Comparative Analysis of Protective Cultures: Application in Heated Meat Products and Their Plant-Based Alternatives

1. Introduction

The significant negative impact of food waste, particularly in terms of its contribution to the global carbon footprint and climate change, highlights its relevance to the Sustainable Development Goals (SDGs). Not only is addressing this issue a global priority, it is also the responsibility of the food industry to adopt sustainable practices (United Nations, https://sdgs.un.org/topics/chemicals-and-waste; Bhatia et al., 2023).

SDG Target 12 explicitly calls for ‘sustainable consumption and production patterns’ and commits Member States to halving per capita food waste at retail and consumer levels, and reducing food losses across production and supply chains, including post-harvest stages, by 2030 (United Nations, https://sdgs.un.org/topics/chemicals-and-waste).

Consumers and retailers value information about ingredients and additives and are interested in food production. The trend toward products with clean labeling and particularly fresh, minimally processed products is also evident in discount stores and retail chains’ private label programs, as foods that contain many ingredients that must be declared with E numbers (e.g., sorbic acid – potassium sorbate E 200 – 202) (European Commission; https://ec.europa.eu/food/food-feed-portal/screen/food-additives/search; Karbowiak et al. 2023).

At the same time, there is a strong demand for products with a long shelf life. Manufacturers should allocate more resources to this area by monitoring the production of their products (Hazard Analysis and Critical Control Points – HACCP and Good Hygienic Practices – GHP) and improving their risk analysis of products placed on the market until the end of shelf life and beyond. This enables particularly sustainable but also cost-saving production (Witkowski et al., 2022; Kaveh et al., 2023).

The rapid global movement of goods, a defining feature of globalization, has further emphasized the need for stable products. This has driven the demand for technological advancements to combat microbial spoilage and contamination by food-pathogenic microorganisms. Starter and protective cultures play a critical role in enhancing food stability and shelf life, acting as biological preservatives by outcompeting spoilage microorganisms and pathogens. It is evident that they constitute a pivotal element within the framework of hurdle technology, thereby serving as a substitute for chemical preservatives (Kruse, 1999; Fischer and Titgemeyer, 2023).

In addition to extending shelf life to reduce food waste, lowering meat consumption has emerged as a critical strategy for cutting CO₂ emissions. Animal product production is particularly carbon-intensive, driving increased efforts to develop plant-based and cultured meat alternatives. The growing market for these products is especially notable, with plant-based alternatives projected to capture 25% of the market and cultured meat 35% by 2040 (Statista, https://de.statista.com/outlook/cmo/lebensmittel/fleisch/weltweit#umsatz; Gerhardt et al., 2019).

Regulatory progress is also underway: approvals for the sale of cultured meat have already been granted in Israel, the UK, Singapore, Australia, New Zealand, and the US (Supplement Table S1). In Europe, the European Food Safety Authority (EFSA) has received its first four applications for the approval of cultured meat products, including beef, beef fat, foie gras, and hot dogs (GFI, https://gfieurope.org/wp-content/uploads/2023/05/GFI-Europe-Sustainable-Proteins-in-Germany-Summary-EN.pdf; transgen.de, https://www.transgen.de/lebensmittel/2700.fleisch-zellkultur-biotechnologie.html).

Currently, the distribution of cultured meat products is still extremely limited, while plant-based meat alternatives (PBMAs) are available in almost all supermarkets (Gerhardt et al., 2019). Given the increasing market relevance of PBMAs, the potential application of protective cultures in this area is a topic of interest.

Despite extensive research into the use of protective and starter cultures in meat and processed meat products, the knowledge and application of protective cultures in plant-based alternatives and cultured meat remain limited (Chr. Hansen, 2024; M-Food, https://www.meatcracks.de/media/m-cultures_meat_de_ex.pdf). Consequently, the use of raw materials and additives, production technologies, and the market landscape in this sector are highly dynamic. It is especially important to consider the matrix, the microbiome, and the manufacturing processes when using protective cultures in plant-based meat alternatives. (Margerin, 2018). To date, no published studies have investigated the use or effectiveness of protective cultures in PBMA.

Since cultured meat is not yet widely available and is expected to behave similarly to conventional meat, this study focuses on heated meat products and their plant-based alternatives (Habowski & Sant’Ana, 2024). This review seeks to highlight the potential of protective cultures and bacteriocins, illustrate the diversity of existing research, and identify knowledge gaps related to cooked meat products and the rapidly growing market for plant-based alternatives.

By undertaking this approach, it is possible to gain valuable insights and establish key areas that require further investigation.

2. Systematic Literature Review – Workflow

The main question aims to clarify the current scientific and economic status of protective cultures and bacteriocins for cooked meat products and their plant-based alternatives. In particular, the influence of protective cultures and bacteriocins on product safety, quality, and longer shelf life was examined. Further research also addressed microbial diversity, the safety of protective cultures, and technological information on the production of meat-based products and plant-based alternatives (Supplement Figure S1).

For primary web-based research, Scopus, PubMed and Web of Science were prioritized, to access systematic reviews or bibliometric analyses. Google Scholar research was applied to achieve broader coverage that includes grey literature, preprints, and conference papers. As secondary sources, ScienceDirect, SpringerLink, and Wiley Online Library provide full-text access to a wide range of articles and book chapters. Legal texts and guidelines and outbreaks (n=26) from the relevant authorities were also reflected. Websites and guidelines of manufacturers of protective cultures and trade fair reports (n=6) and market analyses (n=6) were ultimately considered in the review process.

When developing search strings, we applied Boolean operators AND and OR, to refine the search and capture all relevant variations of keywords. After an initial search the number of articles was 1,320. After removing duplicates, 1,108 studies remained. The aim of the studies and the conclusions of the literature references were examined, and studies that were not related to starter cultures, protective cultures, meat, and plant-based products were excluded (n = 875). After this step, 233 studies remained. In the next step, studies on raw fermented products and/or starter cultures were examined in more detail with regard to their thematic relevance for this review. In the next step, studies for starter and fermented products were excluded if the cultures tested in the latter studies had no proven protective effect (remaining studies n=133). As the study design on protective cultures and bacteriocins was very heterogeneous, a meta-analysis was not possible because the results are not statistically comparable. To contribute to the innovative character of the paper, 48 additional scholarly sources were incorporated. These sources addressed topics including general bacterial resistance, encapsulation technologies, mechanical processing and protein technologies, microbial diversity in production environments, cultivated/microbial meat, sustainable food systems, and specialized academic literature.

3. Starter Cultures and Protective Cultures-Definition

Starter and protective cultures are used as single cultures or mixed cultures with defined physiological and technological properties (Bintsis and Athanasoulas, 2015) (Figure 1). The fundamental difference between starter and protective cultures lies in their different technological applications.

