Exploring the Potential of Lactic Acid Bacteria Fermentation as a Clean Label Alternative for Use in Yogurt Production

Abstract

The demand for healthier, more natural, and sustainable foods has increased, which drives the development of clean label food products. The clean label trend is associated with developing food products with as few ingredients as possible, free of synthetic additives, and with ingredients that customers understand and consider healthy. Yogurt is a fermented food with numerous health benefits, and is an excellent source of proteins, vitamins, and minerals. However, yogurt may contain chemical additives (including preservatives) that concern consumers as they are associated with potential health risks. Lactic acid bacteria (LAB) are Gram-positive, non-spore-forming, catalase-negative, and non-motile, with antimicrobial activity due to metabolites produced during fermentation. These metabolites include bacteriocins, organic acids, and exopolysaccharides, among others. Thus, in addition to its use in several technological and industrial processes in the food field, LAB present good potential for application as a clean label component for preserving foods, including yogurts. This review article provides an overview of the potential use of LAB and its compounds obtained from fermentation to act as a clean label ingredient in the preservation of yogurts.

1. Introduction

Fermented foods have received attention because of their health benefits [1]. Yogurt is a traditional fermented dairy product made by adding yogurt bacteria, such as Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus, to standardized milk [2]. Depending on the type of yogurt, additives are used to improve its physical, textural, sensory, and rheological characteristics. These additives serve different technological purposes such as stabilizing, thickening, adding flavor, preserving, and coloring the yogurt [3].

Potassium sorbate and sodium sorbate are additives widely applied in the food industry in yogurts as preservatives because of their high stability, solubility, and ease of use [4,5]. Although potassium sorbate is considered safe and has low toxicity, long-term consumption can cause harm to health because it shows cytotoxic and genotoxic effects [6,7]. Therefore, the negative impact of chemical additives on human health drives consumers to look at products with natural preservatives added to them [8,9]. Moreover, the increase in chronic diseases has generated more consumer and government concerns about synthetic additives [6]. In this sense, the clean label trend stands out, which is associated with food products that are additive-free, have undergone minimal processing, and have short ingredient lists [10].

Lactic acid bacteria (LAB) are a group of microorganisms known for their use as starter cultures for producing fermented foods [11]. These bacteria gained prominence for their probiotic capacity, physiological properties, and survival. LAB can contribute to improved nutritional and sensory aspects of food products and the stability of raw materials [12,13]. Furthermore, the compounds produced by LAB can act against pathogenic and spoilage microorganisms, by interrupting the function of the cell membrane, causing its permeability, cell lysis, and consequent loss of cell contents and death [14]. Thus, these antimicrobial compounds boost the investigation of LAB to act as potential replacements to food chemical preservatives in the clean label trend.

Among LAB’s antimicrobial metabolites, bacteriocins, organic acids, and exopolysaccharides stand out. Several studies have reported the antibacterial activity of bacteriocins, while only a few have examined their antifungal activity [15]. Bacteriocins are effective against foodborne pathogens and bacteria-causing food spoilage. Lactic acid is an organic acid produced from the breakdown of lactose by LAB through metabolic activity. Depending on the specific strains of LAB and their fermentation pathways, other organic acids may also be synthesized [16,17]. EPS is another metabolite produced by LAB that can be found in the extracellular environment or bound to the cell surface. In addition to their potential to act as a preservative, EPS may be a substitute for synthetic additives for enhancing the rheological properties, texture, volatile substances, and taste of dairy products [18].

Therefore, the antimicrobial properties of these compounds give LAB a high ability to inhibit and control the growth of pathogenic microorganisms within the clean label concept. In this context, a comprehensive and extended literature review was performed mainly on the most recent papers (last 2–5 years) providing an overview on the antimicrobial metabolites produced by LAB. This review article highlights the potential use of LAB and its compounds obtained from fermentation to act as a clean label ingredient for yogurt preservation. This article also addresses the processing of yogurt, its physicochemical, sensory, textural, and rheological properties, as well as the main challenges of using LAB and its metabolites as a clean label preservative, and their dependence on suitable processing procedures, allowing us to minimize the gap between in vitro and real food matrices.

2. Yogurt

2.1. Yogurt Production

Yogurt is a dairy product available on the market in different textures, flavors, and forms to satisfy consumers’ needs. According to legislation, yogurt is a coagulated product obtained by lactic fermentation due to the action of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in milk or dairy products indicated in n° 1 of n° 6 with or without the products indicated in n° 7, and specific microbiota must be alive and abundant in the final product (5 × 10⁷/g) [19].

Yogurts can be produced in three ways: to make set, stirred, and drinkable products (Figure 1) [20]. These products are classified according to their physical nature (fluid-like, semi-solid, or solid), chemical composition (whole-, low-, and non-fat), flavor (plain and flavored), and style (e.g., Greek yogurt). Although there are differences in the production of these yogurts, the main steps are the same: milk standardization, homogenization, heat treatment, milk incubation and fermentation, cooling, and storage. Yogurts can contain chemical additives in their formulations to improve various properties, including physicochemical, textural, sensorial, rheological [3], and microbiological characteristics. Stabilizers, flavors, preservatives, thickeners, and colorants are some examples of synthetical additives added to yogurt, which are used to address the challenges encountered in its production and satisfy consumers’ needs. As observed in Figure 1, additives and fruit preserves can be added to yogurt at different stages of the production process [20].

