Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-05-04
2004-04-20
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S041000, C435S243000, C435S252100, C435S254100, C435S260000, C435S822000
Reexamination Certificate
active
06723526
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
REFERENCE TO SUBMISSION ON COMPACT DISC
Not applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a composition for preserving determined and reproducible amounts of micro-organisms.
It relates further to lens-shaped pellets comprising such a composition.
It has also as an object a process for producing such pellets.
2. Description of Related Art
The presence of benzene in mineral water Perrier, Listeria in cheese or epizooty of bovine spongiform encephalopathy have made quite a stir. The companies in the field of water, foodstuffs or health, positioned in sensitive sectors, have customers quite attentive to the quality of the goods delivered to them. Thus, the prevention of crisis situations is vital. In fact, any incident, such as a contamination, may have serious consequences for companies (waste production, image degradation). Consequently the danger of a contamination is a major concern for the authorities who edict increasingly stronger national, European or international regulatory constraints in terms of microbiological safety and for the industries who establish quality assurance plans including in particular the search of critical contamination points in the processes.
Consequently quite a number of public or private laboratories carry out each year many thousands of chemical and microbiological analysis in the fields of foodstuffs, environment or health.
Inter-laboratory studies have clearly showed that the laboratories generally worked with quite a good precision, but important variations existed between the results obtained from different laboratories.
Thus, excess results may be observed in laboratories having badly adjusted stoves, whereas default results are observed in laboratories having culture media of a poor quality.
Such erroneous results may have an great economical impact upon public health. The excess results may lead to refuse wrongly a product batch, to programme unnecessarily sanitizing operations to improve a beach quality or to disturb people by prohibiting tap water consumption.
In contrast, default results may incur a risk for the consumers by authorizing sales of contaminated products, distribution of poorly disinfected water or bathe in polluted water.
To secure reliable measurements, the checking laboratories have been asked to be accredited. Initially such accreditation only defined obligations of means (qualified personnel, adapted rooms, maintenance of measurement apparatus, control of raw materials and writing of the analytical procedures). At the present time, it also requires obligations of results. Such a new requirement imposes the implementation of a quality system including internal controls and participation to inter-laboratory tests.
For controlling the analytical chains internally and organize inter-laboratory tests, it is imperative to use reference materials. Such reference materials must be stable in time and should allow to reconstitute homogenous control samples containing an exact amount of the product to be dosed.
In chemistry, such reference materials have been existing for many years. In contrast, in the microbiological field, it is particularly difficult to preserve exact amounts of micro-organisms. In fact, the number of micro-organisms may either increase, if the preservation media enables their multiplication during manufacture, transport or storage, or decrease, if they die during manufacture, transportation or storage or at the time of the reconstitution of the control sample before use.
Methods for preserving micro-organisms, such as lyophilization and cryoconservation, have been already described in the all. However, such methods lead upon the conditioning step to high mortality rates amongst the micro-organisms being preserved, and consequently do not allow to master accurately the final quality of the surviving micro-organisms.
Thus, two patents appealing to a rude freezing method in liquid nitrogen do not describe the preservation of bacteria or micro-organisms and do not address to the problem of the quantitative dosages.
U.S. Pat. No. 5,364,756 discloses a method of cryoconservation for eucalyote cells. A suspension of such cells is prepared in a buffer comprising cryoprotecting agents, and then the solution is nebulizated with ultrasounds so as to form microdroplets that are rapidly cooled and dried.
U.S. Pat. No. 5,405,616 relates to the preservation of pharmacologically active molecules. The particles comprising such molecules are predominantly made of a skeleton-forming water-soluble hydrophilic macromolecule. Various proteins are mentioned, mainly proteins from vegetal origin.
Exact amounts of micro-organisms may be obtained from naturally or artificially contaminated samples. However, the preservation of such samples cannot go beyond 24 hours and requires a lower temperature than 10° C.
Exact amounts of various bacteria are also obtained after freezing in a medium containing serum and inositol (1993 Peterz and Steneryd, J. of Appl. Bacteriol., 74, 143-148) or skimmed milk (1994 Schijven, Havellaar and Bahar, Appl. And Environ. Microbiol., 60, 4160-4162). It seems however that such frozen bacterial suspensions are not stable beyond three months for the first type of preparation and one year for the second one. Moreover, they require very demanding transportation conditions (short deliveries and maintenance of the temperature below −70° C.). Finally their stability in case of successive thaw-freeze has not been demonstrated.
Presently only atomization manages to conciliate the preservation, stability and transportation constraints with the requirement of an exact amount of micro-organisms.
Suspensions of
Bacillus cereus
in concentrated milk atomized and encapsulated in gelatine have been used as reference materials after transportation at room temperature (Paul H. In't Veld, 1993, Int. J. of Food Microbiol., 20, 23-36).
However, such technique is difficult to be implemented, because it requires a dissolution of the gelatine in a reconstitution medium maintained a 37° C. Moreover, some species like
Aeromonas hydrophila, Pseudomonas aeruginosa
and
Campylobacter jejuni
cannot be preserved stably by atomization. Such a process is not recommended either for preserving respiratory tract pathogens being transmissible with aerosols like
Legionella pneumophila
. Finally the reference materials prepared by atomization have a high cost.
DESCRIPTION OF THE INVENTION
It appears thus from the art that no process was known which would be easy to be carried out and cheap allowing to preserve stably exact amounts of micro-organisms.
The Assignee has solved this problem by finding a particular composition being able to hold viability of micro-organisms.
The present invention thus relates to a composition for preserving predetermined and reproducible amounts of micro-organisms comprising in combination efficient amounts of at least one material being able to form the skeleton of lens-shaped pellets and at least one saturating material as well as micro-organisms.
For the present invention it is meant by reproducible amounts of micro-organisms variations of the ratio between two measurements comprised, in 95% of the cases, between 0.25 and 4, more preferably between 0.5 and 2.
Said composition comprises from about 10 to 60% of substances being able to form the skeleton of the lens-shaped pellets. Preferably a mixture of albumin and starch is used containing from about 20 to 40% albumin and from 2 to 5% starch (in total weight of the composition). After freezing and drying, these molecules form a highly porous and simultaneously mechanically stable skeleton that is dissolved rapidly in water.
Advantageously albumin belonging to such a composition is ovalbumin. Ovalbumin may appear in particular as an egg white preparation. Egg white is a sterile medium rich in protecting proteins within which it is possible to disperse the micro-organisms homog
Hernandez Jean-François
Oudart Eric
Paluszkiewicz Fabrice
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur de Lille
Naff David M.
Ware Deborah K.
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