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

Name Dosage per kg

• f food

Microbial targets Food Applications Comments

Nisin 5‐200 mg/kg Gram+ bacteria Canned foods dairy products and cook meats, poultry GRAS notice #GRN 000065 http://www.biocaxis.com/food%20additives/ NISIN.htm http://tblsucralose.en.made‐in‐ china.com/product/qeZJLOESCAUh/China‐ Nisin‐Food‐Additive.html Lauryl arginate 30‐200 mg/kg Gram+/‐ bacteria, fungi Meat and poultry products GRAS notice GRN#000164 http://www.lamirsa.com/vedeqsa_inc_produc tos.php?idioma=uk ε‐Poly‐lysine 100‐1000 mg/kg Gram+/‐ bacteria, fungi Sliced fish, rice, noodles, cooked vegetables (Japan) GRAS Notice#000135 http://www.chisso.co.jp/yokohama/english/re seach/polylysine/food/index.html Lysozyme 125‐250 mg/kg Gram+ bacteria Used in milk, wine and in casings for frankfurters GRAS Notice# 000191

• P. Zeuthen, Leif Bøgh‐Sørensen, Food

preservation techniques. Bacteriophage P100 1010‐1013 pfu/kg

• L. monocytogenes

Cheeses; approved to be used in a variety of products GRAS Notice#000218

Examples of naturally‐derived food preservatives

• produced by Lactococcus lactis
• 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.

Nisin

• 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 you put it into your mouth Think twice BEFORE 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

Amino acid sequence of subtilosin A. The positions of the posttranslational formed linkage are indicated by solid lines. Kawulka et al. (2008)

Pathogen MIC (μg/mL) Listeria monocytogenes 12.5 Gardnerella vaginalis 9.2 Pseudomonas aeruginosa 50.0 Staphylococcus aureus 100.0

milk milk

?

• 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:

10 20 30 40 50 60 70 10 20 30 40 50

[Substance B]

[Substance A]

FICindex= FICbacteriocin + FICantimic. = [bacteriocin]/MICbacteriocin + [antimic.]/MIC antimic.

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.

http://www.curcuminoids.com/images/curcumin2.gif

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

Curcuminoids

• 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

‐Poly‐L‐lysine

*doi: 10.1016/j.bbrc.2013.08.001

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

Zinc lactate

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

Listeria monocytogenes

Lauric arginate

The figure depicts microbial growth with no added antimicrobials (○), in the presence of nisin (150 IU ml−1, ▪), poly‐lysine (5 μg ml−1, •) or both (□).

Nisin and ε‐L‐poly‐lysine synergize against Listeria monocytogenes

Badaoui Najjar et al. 2007

Organism

• L. monocytogenes ScottA
• L. monocytogenes NR30 (Nisr)

Antimicrobials (µg/ml) Mixture FIC index Inter action Mixture FIC index Inter action Encapsulated curcumin *15+60 0.74 Additive *57.5+20 0.95 Additive 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

FIC indices of antimicrobial combinations tested against Listeria monocytogenes strains grown in BHI broth

* Subtilosin concentration

Amrouche et al. 2010

FICindex= FICsubtilosin + FICantimic. = [subtilosin]/MICsubtilosin + [antimic.]/MIC antimic.

http://www.oasiscorpinc.com/userfiles/1/product_images/EXC641.jpg

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

Formulated antimicrobial Control‐released antimicrobial Resulted (final) concentration

Time C (concentration)

a b c

At any given time: Cc=Ca+Cb

Combination mode of delivery

Release rate of nisin to a 200 mL system (1.53x10‐10 cm2/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

Time CFU/ml lag exponential stationary

Target release rate

Microbial growth rate Time Effective control of microbial growth

≥ MIC

Time slow rate Constant rate fast rate Amount released Antimicrobial release trend from a vehicle Microbial growth trend

• A. Balasubramanian and K. Yam 2010

http://i.dailymail.co.uk/i/pix/2012/05/06/article-0-12F54C35000005DC-516_634x453.jpg

Three‐times world sumo champion Byambajav Ulambayar shows some young American kids his moves in Los Angeles

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 cm2/s, (●) release profile using diffusivity of 3.83x10‐11 cm2/s, (◊) release profile using diffusivity of 6.13x10‐12 cm2/s, (+) release profile using diffusivity of 1.53x10‐12 cm2/s.

