Antibiotics

Milk should not contain antibiotic residues.  Milk production is regulated by the Dairy Products (Hygiene) Regulations 1995.  These regulations include the standards for raw milk.  Prior to 1990 milk was deemed to be contaminated if an antibiotic concentration of > 0.01 international units (iu) /ml was present, the standard has now been increased to 0.006 iu/ml.  Manufacturers buying milk from producers impose stringent financial penalties on farmers producing contaminated milk and have procedures to exclude this from the food chain.  Despite legislation and financial penalties, there is evidence to suggest that residues occasionally still cause problems.  In a survey of the causes of slow acid production by cheese starters in the

UK (Boyle and Mullan, 2000, unpublished results) found that, some 28 % of respondents attributed slow acid problems to antibiotics. 

Antibiotics gain entry to milk because of mastitis treatment; mastitis means inflammation of the udder.  Although this term includes all inflammatory conditions of the udder, it isdefined here as a bacterial infection of the udder.  The common causative organisms of mastitis in the UK are Str. agalactiae, Str. dysgalactiae, coagulase-negative staphylococci and Staphylococcus aureus. 

The antibiotics used in veterinary medicine belong to six major groups:-

Aminoglycosides e.g. gentamicin
Penicillins and cephalosporins (ß-Lactams) e.g. cloxacillin
Macrolides-e.g. erythromycin
Quinolones and fluroquinolones see World Health Organisation
Sulphonamides e.g. trimethoprim
Tetracyclines e.g. tetracycline

In the recent past, penicillin G was one of the most commonly used antibiotics.  While penicillin G still has value, particularly in treating streptococcal infections, the emergence of penicillin resistant strains, the finding of new, penicillin G insensitive causal agents of mastitis and the continual search for, and introduction of, new therapeutic agents has resulted in the use of a large number of antibiotics.  Penicillin G, a member of the ß-lactam group of antibiotics is rapidly inactivated by ß-lactamase, an enzyme produced by staphylococci and other bacteria.  However, ß-Lactams can be chemically modified to produce semi-synthetic penicillins that are resistant to most ß- lactamases e.g. cloxacillin.

Antibiotics are frequently administered to infected animals in a solution or suspension that is infused into the infected quarter or quarters of the udder.  The antibiotic preparations available in the United Kingdom for intramammary infusion include penicillin G, erythromycin, ampicillin, cloxacillin, streptomycin, aureomycin, neomycin, and novobiocin.  The sensitivity of some lactococci to antibiotics is given in table 2.  Depending upon formulation, quick or slow release of antibiotic may be obtained.  Withholding times, during which the milk will contain the antimicrobial agent and must not be added to the uncontaminated milk of the remainder of the herd, are given on the manufacturer’s instructions.

The level and duration of antibiotic diffusion into milk depends upon several factors including the particular antibiotic, its concentration and method of preparation (aqueous solution, suspending medium).  The method of preparation markedly influences retention and can affect adhesion of the antibiotic to equipment and pipelines.

The amount of antibiotic excreted into milk may vary from eight to 80%; usually it averages about 50%.  It is therefore difficult to know the exact amount of antibiotic residue in milk at different milkings after treatment.  Generally, the concentration of antibiotic in milk decreases rapidly with successive milkings, usually at an exponential rate.

The extent to which starter inactivity is related to residual antibiotics in milk is not precisely known but as discussed earlier, antibiotics are still thought to be responsible for acidification problems. 

Meanwell (1962) made an interesting observation that is still relevant to those factories manufacturing their own bulk starter.  Low levels of penicillin in the bulk starter milk had a more pronounced affect on acid production when the culture grown in that milk was used in cheese manufacture compared with acid production by a normal starter inoculated into cheese milk containing a high level of penicillin.  For this reason, it is imperative that milk used for starter production is antibiotic free.  This is one of the many reasons why so many companies use commercial frozen or freeze-dried starter cultures for direct vat inoculation rather than manufacture their own bulk starter.

