Application of metabolomics in the study of plant volatile organic compounds biosynthesis mechanism and antibacterial activity

Baiqu Metabolomics Information: Biosynthetic Mechanism and Antibacterial Properties of Plant Volatile Organic Compounds IF14.
46
Food microbial safety has always been a topic of concern
.
Traditional chemical preservatives are still dominant in practical applications.
With the increasing demand for microbial and chemically safe food, traditional chemical preservatives are gradually fading out of the sight of researchers and consumers.
Therefore, natural products are considered are promising alternatives, especially plant volatile organic compounds (VOCs) with biocompatibility, availability and utility, which have been found to be effective against food-borne pathogens and spoilage organisms
.


On May 13, 2022, Trends in Food Science & Technology (IF=14.
46) published online the paper titled "Rethinking of botanical volatile organic compounds applied in food preservation: Challenges in acquisition, application, microbial Inhibition and stimulation" review article
.



This article aims to comprehensively review the biosynthesis and antibacterial mechanisms of VOCs, and summarize the current and potential applications of VOCs in food pathogen control from both applied and basic research
.
The biosynthesis and rate-limiting steps of VOCs are systematically described, providing guidance for synthetic biology of VOCs production
.
Meanwhile, this paper makes a conceptual distinction between VOCs and essential oils, where the synergistic and antagonistic effects of each VOC component require more research
.
Synthetic biology techniques can replace traditional extraction techniques and realize large-scale production of VOCs.
However, the biosynthesis and regulation pathways of VOCs remain to be cracked
.
Modified atmosphere packaging, emulsions, coatings, etc.
are multifunctional platforms for VOC applications and solutions to challenges that affect practical VOC applications
.
Due to its potential toxicity, stringent regulatory and safety assessments are required
.


01 The biosynthesis
of VOCs can be divided into volatile phenylpropanoids (Volatile phenylpropanoids/benzenoids, VPBs), volatile terpenoids (volatile terpenoids, VTPs), and volatile aldols according to different biosynthetic pathways and chemical structures.
Compounds (volatile alcohols/aldehydes, VAAs)
.
The chemical structure of VTPs is represented by the C5 isoprene unit, and these basic C5-isoprene building blocks are produced by two distinct pathways, the cytoplasm-localized MVA pathway and the plastid-localized MEP pathway
.
Prenyl diphosphate (IPP) and its allyl isomer dimethylallyl diphosphate (DMAPP) are common five-carbon terpenoid precursors, which are produced by MEP with acetyl-CoA as a substrate.
pathway and the MVA pathway using pyruvate as a substrate
.
The precursors for the synthesis of volatile sesquiterpenes (C15) are mainly provided by the MVA pathway, while the MEP pathway provides precursors for the synthesis of volatile hemiterpenes (C5), monoterpenes (C10) and diterpenes (C20)
.
3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered to be the rate-limiting enzyme in the MVA pathway; pyruvate and glyceraldehyde-3-phosphate serve as substrates for the MEP pathway, which are metabolites produced by primary metabolism Therefore, the MEP pathway is limited by the rate of primary metabolism
.
The biosynthesis of VPBs begins with the essential amino acid phenylalanine (Phe) as a substrate
.
Similar to terpenoid biosynthesis, primary metabolism controls the influx of carbon (phosphoenolpyruvate) into VPBs biosynthesis, which is mediated by 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHAPS) , which controls the shikimate pathway to produce phenylalanine
.
Compared with VTPs and VPBs, VAAs such as (Z)-3-hexenol, nonanal, and methyl jasmonate (MeJA) are produced by catabolism, and polyunsaturated fatty acids (PUFAs) such as C18 unsaturated fatty acids, sub- Oleic acid, or linolenic acid, is the source of VAAs production
.
The cellular content of PUFAs depends on the acetyl-CoA plastid pool produced by pyruvate, and part of the PUFA entering the lipoxygenase (LOX) pathway is converted to α-hydro(pero)xy PUFAs by α-dioxygenase, and the other part is converted into α-hydro(pero)xy PUFAs.
autoxidation products
.
These lipid peroxide-derived species will be further processed through two branched pathways: the allene oxygenase (AOS) branch and the hydrogen peroxide lyase (HPL) branch, ultimately yielding a wide variety of VAAs
.



