Introduction

Bacteriocins are defined in a broad sense as ribosomally synthesised antimicrobial peptides or proteins of bacterial origin [55]. Research on bacteriocins has provided relevant information on peptide structure, biosynthesis, protein secretion, mode of action, ecological function, and applications in food preservation and health [19, 27–29, 31, 41, 42, 67, 82, 83, 88, 90, 95]. Among the different bacteriocins studied to date, AS-48 stands, together with nisin, as one of the best-studied antimicrobial peptides. AS-48 is a cyclic peptide produced by several enterococcal isolates [35], including Enterococcus faecalis strains (such as S-48, EFS2, INIA 4, or 39-5) [37, 57, 64, 98] and Enterococcus faecium strains 7C5 and RJ16 [5, 34]. The physico-chemical properties, mode of action, and genetic determinants of this bacteriocin have been reviewed [66].

The structure of AS-48 is based on a 7.14-kDa cationic cyclic peptide linked by a head-to-tail peptide bond and folded into five alpha-helix regions [43, 86, 87] (Fig. 1). Such folded structure confers bacteriocin molecules a remarkable stability to extremes of pH, heat, and denaturing agents [26], a reason why AS-48 remains stable in food environments. Interestingly, AS-48 adopts different oligomeric conformations according to physiochemical conditions, existing in monomeric form at pH below 3.0 and in dimeric forms in the pH range of 4.5–8.5 [2, 87]. The dimeric states can form either by hydrophobic interactions (DF-I) or by hydrophilic interactions (DF-II). Bacteriocin oligomerization is thought to be responsible for the high water solubility of this peptide (which is in the mg ml−1 range) [4].

Fig. 1
figure 1

The cyclic structure of enterocin AS-48

Full size image

AS-48 is a pore-forming peptide, which disrupts the bacterial cytoplasmic membrane and leads to dissipation of proton motive force [40]. It has been proposed that transition from dimeric state DF-1 to DF-II is responsible for insertion of AS-48 molecules into the bacterial membrane [87]. By contrast, AS-48 does not have any effect on any of the eukaryotic organisms tested so far, such as Saccharomyces cerevisiae, Naegleria fowleri or Acanthamoeba sp., even at concentrations as high as 100 μg ml−1 [38] nor the HeLa and MCDK cell lines nor erythrocytes [66], a reason why it is considered a safe antimicrobial for application in foods.

A relevant feature of AS-48 is the broad spectrum of antibacterial activity, involving most of the Gram-positive bacteria tested and also some Gram-negatives [38, 39]. In culture broths, addition of AS-48 alone or in combination with chemical preservatives or moderate heat treatments shows a remarkable bactericidal activity against foodborne pathogens such as Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, or Salmonella choleraesuis [1, 3, 6, 78]. Furthermore, control of foodborne pathogens has been demonstrated in several food products of animal origin, including L. monocytogenes and S. aureus in sausages [7, 8] and cooked ham [11], B. cereus in cheese [80], and L. monocytogenes and/or S. aureus in skim milk and dairy products [12, 81]. Extensive work has been carried out on application of AS-48 for preservation of vegetable foods and drinks, as will be described further in this review.

Compared to meat and dairy products, vegetable foods and beverages still remain an emerging field for application of bacteriocins, where most trials have been based on inoculation with bacteriocinogenic cultures (mainly in fermented foods) and much less on addition of bacteriocin preparations, mainly nisin [41, 90]. The purpose of this review is to provide an overview of the recent progress on application of AS-48 for preservation of vegetable foods and drinks and the benefits of using this bacteriocin as a natural preservative.

Fruit Juices and Beverages

The thermophilic endospore former Alicyclobacillus acidoterrestris can withstand pasteurisation temperatures commonly applied during food processing and spoil freshly made juices as well as processed juices [91]. Laboratory trials have shown that addition of low AS-48 concentrations (2.5 μg ml−1) to juices artificially contaminated with vegetative cells and endospores of this bacterium caused complete bacterial inactivation and afforded protection for up to 14 days in freshly made orange and apple juices and for up to 60–90 days in several commercial fruit juices [45] (Table 1). Treatment of endospores with AS-48 caused inhibition of germination and disorganisation of endospore structure [44, 45] (Fig. 2). Another report indicated that AS-48 addition caused rapid inactivation of the thermophilic sporeformer Geobacillus stearothermophilus in coconut milk and coconut water [71] (Table 1), strengthening the value of this bacteriocin for application in the preservation of fruit juices.

