC50 ¼ 14.25 mgml ). Moreover, the Kumis 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.

1. Introduction Rahman et al., 2009) and Enterobacteriaceae (Gadaga et al., 1999; Kebede et al., 2007) are often found as contaminating bacteria. Colombian kumis is a fermented cow milk, widely consumed in In general, research on the microbiota of fermented milk rural and urban areas in South West Colombia. Traditionally, kumis products is addressed to dominant populations as yeasts and lac- is a home-made beverage produced by spontaneous fermentation tobacilli, while studies on Enterococcus population are limited. of raw whole milk for 2 or 3 days depending on room temperature Enterococcus species are often found in fermented milk products and milk producers. The product of this fermentation is a low and may be present in relevant numbers (Giraffa, 2003), indicating alcoholic (1e2%), creamy and sparkling beverage, with a slight that they can influence sensory characteristics as well as the safety degree of sourness. It is stored at about 4e10 C and consumed of the product. In fact, enterococci seem to play an important role in within 3 days, adding sugar cane and cinnamon before serving. The the development of the sensory profile of milk products, due to remaining beverage is used as an inoculum for the following day. their metabolic activity on citrate and proteins. Moreover, they can In traditional fermented milk products like Colombian kumis, exhibit probiotic activity (Foulquié Moreno et al., 2006) and some fermentation has a symbiotic origin and depends on the action of strains have been used to produce fermented milk with hypoten- two distinct microbial groups: 1) lactobacilli that are reported to sive and/or Angiotensin-I-converting enzyme (ACE) inhibitory play a major fermentative role affecting aroma, texture and acidity of characteristics (Muguerza et al., 2006; Quiros et al., 2007; Regazzo the product, as well as being of some benefit to human health et al., 2010). On the other hand, enterococci can also be opportu- (Montanari et al., 1996) and 2) yeasts, whose presence is crucial for nistic pathogens in humans, possessing some virulence factors the desirable properties of carbon dioxide and ethanol (Narvhus and (Mundy et al., 2000) allowing adherence to host tissue, invasion, Gadaga, 2003). Moreover, also enterococci (Obodai and Dodd, 2006; and resistance to host defence mechanisms (Giraffa, 2003). In addition, enterococci can produce amino acid decarboxylases, and

* Corresponding author. Tel.: þ39 0 861 266913; fax: þ39 0 861 266915. therefore are able to form biogenic amines (BAs) (Bover-Cid et al., E-mail address: [email protected] (C. Chaves-López). 2001; Suzzi and Gardini, 2003). The production of biogenic

