Evaluation of antimicrobial activity of water-soluble flavonoids extract from Vaccinium bracteatum Thunb. leaves

Abstract

Aqueous extract of Vaccinium bracteatum Thunb. leaves (VWFE) is traditionally used for food preservation in China, which is rich in flavonoids compounds. VWFE could effectively inhibit the growth of both Gram negative (Escherichia coli) and positive bacteria (Staphylococcus aureus and Bacillus subtilis), however, no inhibition effects were observed on mold and yeast. The minimum inhibitory concentration of VWFE were 2.06 mg/ml, 1.03 mg/ml, and 4.11 mg/ml for E. coli, S. aureus and B. subtilis, respectively, which were 13%, 13%, and 26% of sodium benzoate and 23%, 11%, and 46% of potassium sorbate. Cell membrane permeability assays indicated that cell membrane disruption was one of the antibacterial mechanisms of VWFE. VWFE showed a good thermal stability. The expiration date of VWFE was 6 months at 25 °C, which was predicted using the accelerated aging method. This present work indicated VWFE is a potential natural antibacterial preservative.

Introduction

Food could be preserved for a long time when the basic causes of its spoilage are controlled. The methods for preserving food are varied, and adding chemical preservatives is a conventional method for enhancing food safety, since food pollution and spoilage are commonly caused by the action of microorganism among other factors (Jang and Kim, 2017). However, consumers today are increasingly concerning about chemicals which residues in food and tend to choose natural, healthful, and safe food (Caleja et al., 2016). Consequently, there is growing interest in using natural antimicrobial compounds, such as extracts of spices and herbs for food preservation (Bag and Chattopadhyay, 2015; Martínez-Graciá et al., 2015). Specially, the extracts of various medicinal plants containing flavonoids have been reported to possess strong antimicrobial activity (Bitis et al. 2017; Seleem et al., 2016).

The flavonoids that belong to polyphenols and generally occur as glycosylated derivatives constitute, which are a large group of secondary plant metabolites (Bag and Chattopadhyay, 2015; Wang et al., 2018). As dietary compounds, they are widely known as antioxidants that can inhibit the oxidation of low-density lipoproteins and reduce thrombotic tendencies. Many studies have shown that plant flavonoids have many biological activities such as antimicrobial activity (Sousa et al., 2009), and antioxidant (Youn et al., 2019), and hypolipidemic (Sharma et al., 2008).

Vaccinium bracteatum Thunb. (VBT, named as Wu Fan Shu in China) that belongs to the genus of Rhododendron is a resource of traditional Chinese herbal medicine. It is grown massively in China but can be found worldwide. Some physiological functions of VBT leaves (VBTL) had been recorded in Compendium of Materia Medica, such as anti-fatigue, digestion resistibility, and antioxidant activity. The antioxidant-active compounds isolated from VBTL were determined as quercetin, chrysin, apigenin, kaempferol, and lutelin, in our previous research (Wang et al., 2007). Recent reports showed that VBTL are rich in phenolic and flavonoid compounds (Wang et al., 2011; Chen and Zhang, 2014).

In eastern coastal region of China, aqueous extract of VBTL is used to dyeing rice, and the product could be stored for more than 3 months without any preservative treatments. However, there are few reports about the antimicrobial activity it. In this presented work the antimicrobial activities of VBTL water-soluble flavonoids extract (VWFE) against several food-related microorganisms were investigated.

Materials and methods

Materials

VBTL were collected from Liyang county, Jiangsu province of China. The VBTL was identified and authenticated by Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Jiangsu, China. A voucher specimen (Number 00196872) was deposited in the herbarium of the Institute. O-Nitrophenyl-β-d-Galactopyranoside (ONPG, analytical grade) was purchased from Sigma Chemical Co (St Louis, MO, USA). All other chemicals used were of analytical grade.

VBTL (50 g) that had been ground to pass 60 mesh were homogenized by a crusher and soaked with deionization water (1000 ml) under 50 °C for 2 h, and then centrifuged at 3000×g for 30 min. The residues were re-extracted two more times under the same conditions. The water extract was concentrated by rotary evaporation at 55 °C. Then, flavonoids of the concentrate were separated by adding ethanol to a final concentration of 75% (v/v) (Ghani et al., 2008). The supernatant was concentrated by rotary evaporation at 55 °C under reduced pressure to remove ethanol and the VWFE were remained.