Table 1.  Sensitivity of starter cultures to antibiotics

 * Concentration expressed in International units ml^-1.  Concentration of other antibiotics in mg ml^-1.⁽¹⁾  Determined using an agar diffusion method.⁽²⁾  Markedly strain dependent.⁽³⁾ Ranging from a reduction in acid production of 80% to complete cessation of acid production except for (1).Table taken from Haverbeck et al. (1983)

 The effects of residues other than penicillin on starter activity during cheese manufacture has not been well documented.  One of the difficulties is many current tests for antibiotics have been originally designed to detect penicillin and quality assurance tests using these methods may not detect other antibiotics or else give a false low indication of their concentration (see Haverbeck et al., 1983).  In addition, it is possible that milk may contain several different antibiotics, and that they operate synergistically to inhibit starter growth although present in low concentration.

In the past, some manufacturers of cheese and yoghurt added penicillinase to milk intended for starter manufacture.  This treatment will inactivate the natural penicillins but not synthetic penicillins, for example, cloxacillin or non ß-Lactams.

On farm, testing of milk tanks combined with individual tanker load sampling is being used to detect and prevent contaminated milk from entering the supply chain.  This practice combined with the extensive bulking of milk at modern dairies will normally prevent major acidification problems due to antibiotic contamination.

Production of nisin

Raw milk contains lactococci.  If favourable conditions for growth occur then high numbers will result.  Some Lc. lactis strains produce the antibiotic nisin.  Nisin is a broad spectrum antibiotic and if produced it will inhibit some starter cultures.  Providing there is adequate control of temperature during the production, storage and distribution of milk nisin production should not be a problem.

Sterilant and detergent residues

Sterilant and detergent residues may inhibit the growth of starter bacteria.  The minimum concentration required for inhibition varies with the different anti-microbial agents and between different strains of starter bacteria.  Residues gain entry to milk at the (a) farm, (b) during transport to the factory and (c) the factory due to careless use of sterilants or detergents, incomplete draining or inadequate rinsing of equipment.

The concentration of sterilants required to inhibit the lactococci and other LAB have been studied.  In general, the concentration of these compounds in properly produced milk should not markedly inhibit starters.  However, the situation with quaternary ammonium compounds is less clear.  These compounds are stable in milk and can be difficult to rinse off surfaces.

Nevertheless, quality assurance staff in some plants has ascribed some instances of slow acid production to sterilant and detergent residues.  Boyle and Mullan (2000) found that some 17 % of cheese factories attributed acid production problems to these residues.

The inhibitory effects of sterilant and detergent residues are prevented by the correct and ethical use of these materials.  Proper use includes the use of the chemical at the correct concentration and adequate rinsing and draining.  Their presence is mitigated by dilution with uncontaminated milk.

Free fatty acids

Free fatty acids are present at low concentration in freshly drawn milk.  Their concentration may increase due to the activity of milk lipase or microbially produced lipases.  Pseudomonas sps.  if permitted to grow in refrigerated milk will produce lipases and high concentrations of.  free fatty acids.  However, such milk normally contains a total bacterial count in the region of 1 x 10⁷ CFU/ml.

Fatty acids are inhibitory to lactococci and in particular to Lc. lactis subsp. cremoris.  However, relatively high levels of fatty acids are required; 0.1% butyric, decanonic, hexanoic and oleic acid were required for the inhibition of Lc. lactis subsp. cremoris.  Such high concentrations of free fatty acids do not normally occur in modern hygienically produced milk that has been held at correct storage temperatures.

Natural indigenous antimicrobial proteins

The ability of raw milk to inhibit the growth of many bacterial species has been known for many years and one of the earliest reports was authored by Hesse in 1894.  Jones and his co-workers around 1920 termed the heat labile inhibitors in milk as ‘lactenins’.  The early work has been comprehensively reviewed by Reiter and MØller-Madsen (1963) and their paper in the Journal of Dairy Research is recommended reading.  Recent work has shown that these inhibitors include:

The lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system
Immunoglobins
Lysozyme
Lactoferrin
Vitamin binding proteins.

