Antibacterial activity and mechanism of action saponins from Chenopodium quinoa Willd. husks against foodborne pathogenic bacteria

https://doi.org/10.1016/j.indcrop.2020.112350Get rights and content

Highlights

  • Six saponins are mainly distributed in quinoa husks.

  • Low-polarity saponins have high inhibitory activity against foodborne pathogens.

  • Saponins in quinoa can cause the bacterial biofilm system to collapse.

Abstract

The present study investigated the chemical composition of quinoa saponins and examined their possible antimicrobial modes of action against foodborne pathogenic bacteria.

Six different compounds (Q1-Q6) were extracted from quinoa husks, separated/purified by column chromatography and identified by HPLC/MS and NMR. The anti-bactericidal effects against six types of bacteria, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Salmonella enteritidis, Pseudomonas aeruginosa, and Listeria ivanovii, were determined by the disk diffusion method and assessment of the MIC/MBC. The changes in membrane integrity were tested using a microplate reader, a flow cytometer, and a transmission electron microscopy.

All the compounds exerted anti-bactericidal effects against S. aureus, S. epidermidis and B. cereus. Q4 showed the strongest activity against S. aureus and S. epidermidis, with an MIC value of 0.0625 mg/mL and an MBC value of 0.125 mg/mL. The concentration of quinoa saponin Q4 exhibited a dose-dependent relationship with its anti-bactericidal effect. In fact, a dose-effect relationship was found between the concentration of all quinoa saponins and their bacteriostatic effects. In addition, the release of nucleic acids and proteins from B. cereus increased gradually with increases in the Q4 concentration, and the membrane structure of B. cereus was destroyed by Q4.

Notably, quinoa saponins caused severe damage to the tested bacteria through degradation of the cell wall followed by disruption of the cytoplasmic membrane and membrane proteins, which resulted in leakage of the cell contents.

Introduction

The incidences of food poisoning and foodborne diseases caused by microbial spoilage during processing and storage are increasing throughout the world and becoming extremely complicated public health issues (Patra and Baek, 2016). Foodborne pathogens are one of the main causes of morbidity and mortality worldwide (Yang et al., 2014). As many as 250 types of foodborne diseases, most of which are transmitted through contaminated food, have been identified (Nimri et al., 2014). In the United States, it has been estimated food diseases affect 76 million people each year and are responsible for 5000 deaths each year (Rajeshkumar and Malarkodi, 2014). In addition, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Salmonella, Shigella, Campylobacter, Escherichia coli, Yersinia and Clostridium have been identified as responsible for foodborne diseases (Nimri et al., 2014; Shahbazi, 2015; Scallan et al., 2015).

Synthetic additives and antimicrobial agents are being used in food processing to reduce the growth of microorganisms with the aim of avoiding pollution and prolonging the shelf life of food (Patra and Baek, 2016). Unfortunately, synthetic chemical additives have certain toxic effects, and the use of preservatives has been shown to cause respiratory or other health problems, e.g., the commonly used addictive sodium benzoate, might cause cumulative toxicity and induce asthma and nitrate and nitrite might convert ingested substances into toxins (Chen et al., 2016). More seriously, resistance to penicillin started emerging since the drug started being used (Rajeshkumar and Malarkodi, 2014). At present, the re-emergence of multidrug-resistant strains of foodborne pathogens makes it more difficult to provide a safe food supply, and microorganisms are more resistant to conventional preservatives (Economou and Gousia, 2015). Due to the increasing attention that consumers are paying to processed foods and natural foods containing antibiotics, there is a growing demand for the use of natural inhibitors as alternatives to artificial antimicrobial agents (Cetin-Karaca and Newman, 2015; Ozogul et al., 2015).

The emergence of modified atmosphere packaging (MAP), vacuum packaging (VP), electron beam, heat treatment, acidified sodium chlorite, lactic acid bacteria, antibacterial ice, freezing, irradiation and high pressure has effectively reduced the infection of foods with microorganisms (Alboofetileh et al., 2014). However, some of these technologies have a negative impact on food quality and costs, and some do not remove harmful pathogens, such as monocytogenes, from food (Ozogul et al., 2015; Alboofetileh et al., 2014). Therefore, the identification of alternatives remains a classic approach for reducing or eliminating food-related microorganisms during food shelf life (Alboofetileh et al., 2014).

The extraction of bioactive ingredients from plant resources has always been a hot topic in scientific research (Ozogul et al., 2015; Donato et al., 2015). Saponins are the active components and the main secondary metabolites of the plants used in many ethnic medicines (Ozogul et al., 2015; Yao et al., 2014). The biological activities of various saponins, such as antibacterial, anti-inflammatory, antifungal and antiviral activities, are based on their chemical structures. It has been reported that a type of Rehmannia glutinosa saponin, denoted 3-O-glucopyranose oleanolic acid, exhibits anti-inflammatory activity in the inflammatory exudation and proliferation phase of Randia dumetorum Lam (Yao et al., 2014; Ma et al., 1989). Saponins exist in all types of plants in nature, such as ginseng, lentil, Cassia obtusifolia, Croton macrostachyus, and fenugreek (Kachur and Suntres, 2016; Voukeng et al., 2016; Del et al., 2020). The contents of saponins vary among different plants, but higher abundances are found in quinoa and fenugreek (Del et al., 2020).

