Bacteriocins and naturals: step deeper into the food Michael L. - PowerPoint PPT Presentation

Bacteriocins and naturals: step deeper into the food Michael L. Chikindas Health Promoting Naturals, Rutgers Center for Digestive Health, New Jersey Institute for Food, Nutrition and Health

Nature is good

Examples of naturally‐derived food preservatives Dosage Name Microbial targets Food Comments per kg Applications of food Nisin 5‐200 Gram+ bacteria Canned foods dairy GRAS notice #GRN 000065 mg/kg products and cook http://www.biocaxis.com/food%20additives/ meats, poultry NISIN.htm http://tblsucralose.en.made‐in‐ china.com/product/qeZJLOESCAUh/China‐ Nisin‐Food‐Additive.html Lauryl arginate 30‐200 Gram+/‐ bacteria, Meat and poultry GRAS notice GRN#000164 mg/kg fungi products http://www.lamirsa.com/vedeqsa_inc_produc tos.php?idioma=uk ε‐Poly‐lysine 100‐1000 Gram+/‐ bacteria, Sliced fish, rice, GRAS Notice#000135 mg/kg fungi noodles, cooked http://www.chisso.co.jp/yokohama/english/re vegetables (Japan) seach/polylysine/food/index.html Lysozyme 125‐250 Gram+ Used in milk, wine GRAS Notice# 000191 mg/kg bacteria and in casings for P. Zeuthen, Leif Bøgh‐Sørensen, Food frankfurters preservation techniques. Bacteriophage 10 10 ‐10 13 L. monocytogenes Cheeses; approved GRAS Notice#000218 P100 pfu/kg to be used in a variety of products

• produced by Lactococcus lactis Nisin • 34 amino acids • contains unusual amino acids: dehydroalanyne, lanthionine and  ‐methyl lanthionine • resistant to pronase, trypsin • sequenced and cloned • prevents spore outgrowth • kills vegetative cells • heat stable • FDA approved GRAS status as a preservative for certain applications • used for more than 50 years world‐wide, with no limit in some countries.

L. Nilsson, Y. Chen, M. L. Chikindas, H. H. Huss, L. Gram, and T. J. Montville. 2000. Carbon dioxide and nisin act synergistically on the cytoplasmic membrane of Listeria monocytogenes . Applied and Environmental Microbiology 66: 769‐774.

Think twice BEFORE Think twice BEFORE you put it into your mouth you put it into your mouth

Bacterial Isolation A single, pure culture of an unknown organism was isolated from the dairy beverage The organism did not appear to be a Lactobacillus species

Identification • Gram‐positive organism • Motile, spore‐forming rod • Ribotyping analysis: Organism is 88% similar to Bacillus subtilis ATCC 6051 • 16S rRNA analysis: Organism was identified as Bacillus amyloliquefaciens , a very close relative of B. subtilis • Draft genome sequencing re‐classified as B. subtilis

Subtilosin A • 35 amino acids, negative net charge, cyclic • Food origin • Antimicrobial activity against human pathogens Pathogen MIC ( μ g/mL) Listeria monocytogenes 12.5 Gardnerella vaginalis 9.2 Pseudomonas 50.0 aeruginosa Amino acid sequence of subtilosin A. The positions of the posttranslational formed linkage are indicated by solid lines. Staphylococcus aureus 100.0 Kawulka et al. (2008)

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• Advantageous to use the “multiple hurdle” approach • Synergistic compounds allow for use of lower concentrations of each individual substance • Find each compound’s individual MIC • Plot MICs on an isobologram OR: FIC index = FIC bacteriocin + FIC antimic. = [bacteriocin]/MIC bacteriocin + [antimic.]/MIC antimic. 70 60 [Substance A] 50 40 30 20 10 0 0 10 20 30 40 50 [Substance B]

http://www.curcuminoids.com/images/curcumin2.gif Curcuminoids A colorless hydrogenated product derived from the yellow curcuminoids, (the biologically active principles from the rhizomes of Curcuma longa ), function as efficient antioxidant compounds and is useful in achromatic food and cosmetic applications that currently employ conventional synthetic antioxidants At the molecular level, the curcuminoids have been shown to inhibit nuclear factor NF kappaB (NFκB) a transcription factor that triggers inflammatory mediators. NFκB is implicated in a variety of chronic disease conditions ranging from cardiovascular diseases to cancer. Curcuminoids offer antioxidant support, anti‐inflammatory support, support a healthy immune system, and potentially prevent connective tissue break down through inhibiting destructive enzymes , with benefits in healthy aging.

