Studies the effect of ultra-

high pressure

homogenization and

temperature on some spore forming bacteria

B. licheniformis, B. subtilis, B. cereus and

Geobacillus stearothermophilus

Mostafa A. Shalaby

Abstract

The aim of this study is to evaluate the effects of combination of high

homogenization pressure (HPH) and temperature processes on some spore

forming bacteria (B. licheniformis, B. subtilis, B. cereus and Geobacillus

stearothermophilus) to establish alternative non-thermal methods to obtain

safe milk. High pressure homogenization (HPH) is based upon common milk

homogenization processes, though it uses 10-15 times higher pressures, which

makes the process able to promotes Bacillus reduction.

Milk plays a significant role for human nutrition and stands for one of the

most frequently sold types of food worldwide. The nutritional composition,

high water activity and neutral pH turn milk into an adequate media for

microbial development which can lead growth of enterobacterias, lactic

acid bacteria, Pseudomonas, Staphylococcus and Listeria and also

sporulated microorganisms as Bacilli and Clostridia, which are

thermoresistant and important for milk deterioration.

Introduction

Bacillus is a genus of Gram-positive, rod shaped

(bacillus), bacteria and a member of the phylum Firmicutes. Bacillus species

can be obligate aerobes (oxygen reliant), or facultative anaerobes (having

the ability to be aerobic or anaerobic). They will test positive for the

enzyme catalase when there has been oxygen used or present. Ubiquitous in

nature, Bacillus includes both free-living (non-parasitic)

and parasitic pathogenic species. Under stressful environmental conditions,

the bacteria can produce oval endospores that are not true spores but which

the bacteria can reduce themselves to and remain in a dormant state for very

long periods. These characteristics originally defined the genus, but not all

such species are closely related, and many have been moved to other

genera of Firmicutes.

Many species of Bacillus can produce copious amounts of enzymes which are

made use of in different industries. Some Bacillus species can form intracellular

inclusions of polyhydroxyalkanoates under certain adverse environmental

conditions, as in a lack of elements such as phosphorus, nitrogen, or oxygen

combined with an excessive supply of carbon sources.

B.subtilis has proved a valuable model for research. Other species

of Bacillus are important pathogens, causing anthrax and food poisoning.

Introduction

Bacillus spp. spores

Species B. boroniphilus B. globigii B. marinus B. pumilus

B. acidocaldarius B. caldolyticus B. globisporus B. megaterium B. safensis

B. alcalophilus B. centrosporus B. infernus B. mesentericus B. schlegelii

B. aminovorans B. cereus B. insolitus B. mucilaginosus B. sphaericus

B. amyloliquefaciens B. circulans B. larvae B. mycoides B. sporothermodurans

B. aneurinolyticus B. coagulans B. laterosporus B. natto B. stearothermophilus

B. anthracis B. fastidiosus B. lentimorbus B. pantothenticus B. subtilis

B. aquaemaris B. firmus B. lentus B. pasteurii B. thermoglucosidasius

B. atrophaeus B. flavothermus B. licheniformis B. polymyxa B. thuringiensis

B. azotoformans B. fusiformis B. macerans B. popilliae B. vulgatis

B. badius B. galliciensis B. macquariensis B. pseudoanthracis B. weihenstephanensis

Scientific classification

Domain:

Bacteria

Division: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Cohn, 1872[1]

We will work in four of strains (1)B. licheniformis, (2)B. subtilis, (3)B. cereus

(4)Geobacillus stearothermophilus

Introduction

(1)

Bacillus licheniformis is a bacterium commonly found in

the soil. It is found on bird feathers, especially chest and

back plumage, and most often in ground-dwelling birds

(like sparrows) and aquatic species (like ducks), and its part

of the subtilis group along with Bacillus subtilis and Bacillus

pumilus.

These bacteria are commonly known to cause food poisoning and food

spoilage. B.licheniformis also is known for contaminating dairy products.

Food borne outbreaks usually involve cases of cooked meats and

vegetables, raw milk, and industrially produced baby food contaminated

with B. licheniformis

Introduction

Bacillus subtilis Originally named Vibrio subtilis in 1835, this organism was

renamed Bacillus subtilis in 1872. Other names for this bacteria also

include Bacillus uniflagellatus, Bacillus globigii, and Bacillus natto. Bacillus

subtilis bacteria were one of the first bacteria to be studied. These bacteria are a

good model for cellular development and differentiation (Entrez Genome

Project).

(2)

Bacillus subtilis cells are rod-shaped, Gram-positive

bacteria that are naturally found in soil and

vegetation. Bacillus subtilis grow in the mesophilic

temperature range. The optimal temperature is 25-35

degrees Celsius (Entrez Genome Project). Stress and

starvation are common in this environment,

therefore, Bacillus subtilis has evolved a set of strategies

that allow survival under these harsh conditions. One

strategy, for example, is the formation of stress-resistant

endospores.

Another strategy is the uptake of external DNA, which allow the bacteria to

adapt by recombination. However, these strategies are time-consuming. Bacillus

subtilis can also gain protection more quickly against many stress situations such

as acidic, alkaline, osmotic, or oxidative conditions, and heat or ethanol. The

alternative sigma factor ?B is a global regulator of stress response. Heat, acid, or

ethanol and glucose or phosphate starvation are all stimuli that

activate (Bandow 2002).

Introduction

Bacillus cereus is a large, 1 x 3-4 µm, Gram-

positive, rod-shaped, endospore forming, facultative

aerobic bacterium [2]. It was first successfully isolated

in 1969 from a case of fatal pneumonia in a male

patient and was cultured from the blood and pleural

fluid [5]. 16s rRNA comparison reveals Bacillus

cereus to be most related to Bacillus anthracis, the

cause of anthrax, and Bacillus thuringiensis, an insect

pathogen used as pesticide [3]. Although they have

similar characteristics, they are distinguishable as B.

cereus is most motile, B. thuringiensis produces crystal

toxins, and B. anthracis is nonhemolytic [4].

