PURIFICATION OF BACTERIOCINS

To develop bacteriocins for various purposes, the most important step is to obtain them on a large scale in purified form as they, being secreted into the media, may contain undesirable media components. This is generally achieved by concentrating the bacteriocins from the culture supernatant

Lactic Acid Bacteria

Media

pH

Temp

erature

Inoculum

Incubation Agi- Duration tation (hr)

NaCl

Molecular

Weight

References

Lactobacillus

lactis

MRS

6.0

30°C

5%

48 hr

1.5%

94 kDa

Rajaram et al., 2010

Lactobacillus

fenneutum

SBS001

MRS

9.0

35 °C

24 hr

2%

78KDa

Singh et al., 2013

Lactobacillus ru minus AU06

MRS

6.5

35 °C

1%

48 hr

21KDa

Elayaraja et al., 2014

Lactobacillus

plantarum

LPCOIO

MRS

6.2

22— 27°C

1073 to 107J CFU/ ml

  • 2.3-
  • 2.5%

Sanchez et al., 2002

Lactobacillus

plantarum

ST194BZ

  • (i) MRS broth, without organic nutrients, supplemented with tryptone 20 g/L, meat extract 20 g/L, yeast extract 20 g/L, tryptone 12.5 g/L plus meat extract 7.5 g/L, tryptone
  • 12.5 g/L plus yeast extract
  • 7.5 g/L, meat extract 10 g/L plus yeast extract 10 g/L, or a combination of tryptone 10 g/L, meat extract 5 g/L and yeast extract 5 g/L, respectively; (u) MRS broth with glucose 20 g/L;

5.5, 6.0 or 6.5

u>

о

о

О

2%

20 hr

Two

bacteriocins; ST194BZ(a) of 3.3 kDa and ST194BZ(b) of 14.0 kDa

Todorov et al., 2005

Lactic Acid Bacteria

Media

pH

Temperature

Inoculum

Incubation Agi- Duration tation (hr)

NaCl

Molecular

Weight

References

  • (ill) MRS broth without glucose, supplemented with 20 g/L of fructose, sucrose, lactose, mannose, and maltose, respectively;
  • (iv) MRS broth with 0.5-40 g/L of maltose as sole carbon source;
  • (v) MRS broth with 2 g/L of K,HP04 or 2-50 g/L of KH,P04; pH was corrected to pH=6.5 with 0.1 M HC1; and
  • (vi) MRS broth supplemented with 1-50 g/L of glycerol.

Lactobacillus

plantarum

ST13BR

(a) MRS broth, without organic nutrients, supplemented with tryptone (20 g/L), meat extract (20 g/L), yeast extract (20 g/L), tryptone (12.5 g/L) plus meat extract (7.5 g/L), tryptone (12.5 g/L) plus yeast extract (7.5 g/L),

  • 4.5, 5.0,
  • 5.5, 6.0 and 6.5

30°C

2%

20 hr

Bacterio-cin

ST13BR

-lOkDa

Todorov et al„ 2004

Lactic Acid Bacteria

Media

PH

Temperature

Inoculum

Incubation

Duration

(hr)

Agitation

NaCl

Molecular

Weight

References

meat extract (7.5 g/L) plus yeast extract (7.5 g/L), or a combination of tryptone (10.0 g/L), meat extract (5.0 g/L) and yeast extract (5.0 g/L), respectively;

(b) MRS broth, i.e., with 2% (v/v) glucose;

(c) MRS broth without glucose, supplemented with 2% (v/v) fructose, sucrose, lactose, mannose, and maltose, respectively;

(d) MRS broth with 0.05- 4.0% (w/v) maltose as sole carbon source;

  • (e) MRS broth with 2 g/L K,HP04 or 2-50 g/L KH,P04; and
  • (f) MRS broth supplemented with 0-50 g/L glycerol.

Lactic Acid Bacteria

Media

PH

Temp

erature

Inoculum

Incubation

Duration

(hr)

Agi

tation

NaCl

Molecular

Weight

References

Lactobacillus brevis OG1

MRS supplemented with tryptone (0.0-3.0%), yeast extract (0.0-3.0%), beef extract (0.0-3.0%), triammonium citrate (0.0-0.2%) sodium acetate (0.0-0.5%), MgS04-7H,0 (0.0-0.5%), MnS04-4H~0 (0.0-0.1%), K,HP04 (0.0- 0.2%), NaCl (6.0-0.3Н), glucose (0.0-3.0%) and tween 80 (0.0-1.0%).

5.5

30- 37°C

1%

48 hr

1-2%

Ogunbanwo et al„ 2003

Lactobacillus

acidophilus

ACC, L. acidophilus IBB 801, L. caseilmunitas, L. caseiYTT 9029,

L. gassetiKJ, L. johnsoniiLal, and L. rhamnosus GG.

MRS medium; milk medium (skimmed milk powder reconstituted to 10% w/v); milk medium supplemented with yeast extract (0.0, 0.3, 0.5, or 1.0%)

6.5

37°C

1 % (v/v)

100 rpin

Avonts et al„ 2004

Lactic Acid Bacteria

Media

pH

Temperature

Inoculum

Incubatioi

Duration

(hr)

l Agitation

NaCl

Molecular

Weight

References

Lactococcus lactis subsp. lactis A164

M17 broth supplemented with carbon source (glucose, lactose, sucrose, xylose, fructose, maltose, galactose, arabinose, or raffinose) at 0.5% level.

