contents and acknowledgement

AB4426 Masters Research Project 2010

Spoilage of Food: Effect of Nisin on foodborne pathogens and spoilage microorganisms.

A thesis presented in part fulfillment of the

Requirements for the degree of

Master of Science of the

University of Wolverhampton

By

Nikhil Taduka, MSc

0924841

University of Wolverhampton June 2011

AB 4426 Masters Research Project

Spoilage of Food: Effect of Nisin on foodborne pathogens and spoilage microorganisms.

Nikhil Taduka 0924841

Project Supervisor: Dr. Roy Protheroe

Internal Assessor: Dr. Dave Hill

External Examiner:

This project investigation was carried out during March to May 2011

Mode of Attendance: Full Time

Grade Point Awarded:

Table of Contents

Acknowledgment

Declaration

Abstract

S.No Title Page no.1 Introduction 1

1.1 Nisin 6

1.2 Production of nisin 101.3 Nisin and foodborne pathogens 121.4 Escherichia coli 131.5 Listeria innocua 141.6 Staphylococcus aureus 15

1.7 Project aims 16

2 Materials and Methods 172.1 Microorganisms 182.2 Media preparation 182.2.a TSA preparation 18

2.2.b Double strength TSB preparation 182.2.c Preparation of Milk model media 192.2.d Preparation of Nisin stock solution 192.3 Minimal Inhibition Concentration 21

2.3.a Procedure for Minimal inhibition concentration technique

22

2.4 Miles and Misra 222.5 Well inhibition test 232.5.a Procedure for Well inhibition test 232.6 Spiral plate technique 232.6.a Plating Procedure for sample 24

2.7 Bioscreen 28

2.7.a Procedure for Bioscreen C technique 29

3 Experimental Results 313.1 Results for Minimum Inhibitory

Concentration (MIC) in TSB32

3.2 Results for Minimum Inhibitory Concentration (MIC) in Milk media

35

3.3 Results for Miles and Misra of Milk media with nisin

38

3.4 Results for Miles and Misra of Milk media without nisin

40

3.5 Results for Well Inhibition Method 423.6 Results for Spiral Plate Technique 433.7 Results for Bioscreen C method when done

with TSB45

3.8 Results for Bioscreen C method when done with Milk media

48

4 Discussion 52

5 Conclusion 56

References 68

Declaration

Hereby I declare that this thesis regarding, “Inhibitory efficacy of nisin against the growth of

different indicator organisms” and the work reported here is of my Own. All the external help

has been duly acknowledged and all the sources of information have been cited accordingly.

Name: NIKHIL TADUKA

Signature :

Date:24-06-2011

Acknowledgement

I would like to express my whole hearted thanks to Dr. Roy Protheroe for his continued, untiring

help and advice throughout the project, technical support for their unavoidable assistance. I

would like to thank laboratory technicians at the University of Wolverhampton for their

suggestions and help throughout the project session. I would also like to thank my parents for

their valuable support since my academic year. This project work wouldn’t be possible without

above stated persons.

NIKHIL TADUKA

0924841

Spoilage of Food: Effect of Nisin on foodborne pathogens and spoilage

microorganisms.

Abstract: The main aim of this study is to determine the inhibitory efficacy of nisin against

several gram positive and gram negative food spoilage pathogenic organisms like S. aureus, L.

innocua and E. coli. The inhibitory efficacy of nisin was determined against organisms using

MIC, Miles and Misra, Well Inhibition method and Bioscreen C technique. In this experiment,

different concentrations of nisin 3g/100ml, 5g/100ml and 10g/100ml i.e. 3%, 5% and 10% of

nisin was used.

When higher concentrations of nisin were used, it was found to be more effective against gram

positive microorganisms. Gram negative organism E. coli is found to be more resistant towards

nisin. Nisin at higher concentration showed only suppressed growth of gram negative organism

instead of showing growth.

However, the inhibitory efficacy of nisin is more effective on S. aureus as there is no growth

seen even after 48 hrs. A computerized Bioscreen C incubator was used, which incubates the

bacterial culture, process data and provides printouts of the results. With the help of Bioscreen C,

it is confirmed that growth of different indicator organisms are inhibited by the effect of nisin.

It can be concluded that nisin may be useful to improve the microbial quality and ultimately the

safety and shelf life of vegetarian foods since nisin has nature of inhibiting the food spoilage.

CHAPTER- 1INTRODUCTION

1

Food that is not favorable for human consumption is considered as food spoilage. The growth of

food spoilage and food-borne pathogens on/in food can decrease nutritional quality of the food

by consuming fat, protein and carbohydrate that are present in the food, subsequently causes

food discoloration, heating, mustiness, biochemical changes, weight loss and toxicity. Some

species of food borne pathogens like lactic acid bacteria, coliforms, Pseudomonads, Brochothrix

thermosphacta, etc. produce toxic compounds that can adversely affect the health of humans for

example, Staphylococcus toxin that has formed in a food or poisonous mushrooms. (Li and Yi,

2003 , You, 2006).

Visible growth of microorganisms, slime formation and physical damage of food like more

ripening confirms food spoilage. Food spoilage causes enormous financial loss that is observed

throughout the world, a wide variety of imported foods and travel by tourists have also increased

the risk of illness to more affluent persons, with a world-wide economic impact of possibly

$20,000 million. Foodborne disease costs may be as high as $1000/case which means billions of

dollars for many countries. It is believed that the social cost for foodborne illness in developing

countries is greater than in industrialized countries, but because the cost of living is lower an

absolute dollar value may be misleading. Public health authorities are urged to put more effort

into preventing such diseases from occurring through encouraging proper food handling

practices. Microbial activity plays a major role in food spoilage as it affects food supply and one

fourth of worlds food supply is lost due to it alone. Psychotropic microorganisms, yeasts,

moulds, rodents and other animals are causative agents of food spoilage. Range of preservation

techniques and modern food technology could not control the food spoilage adequately. (

2

Ewen C. D. Todd, 2002).

Spoilage of foods occurs very common in our daily life. Foods that are usually affected by

microorganisms are bread, vegetables, wine, fish, meat, juice, fruits and eggs. Microorganisms

that are involved in food spoilage are pseudomonas species, lactic acid bacteria,

Enterobacteriaceae, Phobacterium, Bacillus, Erwinia, Brocothrix, Clostridia, S. putrefacium, P.

phosporium, Aeromonas spp, L. sake, L. curvatus, P. putida, P. fragi, Pseudomonas aeruginosa is

the most common organisms that cause spoilage in foods (Mellor et al., 2011). Especially in

foods that are aerobically stored and with a high water content and natural pH, e.g., red meat

(Dainty and Mackey, 1992; Dainty. 1996; Borch et al., 1996), fish (Gramand Huss, 1996),

poultry (Lahellec and Colin, 1979; C&l10 et al., 1988; Regez et al., 1988), milk and dairy

products (Walker and Stringer, 1990; Craven and Macauly, 1992a; Craven and Macauly, 1992,

Craven and Macauly, 1992). When pH is high, Pseudomonas species and a few other gram

negative psychotropic organisms will dominate protienaceous food stored aerobically at chill

temperature (fish and dark firm dry meat). In the case of marine and iced fish S. putrefaciens

like microorganisms develops in parallel and may become the dominant spoilage

microorganism.

Pseudomonas can cause food spoilage in many ways. NPN will be the first to be metabolised in

foods that are of animal origin. Lipases and protease production may release fatty and amino

acids that after metabolism may result in off odours, off flavours and rancidity. Then

extracellular slime production and pigmented growth development that often becomes visible.

Pseudomonas is specific spoilage organism in ready to eat vegetables (Pitt and Hocking 1997).

This microorganism causes food spoilage in foods like fish, meat, milk, raw vegetables and eggs.

3

In pasteurised milk, Pseudomonas arises from post process contamination (Eneroth et al., 2000).

This microorganism can be inhibited of food spoilage by vacuum packing.

Vegetables are spoiled by gram negative bacteria which can degrade pectin-vegetable polymer

(Liao, 1989; Liao et al., 1997). Eriwinia and pseudomonas are specific spoilage organisms of

ready to eat vegetables. Fungi that can degrade pectin can also contribute to vegetable spoilage.

