Antioxidant Properties of Food‐Derived Lactic Acid Bacteria: A Review

ABSTRACT

The mechanisms of antioxidant action of lactic acid bacteria (LAB) have not been fully explained. This review aimed to characterise the antioxidant properties that can be presented by LAB strains isolated from food. The review presents a definition and classification of the antioxidants, mechanisms of antioxidant action of LAB, discusses the most popular antioxidant assays, taking into account the mechanisms underlying each test and the practice of assessing antioxidant capacity, and presents examples of studies of food‐derived LAB and fermented food with antioxidant properties. LAB are an important part of the human microbiota, and their role in antioxidant processes is extremely important. They can respond quickly and effectively to free radicals by enhancing antioxidant activity, chelating metal ions, producing antioxidant enzymes and other metabolites, and thus mitigating the damage caused by oxidative stress. This review also presents methods for testing antioxidant properties that can be used for LAB screening. The most commonly used methods are the classical methods of testing antioxidant activity, such as DPPH (2,2‐diphenyl‐1‐picrylhydrazyl), ABTS (2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)), or FRAP (Ferric Reducing Antioxidant Power) assays. We recommend using at least three different assays. It is important to consider whether to test live or inactivated cells, post‐culture supernatant, cell lysates, protein fractions or purified exopolysaccharides. In conclusion, due to their properties, lactic acid bacteria strains may prove to be an interesting and natural alternative to synthetic antioxidants used in food production. Lactic acid bacteria have been shown to be not only useful as microorganisms that support the proper functioning of the digestive tract or as probiotics, but also allow their antioxidant properties to be noticed and strengthen the defence against oxidative stress.

The graphical abstract shows an example of the division of the mechanisms of action of lactic acid bacteria in the context of antioxidant properties. Point 1 describes the scavenging of free radicals. Point 2 describes the chelation of metal ions. Point 3 describes the accumulation of manganese and the production of antioxidant enzymes. Point 4 describes the effect on the intestinal microbiota.

1. Introduction

Recently, there has been an increasing interest in free radicals, oxidative stress in biological systems and their impact on the physiology of the human body. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the body to remove them (Dash et al. 2024). Physiologically, free radicals are formed as a side effect of reactions naturally occurring in the human body, such as cellular respiration or metabolic processes. The human body, in self‐defence against the accumulation of ROS, produces free radical scavengers that prevent or delay cell damage (Chandra et al. 2020; Leyane et al. 2022). Free radicals are molecules that have one or more unpaired electrons in their outer orbit. As a result of rapid reactions with neighbouring molecules, an unpaired electron is transferred, which leads to the formation of further free radicals or ROS (Kaur and Geetha 2006). Table 1 below shows the types of reactive species that can occur in living organisms.

TABLE 1.

Reactive oxygen, nitrogen, sulphur and chlorine/bromine species.

Types of reactive species	Radical	Non‐radical
Reactive oxygen species (ROS)	Superoxide, O2 −•• Hydroxyl, •OH Hydroperoxyl, HO2 • Carbonate, CO3 •− Peroxyl, RO2 • Alkoxyl, RO• Carbon dioxide radical, CO2 •− Singlet, 1O2	Hydrogen peroxide, H2O2 Ozone, O3 Singlet oxygen 1O2 Hypobromous acid, HOBr Hypochlorous acid, HOCl Hypoiodous acid, HOI Organic peroxides, ROOH Peroxynitrite, ONOO− Peroxynitrate, O2NOO− Peroxynitrous acid, ONOOH Peroxomonocarbonate, HOOCO2 − Carbon monoxide, CO
Reactive nitrogen species (RNS)	Nitric oxide, NO• Nitrogen dioxide, NO2 • Nitrate radical, NO3 •	Nitrous acid, HNO2 Nitrosyl cation, NO+ Nitroxyl anion, NO− Dinitrogen trioxide, N2O3 Dinitrogen tetroxide, N2O4 Dinitrogen pentoxide, N2O5 Alkyl peroxynitrites, ROONO Alkyl peroxynitrates, RO2ONO Nitryl chloride, NO2Cl Peroxyacetyl nitrate, CH3C(O)OONO2
Reactive sulphur species (RSS)	Thiyl radical, RS•	Hydrogen sulphide, H2S Disulphide, RSSR Disulphide‐S‐monoxide, RS(O)SR Disulphide‐S‐dioxide, RS(O)2SR Sulfenic acid, RSOH Thiol/sulphide, RSR
Reactive chlorine/bromine species (RCS/RBS)	Atomic chlorine, Cl Atomic Bromine, Br	Chloramines, NH2Cl Chlorine gas, Cl2 Bromine gas, Br2 Bromine chloride, BrCl Chlorine dioxide, ClO2

Note: Adapted from Graves (2012) and Martemucci et al. (2022).

So far, based on scientific research, it has been proven that chronic oxidative stress accelerates the ageing process and also affects the development of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Down syndrome, cardiovascular diseases, diabetes, depression, chronic kidney disease, metabolic syndrome, and colon cancer (Jin and Kang 2024; An et al. 2023; Bader et al. 2024; Jomova et al. 2023; Masenga et al. 2023; Bardelčíková et al. 2023; Son et al. 2023).

To counteract the accumulation of ROS and its harmful effects, organisms produce a complex antioxidant defence system. Antioxidant defence lines are a set of mechanisms that protect these microorganisms from the harmful effects of reactive species. The first line of the antioxidant defence system consists of antioxidant enzymes, such as superoxide dismutases (SOD: Mn‐SOD, Cu‐SOD, EC‐SOD), catalase (CAT) and glutathione peroxidase (GPx). Bacteria do not always possess the full complement of enzymes typical of aerobic organisms, but they have developed some key mechanisms. This is the most powerful line of antioxidant defence against oxidative stress and it prevents the formation of new free radicals. The second line of antioxidant defence consists of non‐enzymatic antioxidants, such as vitamins C and E, carotenoids, flavonoids and low‐molecular‐weight thiol compounds. Their function is to scavenge free radicals to prevent oxidative chain reactions. The third line of antioxidant defence is an adaptive and regulatory function and consists of enzymes, present in the cytosol of cells and mitochondria, that remove oxidised biomolecules through a variety of oxidant‐induced enzyme systems (DNA repairs enzymes, lipases, proteases, transferases and methionine‐sulfoxide reductases). Their task is to recognise, degrade and remove proteins modified by oxidative stress and prevent their accumulation in cells. In addition, there is increased expression of defence genes under stress conditions, and changes in metabolism occur, preferring metabolic pathways that generate fewer reactive species (Pisoschi and Pop 2015; Jomova et al. 2024; Dissanayake et al. 2025).