A basal medium (lactose 5 g 1-1; ascorbic acid 0.5 g 1-1; MgS04 0.25 g 1-1; Disodium glycerophosphate 19 g 1-1) supplemented with nitrogen sources (beef extract, tryptone. soytone. yeast extract, peptone, casitone, proteose peptone, or casein) added at 1.0% level.

6.0

30°C

1%

20 hr

100 rpin Without aeration

Cheigli et al„ 2002

Bacillus lichenifonnis strain P40

Feather meal (10 g H), grape bagasse (30 g 1_1), an industrial fibrous soybean residue (30 g H) and cheese whey (70 g l'1)

6.0-7.0

30°C

1%

24 hr

Under

shaking

condition

(125

cycles

min-1)

Olivera et al„ 2004

Lactic Acid Bacteria

Media

pH

Temperature

Inoculum

Incubation

Duration

(hr)

Agitation

NaCl

Molecular

Weight

References

Carnoba- cteriumpiscicola strain A9b

All Purpose Tween (APT). Trypticase Soy Broth (TSB), BHI broth and de Man. Rogosa, and Sharpe (MRS) Broth; modifications of MRS-MRS without glucose (MRS7-G); without Tween 80 (MRS7-T); medium adjusted to pH 7.2 ± 0.2 using 2 N NaOH before autoclaving (MRS7 m); sodium acetate trihydrate was used in place of the anhydrous sodium acetate (MRS7)

7.0±7.2

25 °C

1%

24 hr

1-3%

Himelbloom et al„ 2001

Lactobacillus Plantarum ST31

MRS medium supplemented with varying concentrations of: Tiyptone, Meat extract, Yeast extract, Tiyptone + Meat extract, Tiyptone + Yeast Extract, Maltose, Fructose, Lactose, Saccharose, Glucose, K,HP04. KH,P04, Glycerol

6

30°C

2%

24 hr

Todorov et al.. 2000

Lactic Acid Bacteria

Media

pH

Temperature

Inoculum

Incubation Agi- Duration tation (hr)

NaCI

Molecular

Weight

References

Brevibacteriwn linens ATCC 9175

TSB-modified medium (10 g H trypticase peptone, 3 g h'bacto-peptone, 2.5 g 1_1

yeast extract, 5 g H glucose, 2.5 g H K,HP04, 0.2 g H MgS04.7H,0)

25,30 and 37°C

24 hr

Rotary

shaker

125

cycles

mhr1

  • 40, 80,
  • 120 g

l-1 NaCI

Motta et al., 2003

Lactobacillus

pentosus

ST151BR

MRS broth, ВШ broth, M17 broth, soy milk (10%, w/v, soy flour) and molasses (2-10%, w/v)

5.5, 6.0 and 6.5

  • 30°C,
  • 37°C

2%

28 hr

Without

agitation

  • 3.0 kDa
  • (bacteriocin

ST151BR)

Todorov et al., 2004

Lactobacillus

salivariusCRL

1328

LAPTg; MRS

MRS-6.5

37°C

Inoculum optical density (O.D.) of 1.4 at 540 mu

6 lir

Tomas et al., 2002

LAPTg-

6.5 or 8.0

37°C

Inoculum optical density (O.D.) of 1.4 at 540 mu

6 lir

Lactic Acid Bacteria

Media

pH

Temperature

Inoculum

Incubation

Duration

(hr)

Agitation

NaCl

Molecular

Weight

References

Enterococcus faecium BS13

Agro-industrial byproduct based carbon sources (whey, potato starch liquor, kinnow peel, deoiledrice bran, and molasses), nitrogen sources (soya okra, pea pod, and corn steep liquor)

6.5

37°C

1% (vv-1)

12, 18,24, 30 hr

100 rpm

Bali et al., 2014

(Garsa et al, 2014). This is necessary to cut down its cost and enhance its applications in the food industry (Jamaluddin et al., 2017).

