Skip to main content
NCBI home page
Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2020 Aug 19;58(6):2216–2226. doi: 10.1007/s13197-020-04732-8

Molecular characterization of non-biogenic amines producing Lactobacillus plantarum GP11 isolated from traditional pickles using HRESI-MS analysis

V Priyanka 1, A Ramesha 1, Devaraja Gayathri 1,✉, M Vasudha 1
PMCID: PMC8076391  PMID: 33967318

Abstract

Fungal spoilage and toxic biogenic amine production is a major risk in fermented products. Therefore, the selection of nontoxic biogenic amines producing probiotic bacteria plays a vital role in the fermentation process. In the present study, a total of 18 bacterial isolates were isolated from eight different homemade pickle samples and 15 lactic acid bacteria (LAB) were identified based on biochemical tests. Out of which only seven isolates (GP1, GP2, GP3, GP4, GP5, GP9, and GP11) exhibited antifungal activity against pickle contaminant Aspergillus sp and Penicillium sp. Among the potential LAB isolates, GP11 showed the highest antifungal activity against Aspergillus sp and Penicillium sp with a zone of inhibition 28.33 ± 0.57and 19.66 ± 0.57 mm respectively. The potent LAB isolates were tested for amino acid decarboxylase activity, in which GP2, GP3, GP4, and GP5 exhibited to produce tyramine, cadaverine, and phenylethylamine while GP1 and GP5 have produced tyramine and phenylethylamine respectively. However, highly potent antifungal active isolate GP11 did not produce biogenic amine. Further, GP1, GP9, and GP11 were subjected to confirmation of biogenic amines production using HRESI-MS. HRESI-MS analysis of the GP1 and GP9 sample confirmed the presence of phenylethylamine and tyramine respectively. Interestingly, GP11 isolate did not show any biogenic amines production and GP11 was further subjected to 16S rRNA typing and identified as Lactobacillus plantarum. On in situ pickle sensory evaluation, GP11 lactopickle was graded as very good quality when compared to traditional one. Therefore L. plantarum GP11 could be developed as an ideal starter culture for the fermented production of a pickle.

Keywords: Antifungal activity, Biogenic amines, Non biogenic amines, Lactic acid bacteria, Lactobacillus plantarum GP11

Introduction

Pickles provide distinct health benefits such as improved immunity, increased gut health and would act as antidepressants. Hence they have found a permanent place in most of menu cards of cuisines across the world with special reference to kitchens in India. Pickle being a fermented product, is being the risk of microbial spoilage. Fungi keep posing a considerable threat in spoilage of fermented food products and produce a variety of mycotoxins (Bryla et al. 2016). Food and Agricultural Organization (FAO) has reported that almost 25% of agricultural and fermented foods are contaminated with fungi or mycotoxins worldwide (Kalagatur et al. 2018). The chief fungal intoxication of pickle may be due to mycotoxins such as aflatoxin, ochratoxin, and patulin (Barkai-Golan. 2008). Not all microorganisms harmful, there are some organisms that are health promoting called probiotics, according to the FAO, and World Health Organization “probiotics are live microorganisms which when administered in adequate quantity confer a health benefit on the host” (De Llano. 1998; Dangmance et al. 2015). Lactic acid bacteria (LAB) are a group of bacteria present naturally in the gastrointestinal tract of animals and fermented foods including meat, beverages and dairy products (Yu et al. 2012; Dallal et al. 2017). LAB are nutritionally fastidious bacteria, chiefly require media enriched with vitamins, amino acids, and lipids (Rodriguez et al. 2014).

Biogenic amines (BA) are the low molecular weight organic bases, which are produced by the metabolism of microorganisms. Biogenic amines are present in a variety of fermented and non-fermented foods, including fish, meat, cheese, vegetables, and wines, and described as organic bases with aliphatic, aromatic and heterocyclic structures (Önal 2007; Shiling et al.2015). Moreover, these are obtained from amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamine by decarboxylation. Decarboxylation of amino acid by removal of the α-carboxyl group from the corresponding amino acid structure would lead to the formation of corresponding amines (Ono et al. 2014; Priyadarshani and Rakshit.2014). However, these biogenic amines (tyramine and histamine) would have adverse effects on human health causing allergies, hypertension, and headache. Poor quality processing and wrong selection of microbial strains for fermented pickle products favour the contamination of tyramine, cadaverine, phenylethylamine, and histamine. Microorganisms with aminoacid decarboxylase activity can too cause high bio-amine content in a pickle, for example, starter culture like Lactobacillus curvatus possessing the capability to form biogenic amines (Singh et al. 2012). Biogenic amines such as catecholamines; dopamine, norepinephrine, and epinephrine would mediate a variety of neuronal functions such as motor control, cognition, emotion, memory processing, and endocrine modulation (Kongkiattikajorn. 2015). In addition, some of the biogenic amines such as tyramine, cadaverine, phenylethylamine, and histamine could cause allergic/neurological complications (Singh et al. 2012).