Please cite this article in press as: Chaves-López, C., et al., Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population, Food Microbiology (2011), doi:10.1016/j.fm.2011.02.006 2 C. Chaves-López et al. / Food Microbiology xxx (2011) 1e7 amines and in particular histamine and tyramine is an undesirable E. faecium, the gene 16S rDNA of the isolates KE 03, KE 12, KE 68 was property for selection of starter cultures (Buchenhüskes, 1993; amplified as previously described (Marchesi et al., 1998) and then Chamba and Jamet. 2008), due to the toxicological effect deriving sequenced (BMR Genomics, Padua, Italy). For species attribution, from their vasoactive and psychoactive properties (Suzzi and the obtained sequences were matched with those present in the Gardini, 2003). databank of NCBI (National Centre for Biotechnology Information), To the best of our knowledge, studies on the Enterococcus spp. by means of BLAST (Basic Local Allignment Search Tool) program. involved during Colombian kumis production have never been Identification and presence of virulence factors as aggregation performed so far. Thus, the aim of the present study was to identify substance (asa1), gelatinase (gel E), cytolysin (cyl), enterococcal and characterise the Enterococcus population in samples of tradi- surface protein (esp) and hyaluronidase (hyl), and VanA or VanB tional Colombian kumis collected from different areas of South genetic determinants for vancomycin resistance were performed as West Colombia. In particular we investigated safety and positive previously described (Serio et al., 2007). aspects, like the production of peptides with Angiotensin I-con- Frequency percentage analysis. In order to evidence the most verting enzyme (ACE) inhibitory activity, as well as for potential frequent Enterococcus species in the samples, the method previ- negative traits, such as the presence of several putative virulence ously reported (Osorio-Cadavid et al., 2008) was performed. Briefly, factors, vancomycin resistance and the production of BAs. colonies randomly collected from plates at the highest dilution give a high probability to pick up strains belonging to the dominant 2. Materials and methods species (Pulvierenti et al., 2004). In order to study distribution species in our samples the method proposed by Solieri et al. (2006) 2.1. Samples collection has been performed. It was considered how many times each species was detected in the samples, without considering the strain Samples of traditional kumis fermented naturally, were number belonging to the species. In this way it was obtained the collected from 13 different production sites located in the Valle del number of positive samples to each species and the corresponding Cauca (South West Colombia). All samples were collected at the end frequency, defined as the number of sample positive to a species of fermentation. Each sample (approximately 150 ml) was asepti- divided by the total number of samples expressed in percentage. cally transferred to a 250 ml sterile screw-capped bottle and kept at Screening of skim milk proteolysis by Enterococcus strains.To 4 C until a maximum of 12 h before the analyses. evaluate the capability of the 72 strains to hydrolyse milk protein and produce peptides with ACE inhibitory activity, purified colonies 2.2. Characterization of Colombian kumis were transferred into 4 ml of reconstituted skim milk (Biolife, Milan, Italy) and incubated overnight. Each pre-culture sample, Ten ml of each sample were mixed with 90 ml of 0.85% (w/v) prepared with a single bacterial strain, was inoculated (5% v/v) into sterile physiological saline, and homogenised in a Stomacher Lab- triplicate 100 ml of pasteurised skim milk. Samples were analysed blender 400 (Seward, UK) for 2 min. Appropriate serial dilutions in to determine pH, proteolysis (measured as free-NH3 groups by OPA the same diluent were prepared. Lactic acid bacteria were method), ACE-inhibitory activity at 48 h of incubation at 28 C. enumerated on MRS agar (Oxoid Ltd, Cambridge, UK) and entero- Bacterial growth was determined by plating on M17 agar. The cocci on Kanamycin Azide Agar (KAA), both incubated anaerobically fermentation process was stopped by pasteurisation of fermented by means of anaerobic jars and BBL GasPak anaerobic system milk at 75 C for 1 min. 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, 2.3. Dynamics of peptides with ACE inhibitory activity and Violet Red Bile Glucose Agar (VRBGA) (Oxoid), incubated at production and BA accumulation in vitro 37 C for 24 h, was used for Enterobacteriaceae. Colonies grown on different substrates were randomly selected or all sampled if the Among 72 Enterococcus strains,3E faecalis and 2 E. faecium were plates contained less than 10 colonies. The purity of the isolates was selected to evaluate the dynamics of ACE inhibitory activity checked by streaking again and subculturing on appropriate media. production and BA accumulation. These strains were chosen due to .For long term maintenance of isolates, stock cultures were stored their ability to produce fermented milk with high ACE inhibitory at 80 C in 25% (v/v) of glycerol. activity (> 63%), in absence of virulence factors and vancomycin The pH of each sample was measured by Mettler Toledo MP 220 resistence. Samples (250 ml of skim milk) were prepared as above pHmeter (Mettler Toledo, Spain). described, and aliquots of 4 ml were periodically collected (0, 6, 10, The occurrence of BAs in kumis samples was determined as 18, 24, 30, 36, and 48 h) for the following analyses: cell counts, pH, described afterwards. peptide content, ACE inhibitory activity and BAs accumulation. Assay for ACE inhibitory activity and peptides quantification. The Identification and characterisation of presumptive measurement of ACE inhibitory activity was carried out spectro- enterococci from kumis photometrically using the pH 4.6 soluble fraction obtained by centrifugation (10.000 g 10 min at 4 C) as previously reported Representative colonies on KAA were subcultured onto M17 agar (Pan et al., 2005). Briefly the supernatant was filtered by ultrafil- media and tested for their morphology, Gram reaction, catalase, tration and then freeze-dried. The residual was dissolved in 1.0 ml oxidase activity and growth at 10 and 45 C by standard methods. of 0.1 M borate buffer containing 0.3 M NaCl (pH 8.3), and centri- The DNA was extracted by means of Chelex 5% (Sigma Chemicals, St. fuged. The supernatant was filtered using 0.45 mm PVDF filters. Louis, Mo.) as previously described (Serio et al., 2007) Specific-PCR Then, 200 ml of HHL buffer (5 mM Hip-His-Leu in 0.1 M borate reactions with E. faecalis and E. faecium species-specific primers, as buffer containing 0.3 M NaCl, pH 8.3) were mixed with 80 mlof previously described (Dutka Malen et al., 1995), were performed. As sample solution and pre-incubated for 3 min at 37 C. After addition reference strains, E. faecalis ATCC 19433, E. faecium ATCC 19434, of 20 ml of ACE (Rabbit lung ACE, dissolved in distilled water, 0.1 E. durans ATCC 19432 and E. italicus DSM 15952 were employed. units/ml), the mixture was incubated for 30 min at 37 C. The In order to confirm the obtained results and to identify the reaction was stopped by adding 250 ml of 1.0 N HCl and mixed with isolates which did not belong to the species E. faecalis and 1.7 ml of ethyl acetate. The hippuric acid formed was extracted with

Table 1 Cell numbers (log CFU g 1) of the most important microbial groups founds in the traditional Colombian kumis.