Slime production is observed where hydrolysed polymers are spoilage products and pectin is

substrate (Lund 1992).

S. putrefaciens, P. phosphoreum and aeromonas species are the causative organism of spoilages

in fish, fishy off odour was seen as the trimethylamine is spoilage product and trimethyalamine

oxide is substrate (Shaw and Shewan 1968, Daal Gard et al., 1993, Gram et al., 1990). Spoilage

of fish was also due to some other microorganisms like enterobacteriaceae, lactic acid bacteria,

and L.sake, L.curvatus, P.fragi and pseudomonas species. Sulphide off odour, sulfhydryl off

odours, fruity off odour confirm the spoilage as the spoilage products and substrates are

(CH3)2S2, esters, H2S and methionine, cysteine respectively (Chai et al., 1968, Herbert and

Shewan 1976, Dainty and Mackey 1992, Miller et al., 1973). These can be prevented by vacuum

packaging (Lyhs et al., 1998; Truelstrup-Hansen and Huss, 1998; Joffraud et al., 2001).

Microorganisms that were responsible for spoilage of milk are B.cereus, P.fragi, P.putida and

Y.intermedia(Cormier et al., 1991, Whitfield et al., 2000). Most of the microorganisms do not

survive in the pasteurised milk but spores of psychotropic bacillus survive from the heat

treatment to cause spoilage (Ternstrom et al., 1993). Formation of sweet curdling indicates the

milk spoilage in which the spoilage substrate and product are phospholipid and protienaceous fat

particles (IDF 1992).

4

Meat is spoiled due to microorganisms like Enterobacteriaceae, (LAB) Lactic acid bacteria, B.

thermosphacta, L. sake, L. curvatus, L. plantarum, homofermentative LAB and Pseudomonas

species (Dainty and Mackey 1992). Greening, Cheese off odour and acid off odour implies the

meat is spoiled. Spoilage products were H2S, CH3S2, Biogenic amines, acetoin, diacetyl and 3-

methylbutanoyl where amino acids, glucose, cysteine and methionine are spoilage substrates

(Jorgensen et al., 2000b).

Other foods like fruits, mayonnaise salads, eggs, beer, wine, juice, cereals and nuts are also

affected by microorganisms. Yeasts and filamentous fungi are the cause of spoiled fruits, cereals

and nuts, as LAB, Pediococcus species and yeasts could spoil mayonnaise salads, beer and wine

(Jespersen and Jakobsen 1996). Egg spoilages were due to Enterobacteriaceae and

pseudomonas. Wine can also be spoiled due to P. brevicompactum and A. flavis where substrate

used is 2, 4, 6 tri chloro phenol that effects to make product tri chloroanisol. Musty odour was

observed in wine spoilage (Filtenborg et al., 1996). Medicine off odour confirms juice spoilage

caused by A. Acidoterrestris where sugars are substrate and products are 2methoxy-phenol,

sediments (Walls and Chuyate, 2000). These are some other sources of food spoilage.

To overcome all this microorganisms invasion in food industry, many different techniques are

followed for the preservation and shelf life extension of food like chemical preservatives, heat

processing, vacuum packing, modified atmosphere packing and refrigeration (Alexander et al.,

2003). The method of microbial growth inhibition most appropriate to food is the use of food

preservatives. An ideal food preservative must be inexpensive, corrosion-free, low in toxicity,

and have good antimicrobial activity. The inhibitors available for practical use today are mainly

chemical preservations. However, the safety problems with chemical preservatives are receiving

5

growing attention and consequently natural preservatives for foods have high potential for the

food industry (Li & Yi, 2003; Xie, Zhong, Xu, & She, 2001).

Preservative methods are modified, use of antimicrobial film during refrigeration and vacuum

packing showed delay of microbial growth (Ouattara et al., 2000).Application of anti-microbial

compounds in the food showed good results in inhibiting microbial growth. Various

antimicrobial agents, such as organic acids, bacteriocins, and spice extracts have been shown to

delay spoilage (Abugroun et al., 1993, Miller et al., 1993, Hotchkiss, 1995; Ouattara et al.,

1997a, Ouattara et al., 1997b).Many extracts from medicine plant have been known to possess

antimicrobial effects and used for the purpose of food preservation and medicinal purposes

(Cowan, 1999; Lee, Chang, & Su, 2007; Rıo’s & Recio, 2005; Shelef, Smith, Stewart, & Fyte,

1998;Tassou, Koutsoumanis, & Nychas, 2000; Valero &Salmeroj, 2003). Application of the

extracts of E. lindleyanum DC in food industry not only facilitates anti-microbial and anti-

microbial activities, but also contributes to such pharmacological activities as food anti

oxidation, healthcare as well as food nutrients (Wang, Xiao, Xiao, & Lin, 2002).

1.1 NISIN:

Nisin is a Lantibiotic of class I bacteriocins that contain unusual amino acids (dehydroalanine

(Da), dehydrobutyrine (Db), lanthionine, and 3-methyllanthionine), exists as two variants; Nisin

A and Nisin Z that are differentiated by single amino acid. Histidine and asparagine are present

at position 27 in nisin A and nisin Z respectively. Difference in structure may not affect

antimicrobial activity but solubility and diffusion were affected as higher solubility and diffusion

were observed in Nisin Z when compared to nisin (De Vos et al., 1993 and Laridi et al., 2003).

Recently another variant of nisin was discovered and named as Nisin Q (Zendo et al., 2003).

6

Figure 1: Molecular structure of nisin. (Source : en.wikipedia.org)

Nisin is an effective bactericidal agent active against Gram-positive bacteria but inactive

against Gram-negative bacteria due to its lipopolysaccharide composition present on its outer cell

wall (Boziaris and Adams, 1999). Nisin can inhibit strains of Lactococcus, Streptococcus,

Micrococcus, Pediococcus, Lactobacillus, Listeria and Mycobacterium (Sahl et al., 1995). Nisin

susceptible Gram-positive spores like Bacillus and Clostridium spp. were more sensitive

than vegetative cells (Delves-Broughton et al., 1996). In spite of nisin’s property to inhibit gram

positive bacteria, they show resistance to nisin due to their capability of producing nisinase

enzyme which could inactivate (Abee et al., 1995).

In vegetative cells nisin effect is exerted at the cytoplasmic membrane. Pore produced due to

Nisin can disrupt the proton motive force and the pH equilibrium leads to ion leakage and

ATP hydrolysis causing cell death. Nisin can also bind to lipid II, a peptidoglycan precursor,

which makes nisin interfere with cell wall biosynthesis where it inhibits cell wall biosynthesis.

7

Such binding is also intrinsic to the ability of nisin to form pores (Bauer and Dicks, 2005,

Deegan et al., 2006).

Normally, lipopolysaccharide (LPS) acts as a barrier to the action of the nisin on the cytoplasmic

wall in Gram-negative cells which are resistant to nisin. In composition of gram negative

microorganisms outer layer which on the other hand, chelating agents, such as EDTA, confine

the divalent magnesium and calcium ions of the LPS causing destabilization of the layer. Thus,

the bacteriocin can be transported through the LPS layer and create pores in the cytoplasmic

membrane, causing a loss of the proton motive force and a leakage of intracellular nutrients

(Millette, Smoragiewicz, & Lacroix, 2004).

Vessoni Penna, Jozala, Gentille, Pessoa, and Cholewa (2006) confirmed chelating effect of

EDTA, reporting that the presence of EDTA was essential to improve nisin activity on E.

coli DH5-α expressing the recombinant green fluorescent protein (Gram-negative) growth

inhibition. Other studies have reported the nisin anti-bacterial activity in combination with

chelating agent toward Gram-negative bacteria (Fett, 2004) (Belfiore et al., 2007).

Nisin action against spores is caused by binding to sulfhydryl groups of protein residues (Morris,

Walsh, & Hansen, 1984). It was observed that spores became more sensitive to nisin the more

heat damaged they are, and it is an important factor in the use of nisin as a food preservative in

heat processed foods. For example, spores of the Clostridium anaerobe PA3679 which have

survived heat treatment for 3 min at 121.1 ºC are 10 times more sensitive to nisin than those

which have not been heat damaged (Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996).