Table 1 Antibacterial effects of AS-48 in fruit juices and beverages
Full size table
Fig. 2
figure 2

Electron microscopy examination of Alicyclobacillus acidoterrestris DSMZ 2498 endospores treated with AS-48. Untreated control endospores (a), and samples of bacteriocin-treated preparations after 8 h (b) or 24 h of incubation (c, d). Bar = 0.2 μm [44]

Full size image

AS-48 showed strong bactericidal activity against bacteria causing ropiness and other alterations in apple juice and apple cider (Table 1). Vegetative cells of the rope-forming strain Bacillus licheniformis LMG 19409 isolated from spoiled Normand ciders [60] were rapidly inactivated by bacteriocin addition in fresh-made apple juice and in commercial apple ciders [46]. Although B. licheniformis endospores were resistant to AS-48, the combination of bacteriocin and moderate heat treatments (85–100°C) increased the heat inactivation of endospores in cider, decreasing D and z values [46]. These results suggest that AS-48 could be applied in combination with mild heat treatments to inactivate B. licheniformis endospores in ciders when a high contamination with endospores of this bacterium is suspected.

Lactic acid bacteria (LAB) may cause ropiness of ciders and produce off flavour precursors such as 3-hydroxypropionaldehyde (3-HPA), which may derive to acrolein [17, 18, 32, 89]. Since apple cider is made from fresh apple juice, application of heat treatments to inactivate apple cider-spoilage LAB is not satisfactory. AS-48 was tested against exopolysaccharide-producing lactic acid bacteria strains of Lactobacillus collinoides, Lb. diolivorans, and Pediococcus parvulus as well as 3-HPA-producing Lb. collinoides strains isolated from spoiled apple ciders from the Basque country region of Spain [32, 69]. In fresh-made apple juice, most strains were rapidly inactivated by low bacteriocin concentrations (Table 1), although two strains (Lb. collinoides 5 and Lb. diolivorans 29) as well as mixed cultures of the different strains showed higher bacteriocin resistance.

Representative strains of cider-spoilage LAB were tested in apple juice with sub-inhibitory concentrations of AS-48 in combination with high-intensity pulsed electric field (HIPEF) treatment, a non-thermal process that is gaining acceptance in liquid foods [53, 54, 68, 79]. Based on the original article authors’ assumption (no FIC numbers provided) AS-48 and HIPEF acted synergistically against lactobacilli and pediococci (Table 1). The combined treatments offered better results compared to single treatments, since no viable LAB were detected during storage of the processed samples [72, 73].

Although Gram-negative bacteria are partially or completely resistant to AS-48 because of the protective effect of the bacterial outer membrane, they can be inactivated by application of combined treatments. Addition of AS-48 alone had no effect on the viability of Sal. enterica in apple juice. By contrast, bactericidal activity of HIPEF treatments increased in combination with AS-48 (Table 1). Survival fraction was affected by different parameters such as treatment time and temperature, and AS-48 concentration. The maximum inactivation of 4.5-log cycles was achieved with HIPEF treatment for 1,000 μs in combination with AS-48 at a treatment temperature of 40°C [70]. The synergism reported by authors depended on the simultaneous action of AS-48 and HIPEF, while bacteriocin addition to HIPEF-treated cells had no killing effect [70]. This was attributed to resealing of transient membrane pores after the HIPEF treatment.

Several other combined treatments with AS-48 showed an increased bactericidal activity against Gram-negative bacteria in fruit juices (Table 1). Escherichia coli O157:H7 cells sublethally injured by outer membrane permeabilizing treatments (EDTA, sodium tripolyphosphate, pH 5, and moderate heat) became sensitive to AS-48. Highest bactericidal activity was observed when the bacteriocin was applied in multiple treatments [9].

Other beverage categories in which AS-48 could find potential applications are sport and energy drinks. The low pH and acid content of drinks has been shown to be detrimental for tooth health, causing enamel softening and tooth decay [96, 100, 102]. Therefore, drinks with lower acidity and higher pH values should be recommended. However, raising the pH of drinks may increase the risk of bacterial spoilage, requiring mild preservation treatments to preserve the bioactive components of drinks (such as vitamins and others). In trials carried out with low-acidity drinks, AS-48 rapidly inactivated L. monocytogenes, B. cereus, B. licheniformis, and S. aureus cells [74] (Table 1). The results suggested that AS-48 could be used as a natural preservative in less acidic sport and energy drinks.