• A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

Release rate of nisin to a 200 mL system (1.53x10‐10 cm2/s ) with a trend of initial fast release followed by slow release.

• A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

Effect of nisin’s controlled release on growth of M. luteus in 200 mL TSB at 30°C

(□) cultures in absence of nisin (control), (○) growth of M. luteus with instant addition of 7.45x10‐3 µmol/mL of nisin; (▲) growth for diffusivity of 1.53x10‐10 cm2/s, (●) growth for diffusivity of 3.83x10‐11 cm2/s, (◊) growth for diffusivity of 6.13x10‐12 cm2/s, (+) growth for diffusivity of 1.53x10‐12 cm2/s. Standard error was calculated based on plate counts from 8 plates.

• A. Balasubramanian et al. 2011. Probiotics and Antimicrobial Proteins

System 1: NANOFIBER Polymer Matrix

More than 50 materials can be electrospun Polyvinyl Alcohol (PVA) √ High compatibility and safety √ Good mechanical properties √ Low cost

System 1: NANOFIBER Processing: Electrospinning

Schematic image of electrospinning process Cited from CHRISTINA KRIEGEL et al. (2008)

PVA nanofiber 567 nm ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ PVA/subtilosin nanofibers 278 nm

System 1: NANOFIBER Atomic Force Microscope Image

amount released in 24h 37 amount in formulation = 89.4 （SD±4.1）%

%

System 1: NANOFIBER Subtilosin Content

System 2: PEG‐based hydrogels for administration of bioactives

• PEG‐based

hydrogels formed by covalently cross‐linking 8‐arm PEG‐SH and 4‐arm PEG‐NHS polymers via degradable thioester bonds

• Hydrogel administered as a solution

rapidly undergoes gelation in situ (<10 min for 4‐8% w/v hydrogels)

• Active agents covalently attached to

polymer (nanocarrier‐based hydrogels) or passively entrapped to achieve controlled release

Preparation of hydrogels with subtilosin

8-arm PEG-SH (mg/0.05 ml) 4-arm PEG-NHS (mg/0.05 ml) Time of hydrogel (0.1 ml) formation (min)

4 8 26.7±1.6 6 12 25.6±0.3 8 16 25.6±1.1

• Hydrogels with passively entrapped subtilosin were prepared by mixing subtilosin

with 8‐arm PEG‐SH and 4‐arm PEG‐NHS in buffer (pH 7.4) at RT

• Hydrogels were prepared with 121 μg of subtilosin per 100 μl of hydrogel; verified

using Bio‐Rad protein assay

Release of subtilosin from hydrogels

Release in PBS (pH 7.4) was two‐phase with an initial rapid phase (42‐47% release in 24 h) followed by a slow sustained release phase Average release rate was 4 μg/hr for the first 12 h and 0.25 μg/hr from 12‐120 h

http://blog.jessiehawkins.com/tag/antibiotic‐resistance/ http://www.beva.org.uk/news‐and‐events/news/view/185

How do they fight?

How do they fight?

• In L. monocytogenes, subtilosin caused a partial depletion of the

Ψ, and had a similar minor effect on the  pH. There was no significant efflux of intracellular ATP.

• Subtilosin likely acts upon L. monocytogenes Scott A by perturbing

the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death.

• Subtilosin’s mode of action against L. monocytogenes Scott A

differs from the one previously described for another human pathogen, Gardnerella vaginalis.

van Kuijk et al. 2011

Control of Biofilms: Front Line of the War

• n Pathogens

Time (h)

5 10 15 20 25

OD595

• 0.1

0.0 0.1 0.2 0.3 0.4 0.5

Subtilosin concentration (g/mL)

0.9 1.9 3.9 7.8 15.1

Biofilm integrity %

20 40 60 80 100 120

Log10CFU/mL

2 4 6 8 10

Antimicrobial activity of subtilosin against L. monocytogenes growth. Subtilosin concentrations are as following: 250 µg/mL (●), 125 µg/mL (○), 62.5 µg/mL (▼), 31.25 µg/mL (∆), 15.6 µg/mL (■), 7.8 µg/mL (□), 3.9 µg/mL (♦), 1.9 µg/mL (◊), 0 µg/mL (▲). Inhibition of L. monocytogenes biofilm by subtilosin. Biofilm integrity % ( ▌), Log10 CFU/mL (●) About 80% of L. monocytogenes biofilm formation was inhibited by 15.1 µg/mL of subtilosin Algburi et al. 2017

