LAB bacteriocin applications in the last decade

ABSTRACT

In the early 2000s, the expectations about bacteriocins produced by lactic acid bacteria (LABs) were aimed at food applications. However, the effectiveness of bacteriocins against undesirable micro-organisms opened endless possibilities for innovative research. In the present review, we collected a database including 429 published papers and 245 granted patents (from 2004 to 2015). Based on bibliometric analysis, the progress of bacteriocin research in the last 11 years was discussed in detail. It was found that 164 patents were granted in 2010–2015, which is equivalent to 60% in comparison with previous years (i.e. only 81 patents were granted in 2004–2009). Currently, the research on bacteriocins is still gaining importance. In the realm of therapeutic strategies, about a 37% of the published research was focused on biomedical applications in the last decade. This vein of research is currently seeking for alternative solutions to problems such as cancer, systemic infections, oral-care, vaginal infections, contraception and skincare. On the other hand, food preservation, bio-nanomaterial and veterinary applications represent 29%, 25% and 9%, respectively. All this technology is being applied and will surely grow in the future, since about 31% of the patents granted since 2004 are focused on the biomedical area, 29% on food preservation, 5% on veterinary use; whereas 13% and 16% correspond to patents granted on production–purification systems and recombinant proteins or molecular modifications in the producer strains. This review contributes to the analysis of recent LAB bacteriocin applications and their role in safety, quality and improvement of human health.

KEYWORDS:

• Bacteriocins
• food and pharmaceutical applications
• lactic acid bacteria
• bibliometric study

Introduction

There is a wide variety of bacteria that produce bacteriocins, lactic acid bacteria (LABs) being the most studied ones. LABs belong to diverse phylogenetic groups, mainly Gram-positive, but sharing common characteristics [1,2]. LABs are frequently isolated from nutrient-rich habitats that contain soluble carbohydrates, available vitamins and proteins and have low oxygen tension. Habitats include fermented products such as milk, meat, vegetables, fruit and dairy products. LABs are often localized in manure and wastewater and in the intestinal tract and mucous membranes of mammals [3]. Bacteriocins are small peptides that are ribosomally synthesized. They are secreted to inhibit the growth of competing bacteria, fungi and some parasites [4]. Bacteriocin classifications have been proposed based on producing strains, mechanisms of action, molecular weight and chemical configuration; four groups are identified (Table 1).

Table 1. General classification of bacteriocins.

Type	Classification	Weight	Feature of peptide	Example	References
With post-translational modifications: lanthionine	Ia	Approx. 3.5 kDa	Net positive	Nisin	[5]
	Ib	Approx. 3.5 kDa	Negatively charged or uncharged	Mersacidin	[6]
Without post-translational modifications: non-lanthionine	IIa	3–10 kDa	Strongly cationic, heat stable	Pediocin PA-I, pediocin CP2, pediocin AcH, enterocin A	[3]
	IIb	25 and 65 kDa	Two different peptides	Lactocin Ga1 and Gb	[4]
	IIc	5–7 kDa	Circular	Uberolysin A, carnocyclin, circularin A and AS-48	[7]
			i (Positive net charge)	Gassericin A/reutericin A
			High isoelectric point
			ii (Uncharged)
	III	>30 kDa	Thermolabile, secreted by the sec-dependent system	Enterolysin A, Helveticin J	[8]
	IV	30 kDa	Comprises a mixture of undefined proteins, lipids and carbohydrates		[3]