Fresh-Cut Produce

Freshly cut vegetables and fruits may be involved in the transmission of pathogenic bacteria [30, 33, 52, 63, 84]. Since most of the fruits and a large percentage of the vegetables are consumed without further cooking, it is very important to apply disinfection treatments that, while having little or no effect on the food organoleptic properties, decrease the microbial load, inactivate microbial pathogens, and contribute to prolong the product shelf life. Application of washing treatments with AS-48 solutions caused significant inactivation (P < 0.05) of L. monocytogenes inoculated on whole or sliced fruits (strawberries, raspberries and blackberries, melon, watermelon, pear, and kiwi), but did not avoid proliferation of survivors during storage at abuse temperature [21]. Antilisteria activity of washing treatments increased greatly when AS-48 was applied in combination with several other antimicrobials (Table 2). The combinations of AS-48 (25 μg ml−1) and carvacrol (12 mM) or n-propyl p-hydroxybenzoate (50 mM) also avoided regrowth of the listeria during storage of fruit slices at 22°C.

Table 2 Antibacterial effects of washing treatments with AS-48 in fresh-cut produce
Full size table

Consumption of seed sprouts is considered to have health-promoting effects, but at the same time, sprouts have been shown to act as vehicles for pathogenic bacteria [33, 52, 84]. For this reason, sprouts were chosen as a model food system to test the effects of AS-48 washing treatments against several bacterial species, alone or in combination with chemical preservatives. Immersion treatments (5 min at room temperature) with AS-48 solutions reduced viable counts of L. monocytogenes on artificially contaminated alfalfa and soybean sprouts by approximately 2.0–2.4 log CFU g−1 compared to a control treatment in distilled water [20] (Table 2). After bacteriocin treatments, no viable listeria were detected in most of the samples stored under refrigeration, but listeria proliferation was observed for samples stored at room temperature [20]. Antilisterial activity increased significantly when AS-48 was applied in combination with other antimicrobials, providing several options for decontamination treatments (Table 2).

Washing treatments with AS-48 solutions reduced the viable counts of B. cereus and Bacillus weihenstephanensis cells on sprouts by 1.0–1.5 and by 1.5–2.4 log units, respectively [22]. In both cases, no viable bacilli were detected in samples stored at 6°C although bacterial growth was observed in samples stored at 15 or 22°C (Table 2). Microbial inactivation was enhanced greatly when AS-48 was combined with various antimicrobials or sanitizers (Table 2). The combinations of AS-48 (25 μg ml−1) and sodium hypochlorite (100 ppm), peracetic acid (40 ppm) or hexadecylpyridinium chloride (0.5%) provided the best results [22]. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis cells, no viable bacilli were detected or remained at very low concentrations during one-week storage of samples at 15°C. Results from this study indicate that application of washing treatments containing AS-48 alone can reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature abuse conditions.

Soybean sprouts spiked with Gram-negative bacteria were treated with AS-48 [23]. Washing solutions with AS-48 alone at neutral pH had no effect on Sal. enterica. However, increased bactericidal activity was detected for alkaline bacteriocin solutions and moderate heat [23]. Greatest inactivation (4.7 log cycles) was achieved for sprouts heated for 5 min at 65°C in AS-48 solution adjusted to pH 9.0. Inactivation of Sal. enterica cells increased greatly for washing solutions containing AS-48 in combination with various chemical compounds (Table 2). The combined treatment of AS-48 (25 μg ml−1) and polyphosphoric acid (0.1–2%) was tested against several other Gram-negative bacteria inoculated on sprouts. The bacteria tested showed great differences in sensitivity to polyphosphoric acid, but based on the original article authors’ assumption (no FIC numbers provided) the bacteriocin synergized in its antimicrobial action with polyphosphoric acid in all cases [23]. Combinations of AS-48 and polyphosphoric acid significantly inhibited the populations of Sal. enterica, E. coli O157:H7, Shigella spp., Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila, and Pseudomonas fluorescens on sprouts [23]. Therefore, this combined treatment could be applied to reduce the risks of Gram-negative pathogenic as well as spoilage bacteria in sprouts.