Starter cultures are microorganisms intentionally introduced into food to initiate and control fermentation processes. Their application enhances not only the sensory properties of food but also its nutritional value and digestibility (Gänzle et al., 2024). Starter cultures are often used in the production of fermented meat products, including salami, air-dried sausages, and onion sausages. The main function of these enzymes is to accelerate processes such as lowering the pH, thereby influencing the color, texture, and taste of the products and developing their characteristic aroma. In addition, starter cultures have been shown to improve stability, shelf life, and safety (Leroy et al., 2006).

In contrast, protective cultures are microorganisms that suppress the growth of spoilage organisms (e.g., Brochothrix thermosphacta) and foodborne pathogens (e.g., Salmonella and Listeria monocytogenes) by the production of antimicrobial compounds (Garín-Murguialday et al., 2026; García-Díez and Saraiva, 2021).

The use of protective cultures is a practice applied with the intention of extending shelf life, increasing safety, and reducing spoilage. This phenomenon occurs through three different mechanisms: the synthesis of organic acids that lower the pH-value, the production of bacteriocins (Figure 2), and competing directly with spoilage organisms for nutrients, which can lead to the displacement (Ben Said et al., 2019; Xu et al., 2022).

Protective cultures include specific strains of lactic acid bacteria (LAB), such as Lactococcus lactis (Lc.), Lactobacillus sakei (Lb.), Lb. plantarum, Lb. paracasei, Lb. rhamnosus, and Lb. brevis, as well as Enterococcus faecium (Ec.), Carnobacterium, Bifidobacterium, and Propionibacterium. The use of protective cultures has proven to be an important clean label strategy that offers an alternative to E-number preservatives (Bintsis and Papademas, 2024; Garín-Murgialday et al., 2026; Anumudu et al., 2024; Cocolin, 2025; Karbowiak, 2023).

Protective cultures are also used in ready meals to naturally counteract microbial spoilage and preserve freshness. Spraying protective cultures onto individual sausage slices between cutting and packaging, a critical moment for microbial recontamination, offers innovative new applications (Møller and Zuliani, 2024).

Customized protective or starter cultures and their bacteriocins are used to reduce nitrite concentrations in sausage products. Some strains are also reported to have health-promoting properties, including probiotic potential (Chapter 3.2.) (Petka and Walczycka, 2026). The most commonly used starter and protective cultures in food are provided in Figure 1 (Ammor and Mayo, 2007) (Figure 1).

3.1. Research Focus on Protective Cultures for Heated Meat Products

A comprehensive review of the literature on protective cultures in meat products identified 30 potential cultures or strain combinations that were specifically selected for their antimicrobial properties. Twenty-six of these strains (87%) demonstrated a significant inhibitory effect on target organisms (Table 1). Most studies prioritized the efficacy of protective cultures against L. monocytogenes, the most critical pathogen in ready-to-eat (RTE) meat products. Several investigations employed non-pathogenic surrogate strains to model L. monocytogenes behavior.

L. innocua and L. ivanovii were used due to their physiological similarities to L. monocytogenes and because they pose no direct human health risk (Danielski et al., 2020; Jacobsen et al., 2003; Héquet et al., 2007). While extensive research has been conducted on pathogens, the available literature on spoilage microorganisms remains limited, focusing primarily on B. thermosphacta, a key spoilage organism in refrigerated meat products (Figure 3, Table 1). Other significant spoilage bacteria, such as Pseudomonas, Lactobacillus, and Enterobacteriaceae, as well as fungi, including molds and yeasts, have received comparatively little attention, despite their impact on shelf life and sensory quality.

With the exception of two studies focusing on Pseudomonas and Escherichia coli (E. coli), the target organisms were exclusively Gram-positive bacteria. One potential explanation for this trend is the displacement of Gram-negative bacteria, such as Pseudomonas, by Gram-positive bacteria, such as Latilactobacillus, during the product’s shelf life. Gram-positive bacteria are known to exhibit greater tolerance to lower water activity and/or lower pH (Barcenilla et al., 2024). Additionally, recent studies have identified yeasts as contributors to spoilage, as they were inhibited but not fully eliminated in cooked ham by protective cultures (Palacio et al., 2025).

The dosage of protective cultures is critical and must be tailored to the timing of application (pre- vs. post-heating) and the specific challenges of the meat product (e.g., pH, water activity, competing microbiota). In studies where Lc. lactis DSM 20729, Pediococcus acidilactici LMQS 154 (P.), P. pentosaceus DSM 3313, and Lb. curvatus ACU-1 failed to achieve the desired reduction, this may be attributed to the low initial concentration of the protective cultures used (Bungenstock et al., 2021). The concentration of the protective cultures used should be between 10⁵ log and 10⁷ CFU/g when administered after heating and between 10⁸ and 10¹⁰ CFU/g when administered before heating (Jacobsen et al., 2003; Bredholt et al, 2001; Kotzekidou and Bloukas, 1996).

A review of studies on protective cultures revealed that Berry et al. (1990), Jacobsen et al. (2003), and Kotzekidou and Bloukas (1996) examined their effects prior to heating. In the remaining studies, the cultures were applied exclusively to the surface after heating (Table 1 and Figure 2).

Protective cultures can be applied to sausage meat prior to heating during the emulsification process, brine injection, or tumbling of the ham. Jacobsen et al. (2003) argue that incorporating protective cultures prior to heating is not only advantageous from a process engineering standpoint but also effective in combating Listeria. An innovative method for adding protective cultures before heat treatment is encapsulation. However, it is critical that the capsules containing these cultures do not fully survive the heating process, allowing the protective cultures to proliferate in the final product. A variety of stimuli—such as temperature, pH, mechanical stress, and osmotic pressure fluctuations—have been shown to trigger the release of encapsulated microorganisms. Nevertheless, the efficacy of this approach in heated meat products remains unproven and has, to date, only been demonstrated in high-pressure-treated products (Nedumpilly Puthenveedu Haridas and Bhat, 2026; Anal and Singh, 2007; Lemay et al., 2002; McGillin et al., 2022).

The efficacy of protective cultures can be enhanced by combining them with bacteriocins or by using multiple protective cultures together in products like cooked ham. This synergistic effect has been demonstrated in both heated meat products and vacuum-packed lamb (Gao et al., 2015; Xu et al., 2022). When protective cultures and bacteriocins are used in tandem, the bacteriocin initially inhibits the growth of undesirable bacteria, providing a window for the protective culture to reach a sufficient cell density to effectively suppress the target bacteria. (Schillinger et al., 2001).

Certain microbial communities—comprising environmental, spoilage, and pathogenic organisms—are increasingly adapting to production environments, posing a risk of contaminating food and food ingredients. This adaptation has contributed to a notable rise in the prevalence of Shiga toxin-producing E. coli (STEC), Listeria monocytogenes, Salmonella, and Campylobacter across several European countries (Omelchenko et al., 2026; ECDC: https://www.ecdc.europa.eu/en/food-and-waterborne-diseases-and-zoonoses/surveillance-and-disease-data). Therefore, epidemiological data should be considered when selecting target pathogenic strains. In particular, strains of the clonal complex (CC) of lineage II are particularly common in food. For example, CC 121 and CC9 are known to be persistent in food processing environments (EFSA, 2024). Additionally, data obtained from a single reference strain cannot be generalized to the entire species or even to a specific genetic lineage. Consequently, efficacy tests should always include multiple strains, ideally incorporating field isolates to ensure broader applicability (Gutiérrez et al., 2025).