Sample Yeast Mesophilic LAB Enterococci Enterobacteriaceae pH 1 8.25 0.17aa 9.31 0.27a 8.30 0.14a 3.21 0.16a 4.4 0.2a 2 8.65 0.21a 9.09 0.15a 7.95 0.22a n.da 4.1 0.1b 3 7.00 0.14b 8.60 0.15b 4.91 0.27b n.d 4.3 0.1a 4 8.05 0.21ad 9.01 0.13a 4.29 0.16b n.d 3.9 0.2b 5 7.24 0.15b 7.42 0.53c 6.46 0.21c 2.10 0.21b 4.5 0.1a 6 6.26 0.17c 7.05 0.21c 4.47 0.06b n.d 4.1 0.1b 7 7.03 0.24b 9.59 0.23a 6.59 0.44c 3.70 0.31c 4.3 0.2a 8 7.85 0.12d 9.46 0.21a 4.29 0.29b n.d 4.2 0.1 ab 9 7.95 0.21d 8.56 0.21b 6.34 0.20c n.d 4.3 0.3 ab 10 7.74 0.19d 7.59 0.14c 5.28 0.14d n.d 4.3 0.2 ab 11 6.69 0.17c 8.80 0.14b 6.51 0.05c n.d 4.1 0.2a 12 6.79 0.15b 9.53 0.07a 6.60 0.14c n.d 4.2 0.1 ab 13 8.31 0.15a 9.37 0.34a 5.00 0.28d 3.40 0.10a 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.

ACE inhibitory activity of the peptides produced during kumis 8 fermentation was evidenced in all the samples, with values ranging from 32.21% to 65.72% (data not shown), whereas the total BA 7 1 content ranged from not detectable values to 15.31 mg l .In 6 particular, ethylamine, putrescine, cadaverine and spermidine were detected (Fig. 1). 5

4 3.2. Identification and characterisation of the enterococci isolates 3 Among the different microbial groups, the study focused on enterococci to evaluate their role in kumis. Seventy-two isolates of 2 presumptive enterococci obtained from KAA were phenotypically (number) strains positive 1 characterised. All the isolates showed the typical characteristics commonly used to identify enterococci. By means of species- 0 fi fi 1 lA p lE A p p A B speci c primers, 57 isolates were identi ed as E. faecalis (79.0%) and a y s e l s E sp s c e g cy e el es e an an a + g + V V 12 as E. faecium (17.0%). By sequencing the gene 16S rRNA, the other + + yl + 1 a1 1 c lA sa s sa y fi a a a c isolates were identi ed as 2 E. serioliocida (synonimous of Lacto- + 1 coccus garviae; access numbers: KE03: HM573319; KE12: sa a HM573320) (3.0%), 1 Vagococcus penaei (Access number KE68: Virulence genes HM573318) (1.0%). All strains were analysed for the presence of several known Fig. 2. Occurrence of virulence determinants among enterococcal isolates from virulence determinants, by using PCR. The major part of the isolates Colombian kumis. E. faecalis , E. faecium , E. seriolicida , Enterococcus spp . (42 of 75) were free of virulence factors, while 33 isolates harboured at least one gene (Fig. 2). In particular, all the E. faecium 3.3. Fermentation of skim milk by the Enterococcus species strains from kumis did not possess the virulence genes searched, with the exception of KE13, positive for cyl A gene. The most All the enterococcal strains were tested in skim milk to deter- frequent gene was esp (8 isolates), encoding for an extracellular mine their fermentative capacity and to evaluate the production of protein. In addition, 14 isolates harboured two genes, the most BAs and of peptides with ACE inhibitory activity. widespread combination being asa1 and cyl, present in 8 E. faecalis. The pH decreased during fermentation, with intra and inter- On the other hand, only 5 strains possessed the gene gelE (alone or species variations. In particular, the pH values of skim milk ranged in combination), encoding for gelatinase, while none of the from 4.25 to 5.20 in the samples fermented by E. faecalis, from 4.49 enterococci from kumis had the hyl gene. It is noteworthy that to 4.96 by E. faecium, and from 4.81 to 4.96 by E. seriolicida. The cell 1 enterococci with code from KE50 and KE62, isolated from kumis counts at the end of fermentation ranged from 7.5 Log CFU ml to 1 fi samples of the same region and showing a high homology degree 8.3 log CFU ml (data not shown), con rming that all the strains (data not shown), possessed one or more virulence genes, in carried out fermentation. particular the association asa1 and cylA. Table 2 shows the pH values, the peptide content as OPA index, Among enterococci from kumis, the vancomycin resistance and the ACE-inhibitory activity index (of the eleven strains that detected by VanA- and VanB- specific PCR was very limited. In fact, were shown to possess ACE-inhibitory activity after 48 h fermen- only E. faecium KE49 possessed the gene Van A, encoding for the tation at 28 C). In particular, 9 strains of E. faecalis reduced resistance to vancomycin and teicoplanin, while KE02 and KE 13, between 39.67% and 82.01% the activity of ACE and hydrolysed 1 1 showed the gene VanB, determining resistance to vancomycin but skim milk proteins producing from 1.20 mg ml to 2.45 mg ml not to teicoplanin. 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 16 fermented by E. faecium was limited to the strains KE01 and KE73, with values of 84.35 and 65.83% respectively, and peptide content 1 14 of 2.40 and 1.24 mg ml respectively. The two E. seriolicida strains did not show any ACE-inhibitory activity. 12 Statistical analysis did not show any correlation between free NH groups and ACE-inhibitory activity. ) 3 -1 10 ter i 3.4. ACE-inhibitory activity dynamics in skim milk mg.l