Building the database

A database of 429 published articles and 245 granted patents concerning bacteriocins ranging from 2004 to 2015 was built (provided as Online Supplemental Data). The article database was generated using the online databases Science Direct, Scopus, Springerlink, Ingenta, Wiley, Scielo and Cambridge journals, whereas the patent database was constructed using the online databases Espacenet, PatentScope, Lens, Upsto and Google patents. The articles and patents were organized in a Microsoft Excel™ spreadsheet. Citations were thoroughly revised for repetitions. When duplicated data appeared, these were manually deleted. Data were first classified by publication year and country of origin according to the affiliation of the corresponding author. In order to explore the research topics deeper, the articles were also classified by application topics including: food preservation, systemic infection, oral care, cancer, bio-nanomaterials and packaging, veterinary use, women care, contraception and skin care. The patent database was also classified based on the year of first granting and the classification by application topics including: production and purification of bacteriocin, recombinant proteins and molecular modifications, as well as the applications mentioned for the article database. The patents granted on production processes, characterization, molecular modifications and in situ or ex situ applications in foodstuffs or health matters, have been conferred by institutions such as the United States Patent and Trademark Office, the European Patent Office, the Japan Patent Office, the Republic of China Patent Office, Australia's Intellectual Property and Korean Industrial Property Office.

Bacteriocin studies around the world

The expectations about bacteriocins in the 1990s were aimed at the food industry and food preservation. Due to the effectiveness of bacteriocins against a wide variety of undesirable micro-organisms, many additional applications have been studied nowadays. As an example, in the medical field, bacteriocin applications include the prevention and treatment of multi-drug resistant micro-organisms [1]. As the action mode of bacteriocins is different from that of conventional antibiotics, they are considered new potential effective control agents against microbial pathogens. Alternative applications in both the pharmaceutical and the food industry are studied worldwide (Figure 1).

Figure 1. Global contribution per continent and the most representative countries in bacteriocin studies from 2004 to 2015.

Globally, there is a rise in the studies on bacteriocins as natural inhibition agents to combat bacteria in treatments of systemic diseases; in fact, bacteriocins are potential candidates to replace antibiotics as agents active against multiple-drug-resistant pathogens. Europe, America and Asia are the major continents where the antimicrobial peptides have been studied in the last 10 years; USA, Spain, India, South Africa and New Zealand being the countries that generate most of the research in direct bacteriocin applications.

The increasing interest in bacteriocins, combined with new policies and regulations in international markets concerning food and medical products, has changed the perspectives of the industry in the world. There is an increasing demand for natural products that meet high quality standards and safety, particularly, in health and food derived from ‘clean and safe’ technologies such as the use of natural preservatives and biodegradable materials in food packaging [9]. Many less developed countries have found an opportunity to initiate research in the use of bacteriocins in therapeutic treatment and food processing. In the last decade, 37% of the published research was focused in biomedical applications (Figure 2). Researchers are seeking alternatives to problems such as cancer, systemic, respiratory, oral, stomach and vaginal infections, contraception and skincare; while food preservation, bio-nanomaterials and veterinary uses represent 29%, 25% and 9%, respectively.

Figure 2. Contribution of published papers (%) concerning bacteriocins in different applications from 2004 to 2015.

Frontiers of applications

Diverse applications of bacteriocins are studied because some of them are recognized as GRAS (generally recognized as safe) substances by the United States Food and Drug Administration (FDA) and the European legislation for pharmaceutical and food industry uses. Figure 3 shows the published articles based on bacteriocin applications, such as food preservation, personal care and pharmaceutical treatments, by the most representative countries.

Figure 3. Contributions per continent and the most representative countries in bacteriocin applications from 2004 to 2015.

Food-preservation and intestinal infections

Bacteriocins may act as helper peptides of probiotics strains in the gastrointestinal tract (GI). The peptide acts as a killer against susceptible pathogens. This is facilitated by the narrow cavity of GI, providing a competitive advantage over strains in this environment. Bacteriocins may also serve as signal peptides for other strains and for activation of the immune system [3,10,11].