Understanding the appropriate mechanisms of antioxidant action, as well as new methods for preventing oxidative stress, is still of interest to scientists. Direct measurements of ROS using in vivo methods are difficult to perform due to their short half‐life. Maintaining constant, controlled environmental conditions is easier in vitro. Furthermore, in vitro studies should simultaneously analyse free radical neutralisation while simultaneously conducting digestive processes. This is precisely what happens in living organisms. As we can see, too many factors influence antioxidant processes (Martemucci et al. 2022; Rwubuzizi et al. 2023). Therefore, the level of oxidative stress can be determined indirectly by quantifying the levels of DNA damage, membrane peroxidation and protein modification (Jomova et al. 2024). Microorganisms are, next to plants, an excellent source of biological substances with various metabolic activities. The advantage of microorganisms is cultivation under controlled conditions and a fast growth time, compared with other organisms (Chandra et al. 2020).

A novel approach to developing new microbial cultures involves searching for strains with specific properties. Other features are desirable, not only modulating and improving the balance of the gut microbiome but also, for example, exhibiting antioxidant activity and counteracting oxidative stress. It has been proven that food, especially fermented food, containing microorganisms, such as lactic acid bacteria, can play an important role in preventing oxidative processes. This review aimed to characterise the antioxidant properties that can be presented by LAB strains isolated from food. Examples are given of foods fermented with these bacteria, whose antioxidant potential increases due to fermentation. The review presents a definition and classification of the antioxidants, mechanisms of the antioxidant action of lactic acid bacteria, discusses the most popular antioxidant assays, taking into account the mechanisms underlying each test and the practice of assessing antioxidant capacity, and presents examples of studies of food‐derived lactic acid bacteria with antioxidant properties.

1.1. Classification of Antioxidants

Antioxidants are substances that delay or prevent the oxidation of substrate molecules in low concentrations, causing the transformation of radicals into inactive derivatives (Munteanu and Apetrei 2021). In turn, Huang et al. (2005) defined an antioxidant as ‘a substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxides’. The history of antioxidants began with processes that prevented unsaturated fats from becoming rancid. The turning point was the identification of vitamin E and its role in preventing fat oxidation (Flieger et al. 2021). The removal of electrons or hydrogens can reduce oxidative damage to the cell by directly reacting the antioxidant with free radicals. Antioxidants act as scavengers of free radicals. It is widely believed that the location and number of hydroxyl groups on the aromatic rings of antioxidant compounds may play a key role in antioxidant activity (Houldsworth 2024).

Antioxidants can be produced endogenously or supplied exogenously as food, nutraceuticals or dietary supplements (Munteanu and Apetrei 2021). They can be divided according to different criteria. Figure 1 below presents a classification of the antioxidants with examples.

FIGURE 1.

Classification of antioxidants with examples. The figure shows an example of a division of antioxidants into five different categories with examples provided. In this proposed division, we distinguish antioxidants according to their mode of action, origin, size, solubility, and structure. Based on Munteanu and Apetrei (2021), Ayoka et al. (2022), Flieger et al. (2021), Aziz et al. (2019), Charlton et al. (2023).

Antioxidants act with different activity and have different mechanisms of action. Primary antioxidants directly interrupt the oxidation chain reaction (e.g., during lipid oxidation) by neutralising free radicals. This is most often accomplished by donating a hydrogen atom or electron, which neutralises the radical and converts it to a less reactive form. Secondary antioxidants do not directly inhibit free radical reactions. Their function is to slow down or prevent the initiation of the reaction through environmental modifications. Their mechanisms of action include chelating transition metals, degrading lipid peroxides, regenerating primary antioxidants, and creating physical barriers that impede the access of oxygen or pro‐oxidants to the substrate (Pisoschi and Pop 2015; Shahidi and Zhong 2015; Apak et al. 2016).

Based on the origin of antioxidants, we can distinguish between natural and synthetic antioxidants. Natural antioxidants are produced by living organisms (Shahidi and Zhong 2015). They can neutralise excessive amounts of free oxygen radicals in the body. The best sources of antioxidants are vegetables, fruits, herbs, and spices. The quality and antioxidant activity of natural antioxidants depend on the quality of the natural source, the processes used, and the extraction technology (Gulcin and Alwasel 2022). Synthetic antioxidants do not occur in nature and are produced by chemical synthesis. They are added to food as preservatives to inhibit lipid oxidation. These antioxidants are also widely used in the pharmaceutical industry due to their high reactivity even at low concentrations, low cost, and purity (Gulcin and Alwasel 2023). They are more stable than natural antioxidants (Atta et al. 2017). Both natural and synthetic antioxidants must meet several requirements for non‐toxicity and safety (Xu et al. 2021).

Low‐molecular‐weight antioxidants (< 1000 Da) can diffuse freely throughout cells and tissues. They are present in the cytoplasm, cell membrane and body fluids. They directly neutralise ROS, primarily by donating a hydrogen atom or electron. In contrast, high‐molecular‐weight antioxidants (> 10,000 Da) do not diffuse as freely as small molecules. They are found in specific cellular structures, such as mitochondria and cytoplasm. They act indirectly by enzymatically removing reactive oxygen species and binding metals that catalyse the formation of free radicals (Ayoka et al. 2022; Aziz et al. 2019).

Fat‐soluble antioxidants are mainly found in the cell membrane, whereas water‐soluble antioxidants are found in cellular fluids, such as the cytoplasmic matrix and cytosol (Ayoka et al. 2022).

In another classification, antioxidants can be divided into enzymatic and non‐enzymatic. Enzymatic antioxidants decompose and remove free radicals, which they convert into hydrogen peroxide. This is then metabolised by catalase into water and oxygen in a stepwise reaction in the presence of cofactors. In turn, the task of non‐enzymatic antioxidants is to inhibit reactions involving free radicals (Ayoka et al. 2022; Flieger et al. 2021). Exogenous antioxidants introduced into the body with the diet have become the subject of interest for scientists. At the same time, there is a decrease in the use of synthetic antioxidants.

To sum up, it can be said that antioxidants are used in the prevention and treatment of diseases, as well as in limiting ageing processes in the human body. The exact therapeutic effects require in‐depth research (Martemucci et al. 2022).

1.2. Antioxidant Properties of Lactic Acid Bacteria Derived From Food

Lactic acid bacteria are microorganisms that do not require the presence of oxygen for growth and metabolism. However, they can also grow in the presence of oxygen, which is possible thanks to the regulation of gene expression and changes in metabolic pathways (Zotta et al. 2018). Scientific studies have shown that aerobic cultivation of some LAB increases cell strength due to the improvement of internal pH homeostasis, increased energy, synthesis of antioxidant enzymes and depletion of intracellular O₂. This favours the survival of LAB under oxidative stress conditions. Interestingly, in the studies of Guidone et al. (2013), it was shown that the cultivation of LAB with oxygen allows for an increase in biomass yield during the stationary growth phase when the culture is enriched with haemin and menaquinone. This allows the formation of a respiratory electron transport chain, which translates into an increase in the resistance of bacterial cells to oxidative stress (Guidone et al. 2013).

