Assessment of the Effect of Antimicrobial Substances on Activity of Dairy Starter Cultures
Residue Guideline 30
August 2003
On this page:
- Introduction
- Starter Cultures used in the Australian Dairy Industry
- Effect of Antimicrobial Substances on Starter Cultures
- Test Organisms
- Assessment Methodology
- Reporting of Results
- References
1. Introduction
The residues data requirements for antimicrobials in milk have been reported in Residues Guideline No. 25 - Residues in Milk - Antimicrobials. The Guideline also indicates that the influence of residues on milk processing (including the activity of dairy starter cultures) must be addressed. Dairy starter cultures are lactic acid bacteria used in the production of a range of fermented milk products, including cheese, yogurt, cultured butter and cultured milks.
Cheese and Yogurt Manufacture
The primary role of starter cultures in cheese manufacture is the production of lactic acid from lactose at a consistent and controlled rate, with the consequent decrease in pH affecting a number of aspects of the cheese manufacturing process and ultimately cheese composition and quality. Cultures used in yogurt manufacture produce lactic acid which reduces the pH and imparts the lactic sour flavour, in addition to forming volatile flavour compounds.
Australian Use of Starter Cultures
In Australia, starters are mostly used in the manufacture of cheese, particularly cheddar cheese. Further, in much of the cheddar cheese manufactured, a multiple strain system is used where two to three specific starter strains of Lactococcus lactis subsp. lactis or Lc. lactis subsp. creameries are first grown together in a bulk starter tank at the cheese factory before inoculation into a cheese vat. Alternatively, starters can be supplied as a frozen or freeze-dried concentrate of three or four defined starter strains that are added directly to the vat. Many of the smaller Australian cheese manufacturers as well as yogurt producers use this concentrate system.
Inhibition of Starter Cultures
In cheese manufacture, the time taken to reach the desired pH in the vat is critical for the manufacturer. However there are a number of potential starter culture inhibitors in milk including residues of antimicrobial drugs that affect starter activity. In addition individual starter strains can be inhibited to varying extents influencing the relative proportion of strains in cheese milk during manufacture thereby impacting on cheese flavour profiles.
Inhibition can be overcome during cheese manufacture by increasing the length of time in the vat to reach the final pH, or alternatively, increasing the level of starter culture to compensate for inhibitory substances in milk. Neither of these options is practical or efficient.
Legislative Requirements
According to the AgVet Chemical Code Act 1994 (s14(3)(e)(iii)), the APVMA has to determine that the use of a product -
"would not be likely to have an unintended effect that is harmful to animals, plants or things",
which includes the effect of antibiotics on starter cultures. Therefore, any assessment of antimicrobial product applications with the APVMA for use on dairy cows must include an assessment of the potential effects of antimicrobial substances on micro-organisms used in the manufacture of dairy products. The APVMA takes into account the effects of antimicrobial residues on starter cultures when setting MRLs for antimicrobials in milk.
This guideline has been prepared to provide applicants with a consistent procedure for assessing the potential effect of antimicrobial residues in milk on micro-organisms used for the manufacture of fermented dairy products. It provides information on the range of organisms that should be included in any assessment and the testing procedure for determination of the minimum concentration without effect of antimicrobials.
2. Starter Cultures used in the Australian Dairy Industry
The principal bacteria associated with cheese and yogurt manufacture in Australia are listed in Appendix 1.
Mesophilic Organisms used by the Australian Industry
Mesophilic organisms dominated by strains of Lc. lactis and used primarily in cheeses such as Cheddar have a typical optimum growth temperature of about 30oC.
As mentioned earlier, in much of the cheddar cheese manufactured, a multiple strain system is used where two to three specific starter strains of Lactococcus lactis subsp. lactis or Lc. lactis subsp. cremoris are first grown together in a bulk starter tank at the cheese factory before inoculation into a cheese vat. These are the principal lactococcal species used as starter cultures and generally produce lactic acid as the sole fermentation product. However, some strains of Lc. lactis subsp. lactis are capable of metabolising citrate, producing carbon dioxide, diacetyl and other aroma compounds. These organisms were referred to as either Streptococcus diacetylactis or Sc. lactis subsp. diacetylactis but are now regarded as minor variants of Lc. lactis subsp. lactis, although they are sometimes known as Lc. lactis subsp. lactis biovar diacetylactis.
Another genus of mesophilic lactic acid bacteria often used in dairy fermentations for the production of flavour compounds and gas from citrate are the leuconostocs, particularly Ln. lactis and Ln. mesenteroides. Diacetyl is the main flavour compound in sour cream, lactic butter, fresh cheese and other cheeses such as Gouda and Edam, while CO2 is also important for eye formation in these cheeses.