The microorganisms (Hafnia alvei, Pantoea citrea, Enterococcus faecium and Enterococcus faecalis) associated with sorghum beer, sour milk and sour maize beverage were shown to produce four biogenic amines viz., histamine, putrescine, cadaverine tyramine and claimed to be safer for consumption (Gashe et al. 2013). However, Lactic acid bacteria were found to show antimicrobial activity against some food-spoilage microorganisms from naturally fermented pickled vegetables for example, Lactobacillus coryniformis strain BBE-H3, a non-biogenic amine producer, showed a high level of activity in degrading sodium nitrite and exhibited the ability to eliminate ethyl carbamate and sodium nitrite, carcinogens of food origin (Fang et al. 2016).

However, few lactic acid bacterial isolates can be biogenic amines producers and they are neither beneficial nor harmful (Kongkiattikajorn. 2015; Singh et al. 2012). It is to prevent such a risk, particular starter cultures, possessing either less or no amino acid decarboxylase activity or allergic bacteriocin synthesing capability should have antifungal activity are selected. In this context, ameliorating the lactic acid bacteria from homemade pickle, and to explore antifungal activity with biogenic amino acid-producer/non-producer LAB isolates were evaluated.

Materials and methods

Isolation of lactic acid bacteria

Indian salty and spicy pickle samples were collected from (rural) households of Davangere District villages (Kurki,Tholahunse, Bullapura, Hirethogleri) of Karnataka and stored at 4° C until further use (Table 1). A tenfold serial dilute pour plate technique on MRS media (De Man Rogosa and Sharpe) was carried out, incubated at 37 °C under the microaerophilic condition for 72 h (Sagratini et al. 2012).

Table 1.

Site of Pickle sample collection and Antifungal activity of Lactic acid bacteria isolated from pickle

Pickle. Sl.no Place of sample collection Bacterial isolate tested Zone of Inhibition in mm against pickle spoiled fungi
Aspergillus sp. Penicillium sp.
1

Bullapura

Shivamogga

13.840°N 75.702°E

 GP 1 25.33 ± 0.57g 18.66 ± 1.15de
2 GP 2 13.66 ± 0.57c 12 ± 00b
3 GP 3 15 ± 1c 12.33 ± 1.52b
4 GP 4 18.66 ± 0.57de 13.66 ± 0.57c
5 GP 5 18.33 ± 1.15de 14 ± 00c
6

Hirethogleri, Davangere

14.4666°N 75.9242°E

 GP 6 0a 0a
7 GP 7 0a 0a
8 GP 8 0a 0a
9 GP 9 23.33 ± 1.15f 17.33 ± 0.57d
10 GP 10 0a 0a
11

Kurki, Davangere

14.4666°N 75.9242°E

 GP 11 28.33 ± 0.57h 19.66 ± 0.57e
12 GP 12 0a 0a
13 GP 13 0a 0a
14 GP 14 0a 0a
15

Tholahunse, Davangere

14.4666°N 75.9242°E

 GP 15 0a 0a
16 GP 16 0a 0a
17 GP 17 0a 0a
18 GP 18 0a 0a
Open in a new tab

Values represent mean ± SD of three parallel experiments. In each column, mean values followed by the same letter are not significantly different according to DMRT at P < 0.05

Biochemical characterization of lactic acid bacteria

The bacterial isolates were identified conventionally by Gram staining and catalase test. Gram-positive and catalase-negative isolates were further confirmed using various physiological tests such as oxidase, IMViC, and carbohydrates fermentation tests (Sagratini et al. 2012).

Screening for Antifungal activity of lactic acid bacteria

Antifungal activity of LAB isolates were performed by the agar well diffusion method. In the well diffusion method, the spore suspensions of fungi isolated from spoiled pickles were inoculated on Soyabean casein digest agar (Himedia, India) using the spread plate technique. Later 10 mm agar wells were made using cork borer and inoculated with 100 µl LAB suspension (106cells/ml), incubated (30 °C/72 h) and the zone of inhibition (ZOI) was measured (Cabo et al. 2002;Huh and Hwang 2016; Kalagatur et al. 2018).

Evaluation of amino acid decarboxylase activity of LAB

Production of biogenic amines was carried out using a decarboxylase medium. Decarboxylase broth consisted of 5 g/L tryptone, 5 g/L yeast extract (oxoid), 5 g/L meat extract, 2.5 g/L NaCl, 0.5 g/L glucose, 1 g/L Tween™80, 0.2 g/L MgSO4.7H2O, 0.05 g/L MnSO4.4H2O, 0.4 g/L FeSO4, 2 g/L ammonium citrate, 2 g/L K2HPO4, 0.1 g/L CaCO3, 0.05 g/L pyridoxal-5 phosphate, with 0.06 g/L bromocresol purple (pH indicator) and 10 g/L corresponding amino acid (tyrosine/lysine/phenylalanine/histidine). These components were dissolved in distilled water (pH 5.5) and autoclaved. Then the medium was inoculated with 0.2 ml (106 CFU/ml) of the chosen bacterial isolate and incubated at 37 °C/48-72 h anaerobically. The decarboxylase medium without amino acid was inoculated with bacteria was used as control. The conversion of yellow to purple colour was considered as positive amino acid decarboxylase activity, while, any other results obtained were considered as decarboxylase negative (Hussain et al. 2011; Moracanin et al. 2015).