A review of the actual research reveals several critical gaps:

• Although B. thermosphacta has been the focus of extensive research, other spoilage-causing bacteria, particularly LAB, have received comparatively less attention, despite their substantial impact on sensory deterioration and shelf-life reduction.
• The application of encapsulated protective cultures in heated meat products has been explored in a single study. Consequently, there is an imperative for further research to assess the feasibility, stability, and efficacy of this approach within industrial contexts.
• While the synergistic effects of mixed protective cultures show potential, this strategy is seldom explored. Future studies should prioritize multi-strain combinations to enhance antimicrobial spectra and functional benefits.
• The selection of target organisms for protective cultures should be guided by epidemiological data and a risk-based approach, considering both the prevalence and virulence of pathogens in specific meat products.

3.2. Biological Safety of Starter and Protective Cultures

LAB and their bacteriocins are generally recognized as safe (GRAS) and offer beneficial properties, including antimicrobial, antioxidant, antihypertensive, immunomodulatory, anti-inflammatory, and potential anticancer effects (Carneiro et al., 2024; Castellano et al., 2017). However, careful evaluation of health-related biosafety factors remains essential when selecting them as protective cultures (Carneiro et al., 2024; Castellano et al., 2017).

From a legal perspective, only microbial strains that have been rigorously tested and approved by the European Food Safety Authority (EFSA; https://www.efsa.europa.eu/en/topics/topic/qualified-presumption-safety-qps) or classified as “Generally Recognized as Safe” by the US Food and Drug Administration (FDA) (GRAS; https://www.fda.gov/food/food-ingredients-packaging), may be used as starter or protective cultures in food production.

These approvals guarantee the safety, characterization, and effectiveness of the strains for improving food quality, safety, and preservation (Supplementary Table S2). Regarding product labeling, EU legislation (Regulation (EU) No. 1169/2011) exempts ingredients from being listed if they do not perform a technological function in the final product—such as when they are reduced to natural levels during thermal processing (European Parliament, 2011). However, if the cultures remain active in the final product (as is typically the case with protective cultures), they must be declared on the label.

Additionally, Regulation (EC) No. 852/2004 mandates the verification of food safety measures (European Parliament, 2004). Consequently, every batch of protective cultures produced is tested for bacteriocin production to ensure compliance (AVO, https://www.avo.de/).

Bacteriocin-producing microorganisms, including LAB and other taxa, along with their bacteriocins, show great potential as protective cultures by providing antimicrobial, probiotic, and health-promoting effects. However, the implementation of these technologies necessitates a meticulous evaluation of biosafety concerns. LAB have been observed to harbor antibiotic resistance genes, which have the potential to transfer to pathogens. Furthermore, certain strains of LAB have been found to produce biogenic amines in fermented foods. To ensure safety, strain-specific assessments must be conducted prior to their use. These assessments include testing for minimum inhibitory concentrations (MIC) of key antibiotics and screening for biogenic amine production and virulence factors (Mathur and Singh, 2005; Plavec and Berlec, 2020; D’Silva, 2011). If these risks, costs, and technological challenges—such as maintaining the stability and activity of postbiotic compounds (consist of metabolic byproducts, cell components, or secreted factors that provide health benefits) during food processing—can be addressed, LAB have the potential to enhance food safety, extend shelf life, and improve nutritional and health-promoting properties. The establishment of clear regulatory guidelines could further facilitate their safe and effective integration into food production (Carneiro et al., 2024; Trymers et al., 2026; Anumudu et al., 2024).

3.3. Bacteriocins in Heated Meat Products

Bacteriocins are a heterogeneous group of antimicrobial peptides produced by various protective culture strains (Sugrue et al., 2024). The focus of scientific research is predominantly on bacteriocins produced by LAB, Bacillus or Staphylococcus pasteuri (S.). Bacteriocins demonstrate efficacy even at low concentrations, exhibit high heat stability, maintain stability at low pH values, and do not affect the nutritional properties of food (Amiri et al., 2021; Zendo, 2013; To et al., 2022; Hong et al., 2018).

Bacteriocins are classified into four classes and nine subcategories. A significant distinction is made between post-translationally modified peptides (class Ia) and unmodified peptides (class II) (Favaro et al., 2015; Castellano et al., 2017) (Figure 2). Table 2 provides a comprehensive overview of the bacteriocins utilized in research on meat products. The most prevalent strains were those capable of producing sakacin, followed by those capable of producing pediocin and enterocin (Table 2).

Bacteriozin AS-48, produced by Ec. faecalis subsp. liquefaciens, has been identified as an effective solution for addressing spoilage LAB, B. thermosphacta, and S. carnosus (Parada-Fabían et al., 2025; Baños et al., 2012). The application of a bacteriocin mixture produced by Lb. curvatus, Lc. lactis, P. acidilactici, and Enterococci appeared to be promising as evidenced by the reduction of L. monocytogenes on hot dog sausages (Vijayakumar and Muriana, 2017). It has been demonstrated that Micocin® (a mixture of low-molecular-weight, ribosomally synthesized, antibacterial peptides produced by E. coli) is effective against Clostridium botulinum (Cl.) and L. monocytogenes (Parada-Fabían et al., 2025).

Gao et al. (2015) demonstrated that the combination of bacteriocin and a protective culture (sakacin C and Lb. sakei C2) exhibited a greater degree of effectiveness against L. monocytogenes than either the protective culture or the bacteriocin alone in vacuum-cooked ham.

Just as with protective cultures, the concentration of bacteriocins must be carefully determined to ensure effectiveness. For example, a study found that nisin concentrations of 12.5 mg/kg failed to inhibit Listeria growth, whereas 32 mg/kg demonstrated an inhibitory effect (Laranja et al., 2024). The established correlation between bacteriocin production and optimal cell growth suggests the possibility that not all bacteriocin-producing strains present in specific food matrices actively secrete bacteriocins. The optimal conditions for the formation of bacteriocins for product preservation are contingent upon environmental factors such as temperature, salt concentration, pH, and packaging atmosphere (Yang et al., 2018). The technological processing steps or other recipe components, such as brine, have been demonstrated to exert antagonistic effects (Laranja et al., 2024). Furthermore, it has been demonstrated that bacteriocin resistance, such as nisin resistance in L. monocytogenes, increases with increasing NaCl exposure. In addition to environmental factors, genetic background also plays a decisive role (Bergholz et al., 2013; Malekmohammadi et al., 2017). The resistance mechanisms described are mediated by efflux pumps and regulatory systems (VirAB, VirSR, and the multicomponent system AnrAB). Such common mechanisms can cause co-resistance to nisin, ciprofloxacin, ethidium bromide, bacitracin, cephalosporins, and benzalkonium chloride (Jiang et al., 2019; Rakic-Martinez et al., 2011).