Thermophilic Organisms used by the Australian Industry
Thermophilic organisms, typically species of Sc. thermophilus and Lactobacillus, have optimum growth temperatures of around 42oC. The lactobacillus species used in Australia include Lb. delbrueckii subsp. lactis, Lb. delbrueckii subsp. bulgaricus and Lb. helveticus and, together with Sc. thermophilus, these are generally used for the manufacture of Italian and Swiss type cheeses and yogurt
3. Effect of Antimicrobial Substances on Starter Cultures
Antimicrobials generally belong to well-defined classes of chemical substances and are listed as either antibiotics, which are substances produced by certain micro-organisms that are active against other micro-organisms, or as chemotherapeutics, which are derived synthetically or semi-synthetically. In Australia, antimicrobials are widely used in the dairy industry for the treatment of bacterial infections such as mastitis, footrot and enteritis. However, antimicrobial drug residues are able to enter the milk , although the extent will vary from one antimicrobial to another. The presence of antimicrobial residues in milk can be undesirable from a manufacturing perspective, as they can interfere with starter culture activity and hence disrupt the manufacture process.
The sensitivity of starter cultures to antimicrobial substances present in milk varies considerably (Katla, Kruse, Johnsen and Herikstad, 2001). Even within the same species of culture strain, differences in sensitivity are evident (Packham, Broome, Limsowtin and Roginski, 2001). Further, the response of starter cultures to residual antimicrobials in milk destined for cheese or yogurt manufacture can also be complicated by the presence of other natural potential inhibitors, but an applicant will not be expected to consider these effects when generating data for registration purposes.
4. Test Organisms
The organisms selected for the initial assessment must cover the dairy starter cultures currently used in the Australian dairy industry for the primary acidification of the milk, the bulk of which belong to the genera Lactococcus, Streptococcus, Leuconostoc and Lactobacillus.
- The testing must include a range of single strains of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris, as a greater proportion of cheese manufactured is Cheddar using fresh culture prepared in the cheese factory as bulk starters. The selection of a range of organisms is also important due to the significant individual variation in sensitivity of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris strains to various antibiotics.
- It is recommended that the testing should also include the organism Lactococcus lactis subsp cremoris HP(= ATCC 19257, = DSM 20069, = NCDO 607, = NCIB 8662, = CIP 102301) as a control in determination of the concentration without effect.
- Single and mixed mesophilic cultures that have been prepared for the direct inoculation of the cheese vat must also be included in the assessment as they are used both by the majority of smaller cheese manufacturers and as back up cultures by the major cheese producers.
- Leuconostoc species and citrate positive strains of Lc. lactis subsp. lactis that are utilised for flavour enhancement in cheese and yogurt should be represented.
- Both single strains and defined mixed cultures of thermophilic bacteria representing strains of Sc. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, Lb. helveticus and Lb. acidophilus must also be assessed for their response to antimicrobial substances.
In any assessment of an antimicrobial substance, the applicant should select a range of organisms representing the above groups, justifying the choice of cultures and any omissions made. It must be re-emphasised that organisms must represent those used in the Australian Industry.
5. Assessment Methodology
The determination of the maximum concentration without effect for an antimicrobial substance on a starter culture is based on the guidelines established by The European Agency for the Evaluation of Medicinal Products - Committee for Veterinary Medicinal Products (1999).
As the primary role of starter cultures is the production of lactic acid, then the inhibitory effect of an antimicrobial substance can be determined as the difference in acid production between an inoculated substrate containing the substance and an inoculated control substrate.
Note that all studies should be conducted and reported according to OECD GLP Principles. The assessment of the effect of antimicrobial substances on starter cultures used in the dairy industry is determined as follows:
- Select the species and sub-species to be assessed (See Section 4 ).
- Determine the pH-time curve in milk for an inoculated control under standardised conditions. The determination should be made according to appropriate test guidelines, e.g. International Dairy Federation procedures (IDF, 1980). During the preliminary investigation the pH can be determined at a set time such as 4 hours, or, alternatively, as the time to reach pH 5.0. It is recommended the growth medium used in studies should be reconstituted antibiotic free non-fat milk powder (10 % w/v) as this provides a uniform medium over a long period of time.
- Undertake a preliminary series of incubation runs in milk at several antimicrobial concentrations to monitor the effect on pH. Those starter cultures whose acid production characteristics are most affected are the candidates for more detailed examination. The selection of starter cultures for further examination must be fully justified by the applicant, and should be based on data from preliminary studies. The methods of pre-selection should ensure acceptable growth of all test strains in the blank substrate.