Detection of biogenic amines using High-Resolution Electrospray Ionization Mass Spectrometry (HRESI-MS)

HRESI-MS was used for the determination of biogenic amines (molecular weight) in the sample. Pre-column derivatization of amines by benzoyl- chloride method was used for HRESI-MS (Dapkevicius et al. 2000; Innocente and Dágostin 2002). One ml of the culture sample was mixed with one ml of 6% trichloroacetic acid (for deproteination). Further, 1 ml of 2 M sodium hydroxide was added followed by the addition of 2% benzoyl chloride or dichloromethane in acetonitrile. This reaction mixture was incubated for 20 min at 30 ± 2 °C and the process was stopped by adding saturated NaCl (2 ml) solution and the extraction of biogenic amines was carried out using an equal volume of diethyl ether. The extraction process was repeated and the fine organic layer was transferred into clean centrifuge tubes and then evaporated to dryness using nitrogen gas (Dapkevicius et al. 2000; Alberto et al. 2002; Priyadarshani and Rakshit 2014). Then the samples were analyzed using HRESI-MS for the detection of biogenic amines based on the mass to charge ratio (m/z). HRESI-MS analysis of GP1, GP9 and GP11 isolates were performed using TOF spectrometer with simultaneous electron spray (Micro Mass ESI-TOFMS). Mass spectroscopy method was carried out according to Sagratini et al. (2012).

Genomic DNA isolation, PCR and sequencing

Genomic DNA of selected LAB isolate was separated by phenol:chloroform method with slight modification (Sambrook et al. 1989; Swamy et al. 2016). In brief, for genomic DNA isolation, 10 ml of overnight grown GP11 culture was centrifuged (8000 rpm/10 min), the pellet was washed thrice using 0.1 M PBS and 500 μl of 10 mM TrisHCl. Then 15 μl (10 mg/ml) of lysozyme was added and incubated at 37 °C for one hour. Later 450 μl of lysis buffer and 15 μl of proteinase K (20 mg/ml) was added to the mixture and further incubated in water bath at 50 °C/1 h. Then, 450 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was added and centrifuged (4 °C/10,000 rpm) and the upper aqueous layer was collected. Later, 50 μl of 5 M NaCl was added with two-volume of chilled absolute alcohol and again centrifuged at 12,000 rpm for 15 min/4 °C. The pellet was dissolved in 50 μl TE buffer and stored at -20 °C.

The isolated DNA sample was subjected to PCR amplification using universal 16S rRNA primers 27F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492 R (5′ -GGTTAC CTTGTTACGACTT-3′). 16S rRNA genes were amplified according to Sambrook et al. (1989). The PCR mixtures (50 ml) contained 10 mM Tris–HCl (pH 8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of dNTPs, 0.5 pmol of each primer, 1.25 U of Taq polymerase, and 10 ng of DNA template. PCR amplification was performed and programmed as follows: 5 min of denaturation at 95 °C, followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, with a final extension of 72 °C for 10 min. Further, amplification was ensured through gel electrophoresis and PCR products sequenced were analysed (Sambrook et al. 1989; Swamy et al. 2016).16S rRNA sequences were compared to those available in the GenBank NCBI (National Center for Biotechnology Information databases) using the BLAST and sequence was submitted to NCBI. Multiple sequence alignments of the obtained sequence and reference sequences retrieved from GenBank were used to generate a phylogenetic tree using the online software server phylogeny (Dereeper et al. 2008).

In situ Lacto pickle production using GP11 isolate

Preparation of starter culture

Lactobacillus plantarum (GP11) was serially subcultured on MRS agar and inoculated to MRS broth, then incubated at 37 °C/72 h and the bacterial number was adjusted between 106 to 108 CFU/mL (OD 0.8). For preparation of starter culture, 8% NaCl brine was prepared and inoculated with 1% (v/v) GP11culture under sterile condition and was incubated at 30 °C/48 h. After incubation, the starter culture was used in the Lacto-pickle production.