( 8 The five strains listed in Table 2, characterised by the absence of 6 all the investigated virulence factors and by the production of ACE- inhibitory peptides from 63.20%, were evaluated for the ACE- BAs content 4 inhibitory peptides production dynamics. Subsequent tothe inoculum (5.3e5.8 log CFUml 1) an increase in 2 cell counts was observed for all the strains after 6 h of fermentation Median 1 25%-75% with the maximum levels (7.2e7.6 log CFU ml ) after 30 h for Non-Outlier Range 0 E. faecalis KE06 and KE09 and for E. faecium KE01. The cell growth of Outliers Extremes all the strains determined a low reduction of pH during the first 10 h eth put cad spd tot of fermentation. After this period to 24 h, there was a significant < e Fig. 1. Biogenic amines content in 13 traditional Colombian kumis samples at (p 0.05) pH drop (1.0 1.5 units), particularly for E. faecalis KE09, consuming time (after 3 days of fermentation). E. faecalis KE06 and E. faecium KE01 (data not shown).

Table 2 a 2 Characteristics of skim milk fermented by E. faecalis and E. faecium after 48 h at 28 C and virulence factor of the strains. 1,8

1 ) Strain pH OPA Index (mg ml ) ACE index (%) Virulence factor 1,6 -1

E. faecalis 1,4 KE 02 4.54 0.03 1.38 0.17 76.09 4.21 VanB KE 06 4.45 0.01 1.78 0.22 71.98 2.47 n.d 1,2 KE 09 4.52 0.02 2.45 0.11 82.01 4.35 n.d 1 KE 17 4.38 0.01 1.79 0.31 63.20 2.24 n.d KE 36 4.25 0.03 1.44 0.25 55.40 3.03 gelE asa1 0,8 KE 40 4.34 0.01 1.73 0.19 59.92 2.48 Esp KE 45 4.52 0.01 2.00 0.15 69.73 3.35 asa1 0,6 KE 59 4.57 0.01 1.73 0.27 52.88 1.87 cylA gelE Peptide(mg.mliter content 0,4 KE 61 4.41 0.02 1.20 0.18 39.67 2.41 cylA asa1 E. faecium 0,2 KE01 4.76 0.01 2.40 0.35 84.35 4.28 n.d KE73 4.49 0.01 1.24 0.21 65.83 3.76 n.d 0 0 1020304050 n.d: not detected. Fermentation time (hours)

b 100

A similar trend in the evolution of free NH3 groups was observed 90 in all the strains with an increase until stationary phase, but at 80 a low rate. Also in this case, the most acidifying strains E. faecium 70 KE01 and E. faecalis KE09 showed higher values of free-NH3 groups during the first 10 h (Fig. 3a). 60 The increase in the peptide content was often accompanied by 50 an increase in ACE-inhibitory activity of the product. In fact, in all 40 the samples the ACE-inhibitory activity grew until the late log 30 phase to stationary phase (30 h) with different production rates; ACE inhibition (%) after that, a significant activity decrease was observed (Fig. 3b). The 20 best ACE-inhibitory peptide production, with a significantly higher 10 average content (P < 0.05), was observed in skim milk fermented by 0 the strain E. faecium KE01, whereas the lowest values were deter- 01020304050 mined for E. faecalis KE17. The Spearman’s rank test revealed Fermentation time (hours) a significant positive correlation between values of free NH3 groups and ACE-inhibitory activity only for the strains E. faecalis KE09 and Fig. 3. Evolution of peptide contents and ACEI activity in fermented milks. Changes of ¼ ¼ peptide content (a) and inhibitory activity of angiotensin I-converting enzyme (b) E. faecium KE73 (Sr 0.82; p 0.041). during skim milk fermentation at 28 C. E. faecium KE01:, E. faecalis KE06 , E. faecalis KE09 , E. faecalis KE17 , E. faecium KE73 . 3.5. Hydrolysis under simulated gastrointestinal conditions