B. cereus is mesophilic, growing optimally at temperatures between 20°C and

40°C, and is capable of adapting to a wide range of environmental conditions. It

is distributed widely in nature and is commonly found in the soil as a saprophytic

organism [2]. B. cereus is also a contributor to the microflora of insects, deriving

nutrients from its host, and is found in the rhizosphere of some plants [2].

As a soil bacterium, B. cereus can spread easily to many types of foods such as

plants, eggs, meat, and dairy products, and is known for causing 2-5 % of food-

borne intoxications due to its secretion of emetic toxins and enterotoxins [4].

Food poisoning occurs when food is left without refrigeration for several hours

before it is served. Remaining spores of contaminated food from heat treatment

grow well after cooling and are the source of food poisoning.

Introduction (3)

In addition, Bacillus cereus is an opportunistic human pathogen and is

occasionally associated with infections, causing periodontal diseases and

other more serious infections [5]. Immunocompromised patients are

susceptible to bacteremia, endocarditis, meningitis, pneumonia, and

endophthalmitis [6]. Its potential to cause systemic infections are of current

public health and biomedical concerns. Thus, the genome sequence

of Bacillus cereus is significant in order to establish genetic background

information for future investigations. Sequencing its genome is vital to expand

understanding of its pathogenicity for treatment and for the development of

antimicrobial drugs. Additionally, since Bacillus cereus strains are so genetically

closely related to B. anthracis, genomic comparisons between the two species

are important to the study of B. anthracis virulence.

Introduction

Geobacillus stearothermophilus is a gram positive thermophilic (heat loving) bacteria characterized by a

inner cell membrane and a thick cell wall. G.

stearothermophilus is a rod shaped anaerob found in

thermophilic habitats like thermal vents. Many heat stable

enzymes like xylanase for pulp treatment and

thermolysin-like protease for production of artificial

aspartame have been isolated from this thermophilic

bacteria.

Geobacillus stearothermophilus strain, is an isolated strain that was found in a

hot spring in Yellowstone National Park and has been used in comparative

analysis of thermophiles and mesophiles. Geobacillus stearothermophilus is

constantly used in the biotech industry to test the success of sterilization cycles

of equipment. Due to the bacteria’s high resistance to heat, it is a suitable

Biological Indicator of microbe life after a sterilization cycle (read more below).

Strain Geobacillus stearothermophilus JT2 when grown on blood agar plates are

observed to have an ellipsoidal shape and adhere to each other to form

longitudinal chains containing two or more cells. (6) This strain is observed to be

highly motile and produces a highly temperature stable enzyme α-amylase

(read more below). (6)

Introduction

(4)

* Heat resistance of Bacillus spp. spores

Introduction

* Microbial inactivation at different pressure in milk

Introduction

* Effect of different temperature/ time combinations on the surviving

Introduction

Materials and Methods

Commercial UHT milk will using to guarantee the absence of initial spore counts.

Bacillus spores (B. licheniformis, B. subtilis, B. cereus and Geobacillus

stearothermophilus) was obtained in a culture ******

For the tests, milk will inoculate with ****spores ml-1 and than subjected to

homogenization at pressures of ***, *** and *** MPa and The heat resistance of

spores at ***, *** and ***ºC were measure before and after HPH at ***MPa using

TDT method

In all tests perform the count of spores suspension were determined before and

after the treatment, in order to evaluate the spores reduction (NDR), through

equation 1.

NDR = log initial spores - log spores after treatment

Data will statistically evaluate through variance analysis (ANOVA) and

average test using the software STATISTICA 5.0.

Materials and Methods

A. Determination of thermal resistance of spores

B. Resuscitation of spores

C. Spore survival dynamics

Materials

Mesophilic and thermophilic spore forming

bacteria (B. licheniformis, B. subtilis, B. cereus

and Geobacillus stearothermophilus) will be

obtained from national and/or international

culture collection

Methods Inoculation levels

Each strain will be inoculated in milk at level of 100, 1000, 10000 CFU/ml

Each treatment will be treated at different presser homogenization in combination with different processing temperature

1- 250p/125 C/4 sec

2- 250p/130 C/4 sec

3- 250p/135 C/4 sec

4- 250p/140 C/4 sec

1- 300 p/125 C/4 sec

2- 300 p/130 C/4 sec

3- 300 p/135 C/4 sec

4- 300 p/140 C/4 sec

1- 400 p/125 C/4 sec

2- 400 p/130 C/4 sec

3- 400 p/135 C/4 sec

4- 400 p/140 C/4 sec

Analytical methods Spore forming bacteria will be counted in milk

after inoculation and before the treatment

Sterility evaluation

After treatment, all packed milk will be incubated at optimum temperature for used strain (37 C or 55 C for 5 days, and then the bacterial count will detriment on PCA /48 h and pH will be also measured

The log reduction will be calculated for each treatment

Establishing the D-value and Z-

value for each strain at different

pressure of homogenization

Hence, the decimal reduction time or ‘D-value’, which is the time needed to reduce the size of the treated population by a factor of 10, can be used as a measure of the organism’s or spore’s heat resistance at the corresponding temperature. It is also assumed that the temperature dependence of D is log linear, which produces the ‘z-value’, i.e. the temperature interval at which D will decrease (or increase) by a factor of 10. Although there is growing evidence that the isothermal semi-logarithmic survival curves of micro-organisms and spores are more of a nonlinear nature, this discussion is beyond the scope of this review and we continue to use the widely accepted D and z-concept here.