Sensibility of spores to nisin varies, those of species like Bacillus stearothermophilus and

Clostridium thermosaccharolyticum being particularly susceptible, as all spores which open their

8

coats by mechanical rupture. Otherwise, spores can be sensitized towards nisin by heat

treatment (Immonen, 1999).Sensitivity of nisin to both vegetative cells and spores can vary

between genera and even between strains of the same species (Delves-Broughton, Blackburn,

Evans, & Hugenholtz, 1996).

In view of its strong activity against food pathogens, nisin has been approved by the World

Health Organization as a food preservative for use in the food industry. Nisin is considered as

safe food antimicrobial and has been approved as a generally recognized as safe additive in

pasteurized cheese spread and liquid egg in the US to inhibit Clostridium botulinum spores.

Nisin is primarily active against Gram-positive bacteria but when combined with a chelator, nisin

also can inhibit growth of some Gram-negative bacteria (Stevens et al., 1991; Ray, 1992)

thus itcan potentially be effective against a broad spectrum of bacteria. In food systems, effect of

nisin can be reduced by food characteristics like pH of foods ( Jung et al., 1992).

The direct application of Nisinto food surfaces will result in some loss of nisin activity due to

various food components like water drops when present on the outer layer of food, reduces the

activity of nisin by diluting it. A method to control the release of Nisin over a period of time

would add a microbiological hurdle downstream in the food distribution chain. Han and Floros

(2000) summarized the applications of antimicrobial packaging including nisin containing

packaging. Antimicrobial packaging films can provide a downstream hurdle via controlled

release scenario. There are two general categories of antimicrobialfilms, those in which

the antimicrobial agent migrates from the film and those that the agent remains within the film

material. Due to the nature of food, if the antimicrobial does not migrate from the film at least to

the food surface, it will have limited effect. Nisin containing packaging materials have had

9

potential applications to prevent many food borne pathogenic bacteria and their spores, C.

botulinum, Listeria monocytogenes (Hurst, 1981; Delves-Broughton, 1990; Ray, 1992)

1.2 Production of nisin:

Nisin is synthesized as a precursor peptide of 57 amino acids on ribosomes and biologically

active peptide (34 amino acids) is attained from precursor peptide through enzymatic

modifications by process of post translation. Firstly, some of serine and threonine residues in the

pre peptide are dehydrated to Da and Db, respectively. The double bonds of some of

the Da and Db residues react with the thiol group of a neighbouring cysteine residue to form the

respective thio ether rings of lanthionine and 3-methyllanthionine. Finally, the active nisin is

released and secreted by proteolytic cleavage of the leader peptide (23 amino acids). However,

the mechanism of the biosynthesis has not been satisfactorily characterized, especially formation

of the unusual amino acids and the regio- and stereo-specificities of the modification enzymes.

According to the biosynthetic peculiarity, fermentation method is very preferable for

practical production of nisin to enzymatic method with integration of multi-enzyme steps. L.

lactis IO-1 (JCM 7638) isolated in laboratory produces a peptide antibiotic, which was purified

and identified to be nisin Z, a natural nisin variant. Nisin Z differs from nisin A by a

single amino acid substitution (His27Asn). Because nisin A is hardly soluble at neutral pH, its

use as a preservative is limited at present. Nisin Z is more soluble than nisin A above pH 6

because the asparagine side chain is more polar than the histidine side chain. We have reported

the characterization of pH and heat stability, sensitivity to proteolytic enzymes and antimicrobial

10

spectrum of nisin Z. The results indicated nearly the same characteristics as those of nisin A.

Therefore, nisin Z should be used rather than nisin A as a food preservative. We also investigated

the relationship between the primary metaboliteproduction of nisin Z and lactic acid and cell

growth, which had so far not been clarified in detail.

Nisin is produced by fermentation of fluid milk or whey by strains of Lactococcus lactis, lactis.

Small particles are yielded from the resulting fermentation broth which is subsequently

concentrated and separated, spray dried. (Deegan et al., 2006, Taylor et al., 2007).

Complex media and well controlled parameters such as temperature and pH are essential for the

production of (Lv, Zhang, and Cong, 2005). Commercial media is recommended for the

cultivation of Lactococcus lactis as lowering of pH due to lactic acid accumulation seemed to be

the growth-limiting factor (De Vuyst and Vandamme, 1994). Large scale production is not

possible due to high cost. surplus of proteins (tryptone, peptone, meat extract, yeast extract) are

present in the recommended media for LAB culture and a substantial proportion of which remain

unconsumed, involving superfluous cost and hindering bacteriocin purification (Vasquez,

Gonzalez and Murado, 2006). Hence, search for cheaper formulations for nisin production. Other

options include residual media such as: whey (Mondragon- Parada et al., 2006), sugar molasses

and mussel-processing wastes for bacteriocins productions (Guerra and Pastrana, 2003).

Recently Vessoni Penna and Moraes (2002) have developed new formulations of sucrose with

asparagine’s(7.5–75 g l-1), potassium phosphate (6–18 g l-1), and Tween-80 (1–6 g l-1) in MRS

broth. This study revealed the increase in production of nisin by L.lactis. Another important

observation was the expression obtained with 5 g l-1 of sucrose with 29 g l-1 of asparagine in

11

MRS and a composition of 12.5 g l-1 of sucrose with 75 g l-1 of asparagines in MRS yielded the

same expression which might indicate a stationary phase in the latter case.

1.3 Nisin and Food borne pathogens:

Nisin is the bacteriocins that were authorized to use in the food industry by WHO World Health

Organization and exhibits antimicrobial activity against gram positive bacteria and spores (Arauz

et al., 2009). E.coli has been recognized as food borne pathogens where Entero hemorrhagic E.

coli (EHEC) is more virulent than other strains due to their low infectious dose and infection

severity (Buchanan and Doyle, 1997). As we know that nisin is not active against gram negative

bacteria but when combined with other chelating agents, it inhibits and exhibits sensitivity.

Combination of nisin and curvicin A at low pH show more sensitivity against E.coli (Ganzle et

al., 1999).

Nisin was demonstrated to act upon and kill germinated spores of B. anthracis prior to

development into elongated and dividing bacilli (Gut et al., 2008). Listeria innocua a non-

pathogenic species exhibit similar response to physical chemical or thermal treatments as

L.monocytogenes. Nisin can inhibit L.innocua as it is shown that its population is reduced at low

pH and nisin at 20ºC. Staphylococcus aureus can cause nausea, vomiting, retching, abdominal

cramping, sweating, chill, prostration, weak pulse, shock, shallow respiration and abnormal body

temperature. S.aureus is grown in meat products as they provide a favourable environment. S.

aureus can be inhibited by nisin (Millette et al., 2007). Therefore Nisin is an essential antibiotic

that can be used to control or inhibit many different types of pathogenic organisms.

12

1.4 Escherichia coli:

E.coli is considered to be a common microorganism of human large intestine. Until 1950’s, it

was recognized as non-pathogenic common cohabitants (Olsvik et al., 1991). E.coli is a gram

negative rod shaped bacterium that belongs to the large bacterial family Enterobacteriaceae

Escherichia coli is a zoonotic pathogen that is an important cause of human food borne and

waterborne disease, with a spectrum of illnesses ranging from asymptomatic carriage

and diarrhea to the sometimes fatal hemolytic uremic syndrome. Outbreaks of E.coli disease are

often associated with undercooked beef, but there are other sources of transmission, including

water, produce, and animal contact, which can often be linked directly or indirectly to cattle.

Thus, pre harvest control of this pathogen in cattle production should have a large impact on

reducing the risk of human food borne illness (Berry et al., 2010).

E.coli is an important food borne pathogen and a member of verocytotoxigenic. E.coli is a

common type of bacteria that can cause food poisoning in human being and cause several

diseases and human infections through foods of animal origin and dairy products. This organism

can also be transmitted from one person to another through improper hygiene, contaminated

water, raw milk and spoiled vegetables (Nastasijevica et al., 2009).

E. coli bacteria have been commonly found in recreational waters, and their presence is used to

indicate the presence of recent faecal contamination, but E. coli presence may not be indicative

of human waste.

