different homogenization and pastruzation effect on bacillus resistance in milk
TRANSCRIPT
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.