Lacto pickle production

Freshly harvested tender mangoes were collected from local farms of Davangere, Karnataka, India. Tender mangoes were washed 2-3 times in sterile (autoclaved) water and were sliced into small pieces and were soaked thoroughly in salted warm water for 10 min, later drained and exposed to sun light for 2-3 h. Salt, Chilli powder, Turmeric powder, Fenugreek seeds (soaked in lime juice overnight), Fennel seeds, Black cumin seeds, Mustard seeds with boiled and cooled mustard oil were added to mango pieces in desired amount and mixed well. Later, 8% of NaCl was added, followed by the inoculation of 5% v/v starter culture GP11 (107 CFU) and incubated at room temperature for 3-4 weeks. A control was also maintained in the similar manner without GP11 inoculation. After 3 weeks, the samples were observed for sensory qualities like pH, aroma, maturity and taste.

Sensory evaluation

The consumer’s acceptability of the pickle product was evaluated through a taste testing panel (Xue et al. 2014). This group was composed of 25 general consumers (15 women and 10 men, aged between 25 and 45). The members of the group were asked to assign appropriate score in evaluation sheet to each product tested on a 1 to 9 point Hedonic scale for characteristics color, flavor, texture and overall acceptability of mango pickles with GP11 and without GP11. The scale was arranged as; 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like or dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much and 1 = dislike extremely.

Results

A total of 18 bacterial isolates were isolated from the eight different homemade pickle samples (Table 1). Among them, 15 potential isolates were identified as lactic acid bacteria conventionally by colony characters, Gram’s staining and biochemical characterization. Phenotypically all the isolated colonies showed similar cultural characters with variation including size, shape, colour on MRS agar medium and confirmed the isolates were belonged to the group of lactic acid bacteria.

Antifungal activity of lactic acid bacteria

All 15 bacterial isolates were screened for the antifungal activity. Out of which only seven isolates (GP1, GP2, GP3, GP4, GP5, GP9, and GP11) showed the antifungal activity against selected fungal isolates. The ZOI diameter against Aspergillus sp ranged between 13.66 ± 0.57 mmto 28.33 ± 0.57mm, whereas with Penicillium sp. ranged between 12 mm to 19.66 ± 0.57. Among the potential isolates GP11 showed the highest antifungal activity against Aspergillus sp. and Penicillium sp. with diameter ZOI were 28.33 ± 0.57and 19.66 ± 0.57 mm respectively (Table 1).

Evaluation of amino acid decarboxylase activity of LAB

Seven potential antifungal isolates (GP1, GP2, GP3, GP4, GP5, GP9 and GP11) were tested for decarboxylase activity for biogenic amines production. Among these GP1, GP2, GP3, GP4, GP5, and GP9 exhibited decarboxylase activity. Among these isolates GP2, GP3, GP4, and GP5 produced tyramine, cadaverine, and phenylethylamine while, isolates GP1 and GP5 produced tyramine and phenylethylamine respectively. However, highly potent antifungal isolate GP11 did not produce any biogenic amine (Table 2). Based on these results, GP1, GP9, and GP11 were subjected to confirmation of biogenic amines production using HRESI-MS analysis.

Table 2.

Decarboxylase activity of Lactic acid bacteria isolated from pickle

Sl. No. Bacterial isolate tested Amino acid decarboxylase activity
Tyrosine Lysine Phenylalanine Histidine
1 GP 1 − − + −
2 GP 2 + + + −
3 GP 3 + + + −
4 GP 4 + + + −
5 GP 5 + + + −
6 GP 9 + − − −
7 GP 11 − − − −
Open in a new tab

Determination of biogenic amines using HRESI-MS

After confirmation of decarboxylase activity, the antifungal active cultures GP1, GP9, and GP11 were subjected to mass production and carried out the derivatization process using benzoyl chloride derivatization method. HRESI-MS analysis of the sample, confirmed the presence of phenylethylamine with the molecular weight of 121.0891 (M) from the isolate GP1 (Fig. 1a). While, GP9 sample analysis had confirmed the presence of tyramine with the molecular weight 176.0478 (M + K) (Fig. 1b). Whereas GP11 isolate did not exhibit any tested biogenic amines (Fig. 1c). None of the isolates showed histamine production.

Fig. 1.

Fig. 1

Fig. 1

Open in a new tab

a HRMS of GP-1 LAB isolate, producingphenylethylamine(121.0891 g/mol). b HRMS of GP-9 LAB isolate, producing biogenic amine tyramine. c HRMS of GP-11 LAB isolate, with no amines production

Genomic DNA isolation, PCR and sequencing

The isolate showing intense antifungal activity and which did not show the tested biogenic amines production was identified by 16S rRNA analysis. 16S rRNA gene sequence data of GP11 isolate was related to the sequence of Lactobacillus plantarum strain. Partial sequence data of bacterial isolate was subjected to BLAST analysis. The BLAST analyses of the bacterial isolate sequences implied the dominance of Lactobacillus plantarum strain. BLAST (NCBI) showed 99% sequence similarity with Lactobacillus plantarum (MF966532). Phylogenetic tree clearly showed that GP11 was closely related to Lactobacillus plantarum (Fig. 2) (Flow Chart) and sequences are submitted to NCBI with accession number MN809327.graphic file with name 13197_2020_4732_Figa_HTML.jpg

Fig. 2.