13

1.5 Listeria innocua:

Listeria innocua is a species belonging to the genus Listeria. The species Listeria has an ability

to survive in extreme conditions like pH, temperatures and salt content. Listeria is a gram

positive rod shaped, non-spore forming bacterium. It resembles the pathogenic monocytogenes.

Listeria innocua shows several similar characteristics with that of Listeria monocytogenes like

morphological, biochemical, and molecular characteristics. It is very difficult to differentiate

L.innocua from other species like L.monocytogenes when they both are together in laboratory

(Liu et al., 2003).

The presence of Listeria species in pasteurized products helps them in getting resistance power

against heat L. innocua is used as a biological indicator of L. monocytogenes in food industries as

it consists of majority of strains. Listeria innocua shares the natural environments and can be

easily isolated in the particular food products (Miller et al., 2009).

It is most commonly found in food products it is not surprising that fish, squids, crustaceans and

other seafood have been found to contain the bacteria and in open environment. Listeria innocua

has a natural habitat of decaying plant material where they live as saprophytes. Listeria innocua

can be grown at refrigeration temperatures and are able to survive even at frozen conditions. In

order to inactivate Listeria innocua, ultra sound was used (Aguirre et al., 2009).

Listeria species are resistant to nisin, bacteriocins. It can become sensitive to nisin when other

foods are combined with nisin in food preservation technologies (Miranda et al., 1999) .

14

1.6 Staphylococcus aureus:

Staphylococcus is a gram positive, spherical bacterium. Rosenbach identified S.aureus in 1884.

S.aureus is a facultative anaerobe which is grown by aerobic respiration. It can survive even at

high temperatures ranging from 15oC to 45oC. It is a versatile organism which consists of several

virulent characteristics. It also has a significant nature of causing several infectious diseases in

humans. S.aureus can even cause life threatening infections like bacteremia, endocarditis and

pneumonia (Kanajani and Flower, 2006).

About 20% human population are long term carriers of S.aureus.It is still one of the five most

common causes of nosocomial infections, often causing postsurgical wound infections. Strains

are responsible for food poisoning through the production of an enterotoxin, and pathogenicity is

also associated with coagulase positivity. S. aureus may occur as a commensal on skin.

S.aureus is a significant pathogen used in both human and animal medicine. More than 80% of

S.aureus produces penicillinases. Metacillin is a β lactam antibiotic which is resistant to

penicillinase which is widely used to treat infections caused by S. aureus. In late 1950’s

Metacillin was first introduced in human medicine, which is used for treating penicillin-resistant

Staphylococcal infections (Leonard and Markey, 2008).

15

1.7 Project Aims:

The main aim of this study is to determine the inhibitory efficacy of nisin with particular

reference to two gram positive bacteria i.e., S. aureus and L. innocua and one gram negative

bacteria i.e., E. coli .

To know the inhibitory efficacy, the nisin was subjected to different methods like MIC Minimal

Inhibitory Concentration, Miles and Misra, Well Inhibition method, Spiral Plate Technique and

Bioscreen C. This study further relates the use of nisin against bacterial activity in milk.

16

Chapter 2

Materials and Methods

17

2.1 Microorganisms:

Gram positive strains of Staphylococcus aureus and Listeria innocua and a gram negative strain

of Escherichia coli were used in the experiment. All the bacterial strains were used in indigenous

and were obtained from University of Wolverhampton.

2.2 Media preparation:

Two types of media were used, Trypton soya broth TSA and Trypton soya agar TSA which can

be obtained from university laboratory.

a) TSA preparation-

Weighed out appropriate amounts of TSA i.e., 7.4g TSA into weighing boats. Weighing boats

were pinched to allow transfer of agar into medial flat. 250 ml of distilled water was added using

a measuring cylinder and shaked the bottle. Capped the bottle and sealed with the autoclave tape.

This media was then kept for autoclaving at 121’c for 15 min. The prepared TSA was kept at

50’c and was allowed to cool to 45’c prior pouring. Approximately 15ml of TSA was poured in

each petri dish.

b) Double strength TSB preparation-

Weighed out appropriate amount of TSB i.e., 15g of TSB into weighing boats. Transferred this

measured TSB into a beaker, added distilled water of 250ml to that beaker using measuring

cylinder and add magnetic flea, then place the beaker in magnetic stirrer to dissolve. Transferred

this dissolved TSB into a conical flask and foam bung was added to the foiled fitted cap then

kept for autoclaving at 121’c for 15min.

18

c) Preparation of Milk model media-

Weighed out 0.5g of skimmed milk powder, 2.5g of peptone and 1.5g of yeast extract into

weighing boats. Transferred this into a conical flask and 500ml of distilled water was added

using a measuring cylinder and add magnetic flea, then place the beaker in magnetic stirrer to

dissolve. Transferred this dissolved milk media into a conical flask and foam bung was added to

the foiled fitted cap then kept for autoclaving at 121’c for 15min.

d) Preparation of Nisin stock solution-

Three different concentrations of Nisin stock solution were prepared i.e., 3%, 5%, 10%

respectively. 3% stock solution was prepared by adding 0.45g of Nisin in 15ml of distilled water

and mixed well. This stock solution was filter sterilized. 5% stock solution was prepared by

adding 0.5g of Nisin in 10 ml of distilled water and mixed well. This stock solution was filter

sterilized. 10% stock solution was prepared by adding 1g of Nisin in 10 ml of distilled water and

mixed well. This stock solution was filter sterilized.

19

Methods:

20

2.3 Minimum Inhibition Concentration:

Minimum Inhibition Concentration assay is a technique used to determine the lowest

concentration of a particular antibiotic needed to kill bacteria. This assay is typically performed

on planktonic (free floating) bacterial cells. Therefore, at a particular concentration, the effective

antimicrobial should exhibit its activity against given bacterial cultures. If the given

antimicrobial did not show any effect on particular pathogen, then it can be concluded that it is

not effective agent to be used.

Procedure for Minimal Inhibition Concentration technique:

Prepare 20 test tubes containing 2.5ml double strength TSB, enough tubes to undertake the MIC

in duplicate plus some spares if needed.

Label tubes ½, ¼, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, +ve control and –ve control in duplicates

as well. Aseptically transfer 2.5ml of test sample e.g. effluent water to 1st tube to produce the ½

dilution. Whirl mix tube and transfer 2.5ml to the next tube to produce ¼ dilution.

Repeat the procedure until 1/256 dilution has been reached. Next repeat the whole process for 2nd

set of tubes.

Add 2.5ml of test sample to –ve control tube. This will determine whether you have any bacteria

already present and which may affect the results obtained.

Aseptically transfer 1 drop of reference organism, to each dilution tube and the +ve control, for

both set of the tubes.

21

Do not add any culture to –ve control tubes. The +ve control tube will illustrate normal growth of

reference organism, without any sample influence.

An overnight culture of reference organism will be required to be set up the day before

undertaking of MIC technique.

Incubate these tubes at appropriate temperature for 24/ 48hrs and score tubes accordingly,

utilizing a ‘+’ system i.e. + = low turbidity, ++ = medium turbidity and +++ = high turbidity.

This is an arbitrary system.

Whirl mix them before reading the tubes. If growth is seen in all dilutions, repeat the experiment

starting with a lower sample concentration.

If growth is seen in –ve control tube, then the stock solution is to be filter sterilized using either a

0.45 μm or a 0.22μm Sartorius sterile disposable filter, before repeating MIC experiment.

Compare both sets of tubes with each other, quite carefully, as often a slight discrepancy can

arise between the 2 sets of tubes.

2.4 Miles and Misra:

Mark agar plates into 8 sectors on the base, labelled 10-1 to 10-8. Also label with name and date.

Starting with the most dilute (10-8), transfer 20μl of the suspension onto the sector labelled 10-8.

Repeat for the same dilution. Repeat for each other dilution, using the same tip throughout. Do

not move plate until all droplets have soaked into the agar. Incubate these plates at 37oC for 24

hrs.