Several mechanisms of LAB defence against oxidative stress have been identified (Figure 2). These mechanisms are closely interconnected and complement each other.

FIGURE 2.

Different mechanisms of antioxidant activity of lactic acid bacteria. The figure shows an example of the division of the mechanisms of action of lactic acid bacteria in the context of antioxidant properties. Point 1 describes the scavenging of free radicals. Point 2 describes the chelation of metal ions. Point 3 describes the accumulation of manganese and the production of antioxidant enzymes. Point 4 describes the effect on the intestinal microbiota. Based on Feng and Wang (2020); Bryukhanov et al. (2022); Hu et al. (2023).

Lactic acid bacteria have a direct ability to capture certain types of free radicals, for example, hydroxyl radicals, superoxide anions or hydrogen peroxide (Figure 2. Point 1). The direct scavenging of free radicals consists in their binding or limiting their spread using components contained in the cell wall. These macromolecules forming the cell wall of LAB contribute to maintaining cell integrity during environmental stress. The ability to express some surface components and secrete specific compounds is a functional feature of LAB. The main role in ROS scavenging is played here by peptidoglycan, exopolysaccharides, teichoic, and lipoteichoic acid, as well as proteinaceous filaments called pili and proteins (Sengupta et al. 2013).

Transition metal ions, especially iron and copper ions, participate in Fenton reactions, generating free radicals. In this reaction, Fe(II) catalyses the transformation of hydrogen peroxide into the highly reactive hydroxyl radical. The previously mentioned structures in the LAB cell wall are capable of the chelation of metal ions (Figure 2. Point 2). This is possible through the adsorption of metal ions on the surface of bacteria, which limits their availability in the environment and reduces the possibility of initiating radical reactions (Bryukhanov et al. 2022; Hu et al. 2023).

The third mechanism involves wide‐range manganese accumulation as well as the production of antioxidant enzymes (Figure 2. Point 3), that is, superoxide dismutase, haem‐dependent and haem‐independent catalase, NADH oxidase, NADH peroxidase, and glutathione reductase (Zotta et al. 2018; Bryukhanov et al. 2022).

The antioxidant manganese can enter into redox reactions, but unlike iron or copper, it does not create hydroxyl radicals (Bosma et al. 2021). It has been shown that many LAB (e.g., L. casei and L. paracasei ) have a high demand for manganese accumulation, which is closely related to the protection against the toxic effects of oxygen. Intracellular manganese accumulation allows for the efficient elimination of superoxide anions during aerobic growth. LAB must tightly regulate Mn(II) homeostasis to ensure sufficient levels for manganese‐dependent reactions but also low enough to prevent its cytotoxicity. This is achieved through the control of importers and exporters, mediated by transcription factors and oxidative stress‐responsive riboswitches. This process is facilitated by a system of numerous NRAMP‐type and ABC‐type manganese transport proteins. Manganese is a cofactor of manganese superoxide dismutase (Mn‐SOD) (Bosma et al. 2021). In LAB lacking classical SOD, high concentrations of Mn(II) are found, which replaces SOD. The ability to accumulate manganese is often correlated with the lack of true superoxide dismutase activity (Serata et al. 2018).

Superoxide dismutase (SOD) is a metalloenzyme that catalyses the dismutation of the superoxide anion radical to molecular oxygen and hydrogen peroxide, thereby reducing the intracellular concentration of free metal cations and attenuating the damage caused by H₂O₂ (Feng and Wang 2020). Four types of superoxide dismutases have been described, which differ in the metal atom present in the active site: iron‐containing (Fe‐SOD), manganese‐containing (Mn‐SOD), copper/zinc‐containing (Cu/Zn‐SOD) and nickel‐containing (Ni‐SOD) (Bryukhanov et al. 2022). The most frequently observed superoxide dismutases in LAB are MnSOD, whereas FeSOD and Cu/ZnSOD are much less common. It has been shown that Mn‐SOD activity depends on the intracellular concentration of manganese (Feng and Wang 2020), as mentioned earlier.

Catalase (CAT) is an enzyme from the oxidoreductase group that catalyses the decomposition of hydrogen peroxide into water and oxygen. There are two types of catalases: true haem‐dependent catalase and pseudocatalases, containing manganese in their structure. In general, LAB are catalase‐negative and practically do not produce catalase (Feng and Wang 2020). In some LAB, true catalase occurs only in the presence of haem or haematin in the culture medium. In turn, pseudocatalases are rather rarely found in LAB (Bryukhanov et al. 2022), but some LAB such as L. plantarum can produce pseudocatalase (Peacock and Hassan 2021).

Most often, the LAB antioxidant system is based on the action of NADH oxidase and NADH peroxidase. NADH oxidase uses accumulated oxygen to oxidise NADH to NAD⁺, which causes an increase in the amount of hydrogen peroxide. In turn, the resulting hydrogen peroxide is decomposed to H₂O by NADH peroxidase. This same hydrogen peroxide also has antimicrobial functions, but is toxic to the LAB itself, so it is quickly removed from the surrounding environment (Bryukhanov et al. 2022; Wu et al. 2025).

Thioredoxin reductase (TrxR) is a selenoenzyme, a flavoenzyme, that participates in the oxidation of thioredoxin using a cofactor, NADPH. TrxR forms a complex with a selenium atom, thanks to which it regulates the entire thioredoxin system and regulates the amount of selenium in the body. It plays a role in reductive processes and contributes to the defence of antioxidant cells, protein reduction and nucleic acid biosynthesis (Bryukhanov et al. 2022). The level of expression of thioredoxin genes has a specific effect on the development of various disease states associated with oxidative stress. For example, in the L. casei , the trxB gene is responsible for thioredoxin reductase and the thioredoxin‐thioredoxin reductase system is necessary for its growth in aerobic conditions (Serata et al. 2012).

Because LAB are practically constantly exposed to oxidative stress, they have developed genetically determined antioxidant mechanisms. The basis of these mechanisms is the expression of genes encoding antioxidant enzymes (Feng and Wang 2020; Bryukhanov et al. 2022). The main genes that can be found in LAB are sodA, most often encoding Mn‐SOD; katA, katB, encoding catalase; npr, ahpC, ahpF, encoding peroxidases; gshR, encoding glutathione reductase; and gor, encoding glutathione oxidoreductase (Bryukhanov et al. 2022).

In conditions of oxidative stress, antioxidant enzymes are activated, which allows LAB to resist oxidative stimulation (Tian et al. 2022). Many factors influence the modulation of the gut microbiota (Figure 2. Point 4). The antioxidant activity of LAB is also associated with the production of glutathione, exopolysaccharides (EPS) and short‐chain fatty acids (SCFAs) (Feng and Wang 2020; Tang et al. 2017).