Vegetable Salads

Ready to eat salads are prone to bacterial contamination because of the extensive manipulation of ingredients during manufacture, as well as by cross-contamination [14, 56, 99]. In a Russian-type salad, the concentrations of AS-48 required for inactivation of L. monocytogenes during one week storage at 10°C (Table 3) were much higher compared to other food substrates, most probably due to interaction of bacteriocin molecules with the complex food matrix of the salad [24]. Nevertheless, antilisterial activity of AS-48 in salad was enhanced greatly by addition of various essential oils, bioactive components from essential oils and plant extracts as well as other related antimicrobials of natural origin or derived from chemical synthesis, and by food preservatives (Table 3). Isobolograms for combined treatments clearly indicated synergism for AS-48 and citric acid, lactic acid, and p-hydroxybenzoic acid methyl ester [24].

Table 3 Antibacterial effects of AS-48: vegetable salads and vegetable sauces
Full size table

AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (FIC value, 0.33) and with 2-nitropropanol (FIC value, 0.40) against Sal. enterica serovar Enteritidis in Russian-type salad [25]. In tests carried out in salads stored at 10°C challenged with a cocktail of Salmonella strains (Sal. enterica ssp. enterica serotype Typhi, Sal. enterica ssp. enterica serovar Choleraesuis, Sal. enterica ssp. enterica serovar Enteritidis, Sal. enterica ssp. arizonae serovar Arizonae, and Sal. enterica ssp. salamae), the combinations of AS-48 and p-hydroxybenzoic acid methyl ester or 2-nitropropanol reduced the concentrations of viable Salmonella by up to 4.75 log CFU g−1 during a 7-day storage period (Table 3) [25].

Vegetable Sauces

Vegetable sauces are sold as commercially sterile products, but they are also prepared at home or at restaurants for direct consumption. After handling, contaminated sauces may act as vehicles for S. aureus and lead to food poisoning [58, 61]. When tested in vegetable sauces, AS-48 showed variable effects against S. aureus (Table 3), depending on the bacteriocin concentration, type of sauce and storage temperature [48]. Anti-staphylococcal activity of AS-48 was potentiated by phenolic compounds such as carvacrol, geraniol, eugenol, terpineol, caffeic acid, p-coumaric acid, citral, and hydrocinnamic acid, although the efficacy of the combined treatments depended both on the phenolic compound and the type of sauce (Table 3).

Inactivation of Endospore-Forming Bacteria in Rice-Based Foods and Purees

Endospore-forming bacteria are the main cause of food poisoning and spoilage of vegetable pre-cooked foods, given the abundance of bacterial endospores in the raw materials and the sometimes insufficient heat treatment during cooking process [36, 50, 56, 94]. In boiled rice and in a commercial infant rice-based gruel dissolved in whole milk artificially contaminated with a psychrotrophic enterotoxigenic strain of B. cereus cells, AS-48 addition caused complete bacterial inactivation and avoided enterotoxin production within a temperature range of 6–37°C [47] (Table 4). Activity of AS-48 against B. cereus cells in rice gruel was potentiated by sodium lactate. The heat sensitivity of endospores increased markedly in food samples supplemented with bacteriocin. No survivors were detected after heating with AS-48 for one min at 90°C in boiled rice or at 95°C in rice-based gruel (Table 4).

Table 4 Antibacterial effects of AS-48 against endospore-forming bacteria in cooked and canned foods
Full size table

The effect of AS-48 against aerobic mesophilic endospore-forming bacterial cells was tested in commercial soups and purees (Table 4). B. cereus was completely inhibited by AS-48 in all six vegetable foods tested (natural vegetable cream, asparagus cream, traditional soup, homemade style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22°C [49]. Strains isolated from spoiled purees [51] were slightly more resistant to AS-48, showing a slower inactivation over time. Bacteriocin resistance increased greatly in purees inoculated with cocktails of strains from Bacillus and Paenibacillus species. In the mixed cultures, paenibacilli (along with some B. cereus cells) were the predominant survivors after bacteriocin treatment. Bactericidal activity against the cocktail of strains was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a fast inactivation of bacilli [49].