 ‐Poly‐L‐lysine • Homopolymer of lysine • Characterized by the peptide bond between carboxyl and  ‐amino groups of L‐lysine • Contains approximately 30 L‐lysine subunits • Produced by the soil isolate of Streptomyces albulus • Has a broad range of antimicrobial activity, most likely, due to interaction with the cell membrane, elevated oxidative stress (gene regulation?), etc.* • Certified by the FDA as a GRAS substance safe for human use *doi: 10.1016/j.bbrc.2013.08.001

Zinc lactate • Antimicrobial used in toothpaste • Acts synergistically with subtilosin • Reported as having anti‐viral activity • Food additive in energy drinks (Europe)

Lauric arginate • Na‐lauroyl‐Larginine ethyl ester formulation • A cationic surfactant • Highly potent nature‐derived antimicrobial • Active against Escherichia coli , Salmonella , Listeria monocytogenes

Nisin and ε‐L‐poly‐lysine synergize against Listeria monocytogenes The figure depicts microbial growth with no added antimicrobials (○), in the presence of nisin (150 IU ml −1 , ▪ ), poly‐lysine (5 μ g ml −1 , •) or both (□). Badaoui Najjar et al. 2007

FIC indices of antimicrobial combinations tested against Listeria monocytogenes strains grown in BHI broth L. monocytogenes NR30 (Nis r ) Organism L. monocytogenes ScottA Antimicrobials Mixture FIC Inter Mixture FIC Inter action (µg/ml) index action index Encapsulated *15+60 0.74 Additive *57.5+20 0.95 Additive curcumin Pure curcumin *15+150 0.82 Additive *60 +75 0.82 Additive Poly‐lysine *5+7.5 0.88 Additive *65+0.25 0.79 Additive Zinc lactate (pH 5) *1.5+375 0.67 Additive *2.5+ 25 0.36 Synergy * Subtilosin concentration FIC index = FIC subtilosin + FIC antimic. = [subtilosin]/MIC subtilosin + [antimic.]/MIC antimic. Amrouche et al. 2010

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What is Controlled Delivery About? • Optimum range of release from the carrier – Depends on the vehicle and on the targeted environment • Effective inhibition of microbial growth – Largely determined by the required time of action A. Balasubramanian and K. Yam 2010

Combination mode of delivery At any given time: C (concentration) C c =C a +C b Resulted (final) concentration Control‐released antimicrobial c b Formulated a antimicrobial Time 0

Release rate of nisin to a 200 mL system (1.53x10 ‐10 cm 2 /s ) with a trend of initial fast release followed by slow release. A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

Growth of Listeria monocytogenes Scott A in BHI broth at 10°C. (●) represents cultures in the absence of nisin (control); (  ) represents cultures treated with slow addition of nisin; (  ) represents cultures treated with instant addition of nisin; (  ) represents cultures treated with combined modes of delivery (instant + slow). Chi-Zhang et al. 2004

Advanced Design of the Release Rate Antimicrobial release trend from a vehicle Constant rate slow rate fast rate Amount released Effective control of microbial growth ≥ MIC Target release rate Time lag exponential stationary Microbial growth rate CFU/ml Time Time Microbial growth trend A. Balasubramanian and K. Yam 2010

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Effect of instant addition of nisin on the growth of M. luteus in 200 mL TSB at 30°C ( □ ) cultures in absence of nisin (control), (+) cultures treated with 1.49x10 ‐4 µmol/mL nisin, ( ● ) cultures treated with 2.98x10 ‐4 µmol/mL nisin, ( ■ ) cultures treated with 1.49x10 ‐3 µmol/mL nisin, ( ◊ ) cultures treated with 2.98x10 ‐3 µmol/mL nisin, ( ○ ) cultures treated with 7.45x10 ‐3 µmol/mL nisin. Standard error was calculated based on plate counts from 8 plates. A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

Controlled release of nisin to a 200 mL system predicted by varying diffusivities ( ▲ ) release profile using diffusivity of 1.53x10 ‐10 cm 2 /s, (●) release profile using diffusivity of 3.83x10 ‐11 cm 2 /s, (◊) release profile using diffusivity of 6.13x10 ‐12 cm 2 /s, (+) release profile using diffusivity of 1.53x10 ‐12 cm 2 /s. A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

Release rate of nisin to a 200 mL system (1.53x10 ‐10 cm 2 /s ) with a trend of initial fast release followed by slow release. A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins