Selection of amine oxidizing dairy lactic acid bacteria: enzyme and gene involved in the decrease

39 Pages·2016·1.04 MB·English

AEM Accepted Manuscript Posted Online 16 September 2016
Appl. Environ. Microbiol. doi:10.1128/AEM.01051-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Selection of amine oxidizing dairy lactic acid bacteria: enzyme and gene
2 involved in the decrease of biogenic amines
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5 Rosa Guarcelloa, Maria De Angelisb, Luca Settannia, Sabino Formigliob, Raimondo Gaglioa, w
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6 Fabio Minervinib#, Giancarlo Moschettia and Marco Gobbettib a
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8 aDipartimento di Scienze Agrarie e Forestali, Università degli Studi di Palermo, Palermo, Italy m

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9 bDipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari tt
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10 Aldo Moro, Bari, Italy a
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13 Running title: Lactic acid bacteria for reducing biogenic amine content r
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15 #Corresponding author: Fabio Minervini; e-mail address: [email protected]; Dipartimento ril
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16 di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro, via 2
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17 Amendola 165/a, 70126 Bari, Italy, Tel.: [phone]; fax: [phone]. 9

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18 ABSTRACT
19 Accumulation of biogenic amines (BAs) in cheese and other foods is a matter of public health
20 concern. This study aimed at identifying enzyme activities responsible for BAs degradation in lactic
21 acid bacteria, which were previously isolated from traditional Sicilian and Apulian cheeses.
22 Selected strains would control the concentration of BAs during cheese manufacture. First, 431
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23 isolates, not showing genes encoding for decarboxylases responsible for BA formation, were o
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24 selected using PCR-based methods. Ninety-four out of the 431 isolates degraded BAs (2- lo
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25 phenylethylamine, cadaverine, histamine, putrescine, spermine, spermidine, tyramine or tryptamine) e
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26 during cultivation on chemically defined medium. As shown by RAPD-PCR and partial sequence ro
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27 of 16S-rRNA gene, 78 of the 94 strains belonged to Lactobacillus (Lb. casei, Lb. fermentum, Lb. h
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28 parabuchneri, Lb. paracasei, Lb. paraplantarum and Lb. rhamnosus), Leuconostoc (Ln. lactis and :/
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29 Ln. mesenteroides), Pediococcus pentosaceus, Lactococcus lactis, Streptococcus (S. gallolyticus m
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30 and S. thermophilus), Enterococcus lactis and Weissella paramesenteroides. s
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31 A multi-copper oxidase hydrolyzing BAs was purified from the most active strain Lb. paracasei r
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32 subsp. paracasei CB9CT. The gene encoding for the multi-copper oxidase was sequenced and o
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33 detected also in other amine-degrading strains of Lb. fermentum, Lb. paraplantarum and P. p
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34 pentosaceus. Lb. paracasei CB9CT and another strain (CACIO6CT) of the same species that was 1
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35 able to degrade all the BAs were singly used as adjunct starter for decreasing the concentration of 1
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36 histamine and tyramine in industrial Caciocavallo cheese. The results of this study disclose a b
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37 feasible strategy for increasing the safety of traditional cheeses, while maintaining their typical u
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38 sensorial traits.
39 IMPORTANCE
40 Because high concentrations of the potentially toxic biogenic amines may be found in
41 traditional/typical cheeses, safety of these food items should be improved. Lactic acid bacteria
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42 selected for the ability to degrade biogenic amines may be used during cheese-making for
43 controlling over the concentration of biogenic amines.
44 INTRODUCTION
45 Biogenic amines (BAs) are low molecular weight organic basic compounds formed by microbial
46 decarboxylation of some free amino acids (FAA) or amination and transamination of aldehydes and D
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47 ketones (1, 2). Based on the molecular structure, they are classified as follows: (i) monoamines,
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48 including tyramine, 2 phenylethylamine and tryptamine; (ii) diamines, such as histamine, putrescine a
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49 and cadaverine; and (iii) polyamines (spermine and spermidine) (3, 4). d

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50 Overall, BAs play a role in brain activity, regulation of body temperature and stomach pH, m

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51 gastric acid secretion, immune response, and cell growth and differentiation (5). However, the tt
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52 consumption of food containing high amounts of BAs may cause food intoxication (6). Negative a
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53 effects of BAs on consumer’s health include changes in perception, smooth muscle contractions .
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54 and/or blood pressure (5, 7). The combination of nitrites and BAs in foods results in the formation
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55 of nitrosamines which are potential carcinogens (8). g
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56 Usually, high contents of BAs are found in fermented or spoiled foods. Recently, BAs were
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57 proposed as food safety indicators and as biomarkers for several diseases (9, 10). Within fermented ril
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58 foods, cheeses containing high concentration of BAs may have a serious impact on public health , 2
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59 (11). After fish, cheese is the second most frequently reported food-stuff responsible for histamine 9

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60 intoxication (12, 13). A recent survey on Apulian and Sicilian cheeses revealed that about 50% of y
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61 the cheeses contained at least one BA (especially histamine, followed by tyramine and putrescine) e
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62 at potentially toxic concentrations (> 100 mg kg-1) (14).
63 Enterococci and hetero-fermentative lactobacilli are the main producers of tyramine and
64 histamine, respectively. Other lactic acid bacteria and some Gram-negative bacteria may also be
65 responsible for the formation of BAs in cheeses (15, 16). During cheese ripening, primary and
66 secondary proteolysis release high amount of FAA, some of which (e.g., lys, orn, his, tyr, trp and
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67 phe) are BAs precursors (14, 17). Therefore, the detection of bacteria possessing enzymes that
68 convert precursor FAA into BA allows to estimate the risk of food intoxication. To this purpose,
69 specific molecular probes and/or phenotypic assays were set up (18).
70 The capacity of some lactic acid bacteria to degrade BA to aldehyde, ammonia and hydrogen
71 peroxide by amine oxidases (AOs) was also shown in food processing (3, 19, 20). In particular, two
72 main classes of AOs are described: flavin-containing monoamine oxidases (FlavAO) and copper- D
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73 containing amine oxidases (CuAO), usually found in a wide range of microbes, plants, and animals n
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74 (4, 21). Multi-copper oxidases (MCO) may be also responsible for BAs degradation (18, 22). d
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75 Previously, AOs were characterized mainly in plants and animals (4, 23, 24). Only a few studies f
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76 dealt with bacterial AOs (25).

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77 Nowadays an increasing section of consumers prefers traditional and typical cheeses. Most of p
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78 these cheeses bear either the Protected Geographical Indication (PGI) or the Protected Designation e
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79 of Origin (PDO). However, the manufacture of many traditional/typical cheeses relies on natural a
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80 starter cultures and autochthonous microbiota. Since the members of the traditional/typical cheese .o
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81 microbial community are often undefined and, consequently, their corresponding metabolic /
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82 activities are not known in-depth, it is not unsurprising that those cheeses could accumulate BAs A
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83 (26). Therefore, the selection of autochthonous amine-negative and amine oxidizing lactic acid il
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84 bacteria (to be used as starter, adjunct/attenuated starter) would represent an interesting strategy for 2
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85 reducing the accumulation of BAs in traditional/typical cheeses, while contemporarily maintaining
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86 their sensory attributes (22, 27). Once these lactic acid bacteria will be characterized, they could g
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87 be publicly available and, in view of implementing the Nagoya Protocol (28), used for st
88 increasing the safety of traditional/typical cheeses.
89 In this context, starting from hundreds of lactic acid bacteria previously isolated from 20
90 traditional Apulian and Sicilian cheeses (14), this study aimed at: (i) selecting lactic acid bacteria
91 that do not produce BAs and are able to degrade BAs; (ii) identifying the enzyme and the gene
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92 involved in the degradation of BAs; and (iii) applying the selected lactic acid bacteria as a means
93 for controlling over the concentration of BAs in traditional cheese.
94 MATERIALS AND METHODS
95 Strains and culture conditions. 665 isolates of presumptive lactic acid bacteria were previously
96 isolated from Apulian (Cacio, Caciocavallo Podolico Dauno, Caciocavallo Silano PDO, D
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97 Cacioricotta, Canestrato Pugliese PDO, Caprino di Biccari, Caprino di Castel Fiorentino, Pecorino
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98 Foggiano, Vaccino) and Sicilian (Caciocavallo Palermitano, Ragusano PDO, Caprino Girgentano, a
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99 Fior di Capra, Fiore Sicano, Maiorchino, Pecorino Siciliano PDO, Piacentinu Ennese, Provola dei d

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100 Nebrodi, Tuma Persa, Vastedda della Valle del Belìce PDO) traditional cheeses (14) and used in m

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101 this study. Presumptive lactic acid bacteria were routinely propagated at 25 °C (mesophilic) or 42 tt
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102 °C (thermophilic) for 24 h in either MRS (bacilli) or M17 (cocci) broth (Oxoid Limited, a
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103 Basingstoke, UK). .
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104 Selection of lactic acid bacteria non-producing biogenic amine. Based on a previous study
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105 (14), a molecular approach was used to determine the presence/absence of genes (tyrdcA, hdcA, g
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106 odc, cadA) responsible for BAs formation (tyramine, histamine, putrescine, and cadaverine) in
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107 presumptive lactic acid bacteria. Enterococcus faecium DSM20477, Lactobacillus plantarum ril
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108 ATCC14917 and Lactobacillus paracasei subsp. tolerans LMG9191 were tested for the capacity to , 2
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109 decarboxylate tyr, his, orn and lys detecting, by HPLC analysis, the corresponding BA in the above 9

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110 mentioned strains cultured (30 °C, 48 h) in MRS broth supplemented with precursor amino acid y
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111 (29). E. faecium DSM20477 and Lb. plantarum ATCC14917, capable to decarboxylate tyr and his, e
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112 respectively, were used as positive controls for tyrdcA and hdcA genes. Lb. paracasei LMG9191,
113 capable to decarboxylate orn and lys, was used as positive control for odc and cadA genes. DNA
114 was extracted from bacterial cells cultured for 48 h under appropriate conditions (medium and
115 temperature) by the Instagene Matrix kit (Bio-Rad, Hercules, CA), following the manufacturer’s
116 instructions, and used as template in PCR. For each target gene, Table 1 shows primers (synthesized
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117 by Eurofins Genomics s.r.l., Milan, Italy), PCR conditions, and the size of the expected amplicons
118 as predicted by the software pDRAW32 v1.1.114 (located at http://www.acaclone.com).
119 Amplifications were performed by means of a conventional thermocycler (Esco Healthcare Pte.
120 Ltd., Singapore, Malaysia). PCR products were separated by electrophoresis on 2% (w/v) agarose
121 (Gibco BRL, Cergy Pontoise, France) gel and visualized by UV transillumination after staining
122 with SYBR® safe DNA gel stain (Molecular Probes, Eugene, OR). GeneRuler 100 bp Plus DNA D
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123 ladder (M-Medical, Milan, Italy) was used as a molecular size marker. n
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124 Selection of lactic acid bacteria degrading biogenic amines. Only isolates negative for tyrdcA, d
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125 hdcA, odc and cadA genes were assayed for their capacity to degrade BAs upon cultivation in f
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126 Chemically Defined Medium (CDM), which was prepared as described by Miladinov et al. (36)

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127 with minor modifications. Modified CDM (mCDM) contained (NH4)2SO4 (2 g l-1), Na2HPO4 (6 g l- tp:
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128 1), KH2PO4 (3 g l-1), Na2SO4 (0.011 g l-1), NaCl (1 g l-1), MgCl2 (0.2 g l-1), CaCl2 (0.01 g l-1), and e
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129 FeCl3·7H2O (0.0006 g l-1) (Merck, Milan, Italy). After sterilization, filter sterilized glucose (5 g l–1) .as
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130 (Oxoid), biotin (0.0001 g l–1), folic acid, riboflavin, nicotinic acid, thiamine, and pantothenic acid .o
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131 (0.001 g l–1), pyridoxal (0.002 g l–1), and sodium acetate (2 g l–1) (Carlo Erba Reagenti, Milan, Italy) /
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132 were added. mCDM was singly supplemented with one of the following BAs: tyramine (2.5 mM), A
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133 histamine (1 mM), putrescine, cadaverine, tryptamine, 2-phenyletylamine, spermine, and il
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134 spermidine (0.54 mM) (Sigma-Aldrich, Milan, Italy) (25, 27). mCDM without BA addition was 2
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135 used as the negative control for bacterial growth. Isolates were pre-cultured twice in MRS or M17
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136 broth. Cells were centrifuged at 10,000 × g for 5 min at 4 °C, washed twice in Ringer’s solution and g
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137 inoculated (initial cell density of ca. 7 log cfu g-1) in mCDM broth. After incubation (at either 25 or s
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138 42 °C for 48-72 h), bacterial growth was measured by reading absorbance at a wavelength of 600
139 nm through a 6400 spectrophotometer (Jenway Ltd., Felsted Dunmow, UK). Bacterial growth was
140 confirmed through pour-plating (in MRS or M17 agar media, depending on the isolation medium)
141 mCDM liquid cultures at the beginning and end of incubation. In addition, the capacity to degrade
142 BAs was assessed on 19 isolates by HPLC (25), at ISVEA s.r.l. (Poggibonsi, Siena, Italy), using
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143 ISVEA protocols. The percentage of BA degradation was calculated based on the peak areas
144 corresponding to tyramine, histamine, putrescine, cadaverine, tryptamine, 2-phenyletylamine,
145 spermine, and spermidine soon after inoculation of mCDM and after incubation (27).
146 Genotypic characterization and identification of amine degrading bacteria. Decarboxylase-
147 negative, amine degrading lactic acid bacteria were typed by Random Amplification of
148 Polymorphic DNA-PCR (RAPD-PCR), using primer M13 (37). PCR was performed according to D
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149 the conditions described by Zapparoli et al. (38). PCR products were separated by electrophoresis n
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150 and visualized as described above. RAPD-PCR profiles were analyzed by the pattern analysis d
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151 software package Gelcompare II software version 6.5 (Applied-Maths, Sin Marten Latem, f
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152 Belgium). Calculation of similarities of band profiles was based on the Pearson product moment

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153 correlation coefficient. Dendrograms were obtained by means of the Unweighted Pair Group p
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154 Method using an Arithmetic average clustering algorithm (UPGMA). e
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155 Genotypic identification of lactic acid bacteria was carried out by partial sequencing of 16S a
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156 rRNA gene, as described by Weisburg et al. (39) and Corsetti et al. (40). Primers casei (forward), .o
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157 para (forward), and Y2 (reverse) (Eurofins Genomics) were used to identify species in the /
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158 Lactobacillus casei group (41). PCR products were purified by the QIAquick purification kit A
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159 (Quiagen S.p.a., Milan, Italy) and sequenced by Eurofins Genomics. Comparative searches were il
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160 performed against available sequences in GenBank/EMBL/DDBJ databases using the Basic Local 2
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161 Alignment Search Tool (BLAST) program (42). 16S rRNA partial gene sequences were deposited
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162 in the GenBank database and their accession numbers (AN) are included in Table S4. g
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163 Use of additional primers targeting other genes involved in the formation of putrescine and s
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164 cadaverine. Since putrescine may be produced not only from ornithine but also from agmatine (43),
165 the identified amine degrading strains were further tested using two couples of primers (AgmSq1/
166 AgmSq2 and AgD1/AgD2, Table 1) targeting genes (agu and agdi) involved in the agmatine
167 deiminase pathway. In addition, these strains were tested using an additional couple of primers
168 (Cad2F/Cad2R, Table 1) targeting lys decarboxylase gene. Lactobacillus brevis ATCC367 and E.
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169 faecium ATCC®-BAA-472TM, capable to produce putrescine and cadaverine from agmatine and lys
170 (29), respectively, were used as positive controls.
171 Identification of amine degrading enzyme. Within amine degrading lactic acid bacteria, the
172 most active Lb. paracasei subsp. paracasei CB9CT was selected for the identification of amine
173 degrading enzyme. Cytoplasmic proteins were extracted from cells of CB9CT, according to the
174 method described by Gobbetti et al. (44) with some modifications. In detail, bacterial cells cultured D
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175 (24 h) in 1 l of MRS broth were collected by centrifugation (15,300 × g, 10 min, 4 °C) and washed n
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176 twice with Tris-HCl 50 mM pH 7.5 buffer. Cells were further suspended in the same buffer d
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177 containing sucrose (25% wt/vol) (Sigma-Aldrich). Cell suspension was incubated (37 °C, 30 min) f
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178 under stirring (120 rpm) and finally added (1 g l-1) with lysozyme (Sigma-Aldrich). After cell wall

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179 lysis (37 °C, 1 h, under stirring), the suspension was centrifuged (37 °C, 30 min, 12,000 × g). The p
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180 pellet was suspended in 2 ml of Tris-HCl 50 mM pH 7.5 buffer. After incubation (30 °C, 30 min), e
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181 the suspension was kept in ice bath and ultrasonic treated by a Vibra-Cell Sonicator (Sonic and a
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182 Materials Inc., Danbury, CT, USA) equipped with a microtip setting (sonic power 375 W; output .o
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183 control 5) for 15 min (3 cycles, 5 min/cycle). Subsequently, the suspension was centrifuged (10,170 /
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184 × g, 4 °C, 10 min) and the supernatant, putatively containing cytoplasmic proteins, was added with A
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185 6 ml of cold acetone. After overnight incubation at -20 °C, precipitated proteins were collected by il
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186 centrifugation (19,940 × g, 4 °C, 10 min), washed with acetone and air dried for 10 min. 2
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187 Proteins were re-suspended in sodium acetate 50 mM pH 5.6 buffer and assayed for BA oxidase
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188 activity by the colorimetric assay reported by Foster et al (45), with minor modifications. In detail, g
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189 40 µl of partially purified enzyme, 400 µl of 4-aminoantipyrine (1.5 mM) (Sigma-Aldrich), and s
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190 2,4,6-tribromo-3-hydroxybenzoic acid (1 mM) (Sigma-Aldrich) in potassium phosphate 200 mM
191 pH 7.6 buffer were mixed. Eighty microliters of histamine (100 mM) and 280 µl of peroxidase from
192 horseradish (0.8 U ml-1 of reaction) (Sigma-Aldrich) were added to initiate the enzyme reaction.
193 The reaction mixture was incubated at 30 °C for 45 min and subjected to measurement of
194 absorbance at a wavelength of 510 nm. A positive control was represented by a reaction mixture
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195 wherein partially purified enzyme was replaced by commercial diamine oxidase from porcine
196 kidney (Sigma-Aldrich). A negative control was also included, consisting of a reaction mixture
197 wherein histamine was replaced by a solution of potassium phosphate buffer.
198 Protein extract was applied to a DEAE-cellulose anion exchange column (55 × 1.6 cm, GE
199 Healthcare, Uppsala, Sweden). Proteins were eluted with a linear NaCl gradient (0 – 0.3 M) in
200 sodium acetate 50 mM pH 5.6 buffer, at a flow rate of 42 ml h-1. Fractions with the highest oxidase D
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201 activity were pooled, dialyzed against sodium acetate 5 mM pH 5.6 buffer, 10-fold concentrated by n
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202 freeze drying and subjected to gel filtration on a FPLC Superose 12HR 10/30 column (GE d
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203 Healthcare). Elution with sodium acetate 50 mM pH 5.6 buffer, containing 0.15 M NaCl, was at a f
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204 flow rate of 18 ml h-1. Active fractions were pooled, dialyzed against sodium acetate 50 mM pH 5.6