Figure 1.
Overview of the biosynthetic pathways of plant volatile organic compounds, including VTP, VPB, and VAAs.
02 Mechanisms of VOCs to

defend against pathogens
Affects membrane integrity and pathogen permeability
.
Disruption of the membrane results in leakage of ions, reduction in potential, collapse of the proton pump and cessation of ATP production
.
The hydroxyl groups of phenolic terpenoids have been postulated to function as monovalent cationic transmembrane carriers, carrying H into the cytoplasm and transporting K out (Ben Arfa et al.
, 2006)
.
The structural differentiation of VTPs determines their antimicrobial activity
.
Comparative studies of different VTPs in the control of a range of pathogens, including model bacteria and fungi, have shown that specific structures and the presence of free phenolic hydroxyl groups are critical for antimicrobial activity
.
VPB is also able to change the conformation of polysaccharide, fatty acid and phospholipid layers, thereby coagulating the cytoplasm and disrupting the function of pathogen cell proliferation membranes and cell membranes
.
In addition, VPB was found to induce cell death in Saccharomyces cerevisiae, a model microorganism
.
However, bacterial susceptibility to VOCs can be influenced by factors such as pH, protein, fat, salt, temperature and conditions, which vary widely among various foods
.
Therefore, various methods, treatments, and applicable modalities are needed to reduce adverse effects, which provides guidance for the application of volatile organic compounds in the future
.


Modulation of Host Immunity
Volatile organic compounds are metabolites derived from plant-derived foods that have multifunctional functions in multiple biological pathways in their hosts, including host immune modulation
.
Metabolomics These findings are necessary to understand the underlying mechanisms of how VOCs enhance host resistance to pathogens and suggest that VOCs can be a pretreatment method for foods of plant origin in particular
.
In response to pathogen-associated molecular patterns (PAMPs), such as flagellin, cell wall-degrading enzymes and other toxins secreted by pathogens, plant-derived hosts have evolved a relatively conserved immunity termed PAMPS-triggered immunity (PTI)
.
Likewise, in response to plant PTIs, pathogens have evolved a type III.
secretion system that provides proteins with similar effects to inhibit plant PTI signaling and promote invasion
.
Plant-derived hosts can recognize pathogenic effectors and induce signaling pathways leading to effector-triggered immunity (ETI), which can trigger hypersensitivity responses (HR) and ultimately inhibit pathogen growth
.


Involved in pathogenic metabolism
Some volatile organic compounds that are lethal to pathogenic cells are also toxic to host cells
.
Thus, in nature, many of these VOCs are stored in chemically modified forms that are not toxic to host cells
.
To exert antibacterial effects, these modified VOCs are able to participate in pathogenic metabolism and proceed upon removal of modifications by pathogenic cells, which release toxicity and lead to pathogen lethality
.
VPB is a defense chemical widely distributed in plants and has been shown to be effective against insects, fungi and bacteria, and mutants lacking aniline biosynthesis result in insufficient disease resistance, however, some defense anilines are also toxic to plant cells
.
To avoid toxicity to the host cell, the anisole-like is modified into a glucosylated form, which is not harmful to the host cell
.
Once the tissue is destroyed, the glucosylated form of the aniline is taken up by pathogens and metabolized by β-glucosidases, thereby removing the glucosylation, producing toxic glucosinolates in disease-causing cells
.
These results suggest that VOCs can indirectly participate in pathogenic metabolism and exert toxicity through chemical modification
.



Figure 2.
Antibacterial mechanisms of volatile organic compounds include direct destruction, induction of host immunity and participation in pathogenic metabolism.


03 Practical applications and candidates of volatile organic compounds in the control of food
pathogens , the antibacterial effects of volatile organic compounds have been widely studied
.
This summary is based on the efficiency, mechanism, dosage, and mode of application of volatile organic compounds
.
We summarize the different characteristics of VTPs, VPBs, and VAAs for comparison and propose VOCs as potential candidates for antibacterial agents that have been studied in fields other than food
.


