Broad Spectrum Bacteriocin for Control of Unwanted Bacteria
A composition containing a newly identified bacteriocin produced by a Lactobacillus plantarum strain, isolated from natural corn mash used in commercial fermentation process for production of ethanol. The bacteriocin composition is effective in broad range killing against the vast majority of the lactic acid bacteria (LAB) isolates in ethanol fermentation mash, which include multiple species of Lactobacillus, Lactococcus, Weissella, Leuconostoc, Pediococcus, Enterococcus, and Streptococcus as well as killing activity against non-LAB isolates, such as strains of Staphylococcus, Enterobacter, Bacillus, and Clostridium. The bacteriocin is also combined with broad range bacteriophage to provide synergistic effectiveness again unwanted bacteria in industrial biofuel fermentation process and in human and animal bacterial infections.
This application claims benefit of Provisional Application Ser. No. 61/991,885 filed May 9, 2014, the content, figures and disclosure of which are incorporated herein by reference.
BACKGROUND1. Field
A broad spectrum bacteriocin composition and method for control of unwanted bacteria, particularly lactic acid bacteria found in fermentation processes.
2. Background
Bacteriocins are usually antimicrobial peptides produced by bacteria, particularly Gram-positive bacteria, to inhibit the growth of related species or other genera at high potency. Lactic acid bacteria (LAB) are a group of popular bacteriocin-producing bacteria, with a substantial number of bacteriocins produced by LAB strains discovered and well characterized. The characterized bacteriocins produced from LAB can be classified into three major groups: Small and heat stable bacteriocins containing lanthionine (class I bacteriocin, or lantibiotics), small and heat-stable non-lanthionine-containing bacteriocins (Class II), and large and heat-labile lytic proteis (Class III).
Antimicrobial spectrums of bacteriocins produced by LAB can vary depending on their specific protein structures. Most of the LAB bacteriocins are capable of inhibiting a wide range of LAB species/strains, and some of LAB bacteriocins are also very potent against some Gram-negative bacteria. Bacteriocins showing broad activity spectrum against target bacteria populations can have wide application for industrial antimicrobial use and remediation of human and animal bacterial infection. For example, plant material-based commercial ethanol fermentation process often suffers from LAB contamination and therefore the inhibition of yeast activity, which results in significant product yield loss. The indigenous contaminating LAB population in the fermentation raw materials is control targets since they pose potential risk on yeast fermentation. Bacteriocins produced by the indigenous LAB population are more likely to act towards species and/or strains of the indigenous LAB population and thus can be used as an effective control on contaminating LAB. The contaminating LAB population in ethanol plants has been identified to contain multiple LAB genera and each LAB genus contains different species. A bacteriocin with broad LAB control spectrum is needed for such contamination control. Bacteriocins effective for broad-spectrum LAB remediation could also be effective against LAB species and/or other pathogenic agents associated with dental caries, bovine mastitis and other clinical infections.
In addition the use of bacteriocin derived from lactic acid bacteria as antimicrobials is safe. Bacteriocins from LAB are described as “natural” inhibitors, in regard to the long history f safe use of LAB in food industries. Lactic acid bacteria are ubiquitous in nature and are commonly associated with plant materials and thus commonly present in fermented food. Many bacteriocins produced by lactic acid bacteria are isolated from foods such as meat and dairy products, which normally contain lactic acid bacteria. These bacteriocins have been unknowingly consumed by human for centuries. Some of the broad spectrum bacteriocins produced by LAB are used commercially in food and pharmaceutical industries. Examples include nisin (Class I bacteriocin) produced by Lactococcus lactis and pediocin (Class II bacteriocin) produced by Pediococcus acidilactici. Nisin is granted GRAS (generally recognized as safe) status by FDA (GRAS Notice No. GRN 000065). It is approved for use in over 40 countries and has been used as food preservatives for over 50 years.
The present invention is a composition of a newly identified broad range bacteriocin produced by a Lactobacillus plantarum strain, isolated from natural corn mash used in fermentation process for production of ethanol.