Fig. 2

Open in a new tab

Phylogenetic tree of bacterial isolate GP11 strain

Sensory attributes of Lacto pickle

The mean score for color, flavor, texture and overall acceptability of processed fermented pickles are given in Table 3. A one way analysis of variance was carried out for color, flavor, texture and overall acceptability and result revealed that there is no significance difference (P < 0.01) in the colour of sample (Fig. 3). The panellists accepted the lacto pickle than the control (Traditional).

Table 3.

Sensory score for color, flavor, texture and overall acceptability of processed pickles (P < 0.01)

Sensory attributes Sensory scores (Mean and SD)
Control (without starter culture) Lacto-pickle (with starter culture GP11)
Color 7.13 ± 0.01 7.33 ± 0.01
Texture 7.19 ± 0.01 7.89 ± 0.01
Flavor 7.39 ± 0.01 8.29 ± 0.01
Overall acceptability 7.40 ± 0.01 8.20 ± 0.01
Open in a new tab

Fig. 3.

Fig. 3

Open in a new tab

Sensory score for color, flavor, texture and overall acceptability of processed pickles

Discussion

Pickle is an important part of Indian meals and provides nutritional values including proteins, carbohydrates, dietary fibres, minerals, and vitamins. Traditional methods are used in the process of pickling, preservation, but the maintenance of hygiene of raw materials and the process has found to be challenging or questionable. Preservation using beneficial indigenous microbiota plays a major role in pickle production (De Llano 1998; Dangmance et al. 2015). Fermentation in pickling methods using appropriate probiotic strains would offer several health benefits and prevent fungal spoilage. Probiotic Lactic acid bacteria are commonly used as starter culture in the fermentation of pickles and these bacteria are capable of producing antimicrobial components which can act as a preserving agent with health benefits. Less amount of BA intake are generally not harmful while their increased amount and when detoxification was reduced, then BA can cause health problems (Doeun et al. 2017). Further, those LAB isolates without decarboxylase activity and with antimicrobial activity have to be chosen to reduce or control the harmful biogenic amines and spoilage.

In this context, a total of 18 bacterial isolates were isolated on MRS media from homemade pickles. Among them, 15 isolates typically exhibited lactobacillus characteristics which were confirmed by biochemical tests. Aspergillus sp. and Penicillium sp. were isolated from spoiled pickles. Out of which, only seven bacterial isolates (GP1, GP2, GP3, GP4, GP5, GP9, and GP11) showed antifungal activity against Aspergillus sp and Penicillium sp. Lactobacillus plantarum GP11 exhibited maximum ZOI 28.33 ± 0.57 mm and 19.66 ± 0.57 mm for Aspergillus sp. and Penicillium sp. respectively. While Enterococcus durans inhibited the growth of different species of Aspergillus and Penicillium including Alternaria alternate (MerihKivanc and Pektas 2014). Further similar results were observed with 32 isolates of lactic acid bacteria which inhibited the growth of Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica and thus the shelf life of fermented cheese was improved (Marcia et al. 2018). Similarly, Jeong-dong (2005) screened antifungal activity of lactic acid bacteria isolates obtained from Kimchi, among them, Lactobacillus casei, L. lactis and L. pentosus were highly active against Aspergillus fumigatus. Thus, the present study is in agreement with earlier reports (Kim 2005; MerihKivanc and Pektas 2014; Marcia et al. 2018).

Further, potent antifungal isolates (GP1, GP2, GP3, GP4, GP5, GP9 and GP11) were screened for decarboxylase activity producing harmful biogenic amines tyramine, cadaverine, and phenylethylamine. Among them, GP2, GP3, GP4, and GP5 isolates produced biogenic amines such as tyramine, cadaverine, and phenylethylamine while GP1 and GP5 produced tyramine and phenylethylamine respectively. However, GP11isolate did not show any tested biogenic amine production. The production of biogenic amines by LAB amino acid decarboxylase activity might have an adverse influence on the quality of fermented products when LAB was used as the starter culture (Fang et al. 2016). To corroborate this study, amino acid decarboxylase active isolates obtained from shalgam and decarboxylated the tyrosine, thus produced harmful tyramine (Aysun et al. 2017). In the present study, LAB GP11 isolate did not produce the biogenic amines by decarboxylase activity. However, the determination of the amino acid decarboxylase activity depending on color change perhaps gives false-negative results (Aysun et al. 2017).