Chenopodium quinoa Willd. is the Inca indigenous people’s main traditional food, but its saponins, which are the main component of its husks, are removed during processing (Woldemichael and Wink, 2001; Jarvis et al., 2017). Quinoa saponins have been used as biopesticides against Oncomelania hupensis (Castillo-Ruiz et al., 2018). Previous laboratory studies have shown that oral pathogenic bacteria can be inhibited by crude saponins obtained from quinoa husks (Sun et al., 2019). According to the different saponin skeletons, saponins are divided into triterpenes and sterols. The type of ring formation and the change of the positions of the main functional groups make the aglycon mother core structure subdivided into dozens of species. The number of glycosidic bonds, binding sites and sugar types have resulted in a variety of saponin types. For example, the main types of ginsenosides are tetracyclic triterpenoid dammarane-type saponins (Kim and Zhang, 2015; Rahimi et al., 2019; Xue et al., 2019). Quinoa saponins belong to the triterpenoid saponin oleanolic acid type and are mainly composed of phytolaccagenic acid (PA), oleanolic acid (OA), serjanic acid (SA), and hederagenin (Hed) (Li et al., 2006; Xue et al., 2019). Because few studies have investigated the effect of quinoa saponins on foodborne pathogenic bacteria, the structure-activity relationships of quinoa saponin monomer compounds and the mechanisms of action underlying their bacteriostatic effects remain unclear. Therefore, in this study, we extracted and purified saponins from quinoa husks and studied their inhibitory effects on foodborne pathogenic bacteria as well as their structure-activity relationships and related mechanisms.

Section snippets

Chemicals and instrument

Chenopodium quinoa Willd. husks were purchased from Shanxi Yilong Quinoa Development Co., Ltd. Jingle County, Shanxi Province ground through a 60-mesh screen and set aside. Silicone board G was purchased from Qingdao Ocean Chemical Co., Ltd. Penicillin 800000 units, purity ≥ 98 %) and cefixime (100 mg/tablets, purity ≥ 98 %) were purchased from Shandong Lukang Pharmaceutical Co, Ltd. Staphylococcus aureus (ATCC-6538), Staphylococcus epidermidis (BNCC-102555), Salmonella enteritidis

Liquid chromatogram of saponins from Chenopodium quinoa husks

The liquid-phase spectrum of the prepared solution of crude saponins of quinoa husks is shown in Fig. 2A. The peaks of crude saponins from quinoa mainly consisted of Q1∼Q6, and subsequent separation and purification experiments were then performed to isolate these six saponin monomers (Fig. 2B).

Enrichment results of crude saponins from Chenopodium quinoa husks

The flow chart and content of crude saponins from quinoa husks enriched using macroporous resin are shown in Fig. 1B. As shown, the mass of component I was 2.06 g, and thus, component I accounted for

Discussions

As an important public health issue, foodborne diseases have attracted much attention. Antibiotics constitute very important and necessary strategies for the inhibition of pathogenic bacteria. But bacteria have developed resistance to these drugs. To address this problem, scientists are searching for new antibiotics from natural sources (Rajeshkumar and Malarkodi, 2014). Natural products are relatively safe and effective alternatives to antibiotics. Recent studies have shown that saponins,

Conclusion

Triterpenes or steroids are used as glycosidic skeletons, and sugars bind to one or more points of the structure to form branching chains. This type of structure makes saponins lipophilic and hydrophilic. As a result, the effect of saponins on the biofilm system is mainly reflected in two aspects. First, due to surfactant characteristics, saponins can reduce the surface tension in aqueous solution and form micelles. When the micelle concentration reaches a critical point, certain structural

Funding

This study was funded by project No. ZR2019PH043 supported by Shandong Provincial Natural Science Foundation.

Informed consent

Clinical experiments were not included in this study and there is no informed consent involved.

CRediT authorship contribution statement

Shixia Dong: Methodology, Software, Writing - original draft. Xiushi Yang: Resources, Writing - review & editing. Lei Zhao: Methodology, Data curation. Fengxiang Zhang: Writing - review & editing, Supervision, Data curation. Zhaohua Hou: Writing - review & editing. Peng Xue: Conceptualization, Funding acquisition, Resources, Supervision, Project administration, Writing - review & editing.

Declaration of Competing Interest

There are no conflicts of interest.

Acknowledgements

This work was supported by the Doctoral research start-up funds of Weifang Medical College and project No. ZR2019PH043 supported by Shandong Provincial Natural Science Foundation.

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