Glutathione (GSH) is a non‐protein thiol found in almost all living organisms. It plays a role in the response to oxidative stress by protecting cells from free radicals and stabilising redox in the intracellular environment (Bryukhanov et al. 2022). GSH regenerates other antioxidants, for example, vitamins C and E, maintains –SH groups in proteins in a reduced state, and participates in the detoxification of xenobiotics. An adequate and stable amount of glutathione in the body does not allow the accumulation of hydrogen peroxide, which can result in cell damage (Brodzka et al. 2024).

Exopolysaccharides are long‐chain macromolecular biopolymers, linked by a glycosidic bond with an α or β structure, secreted by the cell to the outside. In terms of chemical composition, they are divided into homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Most LAB can produce EPS in greater or lesser amounts (Zhang et al. 2024), and as LAB have GRAS status, EPS derived from them are also generally considered safe (Tarannum et al. 2023). It has been shown that EPS derived from LAB can scavenge free radicals DPPH (Khalil et al. 2022) or hydroxyl radicals (Tarannum et al. 2023).

Short‐chain fatty acids are a by‐product of the fermentation of carbohydrates and dietary fibre. The most commonly produced by LAB are acetic acid, propionic acid, and butyric acid. SCFAs can directly neutralise reactive oxygen species, contributing to reducing oxidative stress. Particularly important features are attributed to butyric acid, which affects the activation of genes responsible for the production of antioxidant enzymes. It also has a special effect on various signalling and epigenetic pathways in cells (Kang et al. 2021).

As presented in the previous chapter, LAB have developed various complex and multifaceted mechanisms of antioxidant action. The antioxidant capacity of LAB is closely linked to their activity in the host's gastrointestinal tract. The variation in LAB antioxidant activity indicates that they are strain‐dependent, which opens up the possibility of selecting bacteria with specific implementation and health‐promoting potential, as discussed in the following chapters.

1.3. Methods of Measuring Antioxidant Activity in Lactic Acid Bacteria

It is necessary to distinguish two definitions, often confused as the same. The first one is that antioxidant activity is related to the kinetics of antioxidant action to quench reactive molecules. It is expressed as the reaction rate or percentage of scavenging per unit time. Another definition is the antioxidant capacity, which is the thermodynamic efficiency of the conversion of reactive molecules by antioxidants. This is the number of moles of reactive molecules scavenged by 1 mol of antioxidants in a given time (Apak et al. 2016). The definition of ‘antioxidant activity’ describes the properties of a single compound in a given test (Sadowska‐Bartosz and Bartosz 2022). Most tests are based on the generation of a synthetic coloured radical or redox‐active compound, and their changes are monitored spectrophotometrically at different wavelengths. Antioxidant capacity is determined as a percentage of inhibition or using an appropriate standard (Floegel et al. 2011). According to Huang et al. (2005), the term ‘antioxidant capacity’ is interchangeably referred to as ‘efficiency’, ‘power’, ‘parameter’, ‘potential’, ‘potency’, or ‘activity’. Today we know that these terms do not always mean the same thing. As antioxidant activity is defined as a single test that reflects only a chemical reaction under specific conditions, it is a mistake to define it as ‘total antioxidant activity’.

A feature of the methods of measuring antioxidant activity is in particular the impossibility of comparing methods with each other. This is due to the specificity of each test: the type of free radicals, different systems generating radicals, or the mechanism of action. The antioxidant capacity depends on the generated radical to be removed, so it must always be determined and carefully selected (Ghiselli et al. 2000). Antioxidant assays can target a single, specific compound or target total antioxidant capacity, which is the combined capacity of all antioxidant compounds in a sample (Danet 2021). In the case of biological samples, antioxidant capacity better represents antioxidant status than antioxidant activity (Ghiselli et al. 2000). The antioxidant standards such as ascorbic acid, butylated hydroxyl toluene (BHT), α‐tocopherol, butylated hydroxyl anisole (BHA), gallic acid, and Trolox (6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid) are commonly used (De Menezes et al. 2021).

The first methods of measuring antioxidants were based on the measurement of lipid oxidation. Over time, these methods have been improved. The sensitivity of the tests and the automation of the processes have increased (Munteanu and Apetrei 2021). The study of antioxidant activity is a very complex process, and a single method is not able to fully describe the reactions taking place. This is especially true for in vivo studies. Moreover, it is better to choose different test methods, even those that are not strictly correlated with each other. This will allow for a better understanding of the mechanisms that play a key role in the antioxidant effect (Rumpf et al. 2023).

A still‐existing problem that will be difficult to solve is the fact that there is no single, proven and universal method for checking the antioxidant activity of both food and biological matrices (Bibi Sadeer et al. 2020). Another problem with these methods is the lack of standardisation and procedures for carrying out the tests. In vitro studies do not take into account many biological parameters. However, because in vivo studies are usually associated with ethical issues as well as high costs, the importance of in vitro tests in science is emphasised (Nwachukwu et al. 2021). As emphasised by Danet (2021), in vitro antioxidant tests and the determination of total phenolic content are used to assess potential beneficial antioxidant effects, as well as for food quality control, where samples can be compared with reference material. Their application is very wide. The inappropriate selection of the method can lead to interferences in reaction mixtures, resulting in an underestimation or overestimation of results (Bibi Sadeer et al. 2020). As emphasised by Gulcin and Alwasel (2023), antioxidant activity should always be measured by at least three different methods.

There are various ways of classifying methods for measuring antioxidant activity. One example is the division based on the chemical reactions that occur (Table 2).

TABLE 2.

Most commonly used methods for screening the antioxidant activity of lactic acid bacteria.