Resistance mechanisms in bacteria, such as those mediated by efflux pumps (e.g., VirAB) and regulatory systems (e.g., VirSR, AnrAB), can lead to cross-resistance not only to bacteriocins like nisin but also to a range of antimicrobials, including ciprofloxacin, ethidium bromide, bacitracin, cephalosporins, and benzalkonium chloride (Jiang et al., 2019; Rakic-Martinez et al., 2011). Beyond genetic co-resistance, exposure to benzalkonium chloride—a common disinfectant in food processing—may further promote co-selection. This is supported by the frequent co-localization of benzalkonium chloride resistance genes (e.g., bcrABC, emrC) with genes conferring acquired antibiotic resistance (Yu et al., 2018; Kawacka and Olejnik-Schmidt, 2024; Moura et al., 2024).

The gadD3 gene (part of the GAD system) and mutations in mpt/manR contribute to resistance against nisin and class IIa bacteriocins (Lakicevic et al., 2022; Gravesen et al., 2002; Zawiasa and Olejnik-Schmidt, 2025). Resistance also depends on clonal complex (CC), with SSI-1 (in CC3, CC5, CC7, CC9) enhancing resistance under salt stress (Malekmohammadi et al., 2017). Additional mutations (e.g., RsbU G77S, PBPB3 V240F) in CC7 and CC121 further increase resistance and may boost virulence (Wambui et al., 2020; Centorotola et al., 2021). CC2, CC3, CC6, CC7, CC14, CC199, and CC403 show particularly high resistance, emphasizing the role of genetic background (Malekmohammadi et al., 2017; Wambui et al., 2020).

However, the inherent resistance of L. monocytogenes to nisin significantly restricts its effectiveness, necessitating its application in conjunction with other additives or technological measures (Wu et al., 2023). Nevertheless, combination strategies should not be based on the simultaneous use of other bacteriocins. This recommendation is substantiated by the existence of cross-resistance mechanisms, whereby strains that are resistant to nisin are frequently also resistant to class IIa bacteriocins (e.g., pediocin), and vice versa (Gravesen et al., 2002b). The effectiveness of bacteriocins is heavily influenced by the food matrix. For instance, Danielski et al. (2020) found that C. maltaromaticum produced only minimal bacteriocin levels in cooked ham, likely due to limitations imposed by the food matrix. Similarly, the antimicrobial activity of Ec. faecium bacteriocins can vary significantly depending on the food type, processing conditions, and composition.

While the efficacy of Ec. faecium bacteriocins has been well-documented in cheeses and fermented soybeans, their performance in processed meats and other complex foods has proven less consistent (Ben Braiek & Smaoui, 2019).

The bio conservative effect of bacteriocins in meat and meat products is currently only partially exploited commercially, with nisin and pediocin PA-1 being commercially available for food preservation. The prevailing legal framework in both the US and the EU presently permits the utilization of nisin (E 243) exclusively as a pure bacteriocin (Barcenilla et al., 2022; European Parliament, 2008; Annex II of EU Regulation 1333/2008; Food & Drug Administration (FDA), https://www.law.cornell.edu/cfr/text/21/184.1538; Wu et al., 2023). In the United States, certain pediocin-based preparations have received regulatory approval (FDA, https://downloads.regulations.gov/FDA-2025-N-1927-0046/attachment_1.pdf).

In order to achieve the full potential of bacteriocins, several challenges must be overcome. These objectives include the optimization of methods for identifying bacteriocin genic strains and the validation of their activity in situ, as well as the coordination of political and regulatory processes to establish harmonized frameworks that facilitate the appropriate and efficient utilization of bacteriocin-producing strains and bacteriocins (Cocolin, 2025; Castellano, 2023). Presently, bacteriocins are required to meet specific approval criteria, encompassing proper identification, characterization of their chemical composition, safety assessment, and indication of the recommended application concentration. Despite the challenges associated with large-scale application, bacteriocins have significant potential for improving food safety and extending shelf life (Parada-Fabían et al., 2025).

3.4. Application of Protective Cultures in Pbmas

Chen et al. (2025), Lindberg et al. (2025), and Xie et al. (2024) have conducted systematic reviews examining the environmental, processing, nutritional, health, and future perspectives of plant-based meat alternatives (PBMAs), while Munialo et al. (2025) provide a comprehensive overview of meat substitutes, including protein sources, quality characteristics, analytical methods, and consumer perceptions. However, the application of protective cultures in PBMAs has yet to be explored in scientific detail. Key challenges and future directions involve selecting the right protective culture, ensuring its feasibility for industrial implementation, and combining these cultures with emerging protein sources to create safe, high-quality food products (Schreuders et al., 2021).

A promising approach in extruded PBMAs involved a combination of 1% chitosan, 2.5% tea polyphenols, and 0.04% nisin as an alternative to conventional preservatives, which had a shelf life four times longer than the untreated control samples (Dai et al., 2022).

In the field of flaxseed oil cake–based plant milk alternatives for the development of innovative probiotic beverages, the incorporation of Lb. rhamnosus in combination with sucrose-driven acidification has been shown to effectively inhibit pathogenic microorganisms such as Bacillus cereus (Bc.) (Łopusiewicz et al., 2021). Lb. rhamnosus has been demonstrated to exert a protective effect against E. coli 0157:H7 in the context of fermented raw sausages (Erkkilä et al. 2000).

Elhalis et al. (2023) provide a comprehensive overview of the pivotal factors influencing the development of protective cultures in PBMAs. Key aspects that should be considered include the ability to displace the natural microflora, the detoxification of mycotoxins and biogenic amines, cost-effectiveness, and processing and storage stability. The cultures must demonstrate resilience to rigorous processing techniques, such as extrusion, while enhancing flavor, texture, and safety attributes. The current gaps in the field include limited strain-specific data for plant-based proteins (soy, peas, mycoprotein), as well as challenges related to scalability.

Starter and protective cultures often overlap in terms of their species and functions, so factors such as adaptability, detoxification, and stability apply to both. An example is tofu, a traditional soy-based meat alternative in which nisin, in combination with protective cultures (Ec. faecium BFE 900-6a or Lc. lactis BFE 902) L. monocytogenes completely inhibit (Malav et al., 2015). Nisin initially suppresses Listeria until the protective cultures reached a sufficient density to take over long-term inhibition (Schillinger et al., 2001). This approach demonstrates how hurdle technology—the combination of bacteriocins and protective cultures—can enhance the safety of plant-based meat alternatives. The principle could be applied to extruded or moisture-rich plant-based products to extend shelf life and improve safety without relying on synthetic preservatives.