The flavonoids was measured using a previously reported method with rutin as standard (Meda et al., 2005). It was expressed as rutin equivalents in mg per ml extract.

Strains and media

Three fungi, Aspergillus niger 3.3928, Aspergillus oryzae 3.5232, Saccharomyces cerevisiae 2.1882 and three bacterial, Escherichia coli K12, Staphylococcus aureus 1.89, Bacillus subtilis 168, which are very common in foods, are all stored at School of food science and technology, Jiangnan University, Wuxi, China. LB medium is used for cultivation of E. coli, S. aureus, and B. subtilis. PDA medium is used for A. niger and A. oryzae, and S. cerevisiae is cultured with YPD medium. The tested bacteria strains were cultured at 37 °C, and fungi were cultured at 28 °C. The cultivation was stopped at 0.8–1.0 OD ranges (equivalent to 10⁸ CFU/ml) for antimicrobial tests. The optical density (OD) of fermentation broth was determined at 625 nm (Rauha et al., 2000).

Antimicrobial assay

Oxford cup assay was employed to determine the extract’s antimicrobial activity (Zampini et al., 2005). 0.1 ml culture suspension was spreaded on the surface of plates. Two Oxford cups (φ5 mm) were arranged on the plate surface. The VWFE of 0.25 ml was added to the one of the cup, while sterile water was added to the other as a control. The bacteria plates were incubated at 37 °C for 24 h, while the fungi plates were incubated in 28 °C for 48 h. After incubation, the inhibition zones were measured. The result was reported as the average of six replicates.

The minimum inhibitory concentration (MIC) was defined as the lowest concentration to prevent visible growth. MIC values were determined using tube double dilution method (Zampini et al., 2005). VWFE were diluted with sterile deionization water by two-fold serial dilutions. The dilution of 1 ml was incorporated into 9 ml of agar media. After solidification, 0.1 ml microbe suspension was coated. The bacteria plates were incubated at 37 °C for 24 h, while the fungi plates incubated in 28 °C for 48 h.

Time-kill assay

The time-kill assay was performed according to the reported method (Lou et al., 2010). The liquid media with VWFE concentrations of 1 × MIC, 3 × MIC, and 5 × MIC were prepared, respectively. The microorganism was inoculated with 10% inoculum (v/v). The cultivation for bacterial was performed in 250 ml shake flasks containing 50 ml medium at 37 °C and 150 rpm, and for fungi was cultured in 250 ml shake flasks containing 25 ml medium at 28 °C and 150 rpm.

During cultivation, 1 ml samples were drawn periodically and diluted with sterile deionization water by 10 folds serial dilution. Each dilution was coated on agar plates (100 μl) and incubated at 28 °C or 37 °C for 24 h, and then the colonies were counted. Only plates containing colonies between 30 and 300 counts were counted. Bactericidal activity was defined as a ≥ 3 log₁₀ reduction (≥ 99.9% reduction) in CFU/ml. All samples were made with 3 replicates.

Analysis of cell membrane disruption

The integrity of cell membrane was indicated by the activity of β-D-galactosidase which is produced by living cell (Gao et al., 2007). The substrate of ONPG can be hydrolyzed to form o-nitrophenol (ONP) under catalysis of β-d-galactosidase. The product, ONP, has a characteristic absorption at 420 nm, so it is possible to estimate whether the cell membrane of microorganisms is disrupted through examining the extent of the above enzyme-accelerating reaction.

Cells suspension (0.75 ml, 10⁷ CFU/ml) of S. aureus (Gram-positive, G⁺) and E. coli (Gram-negative, G⁻) and ONPG solution (0.75 ml, 25 mM) were added simultaneously into sodium phosphate buffer (7.5 ml, pH 7.4). After shaking for 1 min under 37 °C, VWFE (1 ml) were added, and then the changes of absorption at 420 nm were measured continuously with time varying.