22

2.5 Well inhibition test:

It is a method used in measuring the effectiveness of an antimicrobial agent against bacteria

grown in culture. This test is also called as Zone of inhibition test. This test is performed in order

to know the antimicrobial activity of nisin to inhibit the growth of gram positive and gram

negative bacteria.

Procedure for well inhibition test:

A microbial suspension was spread by a sterile swab, evenly, over the face of a sterile agar plate.

The antimicrobial agent about 40μl is applied to the well made on the agar plate (in a fashion

such that the antimicrobial doesn't spread out from the centre) and incubated at 37 oC for 24 hrs.

If substantial antimicrobial activity is present, then a zone of inhibition appears around the test

product.

The zone of inhibition is simply the area on the agar plate that remains free from microbial

growth.

The size of the zone of inhibition is usually related to the antimicrobial activity present in the

sample or product - a larger zone of inhibition usually means that the antimicrobial is more

potent.

2.6 Spiral plate technique:

A method is described for determining the number of bacteria in a solution by the use of a

machine which deposits a known volume of sample on a rotating agar plate in an ever decreasing

23

amount in the form of an Archimedes spiral. After the sample is incubated, different colony

densities are apparent on the surface of the plate. A modified counting grid is described which

relates area of the plate to volume of sample. By counting an appropriate area of the plate, the

number of bacteria in the sample is estimated. This method was compared to the pour plate

procedure with the use of pure and mixed cultures in water and milk.

Spiral platers are used extensively in the food industry to test for safe levels of bacteria - non-

food applications include pharmaceuticals, cosmetics, water and mouth and skin bacteria. The

benefits of spiral plating compared to conventional methods include minimal set up time, the

elimination of serial dilutions, up to 69% labour saving, a reduction in consumable costs and

laboratory waste with up to a two thirds reduction in incubator space requirements.

a) Plating Procedure for sample

Turn on vacuum.

Turn on power, ready light on, and set unit to automatic.

Check stylus tip angle daily and adjust as necessary.

1. Tip of stylus touches back of arc marking the starting point on the turntable, tip OK

2. Tip of stylus does not touch back of arc marking the starting point on the turntable, adjust tip

and check using

Use vacuum to hold a microscope cover slip or equivalent against the face of the stylus and hold

stylus/cover slip about 1 mm above platform surface, if parallel using level gauge proceed, if not

adjust and recheck. Then run dye solution (item 16) to assure spiral plater was dispensing liquid

24

uniformly over plate surface. Filled one 5 ml beaker (or approved equivalent) with sterile water

and another with 5% Sodium hypochlorite solution (or approved equivalent). Stylus tip was

cleaned by rinsing for 1 second in sodium hypochlorite solution (item 12) 3x and then in sterile

water 3x prior to introducing each sample. Labelled plate with sample information and make a

vertical mark on the side of the plate bottom to indicate the start of sample deposition. Then tip

was inserted into agitated sample in rigid container, or poured into sterile 5 ml beaker, or

approved equivalent, avoiding foam. Open vacuum filling valve.

a. Draw up sample through sight glass and close valve.

b. Assure that there is a solid column of sample in the sight glass, i.e. no bubbles.

c. Lift stylus out of sample and touch off excess sample onto dry area of sample container.

d. Place agar plate on platform and remove cover.

e. Place stylus tip on agar surface and start motor.

f. After inoculation, when stylus lifts from agar surface and moves to starting position

immediately remove plate and replace lid.

g. Repeat f - n for each sample to be tested.

25

h. After absorption of liquid, invert plate and place in 32C incubator within 20 minutes.

After all samples and controls have been plated, repeat step. Turn off power and vacuum (as

advised by Andy Brooke, Senior Lab Technician, Wolverhampton University).

The spiral plate method gives the counts that were higher than counts obtained by the pour plate

method. The time and materials required for this method are substantially less than those

required for the conventional aerobic pour plate procedure.

Figure 2: spiral plater equipment (Source: googlescholar.com)

26

Counting grid: The counting grid shown in Fig. is used to relate the colonies on a spiral plate to

the volume in which they were contained. The grid covers the area of an agar plate and was

prepared by dividing the area into four concentric circles and into eight wedges or octants. Thus,

each octant is subdivided into four arcs. The relation of liquid volume to area was determined for

different portions of an octant and is shown as the shaded areas in Fig. On each plate, only an

area with well-separated colonies is counted and the area which is counted is dependent upon the

number of colonies. Colonies are counted in one octant from the outer edge towards the center

until at least 25 colonies are observed. The remainder of that arc where the 25th count occurred

must be counted. A like area of an opposite octant is counted, and that count is added to the first

count. If there are not 25 colonies on one octant, then the colonies on the total plate are counted.

The colony count is divided by the liquid volume corresponding to the area over which the

colony count was obtained, resulting in the bacterial concentration of the sample.

Figure 3: counting grid for spiral plater results (Source: google.co.uk)

27

2.7 Bioscreen:

Bioscreen C is a well-controlled incubator equipped with 8 filters attached to a computer. This

incubator has a very sensitive than a spectrophotometer. Bioscreen C is a multipurpose

instrument consists of a various instruments together like incubator, shaker, and turbidity reader.

Bioscreen C incubator is extremely versatile since any microorganisms can be grown with any

liquid growth medium and with any chemical. All these activities can be processed by using user

created test protocol.

Bioscreen was used to compare cell numbers of microorganisms after growth in test and control

broths. The software converts kinetically measured optical density (O.D.) data into growth

curves and, by constructing appropriate calibration graphs, the data can be interpreted as

bacterial counts. The Bioscreen sample plates are sterile, disposable and can take up to 200

samples per test, enabling replication to be easily performed. This micro plate is also called as

“Honeycomb” which is highly suitable for accurate temperature control. This method eliminates

the need for time consuming serial dilutions, whilst also reducing the amount of test materials

required. O.D. measurement proved to be a cheap, fast and reliable method for assessing the

quality of liquid media. Bioscreen C incubator is used to calculate the growth of pure and also

mixed bacteria culture.

28

Figure 4: Bioscreen C equipment (Source: oxfordjournal.org)

a) Procedure for Bioscreen C technique:

One honeycomb plate with 100 wells was used to find the inhibitory efficacy of different

concentrations of nisin. Then 10 fold serial dilutions were performed with nisin and TSB and

nisin and milk model media. Ten test tubes were taken for the first set and all these test tubes

were added with 2.5ml TSB and second set were added with 2.5ml milk model media. To the

first test tube, different concentrations of nisin solution were added and serially diluted to 10 -8

dilution.

The same steps were followed by taking different concentrations of nisin solutions for different

sets of test tubes. Different indicator organisms were taken and inoculated into different sets of

29

test tubes containing different concentrations on nisin solution. 100μl of bacterial culture from

first test tube was transferred to first well. All the culture samples from first 10 test tubes without

inoculum were transferred into initial ten wells in honeycomb plate. Different cultures of

indicator organisms were transferred to the next set of wells from different test tubes by

following the same procedure. To avoid contamination, the entire process was done in the

microbiology chamber in a sterile atmosphere.

After all the wells were filled, the plate was filled with a honeycomb lid. Bioscreen C incubator

was switched on and the computer attached to the Bioscreen C should be turned on before

starting the incubation process (as advised by Andy Brooke, Senior Lab Technician,

Wolverhampton University).

Place the honeycomb plate with the culture in the sample tray. Open the excel sheet in the

computer attached to the Bioscreen C and adjust the temperature, running time and optical

density 420nm based on the experiment protocol. After checking all the requirements, the

experiment was started. All the samples in the honeycomb plate are placed in the sample tray and

incubation process at 37oC for 12 hrs.

30

Chapter 3RESULTS

31

3.1 Results for Minimum Inhibitory Concentration (MIC) in TSB:

The Minimum Inhibitory Concentration is a test performed to determine the inhibitory effect of

bacteriocin Nisin against three different microorganisms like L. innocua, S. aureus and E. coli.

MIC was done at different concentrations of Nisin. The concentrations used in this experiment

are 15 mg/ml, 25 mg/ml and 50 mg/ml of Nisin. In the initial test, 15 mg/ml Nisin solution is

used. After 24hr incubation, the samples were tested for the turbidity of culture grown in the test

tubes.

