ABMI

Appendix


Definitions Aseptic Filling

We agree on aseptic filling in contrast to hot filling and call it


  • This technology is the technical precondition which allows aseptic filling of microbiologically sensitive beverages.
  • In the process, a pasteurised, sterilised or aseptically filtered product is filled contamination-free into sterilised containers and sealed.
  • Finished packs sealed according to this procedure have permanent microbiological stability.

Initial Bioburden of Packaging Material

Microbiologic raw packaging material evaluation:

  • Before using packaging materials such as preforms, caps or bottles, the initial bioburden will be evaluated. This evaluation will be carried out on 10 units of the packaging material sampled in accordance with good laboratory practice.
  • Preforms or caps will be placed in sterile vials filled with a sterile recovery liquid based on pure sterile water containing 1 ‰ of Tween 80.
  • Inside of the bottles: fill 100 ml of the recovery agent in the bottle, and close it with a sterile cap. Complete bottles: cut the bottles in different parts under aseptic conditions in order to be able to place the pieces in a sterile container. Then proceed as with caps or preforms.
  • After shaking, the recovery liquid is filtered on a membrane that will be incubated on a Plate Count Agar during 5 days at 30°C.
  • After incubation the CFU’s will be counted.


Definition CIP/COP and SIP/SOP

CIP = cleaning in place
CIP means automatic cleaning of internal parts of pipes, vessels, etc. by liquid products (e.g. appropriate chemicals)

COP for us means: cleaning of external surfaces inside an isolator by liquid products (appropriate chemicals)

SIP = sterilization in place
SIP means automatic sterilization of internal parts of pipes, vessels etc. by
appropriate methods

SOP = automatic sterilization of external surfaces inside an isolator by means of
sterilants, disinfectants or other appropriate methods


Integrity Test

Procedure using electrical conductivity

The packaging tightness test is performed to detect potential leaks on bottles closed with caps or aluminium foils using the measurement of the electrical resistance.

Equipment, instruments and reagents used

  • City water that has a measurable conductivity
  • Tester to measure the resistance. The apparatus needs to reach 2000 MΩ

Prerequisites

  • Define the quantity of bottles required for performing the test, at least 100
  • Define cap application torque using a specific dynamometer
  • Each bottle must be cut without damaging the closure

Procedure

Each bottle must be cut and soaked in standard city water


Introduce inside every bottle approximately 100 ml of the same standard city water


With a tester, set the resistance measurement modality on 2000 MΩ and insert one of the electrodes inside the bottle in contact with the city water and the other one inside the bath outside of the bottle.

Since the two environments, the one inside and the one outside the bottle are separated, the tester shows an infinite resistance called "not readable/out of range".

The first measurement must be carried out 10 minutes after start. The bottles stay in the bath for 72 hours During that period some measurements are performed before the bottles are removed.

If the tester shows a value that is readable there is a connection between the inside and outside of the bottle. This is considered as an untight package.

No leak is accepted in this trial of 100 bottles


Inoculation methods

Preparation of spore suspension

Inoculation methods for packaging material need to be carried out in a professional way in order to achieve results that are indisputable.

General consideration regarding spores

  • Spores of bacteria's or moulds should be from a germ which is listed in an official type culture collection and commercially available.
  • For spore suspensions, there should be different, independent suppliers available.
  • The spores need to be prepared in a water suspension free from salts and organic material that could provide a residue during drying.
  • Spore dilutions need to be prepared in pure sterile water. No ringer solution may be used.
  • Regarding spores of moulds, they need to be free from organic material coming from cultivation. Normally the spore suspensions are washed 3-4 times and centrifuged in order to remove any kind of foreign matter.

There are three different methods for the inoculation of the packaging material:

  • Inoculation with a single dot:

    The dot has a volume of 10µl and is placed with a micro pipette on the surface of the material that needs to be tested. When starting from a defined concentrated spore suspension (e.g. 108 spores/ml), dilutions will be prepared in order to have the final correct concentration level of spores. The inoculation area will be marked with a circle.
  • Inoculation with multi dots:

    The dots will have a total volume of 10ìl and are placed with a micro pipette on the surface of the material that needs to be tested. When starting from a defined concentrated spore suspension (e.g. 108 spores/ml), dilutions will be prepared in order to have the final correct concentration level of spores. The sum of the individual dots concentration will be equal to the concentration level that is required. If the final level is 10,000 spores and 10 dots are placed on the material, each dot will have an amount of 1,000 spores. The inoculation area will be marked with circles.
  • Spray inoculation:

    A sprayer that delivers a constant volume and a homogeneous fine film will be used to inoculate the inside of the packaging material. The difficulty is to carry out constantly the same distribution of inoculation. Training of the operators needs to be carried out.