There are three major techniques for the purification of bacteriocins. The first technique involves a conventional multi-step method that includes a series of steps involving hydrophobic interaction chromatography, ammonium sulfate precipitation, gel-filtration chromatography, reversed-phase high-pressure liquid chromatography (HPLC), and ion-exchange chromatography. However, these conventional methods are laborious and have some disadvantages as the protein yields are low in this method (Garsa et al., 2014). This is probably due to multistage operations leading to low yield, becoming the main problem in the purification of bacteriocins at industrial levels. Moreover, these methods are also time-consuming and expensive (Jamaludddin et al., 2017). So, there was a requirement for protocols with efficient recovery, less tune consuming and that can be scaled up. The second technique is a simple three-step protocol which consists of the following: ammonium sulfate precipitation, alcohol/solvent precipitation, and reversed- phase HPLC (Duhan et al., 2013; Garsa et al., 2014) for purification at large scale. The third technique involves bacteriocin isolation by a unit operation known as expanded bed adsorption (EBA) chromatography which uses a hydrophobic interaction gel (Duhan et al., 2013). This is the only unit operation by which product concentration, clarification, and initial purification can be achieved (Jamaluddin et al., 2017). The main advantages of the EBA are that it shortens the total processing time, reduces the number of purification steps, and increases productivity. The operation runs with a high flow rate and high processing volume and can be used in a large-scale process. It also provides a cost-effective alternative process over other purification methods (Garsa et al., 2014). The latter two methods are faster than the first method and are successfully being used for the purification of several economically potential bacteriocins. The different bacteriocin purification techniques are described in Table 8.2.

TABLE 8.2 Different Strategies Used for the Bacteriocin Purification

Producer Strain(s)

Bacteriocin

Purification Method

References

Genus Lactobacillus

Lb. Acidophilus M46

Acidocin В

Centrifugation Isopropanal extraction Reversed-phase HPLC

Acedo et al., 2015

Producer Strain(s)

Bacteriocin

Purification Method

References

Lb. cun’atus ACU-1

Sakacin G

Ammonium sulfate

fractionation

Centrifugation

Cation-exchange

chromatography

Reversed-phase HPLC

Mechoud et al., 2017

Lb. coiynifonnis MXJ 32

Lactocin MXJ 32 A

Ammonium sulfate

precipitation

Dialysis

Ion-exchange

chromatography

Reversed-phase HPLC

Lu et al., 2014

Lb. murinus AU06

Bacteriocin

Centrifugation

Ammonium sulfate

precipitation

Cation-exchange

chromatography

Hydrophobic

interaction

chromatography

Elayaraja et al., 2014

Lb. paracasei FX6

Bacteriocin F1

Ammonium sulfate precipitation Chloroform/methanol extraction'precipitation Reversed-phase HPLC

Miao et al., 2014

Lb. casei TN-2

Caseicin TN-2

Ammonium sulfate precipitation Ion-exchange chromatography Reversed-phase HPLC

Hu et al., 2014

Lb. plantarum ZJ008

Plantaricin

ZJ008

Centrifugation Cation-exchange chromatography Gel chromatography Reversed-phase HPLC

Zhu et al., 2014

Lb. brevis UN

Gel exclusion chromatography

Gautam et al., 2014

Lb. buchneri

Silicic acid adsorption/ desorption Cation-exchange chromatography

Sahnan et al., 2016

Producer Strain(s)

Bacteriocin

Purification Method

References

Lb. plantarum N1326

Plantaricyclin A

Centrifugation Methanol extraction/ precipitation Reversed-phase HPLC

Borerro et al., 2017

Lb. sakei MBSal

Centrifugation Cation-exchange chromatography Reversed-phase HPLC

Barbosa et al., 2014

Lb. bulgaricus K41

Adsorption/desorption

Cation-exchange

chromatography

Zaeim et al., 2014

Lb. cuiratus LB65

Curvaticin LB65

Ammonium sulfate precipitation Cation-exchange chromatography Reversed-phase HPLC

Abdelbasset et ah, 2014

Lb. paraplantaivm FT259

Centrifugation Methanol extraction/ precipitation Reversed-phase HPLC

Winkelstroter et ah,2015

Lb. plantarum 510

Plantaricin Y

Centrifugation Adsorption Gel electrophoresis Reversed-phase HPLC

Chen et ah, 2014

Genus Enterococcus

E. faecalis KT2W2G

Ammonium sulfate precipitation Cation-exchange chromatography Reversed-phase HPLC

Aran et al., 2015

E.faecium T136

Enterocin A

Ammonium sulfate

precipitation

Gel filtration

Cation-exchange

chromatography

Hydrophobic

interaction

chromatography

Reversed-phase HPLC

Jimenez et al., 2014

E. faecium FL31

BacFL31

Centrifugation Ammonium sulfate precipitation Reversed-phase HPLC

Chakchouk- Mitbaa et ah, 2014

Producer Strain(s)

Bacteriocin

Purification Method

References

Genus Laetoeoceus

L. lactis TW34

Nisin Z

Centrifugation Ethanolic precipitation Cation-exchange chromatography Gel electrophoresis

Sequeiros et al., 2015

L. gatvieaeLG34

Garviecin LG34

Ethanol precipitation

Cation-exchange

chromatography

Gao et al., 2015

Genus Leuconstoc

Leu. MesenteroidesWi

Adsorption/desorption Cation-exchange chromatography Mass spectrometry

Dundar et al., 2014

Genus Streptococcus

S. hyointestinalis DPC6484

Nisin H

Reversed-phase HPLC Ammonium sulfate precipitation Methanol extraction

O’Connor et al., 2015

 
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