Biogenic amines (BAs) are low molecular weight organic compounds that have been identified as toxicological agents in various foods, such as fishery products, dairy, meat, wine, and so on [1
]. The ingestion of foods containing relatively high concentrations of BAs could lead to several health hazards, such as headaches, hypotension, respiratory distress, heart palpitations and digestive problems, particularly when alcohol is present [3
]. Histamine, which is well-known because of its implication in many food poisoning cases, has a potent vasodilatory action that could cause important drops in blood pressure [5
]. Tyramine, as one of the vasoconstrictor amines, can provoke a release of noradrenaline resulting in an increase of arterial pressure [5
]. Even though there are no accurate regulations for BAs, several countries including France, Germany and Australia have set regulations and limits for histamine and many wine importers in the EU require a BA analysis [4
]. The presence of BAs is considered a marker of poor wine quality and bad winemaking practices [4
BAs are synthesized in fermented food by decarboxylation of corresponding amino acids by microorganisms [1
]. According to the previous studies, BAs could be formed by lactic acid bacteria in wine [8
], Chinese rice wine [10
] and Korean rice wine [1
]. As a traditional alcoholic beverage, Chinese rice wine (CRW), which has been popular in China for thousands of years [11
], has high nutritional values, and thus, it has been used as an ingredient in traditional Chinese medicine [12
]. Since the brewing process of CRW is the typical open semisolid-state fermentation, lots of microorganisms (molds, yeast, bacteria) are brought in the glutinous rice with the addition of Chinese koji [3
], and the system is favorable to BAs generation combining with the high amount of free amino acids [2
]. The abundant bacteria in CRW, mainly originating from Chinese koji, the surroundings and the surfaces of the equipment, could be one of the main reasons for the formation of BAs [10
Histamine, tyramine, putrescine, cadaverine and phenylethylamine are the most representative BAs detected in the wine [6
]. Histamine and tyramine have been considered as the most toxic products in wine, and putrescine and cadaverine could potentiate these effects [4
]. The formation of BAs was traditionally controlled by avoiding the growth of spoilage bacteria, decreasing the amino acid precursors and inoculating starter cultures with negative decarboxylase activity [6
]. Driven by greater awareness of the importance of food quality and safety by consumers, the methods for degradation of BAs in fermented foods have been explored. Biological enzymatic degradation of BAs would be a safe and economic way while avoiding the production difficulties. Two Lactobacillus plantarum
strains (named NDT 09 and NDT 16) isolated from red wine were able to degrade 22% of tyramine and 31% of putrescine, respectively [14
]. Three different strains of Brevibacterium linens
were utilized to eliminate tyramine and histamine in cheese [6
], and the strain K. varians
LTH 1540, it was also found, could degrade tyramine during sausage ripening [15
]. Two lactic acid bacteria were used to degrade 50%–54% of histamine in fish silage [16
]. However, the relationship between BAs degradation and microbiological enzymes of the strains has not been explored yet.
In this work, a Lactobacillus plantarum was obtained from CRW which could degrade BAs. The optimal industrial conditions of the selected strain were analyzed, and the microbiological amine oxidase enzymes were identified and biochemically characterized. Our results could receive considerable interest by providing a green industrial strategy to control the BAs contents in the rice wine and improve the safety consumption of the fermented foodstuffs.
2. Materials and Methods
Man Rogosa Sharpe agar (MRS) medium was obtained from Oxoid. Ltd. (Basingstoke, Hants, UK). The BA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bacterial genomic DNA extraction kit was obtained from Tiangen (Beijing, China). Ultra-pure water was obtained from a Millipore purification system (>18.3 MΩ·cm). Formic acid, methanol and acetonitrile used in the preparation of the mobile phase were of LC-MS grade. All other chemicals used were of analytical grade.
2.2. Strains Screening and Identification
Fermentation broths were collected at the later stage from a typical rice wine production process in Shaoxing (Zhejiang, China). The suspension was filtered through four layers of sterile gauze to remove the unliquefied rice and sealed in a sterile plastic bottle. One gram of fermentation broths was diluted 10-fold by a 0.85% NaCl solution and routinely subcultured 5 to 10 times on MRS medium to obtain purified clones. The screening medium designed was based on the method of Landete [17
] to obtain the bacteria that could decrease biogenic amine content. These strains isolated were kept frozen at −20 °C in a sterilized mixture of culture medium and glycerol (50:50, v
) according to the methods described by García-Ruiz [18
], and further identified by 16S rRNA gene sequencing.