The remainder of this section will deal in summary form with the two inhibitors that affect cheese starters, namely the LP system and immunoglobulins. For more information on these inhibitors please see the section on the exploitation of antimicrobial proteins. Note that milk can also contain bioactive peptides some of which may be antimicrobial.

The LP system

Lactoperoxidase is a basic glycoprotein that contains a heme group.  It has been well characterised and will be discussed further in the application section of this website.  As with peroxidases generally lactoperoxidase catalyses reactions in which hydrogen peroxide is reduced and a suitable electron donor is oxidised.  A wide variety of electron donors can be oxidised and some can be used to assay the enzyme. 

 Bovine milk, but not human milk, contains a high concentration of lactoperoxidase (ca. 30 µg/ml).  It constitutes about 1% of the total serum proteins.  This enzyme is synthesised in the mammary gland and is a normal constituent of milk.  Wright and Cramer (1957) first showed that the inhibition of a particular strain of Lc. lactis subsp. cremoris (Str. cremoris 972) was associated with lactoperoxidase.  Later workers demonstrated that hydrogen peroxide and thiocyanate were required in addition to lactoperoxidase for inhibition of acid production to occur. 

It is now known that hydrogen peroxide forms a complex with lactoperoxidase; this complex oxidises thiocyanate (SCN^- ­) to sulphate, carbon dioxide, ammonia, and water via an unstable intermediate oxidation product that is inhibitory to some lactococci, and other LAB bacteria, but kills other bacteria including some pathogens. 

It appears likely that the antimicrobial effects are due to oxyacids of thiocyanate e.g. OSCN^- .  The inhibitor(s) is/are heat labile and are inactivated by sulphur containing reducing agents e.g. cysteine, glutathione.  Catalase, which breaks down hydrogen peroxide and is present in milk, does not inhibit the lactoperoxidase system unless deliberately added to milk, to give high un-physiological levels. 

The mechanism whereby the LP system inhibits or kills bacteria has been well studied.  In their study of the mechanism of the inhibition of lactococci Oram and Reiter (1966) found that hexokinase, the enzyme which catalyses the first step in the glycolytic pathway, was completely inhibited.  Glucose 6 — phosphate dehydrogenase and aldolase were partly inhibited while phospohexokinase was only slightly affected.  Lactococci resistant to the lactoperoxidase system possess an enzyme, which in the presence of NADH₂ reduces the intermediate oxidation product and renders it non-inhibitory.  Other studies have confirmed that the LP system oxidises essential SH-groups in vital metabolic enzymes and depletes reduced nicotinamide adenine nucleotides,

The thiocyanate content of milk depends largely on the feeding regime and varies from 0.02 mM (during the winter months) to 0.25 mM (during the summer months).  Increased levels of SCN^- can result from the detoxification of cyanide present in clover or from glucosides contained in certain plants, e.g. SCN^- is produced because of the action of rhodanese on glucosides. 

Hydrogen peroxide, the third component of the system, is supplied metabolically by the lactococci.  In fact, H₂O₂ production by starter cultures is one of the factors that may limit biomass in the production of starter concentrates. 