SUMMARYThis invention is a composition of a newly identified bacteriocin produced by a Lactobacillus plantarum strain, isolated from natural corn mash used in commercial fermentation process for production of ethanol. The bacteriocin composition is effective in broad range killing against the vast majority of the LAB isolates in ethanol fermentation mash, which include multiple species of Lactobacillus, Lactococcus, Weissella, Leuconostoc, Pediococcus, Enterococcus, and Streptococcus as well as killing activity against non-LAB isolates, such as strains of Staphylococcus, Enterobacter, Bacillus, and Clostridium. The bacteriocin is also combined with broad range bacteriophage to provide synergistic effectiveness again unwanted bacteria in industrial biofuel fermentation process and in human and animal bacterial infections.
Bacteriocins need to be produced at a large scale for commercial application. The produced bacteriocin can be used either in crude preparations or purified forms. Bacteriocins can be produced in several ways: (1) using bacteriocin-producing strains directly; (2) using recombinant protein over-expression systems. Alternatively, bacteriocin may be produced in situ utilizing the above-mentioned systems during the treatment process.
(1) Bacteriocin Production in Native Host StrainsThe culturing conditions of the bacteriocin producing strain are first optimized in a small volume batch culture to achieve the maximum yield of bacteriocin production. The optimized parameters include the media composition, culture temperature and pH, anaerobic conditions, etc. In addition to maximizing bacteriocin yields, the optimized parameters will also consider the culture volume scale up and downstream concentration and purification of bacteriocin if applicable. Production of bacteriocin at a large scale will be achieved in large volume culture vessels or commercial fermentors. Bacteriocin production can also be achieved in fed-batch cultures where the determined limiting nutrient substrates are fed to the culture to sustain the high level bacteriocin production for a longer time.
It is possible to obtain bacteriocin over-producing mutants of the host strain via natural mutation events. With the identification of the genetic determinants of bacteriocins through genome sequencing, it is possible to obtain bacteriocin over-producing mutants of the host via specific genetic manipulations. The optimal growth conditions of these bacteriocin over-producing mutants can be determined for bacteriocin production at a much higher level.
(2) Bacteriocin Over-Expression Using Non-Native Expression HostsEither using the native bacteriocin-producing strains or using recombinant protein over-expression systems in non-native strains, bacteriocin produced can be used in its crude forms for application. The bacteriocin produced can also be concentrated and purified for application. Alternatively, bacteriocin-producing strains may be introduced to the systems to be treated, and the bacteriocin of interest may be produced in situ utilizing the above-mentioned systems during the treatment process.
Bacteriocin can be used as the only active ingredient for anti-microbial application. Alternatively, bacteriocin can be used together with other agents or adjuvants for enhanced activity. For example, bacteriocin can be used together with bacteriophage to achieve broader range of control and with greater effects.
Bacteriocin ApplicationsEither used alone or in combination with other antimicrobial agents, the two-peptide lantibiotic GP15cin described herein can be used in the following practices where either specific or general lactic acid bacteria are the controlling targets:
Biofuel Fermentation ProcessGP15cin and other bacteriocins targeting LAB can be used to control bacterial contamination in various biomass-refining processes, including but not limited to ethanol biofuel production. Commercial ethanol plants use starch or sugar-based raw plant materials and rely on yeast fermentation for ethanol production. This microbiological process uses non-sterile substrates and thus is inevitably subjected to bacterial contamination, often to the detriment of fermentation performance ultimately resulting in significant ethanol yield loss. Lactic acid bacteria, especially Lactobacillus spp., are the predominant contaminating bacterial group identified worldwide. Common approaches used by fermentation plants to reduce LAB contamination include sanitization and the copious use of antibiotics such as virginiamycin and penicillin. Compared to current controlling products, bacteriocins originated from LAB are of GRAS (generally recognized as safe) status as designated by the U.S. Food and Drug Administration (FDA) and pose no environmental hazards.
GP15cin described herein were produced by a L. plantarum strain, which was isolated from the indigenous LAB populations present during commercial ethanol fermentation. GP15cin can act towards species and/or strains of similar indigenous LAB populations and thus can be used as an effective control for contaminating LAB.
