Projects per year
Abstract
Nisin, a 34-amino acid polypeptide produced by certain Lactococcus lactis strains, is a bacteriocin used as a food preservative, and it is the only bacteriocin that has received FDA approval for use in purified form. Nisin is known to exert a high antibacterial activity against Gram-positive bacteria, however, is rather ineffective towards Gram-negative bacteria. The latter has been shown to be due to the outer membrane of these bacteria, which is impermeable to nisin and thus prevents it from reaching its target, lipid II, in the inner membrane. In recent years, nisin has been considered as an alternative to the classical antibiotics used to treat infections, especially caused by multi-drug resistant bacteria, and nisin has even been shown to excert an anti-cancer effects. One challenge that may conflict with its more broad application, is that nisin is quite expensive to produce; costly substrates are needed, production yields are low and the purification process is costly. Genetic engineering can be used to improve the nisin production, however, genetically engineered microorganisms (GMO’s) are generally not allowed in foods and production based on recombinant microorganisms is far from easy due to regulation. Therefore, isolation of natural, non-engineered strains that produce nisin with high yield, and development of new low-cost media is crucial if efficient industrial production of nisin is to be established. Furthermore, finding natural ways to enhance the antibacterial action of nisin against Gram-negative bacteria, such as food-borne Escherichia coli, can expand its application in food preservation.
Like other lactic acid bacteria, L. lactis relies on glycolysis as its main source for generating the ATP needed for growth. The major fermentation end product, lactic acid, gradually accumulates during growth, and eventually causes growth arrest, even in pH-controlled fermentations. This is a challenge when producing nisin, as its production is biomass-dependent. In chapter 2, we describe the nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, which has a low lactate dehydrogenase activity. The ability of Ge001 to produce nisin using dairy waste as the fermentation substrate was assessed. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12084 IU/mL nisin was attained, which is comparable to the highest titers reported using expensive, rich media.
Nisin-producing L. lactis strains can protect themselves against nisin through their immunity system, which is comprised of the lipoprotein NisI and the ABC transporter NisFEG. Despite the effectiveness of these immunity mechanisms, producing strains are not fully recalcitrant towards nisin, and nisin resistance thus is another bottleneck in nisin production. In chapter 3, it is described how adaptation of the nisin producer ATCC11454 to the cationic disinfectant chlorhexidine can lead to enhanced nisin production. The chlorhexidine resistant strain AT0606 had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Subsequently, we explored the potential of using AT0606 for cost-efficient production of nisin, and were able to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production.
Foodborne and pathogenic E. coli strains pose a serious public health threat worldwide, and measures to control the growth of these harmful pathogens in foods are urgently needed. As mentioned above, nisin has difficulties penetrating the outer membrane of Gram-negative bacteria. Phytic acid (PA) is a natural plant antioxidant that potentially can destabilize the outer membrane of E. coli. In chapter 4, we study the antibacterial effect of nisin and PA cocktails against pathogenic E. coli O157:H7. It was found that nisin and PA, when combined, had a stronger bactericidal effect, and that the two compounds acted in a synergistic manner, both in the planktonic and the biofilm state, which was demonstrated by live/dead staining and plate counting. Electron scanning microscopy studies revealed that the nisin plus PA treatment had a severe effect on E. coli O157:H7, which after treatment appeared deflated with large invaginations in the cell envelope. The effect of nisin plus PA against E. coli O157:H7 was furthermore assessed on cold-stored beef, where the same synergistic effect was observed.
Summing up, in this thesis, several important limitations of nisin have been addressed. We have demonstrated that it is feasible to greatly enhance the nisin production capacity of L. lactis by using natural approaches such as classical mutagenesis, natural conjugation and adaptive laboratory evolution. Two low cost media were explored, both of which were found to support excellent nisin production. Finally a major limitation of nisin was overcome; its low efficiency towards Gram-negative bacteria, where we found that nisin, when used in combination with PA, efficiently could eradicate a dangerous foodborne pathogen on cold stored beef.
Like other lactic acid bacteria, L. lactis relies on glycolysis as its main source for generating the ATP needed for growth. The major fermentation end product, lactic acid, gradually accumulates during growth, and eventually causes growth arrest, even in pH-controlled fermentations. This is a challenge when producing nisin, as its production is biomass-dependent. In chapter 2, we describe the nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, which has a low lactate dehydrogenase activity. The ability of Ge001 to produce nisin using dairy waste as the fermentation substrate was assessed. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12084 IU/mL nisin was attained, which is comparable to the highest titers reported using expensive, rich media.
Nisin-producing L. lactis strains can protect themselves against nisin through their immunity system, which is comprised of the lipoprotein NisI and the ABC transporter NisFEG. Despite the effectiveness of these immunity mechanisms, producing strains are not fully recalcitrant towards nisin, and nisin resistance thus is another bottleneck in nisin production. In chapter 3, it is described how adaptation of the nisin producer ATCC11454 to the cationic disinfectant chlorhexidine can lead to enhanced nisin production. The chlorhexidine resistant strain AT0606 had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Subsequently, we explored the potential of using AT0606 for cost-efficient production of nisin, and were able to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production.
Foodborne and pathogenic E. coli strains pose a serious public health threat worldwide, and measures to control the growth of these harmful pathogens in foods are urgently needed. As mentioned above, nisin has difficulties penetrating the outer membrane of Gram-negative bacteria. Phytic acid (PA) is a natural plant antioxidant that potentially can destabilize the outer membrane of E. coli. In chapter 4, we study the antibacterial effect of nisin and PA cocktails against pathogenic E. coli O157:H7. It was found that nisin and PA, when combined, had a stronger bactericidal effect, and that the two compounds acted in a synergistic manner, both in the planktonic and the biofilm state, which was demonstrated by live/dead staining and plate counting. Electron scanning microscopy studies revealed that the nisin plus PA treatment had a severe effect on E. coli O157:H7, which after treatment appeared deflated with large invaginations in the cell envelope. The effect of nisin plus PA against E. coli O157:H7 was furthermore assessed on cold-stored beef, where the same synergistic effect was observed.
Summing up, in this thesis, several important limitations of nisin have been addressed. We have demonstrated that it is feasible to greatly enhance the nisin production capacity of L. lactis by using natural approaches such as classical mutagenesis, natural conjugation and adaptive laboratory evolution. Two low cost media were explored, both of which were found to support excellent nisin production. Finally a major limitation of nisin was overcome; its low efficiency towards Gram-negative bacteria, where we found that nisin, when used in combination with PA, efficiently could eradicate a dangerous foodborne pathogen on cold stored beef.
Original language | English |
---|
Place of Publication | Kgs. Lyngby |
---|---|
Publisher | Technical University of Denmark |
Number of pages | 129 |
Publication status | Published - 2022 |
Fingerprint
Dive into the research topics of 'Overcoming constraints in production and target range of nisin by using natural approaches'. Together they form a unique fingerprint.Projects
- 1 Finished
-
Characterization of novel lantibiotics in actinobacteria
Zhao, G. (PhD Student), Kuipers, O. (Examiner), Jensen, P. R. (Main Supervisor) & Solem, C. (Supervisor)
01/01/2019 → 16/01/2023
Project: PhD