To select strains of enterococci for functional kumis production, (82.01 4.35), and the best activity power, as evidenced by the low m 1 the stability of the peptides and of the ability to form new ACE- IC50 values (14.25 gml ). inhibitory peptides after a simulate physiological digestion were determined in all the five strains. With this aim, pepsin and 3.6. Kinetics of biogenic amines production pancreatin digestion of supernatant of fermented skim milk was performed (Fig. 4). Except for E. faecalis KE09, all strains showed A matter of concern with respect to the possible negative treats a significant increase of ACE-inhibitory activity after hydrolysis of enterococci is indeed the production of BA. For this reason, BA with pepsin. After the subsequent incubation with pancreatin, ACE- production in skim milk during 48 h fermentation by five selected inhibitory activity slightly increased in the samples inoculated with Enterococcus strains was investigated. The preliminary screening for strains E. faecium KE01 and E. faecalis KE09 and KE17, and decreased decarboxylase activity (data not shown), carried out in synthetic with strains E. faecalis KE06 and E. faecium KE73. Mainly, under medium in presence of the BA precursors, gave a positive result for simulated gastrointestinal conditions, the skim milk samples fer- phenylalanine, ornithine, tryptamine and lysine decarboxylation in mented by E. faecium KE01, E. faecalis KE06, KE09 and KE17 all the strains. Tyrosine was decarboxylated by all the strains except possessed an increased ACE-inibitory activity. for E. faecalis KE09 and E. faecium KE73. Finally, histidine decar- To evaluate the power of peptide activities after simulated boxylation test was negative for all the strains. fi gastrointestinal digestion, IC50 was determined. Although this The quanti cation of BAs produced during skim milk fermenta- value can be overestimated in presence of free amino acids inter- tion evidenced the presence of very low quantities of these 1 fering with the calculation of the peptide concentration and, more compounds (lower than 8.43 mg L ) and in particular of spermi- generally, due to the possible breakdown of the large peptides dine, ethylamine, phenyletylamine and tyramine. Fig. 5 shows the resulting from the ACE activity (Minervini et al., 2003), this value kinetics of production of tyramine, the most abundant BA, by the can be considered a useful tool to compare the different fermented selected Enterococcus isolates during 48 h of skim milk fermentation. milks. Before digestion, the IC50 values for E. faecalis KE06, KE09 and KE17 were 18.12 0.73 mgml1, 14.25 1.0 mgml1, and 4. Discussion 28.12 0.8 mgml 1 respectively, while those for E. faecium KE01 and KE73 were 15.63 1.11 mgml1 and 16.44 1.2 mgml1 In this study we report that enterococci constitute together with respectively. It is interesting to point out that the sample fermented other LAB and yeasts the main microbial groups of traditional by E. faecalis KE09 showed the highest ACE-inhibitory activity Colombian kumis. In other traditional fermented milks, such as

100 Proteolysis of milk by hydrolysis or fermentation involves the

90 production of peptides that may exhibit different biological activities, such as antihypertensive, immunomodulating, osteoprotective, 80 antilipemic, opiate, antioxidative and antimicrobial activities 70 (Möller et al., 2008). Among the antihypertensive peptides, ACE-

60 inhibitory peptides have been proved to reduce blood pressure (Mundy et al., 2000), for this reason, foods containing these 50 peptides can be regarded as co-adjuvant in the treatment of mild 40 hypertension (Seppo et al., 2003). In this context, some Colombian 30 kumis samples showed values of ACE-inhibitory activity up to 65%,