The results for the nisin concentration of 3% against all three indicator organisms are shown in the table 1.

Dilution 1:1 1:2 1:4 1:16 1:32 1:64 1:128 1:256+ve control

-ve control

Con. of Nisin in dilutions (mg/ml)

15 7.5 3.75 1.875 0.9375 0.4687 0.2343 0.1171

E.coli +++ +++ +++ +++ +++ +++ +++ +++ +++ -

S. aureus - ++ ++ +++ +++ +++ +++ +++ +++ -

L. innocua - ++ ++ +++ +++ +++ +++ +++ +++ -

Table 1: Shows MIC of Nisin solution 3% against different organisms.

Where - = Clear, ++ = Slight Turbidity, +++ = Turbidity.

32

From table 1 results, it can be concluded that Nisin did not show much effect on all indicator

organisms at 15 mg/ml concentration. In the first test tubes of all three microorganisms, there is

no growth seen. It is shown that Nisin can inhibit the growth of all organisms except E.coli. But

in the rest of the tubes, no inhibition is seen. In the second dilution of both gram positive

organisms L. innocua and S. aureus, slight turbidity is seen which says that Nisin at 7.5mg/ml

can inhibit the growth to some extent.

When the test is performed with 5% Nisin solution, the results obtained are shown in the table 2.


-ve control


25 12.5 6.25 3.125 1.562 0.7812 0.3906 0.1953

E.coli +++ +++ +++ +++ +++ +++ +++ +++ +++ -

S. aureus - - - ++ ++ +++ +++ +++ +++ -

L. innocua - - - ++ ++ +++ +++ +++ +++ -

Table 2: Shows MIC of Nisin solution (25 mg/ml) against different indicator organisms.


The Nisin solution shows good inhibitory effect at concentration of 25 mg/ml against both gram

positive organisms. According to MIC results obtained in the above table, it clearly explains that

Nisin is more effective against L. innocua and S. aureus. Both gram positive organisms showed

33

effect at 25mg/ml, 12.5 mg/ml, 6.25 mg/ml. Slight turbidity is seen which says that Nisin at

3.125 mg/ml can inhibit the growth to some extent.

When the test is performed with 10% Nisin solution, the results obtained are shown in the table 3.


-ve control


50 25 12.5 6.25 3.125 1.562 0.7812 0.3906

E.coli - ++ ++ +++ +++ +++ +++ +++ +++ -

S. aureus - - - - ++ ++ +++ +++ +++ -

L. innocua - - - ++ ++ ++ +++ +++ +++ -

Table 3: Shows MIC of Nisin solution 50 mg/ml against different organisms.


From table 3, it can be concluded that Nisin show effect on all indicator organisms at 50 mg/ml

concentration. In the first test tubes of all three microorganisms, there is no growth seen. It is

shown that Nisin can inhibit the growth of all organisms even gram –ve E.coli at 50 mg/ml conc.

But in the rest of the tubes of E. coli, no inhibition is seen. In the next dilutions of both gram

positive organisms L. innocua and S. aureus, clear solution is seen in the next two tubes which

says that Nisin at 50 mg/ml can inhibit the growth even at 6.25 mg/ml. It is concluded that Nisin

is more effective against S. aureus than that of L. innocua.

34

3.2 Results for Minimum Inhibitory Concentration (MIC) in Milk media:

The Minimum Inhibitory Concentration is a test performed to determine the inhibitory effect of

bacteriocin Nisin against three different microorganisms L. innocua, S. aureus and E. coli. MIC

was done at different concentrations of Nisin. The concentrations used in this experiment are 15

mg/ml, 25 mg/ml, and 50 mg/ml Nisin. In the initial test, 15 mg/ml Nisin solution was used.

After 24hr incubation, the samples were tested for the turbidity of culture grown in the test tubes.

In this experiment, milk model media is used instead of TSB.

The results for the nisin concentration of 15 mg/ml against all three indicator organisms are shown in the table 4.


-ve control


15 7.5 3.75 1.875 0.9375 0.4687 0.2343 0.117

E.coli +++ +++ +++ +++ +++ +++ +++ +++ +++ -

S. aureus - - ++ ++ +++ +++ +++ +++ +++ -

L. innocua - ++ ++ +++ +++ +++ +++ +++ +++ -



From the table 4 results, it can be concluded that Nisin did not show much effect on all indicator

organisms at 15 mg/ml concentration. In the first test tubes of all indicator microorganisms, there

35

is no growth seen. It is shown that Nisin can inhibit the growth of all organisms except E.coli.

But in the rest of the tubes, no inhibition is seen. In the second dilution of both gram positive

organisms L. innocua and S. aureus, slight turbidity is seen which says that Nisin at 7.5mg/ml

can inhibit the growth of L. innocua to some extent and 3.75 mg/ml can inhibit the growth of S.

aureus to some extent.

When the test is performed with 25 mg/ml Nisin solution, the results obtained are shown in the table 5.


-ve control


25 12.5 6.25 3.125 1.562 0.7812

0.3906 0.1953

E.coli ++ ++ +++ +++ +++ +++ +++ +++ +++ -

S. aureus - - - ++ ++ +++ +++ +++ +++ -

L. innocua - - ++ ++ ++ +++ +++ +++ +++ -

Table 5: Shows MIC of Nisin solution (25 mg/ml) against different indicator organisms.


The Nisin solution shows good inhibitory effect at concentration of 25 mg/ml against both gram

positive organisms. According to MIC results obtained in the above table, it clearly explains that

Nisin is more effective against L. innocua and S. aureus. Both gram positive organisms showed

36

effect at 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml. In this experiment, S. aureus is

more inhibited than L. innocua at the least concentration.

When the test is performed with 50 mg/ml Nisin solution, the results obtained are shown in the table 6.

Dilution

1:1 1:2 1:4 1:16 1:32 1:64 1:128 1:256+ve control

-ve control


50 25 12.5 6.25 3.125 1.562 0.7812 0.3906

E.coli - - ++ ++ +++ +++ +++ +++ +++ -

S. aureus - - - - ++ ++ ++ +++ +++ -

L. innocua - - - - ++ ++ ++ +++ +++ -



From the table 6 results, it can be concluded that Nisin show effect on all indicator organisms at

50 mg/ml concentration. In the first test tubes of all three microorganisms, there is no growth

seen. It is shown that Nisin can inhibit the growth of all organisms even gram –ve E.coli at 50

mg/ml conc. But in the rest of the tubes of E. coli, no inhibition is seen. In the next dilutions of

both gram positive organisms L. innocua and S. aureus, clear solution is seen in the next two

tubes which says that Nisin at 50 mg/ml can inhibit the growth of S. aureus even at 3.125mg/ml

and can inhibit growth of L. innocua at 6.25 mg/ml.

37

It is concluded that Nisin is more effective against gram positive organisms that of gram negative

organisms in milk media than in TSB because milk media as got more nutrients.

3.3 Results for Miles and Misra of Milk media with nisin:

Growth of indicator organisms in Milk media with Nisin: The growth of indicator organisms

like L. innocua, S. aureus and E. coli in Milk media were determined by using viable cell count

method. In this experiment, 50 mg/ml of Nisin is added and the viable count of the growth of

different indicator organisms is calculated after 24 hrs. All the results were taken by using Miles

and Misra technique. From the results obtained, the viable count of the dilution 10 -3 is considered

and obtained valued are shown. The calculations for viable count are done as follows:

Viable count calculations:

For E. coli:

Volume delivered on to 10-3 dilution = 20μl (0.02cm3)

Colonies found in 0.02 cm3 of 10-3 dilution = 98

Colonies present in 1.0cm3 of 10-3 dilution = 98×1/0.02×1/10-3

= 98×50×103

= 98×5×104

= 490×104

= 49×105

Log values of 49×105= 6.69 Log CFU/ml.

38

ForS. aureus:




= 42×50×103

= 42×5×104

= 210×104

= 21×105

Log values of 21×105= 6.32 Log CFU/ml

For L. innocua:




= 60×50×103

= 60×5×104

= 300×104

= 30×105


All the viable count calculations are taken in Colony Forming Units method. The values are

taken in consideration from 10-3 dilutions and the obtained graph is shown in the graph below. In

the graph, it shows that all the indicator organisms grow in similar growth value in the 10 -3

dilutions. Very little difference can be seen in the growth of gram –ve organisms and gram +ve

organisms.