VWFE stability to pH and temperature

To investigate the effect of pH on VWFE stability the pH of VWFE solution was adjusted to pH ranging 4–9, respectively, with HCl (0.1 M) or NaOH (0.1 M). The samples were incubated at 37 °C for 24 h, and then the remained antimicrobial activities of VWFE against S. aureus were determined under standard assay condition.

The temperature stability was studied by incubating VWFE at 60 °C, 80 °C, 100 °C and 120 °C for 20 min, respectively, and then the residual antimicrobial activity against S. aureus were determined according to the procedure described above.

Expiration date prediction of VWFE

The expiration date of VWFE was predicted by the accelerated aging method (Zheng et al., 2017). The expiration date is defined as the time when the total flavonoid contents is 90% of the initial concentration. The accelerated test was conducted at temperatures of 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C. The content of total flavonoids was detected at 0 day, 2 day, 4 day, 6 day, and 8 day, respectively. Under certain temperature, the equation can be represented as Eq. (1).

$$\text{Log}{\text{C}}_{\text{t}}=log{\text{C}}_0-{\text{k}}_{\text{T}}·\text{t}/2.303$$ 1

C₀ is the initial total flavonoid contents of VWFE (defined as 100%), and C_t is the relative content of total flavonoid at time t. k_T is the rate constant under the temperature T. The k_T at each temperature could be obtained based on the linear regression between log C and t.

Arrhenius exponential law is the theory evidence of accelerated aging test, of which the logarithmic form can be expressed as Eq. (2).

$$\text{Log}{k}_T=\left(-\text{Ea}/2.303\text{R}\right)·\left(1/\text{T}\right)+log\text{A}$$ 2

where k_T is the rate constant calculated from Eq. (1) at temperature T, Ea stands the activity energy, and R is gas constant. A regression equation could be obtained based on the linear relation between log k_T and 1/T. For example, the k₂₅, the rate constant at 25 °C, could be calculated from log k₂₅. Thus, the period for 10% degradation of total flavonoids in VWFE at 25 °C could be calculated according to the Eq. (1), namely, the expiration date of VWFE as a natural preservatives.

Results and discussion

Antimicrobial activity assays of VWFE

The aqueous extract of VBTL is traditionally used for food preservation in China, in which flavonoids are the main activity compounds. The total flavonoid content in the VWFE was measured as 16.46 mg/ml. The antimicrobial activities of VWFE was detected by Oxford cup assay. VWFE showed obvious antimicrobial activity against all tested bacteria. The inhibition zone diameter were 22.9 ± 0.5 mm, 24.6 ± 0.6 mm, and 18.5 ± 0.9 mm, respectively, for E. coli, S. aureus, and B. subtilis. Moreover, some bacteria isolated from fermented rice were tested. 6 strains of E. coli, S. aureus, and B. subtilis, respectively, were selected to perform the Oxford cup assay, which were identified by 16S rDNA sequence analysis. The inhibition zone diameter resulted by VWFE were 23.6 ± 1.1 mm, 25.3 ± 1.6 mm, and 19.9 ± 1.8 mm for E. coli, S. aureus, and B. subtilis, respectively. This result showed that VWFE could inhibit most common pollution bacteria from rice. That is also according to the long-history experience of VBTL application in China. S. aureus was the most sensitive to VWFE. The MIC of VWFE are 2.06 mg/ml, 1.03 mg/ml, and 4.11 mg/ml for E. coli K12, S. aureus 1.89, and B. subtilis 168, respectively, which are only 13%, 13%, and 26% of sodium benzoate and 23%, 11%, and 46% of potassium sorbate.