Biogenic amines of food products also determined using various chromatographic and mass spectroscopic methods such as RP-HPLC, GC–MS and LCMS (Nalazek-Rudnicka and Wasik 2017; Bonczar et al. 2018). In recent times, a number of novel techniques have been proposed to detect biogenic amines using simplified analytical procedures. In this regard, the MS/MS such as HRESIMS has found to be better and attractive since its analytical performance provides sensitivity. The positively charged amines make them appropriate for the use of positive ionization often with electrospray sources (Sentellas et al. 2016). Similarly, Nalazek-Rudnicka and Wasik (2017) developed the LC–MS method to determine seventeen biogenic amines in beers and wines and reported that the use of tandem mass spectrometric detection of biogenic amines was most convenient and resulted in high sensitivity and selectivity. Therefore, the biogenic amine production capacity of the highly potent isolates was further confirmed by HRESI-MS analysis. The potent antifungal isolates GP1, GP9, and GP11 were subjected to the production of biogenic amines and HRESI-MS analysis. HRESI-MS analysis of the GP1 sample confirmed to produce the phenylethylamine with the molecular weight of 121.0891 (M) while GP9 isolate confirmed to produce tyramine with the molecular weight 176.0478 (M + K). However, GP11 isolate did not produce any tested biogenic amines coinciding with previous decarboxylase activity.

Similarly, Cunha et al. (2011) used GC MS to analyze biogenic amines in 24 grape juice samples and found fifteen different types of biogenic amines. Among them putrescine was the most abundant followed by ethylamine and methylamine. Whereas Capozzi et al. (2012) conducted HPLC analysis to analyze production/degradation of tyramine and putrescine by Enterococcus faecium OT23 and L. brevis IOEB 9809 and these strains lowered concentration of tyramine and putrescine to 29.62 and 38.17% respectively. Similarly, Acetic acid bacteria, lactic acid bacteria, Oenococci, and yeast have shown to produce biogenic amines (Garai et al. 2007; Fang et al. 2016). However, Moret et al. (2005) reported consumption of high amount of biogenic amines containing fermented food leads to foodborne poisoning and causing various diseases.

Bio-preservation is one of the strategies to prevent food spoilage using various probiotics such as Bacillus, Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Carnobacterium, Aerococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella. When these probiotic LAB were used, some of the species tend to produce biogenic amines tyramine, cadaverine and phenylethylamine (Garai et al. 2007) that would result in allergies, hypertensive crisis, headache, neurological disease manifestation (Singh et al. 2012). In the present study, GP1 and GP9 produced biogenic amines phenylethylamine and tyramine as confirmed by decarboxylase activity and HRESI-MS analysis. Therefore, there is a need to identify non biogenic amine producer in order to use them as bio preservative or for the improvement of flavour and shelf life of the food. In this context, GP11 has been found to be one of the potential non-biogenic amine producers, promising LAB and was identified as Lactobacillus plantarum by 16S rRNA analysis. On sensory evaluation, all the panellists preferred the GP11 fermented mango pickle rather than the pickle without GP11. Lactobacillus plantarum GP 11 strain exhibited potent antifungal activity and it could be further developed for industrial scale Lactopickle production.

Conclusion

The harmful Biogenic amines can be controlled by adopting good manufacturing practices. The non-biogenic amine producer Lactobacillus plantarum GP 11 strain can be used as a potential starter culture for the production of fermented pickle (Lactopickle) in large scale and which can act as bio-preservative with significant antifungal activity.

Author’s contribution

P V Research student, executed the planned experimental work. A R Helped in analysing data. D G Research supervisor, developed the idea, planned the experimental design and prepared the manuscript. M V Helped in the experimentation process and manuscript preparation.

Compliance with ethical standards

Conflict of interest

Authors declare that ‘There is no conflict of interest among authors or instition or any other materials’.