39

3.4 Results for Miles and Misra of Milk media without nisin:

Growth of indicator organisms in Milk media without nisin: The growth of indicator

organisms like L. innocua, S. aureus and E. coli in Milk media were determined by using viable

cell count method. In this experiment, nisin is not added and viable count of the growth of

different indicator organisms is calculated after 24 hrs. All the results were taken by using Miles

and Misra technique. From the results obtained, the growth count of the dilution 10 -5 is

considered and obtained valued are shown. The calculations for viable count are done as follows:

Viable count calculations:

For E. coli:

Volume delivered on to 10-5 dilution =20μl (0.02cm3)



= 32×50×105

= 16×107

Log values of 16×107= 8.20 Log CFU/ml.

ForS. aureus:



Colonies present in 1.0cm3 of 10-5dilution = 30×1/0.02×1/105

= 30×50×105

= 30×5×106

= 150×106

= 15×107


40

For L. innocua:




= 28×50×105

= 28×5×106

= 14×107


E.coli S.aureus L. innocua0

1

2

3

4

5

6

7

8

9

milk media with nisin

milk media without nisin

organisms

Log

of v

iabl

e co

unt (

CFU

/ml)

Figure 5: Analyzing the growth of E.coli, S.aureus and L.innocua with and without nisin in milk media

stored at 37oC for 24 hrs. (n=2).

41

Generally, TSB is used to grow all types of bacteria and is called as an ideal medium. All the

indicator organisms are inoculated in TSB at 370C for 24hrs. After incubation period, the viable

count of bacteria was determined by using Miles and Misra technique. According to the results

shown in graph 4 and graph 5, there is a difference in the growth of microorganisms growing in

milk media with nisin and milk media without nisin. Among the indicator organisms, E. coli

shows more growth. From the results, it is showed TSB is more favorable for microorganisms

than milk media. All these results are taken from 10-3 and 10-5 dilutions and the log values are

taken for the viable count obtained from the result.

3.5 Results for Well Inhibition Method:

Zone of Inhibition is a method used to know the inhibitory efficacy of Nisin by the formation of

zones on agar plates. The agar plates containing of desired cultures are incubated for 24hrs. After

incubation, clear zones are formed around the wells that are on the agar plates. These zones are

called as “Zones of Inhibition”. The size of zones of inhibition formed on agar plates is directly

proportional to the effect of bacteriocin Nisin. The larger the clear area around the well more is

the effectiveness of the compound. 15 mg/ml, 25 mg/ml and 50 mg/ml of Nisin is used in this

test and only S. aureus is used in this test to know the inhibitory efficacy of Nisin.

Organism Conc of Nisin Diameter of zone of inhibition in cm

S. aureus 15 mg/ml 0.9cm25 mg/ml 1.0cm50 mg/ml 1.2cm

Table7: Shows the diameter of zone of inhibition formed by Nisin around the well

42

Only S. aureus is used in this experiment. For this test, 40μl of nisin solution is used to find the

efficacy of Nisin. The growth of gram negative organisms can be inhibited if Nisin is used in

high concentrations. For S. aureus, 3% Nisin showed 0.9cm of zone of inhibition, 5% Nisin

showed 1.0cm of zone of inhibition and 10% Nisin showed 1.2cm of zone of inhibition.

3.6 Results for Spiral Plate Technique:

Pearson and Hartley have presented the test and tables used to examine the assumption of

normality in the spiral plate technique.

Spiral plate technique results are shown in table 8:

Plate Conc of Nisin Organism CountColony forming unit/ml of culture in TSB (cfu/ml)

1 3% E. coli

2 3% S. aureus 35 colonies 2.8×103

3 3% L. innocua 69 colonies 1.3×105

4 5% E. coli

5 5% S.aureus 35 colonies 7.7×103


7 10% E. coli

8 10% S. aureus 51 colonies 1.0×103


10 3% 10-10S. aureus 37 colonies 7.4×102



43

In this technique, the results that were observed are not accurate as there is a technical error with

the spiral plater equipment, there was no further progress in this experiment. This is an attempt to

use spiral plater in the experiment which reduces the time and work in viable counting. But the

results of spiral plater are a bit similar to that of the other techniques in this experiment.

44

3.7 Results for Bioscreen C method when done with TSB:

The results of bioscreen C explains the inhibition of growth pattern of different organisms

obtained through the experiment. The growth curves of indicator organisms were plotted with

respect to time in min against optical density. The OD values on Y axis measures the growth of

bacteria.

0 100 200 300 400 500 600 700

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

25 mg/ml12.5 mg/ml6.25 mg/ml3.125 mg/ml1.562 mg/ml0.7812 mg/ml0.3906 mg/ml0.1953 mg/ml+ve control-ve control

Time (min)

O.D

. at 4

20 n

m

Figure 6: Effect of nisin on the growth of E.coli in TSB with the scale time on X-axis and O.D. on Y-axis (n=2)

The effect of nisin on growth of gram negative and gram positive organisms like E. coli, S.

aureus and L. innocua are estimated by using Bioscreen C. The effect of nisin concentration of

25 mg/ml is estimated on the growth of E. coli. Nisin along with TSB is serially diluted and E.

coli is introduced to the test tubes and the samples are transferred into honey comb plate in wells

21 to 30. Positive and negative controls are in 29th and 30th wells. These positive and negative

45

controls are taken for each organism and the growth value of culture in different concentration is

compared to positive control. In the above figure, shows the results for E. coli against 25 mg/ml

Nisin in TSB. As there is growth of the organism at all times, it says that there is no effect of

Nisin on E. coli. 25 mg/ml con. of nisin killed the bacteria but lower con. levels of nisin inhibited

the bacterial growth.

0 100 200 300 400 500 600 700

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Time (min)

O.D

. at 4

20 n

m

Figure 7: Effect of nisin on the growth of S.aureus in TSB with the scale time on X-axis and O.D. on Y-axis (n=2)

Fig. 7 shows the effect of nisin on S. aureus in TSB. Positive control shows that the growth of S.

aureus in the absence of nisin and the negative control shows the presence of nisin in TSB not

with any culture in it. . Nisin along with TSB is serially diluted and S. aureus is introduced to the

test tubes and the samples are transferred into honey comb plate in wells 51 to 60. The highest

46

concentration of nisin i.e., 25mg/ml shows the effect of nisin by inhibition as there is some

growth in initial hours and the growth stops and comes to log phase. Even 12.5 mg/ml, 6.25

mg/ml and 3.12 mg/ml concentrations of nisin is effective against S. aureus as there is no growth

seen at that concentration. There is some inhibition of growth at lower concentration.

0 100 200 300 400 500 600 700

-0.400

-0.300

-0.200

-0.100

0.000

0.100

0.200

0.300

0.400

0.500


Time (min)

O.D

. at 4

20 n

m

Figure 8: Effect of nisin on the growth of L.innocua in TSB with the scale time on X-axis and O.D. on Y-axis (n=2)

Fig. 8 shows the effect of nisin on L. innocua in TSB. Positive control shows that the growth of

L. innocua in the absence of nisin and the negative control shows the presence of nisin in TSB

not with any culture in it. Nisin along with TSB is serially diluted and L. innocua is introduced to

47

the test tubes and the samples are transferred into honey comb plate in wells 81 to 90. At

12.5mg/ml, 6.25mg/ml concentrations, nisin inhibited the growth of L. innocua completely. As

the concentration of nisin decreases, the growth of the organism increased.

3.8 Results for Bioscreen C method when done with Milk media:

0 50 100 150 200 250 300 350 4000

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Time (min)

O.D

. at 4

20 n

m

Figure 9: Effect of nisin on the growth of E.coli in milk media with the scale time on X-axis and O.D. on Y-axis (n=2)

The effect of nisin concentration of 25 mg/ml is estimated on the growth of E. coli in milk

media. Nisin along with milk media is serially diluted and E. coli is introduced to the test tubes

and the samples are transferred into honey comb plate in wells 21 to 30. Positive and negative

48

controls are in 29th and 30th wells. These positive and negative controls are taken for each

organism and the growth value of culture in different concentration is compared to positive

control. In the above figure, shows the results for E. coli against 25 mg/ml Nisin in milk media.