Plant extracts are always important source for food and pharmaceutical application considering their excellent antimicrobial effects, antioxidant capacity and other activities. Phenolic acids and flavonoids of peanut by-products were reported antimicrobial effects against gram-positive and gram-negative bacteria. The MIC of water extraction of peanut skin are > 5.0 mg/ml, > 5.0 mg/ml, and 2.6 mg/ml for E. coli, S. aureus, and B. subtilis, respectively (Tamura et al., 2016). However, lower MIC were obtained by using meal from dry-blanched peanuts probably due to the difference of phenolics (Camargo, et al., 2017). Moreover, some bioactive flavonoids, such as gliricidin-7-O-hexoside and quercetin-7-O-rutinoside derived from Asplenium nidus (fern), possess potent inhibitory against multidrug-resistant bacteria. (Jarial et al., 2018). Besides, some plant exacts showed antifungal activity (Devi and Ganjewala, 2009; Khan et al., 2017). The MIC of VWFE is similar to that of flavonoid extract from other plants, such as Eriocaulaceae (Silva et al., 2009), Rosmarinus officinalis (Outaleb et al., 2015), walnut green husk (Han et al., 2015), and peanut by-products (Tamura et al., 2016). However, no inhibition effect was observed on mold and yeast with VWFE.

In vitro time-kill assays are expressed as the rate of killing by a fixed concentration of an antimicrobial agent and are one of the most reliable methods for determining tolerance (Hevesi et al., 2012). The time-kill curves of VWFE against bacteria are presented in Fig. 1. Generally, the effect of the VWFE on the test bacteria is time and concentration dependent. The VWFE of 1 × MIC exhibited 2.53 log CFU/ml and 3.53 log CFU/ml drop for E. coli and B. subtilis after 12 h incubation, respectively, indicating 99.7% and 99.9% reduction in cell numbers. Specially, after 12 h incubation complete eradications of three strains were obtained with VWFE of 3 × MIC.

Fig. 1.

Time kill kinetics of VWFE against three bacteria. (A) E. coli; (B) S. aureus; (C) B. subtilis. Triangle, 5 × MIC; square, 3 × MIC; diamond, 1 × MIC

Most active component from plants could inhibit the growth of Gram-positive bacteria while few are active against Gram-negative strains (Alencar et al., 2007; Kuete et al., 2006; Murthy et al., 2006). In this research the VWFE showed a remarkable antibacterial activity both on both Gram-positive and Gram-negative bacteria. Specially, VWFE exhibited stronger effect against S. aureus when compared with that of E. coli and B. subtilis. This result was similarly with the earlier reports about some other plant extracts (Hori et al., 2006; Usman et al., 2009). A possible explanation for it may lie in the differences in the cell membranes and is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside. (Duffy and Power, 2001; Shan et al., 2007). Specially, the resistance of some Gram-negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier to the penetration of numerous antibiotic molecules. However, Gram-positive bacteria do not have such an outer membrane and cell wall structure, so it is more sensitive to some antibacterial substances (Shan et al., 2007).

Effect of VWFE on cell membrane

It was known that β-d-galactosidase has special hydrolyzing activity for β-d-galactose glycoside bond in sugar fat and lactose. If the bacteria cell was broken, the β-d-galactosidase will leak out of the cells, and then catalyzes the hydrolysis of ONPG in the solution (Sun et al., 2014). This unique property therefore can be employed as an indication for any alterations of cell wall and cytoplasmic membrane integrity. As shown in Fig. 2, there was little absorption of the treat without VWFE. The absorption increased with the time extending when VWFE was added into the cell suspension, indicating the cell membrane was disrupted by VWFE and then resulted in the releasing of intracellular β-d-galactosidase. Specially, the absorption of S. aureus was higher than those of E. coli and B. subtili after treated for the same period. Those results were according to the MIC for three strains.

Fig. 2.

Time curves of the absorption of ONP. (A) E. coli; (B) S. aureus; (C) B. subtili. Diamond, the blank control; square, treatment by VWFE

The antibacterial mechanism of flavonoid compounds is based on it ability of destroying the cell membrane of bacteria, it was according with the analysis of cell permeability in this research. The difference sensitive of bacteria strains to flavonoid compounds was due to the difference structures of cell wall and membrane, and then resulted in the difference integrity of cell membranes (Fig. 2) and viable cell (Fig. 1). Thus, the highest sensitivity of S. aureus may be due to the structure of its cell wall and outer membrane (Lee et al., 2008).