Application and potential of VTPs in food preservation
VTPs are widely used in the preservation of meat products, which are always rich in lipid compounds and are susceptible to contamination by foodborne pathogens
.
The effective lethality of VTPs against foodborne pathogens is one of the benefits of their use in meat products; in addition, during storage, oxidation of lipid compounds can produce unexpected flavors, and many terpenoids have strong antioxidant properties ability to rescue odor conditions during storage
.
For plant hosts, VTPs can stimulate the immunity of plant-derived hosts, enhance their resistance to microorganisms, and at the same time can directly destroy their membrane structure, causing direct killing, but the direct lethal ability of non-plant-derived humus host pathogens limited
.


Application and potential of VPBs in food preservation
VPBs have good anti-pathogenic effects on food-borne pathogens and microbial toxin
.
The MICs of many VPBs were lower than those of VTPs and VAAs, suggesting that VPBs have broad application prospects as an antibacterial drug against foodborne pathogens
.
Although many VPBs have strong antibacterial and antioxidant capabilities, VPBs are prone to produce unpleasant flavors when used in meat products, and the combination of encapsulation technologies such as emulsions will be a solution
.
For plant-derived foods, VPBs have outstanding applicability because many VPBs are part of the volatile odor and taste components of plant-derived foods
.
In addition, for some deep-processed products, such as cheese, VPBs can enhance their flavor, which suggests that VPBs can be used as food additives to enhance flavor and solve microbial problems
.


The application and potential of VAAs in food preservation
Many studies have generally confirmed the direct destruction of VAAs and their derivatives on plant pathogens, but they are rarely studied in the field of food, which indicates that VAA is an unexplored natural antibacterial agent
.
Many VAAs have a strong odor due to their high volatility, which may affect the flavor of the food, however, due to their homology, they are compatible with plant-derived foods
.
For some processed plant-derived foods, such as juice, VAAs can be added as a flavor additive that also has antibacterial properties
.
Although aldehydes are the most antibacterial VAA compounds, they are not suitable for use in lipid-rich products due to their oxidative properties
.


04 Application challenges of volatile organic compounds in food microbial control: rethinking perspectives
Although volatile organic compounds have significant activity in controlling food pathogens and have broad application prospects as food preservatives, according to the current review, in Challenges remain for practical applications in the food industry
.


Potential risks to food quality
Through metabolomic studies, it was found that although volatile organic compounds have good biocompatibility, their limitations include: (1) effects on sensory properties; (2) damage to fruit and vegetable product tissue; (3) oxidation of lipid-rich products lipids in products, these are potential side effects of volatile organic compounds on food quality
.
Due to their high volatility, VOCs exhibit strong odors and affect organoleptic properties, but sometimes some VOCs are compatible with the food used, and some even help to improve taste, since VOCs are inherently are present in these foods with little effect on their organoleptic properties
.
However, for non-plant foods, especially muscle foods and some plant foods, the effect of VOCs on flavor sets up obstacles for their practical application, and a comprehensive sensory evaluation is required
.
Inappropriate concentrations or types of plant-derived volatile organic compounds can often cause serious harm to food
.
Encapsulating VOCs can avoid direct contact of VOCs with the food matrix, control their release, and reduce their impact on food flavor
.
In addition, encapsulating VOCs can protect their chemical properties, reduce their exposure to environmental conditions and control their mobility
.
Encapsulation can be accomplished by a combination of emulsions and coatings/biofilms
.
Emulsions can encapsulate volatile organic compounds into droplets, providing a possible carrier for the long-term utilization of volatile organic compounds
.


Escape Mechanisms of Microorganisms to VOCs Toxicity
Pathogens have co-evolved over countless years to form a response mechanism to adversity, including circumventing the toxicity of VOCs
.
Studies have confirmed that pathogens are able to detoxify VOCs that cause cytotoxicity through efflux mechanisms.
In addition, some pathogens have deciphered VOCs and manipulated them to recruit allies, and some pathogens have developed mature transformation systems capable of assimilating VOCs as their own nutrients
.
In vitro studies have shown that exogenous terpene treatment activates a series of terpene-related genes in fungi, and studies have shown that a class of ABC transporters pumps excess monoterpenes out of cells, and this protein is widely associated with various in microorganisms
.
VOCs can even be utilized by pathogenic bacteria as a nutrient source, and Penicillium is able to convert limonene to other nontoxic terpenoids such as alpha-terpineol during early to mid-growth stages, suggesting that Penicillium may be able to avoid this pathway.
Opens the attack of limonene from citrus
.