Bacteriocins as food preservatives have been the most studied area (Figure 2). An antimicrobial peptide can be added in situ (e.g. by adding bacteriocin-producing cultures) or in the form of ex situ preparations (e.g. addition of purified or semi-purified peptides) [12]. Some crude-extract preparations have been tested and added ex situ in fresh vegetables and meat, for instance. Crude extracts allow wider ex situ applications and the preparations can be used as food additives. Oliveira et al. [13] and Randazzo et al. [14] reported a reduction in aerobic mesophilic and psychrotrophic bacteria when crude extracts are added on lettuce samples, especially when combining with safe processing methods. De Souza Barbosa et al. [15] used an extract with sakacin produced by Lactobacillus curvatus. This extract was used during salami maturation, inoculated with Listeria monocytogenes. This test showed a 2 log count reduction of the pathogen at the end of 30 days of salami maturation, compared with the control. Jofré et al. [16] and De Alba et al. [17] have compared the efficiency of bacteriocins (e.g. nisin, enterocin A and B and sakacin K) in combination with high-pressure-processing technologies (HPP); their results suggest that bacteriocins could be effective for protection against certain pathogens in meat, vegetables and dairy products (Table 2).

Table 2. Bacteriocins in food and biomedical applications

Bacteriocin	Bacteriocin concentration	Target	Application	Conditions	Reduction of target cell concentration	References
Nisin	100 IU/g	Escherichia coli O157:H7	Food technology: Surface of dry -cured ham	500 MPa 400 MPa	4 log with nisin 3 log with pediocin and nisin	[17]
Pediocin	5000 BU/mL	Clostridium perfringens Listeria monocytogenes	Food technology: frankfurters	Prepared frankfurters with bacteriocins and indicator strain were stored at 4, 10 and 15 °C for 30–60 days with continuous sampling.	L. monocytogenes and C. perfringens counts were reduced by 2 log cycles in the treated frankfurters, after storage at 10 °C for 60 days	[18]
Nisin Z	320 AU/mL in the film solution	Salmonella typhimurium, S. enteritidis, L. monocytogenes, Staphylococcus aureus and E. coli	Food technology: raw turkey breast, deli ham, and raw beef	The bacteria-inoculated poultry samples were wrapped with pullulan films containing lauric arginate and/or nisin Z, then vacuum-sealed and kept at 4 ºC for 28 days to be sampled.	Approx. 5 log/cm^2 S. aureus and L. monocytogenes Approx. 3 log/cm^2 S. typhimurium and S. enteritidis Approx. 4.5 log/cm^2 E. coli	[19]
Sakacin P	200 AU/mL	L. monocytogenes	Food technology: salami	Ambient temperature	1.7 log	[15]
Bovicin HC5	100 AU/mL in the presence of ethylenediamine-tetraacetic acid (EDTA)	S. typhimurium	Food technology	The assays initiated with 5 log CFU/mL, bovicin HC5 (100 AU/mL) and EDTA (1.6 mmol/L), incubated at 37 °C	3.5 log CFU/mL; there were evident important effects on the size and morphology of the cells	[20]
Nisin	Nanofibers prepared from poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO), involving the use Nisin, 15 mg/mL, and dihydroxybenzoic acid (DHBA), 50 mg/mL	S. aureus strains Xen 31 and Xen 30 (both methicillin resistant) and strains Xen 36 and Xen 29	Biomedical application: a combination of nisin and DHBA was incorporated into nanofibers prepared from a blend of PDLLA and PEO	log10 6.7 CFU/mL bacteria were grown in TSB in microtiter plates in the presence of nanofibers (0.5 cm^2) at 37 °C for 24 h	When using the nanofibers + nisin + DHBA, there was a decrease of approx. 88% in bacterium biofilm formation in the culture	[21]
Nisin	50 mg/kg of body weight, in the presence of doxorubicin (DOX) 10 mg/kg	Cancer cells of skin tissue in female mice induced by dimethylbenz (a) anthracene applications	Biomedical application: animals in the test groups were administered nisin subcutaneously and DOX intravenously	Nisin + DOX, twice a week for one month	Nisin acts as DOX potentiator rendering a reduction of both tumour size and tumour burden (58.16% and 66.82%, respectively), probably by inducing an increase in the rate of apoptosis and might involve some non-oxidant-antioxidant based mechanism	[22]
Nisin ZP and AP	Nisin Z and nisin A (95% ultrapure) doses in vitro 100 to 800 μg/mL; in vivo 400 or 800 mg/kg of body weight/day	Four human HNSCC (head and neck squamous cell carcinoma) cell lines. Also, an oral cancer floor-of-mouth mouse model was used	Biomedical application: nisin water solutions were applied to the cell cultures and by oral gavage to be tested	In vitro: cultures were grown at 37 °C from 6 to 48 h, and in vivo experiments were conducted from 3 to 9 weeks; In vivo: mouse model was used; oral cancer was induced.	In vitro: ultrapure nisin significantly increased the percent of apoptotic HNSCC cells reducing the viable cells during culture; In vivo: both nisin ZP and nisin AP, significantly reduced the tumour volume after three weeks	[23]
Fermenticin HV6b	0–500  μg/mL approx.	Human spermatozoa (among other bacterial cells and human cancer cells)	Biomedical application: contraception	The effect on the motility and immobilization of human spermatozoa was evaluated	Reduced motility of the human spermatozoa, beginning after 30 seconds	[24]
Lacticin 3147	In vivo: 40–50 mg/kg second doses 25–21 mg/kg approx., subcutaneously	S. aureus Xen 29 within a mouse peritonitis model (1 × 10^6 CFU)	Biomedical application: systemic infection	Mice received the 1 × 10^6 CFU dose; then bacteriocin doses were intraperitoneally applied 3 and 5 h post-infection	Lacticin 3147 reduced S. aureus Xen 29 growth and prevented dissemination of the pathogen; a significant reduction (1–2 log10) in CFU was observed in the spleen, liver and kidney of the mice model	[25]
Fermenticin HV6b	20–500 μg/mL	Cell lines from hepatocarcinoma (HepG2), breast carcinoma (MCF-7), perpetual cervical (HeLa), spleen lymphoblast (Sp2/0) and kidney embryonal (HEK-293) cells	Biomedical application: anti-cancer activity	Tissues were exposed to fermenticin for 4, 24 and 48 h	The half-inhibitory concecntration (IC-50) of fermenticin was 0.9 μg/mL in MCF-7, 0.5 μg/mL in Sp2/0, 0.1 μg/mL in Hep-G2; 0.6 μg/mL in HEK-293 and 0.4 μg/mL in HeLa	[24]