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205 buffer, and, finally, applied to a FPLC Mono-Q HR 5/5 column (GE Healthcare) eluting with a p
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206 linear NaCl gradient from 0 to 0.5 mM in the same buffer at a flow rate of 24 ml h-1. Protein e
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207 concentration during the purification steps was determined according to Bradford (46). a
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208 The active fraction was digested by trypsin (from bovine pancreas suitable for protein .o
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209 sequencing) (Sigma-Aldrich) and the resulting peptides were identified by LC-MS/MS on Q- /
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210 Exactive Orbitrap at Bioproximity, LLC (Chantilly, VA, USA). Protein identification was carried A
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211 out by using the Amazon Web Services-based cluster compute instances using the Proteome Cluster il
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212 interface. The protein sequence was analyzed by a BLAST search in GenBank/EMBL/DDBJ 2
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213 (RefSeq) and UniProtKB databases (www.uniprot.org/help/uniprotkb), and aligned by ClustalW
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214 version 1.83 (47) to similar proteins from lactic acid bacteria. The alignment output files were g
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215 formatted using BioEdit version 7.0.9 (http://www.mbio.ncsu.edu/BioEdit/). Protein domains were s
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216 located using the Pfam (48) (http://pfam.sanger.ac.uk), Simple Modular Architecture Research Tool
217 (SMART) (49) (http://smart.embl-heidelberg.de) and Conserved Domain Database (CDD)
218 (http://www.ncbi.nlm.nih.gov) databases.
219 Detection of genes encoding for amine degrading enzyme. Based on the amino acid sequence
220 of amine degrading enzyme, four couples of primers (Eurofins Genomics) were designed (Table 1).
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221 Primers mapping on internal regions (BC2-4) were used in nested amplifications, in order to verify
222 the nature of the identified gene and to obtain its complete sequence. To detect the presence of the
223 identified amine degrading enzyme of Lb. paracasei CB9CT in the other amine degrading strains,
224 genomic DNA from selected strains (one for each species) were used as templates for amplification.
225 PCR products were separated, visualized and identified by sequencing as described above.
226 Additionally, degenerated primers (Eurofins Genomics), targeting for AOs genes from D
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227 Firmicutes and other bacteria, were designed and tested in different combinations (see n
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228 Supplemental material). d
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229 Experimental cheese manufacture and analysis of biogenic amines contained in cheese. f
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230 Caciocavallo-type cheese was manufactured at the industrial plant Ignalat (vat of about 200 liters),

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231 located in Noci (Bari, Italy). Pasteurized cows’ milk was hold at 37 °C and used to manufacture six p
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232 types of experimental cheeses using different cultures combinations: (i) commercial Streptococcus e
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233 thermophilus (Sacco, Cadorago, Como, Italy), and histamine producer Lactobacillus fermentum a
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234 CCS12BT; (ii) S. thermophilus and Lb. paracasei subsp. paracasei CB9CT (capable of growing on .o
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235 mCDM added with tyramine, histamine, tryptamine, or spermine); (iii) S. thermophilus, Lb. /
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236 paracasei CB9CT and Lb. fermentum CCS12BT; (iv) S. thermophilus and Lb. paracasei subsp. A
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237 paracasei CACIO6CT (capable of growing on mCDM added with any of the BAs); (v) S. il
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238 thermophilus, Lb. paracasei CACIO6CT and Lb. fermentum CCS12BT; and (vi) S. thermophilus 2
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239 only (control cheese). All strains were inoculated at ca. 7 log cfu ml-1 of milk. 30 minutes after 9
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240 addition of liquid calf rennet (0.0001%, vol/vol), coagulation took place. The coagulum was first g
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241 cut coarsely by hand, held under whey at 37 °C for 2 h, and then reduced to particles of 1.5 to 2 cm. s
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242 When the curd reached a pH of 5.25 (ca. 5 h, at room temperature), it was stretched (ca. 65 °C, 5
243 min) in hot (80 °C) water. Cheeses were salted in brine (30% [wt/vol] NaCl) for 12 h. Ripening was
244 at ca. 10 °C and a relative humidity of 83% for 60 days. The weight of the cheese was
245 approximately 1.5 kg.
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