Safety and management standards for VOCs in the food industry
Due to the broad potential of VOCs utilization, risk assessment of VOCs is necessary for their future applications
.
Indeed, comprehensive studies around cytotoxicity, metabolic toxicity, and skin toxicity, as well as the underlying mechanisms of protein adducts, DNA adducts, and cytoskeletal morphology, have been extensively studied
.
The direct toxicity of most VOCs exists only at very high concentrations, indicating that VOCs are basically safe if the applied concentration of VOCs is strictly controlled
.
The first regulatory aspect that should be strictly considered is concentration
.
Another aspect is processing and storage toxicity: food is always processed or stored under high temperature, high osmotic pressure, acidic or alkaline conditions
.
High temperatures or other extreme conditions can lead to VOC degradation, which can produce toxic VOC derivatives
.
In addition to processing toxicity, interactions or chemical reactions between volatile organic compounds and food ingredients can also produce toxic compounds
.
Therefore, a series of systematic safety assessments should be carried out before the final application of VOCs
.


Green technologies such as microwave-assisted extraction, enzyme-assisted extraction, supercritical fluid extraction, and ultrasonic-assisted extraction for the production of VOCs
have been widely used in the extraction of biologically active substances from plant-derived food by-products.
Substances with volatile characteristics are extracted from food and its by-products, and the reuse of these by-product extracts creates a low-cost biopreservative technology
.
Among them, supercritical fluid extraction is the best technique for the extraction of volatile organic compounds because the biological activity remains unchanged under the extraction conditions
.
The rapid development of molecular biology and computer science has provided support for the construction of "cell factories" that can produce VOCs on a large scale
.



Figure 3.
ABC transporters transforming extracellular monoterpenes preserved in microorganisms (A), representative hepatocellular lesions caused by high levels of d-limonene (B), challenges, future prospects and potential solutions, using For perspective studies and practical utilization of plant VOCs (C)

Plant volatile organic compounds are versatile in the control of foodborne pathogens and spoilage organisms
.
According to chemical structure, volatile organic compounds are classified into VTP, VPB and VAAs, which are rate limited by HMGR, DAHP and LOX enzymes, respectively
.
The preliminary biosynthetic pathways of VOCs were identified, enabling synthetic biology research to synthesize VOCs on a large scale
.
Mechanistic studies have found that volatile organic compounds can directly change the function of pathogenic cell membranes and impair energy metabolism
.
Volatile organic compounds can also trigger SAR for fresh produce
.
Meanwhile, volatile organic compounds can participate in or interfere with pathogenic metabolism after ingestion by pathogens and produce harmful compounds, thus elegantly preventing toxicity to the host
.
Many volatile organic compounds have high potential as natural antibacterial agents in basic research but have been neglected in applied research
.
We found that different volatile organic compounds have different antibacterial properties
.


VTP is effective in controlling both spoilage organisms and food-borne pathogens, and is particularly prominent in protecting food-borne pathogens, and VAAs have mostly untapped potential
.
Looking ahead, metabolomics challenges still hinder the development of VOCs in food preservation
.
Applicable challenges relate to: (1) adverse effects, including reduced organoleptic properties, damage to plant-derived foods, and oxidation of lipids; (2) toxicity of volatile organic compounds that escape pathogens or utilize volatile organic compounds as a source of nutrition; (3) ) potential toxicity of volatile organic compounds to human body; (4) obtaining volatile organic compounds through green extraction technology and synthetic biology, which can be solved by diversified delivery systems and strict regulation of applied concentration
.
After considering and solving these problems, we believe that plant volatile organic compounds can be a "green solution" for the control of food microbial problems
.

Article/Aqu Metabolomics