Milk standardization consists of standardizing fat and protein content. Milk powders, including non-fat dry milk, whey protein concentrate, or milk protein concentrate, are incorporated into the milk to achieve the desired characteristics. Generally, milk homogenization occurs using 10–20 MPa e 5 MPa in first- and second-stage pressure, respectively, and at 55–65 °C. The milk fat globules fragment into smaller molecules, increasing the surface area and preventing yogurt fat separation (creaming) during fermentation and storage [3].

After homogenization, milk undergoes heat treatment to destroy microorganisms that may change the attributes of yogurt and inactive enzymes [21]. Next, the milk is cooled to the incubation temperature (around 40–45 °C) to allow the start culture to grow. During the fermentation, lactic acid bacteria (LAB) convert lactose into lactic acid. Consequentially, the pH of the milk drops from 6.7 to ≤4.6 [3]. Acidification is responsible for coagulation (protein denaturation) and consequently gel formation. Casein begins destabilizing at pH 5.3–5.2, leading to precipitation and denaturation when pH is 4.7–4.6 [22]. When the yogurt reaches the desired pH (~4.6), it is cooled (<5 °C) to stop the fermentation process and thereby prevent a rise in acidity [3].

2.2. Fruit Preparations

Fruit preparations used in yogurts are defined as suspensions of fruit particles or pure pulp stabilized in a sweetened and acidified matrix, which additionally can have flavors/coloring material. They are added into yogurt products within the 10–20% level in the final product [23]. The combination of fruit pulp or fruit preparations with yogurt represents an approach to masking the acidity of this product [24]. Additionally, diverse fruits can be added to yogurt, such as berries, strawberries, bananas, mango, lemon, and apple [25].

Yogurts, being high-water content acidic media, are susceptible to spoilage by molds and yeasts introduced throughout production and processing. Moreover, adding fruits or fruit jams constitutes other potential fungal and bacterial contamination sources. This microbial growth in yogurt interferes with the flavor, texture, odor, and even color, making the product unacceptable to the consumer [26]. Therefore, for yogurts to have a better quality and extended shelf life, they may contain different additives, such as emulsifiers, preservatives, color, acidifiers, sweeteners, thickeners, and flavors [27]. Also, fruit preparations can contain preservatives like potassium sorbate to inhibit microbial growth [23].

Incorporating fruits into yogurt contributes to improving its nutritional profile and its sensory properties, increasing acceptability, since not all consumers appreciate yogurt in its natural form [28]. Nevertheless, it affects many physicochemical properties, including the texture, pH, acidity, and syneresis. It also influences the stability of the yogurt, increasing or decreasing compared to plain yogurts. In addition, depending on the concentration of the fruit flavor used in the yogurt, changes in the product’s characteristics may occur [25]. A study performed by Kamber [29] evidenced that adding fruit to yogurt affected the pH and acidity and influenced the development of microflora. Likewise, Wang et al. [30] related the influence of the addition of different concentrations of apple pomace on the physicochemical properties of yogurt. Thus, optimizing the physicochemical characteristics of yogurt to be well-accepted by consumers and simultaneously improve its quality is essential.

2.3. Physicochemical and Sensory Properties

The physicochemical properties generally measured in yogurt are the protein and fat content, pH value, titratable acidity, and syneresis. According to the regulations stated by Codex Alimentarius, the minimum milk protein content allowed in yogurts is 2.7%, increasing to 5.6% in the case of concentrated yogurts. The fat content must be below 15%, and titratable acidity must be 0.6% at the minimum [31]. Titratable acidity is the total acid concentration in food expressed as a percentage of lactic acid in yogurt [32]. Most yogurts have 0.8–1% lactic acid and a pH of 4.6 [22]. Syneresis or whey separation consists of the formation of whey on the surface of yogurt due to the shrinking milk protein gel that reduces the size of the casein aggregates. Furthermore, syneresis is the main disadvantage regarding the sensorial attractiveness of yogurt [33] because it is usually linked to low-quality products by consumers [34].

The main quality characteristics of yogurt are its texture, taste, aroma, and flavor [22]. In addition, color is another quality characteristic of yogurt, which influences the acceptability of the product to consumers [35]. Yogurt’s characteristic flavor comes from the production of lactic acid by LAB, the presence of aromatic compounds naturally found in milk and produced during fermentation [22]. LAB create flavor precursors that are then transformed into flavor compounds. Many enzymes hydrolyze carbohydrates, proteins, and lipids. Glycolysis, lipolysis, and proteolysis are metabolic processes of LAB responsible for the formation of volatile compounds [36]. The flavor of yogurt is mainly defined by the presence of acetaldehyde, diacetyl, ethanol, acetone, and 2-butanone, which are important volatile compounds. However, acetaldehyde contributes to its tart and green-apple-like flavor, allowing yogurt to distinguish itself from other fermented dairy products, as does diacetyl [22].

