Fate of the Two-Component Lantibiotic Lacticin 3147 in the Gastrointestinal Tract

Abstract

The component peptides of lacticin 3147 were degraded by ␣-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.Lacticin 3147 is a lantibiotic produced by Lactococcus lactis (16,23). Its antimicrobial activity is mediated by the combined action of its component peptides, LtnA1 and LtnA2 (29). Like other bacteriocins, lacticin 3147 has a number of potential applications for both food and biomedicine (8). In particular, it offers advantages for use in foods, and previous studies have demonstrated its efficacy, both as a food biopreservative and in improving food quality (9, 27). While its broad spectrum offers certain advantages, it may also be a cause for concern, given that it has inhibited all gram-positive bacteria tested to date, including prominent members of the resident gut microbiota such as Lactobacillus, Enterococcus, and Streptococcus spp. (23). Indeed, the direct addition of lacticin 3147 to a human fecal fermentation gut model causes a dramatic reduction in gram-positive populations after only 30 min (22). Consequently, the ingestion of lacticin 3147 or other broad-spectrum bacteriocins in foods has the potential to perturb the commensal gut microbiota. Therefore, as part of a risk assessment for bacteriocins with potential applications in foods, it is important to determine their fate in the gastrointestinal tract (GIT) in vivo. This type of information is also important when considering potential therapeutic applications of orally delivered bacteriocins. This study evaluates the fate of lacticin 3147 in the digestive tract, initially by means of in vitro/ex vivo simulation studies and subsequently in vivo using the pig as a model. A food-grade lacticin 3147 ingredient developed in our laboratory to facilitate food applications was employed (17,18). This spray-dried lacticin 3147 skim milk powder was manufactured as described previously (22) and reconstituted (10%, wt/vol) in sterile distilled water to prepare the reconstituted lacticin 3147 skim milk (RLSM) used in experiments.To test lacticin 3147's sensitivity to a range of proteolytic enzymes representative of those found in the mammalian GIT, RLSM was incubated at 37°C for 3 h with 25 mg/ml trypsin,

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract

“…In another example, bacteriocinproducing cultures have also been shown in livestock to alter rumen flora and decrease shedding of bacterial pathogens (for a review see Diez-Gonzalez, 2007). The lantibiotics nisin and lacticin 3147 have both been shown to have anti-C. difficile activity (Bartoloni et al, 2004;Rea et al, 2007). However, in vivo studies showed that both these bacteriocins are degraded by digestive enzymes in the upper mammalian GIT.…”

“…The peptides were then purified using RP-HPLC and the active fractions identified using MALDI-TOF MS as previously described (Rea et al, 2010). Biological activity was assessed using the WDA with C. difficile ATCC 59635 as the target organism (Rea et al, 2007).…”

“…The purified thuricin peptides Trn-a and Trn-b (35 mg ml 21 ), separated by reversed phased high pressure liquid chromatography (RP-HPLC) as described (Rea et al, 2010), were treated with 77 mg pepsin ml 21 (in 10 mM HCl) or 77 mg a-chymotrypsin ml 21 as previously described (Gardiner et al, 2007). The presence of the intact peptides following exposure to pepsin and a-chymotrypsin was determined over time using MALDI-TOF MS. Lacticin 3147 Ltn-a (degraded by achymotrypsin) and the cell-free supernatant of Lactobacillus gasseri DPC 6480 were used as positive controls for a-chymotrypsin and pepsin, respectively.…”

“…While the emergence of resistant strains is infrequent, recent studies have shown that there is a trend towards decreased susceptibility (Brazier, et al, 2008;Baines, et al, 2008;Huang et al, 2009;Gerding, 2010). Bacteriocins such as lacticin 3147 and nisin have previously been shown to inhibit C. difficile in vitro (Bartoloni et al, 2004;Rea et al, 2007). However, lacticin 3147 has been shown not to survive transit through the mammalian gut (Gardiner et al, 2007).…”

“…Bacteriocins such as lacticin 3147 and nisin have previously been shown to inhibit C. difficile in vitro (Bartoloni et al, 2004;Rea et al, 2007). However, lacticin 3147 has been shown not to survive transit through the mammalian gut (Gardiner et al, 2007). More recently thuricin CD, produced by Bacillus thuringiensis DPC 6431, has been found to have a narrow inhibition spectrum in vitro, with potent activity against a wide range of clinical isolates of C. difficile, including the virulent ribotype 027 (Rea et al, 2010).…”