Antimicrobial activity in packaging and nanotechnology

The demands for high quality food with long shelf life, together with environmental concerns to reduce solid waste, encourage the research on edible coatings incorporating antimicrobial additives. Film-bacteriocin packaging allows the inhibition of undesirable micro-organisms during storage and distribution [26]. Diverse bacteriocin/biopolymers systems have been studied; e.g. nisin/chitosan combinations showed effective antilisterial action, which was higher on the covered food surfaces [26,27]. Bacteriocins can be immobilized via covalent linkages into packaging systems providing stability against proteolytic enzymes [28,29].

On the other hand, bacteriocin nano-capsules obtained through nanoemulsions, nanoliposomes, nanoparticles and nanofibers have shown attractive possibilities in food and medical applications (Table 2). Nanoemulsions including bacteriocins (e.g. nisin, pediocin and subtilosin) have been tested in combination with curcumin, carvacrol and cymene against L. monocytogenes, Salmonella typhimurium, Candida lusitaniae and E. coli. [2,30,31]. De Mello [32] tested nano-vesicles encapsulating nisin, pediocin and BLIS P34 and evaluated the stability of emulsions and their activity against L. monocytogenes. Nano-fibres with ethylene oxide and poly(D,L-lactic acid) including nisin or plantaricin could be used in deep wounds infected with Staphylococcus aureus [33,34] although this technology has to be optimized.