2.4. Textural and Rheological Characteristics

The analysis of yogurt’s structure involves examining its texture, rheological properties, and microstructure. From a rheological perspective, yogurt is classified as a non-Newtonian fluid and can be characterized as a shear-thinning substance, meaning its viscosity decreases as the shear rate increases [37]. The structure of the yogurt gel results from protein aggregate formation because of milk acidification, which results in a porous protein network that retains the whey [38,39]. Whey proteins undergo denaturation at elevated temperatures. Subsequently, they bind to casein micelles, increasing the protein’s density [34,38]. In the case of stirred yogurt, the gel network is broken and stirred until it reaches a homogeneous consistency after fermentation. This leads to a different mouthfeel when compared to set yogurt. Due to the mixing process, viscosity decreases, which then increases during storage [37]. This phenomenon is called rebodying, i.e., the microgels are capable of reaggregation [38].

Yogurt behaves as a weak gel, resulting in storage modules G′ that are higher than viscous modules G′; viscoelastic functions slightly depend on the oscillation frequency [34]. In general, the rheological experiments used on stirred yogurt (1) measure the apparent viscosity at a specific shear rate; (2) measure the viscoelastic properties of the stirred gel (complex viscosity, G′, G″, etc.); and (3) include texture profile analysis tests to measure the firmness [38] that is the needed to allow for a given deformation. This technique also evaluates the adhesiveness (the force needed to separate the material that sticks to the teeth while eating) [39] and cohesiveness (the amount of deformation a material can withstand before breaking) [40].

The textural and rheological characteristics of yogurt are essential in determining its characteristics, shelf life, and consumer acceptability [40]. Many factors can affect these characteristics (

Table 1. Influence of various factors on yogurt texture and rheology and syneresis.

Type of Yogurt	Study Factor	Textural and Rheological Properties	Syneresis	Reference
Greek-style	Retrograded starch	Increased consistency, firmness, and water-holding capacity.	Decreased	[41]
Stirred	Mulberry pomace	Increased titratable acidity, water-holding capacity, consistency, and viscosity. Decreased firmness. Improved microstructure.	Decreased	[42]
Low-fat stirred		Increased apparent viscosity.	-	[43]
Stirred	Post-heating treatment	Treatments below 65 °C/25 s increased gel strength, firmness, and viscosity. Treatments above 65 °C/25 s–Decreased these properties.	-	[44]
-	Concentrated strawberry pulp	Decreased viscosity.	-	[45]
Unstirred	Exopolysaccharides	Increase gel strength and viscosity.	Decreased	[46]
Stirred	Heat exchanger type	Plate heat exchanger. Tubular heat exchanger, firmer yogurt gel.	Decreased	[47]
			-
	Fat content	Increased firmness and viscosity.	Decreased
Stirred	Smoothing temperature	Yogurt at 42 °C: heterogeneous microstructure.	Increased	[48]
		Yogurts at 20 °C and throughout a 42 °C-to-20 °C cooling ramp: homogeneous network.	Decreased
		The apparent viscosity was higher at 20 °C than at 42 °C.	-

), such as homogenization, polysaccharide presence, storage period, smoothing temperature [38], milk composition (mainly total solids, protein, and fat content), starter culture selection, etc. [37].

3. Sorbates and Benzoates

Potassium sorbate (PS), and sodium benzoate (SB) are used as preservatives in the food industry. Based on European nomenclature, PS is also known as E202, and SB as E211. They have antifungal and antimicrobial properties, inhibiting the growth of bacteria, yeast, and mold. Moreover, PS and SB are generally recognized as being safe (GRAS). PS is a potassium salt of sorbic acid, while SB is a benzoic acid salt, with both of them odorless and soluble in water [5,6]. Some applications of PS include cheese, fruit juice, margarine, bakery, canned tomato paste [5], and yogurt [49]. SB is used to conserve acidic pH foods such as fruit pulp and purees, jams, beer, fruit yogurts, margarine, and salads [6,50].

According to the World Health Organization and the Food and Agriculture Organization of the United Nations, the recommended daily intake limit for PS is 25 mg/kg body weight [7]. In March 2019, the European Food Safety Authority [51] changed the temporary acceptable daily intake value to 11 mg sorbic acid/kg body weight per day for potassium salt, based on new data about reproductive toxicity. Regarding SB, the limit of daily is 5 mg/kg body weight [5]. For example, a person who weighs 70 kg can safely ingest 770 mg and 350 mg of PS and SB per day, respectively, without suffering negative effects [50]. Mazdeh et al. [4] reported values of 178.75 ± 1.75 and 23.31 ± 4.11 mg/kg of yogurt for potassium sorbate and sodium benzoate mean amounts in yogurts, respectively, namely, in fruit concentrate yogurts, and it is not difficult to exceed the acceptable daily intake recommended by the authorities. Therefore, it is important to look for alternatives to synthetic preservatives to reduce their use or even replace them entirely.