Ethical approval

There is no ethical issues exist in this research work as there are no animals involved in the studies.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Alberto MR, Arena ME, de Nadra MCM. A comparative survey of two analytical methods for identification and quantification of biogenic amines. Food Control. 2002;13:125–129. doi: 10.1016/S0956-7135(01)00051-2. [DOI] [Google Scholar]
  2. Aysun CSM, Demirkol O, Ayhan K. Amino acid decarboxylase activities and biogenic amine formation abilities of lactic acid bacteria isolated from shalgam. Int J Food Prop. 2017;20:171–178. doi: 10.1080/10942912.2016.1152479. [DOI] [Google Scholar]
  3. Barkai-Golan R. Penicillium mycotoxins. Mycotoxins Fruits Vegetables. 2008 doi: 10.1016/b978-0-12-374126-4.00007-3. [DOI] [Google Scholar]
  4. Bonczar G, Filipczak-Fiutak M, Pluta-Kubica A, Duda I, Walczycka M, Staruch L. The range of protein hydrolysis and biogenic amines content in selected acid- and rennet-curd cheeses. Chem Pap. 2018;72:2599–2606. doi: 10.1007/s11696-018-0490-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bryla M, Wáskiewicz A, Podolska G, Szymczyk K, Jedrzejczak R, Damaziak K, Sułek A. Occurrence of 26 mycotoxins in the grain of cereals cultivated in Poland. Toxins. 2016;8:160. doi: 10.3390/toxins8060160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabo ML, Braber AF, Koenraad PMFJ. Apparent antifungal activity of several lactic acid bacteria against Penicilliumdiscolor is due to acetic acid in the medium. J Food Protec. 2002;65:1309–1316. doi: 10.4315/0362-028X-65.8.1309. [DOI] [PubMed] [Google Scholar]
  7. Capozzi V, Russo P, Ladero V, Fernández M, Fiocco D, Alvarez MA, Grieco F, Spano G. Biogenic amines degradation by lactobacillus plantarum: toward a potential application in wine. Front Microbiol. 2012;3:122. doi: 10.3389/fmicb.2012.00122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cunha SC, Faria MA, Fernandes JO. Gas chromatography-mass spectrometry assessment of amines in Port wine and grape juice after fast chloroformate extraction/derivatization. J Agric Food Chem. 2011;59:8742–8753. doi: 10.1021/jf201379x. [DOI] [PubMed] [Google Scholar]
  9. Dallal MS, Zamaniahari S, Davoodabadi A, Hosseini M, Rajabi Z. Identification and characterization of probiotic lactic acid bacteria isolated from traditional Persian pickled vegetables. GMS Hyg Infect Control. 2017;12:1–8. doi: 10.3205/dgkh000300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dangmance N, Chumkeaw N, Munkhun R, Boonson J. Isolation of lactic acid bacteria from pickled cabbage for probiotic characterization. BHST. 2015;13:63–66. [Google Scholar]
  11. Dapkevicius MLE, Nout MR, Rombouts FM, Houben JH, Wymenga W. Biogenic amine formation and degradation by potential fish silage starter microorganisms. Inter J Food Microbial. 2000;57:107–114. doi: 10.1016/S0168-1605(00)00238-5. [DOI] [Google Scholar]
  12. De Llano DG. Biogenic amine production by wild lactococcal and leuconostoc strains. Lett Appl Microbial. 1998;26:270–274. doi: 10.1046/j.1472-765X.1998.00320.x. [DOI] [PubMed] [Google Scholar]
  13. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie J-M, Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008;36:W465–469. doi: 10.1093/nar/gkn180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Doeun D, Davaatseren M, Chung M-S. Biogenic amines in foods. Food Sci Biotechnol. 2017;26:1463–1474. doi: 10.1007/s10068-017-0239-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fang F, Feng T, Du G, Chen Evaluation of the impact on food safety of a Lactobacillus coryniformis strain from pickled vegetables with degradation activity against nitrite and other undesirable compounds. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2016;33:623–630. doi: 10.1080/19440049.2016.1156774. [DOI] [PubMed] [Google Scholar]
  16. Garai G, Dueñas MT, Irastorza A, Moreno-Arribas MV. Biogenic amine production by lactic acid bacteria isolated from cider. Lett Appl Microbiol. 2007;45:473–478. doi: 10.1111/j.1472-765X.2007.02207.x. [DOI] [PubMed] [Google Scholar]
  17. Gashe BA, Mpuchane S, Matsheka MI, Magwamba CC. Biogenic amine producing bacteria associated with three different commercially fermented beverages in Botswana. Afri J Microbiol Res. 2013;7:342–350. doi: 10.5897/AJMR12.1645. [DOI] [Google Scholar]
  18. Huh CK, Hwang TY. Identification of antifungal substances of Lactobacillus sakei subsp. ALI033 and antifungal activity against Penicillium brevicompactum strain FI02. Prev Nutr Food Sci. 2016;21:52. doi: 10.3746/pnf.2016.21.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hussain H, MohdFuat AR, Vimala B, Ghazali HM. Screening method for detection of immediate amino acid decarboxylases-producing bacteria implicated in food poisoning. Trop Biomed. 2011;28:351–361. [PubMed] [Google Scholar]