4. Process Differences Between Heated Meat Products (Hmp) and Pbmas

To select the optimal protective culture, the manufacturing process must first be described (Figure 4), as well as the process step in which the culture is to be used (Chapter 3.1). The type of packaging is important, as products are sold either in slices or whole, and either vacuum-packed or under a modified atmosphere, depending on market requirements. The added protective culture must have an advantage over competing flora in the respective packaging (Castellano et al., 2017). When using protective cultures, it is essential to check their compatibility with starter cultures used at the same time (e.g., S. xylosus, Lb. sakei) to avoid unintended inhibition. Pilot-scale trials or in vitro co-culture tests are recommended prior to large-scale application (Laranjo et al., 2019).

4.1. Emulsion Type Sausage

Cooked sausages are food products made from a mixture of meat, fat, water, salt, spices, and additives, with fat distributed within a homogenized protein matrix (Peña-Saldarriaga et al., 2020). They can be classified based on raw materials, additives, degree of mincing, size, and consumption method (Bahrami et al., 2023).

The production process typically involves grinding ingredients into a fine mixture (sausage meat or emulsion), facilitated by the breakdown of sarcomeres and activation of muscle proteins like myosin and actin, which act as emulsifiers. Salt (NaCl) enhances protein unfolding by increasing electrostatic repulsion, allowing proteins to stabilize lipid and aqueous phases (Feiner, 2006; Schilling, 2019). The mixture is then stuffed into natural or artificial casings (e.g., collagen, cellulose, plastic, or co-extruded alginate-collagen hybrids) (Suurs & Barbut, 2020).

Heat treatment (>65 °C) denatures proteins, converting the emulsion into a solid gel (Ghebremedhin et al., 2022; Dickinson & Chen, 1999). After heating, cooling stabilizes the product microbiologically (Ryland et al., 2006) (Figure 4).

4.2. Cooked Ham

In traditional cooked ham production, whole legs are used, though modern processes may employ alternative cuts or smaller meat pieces (Tomović et al., 2022). The process begins with brine injection (containing sodium chloride, nitrite, spices, phosphates, salts, ascorbate, and sugar) via a needle injector (Casiraghi et al., 2007). This is followed by tumbling, where meat is lifted and dropped in a rotating drum, converting kinetic energy into potential energy. The combination of drop height and force improves tenderness, water-holding capacity, and yield (Pietrasik & Shand, 2005; Cassidy et al., 1978). Tumbling also disrupts meat cells, releasing myofibrillar proteins that act as natural binders (Siegel et al., 1978), while LAB counts increase by up to 3 log CFU/g (Veselá et al., 2022).

The ham is then heated to a core temperature of 70 °C ± 5 °C, reducing bacterial populations by ~50%, though heat-stable species may reappear after a latency phase (Mustedanagic et al., 2023; Zagdoun et al., 2020). As with cooked sausages, rapid cooling follows heating to stabilize microorganisms and prevent regrowth (Ryland et al., 2006).

4.3. Plant-Based Sausage Processing

Plant-based proteins that share a structural similarity with myofibrillar meat proteins are crucial for creating meat-like textures in meat alternatives (Supplement Table S3). Common sources include soy, peas, chickpeas, lentils, and mushrooms, which are used in textured (e.g., TVP, extrudates) or non-textured forms (e.g., concentrates) to mimic muscle fibers (Schilling, 2019).

Vegetable oils (rapeseed, coconut, palm) replace animal fats, while thickening agents (carrageenan, xanthan gum, methylcellulose) stabilize emulsions and improve moisture content and mouthfeel (Mazumder et al., 2023; Kyriakopoulou et al., 2021; Malav et al., 2015). The addition of ingredients such as wheat gluten or pectin has been shown to result in a fibrous texture (Schreuders et al., 2019). Edible mushrooms (e.g., Pleurotus ostreatus, Lentinula edodes) offer umami flavor and fibrous texture. Genetic modifications (e.g., enhanced protein expression or reduced bitterness) could optimize their scalability and sensory profile for meat substitutes (Maini Rekdal et al., 2024; Malav et al., 2015). Research initiatives, nascent businesses and established corporations are engaged in the development of alternative protein sources as meat substitutes on a microbiological basis. These include bacteria, microalgae, fungi, and yeasts (Cardoso Alves et al. 2023).

The texture of sausage products poses a particular challenge therefore, extrusion and/or fermentation of plant-based raw materials is an important step toward improving chewability in the production of PBMAs (Maung et al., 2020). The extrusion of pea proteins at high moisture content and lactic acid fermentation reduce the pea-like odor caused by n-hexanal (Valtonen et al., 2023; Schindler et al., 2012). The key parameters for extrusion are the moisture content of the protein mass and the cylinder temperature (Sun et al., 2022). Shear cell technology can serve as an alternative with similar critical variables (temperature, shear duration, pressure, and the resting phase after heating) for achieving a meat-like structure (Manski et al., 2007; Sha and Xiong, 2020).

Fermentation of PBMAs improves their nutritional value and organoleptic properties, such as reducing the bitterness and increasing the juiciness of soy proteins (Razavizadeh et al., 2022; Boukid et al., 2023). Bc. subtilis improves the fermentation of extruded plant-based proteins, thereby enhancing the product’s texture and nutritional value (Maung et al., 2020). Off-flavors that may occur are associated with an earthy, bitter, astringent, or bean-like taste. To counteract these deviations, the fermentation of plant matrices with LABs is employed (Erem and Kilic-Akyilmaz, 2024).

Lb. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lb. plantarum, and Lb. fermentum, play a crucial role in improving plant-based ingredients like field beans, hemp, and soybeans by addressing both anti-nutritional compounds and off-flavors. Lb. delbrueckii subsp. bulgaricus, Lb. plantarum and the animal-derived Lb. acidophilus significantly reduce hexanal and hexanoic acid, which are responsible for undesirable grassy or rancid notes, thereby enhancing the overall flavor profile of plant extracts (Monica et al., 2025). The microbial metabolism of LAB degrades antinutritional factors including phytates, which bind essential minerals such as iron and zinc and reduce their bioavailability, phenolic compounds contributing to bitterness and astringency, oxalates that impair mineral absorption, raffinose-family oligosaccharides causing digestive discomfort, and saponins with bitter taste and potential intestinal irritation Additionally, enzymatic activities such as α-galactosidase, phytase, amylases, lipases, and proteases enhance nutrient bioavailability, digestibility, and the release of bioactive compounds (Molina et al., 2025; van de Velde et al., 2026).

The subsequent filling of the PBMAs can be performed using a conventional sausage-filling machine into a casing made of cellulose, plastic, or alginate (Suurs and Barbut, 2020; Valtonen et al., 2023). The heating process leads to denaturation and changes in protein folding (Ghebremedhin et al., 2022). To prevent the germination of heat-stable spore-forming bacteria such as Bacillus spp. and Clostridium spp. after heating, an additional heating step for decontamination is often required (Wild et al., 2014; Kabisch et al., 2023). Heat treatment at 120 °C ± 2 °C for 30 minutes has been demonstrated to yield comparable microbial inactivation and structural modifications in both animal-derived and plant-based food matrices, ensuring effective pathogen reduction and product stability (Yadav et al., 2015). However, the development of thermal processing protocols for novel plant-based products necessitates product-specific optimization to address their distinct physicochemical and sensory attributes.