ACE inibitory activity (%) these values are similar to those reported by Chen et al. (2010) in 20 Koumiss (a traditional fermented mare’s milk). It is reported that 10 the type of bacteria used as a starter can be one of the major factors 0 influencing the synthesis of bioactive peptides in dairy products, E. faecium KE01 E. faecalis KE06 E. faecalis KE09 E. faecalis KE17 E. faecium KE73 and the protein substrate seems to be important for ACE-inhibitors Strain production (Gobetti et al., 2002; Leclerc et al., 2002). Fig. 4. ACEI activity after “in vitro” digestion of fermented skim milk. Inhibitory activity On the other hand, in this work it was evidenced that milk of angiotensin I-converting enzyme during simulated physiological digestion of fer- fermented individually with some strains of E. faecalis (9) and mented skim milk with Enterococcus spp at 28 C by 30 h. Undigested-, with pepsin E. faecium (2), showed ACE inhibition activities with values þ ; with pepsin pancreatine . 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 Gariss (Abdelgadir et al., 2008), Amasi (Gran et al., 2003), Nyarmie activity of the samples after simulated gastrointestinal digestion; (Obodai and Dodd, 2006) and Koumiss (Hao et al., 2010), Entero- these skim milks may contain ACE-inhibitor precursors which can coccus spp. are frequently reported, although their counts are not lead to the release of ACE-inhibitory peptides during the simulated specified. The high levels of this species in the different samples of digestion. Noticeable are the values of IC observed in this study Colombian kumis suggest that enterococci could play an important 50 after digestion at 30 h of fermentation, in fact they were lower than role in the characteristics of this product, probably through prote- those reported previously by Muguerza et al. (2006) using different olysis, lipolysis and citrate breakdown, hence contributing to their strains of E. faecalis (34e59 mgml 1). As evidenced by the data on typical taste and flavour. This is the first report about Enterococcus the production kinetics of ACE-inhibitory peptides, the fermenta- species associated with Colombian kumis, and according to our tion of milk by Enterococcus seems to be prone to a dynamic system findings E. faecalis is the dominant species, followed by E. faecium, where peptides are constantly released; some of them are subse- as also reported in other dairy fermented products (Giraffa, 2003). quently hydrolysed and probably utilised for cell growth, while During milk fermentation, the main chemical changes are lactic others accumulate over fermentation. In fact, for all the strains acid production and proteolysis of some milk proteins. With this a maximum ACE-inhibitory activity was detected after 30 h of respect, although Enterococcus species are not generally considered fermentation, followed by an activity decrease. A major production highly proteolytic microorganisms (Macedo et al., 2000; of ACE-inhibitors for 3 strains of E. faecalis during the first 24 h, with Sarantinopoulos et al., 2001; Serio et al., 2010), in this study the a significant reduction at 48 h has been previously reported (Quirós strains from Colombian kumis seemed to possess a somewhat et al., 2007). On the other hand, the maximum ACE-inhibitory proteolytic activity and the differences of free NH group amounts 3 activity after 3 h of fermentation, and a decrease after 6 h for the measured during skim milk fermentation might be related to the strains of L. casei, S. thermophilus and L. debruekii subsp bulgaricus different proteinases of the strains. In fact, in E. faecalis, proteinases has been reported (Ramachandran and Shah, 2008.). able to hydrolise caseine, bovine seroalbumine, b-lactoglobuline e Recently, several strains of enteroccoci with bioactive potential and a-lactoalbumine were distinguished (Hegazi, 1990; García de satisfying stringent technological characteristics as well, have already Fernando et al., 1991; Pritchard and Coolbear, 1993). 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; 10 Regazzo et al., 2010). However in these studies virulence factors and 9 BAs production were not considered. In our study, 5 strains that did 8

) not harbour any of the virulence factors and did not carry VanA or VanB -1 7 determinants, were able to produce fermented milks showing 6 considerable ACE-inhibitory activity, and produced very low quantities of tyramine in skim milk. Although decarboxylase-positive strains 5 were isolated from kumis samples, only limited quantities of biogenic 4 amines were detected in ﬁnal products; moreover, in spite of the

Tyramine (mg.liter 3 abundance of enterococci population, histamine, tyramine and phe- 2 nyethylamine were never detected. Data regarding BAs in fermented 1 milk are scarce (Maga, 1978), however, it should be underlined that in 0 this type of products casein degradation is lower with respect to 0 6 12 18 24 30 36 42 48 54 cheese, thus resulting in a reduced availability of amino acid Fermentation time (hours) substrates, generally decreasing BAs formation (Shihata and Shah, 2000). In fact, some enterococci are able to form tyramine in milk Fig. 5. Tyramine content during skim milk fermentation. Kinetics of tyramine only when tyrosine is abundant (Martuscelli et al., 2005). production during skim milk fermentation by E. faecium and E. faecalis isolated from ﬁ Colombian kumis at 28 C. E. faecium KE01:; E. faecalis KE06 ; E. faecalis KE09 ; In conclusion, our ndings indicate that the E. faecalis and E. faecalis KE17 ; E. faecium KE73 . E. faecium strains, with their particular metabolic proﬁles,