- Use a range of antimicrobial concentrations that would represent residues found in milk, down to a minimum of half the limit of analytical quantitation (LOQ) and to a maximum of the MRL proposed. The substance used for spiking should be the equivalent of the residue definition. Where there is any doubt, the Veterinary Residues Team should be contacted through 02 6210 4700.
- Determine the concentration without effect values by assessing the effect of antibiotic on the sensitive starters, selected after the preliminary work, when grown in milk. The organism is generally inoculated at the level used in manufacture into milk and the pH profile of each test sample determined by continuous monitoring of the medium for a period of up to 6 hours.
-
The
concentration without effect for the antimicrobial substance
is defined as the highest concentration of the substance in the sample
containing the most sensitive starter culture organism giving rise
to either:
- a pH at 4 hours not differing from the pH in the control sample by more than 0.3 units or
- an increase in time of not more than 1.0 hour to reach pH 5.2 relative to the control sample.
- The concentration without effect is the mean of three separate experimental trials.
Note: The use of a fixed endpoint procedure for determining the activity of starter cultures does not provide any information concerning the length of the lag phase, the timing of the most rapid phase of acid production, the rate of acidification and the terminal pH value. Continuous pH monitoring of starter cultures in a test medium will provide this information and would be the preferable method for determining the effect of inhibitors on culture activity. Monitoring is achieved using either pH probes attached to individual meters or to a multi-channel amplifier connected to a data acquisition system with the only limitations being the available number of probes and data logging channels.
6. Reporting of Results
Results of studies carried out should be reported according to OECD Principles on Good Laboratory Practice, Part 9; Reporting of Results .
7. References
Broome, M.C., Powell, I.B. and Limsowtin, G.K.Y. (2002), Starter Cultures: Specific Properties. In Encyclopedia of Dairy Sciences. Volume 1. (Eds. H. Roginski, J.W. Fuquay and P.F. Fox), Academic Press, London, pp 269-275.
European Agency for the Evaluation of Medicinal Products (1999). Committee for Veterinary Medicinal Products. Note for guidance for the assessment of the effect of antimicrobial substances on dairy starter cultures.
International Dairy Federation Bulletin (1980), Starters in the manufacture of cheese 129.
Katla, A-K., Kruse, H., Johnsen, G. and Herikstad, H. (2001), Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Int. J. Food Microbiol. 67, 147-152.
Packham, W., Broome, M.C., Limsowtin, G.K.Y. and Roginski, H. (2001), Limitations of standard antibiotic screening assays when applied to milk for cheesemaking. Aust. J. Dairy Technol. 56, 15-18.
For more information contact:
APVMA
Residues Contact Point
Phone: (02) 6210 4700
E-mail: residues@apvma.gov.au
Appendix 1. Principal bacteria associated with fermented dairy products
| Species / subspecies | Main uses / Other comments |
|---|---|
|
Lactococcus Lc. lactis subsp. lactis Lc. lactis subsp. lactis biovar diacetylactis Lc. lactis subsp. cremoris |
Mesophilic starter used for many cheese types. Used in Gouda, Edam, sour cream and lactic butter. Mesophilic starter used for many cheese types. |
|
Streptococcus Sc. thermophilus |
Thermophilic starter used for yogurt and many cheese types particularly hard and semi hard high-cook cheeses. |
| Lactobacillus | |
| Lb. acidophilus | Probiotic adjunct culture used in cheese and yogurt. |
| Lb. delbrueckii subsp. bulgaricus | Thermophilic starter for yogurt. and many cheese types, particularly hard and semi hard high-cook cheeses. |
| Lb. delbrueckii subsp. lactis | Used in fermented milks and high-cook cheese. |
| Lb. helveticus | Thermophilic starter for fermented milks and many cheese types particularly hard and semi hard high-cook cheeses |
|
Lb. casei |
Cheese ripening adjunct culture. |
| Lb. plantarum | Cheese ripening adjunct culture. |
| Lb. rhamnosus | Cheese ripening adjunct culture. |
|
Leuconostoc Ln. mesenteroides subsp. cremoris |
Mesophilic culture used for Edam, Gouda, fresh cheese, lactic butter and sour cream. |
|
Brevibacterium Brev. linens |
Used in smear surface-ripened cheeses, Camembert, Stilton and Limburger and as a cheese ripening adjunct culture. |
|
Propionibacterium Prop. acidipropionici Prop. freudenreichii subsp. shermanii |
Used in Gruyère and Emmental cheeses. Used in Gruyère and Emmental cheeses. |
Modified From: Broome, Powell and Limsowtin (2002)