A critical aspect of this process is the establishment of customized thermal profiles that maintain functional properties such as protein denaturation, moisture retention, and texture, while accounting for variations in protein sources and lipid substitutes. Furthermore, post-thermal pasteurization in final packaging, particularly for high-moisture products like plant-based deli slices, is imperative to ensure extended shelf life without compromising organoleptic or nutritional quality (Kong et al. 2026).

The optimization of protective culture incorporation, before or after heating, is important to prevent thermal inactivation or sublethal injury, which could compromise their competitive efficacy. Moreover, the rapid post-thermal cooling is a critical step in minimizing microbial proliferation and preserving structural integrity, in accordance with conventional meat processing standards (Wild et al., 2014; Kabisch et al., 2023).

4.4. Plant-Based Products Available on the Market

The production of PBMA is currently confronted with numerous challenges, including the optimization of flavor, the comparison of nutritional value to minimally processed foods, concerns regarding chemical safety, and social and economic considerations (He et al., 2020). Notwithstanding these challenges, a diverse array of meat and sausage substitutes is currently available in retail establishments and restaurants, encompassing sausages, meat-like products, and ready-to-eat meals (see Supplementary Tables S1 and S3). These products are manufactured on a global scale by traditional meat producers, newly established startups, and specialized subsidiaries. By 2022, GFI Europe reported that over 1,500 companies were engaged in PBMA production (GFI Europe, 2023). For decades, soy protein was the cornerstone of plant-based meat alternatives, but the industry has since expanded to include a diverse range of protein sources, each selected for its sustainability, functional properties, and nutritional benefits. In addition to soy, pea protein has gained prominence due to its high protein content, neutral taste, and suitability for cultivation in temperate climates (Schreuders et al., 2019; EUVEPRO, 2019). Other emerging protein sources that now play a key role include: lentil and chickpea protein, valued for their high fiber content, essential amino acids, and low allergenicity (Boukid et al., 2023). Wheat gluten, which provides elasticity and binding properties that are crucial for achieving the fibrous texture characteristic of plant-based meat alternatives made from whole pieces (“chunks”) (Kyriakopoulou et al., 2021). Rice and potato protein, which is often used as a hypoallergenic alternative and offers clean flavor profiles as well as complementary amino acid profiles when blended with legume proteins (Dimina, 2022). Mycoprotein, which offers a meat-like fibrous structure and high-quality protein, although its fermentation process and potential allergenicity require careful management (Finnigan et al., 2019). Algae-based proteins (e.g., spirulina, chlorella), which offer high protein content, essential fatty acids, and pigments for natural coloring, though challenges regarding scalability and cost-efficiency remain (Bleakley and Hayes, 2017). Sunflower and rapeseed protein, which is establishing itself in Europe as a sustainable, locally sourced option, with research continuing to improve its functional properties (Schwab et al., 2020).

5. Microbiology of Plant-Based Products

Despite the multiple advances of plant-based products, critical knowledge gaps remain, particularly regarding the microbial safety and spoilage potential of these newer protein sources. The initially low bacterial count in PBMAs is primarily attributable to the extrusion process of the raw materials (Dogan et al., 2025; Liu et al., 2023). The accelerated growth can be attributed to the specific physicochemical properties that currently favor the survival and proliferation of microorganisms in plant-based products. These include, in particular, a nearly neutral pH value (which is traditionally somewhat lower in sausage products), a high-water activity (a_W), and a high protein and moisture content (Martín-Miguélez et al., 2025).

The limited research on their microbial flora is partly due to their recent market introduction, leaving significant uncertainties regarding resistance to pathogens, spore-forming bacteria, and optimal inactivation methods (Kabisch et al., 2023; Geeraerts et al., 2020).

To address these challenges, targeted microbial characterization is essential, focusing not only on the total plate count but also on spore-forming bacteria and heat-resistant pathogens that can compromise product safety and shelf life (Kyrylenko et al., 2023). The development of tailored processing techniques—such as optimized heat treatments, fermentation protocols, or natural preservation systems—will be crucial to ensuring the safety, stability, and consumer acceptance of these innovative protein sources. There is an urgent need for research to address these gaps and establish scientifically sound safety standards for the next generation of plant-based foods.

5.1. Microbiological Diversity of Pbmas

The level of microbial contamination in plant-based protein raw materials—even among raw materials of the same type—can vary significantly, influenced by a complex interplay of various factors along the supply and processing chain. These factors include geographic origin, environmental conditions (e.g., temperature, humidity, and precipitation during cultivation), agricultural practices (e.g., use of pesticides and fertilizers), harvesting techniques, storage conditions, transport logistics, and subsequent processing methods (Kyrylenko et al., 2023). While plant-based protein raw materials are generally well-suited for PBMAs due to their functional and nutritional properties, their microbial profiles reveal both opportunities and challenges. Enterobacteriaceae (e.g., E. coli, Salmonella spp., Klebsiella spp., Enterobacter spp.) are frequently present in soil and irrigation water, particularly in organically fertilized fields (Kabisch et al., 2023). Wild animals, livestock, or insects can transmit Enterobacteria to plants via fecal contamination (Geeraerts et al., 2020). High humidity or improper drying can promote bacterial growth, particularly in high-moisture ingredients (e.g., pea protein concentrates, soy flour) (Wild et al., 2014). Enterobacteria can be introduced by harvesting machinery and also during processing, such as grinding, extrusion, or mixing, if the equipment or processing environment is not adequately disinfected. Poor-quality process water (e.g., for washing or rehydration) can lead to recontamination (Kyrylenko et al., 2023; ILSI, https://ilsi.eu/publication/the-enterobacteriaceae-and-their-significance-to-the-food-industry/).

Salmonella, as a foodborne pathogen, is relevant in the risk analysis of PBMAs because both contaminated raw materials play a role and the pathogen exhibits high tenacity and survivability in low-moisture foods (LMF). Recalls occurred for plant-based chicken meat alternatives, soy-based meat substitutes, cashew cheese and wild berry diet supplements (https://www.fsis.usda.gov/recalls; https://www.fda.gov/food/outbreaks-foodborne-illness/outbreak-investigation-salmonella-jules-cashew-brie-april-2021; https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts/superfoods-inc-dba-live-it-recalls-live-it-super-greens-because-possible-health-risk).