In acidic foods such as yogurt, PS inhibits the growth of bacteria, fungi, and yeast [52]. However, using PS in corrupted and alkaline foods is not recommended [6]. The antimicrobial activity of PS and other preservatives, such as sorbic acid, is associated with the carboxyl group (-COOH) and the number of carbon atoms in its structure. Sodium acetate and potassium sorbate have higher antimicrobial properties compared to other additives. This fact relates to their shorter carbon chains and the ability to cross cell membranes [52]. Moreover, the antimicrobial effect of PS depends on the dissociation of sorbic acid and consequently on the pH. However, the antimicrobial mechanism of the preservative is not yet fully understood [6]. The minimum inhibitory concentration (MIC) values of potassium sorbate and sodium benzoate are 5 mg/mL for Escherichia coli, 10 mg/mL for Bacillus mucoides, Bacillus subtilis, and Staphylococcus aureus, and 50 mg/mL for Aspergillus flavus [6]. In the case of Candida albicans, the MIC is 50 and 2.5 mg/mL for potassium sorbate and sodium benzoate, respectively. In other words, microorganisms with a lower MIC are more sensitive to preservatives, meaning a small amount is enough to inhibit their growth. In turn, the higher the MIC, the greater the microorganism’s ability to resist the preservative, and a greater amount is needed to inhibit it, as it the case of Aspergillus flavus and Candida albicans for PS.

While potassium sorbate is generally regarded as a safe and low-toxicity food additive [53], there is significant concern about its potential negative impact on human health. The lethal oral dose of PS is 500 mg/kg body weight. Prolonged ingestion of PS can lead to shortness of breath, headache, chest pain, mucosal irritation, and pulmonary edema [6]. Also, when the consumption of sodium benzoate goes beyond the recommended limits, it can cause health problems like nausea, vomiting, abdominal pain, and central nervous system depression [54]. Therefore, several studies have been conducted on the risks of this compound and other food additives to human health. Mohammadzadeh-Aghdash et al. [52] investigated the cell toxicity and genetic toxicity of potassium sorbate in human umbilical vein endothelial cells. Based on the results, this food additive does not present significant cyto-genotoxic effects at low concentrations. Other studies have reported the importance of the interaction between food preservatives and gut microbiota. Gut microbiota play an important role in helping to regulate several physiological processes in the host organism. According to Peng et al. [53], PS affected the gut health of zebrafish, reducing the amount and diversity of bacteria in the gut, and hampering their regular metabolism. Nagpal et al. [54] evaluated the effect on mice gut microbiota diversity and potassium sorbate, benzoic acid, and sodium nitrite composition in doses equivalent to those indicated as safe. This study revealed that PS resulted in lower gut microbiota diversity in the host that consumed it compared to the other two preservatives. Xiao et al. [7] observed negative effects of PS intake, such as infiltration of inflammatory cells in the liver and effects on gut microbiota, when this compound was taken for 10 weeks. These effects were abolished after a 5-week washout period, and a healthy gut microbiota was again established [7]. Thus, since potassium sorbate presents a potential health risk, searching for natural components to replace this chemical preservative in various foods is crucial.

4. Clean Label Concepts in Yogurt Production

Consumers are now more aware of their health, so the demand for healthy foods has increased. In addition, they are looking for more natural, healthy, sustainable, and tasty food. Consumers consider yogurt a food that is easy to purchase, rich in nutrients, and associated with the perception of a healthy diet [55]. Yogurt is an excellent source of vitamins, such as vitamins A, B2, and B12, as well as minerals, including calcium (in its bio-available form), phosphorus, and potassium. Furthermore, it is a good source of essential fatty acids and proteins, providing high biological value [56]. Incorporating natural compounds into yogurts enhances its organoleptic properties and promotes consumer health benefits [57].

The popularity of yogurt is mainly due to its numerous health benefits. Some benefits are related to the easy digestion of protein and sugar compared to milk, the improvement of lactose digestion and tolerance, and the decrease in the risk of osteoporosis [57]. However, yogurt contains preservatives that concern consumers, as despite being authorized, their safety has been questioned. Therefore, consumers’ demand for clean label products has increased recently [9].

The definition of the clean label concept differs among consumers and within the food industry, lacking a clear and consistent objective. For example, a clean label product is a product that has few ingredients, all of them easily recognizable, understandable, and perceived as healthy by consumers [58]. Thus, for foods to receive a clean label, they must be “additive-free”, involve “minimal processing”, and have a “short ingredient list” [10]. The food industry has made a great effort to develop products, for example, with less sugar, without artificial preservatives and easy-to-recognize ingredients to meet consumers’ needs [9]. Applying natural preservatives is a promising approach to developing healthier and clean label food products while maintaining their safety and shelf life. Natural preservatives from metabolites produced by microorganisms have antimicrobial and antioxidant activity, representing an effective alternative to synthetic preservatives [59].