The application of bacteriocin and/or bacteriophage to control LAB in common ethanol production plants using starch feeds is well documented in the patent literature including published patent applications 2014/0148379, published May 29, 2014 and 2009/0104157, published Apr. 23, 2009, the disclosure and figures of which are incorporated herein by reference. Application of the present invention bacteriocin GP-15cin can be utilized in the same way as described in these applications.
As noted above, GP-15cin may be applied alone or in combination with other bacteriocin and/or bacteriophage virulent for unwanted bacteria in the same system. Bacteriocin producing hosts are immune to the bacteriocins produced by themselves. GP-15cin is therefore not active against its own production host, GP15cin. Though the bacteriocin produced by Lactobacillus GP15 exhibits broad host range in inhibiting various indigenous LAB, a very small number of strains of LAB present in natural fermentation mash may not be sensitive to this bacteriocin. Therefore it is desirable to combine GP-15cin with a different bacteriocin in a cocktail that will kill the bacteria insensitive to GP15cin. Additionally GP-15cin may be combined with other antimicrobial agents to broaden the scope of effectiveness against the range of unwanted bacteria that are targeted. For example the combination of bacteriocin and bacteriophage (phage) ensures a broader inhibition spectrum and more efficient control of the bacteria contamination. Phage targeting these non-sensitive strains can be isolated, and be combined with the bacteriocin for a complete control of contaminating bacteria.
The Synergistic Effect Between Bacteriocin and BacteriophageThe combination of the GP-15cin bacteriocin and some bacteriophage has been found to have a synergistic effect in remediation of unwanted LAB bacteria isolated from ethanol fermentation plants. Bacteriocin alone, bacteriophage alone, and bacteriocin combined with phage were tested against Lactobacillus in batch culture systems. To be specific, logarithmic growth phase cultures of Lactobacillus fermentum strain 0315-25 (OD600 being 0.1) were treated either with single agent at different doses, such as bacteriocin GP15cin alone at different doses (0.1%, 1%, and 10%, v/v), or a phage designated herein as LferInf alone at different MOIs (0.1, 1, and 10), or two agents combined together at different doses, such as 0.1% GP15cin plus LferInf at MOI of 0.1, 0.1% GP15cin plus phage LferInf at MOI of 1, 1% GP15cin plus LferInf at MOI of 0.1, and 1% GP15cin plus phage LferInf at MOI of 1. Culture without any treatment was served as control. LferInf is a bacteriophage isolated from wastewater and found to be effective against a broad host range of Lactobacillus and described in more detail below. The growth of bacteria was monitored for 24 h, and the treatment effects were compared. The results are shown in
This synergistic effect between two antimicrobial agents in inhibiting bacterial growth has great application potentials. This synergistic effect between bacteriocin and phage allows effective suppression of target bacteria, as well as significantly reduced production cost, since the synergistic effect allows each agent to be used at a much lower dose than that required when a single agent is used. Bacteriocin and phage can be added to ethanol fermentation at the same time, or added sequentially depending on the growth kinetics of their respective target hosts. Bacteriophage virulent for LAB bacteria useful for this invention may be identified, isolated, purified, encapsulated, and commercially produced by means and methods known in the art. See for example U.S. application Ser. No. 13/465,700 filed May 7, 2012, now published application US 2013/0149753; U.S. application Ser. No. 13/466,272 filed May 8, 2012, now published application US 2013/0149759; Published application US 2009/0104157, published Apr. 23, 2009 and WO 2006/050193. The disclosures of these patents and patent applications are incorporated herein by reference for all purposes. In this invention any of the bacteriophage virulent for LAB found in the fermentation of starches and sugars to biofuel processes may be used, as well as bacteria that produce the phages in situ.
In general it is not preferred to add the bacteriocin-producing host bacteria to the fermentation reactors, mainly because of their acid production and also because of the nutrient competition with fermentation yeasts. GP-15cin may be added by growing up the host GP-15 to produce GP15cin, and then add the cell-free GP15cin to the system to be treated. It is also possible to add the host bacteria to the system to be treated and use its bacteriocin production directly in situ, but its acid production has to be limited or deleted. Acid deficient host strains thus need to be developed. This is usually done by either screening for naturally derived or artificially introduced genetic mutation. This way the host can produce bacteriocin in situ, but not producing harmful other products (such as acids) to inhibit yeast.