Canned Foods

Low-acid canned foods can be spoiled by endospore-forming bacteria if heat treatments fail to inactivate the bacterial endospores [56, 65]. Addition of AS-48 in three low-acid vegetable canned foods (tomato paste, syrup from canned peaches, and juice from canned pineapple) caused complete or partial inactivation of Bacillus coagulans cells [62] (Table 4). Microbial inactivation increased upon addition of lactic acid, glucose, or sucrose. Although AS-48 had no significant effect on B. coagulans spores, the combined application of AS-48 and heat (80–95°C for 5 min) significantly increased endospore inactivation. These results were similar to those reported previously for B. cereus spores [47].

Thermophilic spoilage bacteria could also be controlled by bacteriocin addition. In samples from canned corn and peas supplemented with AS-48 and inoculated with vegetative cells of two G. stearothermophilus strains, no viable bacilli were detected during storage of samples at 45°C for 30 days [71]. In the canned food samples inoculated with intact G. stearothermophilus endospores, no viable cells were detected shortly after bacteriocin addition and during storage (Table 4). Through trypsin rescue experiments, it was shown that intact endospores were in fact resistant to AS-48, but they avidly adsorbed bacteriocin molecules which acted at later stages during germination [71].

Bakery Products and Ingredients

Wheat doughs are a frequent source of bacterial endospores which, after germination, may cause bread defects such as ropiness and produce enterotoxins [85, 92, 93]. AS-48 was tested against rope-forming Bacillus subtilis and B. licheniformis, as well as on B. cereus and Bacillus pumilus strains in experimental dough from wheat flour [77] (Table 5). In doughs supplemented with AS-48 and inoculated with B. subtilis cells, no viable bacilli were detected after 24 h provided that initial bacterial contamination levels were kept below 4 log CFU g−1. However, samples inoculated with endospores activated to germinate required higher bacteriocin concentrations to achieve the same effects. B. cereus and B. licheniformis cells were inactivated by AS-48 in doughs, but only partial inactivation was observed for B. pumilus strains.

Table 5 Antibacterial effects of AS-48 in bakery products
Full size table

In bakery ingredients, inhibition of S. aureus by AS-48 greatly depended on the food substrate, ranging from complete inactivation in liquid caramel to non-significant inhibition in vanilla or chocolate creams [76]. Significant reductions (P < 0.05) of viable counts were also achieved in substrates like pumpkin comfiture or diluted almond cream (Table 5). Anti-staphylococcal activity increased markedly when AS-48 (50 μg ml−1) was applied in combination with eugenol (0.1%), 2-nitropropanol (0.5%), or Nisaplin (3%) [76].

Various types of desserts were contaminated with S. aureus, B. cereus, and L. monocytogenes and then treated with AS-48 [75]. Greatest inactivation of S. aureus was observed in baker cream, while lowest activity was detected in soy-based desserts and in gelatin puddings, in which the efficacy of AS-48 greatly depended on inoculum size (Table 5). For L. monocytogenes, added AS-48 caused rapid bacterial inactivation and avoided regrowth of survivors. The lowest activity was also detected in soy-based desserts. In tests carried out in instant pudding, no viable B. cereus cells were detected after AS-48 treatment, although an increased bacteriocin resistance was observed in the soy pudding. Bacteriocin addition in gelatin pudding inactivated B. cereus cells and inhibited gelatin liquefaction caused by the proteolytic activity of this bacterium (Table 5). According to these results, AS-48 could find applications in desserts in combination with good hygienic practices to reduce the food microbial load. Bacteriocin activity seems to be negatively affected by fat content and by certain other components of raw materials (like soy derivatives). Under those circumstances, the efficacy of treatments could improve considerably in combination with other antimicrobial substances.

Concluding Remarks

AS-48 is a promising candidate for preservation of vegetable foods and drinks, either alone or in combination with other hurdles. In fruit juices, the bacteriocin is highly active against endospore-forming spoilage bacteria as well as spoilage lactic acid bacteria. These effects could be achieved at low bacteriocin concentrations similar to those reported in studies carried out with nisin against endospore formers [59, 101]. The efficacy of other bacteriocins (such as pediocin PA-1/Ach) in fruit juices has not been tested so far, nisin being the only licensed biopreservative for this purpose. Therefore, AS-48 could be used as an alternative to nisin avoiding proliferation of specific nisin resistant strains. Of particular interest is the effect of AS-48 against 3-HPA and EPS-producing spoilage bacteria in apple cider, where no published studies are yet available on the activities of other bacteriocins such as nisin or pediocins. Depending on the target bacteria, AS-48 could be used in drinks alone or in combination with physico-chemical treatments such as chelators or HIPEF treatment, increasing inactivation of Gram-negative bacteria and decreasing the risks for proliferation of survivors during the commercial shelf life of the product. Bacteriocin addition could ameliorate the problems associated to heat treatments, achieving a better preservation of nutrients, vitamins and organoleptic properties. It could also help the food industry to design new food products such as less acidic drinks or more naturally preserved beverages.