Systemic infections and oral care

S taphylococcus aureus is an important pathogen in many respiratory-tract infections [35], so development of effective anti-staphylococcal agents is very important. De Kwaadsteniet et al. [36] evaluated L. lactis F10-nisin F in treatments of respiratory infections in both non-immunosuppressed and immunosuppressed rats, which were inoculated with 4 × 10⁵ viable cells of S. aureus K and treated with 8192 arbitrary units (AU) of nisin F intranasally. De Mello et al. [32] studied mice infected intraperitoneally with S. aureus Xen 36 (a strain with bioluminescent activity) and treated with nisin (2139 AU) for seven days; interestingly, the bioluminescent activity decreased during the first 3.5 h. That result did not indicate a bacteriostatic action, but nisin had an effect on the immune system as compared with the control. Also, the pathways of inflammation are being evaluated with bacteriocins produced by Lactobacilli sp.; inhibition studies have reported up to 45% inhibition of cyclooxygenase (COX), an enzyme responsible for the formation of prostanoids [37].

Pseudomonas aeruginosa is a pathogenic bacterium related to otitis media in infants that can cause long-term hearing difficulties and speech delay, as well as lung infection [34]. Enterococcus mundtii produces a class IIa peptide, ST4SA, which acts against P. aeruginosa lung infections causing cellular damage to pseudomonas but also to S. pneumoniae, Acinetobacter baumannii, E. faecalis, E. faecium, S. aureus and other Gram-positive bacteria isolated from patients with middle ear infections [38]. Streptococcus salivarius K12 colonises the oral cavity producing salivaricin A and B [39], which have important action against the formation of biofilms that cause oral malodour arising from Streptococcus mutans [40,41] and also show activity against S. pyogenes and S. pneumoniae that cause respiratory problems [39]. Ishijima et al. [42] evaluated the effect of salivaricin in pathogenic biofilm formation in candidiasis, causing severe inflammation affecting elderly and immune-compromised individuals. In vitro and in vivo evaluations were conducted against Candida albicans in the oral cavity of a mouse model, where salivaricin did prevent candida colonisation. It is not yet clear what the specific mechanisms involved are; however, there is a potential use in oral health [43–45]. Other examples are listed in Table 2.

Spermicide action and woman care

Among the methods to control the human population, contraceptive chemical spermicides are available; however, they have side effects such as vaginal infection due to flora removal, weakening the natural protection and so promoting urinary tract infections, too [46]. Bacteriocins are potential spermicidal agents due to their ability to affect the sperm motility [47,48]. Lactacin and fermenticin HV6b have shown a significant reduction in the motility of human spermatozoa; moreover, at higher bacteriocin concentrations, the sperm tails become curved or coiled, indicating that cells were damaged beyond simple restriction of movement [24,49]. Fermenticin HV6b has also been demonstrated to inhibit pathogenic vaginal bacteria such as Gardnerella vaginalis [24,50]. Other bacteriocins, such as SB83 produced by Pediococcus pentosaceus SB83 [51], enterocin 62-6, two peptides produced by E. faecium [52] and lactocin 160 (a peptide like-bacteriocin) produced by Lactobacillus rhamnosus [53] have exhibited effective action against G. vaginalis and Prevotella bivia, Bacteroides, Peptostreptococcus and Mobiluncus spp.

Veterinary use

In dairy animals, nisin A, lacticin 3147, aureoicin A70, nisin Z and macedocin ST91KM have been tested to control mastitis which causes great economic losses in the dairy industry [54–56]. Other groups have evaluated divercin AS7, produced by Carnobacterium divergens, as a food additive in poultry, controlling the gastrointestinal tract microflora against Salmonella spp., Campylobacter jejuni, Salmonella enteritidis, S. wien, Shigella flexneri, P. aeruginosa and P. stutzeri [57,58].

Skin care

Bacteriocins for skin care have been marketed by LABs probiotic producers, in presentation of topical formulations also arguing anti-aging benefits [59]. There are available different commercial options to prevent and treat skin diseases, including the external signs of aging, acne, rosacea, bacterial and yeast infections, psoriasis and dermatitis. Concerning bacteriocins, current research suggests that they contribute to the modulation of the skin microflora, skin lipids and the immune system, leading to the preservation of natural skin homeostasis [60]. Salivaricin, nisin A, mersacidin, lacticin 3147 and leucocin A may represent a lead to cure infections caused by multiresistant bacteria; they have also been used against Propionibacterium acnes responsible for pathogenic acne vulgaris and as immune modulators in the treatment of hospital infections of skin and mucosal wounds [61,62]. ESL5, a bacteriocin produced by E. faecalis SL-5, has been applied as a lotion in a patient with inflammatory acne lesions caused by P. acnes, rendering significantly reduced inflammatory lesions and pustules compared with a placebo lotion [63].