Assay	Mechanism	Method	Principle of the method	Advantages	Disadvantages	Literature
ORAC	HAT	Fluorescence spectroscopy	The superoxide radical reacts with a fluorescent probe to form a non‐fluorescent product, the quantity of which can be determined by fluorescence. Antioxidant activity is determined by the reduced rate and quantity of product formed over a given time.	Standardisation, high precision and reproducibility, application in many types of matrices, taking into account the reaction time, use of biologically active antioxidants.	Temperature‐sensitive, pH‐sensitive, interference from reducing compounds and metal chelators, expensive, specialised device required (fluorimeter).	Cao et al. (1993); Prior (2015); Mendonça et al. (2022)
HORAC	HAT	Fluorescence spectroscopy	The method is based on the generation of a hydroxyl radical by a Co(II)‐mediated Fenton‐like reaction which is confirmed by the hydroxylation of p‐hydroxybenzoic acid. The fluorescence decay curve of fluorescein is monitored in the absence or presence of an antioxidant.	Validated, precise and accurate method, representative of food, direct measurement of antioxidant capacity.	Sensitive to environmental conditions and time‐dependent, specialised device required (fluorimeter), long reaction times, requires appropriate analytical techniques.	Ou et al. (2002); Echegaray et al. (2021)
TRAP	HAT	Chemiluminescence	The method is based on the ability of antioxidants to inhibit the reaction between radicals and the target molecule. Consumption of oxygen molecules in the peroxidation process triggers the thermal decomposition of the azo‐compound 2,2′‐diazobis (2‐amidinopropane) hydrochloride (ABAP), which is monitored by the linear decrease of the chemiluminescence quenching probe R‐phycoerythrin.	Fast and convenient, high sensitivity, applicable to hydrophobic antioxidants, but can be adapted to hydrophilic, chemicals are widely available, suitable for in vivo evaluation.	Time‐consuming, laborious, relatively complex method that requires experience in laboratory techniques, the possibility of re‐estimating the results.	Prior et al. (2005); Silvestrini et al. (2023)
TOSC	HAT	ROS scavenging	It is based on the reaction of peroxide radicals generated by the thermal homolysis of 2,2'‐azobis‐amidinopropane (ABAP) with α‐keto‐γ‐methiolbutyric acid (KMBA). KMBA is subsequently oxidised to ethylene, the amount of which is determined by gas chromatography.	Repeatability, efficacy for hydrophilic and lipophilic antioxidants, ability to distinguish between fast and slow‐acting antioxidants.	Long analysis time, instability of test solutions, not readily adaptable for high‐throughput analyses.	Regoli and Winston (1999); Prior et al. (2005)
F‐C test	SET	Spectrophotometric	It consists of using the ability of polyphenols to form coloured complexes with Folin–Ciocalteu reagent. After the reduction of phosphomolybdic and phosphotungstic acids (F‐C reagent) in an alkaline environment, a blue chromophore is formed with maximum absorption at a wavelength of 760 nm. The colour change of the reagent is proportional to the reducing power of the antioxidant.	Simplicity, reproducibility, reference method, low cost of reagents, a wide range of detected compounds, can screen many samples in time.	Sensitive to reaction conditions (pH, temperature, reaction time), can be interfered with by oxidisable species (proteins, reducing sugars, sulphur dioxide, and ascorbic acid), not specific to phenolic compounds, applicable to water‐soluble antioxidants, risk of overestimating results.	Folin and Ciocalteu (1927); Dominguez‐López et al. (2024); Pérez et al. (2023)
FRAP	SET	Colorimetric	It is used to measure total antioxidant activity and evaluates the ability to reduce ferric ion Fe3+ to ferric ion Fe2+. The reaction produces a bluish complex. Changes in the absorbance of the solution are observed at a wavelength of 593 nm.	Simplicity, quick, low cost of reagents, does not require specialised equipment, high repeatability of results, wide range of applications.	Does not detect all antioxidants, applies only to hydrophilic substances, may give false positive results of iron reduction, not suitable for slowly reacting compounds.	Benzie and Strain (1996)
CUPRAC	SET	Spectrophotometric	The test consists of the reduction of cupric (Cu2+) to cuprous (Cu+). The chromogenic redox reagent is bis(neocuproine)copper(II) chelate, which forms a reaction with polyphenols at pH 7.0. The reaction produces a coloured chelate, orange‐yellow bis(neocuproine)copper(II) chloride, which can be analysed at a wavelength of 450 nm.	Low cost of reagents, widely available, stable reagents, fast chemical reaction, ability to screen lipophilic and hydrophilic samples, tests are performed at pH 7, which is close to physiological pH, environmental conditions do not have a significant impact on the measurement results, not expensive and has specialised measuring devices.	For hydrophilic and lipophilic antioxidants, depending on the test modification, a longer measurement time may be required, the possibility of overestimating results at inappropriate pH, the possibility of overlapping the absorption spectra of the oxidising agent and the tested samples.	Apak et al. (2007); Özyürek et al. (2011); Bibi Sadeer et al. (2020)
DPPH assay	HAT/SET	Spectrophotometric or colorimetric, ROS scavenging	The principle of the method is based on the donation of electrons from antioxidants to neutralise the DPPH radical. An unpaired electron in DPPH• colours the solution dark purple, and when paired with another electron, the solution changes colour to pale yellow.	Speed of analysis, measurement accuracy, repeatability of results, easy availability and low cost of analysis reagents, DPPH is relatively stable as a free radical and does not need to be prepared fresh, wide application.	Limited to hydrophobic solvents only, narrow pH range, high impact of environmental conditions on results, DPPH is very susceptible to interference from coloured samples due to its wavelength.	Blois (1958); Rumpf et al. (2023); Flieger et al. (2021); Bibi Sadeer et al. (2020)
ABTS assay	HAT/SET	Colorimetric, ROS scavenging	The test consists of measuring the ability of antioxidants to capture the stable radical cation ABTS•+. ABTS•+ is formed by oxidation with potassium persulphate and then is reduced in the presence of hydrogen‐donating antioxidants. The blue‐green solution has an absorption maximum at 734 nm, and the colour intensity decreases in the presence of the antioxidant.	Fast, simple, high precision, wide pH range tolerance, wide range of applications, for both hydrophilic and hydrophobic antioxidants, repeatability of results, adaptable to high‐throughput methods.	The ABTS•+ radical does not occur naturally (does not occur in any biological system) and is chemically synthesised, risk of overestimating results, reaction kinetics with some antioxidants can be slow.	Re et al. (1999); Bibi Sadeer et al. (2020)
O2 •‐ assay	ND	ROS scavenging	Superoxide anion is converted by superoxide dismutase to oxygen and hydrogen peroxide. The method is based on the kinetics of the competitive reduction of O2 •‐ cytochrome c and the O2 •‐ scavenger.	Rapid analysis, the ability to analyse many samples simultaneously, the ability to measure antioxidants in complex biological systems, precision, rather low cost of reagents.	Reducing compounds and metal chelators can interfere with the assay, temperature sensitivity, not reflected in in vivo tests, lack of specificity.	Huang et al. (2005); Apak et al. (2016); Bibi Sadeer et al. (2020)
H2O2 assay	ND	ROS scavenging	Hydrogen peroxide is converted by catalase to oxygen and water. The test is based on the measurement of the hydrogen peroxide scavenging capacity of antioxidants, using horseradish peroxidase to oxidise scopoletin to a non‐fluorescent product.	Easy, rapid analysis, hydrogen peroxide is relatively stable, ability to analyse a very large number of samples simultaneously, low impact of environmental conditions on the analysis results.	Possibility of interference and disturbances from some compounds, such as thiols and ascorbate, high cost of horseradish peroxidase, substrates are pH‐sensitive.	Huang et al. (2005); Kaur and Geetha (2006)
ROO• assay	ND	ROS scavenging	Peroxyl radicals are formed by the thermal decomposition of 2,2′‐azobis(2‐methylpropionamidine) dihydrochloride and 2,2′‐azobis(2,4 dimethylvaleronitrile). The method is based on the inhibition reaction of the probe signal to assess the scavenging activity of peroxyl radicals. Closely related to ORAC.	ROO• are common in food and biological samples, highly precise when temperature is under control, repeatable, low cost.	Temperature sensitivity, reducing agents and metal chelators may interfere with the test, naturally occurring pigments and fluorophores may interfere with absorbance and fluorescence readings.	Kaboggoza et al. (2023)
HO• assay	ND	ROS scavenging	Hydroxyl radical is formed in the Fenton reaction when hydrogen peroxide reacts with Fe(II). It is the most reactive of the oxygen radicals, causing lipid peroxidation and destruction of cell membranes. The method is related to HORAC.	Sensitive, reactive, useful in many applications.	Hydroxyl radicals are very reactive and have very short lifetimes, high cost of equipment, limited throughput.	Huang et al. (2005); Kaur and Geetha (2006)
1O2 assay	ND	ROS scavenging	Singlet oxygen is formed in the presence of light and photosensitisers. The method involves measuring the rate of decay of light intensity in measuring the 1O2 quenching activity.	High sensitivity, can be harnessed for many applications, easy, fast method, multi‐sample measurement.	Singlet oxygen is an unstable molecule, expensive instruments, disturbance by colour, photodegradation of sample.	Huang et al. (2005); Kaur and Geetha (2006); Takajo and Anzai (2021)
ONOO− assay	ND	ROS scavenging	Peroxynitrite is formed by the reaction of superoxide and nitric oxide. The method relies on the ability to remove peroxynitrite by inhibiting the nitration of tyrosine by peroxynitrite or inhibiting the oxidation of dihydrorhodamine.	Physiological similarity of ONOO− to the human body, ONOO− is a stable species, the final fluorescence intensity is stable over time, relatively high sensitivity.	Lack of specificity, ONOO− quickly decomposes, requires expensive equipment (fluorescence spectrophotometer), high cost of reagents, may not be representative of in vivo conditions.	Huang et al. (2005); Bibi Sadeer et al. (2020)