Decontamination of fresh-cut fruits and vegetables is an interesting field for application of AS-48. Fresh-cut products are highly perishable and can only withstand limited types of treatments for decontamination. Washing treatments with AS-48 are useful to reduce the microbial load of listeria and bacilli, and they can also afford protection for samples stored under refrigeration. A broad range of combinations of AS-48 and other antimicrobial substances provided increased bactericidal effects, affording protection under temperature abuse conditions. Among them, the combinations of AS-48 and polyphosphoric acid are of greatest interest, since they also afforded protection against Gram-negative bacteria. The results obtained for washing treatments with AS-48 against L. monocytogenes are superior to those reported for nisin (50 μg ml−1) and pediocin (240 μg ml−1) [13] because of the lower concentrations of enterocin required (25 μg ml−1). Protection of treated samples during storage has not been reported for nisin or pediocin, being this a key issue for the safety of the treated produce. There are no results available on the activity of nisin or pediocin on other bacteria of concern in fresh produce such as endospore-forming bacilli or pathogenic enterobacteria. Application of AS-48 in combined treatments that avoid transmission of both Gram-positive and Gram-negative bacteria in fresh-cut produce would be beneficial to the vegetable food industry.

Application of essential oils and their bioactive phenolic compounds in food preservation has been pursued for a long time, but it has been limited by the strong impact they have on the food organoleptic properties [15]. Nevertheless, the strong bactericidal effects reported for these antimicrobials in combination with AS-48 opens new possibilities for application in vegetable foods, such as vegetable salads or sauces, where special flavours may be desired. Also, because of the lower concentrations of antimicrobials required for inhibition of target bacteria in the combined treatments, the impact on food is decreased.

Control of endospore-forming bacteria in processed vegetable foods (such as rice-based foods, soups, puree, and canned vegetables) is an interesting field for application of this bacteriocin. The studies carried out have shown that AS-48 can inactivate different species of endospore-forming bacteria in foods substrates, at varying concentrations depending on the food and bacterial strain. Most interesting, it has been shown that addition of AS-48 reduces the intensity of heat treatments necessary for inactivation of endospores in vegetable foods. Nisin is also highly effective against endospore formers in canned vegetable foods [97], but there are scarce or no reports on the effects of other bacteriocins in this field [16]. Since contamination of raw material of vegetable origin with bacterial endospores is one of the main problems in the food industry, AS-48 could find interesting applications as an additional hurdle alternative to nisin in processed vegetable foods not only to increase the efficacy of heat treatments (allowing a reduction of heat intensity) but also to control germinating spores in the finished product.

Endospore-forming bacteria may cause several problems in the bread and bakery industry, since they are often present in flours. Although the effect of AS-48 in flours is limited by the low water availability, this bacteriocin could be incorporated in doughs to prevent proliferation of bacilli responsible for ropiness and enterotoxin production. Similarly, AS-48 could find applications in the bakery industry, controlling the proliferation of foodborne pathogens such as L. monocytogenes, B. cereus, or S. aureus. However, the strong interference observed for high-fat components such as chocolate or soy products with bacteriocin activity is a clear limitation as to the type of substrates where it could be used, unless other antimicrobials acting synergistically with AS-48 are included.

In conclusion, AS-48 could find different applications in vegetable foods either as an additive or as a natural sanitizer. At present, the bacteriocin can be obtained at pilot scale by cultivation of producer strains on food-grade by products such as whey or whey permeates, and can be concentrated and stabilised in different ways, e.g. by cation exchange chromatography, tangential flow ultrafiltration, or spray drying [10]. Hopefully, the availability of commercial preparations of AS-48 together with the body of knowledge already available on the efficacy of this antimicrobial peptide in vegetable food systems should pave the way for its commercial application in food industries.