Time (h)

5 10 15 20 25

OD595

• 0.2

0.0 0.2 0.4 0.6 0.8 1.0

Subtilosin concentration (g/mL)

0.98 1.95 3.90 7.8 15.6

Biofilm integrity %

20 40 60 80 100 120

Log10 CFU/mL

2 4 6 8 10 12

Antimicrobial activity of subtilosin against E. coli growth. Subtilosin concentrations are as following: 250 µg/mL (●), 125 µg/mL (○), 62.5 µg/mL (▼), 31.25 µg/mL (∆), 0 µg/mL (■). Inhibition of E. coli biofilm by

• subtilosin. Biofilm integrity % ( ▌),

Log10 CFU/mL (●) More than 60% of Escherichia coli biofilm formation was inhibited by 15.1 µg/mL of subtilosin Algburi et al. 2017

Subtilosin concentration (g/mL)

1.9 mg 7.81 15.625 62.5 125

Violacein production (%)

20 40 60 80 100 120

Cells integrity %

30 60 90 120

Subtilosin concentration (g/mL)

1.9 3.9 7.8 15.1

AI-2 production %

20 40 60 80 100 120

Cell integrity %

20 40 60 80 100

Inhibition of violacein production by

• subtilosin. Violacein production % ( ▌),

Cell integrity % (●) Effect of subtilosin on AI‐2 production by L. monocytogenes. AI‐2 production % ( ▌), Cell integrity % (●) Subtilosin did not influence AI‐2 production by L. monocytogenes at sub‐MICs of 0.95‐15.1 µg/mL Subtilosin at 7.8‐125 µg/mL showed significant reduction in violacein production without any inhibitory effect on the growth of Chromobacterium violaceum. Algburi et al. 2017

Control of Biofilms: Front Line of the War on Pathogens

Product name Microorganisms Bacteriocin Manufacturer Application

BioSafe™ Lactococcus lactis subsp. lactis BS‐10 Nisin A

• Chr. Hansen

Cottage, feta, and ripened cheeses, prevention of late blowing and off‐flavors due to clostridia HOLDBAC™ (formerly “Bio Profit” by Valio, with same species but different strains) Propionibacterium freudenreichii subsp. shermanii DSM 706 and Lactobacillus rhamnosus DSM 7061 Undefined bacteriocins – see US20150150298 A1, publication date: June 4, 2015 Dupont Nutrition Biosciences Aps Inhibition of mold and psychrotrophes in cottage cheese Bactoferm™ F‐LC Pediococcus acidilactici, Lactobacillus curvatus and Staphylococcus xylos

• L. curvatus is producing

sakacin A and P. acidicactici is likely to produce pediocin PA‐ 1/AcH

• Chr. Hansen

Control of Listeria monocytogenes and as a meat starter ALCMix1 Lactobacillus plantarum and Staphylococcus carnosus Produce plantaricin and carnocin bacteriocins, respectively Danisco DuPont Anti‐listerial cultures for fermented sausages and cooked ham Bactoferm™ B‐SF‐43 Leuconostoc carnosum Leucocin

• Chr. Hansen

Control of listeria in vacuum and modified atmosphere stored meat products Bactoferm™ B‐2 Lactobacillus sakei Sakacin

• Chr. Hansen

Control of listeria in vacuum and modified atmosphere stored meat products Bactoferm™ B‐FM Staphylococcus xylosus and L. sakei Sakacin

• Chr. Hansen

Control of listeria in vacuum and modified atmosphere fresh meat products

Examples of commercially available bacteriocin‐producing food‐grade microorganisms

Conclusion

• Bacteriocins play an integral, multifaceted

role in microbial ecology

• Targeted and controlled delivery should

improve the efficacy of bacteriocins

• Bacteriocins’ diverse functions inspire their

multidimensional applications

Acknowledgements

• Granting agencies:

– NIH – Bill and Melinda Gates Foundation

• Former and present lab members:

– Katia Sutyak – Dimitri Kashtanov – Ammar Algburi – Yundong Chi‐Zhang – Tahar Amrouche – Sandra van Kuijk – Richard (Matt) Weeks

• Collaborators:

– Kit Yam and Aishwarya Balasubramanian – Leon Dicks – Djamel Drider – Patrick Sinko – Andrey Karlyshev – Vyacheslav Melnikov