5. Lactic Acid Bacteria

5.1. General Characteristics

Lactic acid bacteria (LAB) are Gram-positive, non-spore-forming, catalase non-producing, and non-motile microorganisms [60]. The main characteristic of these bacteria is lactic acid production, which is a key to fermentation products. In addition, they have a high tolerance to acidic pH, are aerotolerant, and can appear as cocci or rods [61]. Even though LAB can be present naturally in some dairy products, they are often deliberately incorporated as a starter culture, or sometimes as ingredients or additives to amplify the product’s functionality, particularly their probiotic potential [62]. They can also be present in plants and the animal gastrointestinal tract [22]. Additionally, LAB present GRAS status. They do not pose a risk to human health, allowing for their use in technological and industrial processes in the food sector. In addition, LAB are acid-tolerant and can synthesize antimicrobial compounds [63]. Thus, in addition to its importance for promoting health, LAB presents good potential for use as a clean label ingredient for preserving foods, including dairy products.

LAB are highly diverse, mainly classified under the phylum Firmicutes, class Bacillus, and order Lactobacillales. This order comprises six families (Lactobacillaceae, Leuconostocaceae, Enterococcaceae, Aerococcaceae, Streptococcaceae, and Carnobacteriaceae) encompassing over 30 genera and 300 species. Additionally, the genus Bifidobacterium is also considered a LAB; however, it belongs to the phylum Actinobacteria [61], class Actinobacteria, order Bifidobacteriales, and family Bifidobacteriaceae [64].

Bacteria from the genus Bifidobacterium are Gram-positive, anaerobic or aerotolerant, non-spore-forming pleomorphic bacteria. They are present in the gastrointestinal tract of mammals, birds, and insects. Some examples of Bifidobacterium species are Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis, and Bifidobacterium bifidum. Generally, bifidobacteria have an optimum growth temperature of around 37–41 °C, being considered mesophilic organisms. However, there are a few thermophilic species, such as Bifidobacterium thermacidophilum [65]. Moreover, bifidobacteria represent a significant group of probiotic cultures used in industry. They have shown various beneficial effects, such as a boost in lactose digestibility, a decrease in serum cholesterol level, synthesis of complex-B vitamins, and calcium absorption [66]. However, to recognize the benefits of probiotic intake, the viable cells must be at least 10⁶ CFU/g of the food product [65]. Their optimum growth pH is around 6–7; nevertheless, some strains of Bifidobacterium lactis and Bifidobacterium animalis can grow even at pH 3.5 [66]. Nonetheless, the presence of high oxygen levels affects the growth and viability of these microorganisms. Bifidobacterium animalis subsp. lactis is broadly used in yogurt-like products due to its good tolerance to acid, low pH, and molecular oxygen [67].

For example, Streptococcus thermophilus and Lactococcus lactis enhance flavors in foods like yogurts by producing a tangy, pleasantly acidic taste, characteristic of fermented dairy products [17]. Thus, this fact is positive for using LAB as a clean label ingredient to act as a natural preservative in yogurts, as it will not harm the characteristic acidic flavor of these food products, while also promoting health.

5.2. Mechanism of LAB

There are two fermentation pathways: homolactic fermentation and heterolactic fermentation. In homolactic fermentation, glucose is the carbon source for producing pyruvic acid, which is converted to lactic acid by the lactate dehydrogenase enzyme. Energy is generated through reduced nicotinamide adenine dinucleotide (NADH). Consequently, from one glucose molecule, two lactic acid and two adenosine triphosphate (ATP) molecules are produced through the glycolytic pathway. On the other hand, heterolactic fermentation produces lactic acid, acetic acid or alcohol, and carbon dioxide (CO₂) from one glucose molecule through the phosphoketolase pathway [63]. Glucose 6-phosphate is converted into CO₂, ribulose 5-phosphate, and nicotinamide adenine dinucleotide phosphate (NADPH). The enzyme lactate dehydrogenase plays an essential role in the production of lactic acid because it transforms pyruvate into lactic acid. Furthermore, its stereospecificity determines the configuration of lactic acid. L-lactic acid is catalyzed by L-lactate dehydrogenase, whereas D-lactate is catalyzed by D-lactase dehydrogenase [68]. Additionally, during fermentation, many other metabolites are produced in this pathway, such as aromatic compounds (e.g., diacetyl and acetaldehyde) and different organic acids. LAB can also metabolize other hexoses, such as fructose, mannose, or galactose, as alternative carbon sources [63].

LAB can be categorized as homofermentative or heterofermentative organisms based on their ability to ferment sugars. For example, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are homofermentative bacteria [69]. On the other hand, bifidobacteria are heterofermentative bacteria because they produce acetic and lactic acids; however, generally, they metabolize hexoses using the “bifidus pathway”, where the fructose-6-phosphate phosphoketolase is the key enzyme and CO₂ is not produced [66].

5.3. Antimicrobial Compounds Produced by LAB

LAB have a particular relevance in the biopreservation processes due to their broad spectrum against the growth of unfavorable microorganisms. The LAB activity against microorganisms results from the metabolites produced in the fermentation process. These metabolites include bacteriocins, organic acids, exopolysaccharides, and other molecules like diacetyl, reuterin, and hydrogen peroxide (H₂O₂) [70], which have the potential for use as clean label agents to replace chemical preservatives in food products, especially yogurts.

5.3.1. Bacteriocins

Bacteriocins are small antimicrobial peptides produced by various groups of bacteria, specifically LAB, to defend against other bacteria or pathogens. Bacteriocins are cationic molecules with 20 to 60 amino acids that present hydrophobic characteristics [17,61]. Various classifications of bacteriocins have been proposed based on the first classification proposed by [71], which divided them into four classes. Generally, Class I, lantibiotics, are small thermo-stable peptides post-translationally modified, with uncommon amino acids, lanthionine, and methyl-lanthionine in their primary structure. Class II embraces heat-stable small amphiphilic helical peptides. Regarding class III, this comprises larger bacteriocins, thermo-labile and non-lytic, with complex activity and a complex protein structure. Finally, Class IV bacteriocins are considered more complex due to their lipid or carbohydrate fractions, and are sensitive to glycolytic or lipolytic enzymes. Bacteriocins have different mechanisms of action: after being synthetized and transported, they mainly act through the rupture of cell walls or the inhibition of the synthesis of target molecules [72].

Bifidobacterium animalis is reported to produce the bacteriocin Bifidocinp-A, which showed antimicrobial activity against foodborne bacteria, supporting its use as a natural preservative in yogurt as defined in this paper [73]. Lactacin F from Lactiplantibacillus plantarum has been employed in yogurt to improve its safety [74]. Enterococcus species produce enterocins, which are bacteriocins that are highly effective against spore-forming bacteria of the Bacillus and Clostridium genus [17], such as Bacillus cereus, Clostridium botulinum, and Clostridium perfringens, which can lead to dairy product poisoning due to toxin production and can result in milk and dairy product spoilage [75].

Listeria monocytogenes is cold-tolerant, which implies that it can survive in yogurt stored at refrigerated temperatures. Escherichia coli is very resistant to acid and can survive in yogurt [76]. Staphylococcus aureus is another microbial culture that may also pose a risk in homemade milk and yogurt [77]. Several studies demonstrated that bacteriocins produced by LAB have inhibitory effects on these foodborne pathogens [78,79]. Although best known for its inhibitory effect against bacteria, LAB bacteriocins have also been reported to exhibit antifungal properties. Shehata et al. [80] identified a new bacteriocin with activity against Aspergillus parasiticus and Aspergillus carbonarius, produced by Lacticaseibacillus paracasei, that also inhibited mycotoxin excretion.

Thus, the antimicrobial potential of bacteriocins from LAB makes them promising metabolites that can act as natural preservatives for yogurts. Consequently, it is notable that bacteriocins can be inserted in the clean label trend to increase food safety.

5.3.2. Organic Acids

Organic acids are the main fermentation products of carbohydrate metabolism from LAB. Examples include lactic acid, acetic acid, propionic acid, succinic acid, 2-hydroxybenzoic, and vanillic acid [15]. These acids create an unfavorable environment which inhibits the growth of Gram-negative and Gram-positive bacteria, yeasts, and molds in many food products [81]. Their antimicrobial action primarily involves (1) disrupting the bacterial cell wall and cell membrane structure, (2) lowering intracellular pH; and (3) causing extracellular acidification [82].

The best characterized and effective metabolites are lactic and acetic acid [15]. Acetic acid is known for its effectiveness against different pathogens, particularly fungi [17]. As fermentation time increases, lactic acid concentration rises, and an increased effect of the lactic acid antifungal activity was observed [83]. In addition, synergistic antimicrobial action between lactic and acetic acid can be suggested [16]. In their protonated form, these organic acids are bioactive at low pH [15].

Wang et al. [84] inoculated six strains of Escherichia coli on pea sprouts and then exposed to 0.2 mol/L of ascorbic acid, citric acid, and malic acid for 10 min. This exposition led to cell membrane damage and reduced cell proliferation. Stanojević-Nikolić et al. [85] found that lactic acid was more effective towards bacteria, especially Gram-positive bacteria. Moreover, as the concentration of lactic acid increases, the efficacy of inhabitation against pathogens also increases. Another study conducted by Garnier et al. [86] reported propionic and acetic acids, lactic and acetic acids, and butyric acid as the primarily antifungal compounds in dairy fermentates from Acidipropionibacterium jensenii, Lactobacillus rhamnosus, and Mucor lanceolatus strains. Other organic acids produced by Bifidobacterium adolescentis inhibited Penicillium expansum, a mycotoxin (patulin) producing fungus [87]. It is relevant to highlight that Penicillium and Mucor are the main genera involved in dairy product spoilage, including yogurt [15,88]. The studies prove the excellent antimicrobial activity of organic acids, which enhances the use of LAB as a clean label strategy for developing healthier and additive-free food products. In this sense, using organic acids can contribute to extending yogurt’s shelf life and ensure the food quality within the clean label concept.

5.3.3. Exopolysaccharides

Exopolysaccharides (EPSs), another group of functional components produced by LAB, are biodegradable polymers formed from monosaccharide units linked by a glycosidic bond [63]. These polymers can be divided into homopolysaccharides (HoPSs) and heteropolysaccharides (HePSs). HoPSs are synthesized extracellularly by Lactobacillus, Streptococcus, Leuconostoc, Oenococcus, Pediococcus, and Weissella genera. Meanwhile, LAB-producing HePSs are synthesized intracellularly and belong to Lactobacillus, Lactococcus, Streptococcus, and Bifidobacterium species. Some examples of monosaccharides that compose HoPSs are glucan, fructans, and galactans, consisting of D-glucose, D-fructose, and D-galactose, respectively. In the case of HePSs, these contain rhamnose, glucose, galactose, fructose, and mannose [89,90].

EPSs can be used as a stabilizer, thickener, emulsifier, and gelling agent to improve fermented foods’ appearance, taste, rheological properties and texture. Beyond their positive technological effects, EPSs may also induce beneficial antimicrobial properties, increasing their potential applications in the food industry [91]. A study conducted by Nehal et al. [92] showed that EPSs produced by Lactococcus lactis F-mou exhibited inhibitory activity against various microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Bacillus cereus, Proteus mirabilis, Acinetobacter baumannii, Enterobacter cloacae, and Candida albicans. Likewise, the EPSs isolated from Bifidobacterium longum inhibited the growth of several bacteria, such as Vibrio parahaemolyticus, Salmonella typhimurium, Staphylococcus aureus, and Bacillus cereus [93].

These numerous characteristics related to EPS from LAB highlight the relevance of these microorganisms in the clean label trend by producing antimicrobial metabolites that are responsible for improving the technological and sensory properties of food products. Therefore, it is interesting that more research investigates the potential use of EPS from LAB to replace preservatives in yogurts.

5.3.4. Other Compounds

Other compounds produced by LAB also present antimicrobial effects, which intensify the potential of using LAB as a clean label component in food products. Reuterin (3-hydroxypropionaldehyde) is produced by Lactobacillus reuteri. This compound shows activity toward foodborne pathogens. Reuterin acts by inducing oxidative stress within microbial cells, interacting with thiol groups present in enzymes and other cellular proteins, and damaging their function. This compound also causes microbial cell membrane disruption, decreases intracellular pH, and damages DNA molecules [17]. One study assessed the antimicrobial potential of reuterin and verified that Gram-positive bacteria were more resistant to reuterin’s action than Gram-negative bacteria. In addition, 10 mmol/L of reuterin was enough to completely inhibit the growth of Penicillium expansum [94], while the growth of spores of Fusarium culmorum was reduced to 10% by 4 mmol/L, and by 8 mmol/L in the case of Aspergillus niger and Penicillium expansum [95].

Another compound, diacetyl, has antifungal properties against many molds (such as Penicillium spp. and Fusarium spp.) and Gram-positive and Gram-negative bacteria [15]. It works mainly by lowering the internal pH of microbes, which leads to the loss of molecules and eventual cell death [17]. One study evaluated the antimicrobial effects of LAB on pathogens, Staphylococcus aureus and Listeria monocytogenes, and the results showed antimicrobial activity resulting from diacetyl production [96]. Diacetyl (at a concentration of 75 μg/mL) isolated from Lacticaseibacillus paracasei DGCC2132 inhibited the growth of Penicillium for five days [97], which can be associated with the induction of ROS accumulation, which destroys the membrane structure, resulting in the leakage of cellular materials and cell death [83].

Hydrogen peroxide also presents antimicrobial activity. Some LAB, such as Lactobacillus lactis, Lactobacillus acidophilus, and Lactobacillus delbrueckii subsp. bulgaricus, produce H₂O₂ in anaerobic growth conditions due to a lack of cellular catalase, pseudocatalase, or peroxidase. Its antimicrobial action is ascribed to its potent oxidizing capability, which damages cellular components. H₂O₂ induces bacteriostatic and bactericidal effects; nonetheless, the latter is rarely achieved [98].

Fatty acids produced by LAB also have strong antifungal activity. Fatty acid from Lactiplantibacillus plantarum also exhibited an antifungal effect against Aspergillus fumigatus and Bacillus cereus, with an MIC of 0.21 g/L and 0.25 g/L, respectively [99], while fatty acids from cultures of Lactobacillus hammesii and Lactiplantibacillus plantarum effectively inhibited Aspergillus niger and Penicillium roqueforti [100]. Leyva Salas et al. [101] found that Penicillium commune inhibition in yogurt was associated with higher amounts of four free fatty acids, including C10:0, C12:0, C16:1, and C18:1. Regarding the antimicrobial mechanism of fatty acids, the lipid bilayers of fungal membranes are partitioned by the acids, increasing membrane fluidity and permeability, with the consequent cytoplasmic disintegration of fungal cells [15].