Please cite this article in press as: Chaves-López, C., et al., Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population, Food Microbiology (2011), doi:10.1016/j.fm.2011.02.006 C. Chaves-López et al. / Food Microbiology xxx (2011) 1e7 7 possessed a great variability in ACE-inhibitory production, Macedo, A.C., Vieira, M., Pocas, R., Malcata, F.X., 2000. Peptide hydrolase system of contributing to the ACE-inhibitory activity of Colombian kumis. lactic acid bacteria isolated from Serra de Estrela cheese. International Dairy Journal 10, 764e774. Moreover, after 30 h of milk fermentation with E. faecalis KE09, free Maga, J.A., 1978. Amines in foods. CRC Critical Reviews in Food Science and Nutri- of virulence determinants, significant quantities of peptides with tion 12, 373e403. ACE-inhibitory activity were detected, while very low quantities of Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., Wade, W.G., 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify ethylamine were produced, and no tyramine and histamine were genes coding 16S rRNA. Applied EnvironmentalMicrobiology 64, 795e799. determined. Therefore E. faecalis KE09 could represent an inter- Martuscelli, M., Gardini, F., Torriani, S., Mastrocola, D., Serio, A., Chaves-López, C., esting biotype for potential industrial applications. Schirone, M., Suzzi, G., 2005. Production of biogenic amines during the ripening e fi of Pecorino Abruzzese cheese. International Dairy Journal 15, 571 578. To the best of our knowledge, this is the rst report of ACE- Minervini, F., Algaron, F., Rizzello, C.G., Fox, P.F., Monnet, V., Gobbetti, M., 2003. inhibitory activity in milk fermented with E. faecium. These results Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial peptides from clearly encourage to carry out further research, to evaluate the Lactobacillus helveticus PR4 p.oteinase-hydrolysed caseins of milk from Six species. Applied and Environmental Microbiology 69, 5297e5305. performance of these enterococci strains in presence of LAB and Möller, N.P., Scholz-Ahrens, K.E., Roos, N., Schrezenmeir, J., 2008. Bioactive peptides yeasts populations in real systems. and proteins from foods: indication for health effects. European Journal of Nutrition 47, 4171e4182. Montanari, G., Zambonelli, C., Grazia, L., Kamesheva, G.K., Shigaeva, M.K.H., 1996. References Saccharomyces unisporus as the principal alcoholic fermentation microorganism of traditional koumiss. Journal of Dairy Research 63, 327e331. Abdelgadir, W., Nielsen, D.S., Siddig, H., Jakobsen, M., 2008. A traditional Sudanese Muguerza, B., Ramos, M., Sánchez, E., Manso, M.A., Miguel, M., Aleixandre, A., fermented camel’s milk product, Gariss, as a habitat of Streptococcus infantarius Delgado, M.A., Recio, I., 2006. Antihypertensive activity of milk fermented by subsp. infantarius. International Journal of Food Microbiology 127, 215e219. Enterococcus faecalis strains isolated from raw milk. International Dairy Journal Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M., Vidal-Carou, C., 2001. Amino acid 16, 61e69. decarboxilase activity of bacteria isolated from pork sausages. International Mundy, L.M., Sahm, D.F., Gilmore, M., 2000. Relationships between enterococcal Journal of Food Microbiology 66, 185e189. virulence and antimicrobial resistance. Clinical Microbiology Review 13, 513e522. Bover-Cid, S., Holzapfel, W.H., 1999. Improved screening procedure for biogenic Narvhus, J.A., Gadaga, T.H., 2003. The role of interaction between yeasts and lactic amine production by lactic acid bacteria. International Journal of Food Micro- acid bacteria in African fermented milks: a review. International Journal of Food biology 53, 33e41. Microbiology 86, 51e60. Buchenhüskes, H.J., 1993. Selection criteria for lactic acid bacteria to be used as Obodai, M., Dodd, C.E.R., 2006. Characterisation of dominant microbiota of a Gha- starter cultures in various food commodities. FEMS Microbiology Reviews 12, naian fermented milk product, Nyarmie, by culture and nonculture-based 253e272. methods. Journal of Applied Microbiology 100, 1355e1363. Chamba, J.F., Jamet, E., 2008. Contribution to the safety assessment of technological Osorio-Cadavid, E., Chaves-López, C., Tofalo, R., Paparella, A., Suzzi, G., 2008. microflora found in fermented dairy products. International Journal of Food Detection and identification of wild yeasts in Champús, a fermented Colombian Microbiology 126, 263e266. maize beverage. Food Microbiology 25, 771e777. Chen, T., Wang, Z., Chen, X., Liu, Y., Zhang, H., Sun, T., 2010. Identification of Pan, D., Luo, Y., Tanokura, M., 2005. Antihypertensive peptides from skimmed milk angiotensin I-converting enzyme inhibitory peptides from koumiss, a tradi- hydrolysate digested by cell-free extract of Lactobacillus helveticus JCM1004. tional fermented mare’s milk. Journal of Dairy Science 93, 884e892. Food Chemistry 91, 123e129. Church, F.C., Swaisgood, H.E., Porter, D.H., Catign, G.L., 1983. Spectrophothometric Pulvirenti, A., Solieri, L., Gullo, M., De Vero, L., Giudici, P., 2004. Occurrence and assay and properties using o-phtaldialdehyde for determination of proteolysis dominance of yeast species in sourdough. Letters in Applied Microbiology 38, in milk and isolated milk proteins. Journal of Dairy Science 66, 1219e1227. 