This article discusses the major functions of starters in dairy fermentations. Recent research on the relative importance of the antimicrobial agents produced by starters is included. The importance of undissociated lactic acid (HLac) is discussed with regard to the inhibition of the growth of Listeria monocytogenes, Escherichia coli and Staphylococcus aureus.
The author recommends that regulators should require manufacturers of raw milk cheeses to meet a minimum value for HLac that must be achieved prior to product release for retail sale.
The inclusion of limits for HLac in HACCP plans would provide additional safety for consumers of cheese and in particular raw milk cheeses. An interactive calculator is also provided for the calculation of HLac in cheese. The calculator provides advice on whether the value calculated is sufficient to inhibit the growth of Listeria monocytogenes in cheese and uses an amended value for the pKa of lactic acid that is relevant to the ionic environment in cheese. Further information on starter cultures is available in the section on starters.
It is concluded that a high concentration of HLac in a cheese does not guarantee freedom from pathogens, however, it will normally prevent their further growth and under ideal conditions will contribute to their decline in cheeses that have not been mould-ripened.
Major starter functions
The major functions of starters in dairy fermentations are shown in Table 1.
Table 1. Major functions of starters in milk fermentations
Whey expulsion (syneresis)
Flavour compound production
Formation of diacetyl and acetaldehyde
Lowering of pH and redox potential
Production antibiotic substances e.g. bacteriocins such as nisin
Production of hydrogen peroxide that is inhibitory per se and through activation of the LP system
Formation of inhibitory compounds e.g. D-leucine, diacetyl and reuterin
Production of lactic acid / lactate
Production of acetic acid / acetate
Out competition of other organisms for nutrients
Production of openness to facilitate 'blue veining'
Body and viscosity improvement
Increase cheese yield?
Result in reduced use of milk powder in yoghurt manufacture
Reduce potential for gas and off-flavour development
Make products more acceptable to the lactose intolerant
Lowering of redox potential
Aids flavour development
Although lactic acid production is the major task required of starters in many milk fermentations, starters have other - sometimes equally important - roles.
Cheddar cheese at the start of pressing contains about 0.5% (w/w) lactose. Failure of starters to utilise residual lactose (due to phage-induced cell lysis) during pressing and in the first few weeks of maturation may result in sweet cheese and/or gas production by heterofermentive non-starter bacteria.
Because acid production by the starter influences syneresis, variations in starter activity may result in variations in the moisture content of the final cheese. Obviously these effects have the potential to affect the profitability of dairy plants.
The relative importance and effectiveness of the antimicrobial mechanisms of starters
It has been known for decades that starter bacteria can inhibit the growth of many spoilage and pathogenic bacteria. In some instances it has been demonstrated that lactic acid bacteria may also kill pathogens.
Reuterin, 3-hydroxypropionaldehyde, produced by Lactobacillus reuteri has potent bactericidal activity against E. coli 01575:H7, Listeria monocytogenes and many Gram-negative bacteria. The bactericidal activity of reuterin is enhanced in the presence of the LP system (Arqués et al., 2008). While L. reuteri is not a normal component of defined starters it is present in some artisanal starters. Reuterin is a byproduct of glycerol metabolism and to obtain significant concentrations of this metabolite supplementation of milk with glycerol is required.
While particular strains may demonstrate potent bactericidal activity against particular pathogens there is general acceptance that starter cultures can inhibit the growth, rather than kill, many pathogens and spoilage bacteria.
|Table 2. Organisms inhibited by Streptococcus diacetilactis|
|% Inhibition at 24 hours.|
|Table 3. Experimentally determined values for MICs of undissociated lactic acid|
|Escherichia coli M23||8.32|
|Listeria innocua **||4.9|
|* Minimum inhibitory concentration of undissociated lactic acid (mM)|
|** Determined using sodium lactate|
|Modified from Presser et al. (1997)|
Determination of the concentration of undissociated lactic acid in cheese
In the cheese moisture, weak organic acids such as lactic or acetic exist as a mixture of the dissociated (ionized) and the undissociated molecular species. The proportions of the various molecular species are dependent on the pH of the cheese.
Once the total lactic acid concentration has been determined, there are several methods for measuring total lactic acid, the Henderson–Hasselbalch equation (Equation 1, the brackets indicate the molar concentration of the acid.) can be used to calculate the relative proportions of undissociated and dissociated forms of lactic acids at particular pH values using the pKa of the acid.
The pKa of an acid is the negative log of its dissociation constant. In practice this is the pH at which the acid is 50% dissociated into its constituents.
Equation 1. [dissociated acid] = 10 pH–pKa
Solving for undissociated acid gives equation 2.