Cancer

The potential use of bacteriocins in anti-cancer therapy is due to their inhibition of DNA and membrane protein synthesis, inducing apoptosis or cytotoxicity in tumour cells (Table 2) [5,64]. Nisin has been tested in the treatment of head and neck squamous cell carcinoma (HNSCC), giving place to preferential apoptosis, cell cycle arrest and reducing the cell proliferation in HNSCC cells compared with primary keratinocytes [65,66]. Nisin reduced HNSCC tumorigenesis both in vivo and in vitro at both low (20%) and high (90%) concentrations in the tested conditions [5,23]. Other important bacteriocins in oncology are produced by E. mundtii, where cancer cell lines, such as oral cancer HSC3, breast cancer MCF7, lung cancer H1299 and colon cancer HCT116, showed susceptibility to such bacteriocins [67].

Bacteriocins on the market

The bacteriocins market has experienced an important growth in the last 10 years, basically as a response to the demands of both the pharmaceutical and the food industry [55], involving products that are slowly accepted as GRAS by the FDA and European legislation (Table 3).

Table 3. Bacteriocins produced for commercial use.

Bacteriocin	Commercial name	Company	Applications	Target micro-organisms	References
Nisin A	Nisaplin®	Danisco, Copenhagen, Denmark	Dairy, culinary, meat, bakery products and beverages	Listeria spp., Bacillus spp., Clostridium spp.	[68,70]
Natamycin	Natamax®	Danisco, Copenhagen, Denmark	Cheese, fresh dairy products, processed meat and beverages	Yeasts and molds	[71]
Micocin	Micocin^® II	CanBiocin, Edmonton, Canada	Meat products	Listeria monocytogenes	[72]
Nisin	Chrisin®	Chr. Hansen, Hørsholm, Denmark	Meat, sausages and spore-forming bacteria in cheese	Clostridium botulinum, Listeria monocytogenes	[73]
Pediocin	ALTA® 2351 2341	Kerry Bioscience, Carrigaline, Co. Cork, Ireland	Meat products	Listeria monocytogenes	[68]
Nisin A, Nisin Z	Nisin A® Nisin Z®	Handary, Brussels, Belgium	Dairy products, bakery, beverages, delicacies, meat	Listeria spp., Clostridium spp., Bacillus cereus	[33,74]
Pediocin	Fargo 23®	Quest International, B.V., Naarden, The Netherlands	Meat products	Listeria monocytogenes	[73]
Pediocin, sakacin	Bactoferm F-LC®	Chr. Hansen, Hørsholm, Denmark	Meat products	Listeria monocytogenes	[16,68]

In 1969, nisin (E234) was determined to be safe for food use by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives (JECFA), and in 1983 it was added to the European food additive list. Then, in 1988 it was approved by the FDA [68]. Today, nisin is used in about 60 countries; however, there are differences in national legislation about its adding and the levels of nisin in foodstuffs such as semolina and tapioca, cheese and processed cheese. Commercially, nisin has been used in inhibiting the growth of micro-organisms capable of forming spores, e.g. Clostridium spp. In the United Kingdom, the addition of this preservative in meat products, processed and pasteurized cheeses, fruits and vegetables is regulated by the British Standards Institution Methods, using maximum concentrations of 250 ppm nisin involving good manufacturing practices [69]. Due to this, patents have been granted for bacteriocin applications in specific regions in the world. The United States is the country with leadership in the development of bacteriocins technology with 42% of the granted patents, followed by China and Denmark with 12% and 10%, respectively (Figure 4).

Figure 4. Countries most representative in granted patents for bacteriocins (2004–2015).