5.4. Antioxidant Compounds

Antioxidants play an important role in protecting cells from oxidative stress caused by free radicals, and are closely associated with human health [68]. Interest in the antioxidant properties of LAB has notably increased due to various reports on their capacity to synthesize antioxidant metabolites. Some antioxidant substances are EPSs, bioactive peptides, antioxidant enzymes, and manganese ions [102]. In LAB’s antioxidant domain, the focus is directed to the metabolism of phenolic substances and the limited capability of certain bacteria to synthesize glutathione. For example, Lactobacillus brevis, Limosilactobacillus fermentum, and Lactiplantibacillus plantarum metabolized phenolic acids through the decarboxylase and reductase enzymes [61].

LAB have systems to eliminate free radicals, thereby reducing the risk of radical accumulation during food fermentation and the damage caused by such free radicals to humans [103]. Bifidobacterium, Lactobacillus, and Enterococcus reduced the concentration of free radicals, like DPPH and ABTS, as shown in a study with 15 LAB strains from fermented foods, demonstrating their skill to relieve oxidative stress by scavenging DPPH and ABTS radicals [104].

Knowing that antioxidant compounds are also used as food additives to prevent spoilage, the capacity to produce antioxidant compounds combined with antimicrobial properties helps to emphasize that LAB has a high potential for use as a clean label strategy to replace chemical additives in the preservation of food products.

6. Challenges in Using LAB and Their Metabolites as a Clean Label Approach

Although antimicrobial compounds produced by LAB are promising alternatives to replace chemical preservatives used in the food industry, there are some challenges in their use as clean label ingredients. The efficacy of this biopreservation approach is intricately associated with LAB viability and activity. Diverse aspects, including pH, temperature, and environment, can influence LAB performance. Additionally, incorporating LAB into food products can influence flavor profiles. For example, over-acidification due to high concentrations of organic acids may be considered a defect in food products for individuals [17]. Another limitation is that the antimicrobial activity of bacteriocins can be prejudiced when coming into contact with lipids and proteins, since they are sensitive to lipolytic and proteolytic enzymes [105,106]. Thus, new systems such as nanotechnology emerge to help address these challenges. Using nanoencapsulation becomes an innovative alternative for bacteriocins to enhance their stability when applied to food and contribute to the controlled release of organic acids [106].

The extraction and purification processes for bacteriocins and other LAB antimicrobial metabolites can drive up production costs. Using bacteriocin-producing bacteria can overcome challenges associated with the cost and sensitivity of purified bacteriocins to proteolytic enzymes. Applying this strategy will also contribute to continuously producing the antimicrobial compound in the food matrix [107]. Fermented products derived from bacteriocin production is another strategy for dairy preservation, without the need to initiate a new fermentation process in the dairy product itself. However, this approach presents challenges related to preservation potential, as fluctuations in the concentration and activity of bacteriocins in these fermented products may occur [17].

Only a few bacteriocins (nisin, pediocin, and micocin^®) are approved by the Food and Drug Administration for food preservation [108]. Nevertheless, many other bacteriocins are currently being developed for use in food [81]. Another concern about using bacteriocins as food preservatives is the possible development of resistance by pathogens to bacteriocins. It represents a threat to food preservation and safety, specifically in products fermented by LAB. However, it is necessary to research and understand pathogens’ resistance mechanisms towards these antimicrobial molecules and identify innovative strategies to counteract this issue [61].

The challenges related to using EPS from LAB as a clean label preservative for the food industry include the low yields obtained and the variability between the different bacterial strains that produce it, making it difficult to achieve a consistent production of the antimicrobial compound [17]. Furthermore, understanding the relationship between the structure and function of EPS in foods has posed a challenge [18].

Therefore, the main challenges are related to the efficiency and costs of natural preservatives compared to synthetic preservatives. Achieving the same shelf life at a low cost and keeping food products safe and attractive is crucial to developing clean label products. Thus, it is necessary to determine the appropriate concentration for the incorporation of antimicrobial compounds from LAB so that they are effective and do not compromise food products’ nutritional and technological characteristics.

7. Conclusions

LAB cultures and their antimicrobial metabolites are promising candidates as clean label preservatives capable of inhibiting the growth of pathogenic and spoilage-associated microorganisms. Bioactive compounds from LAB, such as bacteriocins, organic acids, exopolysaccharides, and other molecules like diacetyl, reuterin, hydrogen peroxide, and fatty acids, can act against the growth of several unwanted microorganisms in yogurts. Thus, the use of LAB and its bioactive compounds in yogurts emerges to meet consumer demands for nutritional, sustainable, and additive-free products. Although the antimicrobial potential of LAB and their metabolites is proven, some aspects limit this clean label strategy from being applied in the food industry. Overcoming some of these issues may be directly connected with the processing steps for yogurt and may pass through the addition of LAB-fermented ingredients in a subsequent step after the milk fermentation point, avoiding interference with the main LAB fermentation stage and reducing interference in yogurt characteristics. Challenges related to costs, efficiency, and stability still need to be overcome and clarified so that LAB and its antimicrobial compounds can compete with synthetic preservatives from the food industry.