It is also possible to use the same bacteriocin-producing bacteria strain as the phage propagation host, to obtain the simultaneous production of bacteriocin and phage in one culture.
Medical and Veterinary ApplicationsGP15cin described herein is a potent bacteriocin and showed extremely broad killing spectrum against a list of bacteria, include multiple species of Lactobacillus, Lactococcus, Weissella, Leuconostoc, Pediococcus, Enterococcus, Streptococcus, Staphylococcus, Enterobacter, Bacillus, and Clostridium. The list is likely to expand with new strains being tested. Relying on its broad killing spectrum, GP15cin has utility as antimicrobials for both human medical and animal veterinary applications. In human clinical settings, a list of “superbugs” that are resistant to current treatment antibiotics are emerging. For example, methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis, penicillin resistant Pneumococcus, Propionibacterium acne, and Streptococcus mutans are all significant human pathogens, and the list of Gram-positive antibiotic-resistant strains is expanding. Research data to date indicates that two-peptide lantibiotics may be employed, either alone or in combination with other lantibiotics/ca membrane-acting agents, as an effective therapy to control these drug-resistant pathogens (Lawton E, Ross R P, Hill C, Cotter P D; Two-peptide lantibiotics: a medical perspective. Mini-Reviews in Medicinal Chemistry; 2007, 7, 1236-1247). Little resistance to current lantibiotics has been observed, highlighting the great potential of their medical applications. GP15cin described herein can be used as a viable alternative to conventional antibiotics to control pathogens in both human medical and animal veterinary applications.
The U.S. Pat. No. 7,666,407 describes the use of a specific bacteriocin for control of dental caries, the disclosure of which is incorporated here by reference for all purposes. The same method for control can be used with GP-15cin.
Isolation and Characterization of Phage LferInf (1) Isolation of Phage LferInf and its Killing Spectrum Against LABLAB strains isolated from ethanol fermentation plants were used as host strains for phage isolation and characterization. Using the host strain L. fermentum 0315-25, phage LferInf was isolated from municipal wastewater influent water. The activities of LferInf were tested against LAB strains isolated from different ethanol plants. LferInf showed broad host activities within L. fermentum species. LferInf infected all 12 L. fermentum strains isolated from 8 different plants. LferInf showed activities against some strains of L. mucosae, L. brevis, L. delbueckii, L. reuteri, as well as some undefined Lactobacillus strains.
(2) Morphological Characterization of Phage LferInfFrom transmission electron microscopic (TEM) images, LferInf was observed to belong to Myoviridae family with a contractile tail. The capsid of LferInf was determined to be 88.8 nm (±3.0 nm) in diameter. The tail is 201.7 nm (±4.3 nm) in length and 20.4 nm (±0.9 nm) in width.
(3) Genomic Characterization of Phage LferInfPhage LferInf has a genome of 106,071 bp, which carries 124 putative protein-coding genes of more than 40 amino acids each, 95 genes were detected in the plus strand and 39 on the minus strand, with 48 genes encoding hypothetical novel proteins with no matches detectable by BlastP in the NCBI nr database. Two tRNAs are adjacent and located on the minus strand.
LferInf genome has a GC content of 38.16%.
Several major functional protein modules were identified in the LferInf genome. These include modules responsible for lysis, DNA packaging, head and tail morphogenesis, and DNA replication. Based on its genome features, it was determined that LferInf is one of the SPO-1 like phages in Myoviridae family.