As there is growth of the organism at all times, it says that there is no effect of Nisin on E. coli

even in Milk media as well.

0 50 100 150 200 250 300 350 4000

0.1

0.2

0.3

0.4

0.5

0.6


Time (min)

O.D

. a t

420

nm

Figure 10: Effect of nisin on the growth of S.aureus in milk media with the scale time on X-axis and O.D. on Y-axis (n=2)

Fig. 10 shows the effect of nisin on S. aureus in milk media. Positive control shows that the

growth of S. aureus in the absence of nisin and the negative control shows the presence of nisin

in milk media not with any culture in it. . Nisin along with milk media is serially diluted and S.

aureus is introduced to the test tubes and the samples are transferred into honey comb plate in

49

wells 51 to 60. The highest concentration of nisin in the graph is 0.125mg/ml shows the effect of

nisin as there is some no growth of the organism is seen. Even 0.0625mg/ml, 0.03125mg/ml

concentrations of nisin is effective against S. aureus as there is no growth seen at that

concentration. 0.03125mg/ml conc of nisin shows there is effective growth of the organism for

some time and suddenly the growth of the organism decreased and gone to log phase. It shows

that as the concentration of nisin decreases, the growth of S. aureus is increased according to the

above graph. When S. aureus is treated with 5% nisin in milk media, there is more effect of nisin

in milk media than in TSB.

0 50 100 150 200 250 300 350 400

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

25 mg/ml12.5 mg/ml6.25 mg/ml3.125 mg/ml1.562 mg/ml0,7812 mg/ml0.3906 mg/ml0.1953 mg/ml+ve control -ve control

Time (min)

O.D

. at 4

20 n

m

Figure 11: Effect of nisin on the growth of L.innocua in milk media with the scale time on X-axis and O.D. on Y-axis (n=2)

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Fig 11 shows the effect of nisin on L. innocua in milk media. Positive control shows that the

growth of L. innocua in the absence of nisin and the negative control shows the presence of nisin

in milk media not with any culture in it. Nisin along with milk media is serially diluted and L.

innocua is introduced to the test tubes and the samples are transferred into honey comb plate in

wells 81 to 90. When L. innocua is treated with 5% nisin in milk media, there is more effect of

nisin in milk media than in TSB.

51

Chapter 4Discussion

52

Non-thermal treatments are attracting interest of the food industry due to their capability of

assuring the quality and safety of food. Among them, bacteriocins from lactic acid bacteria, such

as Nisin may be potentially useful for the dairy industry. Bacteriocins are gaining interest

because of their wide antibacterial spectrum with feasible application in foods, such as meat and

fish products, fruits and vegetables, cereals and beverages. Moreover, LAB-derived bacteriocins

are generally recognized as safe and are attractive to the food industry because of their activity

against key Gram-positive pathogens involved in food-borne illnesses, such as Listeria

innocua or Staphylococcus (A. Sobrino-Lopez and O. Martin-Belloso, 2007).

Nisin is an effective bacteriocin used against food spoilage organisms like L. innocua, S. aureus

and E. coli in this study. The effect of nisin on the growth of different organisms was tested by

using MIC, Miles and Misra, Well Inhibition method and Bioscreen C technique. The results

obtained by these experiments explain that nisin shows inhibitory efficacy against gram positive

organisms like S. aureus and L. innocua and stops the growth of these organisms in TSB and

milk media.

The most extensively studied bacteriocin is nisin, it is the only one approved for food

applications and it has gained widespread application in the food industry. The bacteriocin was

first marketed in England in 1953 and since then has been approved for use in over 48 countries.

Significantly, nisin was assessed to be safe for food by the Joint Food and Agriculture

Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives in

1969. Other bacteriocins, such as pediocin (class IIa, of which antilisterial activity is

characteristic), may also have potential applications in food, but they are not currently approved

as antimicrobial food additives(Luciana et al., 2009).

53

According to the results, nisin at higher concentrations i.e., 50mg/ml was more effective in

inhibiting the growth than that of 15mg/ml. The higher concentration of nisin 50 mg/ml showed

complete inhibition of the growth of gram positive organisms in the experiment and gram

negative organism did not show any difference in its growth with the effect of nisin at 15mg/ml

but showed a little difference at 50 mg/ml concentration.

There is significant difference observed between the results obtained from viable count of

bacteria by Miles and Misra and Bioscreen C. By comparing both results, nisin showed similar

inhibitory efficacy on the growth of different gram positive organisms. Even MIC experiment

done showed the same results that are similar to that of the above mentioned.

It has been previously reported that nisin addition reduces L. innocua and S. aureus populations

in milk (Zapico et al., 1999). When milk is treated with 10% nisin and inoculated with L.innocua

the result obtained was 6.47 log CFU/ml and when inoculated with S. aureus the results obtained

was 6.32 log CFU/ml. But when the milk media was not treated with nisin, there was increase in

the viable counts of the microorganisms, when milk media is inoculated with L. innocua, the

viable count of it was 8.17 log CFU/ml and when S. aureus was inoculated in the milk media, the

obtained result was 8.14 log CFU/ml after 24 hrs. This clearly shows that there is a difference in

the values of the viable count of biological indicators in milk media with nisin with that of milk

media without nisin. In the milk media with nisin, there is inhibition of the growth of the

microorganisms as the viable count is less than the viable count of indicator organisms that are

present in milk media without nisin.

According to Jamuna, gram negative organisms found to be more resistant towards the

bacteriocin but showed suppression in the growth of E. coli. It seems that higher concentrations

54

of nisin or the combination of nisin with other bacteriocins may have the ability to inhibit the

growth of gram negative organisms (Jamuna et al., 2005). In milk medium, nisin (50mg/ml) was

very effective against all gram positive indicator organisms compared to gram negative indicator

organism. In case of Gram-negative organisms tested, E. coil was resistant to these bacteriocins

except for suppression of growth until 24 h of incubation at higher concentration The

inhibitory spectra of bacteriocins of lactic acid bacteria generally do not include Gram-negative

bacteria. This is due to the lipopolysaccharide composition present on its outer layer of the cell

wall. This LPS layer acts as a barrier to the action of nisin on the cell wall of gram negative

bacteria. Thus, processes that render these microorganisms sensitive to bacteriocins can greatly

enhance the applications of bacteriocins. Combination of bacteriocins with chelators or physical

disruption of the cells have been successfully applied by (Shefet et al., 1995)

In the present work, E. coli was resistant to the effect of nisin. According to the results obtained

in the Bioscreen experiment, it concludes that nisin can be useful in inhibition of gram positive

organisms but may not be useful in inhibition of gram negative organisms. A greater antilisterial

effect has been observed when a combination of nisin with other bacteriocin was employed,

while a mix of three bacteriocins was found to be effective in preventing the spontaneous

emergence of a bacteriocin-resistant Listeria population in broth and meat system (Vignolo et al.,

2000). More recently, the synergistic action of combinations of two bacteriocins with different

structures produced by same strain has been reported in agar medium (Limonet et al., 2004). Our

results have supported these earlier observations that bacteriocins in combinations can be used

advantageously to design efficient natural food biopreservative(s).

55

Chapter 5Conclusion

56

Nisin is proved to be more effective anti-microbial agent against food spoilage gram positive

organisms like S. aureus and L. innocua. As nisin is nontoxic and does not show any effect on

functional properties of food products, it is most widely used as a food preservative. All these

bacteria can be able to survive and grow in large variety of raw and processed foods like milk,

meat and dairy products. These studies helped in understanding the effect of anti-microbial on

different microorganisms and highlight the importance of hygiene in food industries. Day by day

the resistance of microorganisms against antimicrobials and antibiotics were increasing. New

prevention methods were studied to stop the continuous food spoilage. It is very important to

eliminate the pathogenic organisms and keep the food products safe. Further studies on this

problem helps in preventing the food borne diseases in future.

57

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