Salmonella and Cronobacter sakazakii exhibited similar growth and survival rates when compared in meat juices (from plant-based and animal-based products). Furthermore, L. monocytogenes showed a higher survival rate in plant-based meat substitutes (Bonaldo et al., 2024). L. monocytogenes, persistent in nature and food processing environments is able to grow at refrigeration temperatures, posing a risk in RTE PBMAs. Recalls occurred with vegan beverages, plant based meat alternatives, vegan organic cheese and a foie gras alternative (https://www.canada.ca/en/public-health/services/public-health-notices/2024/outbreak-listeria-infections-recalled-refrigerated-plant-based-beverages.html; https://www.sciensano.be/en/biblio/bsfm-2023-l-monocytogenes-plant-based-foods-insights-a-recent-foodborne-outbreak-linked-vegan#:~:text=Due%20to%20the%20growing%20demand,monocytogenes%20in%20plant%2Dbased%20foods; https://food.ec.europa.eu/food-safety/rasff_en). A 2022-2024 European listeriosis outbreak was linked to contaminated plant-based, fermented vegan cheeses made from cashews and coconut milk (Leclercq et al., 2024).

Gram-positive bacteria, are usually found in low concentrations (<100 CFU/g), particularly in extruded raw materials (Gräfenhahn and Beyrer, 2024). However, the microbial diversity of plant proteins is often dominated by spore-forming bacteria (e.g. Bc. subtilis, Bc. amyloliquefaciens und Bc. licheniformis or Clostridium spp.), which are known for their heat resistance and ability to survive conventional thermal processing (Kyrylenko et al., 2023). A high prevalence of Cl. botulinum was found in vegetarian sausages, suggesting inadequate heat treatment and a link to the raw materials (Pernu et al., 2020). Bc. cereus was detected in over 80% of PBMAs in the Czech Republic (Dušková et al., 2024).

LAB, which, while often beneficial for fermentation and preservation, can also contribute to unintended acidification or flavor changes if handled improperly (Molina et al., 2025). Enterococci and other facultative anaerobic bacteria, which can survive in oxygen-deprived environments and could pose potential safety risks if pathogenic strains are present (Geeraerts et al., 2020). Leuconostoc mesenteroides (Leuc.) and Lb. sakei, Ec. faecium, and C. divergens appear to be the predominant bacteria in PBMAs (Duthoo et al., 2022; Geeraerts et al., 2020; Roch et al., 2024). A study conducted in Finland showed that Leuc. gelidum, Leuc. gasicomitatum, and Leuc. mesenteroides were the predominant microorganisms in a vegetarian carrot sausage causing spoilage (Vihavainen et al., 2008).

Plant proteins can also harbor a range of other microorganisms, including yeasts and molds, which can proliferate under high humidity or improper storage conditions, leading to spoilage, off-flavors, or the formation of mycotoxins (e.g., species of the genera Aspergillus, Penicillium, and Fusarium) (Lin et al., 2023; Wild et al., 2014).

The presence of these diverse microbial populations requires a multi-faceted approach to microbial control, including optimized thermal and non-thermal processing methods (e.g., high-pressure treatment, pulsed electric fields, or cold plasma) to inactivate heat resistant microbes or spores (Ghazali et al. 2025). Fermentation or biopreservation strategies using protective cultures (e.g., L. plantarum) to suppress spoilage microorganisms and extend shelf life. The presence of molds, yeasts, and Enterobacteriaceae in the final product is largely attributable to inadequate process hygiene during the heating process and less so to the quality of the raw material. This can be used as a hygiene indicator (Tóth et al., 2021). Improved hygiene and disinfection protocols throughout the entire supply chain to minimize contamination risks. Regular microbiological monitoring to identify and contain emerging microbial threats and ensure compliance with food safety standards. Addressing these microbial challenges is critical to improving the safety, quality, and consumer acceptance of PBMAs (Grove, 2024).

Figure 5 provides an overview of the CFU/g values for total bacterial count, LAB, and yeasts/molds in various plant-based products. It is important to note that the values are derived from different studies and that the microbial counts were determined at different time points depending on the study. Consequently, the reported minimum and maximum values were systematically considered.

5.2. Risk of Cross-Contamination During Processing

RTE products, including sausages, are considered particularly susceptible to cross-contamination and recontamination throughout the production and logistics chain (Osaili et al., 2011). Unlike animal proteins, which are predominantly colonized by LAB, plant-based proteins primarily contain spore-forming bacteria (Kyrylenko et al., 2023). It is important to note that PBMAs are often produced in facilities that also process animal proteins (GFI Europe 2023). Given the differing microbiomes of the raw materials used, it is essential to exercise caution and prevent cross-contamination between animal and plant-based proteins. Since PBMAs promote the growth of microorganisms, it is essential to implement strict measures to prevent recontamination of these products after heating (Martín-Miguélez et al., 2025). Awareness of the potential for recontamination is essential, as it arises from interactions with other product types, equipment that comes into contact with products, surfaces, or personnel. In particular, contact between heat-treated and non-heat-treated products, as well as inadequate cleaning procedures, pose a risk (Salvat et al., 1995; Osaili et al., 2011). Given the extensive evidence, it is recommended that production processes for animal and plant-based meat products be strictly separated from one another. As an alternative, there are strong arguments in favor of implementing a staggered production schedule. This should be accompanied by the implementation of comprehensive cleaning and disinfection measures between the two product lines.

5.3. Legal Requirements for Pbmas

The regulatory environment for PBMA remains fragmented and in a state of flux, with only a few standardized guidelines specifically addressing their manufacturing, labeling, or microbiological safety.

Regulation (EU) 2015/2283 (Novel Food Regulation) applies to PBMA containing novel ingredients (e.g., mycoprotein, algae-based proteins, or new plant extracts) that were not consumed in the EU to a significant degree prior to May 1997. Requires pre-market authorization and safety assessments by the European Food Safety Authority (EFSA). Regulation (EC) No. 1829/2003 (GM Food and Feed) governs PBMA derived from genetically modified organisms (GMOs). Mandates traceability, labeling, and risk assessment for GMO-derived ingredients (European Commission, 2003) (Seehafer and Bartels, 2019).

There are no EU-wide microbiological criteria for PBMA. This gap is attributed to their recent market introduction and limited empirical data on microbiological risks (Geeraerts et al., 2020). General Food Law (Regulation (EC) No. 178/2002) applies and requires the safe marketing of food, but does not contain any PBMA-specific limits.

Commission Regulation (EC) No. 2073/2005 (microbiological criteria for foodstuffs) applies to ready-to-eat (RTE) foods, but does not explicitly address PBMA.

There are no EU-wide harmonized definitions for the labeling of “vegan” and “vegetarian.” In Germany, the “Guidelines for Vegan and Vegetarian Foods Resembling Foods of Animal Origin” (German Food Codex Commission, 2024) regulate designations and production characteristics and prohibit misleading claims. The Austrian Food Code (A5, Annex 11) establishes labeling requirements for vegan and vegetarian products that refer to animal-based foods (Federal Ministry of Social Affairs, Health, Care, and Consumer Protection, 2024). In France, a law (2020-699) will prohibit the use of meat-related terms (e.g., “sausage,” “steak”) for plant-based products starting in 2024 (Légifrance, https://www.legifrance.gouv.fr/jorf/id/JORFTEXT000049199307). There are currently no specific DGHM (German Society for Hygiene and Microbiology) warning values exclusively for vegan or PBMA products. The general warning values for foodstuffs (DGHM, 2021; https://www.dghm-richt-warnwerte.de/de) can serve as benchmarks for evaluating the hygienic quality and safety of vegan and plant-based products. These values are not legally binding but provide scientifically grounded thresholds for assessing contamination risks.