Note: A systematic search was conducted between 1 December 2024 and 31 May 2025. Electronic bibliographic databases such as Scopus, Google Scholar, PubMed and ResearchGate were used. The searching strategy used the following keywords: antioxidant or antioxidative, activity or capacity or properties, scavenging or reducing or power, DPPH or ABTS or FRAP or ORAC or TRAP or TOSC or Folin and/or Ciocalteu or CUPRAC.

Abbreviations: ¹O₂ assay, singlet oxygen scavenging capacity assay; ABTS assay, 2,2′–azinobis–(3–ethylbenzthiazolin–6–sulfonic acid) assay; CUPRAC, cupric ion reducing antioxidant capacity; DPPH assay, 2,2–diphenyl–1–picrylhydrazyl assay; F–C test, Folin–Ciocalteu test; FRAP, Ferric reduction of antioxidant power; H₂O₂, H₂O₂ scavenging capacity assay; HAT, hydrogen atom transfer; HO^• assay, hydroxyl radical scavenging assay; HORAC, hydroxyl radical antioxidant capacity; ND, not defined; O₂ ^•– assay, O₂ ^•– scavenging capacity assay; ONOO⁻ assay, peroxynitrite (ONOO–) scavenging capacity assay; ORAC, oxygen radical absorption capacity; ROO• assay, peroxyl radical assay; SET, single electron transfer; TOSC, total oxyradical scavenging capacity; TRAP, total peroxyl radical trapping antioxidant parameter.

Hydrogen atom transfer (HAT) reactions involve measuring the ability of an antioxidant to remove free radicals by donating a hydrogen atom. These are usually fast reactions, lasting a few seconds or minutes, and are not very dependent on pH and solvent. Examples include ORAC (Oxygen Radical Absorption Capacity), HORAC (Hydroxyl Radical Antioxidant Capacity), TRAP (Total Peroxyl Radical Trapping Antioxidant Parameter), and TOSC (Total Oxyradical Scavenging Capacity) tests (Bartosz 2010).

Single electron transfer (SET) reactions are based on detecting the ability of an antioxidant to transfer an electron to reduce metal ions, carbonyl groups, and free radicals. They are rather slow reactions and are dependent on the pH value. Examples of this type of reaction are the F‐C test (Folin–Ciocalteu test), FRAP (Ferric Reduction of Antioxidant Power), and CUPRAC (Cupric Ion Reducing Antioxidant Capacity) (Bartosz 2010).

Mixed HAT/SET assays involve the transfer of both a hydrogen atom and a single electron. Examples include the DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl) assay and the ABTS (2,2′‐Azinobis‐(3‐Ethylbenzthiazolin‐6‐Sulfonic Acid)) assay (Rumpf et al. 2023). Some researchers divide these types of assays into HAT and SET only, without indicating mixed assays (Prior et al. 2005; Bartosz 2010).

HAT and SET based methods are most commonly used to determine antioxidant capacity, whereas other methods involving ROS scavenging also need to be designed (Karadag et al. 2009). Huang et al. (2005) indicate that oxidative damage in the human body is caused by six major reactive oxygen species. The damage is mainly caused by superoxide anion (O₂ ^•‐), hydrogen peroxide (H₂O₂), peroxide radicals (ROO^•), hydroxyl radical (HO^•), singlet oxygen (¹O₂) and peroxynitrite (ONOO⁻). Assays using ROS can be used for a comprehensive assessment of antioxidant capacity. Usually, these tests include one oxidant. Table 2 below presents the research methods useful in screening studies of antioxidant activity.

The methods are usually based on simple chemical reactions between an antioxidant and model free radicals. The ABTS and DPPH tests are most commonly used in studies (Gulcin and Alwasel 2023). Due to their simplicity, low cost, lack of requirement for specialised equipment and the ability to analyse many samples simultaneously, these tests have become the basis for the analysis of antioxidant activity. At the same time, in the studies of Dudonne et al. (2009), it was proven that the DPPH and ABTS tests are strongly correlated with the results of the Folin–Ciocalteu method. A strong correlation was also observed between the FRAP method and the total phenol content. These two subsequent tests are often combined with ABTS and DPPH.

As mentioned earlier, we recommend using at least three different methods to test antioxidant activity for LAB screening. Ideally, these tests should represent different mechanisms of antioxidant action. By using several research methods, a more complete and reliable picture of antioxidant activity can be obtained.

1.4. Screening of the Antioxidant Properties of Lactic Acid Bacteria

Bacterial screening involves the selection and identification of bacterial strains with desired, specific biological or technological features. According to Zhou et al. (2022), most methods using free radical scavenging can be successfully used to assess the antioxidant activity of LAB. Both intact cells, cell‐free supernatants, cell‐free extracts, cell lysates and post‐fermentation metabolites can be used for the studies. Enzymes and non‐enzymatic metabolites produced by LAB, such as superoxide dismutase, glutathione reductase and glutathione, occur mainly in the CFS fraction (Wu et al. 2025). In recent years, there has been a significant increase in publications on lactic acid bacteria and their strictly antioxidant properties. Table 3 below presents examples of studies on the antioxidant activity of LAB derived from food.

TABLE 3.

Examples of antioxidant studies performed on lactic acid bacteria strains derived from food.