113e117. Dutka Malen, S., Evers, S., Courvalin, P., 1995. Detection of glycopeptide resistance Pritchard, G.G., Coolbear, T., 1993. The physiology and biochemistry of the proteo- genotypes and identification to the species level of clinically relevant entero- lytic system in lactic acid bacteria. FEMS Microbiology Reviews 12, 179e206. cocci by PCR. Journal of Clinical Microbiology 33, 24e27. Quirós, A., Ramos, M., Muguerza, B., Delgado, M.A., Miguel, M., Aleixandre, A., Foulquié Moreno, M.R., Sarantinopoulos, P., Tsakalidou, E., De Vuyst, L., 2006. The Recio, I., 2007. Identification of novel antihypertensive peptides in milk fer- role and application of enterococci in food and health. International Journal of mented with Enterococus faecalis. International Dairy Journal 17, 33e41. Food Microbiology 106, 1e24. Rahman, N., Xiaohong, C., Meiqin, F., Mingsheng, D., 2009. Characterization of the Gadaga, T.H., Mutukumira, A.N., Narvhus, J.A., Feresu, S.B., 1999. A review of tradi- dominant microflora in naturally fermented camel milk shubat. World Journal tional fermented foods and beverages of Zimbabwe. International Journal of of Microbiology and Biotechnology 25, 1941e1946. Food Microbiology 53, 1e11. Ramachandran, L., Shah, N.P., 2008. Proteolytic profiles and Angiotensin-I con- Gran, H.M., Gadaga, H.T., Narvhus, J.A., 2003. Utilization of various starter cultures verting enzyme and aeglucosidase inhibitory activities of selected lactic acid in the production of Amasi, a Zimbabwean naturally fermented raw milk bacteria. Journal of Food Science 73, 75e81. product. International Journal of Food Microbiology 88, 19e28. Regazzo, D., Da Dalt, L., Lombardi, A., Andrighetto, C., Negro, A., Gabai, G., 2010. García de Fernando, G.D., Hernández, P.E., Burgos, J., Sanz, B., Ordóñez, J.A., 1991. Fermented milks from Enterococcus faecalis TH563 and Lactobacillus delbrueckii Extracellular proteinase from Enterococcus faecalis subsp. liquefaciens. I. Growth subsp. bulgaricus LA2 manifest different degrees of ACE-inhibitory and immu- and extracellular proteinase production under different culture conditions. nomodulatory activities. Dairy Science Technology 90, 469e476. Folia Microbiologica 36, 423e428. Sarantinopoulos, P., Andrighetto, C., Georgalaki, M.D., Rea, M.C., Lombardi, A., Giraffa, G., 2003. Functionality of enterococci in dairy products. International Cogan, M.T., 2001. Biochemical properties of enterococci relevant to their Journal of Food Microbiology 88, 215e222. technological performance. International Dairy Journal 11, 621e647. Gobetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., Di Cagno, R., 2002. Latent Seppo, L., Jauhiainen, T., Poussa, T., Korpela, R., 2003. Fermented milk high in bioactive peptides in milk proteins: proteolytic activation and significant in bioactive peptides has a blood pressure-lowering effect in hypertensive dairy processing. Critical Reviews in Food Science and Nutrition 42, 223e239. subjects. American Journal of Clinical Nutrition 77, 326e330. Hao, Y., Zhao, L., Zhang, H., Zhai, Z., Huang, Y., Liu, X., Zhang, L., 2010. Identification Serio, A., Paparella, A., Chaves-López, C., Corsetti, A., Suzzi, G., 2007. Enterococci of the bacterial biodiversity in koumiss by denaturing gradient gel electro- population of Pecorino Abruzzese cheese: biodiversity and safety aspects. phoresis and species-specific polymerase chain reaction. Journal of Dairy Journal of Food Protection 70, 1561e1568. Science 93, 1926e1933. Serio, A., Chavez-López, C., Paparella, A., Suzzi, G., 2010. Evaluation of metabolic Hegazi, F.Z., 1990. Growth rate, proteolysis and acid production of Streptococcus fae- activities of enterococci isolated from pecorino Abruzzese cheese. International calis subsp. liquefaciens in skim milk with some additives. Nahrung 34, 195e199. Dairy Journal 20, 459e464. Kebede, A., Viljoen, B.C., Gadaga, T.H., Narvhus, J.A., Lourens-Hattingh, A., 2007. The Shihata, A., Shah, N.P., 2000. Proteolytic profiles of yogurt and probiotic bacteria. effect of container type on the growth of yeast and lactic acid bacteria during International Dairy Journal 10, 401e408. production of Sethemi, South African spontaneously fermented milk. Food Solieri, L., Landi, S., De Vero, L., Giudici, P., 2006. Molecular assessment yeast pop- Research International 40, 33e38. ulation from traditional balsamic vinegar. Journal of Applied Microbiology 101, Leclerc, P.L., Gauthier-Sylvie, F., Bachelard, H., Santure, M., Roy, D., 2002. Antihy- 63e71. pertensive activity of casein-enriched milk fermented by Lactobacillus helveti- Suzzi, G., Gardini, F., 2003. Biogenic amines in dry fermented sausages: a review. cus. International Dairy Journal 12, 995e1004. International Journal of Food Microbiology 88, 41e54.