The FDA regulates most PBMAs as “food” under the Federal Food, Drug, and Cosmetic Act (FFDCA). The U.S. Department of Agriculture (USDA) oversees meat alternatives that contain egg or dairy ingredients or are marketed as direct meat product substitutes (Atlas, 2020). The FDA’s Bacteriological Analytical Manual (BAM) contains general testing methods for foodborne pathogens, but there are no PBMA-specific limits (https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam).

USDA-(Food Safety and Inspection Service) FSIS guidelines for ready-to-eat foods may apply to cooked PBMA but are not tailored to plant-based products (https://www.fsis.usda.gov/).

6. Current Availability, Market Situation and Market Development of Protective Cultures for Plant-Based and Heated Meat Products

A growing body of empirical evidence, along with prevailing market trends, underscores the increasing importance of plant-based alternatives in various sectors of the food industry, including fruits, vegetables, dairy products, grains, meat, and seafood (Garín-Murguialday et al., 2026).

In 2025, the global market for protective cultures in the food and beverage sector recorded a revenue volume of $1.5 billion. This sector is expected to exhibit a compound annual growth rate (CAGR) of between 4.3% and 23.6% by 2033 and 2035, respectively. The most significant economic market for functional foods is currently Europe (Mordor Intelligence™ – Industry Reports Source, https://www.mordorintelligence.com/industry-reports/starter-cultures-market; Market Data Forecast, https://www.marketdataforecast.com/market-reports/protective-cultures-market). According to forecasts, dairy products will hold the largest market share, estimated at approximately 55% for 2025.The growth drivers for plant-based products are diverse. The ongoing expansion of the market for protein alternatives (which has a compound annual growth rate (CAGR) of 3.68% globally) is a key factor that deserves attention. As a result, the product range is expanding, but plant-based protein alternatives are also highly perishable, necessitating research into methods to improve their shelf life. Furthermore, the adoption of protective cultures is expected to be driven by demand for improved food safety, reduced perishability, and longer shelf life (Choudhury 2025, https://www.futuremarketinsights.com/reports/food-and-beverage-protective-cultures-market). A number of protective cultures for commercial use in PBMAs have been developed in recent years (Table 3). Suppliers offer both new product lines and conventional starter cultures for the production of sausage substitutes. In the plant-based milk products sector, entire product lines of starter cultures have been available for some time (IFF 2024, https://bioscience.iff.com/solutions/food-and-beverages/plant-based-alternatives; Novonesis, https://www.novonesis.com/en/biosolutions/food-and-beverages/plant-based-foods/plant-based-meats).

The protective cultures available on the market for PBMA products are comparable to the LABs used in products of animal origin. The exact strains of the protective culture products available on the market are often not specified, and in published studies they are designated by internal isolate numbers or exclusively by species names, without providing unique identification numbers for strain classification. Examples of such organisms include S. xylosus DD-34 and Lb. sakei (B-2 Safe Pro®) (Kotzekidou and Bloukas 1995; Comi et al., 2016). While LAB are already well-established in PBMA, probiotics and bacteriocin-producing Bacillus spp. are emerging as potential candidates, although their specificity and efficacy in plant-based matrices still need to be further optimized (Novonesis, https://www.novonesis.com/en/biosolutions/food-and-beverages/plant-based-foods/plant-based-meats; M-Food 2024; To et al., 2022).

The use of protective cultures in PBMA products is intended to inhibit the growth of foodborne pathogens and extend shelf life by controlling spoilage microorganisms. In addition, a growing variety of culinary traditions are being utilized to improve the sensory properties of foods, particularly their texture and aroma. Furthermore, it has been demonstrated that LABs exert a probiotic effect, thereby improving the nutritional value of foods, regulating serum cholesterol levels, preventing intestinal infections, strengthening the immune system, and alleviating antibiotic-associated diarrhea (Gareau et al., 2010; Oelschlaeger, 2010; Muhammed et al. 2025).

It is important to recognize that the development of new food cultures depends on meeting numerous conditions and requirements, thereby transforming a purely scientific approach into a marketable product. First, it is essential to ensure comprehensive health and safety measures, such as those required by GRAS or QPS status. Furthermore, it is essential to ensure that no metabolic activity occurs at low temperatures, as this could potentially lead to undesirable sensory effects for the consumer. At the same time, a sufficient quantity of potentially metabolically active bacteria must be maintained throughout the entire duration of cold storage, even in the event of short-term interruptions in the cold chain. Protective cultures must inhibit the growth of toxic-infectious bacteria within a temperature range of at least 7–8 °C and should ideally suppress a broad spectrum of such target organisms. Furthermore, potential food cultures should be characterized with whole-genome sequencing (WGS) and tested for antibiotic resistance as well as biogenic amines (Elsser 2005; Jůzl et al., 2026).

Current developments point toward a shift away from the general use of food cultures toward tailored, matrix-specific development strategies that also reflect the type of An enhanced protective effect of LAB cultures has been observed under vacuum conditions, whereas this effect appears to be less pronounced in modified-atmosphere packaging (Jůzl et al., 2026; Xu et al., 2021).

The global market for protective cultures for heat-treated foods is currently limited, but it is expected that innovation will expand these markets. The main barriers to growth are regulatory and labeling requirements in certain countries (Choudhury, https://www.futuremarketinsights.com/reports/food-and-beverage-protective-cultures-market; Bourdichon et al., 2021).

7. Data Gaps and Conclusion

There is a notable gap in the existing literature: Currently, there are no specific studies on the use of protective cultures in plant-based cooked sausages and ham products. While products containing protective cultures are commercially available, they are primarily designed for conventional meat preparations or raw sausage products.

Starter cultures for improving texture, taste, and aroma have already established themselves on the market. Given the rapid growth of the plant-based sausage alternative sector, it is reasonable to assume that the development of tailored protective cultures will increase.

To support this, there is an urgent need for scientific research to elucidate the efficacy and mechanisms of action of these cultures in plant-based systems. Comparative studies could highlight the differences between protective cultures in traditional meat products and their plant-based counterparts. Since LABs, are also widespread in plant-based products, it is plausible that similar protective cultures could be effective against the spoilage candidates. However, this hypothesis requires empirical validation.

Further research is required in the domain of protective cultures for plant-based meat products, as well as in the conventional heated meat products sector. In such contexts, protective cultures are primarily employed following the heating process; however, from a technological efficiency standpoint, it would be preferable to inject the brine. Moreover, there is a paucity of scientific findings concerning the encapsulation of protective cultures for incorporation prior to the heating process.

Protective cultures have also been used predominantly as single species and rarely in combination with other protective organisms or bacteriocins. The studies that have carried out such combinations show that a combination of protective culture and bacteriocin in particular can be particularly successful.