Microorganisms	Source	Sample	Methods	Results and conclusions	References
Lactobacillus kunkeei AK1	Bee pollen	Dextran type EPS	DPPH, ABTS, CUPRAC	Relatively strong antioxidant activity was observed for AK1 dextran, determined by DPPH and ABTS radical scavenging and CUPRAC	Yilmaz et al. (2022)
P. pentosaceus MYU 759	Rice	EPS (neutral‐EPS and acidic‐EPS)	ORAC, HORAC, examination of cytoprotective effects against oxidative stress	Acidic‐EPS showed high antioxidant capacity and high N‐acetylcysteine equivalent values were also demonstrated in the total ROS reduction assay	Yamamoto et al. (2019)
L. harbinensis, L. paracasei, L. plantarum	Kefir grains	Supernatant	F‐C, TFC, DPPH, FRAP	High antioxidant activity was demonstrated in terms of total phenolic content, total flavonoid content, FRAP, and DPPH free radical scavenging capacity	Talib et al. (2019)
L. rhamnosus EM1107, L. mucosae CNPC007, L. plantarum CNPC003	Dairy products	EPS	DPPH, ABTS, FRAP	Antioxidant activity has been demonstrated through the ability to scavenge free radicals DPPH and ABTS as well as FRAP	Bomfim et al. (2020)
Lactiplantibacillus, Lactococcus, Lacticaseibacillus, Lactobacillus strains	Raw cow milk	EPS extracts	DPPH, hydroxyl radical scavenging assay, reducing power assay	EPS extracts showed strong antioxidant activity, demonstrated by DPPH and hydroxyl radical scavenging and reducing power capacity	Tarannum et al. (2023)
L. brevis , L. reuteri , L. fermentum strains	Fermented foods	CFS, filter sterilised	ALDH activity, determination of lipid peroxidation, GSH content	Decreased lipid peroxidation and hepatic transferases in ethanol‐induced HepG2 cells, increased ALDH level, regulation of the expression of genes and antioxidant enzymes	Lee et al. (2021)
L. plantarum L6, L. salivarius 1, L. plantarum F53, L. helveticus 611	Bulgarian cheese, fermented milk ‘katak’	Intact cells, CFS, filter sterilised, EPS	DPPH, ABTS	High radical scavenging activity in vitro	Dobreva et al. (2024)
L. paracasei PB‐LP58	Fermented milk	EPS, SCFAs	TAC, ELISA, MDA levels, inflammatory markers	Significant improvement in inflammatory markers, oxidative stress and reduced incidence of pneumonia	Rahimi et al. (2024)
L. plantarum GXL94	Fermented chilli	Intact cells, fermented supernatant, cell‐free extracts	Tolerance to H2O2, DPPH, ABTS, resistance to hydroxyl radicals and superoxide anion radicals, total reducing power assay, antioxidant gene expression	Good tolerance to high concentrations of hydrogen peroxide and strong free radical scavenging properties were found, the mechanism of antioxidant activity has been proven	Zhou et al. (2022)
L. plantarum CRL2130, B2‐producing strain	Sugarcane bagasse	Intracellular bacterial extracts	Cellular studies on N2a lines, IL‐6 production, measurement of intracellular ROS	Reduction of IL‐6 release and ROS formation, reduction of pro‐inflammatory cytokines	Perez Visñuk et al. (2022)
L. plantarum , L. paracasei strains	Fermented pickles	Bacterial culture, supernatant	DPPH	The ability of the strains to capture DPPH radicals was confirmed	Akmal et al. (2022)
P. pentosaceus R, L. fermentum R6	Harbin dry sausages	Intact cells, intracellular cell‐free extract	DPPH, hydroxyl radical scavenging, superoxide radical scavenging, reducing power assay, antioxidant enzyme activity (SOD, GSH‐Px)	The ability of the strains to scavenge various free radicals was confirmed, increased SOD and GSH‐Px activity in the presence of H2O2	Zhang et al. (2021)
L. casei NA‐2	Chinese sauerkraut	EPS	Hydroxyl radical scavenging, superoxide radical scavenging, DPPH, intracellular ROS determination	Protective effects against oxidative damage and potential immunomodulatory and anti‐inflammatory properties have been demonstrated	Xu et al. (2022)
L. plantarum Y44	Turbot	Intact cells	DPPH, ORAC, resistance to H2O2, hydroxyl free radical scavenging, cell cytotoxicity assay (HT‐29), cellular antioxidant activity	The ability to scavenge free oxygen radicals, the ability to inhibit the production of intracellular ROS without causing cytotoxic effects and protection of HT‐29 cells from H2O2 damage have been demonstrated, increased SOD and GSH‐Px activity and reduced MDA levels in HT‐29 cells	Mu et al. (2018)
L. plantarum KM1	Fermented yoghurt	Supernatant, intact cell, cell‐free extract	Resistance to H2O2, ABTS, DPPH, reducing activity, hydroxyl radical scavenging, proteomic analysis	Tolerance to high H2O2 concentration and the ability to capture free radicals were confirmed, the mechanism of antioxidant action was analysed using proteomics	Tian et al. (2022)
B. bifidum MG731, B. lactis MG741, L. plantarum MG989, L. salivarius MG242	Fermented food	Heat‐killed cells	DPPH, ABTS, NO production	High DPPH and ABTS free radical scavenging activity was demonstrated, reduced nitric oxide production was demonstrated by decreased expression of inducible nitric oxide synthase and cyclooxygenase, four strains with high antioxidant potential were selected.	Kang et al. (2021)
L. plantarum, L. paracasei, E. faecium, L. helveticus, W. paramesenteroides, P. pentosaceus strains	Artisanal milk cheeses	CFS	Resistance to hydroxyl radicals, DPPH, ABTS, resistance to superoxide anions, superoxide dismutase activity, resistance to hydrogen peroxide	High antioxidant activity of LAB strains was demonstrated compared with the probiotic strain L. rhamnosus GG	Shi et al. (2019)
Leu. mesenteroides, Leu. citreum, P. acidilactici, P. pentosaceus, W. cibaria, L. brevis, L . curvatus , L. sakei strains	Fermented food	CFS	DPPH, ABTS, NO production, iNOS and COX‐2 expression	The ability of the strains to reduce DPPH and ABTS radicals was confirmed, and a decrease in NO production and iNOS and COX‐2 expression was observed	Kim et al. (2022)
L. plantarum 200,655	Kimchi	Cell‐free extracts	DPPH, ABTS, NO production	The antioxidant activity was confirmed in DPPH and ABTS radical scavenging tests, RAW 264.7 cells treated with cell‐free extracts produced more NO, induced NO synthase and cytokines associated with immune‐enhancing effects (IL‐1β and IL‐6)	Yang et al. (2019)

Note: A systematic search was conducted between 1 December 2024 and 31 May 2025. Electronic bibliographic databases such as Scopus, Google Scholar, PubMed and ResearchGate were used. The searching strategy used the following keywords: antioxidant or antioxidative, and assay and activity or capacity or properties, scavenging or reducing or power and lactic acid bacteria or Lactobacillus or lactobacilli or dismutase or supernatant.