Lactoperoxidase is a relatively heat resistant enzyme; a treatment of 70^º C for 20 minutes reduces enzyme levels by 50%, total inactivation can be obtained by heating to 80^ºC for 5 minutes.  Since these treatments are markedly in excess of that achieved in HTST pasteurisation (72^oC -15s) it should be apparent that lactoperoxidase would normally be present in high concentration in pasteurised milk e.g. cheese milk

The quantitative importance of the lactoperoxidase system as a cause of slow acid production in the dairy industry is not known.  Starters for cheese manufacture, for example, are required to produce acid at a fast rate in pasteurised milk.  Since pasteurised milk contains lactoperoxidase starters resistant to the LP system must be used.  However, resistant starters can give rise to sensitive variants and since starters are generally maintained in high-heat-treated milk, sensitive variants will not be inhibited (no lactoperoxidase present, so there is no pressure to stop the growth of insensitive variants) during normal culturing and may reach high levels.  It is not therefore surprising that starters continuously propagated in such media, although originally resistant to the lactoperoxidase system, have been reported to become susceptible to the inhibitors present in raw milk.  Stadhouders (1961) has shown that most strains of starters are mixtures of lactoperoxidase sensitive and insensitive bacteria.

There have been been several attempts to commercial the LP system. Applications include an anticaries toothpaste, the preservation of milk in developing countries and the manufacture of calf milk replacers containing the Lp system.

Immunoglobulins

It has been known for many years that certain bacteria, including lactococci, agglutinate in raw milk.  Agglutination is caused by relatively specific antibodies that occur in the globulin protein fraction in milk serum, particularly in the euglobulin and pseudoglobulin fractions.  These antibodies, which normally originate from the blood of the cow, are always present in milk but occur at high concentration in the colostrum.

Agglutins inhibit acid production in raw and pasteurised whole or skim-milk.  There is evidence of two types of antibody response.  Response 1 is where sensitive bacteria are attached to fat globules and the second is where antibodies in the skim fraction cause bacteria to ‘stick’ together, form clumps and sediment to the bottom of the vat.  In raw or pasteurised whole milk, the immunogloblins inhibit acid production by facilitating the removal of sensitive bacteria to the cream layer and/or causing sensitive strains to agglutinate, form clumps and sediment to the bottom of the vat.  Inhibition may also occur with some strains in skim milk.  In this medium, the agglutins cause sensitive strains to form clumps that settle on the bottom of the container and localise acid production.

Starter bacteria are only inhibited in milk by agglutins if the bacteria can rise with the cream or fall to the bottom of the vat.  Agglutins are not normally a problem in the manufacture of hard cheeses, but see the comments below concerning long ripening times, since the rennet coagulum immobilises the bacteria in the curd and prevents their movement to the surface with the cream or to the bottom of the vat.  Agglutins, however, cause well documented problems in the manufacture of Cottage cheese (a skim-milk cheese).  Sensitive starters may exhibit slow acid development, `sludge’ on the bottom of the vat; shattered texture and mealy body in the curd; or complete failure to make cheese.  This defect may be avoided by careful selection of cultures and by using simple quality assurance tests to eliminate agglutinin sensitive starters.  Essentially test starters are inoculated into tall tubes of pasteurised skim-milk containing a pH or redox indicator, the control is milk treated with rennet.  Starters sensitive to agglutination will exhibit localised   acid production/redox change at the bottom of the tube.  Control swill show homogenous acid/redox change throughout.

The problems with agglutinins have been well documented and it is generally accepted that because of rennet addition these inhibitors do not cause problems in hard cheese manufacture.  However, this is perhaps, at best, an oversimplification and may not apply to situations in which extended ripening times, in excess of 1 hour, are used. Under these conditions, there may be significant time for agglutinin sensitive starters to display problems.

Agglutinins are inactivated by heat treatment or homogenisation, unlike lactoperoxidase, sometimes known as Lactenin L2, which survives a heat treatment of 71^oC for 20 minutes; immunoglublins are inactivated by this treatment.  HTST pasteurisation (72^oC - 15s) partly inactivates the imunogloblins; 50-75% of the agglutating activity is destroyed by this heat treatment.  Sterilised milk or bulk starter milk does not contain active agglutinins; the agglutinins have been denatured by the heat treatment.

• Inhibitors in milk
• Calf milk replacers
• Shelf life of LP containing milk replacers
• Improved human milk replacers
• Assay of lactoperoxidase, enzyme concentration and biological activity