Application of Phage LferInf in Biofuel FermentationFermentation models simulating bacterial contamination during ethanol fermentation were set up. In these models, yeast was grown on corn mash and the fermentation was challenged with L. fermentum strain 0315-25. The ability of phage to inhibit the contaminating L. fermentum and restore ethanol production by yeast was evaluated. Compared to the infection free control (yeast alone without bacteria), challenging the system with L. fermentum 0315-25 at 107 cfu/ml decreased ethanol yield from 13.7% to 11.7%, increased residual glucose level from 0.44% to 2.87%, increased lactic acid level from 0.19% to 0.53% (w/v), and acetic acid level from 0.08% to 0.28%, in the infection control at the end (72 h) of experiment. Phage treatment mitigated the L. fermentum infection and restored the levels of ethanol to 13.4% (w/v), glucose to 0.04% (w/v), lactic acid to 0.24% (w/v), and acetic acid to 0.08% (w/v). All these levels are comparable to those of the infection free control.
It was clearly demonstrated that adding phage LferInf to L. fermentum-contaminated yeast fermentation models resulted in effective L. fermentum control. Lactobacillus, especially L. fermentum, predominate the contaminating LAB population in commercial ethanol plants. There is great potential of using phage to control Lactobacillus in ethanol fermentation plants. The phage-based products may also be used prophylactically in preventing the contaminating LAB from reaching a high level during ethanol fermentation process.
The mixture of one or more phages to the GP-15cin bacteriocin described herein offers a highly effective antimicrobial cocktail for remediation of unwanted bacteria, particularly in ethanol fermentation processes but also in human and animal bacterial infection control.
While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.
Claims
1. A composition comprising a two-peptide lantibiotic bacteriocin isolated from the indigenous lactic acid bacteria L. plantarum contained in corn mash from an ethanol fermentation process.
2. The composition of claim 1 in an aqueous solution having a pH range of 4-10 and maintained below 65° C.
3. The composition of claim 1 wherein the bacteriocin is GP-15cin as defined in the specification.
4. The composition of claim 1 derived from over-producing system mutants of L. plantarum by natural mutation or genetic manipulation.
5. A method of control of unwanted bacteria in a fermentation process by adding to the feed to the process or to the fermentation vessel a composition comprising a two-peptide lantibiotics bacteriocin produced by Lactobacillus plantarum, isolated from the indigenous lactic acid bacteria from an ethanol fermentation process.
6. The method of claim 5 comprising contacting unwanted bacteria with Lactobacillus bacteria GP-15, as defined in the specification, capable of producing bacteriocin virulent for the unwanted bacteria in a sufficient amount and for sufficient time for the bacteriocin bacteria to produce an effective amount of bacteriocin virulent for unwanted bacteria.
7. The method of claim 5 also comprising contacting unwanted bacteria containing material with other bacteriocin(s) capable of killing the bacteria GP-15 from which the GP-15cin bacteriocin is produced.
8. The method of claim 5 also comprising adding to the bacteriocin a bacteriophage that is capable of acting synergistically with the bacteriocin in broadening the scope and increasing the efficiency of treating unwanted bacteria.
9. The method of claim 5 also comprising adding to the bacteriocin produced by Lactobacillus plantarum the bacteriophage LferInf as described in the specification.
10. The method of claim 5 wherein the bacteriocin producing GP-15 is added to the fermentation reactor after at least an hour of fermentation.
11. A method for treating unwanted bacteria in humans or animals by application of a composition comprising a two-peptide lantibiotics bacteriocin produced by L. plantarum isolated from lactic acid bacteria contained in corn mash from an ethanol fermentation process.
12. The method of claim 11 also comprising contacting unwanted bacteria containing material with other bacteriocin(s) capable of killing the bacteria GP-15 from which GP-15cin is produced.
13. The method of claim 11 also comprising adding to the bacteriocin a bacteriophage that is capable of acting synergistically with the bacteriocin in broadening the scope and increasing the efficiency of treating unwanted bacteria.
14. The method of claim 11 also comprising adding to the bacteriocin produced by Lactobacillus plantarum, GP-15cin, the bacteriophage LferInf as described in the specification.
15. The method of claim 14 wherein the bacteriocin produced by Lactobacillus plantarum, GP-15cin, and the bacteriophage LferInf as described in the specification are formulated into a cocktail effective for reduction of unwanted bacteria.
Type: Application
Filed: Oct 27, 2014
Publication Date: Nov 12, 2015
Inventors: Mei Liu (College Station, TX), Elizabeth J. Summer (College Station, TX)
Application Number: 14/524,747