Abbreviations: ABTS, 2,2′‐azinobis‐(3‐ethylbenzthiazolin‐6‐sulfonic acid) assay; ALDH, aldehyde dehydrogenase; CAT, catalase; CFS, cell‐free supernatant; COX‐2, cyclooxygenase‐2; CUPRAC, cupric ion reducing antioxidant capacity; DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl assay; ELISA, enzyme‐linked immunosorbent assay; EPS, exopolysaccharides; F‐C, total phenolic content assay; FRAP, ferric reducing antioxidant power assay; GSH, glutathione; GSH‐Px, glutathione peroxidase; GST, glutathione S‐transferase; HORAC, hydroxyl radical averting capacity; IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; iNOS, induced nitric oxide synthase; MDA, malondialdehyde; ORAC, oxygen radical absorbance capacity; ROS, reactive oxygen species; SCFAs, short‐chain fatty acids; SOD, superoxide dismutase; TAC, total antioxidant capacity; TFC, total flavonoid content assay.

Fermentation is an effective method for increasing the antioxidant activity of food products (Sarıtaş et al. 2024). In the literature, we can find examples of LAB strains that effectively improve the antioxidant activity of fermented foods, such as meat and meat products, dairy products, meat and milk substitutes, vegetables, fruits, beverages, cereal products, and soy products (Luan et al. 2021; Yang et al. 2021; El‐Sayed et al. 2021; Hunaefi et al. 2013; Zhou et al. 2020; Wang et al. 2014; Li et al. 2021).

Luan et al. (2021) examined the antioxidant activity of L. plantarum CD101 and the effect of bacteria on the formation of bioactive peptides in fermented sausage. The in vitro antioxidant activity of this strain was found to be at the cell surface and in extracellular secretions. Analytical methods identified peptides that are effective in inhibiting oxidative rancidity in fermented sausage.

In turn, the study by Xu et al. (2021) showed a positive effect of L. helveticus 1.0612 on the antioxidant profile of cheddar cheese during ripening. The study aimed to identify antioxidant peptides produced during the manufacturing of cheddar cheese. The addition of L. helveticus 1.0612 had a beneficial effect on the release of antioxidant peptides in cheddar cheese.

The effect of LAB addition on antioxidant capacity was studied in camel milk (El‐Sayed et al. 2021). A classic starter culture (as a control) and strains of L. helveticus, L. casei, L. paracasei and L. rhamnosus were used for milk fermentation. Milk with Lactobacillus bacteria showed higher DPPH radical scavenging activity. Additionally, milk with the L. helveticus strain showed higher total phenolic content and FRAP throughout the storage period.

Yoghurt based on cashew milk showed an increase in total phenolic and flavonoid content and total antioxidant capacity after fermentation with Lactobacillus strains. L. rhamnosus , L. casei, and L. plantarum can be successfully used in the production of cashew yoghurt as a source of antioxidants (Shori et al. 2022).

Vegetables can also be enriched with bacteria and thus enhance antioxidant activity (Hunaefi et al. 2013). Red cabbage was fermented with L. plantarum ATCC 8014 and L. acidophilus NCFM. Fermentation increased antioxidant activity, which was measured by DPPH and TEAC, compared with unfermented cabbage. A slight increase in total phenolic content was also noted, determined by the Folin–Ciocalteu method.

Studies on kiwifruit fermented by L. plantarum showed that the content of phenolic compounds and flavonoids was responsible for the increased DPPH and ABTS scavenging capacity (Zhou et al. 2020). Moreover, fermentation itself had a positive effect on the phenolic profile of this fruit.

Wang et al. (2014) proved that the fermentation of cereal grains with L. plantarum increased the content of phenolic compounds and flavonoids in extracts. Cereal ferments showed stronger DPPH radical scavenging and iron‐reducing activity. The inhibition of LPS‐induced intracellular ROS production without cytotoxic effects in macrophage cells was demonstrated.

In the study by Li et al. (2021), jujube juice was produced with the addition of LAB strains. Jujube juice fermentation with bacteria significantly increased the total phenolic content, measured by the Folin–Ciocalteu method. In addition, the fermented juice showed the most DPPH radical scavenging capacity and higher ferric‐reducing antioxidant power. Antioxidant capacity was significantly improved by LAB fermentation and was positively correlated with the content of caffeic acid and rutin.

Soy products can also be enriched in antioxidant values. Soy milk was fermented by L. plantarum , L. acidophilus , L. casei , L. bulgaricus, and L. helveticus in the study by Wang et al. (2021). Solvent extracts were tested for DPPH and ABTS free radical scavenging and FRAP assay. The selected strains achieved better antioxidant capacities than the compared unfermented soy milk.

The antioxidant potential of LAB is the focus of scientists worldwide. This is related to the growing interest in the role of microorganisms in the production of functional foods and the importance of the microbiome for human health.

2. Conclusions

The antioxidant activity of LAB used in food production, like probiotic properties, is strain‐dependent. This specificity means that different strains may exhibit different antioxidant activity, which directly affects their potential use. Therefore, to select the strains that are the best to be used in functional food production, screening tests are used. Screening aims to isolate strains with the strongest properties, in this case, antioxidant properties. When choosing the best method, many factors must be taken into account, from the analysis conditions to the type of substrate, the type of antioxidant, and the matrix being tested.

One problem is the lack of a single reference method for determining the antioxidant properties of bacteria and food products containing them. The use of one test method cannot reflect the total real antioxidant capacity. This means that as antioxidants have different mechanisms of action, it is important to use more than one test for testing. Understanding the mechanisms of antioxidant action allows for precise planning of the use of a given strain. It can be said that this research approach is comprehensive because it can be used both for food preservation and therapeutic interventions.

LAB are an indispensable element of the human and animal microbiota, as well as a component of fermented food. Undoubtedly, some of the beneficial features of these microorganisms result from their antioxidant properties. Several mechanisms of antioxidant action by LAB are known. Recent scientific research shows promise in the use of LAB strains as protective components of the gastrointestinal tract to reduce the negative effects of oxidative stress, as well as to enhance the overall antioxidant status of the host. However, it should be noted that the mechanisms of antioxidant defence must be fully elucidated. In this study, attention was also drawn to the need for further research to better understand the mechanisms of action and to confirm the health‐promoting effects in well‐designed clinical trials.

Author Contributions

Anna Łepecka: conceptualization, data curation, formal analysis, funding acquisition, project administration, visualization, writing – original draft. Danuta Kołożyn‐Krajewska: conceptualization, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Łepecka, A. , and Kołożyn‐Krajewska D.. 2025. “Antioxidant Properties of Food‐Derived Lactic Acid Bacteria: A Review.” Microbial Biotechnology 18, no. 9: e70229. 10.1111/1751-7915.70229.

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.