Stability analysis and the expiration date prediction of VWFE

As shown in Table 1, the relative antibacterial activity remained more than 50% after incubating for 24 h when the pH was less than 7. However, it was unstable under alkaline conditions, and no inhibition effect was detected when the pH was above 7. As listed in Table 2, the VWFE showed a good thermal stability when it was treated under 100 °C. The basic structural feature of flavonoid compounds is the 2-phenyl-benzo pyrane or flavane nucleus, which consists of two benzene rings linked through a heterocyclic pyrane ring. The hydroxylation at benzene ring of flavonoids is important for the antibacterial activity (Cushnie and Lamb, 2005). When temperature was higher than 100 °C or under alkaline condition, the antibacterial activity of VWFE decreased, indicating the structure might been damaged, especially the hydroxylation at benzene ring of flavonoids (Moriyama et al., 2001).

Table 1.

VWFE stability on pH

pH	The inhibition zone diameter (mm)
	E. coli	S. aureus	B. subtilis
4	22.9 ± 0.5	21.8 ± 1.0	23.3 ± 0.8
5	15.1 ± 0.7	15.9 ± 0.5	22.1 ± 0.7
6	7.4 ± 0.8	9.3 ± 0.8	10.1 ± 0.6
7	–	–	–
8	–	–	–
9	–	–	–

Data are expressed as mean ± SD (n = 6)

“–” means there is no inhibition zone observed

Table 2.

VWFE stability on temperature

Strains	The inhibition zone diameter (mm)
	60 °C	80 °C	100 °C	120 °C
E. coli	27.6 ± 0.9a	25.7 ± 0.5b	24.9 ± 0.9b	23.9 ± 0.6b
S. aureus	27.4 ± 0.8a	25.4 ± 0.8b	24.8 ± 1.0b	25.3 ± 1.2b
B. subtilis	24.3 ± 0.7a	20.8 ± 0.8b	18.6 ± 1.1b	19.6 ± 0.9b

VWFE were treated under 60 °C, 80 °C, 100 °C, and 120 °C for 20 min, respectively. Data are expressed as mean ± SD (n = 6), and different letters are significantly different of LSD test at P < 0.05 for each strain

The accelerated aging method is usually used to predict the stability of pharmaceuticals. As listed in Table 3, the degradation rate of total flavonoids in VWFE increased with the increase of the temperature. Specially, the Log C_t is correlated with the treated time (the correlation coefficients are − 0.9875, − 0.9917, − 0.9948, − 0.9877, and − 0.9856, respectively, for 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C), thus the degradation of total flavonoids in VWFE follows the first order kinetics process. The regression equation under different temperature conditions was obtained as shown in Fig. 3. The rate constant at temperature 25 °C, K₂₅, was calculated as 5.24 × 10⁻⁴ d⁻¹. Therefore, t_0.9, the time when the total flavonoid contents in VWFE is 90% of the initial concentration, is calculated as 201 days according to the Eq. (1). Thus, the expiration date of VWFE as a potential natural antibacterial preservative is predicted as 6 months at 25 °C.

Table 3.

The content of total flavonoids under different temperature conditions

Temperature	Time (days)
	0	2	4	6	8
	Logarithm of the relative content of total flavonoids (Log Ct)
323 K (50 °C)	2	1.996	1.985	1.973	1.961
333 K (60 °C)	2	1.967	1.915	1.898	1.857
343 K (70 °C)	2	1.896	1.778	1.724	1.613
353 K (80 °C)	2	1.836	1.753	1.528	1.301
363 K (90 °C)	2	1.678	1.474	1.033	0.947

Fig. 3.

The regression equation of Log k_T with 1/T

Due to the mistrust of synthetic additives for modern people, the demand for natural preservatives and development of healthy antibacterial compounds is on the rise (Rauha et al., 2000; Caleja et al., 2016). The MIC assay indicated that the antibacterial effect of VWFE was stronger than those of potassium sorbate and sodium benzoate that are usually used in food preservation, indicating that VWFE is a potential additive in food industry. However, a standard product should be developed to expand and facilitate the use of VWFE on food preservation.

Abbreviations

MIC

Minimum inhibitory concentration

ONP

o-Nitrophenol

ONPG

o-Nitrophenyl-β-d-Galactopyranoside

VBT

Vaccinium bracteatum Thunb.

VBTL

Vaccinium bracteatum Thunb. leaves

VWFE

VBTL water-soluble flavonoids extract

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.