2.3. HPLC Determination of Biogenic Amines
Eight biogenic amines of Histamine (HIS), tyramine (TYR), putrescine (PUT), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPM) and spermidine (SPD) were analyzed according to the method of Callejon, Sendra [13
] with slight modifications. The individual strains were cultured on MRS, and 107
cfu/mL were inoculated with the MRS liquid medium contaminated with 50 mg/L of each amine at pH 5.5. After 48 h incubation at 30 °C, the reaction was stopped by adding HCl. Samples were centrifuged at 8000 rpm for 15 min and the supernatant was pipetted into a screw-capped vial. The pre-column derivatization procedure using dansyl chloride as derivatization reagent was performed according to the report of Yongmei, Xin [12
]. The samples were filtered through 0.22 μm millipore syringe filters and analyzed by RP-HPLC using on LC-20A HPLC system (Shimadzu, Kyoto, Japan) with an Agilent C18 column (250 mm × 4.6 mm, 300 A pores, 5 μm particles, Agilent Technologies, Inc., Santa Clara, CA, USA). The column temperature was kept at 30 °C and the detection wavelength was 254 nm with a flow rate of 1.0 mL/min by using water (A) and methanol (B) as eluents. The gradient elution program consisting of a linear gradient from 65% to 70% B in 7 min followed by from 70% to 80% B in 13 min and 3 min isocratic elution.
The percentage of BAs degradation was calculated based on the HPLC data as following,
BAs degradation (%) = (Ccontrol − Cstrain)/Ccontrol
was the concentration of the BAs in the control medium and Cstrain
was the concentration of the BAs in the medium incubated with the strain.
2.4. Bacterial Growth Analysis
The bacterial growth was measured according to the methods described by Cui [19
]. Briefly, the isolated lactic acid bacteria (LAB) strains were diluted to 105
cfu/mL in MRS liquid medium, and the pH and optical density (OD600 nm
) of medium was checked at 28 °C, 33 °C and 37 °C for 36 h, respectively.
2.5. The Bacterial Starter Application in Pilot Scale Fermentation
A pilot fermentation was performed according to the methods described by Zhang, Xue [10
] with modifications (Figure 1
). Glutinous rice (12 kg) was soaked at 18 °C for 20 h and steamed for 30 min. After naturally cooling to room temperature (about 25 °C), the steamed rice was transferred into a 33 L wide-mouth bottle to which 14.5 kg water, 1.5 kg Chinese koji (unique saccharifying agent including molds, yeasts and bacteria, obtained from COFCO Shaoxin wine Co., Ltd., Shaoxin, China) were added. The main fermentation was carried out at 33 °C for 4 days with intermittent oxygen filling, and post-fermentation was then carried out at 28 °C for 20 days. The isolated strain with 105
(low level), 106
(middle level) and 107
(high level) cfu/mL was added into the CRW at the main fermentation and post-fermentation stage, respectively. After filter pressing, clarification, wine frying and sterilization (90 °C for 3 min), finished Chinese rice wines were obtained. Ten milliliters of fermentation broths were taken from different fermentation stages, including addition of starter (AS); main fermentation (MF); post-fermentation 5d (PF5d); post-fermentation 10d (PF10d); and post-fermentation 20d (PF20d)), to analysis the changes in the BAs contents by using the HPLC method. According to the previous studies [20
], pH, alcohol content, total sugar, total acid, non-sugar solid and amino acid nitrogen of CRW were analyzed by using official methods (Chinese National Standard GB/T 13662-2008). Sensory evaluation of CRW was conducted by 30 panelists (15 males and 15 females) who have professional training certificates. The procedure was conducted in a sensory laboratory following GB/T 13662-2008 and ISO 4121. A total of 11 sensory attributes of appearance (color and turbidity), aroma (alcohol, fruit and cereal), taste (sweet, sour and bitter), mouthfeel (astringency, continuation and full body) and harmony were chosen to characterize the sensory properties using quantitative descriptive analysis involving a 0–9 ten-point linear scale (0: none; 1–2: very weak; 3–4: ordinary; 5–6: moderate; 7–8: strong; 9: very strong).
2.6. Separation of the Amine Oxidases
Cell-free extracts were obtained by using the method of Callejon [22
]. The bacterial cells from a 1 L culture were collected by centrifugation at 10,000 rpm, 20 min at 4 °C and washed twice with 50 mM sodium phosphate buffer (PBS), pH 7.4. The samples were resuspended in PBS buffer containing 1 mM of phenyl methylsulfonyl fluoride (PMSF) as protease inhibitor. Cell-free extracts were obtained by disrupting the bacterial cells with 1 g of 106 μM diameter glass beads in a Mikro-dismenbrator®
Sartorius: 10 cycles of 40 s, alternating 5 cycles of disruption with a cooling step of 5 min in ice. The samples were centrifuged at 13,000 rpm for 15 min (PrismR, Labnet, USA), and supernatants were saved at −20 °C until use. The protein content was determined by using the bicinchoninic acid assay kit (BCA, Solarbio, Beijing, China). Monoamine oxidase (MAO) assay kit and diamine oxidase (DAO) assay kit (Jiancheng Institute, Nanjing, China) were both used to determine the amine oxidase activity. The MAO assay kit was based on the ability of MAO to form H2
substrate, which could be determined by a fluorimetric method. The DAO assay kit was based on the oxidation of PUT to pyrroline plus NH3
, which can be determined by the fluorimetric method.