Our review showed that 154 patents were granted concerning bacteriocins in the period 2010–2015, which is 66% more than those in previous years (i.e. in 2004–2009, only 81 patent applications were granted). There has been a remarkable growth of bacteriocins as biotechnology alternatives in recent years. All this technology is being applied and will surely grow in the future, since 31% of patents granted since 2004 are focused on biomedical use, 29% on food preservation and 5% on veterinary uses, whereas 13% and 16% corresponded to patents granted for production–purification systems and recombinant proteins or molecular modifications in producer strains. The minor proportion concerns bio-nanomaterial-packaging and industrial applications (Figure 5). Although the increase in the number of patents granted for applications of bacteriocins is slow, there is a clear upward trend and there is expected to be an increase in the coming years. The broad spectrum offered by bacteriocins alone or in combination, provides powerful alternatives to combat an important number of target organisms. Companies working in this field (e.g. AvidBiotics Corporation (United States); Lanthiopep BV (Netherlands); Novacta Biosystems Ltd. (UK); Danisco, Christian Hansen and Novozymes (Denmark)) have developed broader and innovative applications of bacteriocins in food, biomedicine and other areas. Bactoferm-LC® is a mixed culture (LABs that produce pediocin and sakacinA) anti-listerial preparation produced by Christian Hansen applied to fermented sausages. Likewise, the company produces cultures based mainly on nisin production by mixing Lactobacillus sakei and Leuconostoc carnosum 4010 for use in preservation of meat products [73]. Danisco produces mixtures of protective cultures (HOLDBAC®), chiefly aimed to control Gram-positive pathogens as well as yeasts and molds, heterofermentative bacteria and enterococci. The regulations concerning the use of bacteriocins have had major modifications in the Americas; in 2011, the Health Canada designed a regulation to reduce the risk of Listeria in ready-to-eat foods [72]. The Alberta University released a product based on Micocin®, a Carnobacteria maltaromaticum bacteriocin against L. monocytogenes. Today, in Canada, the United States, Mexico, Costa Rica and Colombia, it is legal to use Micocin® to help to control Listeria in finished products, including meat products [72,75].

Figure 5. Trend in the growth of granted patents concerning bacteriocins from 2004 to 2015 (a) and distribution among different fields of application (b).

Furthermore, there is also a rise in the number of granted patents focused on bacteriocin recombinant proteins derived by directed mutagenesis or insertion of vectors, trans-membrane interactions in the cells and metabolite stability, among others. So far, the biotechnological developments derived from molecular modifications correspond to 18% of the granted patents, according to this review. Several efforts have been made in producing bacteriocin AS-48 due to its ranges of stability and solubility at different temperature and pH conditions, identifying the promoters involved in its expression in order to help to understand the regulation processes [76,77].

Conclusions

There is a constant trend in the development of new policies for both food and pharmaceutical safety applications, thus encouraging the research on new antimicrobial alternatives that can meet these new policies. Particularly, the investment in bacteriocin research and patent development shows a clear upward trend in response to the potential applications of these antimicrobial peptides in the field of both new pharmaceuticals and food products. The trends in the LAB bacteriocin research/application in the last decade reviewed here reflect the need to meet the current demands in food, biomaterials, pharmaceutical and health care Biotechnologies in the twenty-first century. It is envisaged that, in the next 10 years, the patents granted would become doubled, giving an evolutionary spin on the whole biotechnological industry.

Acknowledgments

Authors recognize the valuable contributions of A.G. Calderón-Aguirre (Q.E.P.D.), B. Mendoza-Mendoza, W.Y. Sánchez-Reyes and M.A. López-Ortega, formerly postgraduate members of the Bacteriocin branch-CABA.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by PROMEP under grant Functional Biomolecules Based in Biotechnologies for the Agrifood Sector, 2014–2015, and PROMEP-Posdoc-2014 (MdRLC), as well as by Conacyt [grant number Conacyt-INFR-2014-230138], [grant number Conacyt-INFR-2015-254437], [grant number Conacyt-INFR-2016-269805], [grant number Conacyt-CB-2014-239553].