  20. Innocente N, Dágostin P. Formation of biogenic amines in a typical semihard Italian cheese. J Food Protect. 2002;65:1498–1501. doi: 10.4315/0362-028X-65.9.1498. [DOI] [PubMed] [Google Scholar]
  21. Kalagatur NK, Kamasani JR, Mudili V. Assessment of detoxification efficacy of irradiation on zearalenone mycotoxin in various fruit juices by response surface methodology and elucidation of its in vitro toxicity. Front Microbiol. 2018;9:2937. doi: 10.3389/fmicb.2018.02937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim J-D. Antifungal activity of Lactic Acid Bacteria isolated from Kimchi against Aspergillus fumigatus. Mycobiology. 2005;33(4):210–214. doi: 10.4489/MYCO.2005.33.4.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kongkiattikajorn J. Effect of ginger extract to inhibit biogenic amines accumulation during nham fermentation. J Food Chem Nanotechnol. 2015;1:15–19. doi: 10.17756/jfcn.2015-003. [DOI] [Google Scholar]
  24. Marcia LS, Thierry A, Lemaître M, Garric G, Harel-Oger M, Chatel M, Lê S, Mounier J, Valence F, Coton E. Antifungal activity of lactic acid bacteria combinations in dairy mimicking models and their potential as bioprotective cultures in pilot scale applications. Front Microbiol. 2018;9:1787. doi: 10.3389/fmicb.2018.01787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MerihKivanc KS, Pektas S. Screening of lactic acid bacteria for antifungal activity against fungi. J Food Process Technol. 2014;5:310. doi: 10.4172/2157-7110.1000310. [DOI] [Google Scholar]
  26. Moracanin SV, Stefanovic S, Radicevic T, Borovic B, Djukic D. Production of biogenic amines by lactic acid bacteria isolated from Uzicka sausages. Procedia Food Sci. 2015;5:308–311. doi: 10.1016/j.profoo.2015.09.068. [DOI] [Google Scholar]
  27. Moret S, Smela D, Populin T, Conte LS. A survey of biogenic amine content of fresh and preserved vegetables. Food Chem. 2005;89:355–361. doi: 10.1016/j.foodchem.2004.02.050. [DOI] [Google Scholar]
  28. Nalazek-Rudnicka K, Wasik A. Development and validation of an LC–MS/MS method for the determination of biogenic amines in wines and beers. Monatsh Chem. 2017;148:1685–1696. doi: 10.1007/s00706-017-1992-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Önal A. A review: current analytical methods for the determination of biogenic amines in foods. Food Chem. 2007;103:1475–1486. doi: 10.1016/j.foodchem.2006.08.028. [DOI] [Google Scholar]
  30. Ono H, Shoko N, Tsurii J, Kawamoto T, Sonomoto K, Nakayama J. Monitoring of the microbiota profile in nukadoko, a naturally fermented rice bran bed for pickling vegetables. J Biosci Bioeng. 2014;20:1–6. doi: 10.1016/j.jbiosc.2014.04.017. [DOI] [PubMed] [Google Scholar]
  31. Priyadarshani WD, Rakshit SK. Growth and biogenic amine (histamine and tyramine) potential of probiotic Lactobacillus casei in skim milk. Am J Food Technol. 2014;9:69–79. doi: 10.3923/ajft.2014.69.79. [DOI] [Google Scholar]
  32. Rodriguez MBR, Da Silva CC, Da Silva Feijó MB, Júnior CAC, Mano SB. Bioactive amines: aspects of quality and safety in food. Food Nutri Sci. 2014;5:138. [Google Scholar]
  33. Sagratini G, Fernández-Franzón M, De Berardinis F, Font G, Vittori S, Mañes J. Simultaneous determination of eight underivatized biogenic amines in fish by solid-phase extraction and liquid chromatography-tandem mass spectrometry. Food Chem. 2012;132:537–543. doi: 10.1016/j.foodchem.2011.10.054. [DOI] [PubMed] [Google Scholar]
  34. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  35. Sentellas S, Nunez O, Saurina J. Recent advances in the determination of biogenic amines in food samples by (U) HPLC. J Agric Food Chem. 2016;64:7667–7678. doi: 10.1021/acs.jafc.6b02789. [DOI] [PubMed] [Google Scholar]
  36. Shiling L, Caihong J, Xinglian X, Chengjian X, Kaixiong L, Ruihua S. Improved screening procedure for Biogenic amine production by Lactic Acid Bacteria and Enterobacteria. Czech J Food Sci. 2015;33:1–8. [Google Scholar]
  37. Singh VP, Pathak V, Verma AK. Fermented meat products: organoleptic qualities and biogenic amines–a review. Am J Food Technol. 2012;7:278–288. doi: 10.3923/ajft.2012.278.288. [DOI] [Google Scholar]
  38. Swamy CT, Gayathri D, Devaraja TN, Bandekar M, D’Souza SE, Meena RM, Ramaiah N. Plant growth promoting potential and phylogenetic characteristics of a lichenized nitrogen fixing bacterium Enterobacter cloacae. JBM. 2016;56:1369–1379. doi: 10.1002/jobm.201600197. [DOI] [PubMed] [Google Scholar]
  39. Xue H, Huaxi Y, Lanwei Z, Weiwei H, Yingchun Z, Lili Z, Ming D. Improvement of fermented chinese cabbage characteristics by selected starter cultures. J Food Sci. 2014 doi: 10.1111/1750-3841.12495. [DOI] [PubMed] [Google Scholar]
  40. Yu J, Gao W, Qing M, Sun Z, Wang W, Liu W, Pan P, Sun T, Wang H, Bai N, Zhang H. Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China. J Gen Appl Microbio. 2012;58:163–172. doi: 10.2323/jgam.58.163. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Food Science and Technology are provided here courtesy of Springer

RESOURCES