The cell-free extracts were further ultracentrifuged at 47,000 rpm for 1 h, and the supernatant was precipitated by 75% saturation of ammonium sulfate precipitation [22
]. The protein was redissolved with 50 mM PBS and were loaded onto a Sephadex G-100 column (1.6 cm × 70 cm) followed by a linear gradient elution with a flow rate of 1 mL/min. The protein fraction was collected and measured at 280 nm by using a HD-93-1 spectrophotometer (Purkinje General Instrument Co. Ltd., Beijing, China). There fractions were collected (P1, P2 and P3, Supplement Figure S1
), and were then concentrated and freeze-dried. The degradation ability of the fractions was further evaluated by incubation with 50 mg/L eight biogenic amines at pH 4.0, 33 °C for 2 h.
2.7. Identification of the Amine Oxidases
The fractions separated from the cell-free extracts were digested with trypsin (Promega, Madison, WI, USA) overnight at 37 °C and were identified by LC-MS/MS using the Easy nLC-1000 nano ultra-high-pressure system (Thermo Fisher Scientific, San Jose, CA, USA) coupling with a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The peptide mixture was loaded onto a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE, USA) in buffer A (0.1% Formic acid) and separated with a linear gradient of 4%–50% buffer B (80% acetonitrile and 0.1% formic acid) for 50 min, 50%–100% B for 4 min, and held at 100% B for 6 min at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan for high-energy collisional dissociation (HCD) fragmentation and was searched by using MASCOT engine and Proteome Discoverer 1.3 against the local uniport_lactobocilluspiantarum database.
2.8. Enzymatic Properties of the Amine Oxidases
Effects of temperatures (15, 20, 25, 28, 30, 35, 40, 80 °C at pH 4.0 for 2 h), pH (3.0–5.0) at 30 °C for 2 h, and metal ions (0.2 mol/L, copper ion, ferrous ion, zinc ion, calcium ion and magnesium ion) at 30 °C for 2 h (pH 4.0) on the amine oxidase degradation activity were further investigated.
2.9. Statistical Analysis
All samples were prepared in three independent and each was analyzed in triplicate by the analysis of variance (ANOVA). The results were considered significant at p ≤ 0.05 by the Duncan test.
Biogenic amines are considered as potential health risks since high amounts of them can lead to a series of health problems. The intake of foods with high level of BAs could induce the release of adrenaline and noradrenaline, provoking gastric acid secretion, increased cardiac output, migraine, tachycardia, increased blood sugar levels, and higher blood pressure [23
]. Several researches supported the view that the BAs were formed in winemaking mainly by lactic acid bacteria carrying specific metabolic pathways that convert precursor amino acids into BAs [24
]. In contrast, there is a lack of studies concerning BAs degradation by food sourced micro-organisms in wine, especially in Chinese rice wine.
In this paper, a L. plantarum
CAU 3823, isolated from Chinese rice wine, can degrade more than 40% of the BAs, especially the five major BAs of TYR, PUT, HIS, PHE and CAD in Chinese rice wine. A similar research in grade wine showed that only one strain, L. casei
IFI-CA 52, showed a strong ability to degrade the BAs (54% HIS, 55% TRY and 65% PUT) isolated from wine/ grape cell cultures of 85 strains [18
]. However, the histamine-degrading ability of L. casei
IFI-CA 52 was only 17% when addition of 12% ethanol, suggesting that the ability of L. casei
IFI-CA 52 to reduce amine concentrations in wines would be rare. Regrettably, the ability of this strain to degrade other BAs was not analyzed. Moreover, a pilot scale fermentation, rather than addition of ethanol, would be a better choice to simulate accurately the complicated wine matrix.
In our experiment, pilot scale fermentation tests had proved that L. plantarum
CAU 3823 was competent to be used as an extra starter in CRW industrial producing. Chinese koji was added at the beginning of brewing, which could bring in lots of bacteria, thus the BAs accumulated significantly at the beginning [13
]. The BAs concentration showed a notably increase in the common CRW (the control group) from the starter addition stage to 10-days post-fermentation, indicating the proliferation of bacteria [13
]. The concentration of BAs decreased at the end of post-fermentation, which might be due to the bacteria growth inhibition as the total acid increased during fermentation. According to our results, L. plantarum
CAU 3823 could degrade the BAs in the CRW brewing process, and the formation of biogenic amines was further degraded by increasing the dose of strain. In this study, HIS, TYR, PUT and CAD were degraded significantly during the pilot scale fermentation, especially TYR, which indicated L. plantarum
CAU 3823 could provide a more safety traditional fermented beverage for consumers.