Equation 2. [undissociated acid] = [dissociated acid]
(1 + 10 pH–pKa)
Until recently the pKa of lactic acid was accepted as 3.86 (Dawson,1986). Wemmenhove et al. (2017) have determined that the actual value in Gouda cheese is 3.71. Note it is likely that this lower value probably reflects the true value in most normal fat and medium moisture cheeses. The lower value will also give lower values for HLac in calculations compared with those using the previously accepted value.
Assuming a cheese with a total lactic acid concentration of 1.1%, a pH of 5.25 and a cheese moisture of 37% calculate the concentration of HLac.
This can be done as follows:
Calculate the concentration of lactic acid (M) in the cheese moisture.
1000/37 x 1.1
molecular weight of lactic acid
The concentration of HLac (M) is calculated using equation 2
= 0.00925 M
Converting to millimolar concentration
= 0.00925 x 1000
= 9.25 mM HLac
This calculation would suggest that L. monocytogenes would not be expected to grow in this cheese.
A free calculator for undertaking or checking calculations has been provided.
Note that this value assumes that all the lactic acid (dissociated and undissociated) is present only in the water phase. The situation in reality is more complicated. Undissociated lactic acid would also be expected to be soluble in milk fat. However, since milk fat is protected by the milk globule membrane and a significant proportion of fat will be solid at normal temperatures (around 20% at 20 °C) partition in fat is likely to be low. Nevertheless the calculations described here may slightly over estimate the HLac available for antimicrobial activity.
Are cheeses with high concentrations of undissociated lactic acid free from pathogens?
Pathogens such as E. coli and L. monocytogenes, if present in cheese milk, will normally grow during cheese manufacture and their initial numbers will typically be multiplied by 10 to >10,000 depending on various factors including the strain, the starter used, rate of acidification, and temperatures used in manufacture. There is no evidence that this growth can easily be prevented in cheese, containing normal concentrations of HLac, if these organisms are present in the cheese milk or have been introduced as contaminants during manufacture.
Providing the cheese has been manufactured properly, late phage-induced starter lysis has not occurred and an appropriate concentration of HLac is present further pathogen growth would not be expected. While it may be expected that the numbers of pathogens will decline significantly with storage time, particularly at elevated storage temperatures, it may take many weeks or months for their numbers to decline to their initial concentration in cheese milk. Some strains of E. coli can survive in cheese e.g. Cheddar for many months.
To ensure safety it is paramount that milk for cheese manufacture contains no, or only low concentrations, of pathogens and that pathogens are not introduced during manufacture.
The situation with mould-ripened cheeses, manufactured using milk contaminated with L. monocytogenes is particularly noteworthy.
The pH in mould ripened cheeses rises during storage and can reach pH 6.5 in some weeks. As the pH increases, the concentration of HLac decreases (Table 4).
|Table 4. Effect of ripening time on the pH and concentration of undissociated lactic acid in a mould ripened cheese that initially contained a total lactic acid concentration of 1% (w/w).|
|Time, days||pH||Concentration of undissociated lactic acid (mM)*|
|Cheese had a total lactic acid concentration of 1% (w/w) at manufacture and a moisture content of 45%.|
|*The concentration of undissociated lactic acid was determined using the calculator on this website.|
In the case illustrated in Table 4, the concentration of HLac has dropped to 3.9 mM after 20 days ripening. In the absence of other antimicrobial agents it is likely that any listeria present will commence growth. Since listeria can grow well at the temperatures used in cheese ripening, distribution and storage this high pH, low undissociated acid concentration environment, that is developing with storage may create an ideal environment for the growth of listeria to high cell densities with the high probability of food poisoning and its consequent risks including deaths of consumers.
It should be clear from the above that a high concentration of HLac in a cheese does not guarantee freedom from pathogens, however, it will normally prevent their further growth and under ideal conditions will contribute to their decline in cheeses that have not been mould-ripened.
That regulators should require manufacturers of raw milk cheeses to meet a minimum value for HLac that must be achieved prior to product release for retail sale.
Arqués, J.L., Rodríguez, E., Nuñez, M. et al. (2008).Inactivation of Gram-negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system. Eur Food Res Technol. 227, 77.
Charlier, C., Even, S., Gautier, M., Le Loir, Y. (2008). Acidification is not involved in the early inhibition of Staphylococcus aureus growth by Lactococcus lactis in milk. Intern. Dairy J. 18, 197–203.
Daly, C., Sandine, W.E. and Elliker, P. R. (1972). Interactions of food starter cultures and food-borne pathogens: Streptococcus diacetilactis versus food pathogens. Milk Food Technol. 35, 349-357.
How to cite this article
Mullan, W.M.A. (2017) . [On-line]. Available from: https://www.dairyscience.info/index.php/cheese-starters/225-role-of-starters.html . Accessed: 4 February, 2023. Based on an technical article authored in 2005. Updated September 2018; February 2022.