Identification of functional microorganisms in CRW to reduce the formation of BAs has received more interest. Liu, Yu [13
] utilized an in vivo screening process based on the next-generation sequencing technology to find BA-decreasing microorganism in CRW, and three Lactobacillus
strains were detected that would not form biogenic amines, but only L. plantarum
JN01 could grow under 15% ethanol, and the wine could form an unpleasant rancidity taste and more than 8 g/L total acid when the L. plantarum
JN01 was more than 0.01 gDCW/t. Indeed, high level of functional bacteria could bring about unsatisfactory changes in CRW. A similar trend found in the current study showed that the total acid increased, and alcohol content decreased when 107
cfu/mL (high level) of L. plantarum
CAU 3823 was added into the fermentation mash. Although the sensory scores were also decreased, the whole CRW was within the acceptable range for consumers at high level of the strain. Therefore, L. plantarum
CAU 3823 could be the best choice to date to decrease BAs in CRW.
As a traditional alcoholic beverage, total sugar, alcoholic degree, pH value, total acid, amino acid nitrogen and non-sugar solid would play important roles in the flavor, taste and nutrition of Chinese rice wine [22
]. Although high level (107
cfu/mL) of L. plantarum
CAU 3823 could degrade the BAs maximally, undesirable influence on the acceptability was also noteworthy. Low level (105
cfu/mL) and middle level (106
cfu/mL) of L. plantarum
CAU 3823 could eliminate the negative effect on the qualities of the wine, and what’s more important, similar sensory characteristics were obtained in CRW. Thus, to degrade the content of BAs in CRW to the highest extent, middle level (106
cfu/mL) of the L. plantarum
could be chosen in the CRW fermentation process.
Non-sugar solids, a major nutrition indicator to evaluate the quality grade of CRW, are mainly composed of dextrin, glycerin, non-volatile acid, protein and hydrolysates [25
]. Interestingly, the content of non-sugar solids was increased remarkably when L. plantarum
CAU 3823 was used, especially at middle level (106
cfu/mL), which provided a novel insight that the L. plantarum
CAU 3823 could produce more non-sugar solids in CRW and thus have potential nutritional values.
BA can be converted into products via oxidation by microorganisms which can be used as a carbon and/or energy source or as a nitrogen source [26
]. Limited studies attributed these transformations to amine oxidase activity derived from microorganisms. Yagodina [27
] reported that flavoprotein oxidases existing in some microorganisms could catalyze the oxidation of BAs. Sekiguchi [28
] found a histamine oxidase in the actinobacteria Arthrobacter crystallopoietes
KAIT-B-007 isolated from soil. In this study, the amine oxidases from L. plantarum
CAU 3823 were purified and characterized. Nine amine oxidase proteins, a mixture from L. plantarum
CAU 3823, contributed the most of amine-degrading ability of L. plantarum
CAU 3823. Eight MAOs were identified and thus confirmed a good monoamine oxidase activity shown in fraction 1. Amine oxidases can be divided into two subfamilies based on the cofactor they contain. MAO (EC 126.96.36.199) are a family of enzymes containing flavin that catalyze the oxidation of monoamines, employing oxygen to clip off their amine group [29
]. The amine oxidases containing copper as cofactor (EC 188.8.131.52) are homodimers, which contain three subclass, namely, diamine oxidase, primary-amine oxidase and diamine oxidase [30
]. Amine oxidase [flavin-containing] A and B can catalyze the oxidative deamination of biogenic amines [31
]. Amine oxidase [flavin-containing] B that in humans was encoded by the MAOB gene could preferentially degrade PHE [32
], which confirmed 44.3% PHE degradation in fraction 1. An “aromatic cage” has been found to play a steric role in substrate binding and in flavin accessibility and helps to increase the substrate amine nucleophilicity [33
], which might enhance BA degradation. It is noted that no diamine oxidase was identified although cell-free extracts showed diamine oxidase activity.
To provide a seemingly feasible solution to degrade the BAs in foodstuffs, the biochemical character assays of the amine oxidases mixture from L. plantarum
CAU 3823 were designed. The enzymes were very thermostable, as the activity remained stable at 80 °C, and were fully stable over the pH range of 3–5. Similar results were reported that a putrescine oxidase from Rhodococcus erythropolis
NCIMB 11540 could be stable at 50 °C for 2 h [34
] and a thermostable histamine oxidase was found in Arthrobacter crystallopoietes
]. These results indicated that the amine oxidases could be stable to use in fermented food processing.