Inactivation of alkaline phosphatase as a control of colostrum pasteurization: are you sure to monitor the milk alkaline phosphatase?
First-milking colostrum is an important source of growth factors, vitamins, minerals and maternal antibodies, critical to protect the newborn against infectious diseases in the first weeks and months of life. In human beyond infancy, immune factors in colostrum enhance immune function which is compromised by normal aging, growth factors regulate inflammation, promote tissue growth and healing, and nutrients support cell health and development. As human colostrum cannot be put on the market, the cattle colostrum has become popular as a food supplement for human consumption (Li and Aluko, 2006). It is nearly 40 times richer in immune factors and is biologically transferable to humans with the ability to prevent bacterial and viral diseases, and to improve the gastrointestinal and body condition (Conte & Scarantino 2013). Bovine colostrum is at least 3 times more effective than vaccination to prevent flu and is very cost-effective (Cesarone et al, 2007). Nowadays it is possible to buy the pure bovine colostrum or the products colostrum based, mixed with other components in lyophilized or in liquid form.
However, bovine colostrum and milk could be a vehicle of pathogens for animals and/or human health, such as Salmonella spp., Mycobacterium bovis, Mycobacterium avium subsp. paratuberculosis (Johne’s), Cryptosporidium parvum, Mycoplasma spp., human pathogenic verocytotoxin-producing Escherichia coli, Listeria monocytogenes, Yersinia, enterotoxin producing Staphylococcus aureus, Streptococcus spp., and Campylobacter spp. In the US, the outbreaks and illnesses attributed to raw bovine milk are alarming when one considers the extremely low volume of raw milk consumed (< 1% of total milk) (Headrick, et al., 1998). Based on Center for Disease Control (CDC) data, literature, and state and local reports, FDA (2011) compiled a list of outbreaks that occurred from 1987 to September 2010. During this period, there were at least 133 outbreaks due to the consumption of raw milk and raw milk products. These outbreaks caused 2,659 cases of illnesses, 269 hospitalizations, 3 deaths, 6 stillbirths and 2 miscarriages. Thus the marketing of bovine colostrum and milk must take into account the quality control of raw material and the production chain, till product sale.
In the European Union, the Commission Regulation (EC) No 1662/2006 recognizes that « Colostrum is considered as a product of animal origin but is not covered by the definition of raw milk as referred to in Annex I to Regulation (EC) No 853/2004. Colostrum is produced in a similar way and can be considered as presenting a similar risk to human health as raw milk. It is therefore necessary to introduce specific hygiene rules for colostrum production ». In Annex II (Section IX) the Regulation defines colostrum as «the fluid secreted by the mammary glands of milk-producing animals up to three to five days post parturition that is rich in antibodies and minerals, and precedes the production of raw milk ». Then, in its Chapter I, the Regulation specifies: I) the health requirements for raw milk and colostrum production (animals in « good general state of health », that do not show any « symptoms of infectious diseases communicable to humans through milk and colostrum » or « udder wound likely to affect the milk and colostrum », and to which « no unauthorized substances or products have been administered » ) with special regards to brucellosis and tuberculosis, II) the hygiene requirements for milking equipment, milking, collection and transport and III) the criteria for raw milk and colostrum (plate count, somatic cell count or antibiotic residues). In Chapter II requirements for heat treatment are clearly defined: «When raw milk, colostrum, dairy or colostrum-based products undergo heat treatment, food business operators must ensure that this satisfies the requirements laid down in Chapter XI of Annex II to Regulation (EC) No 852/2004. In particular, they shall ensure, when using the following processes, that they comply with the specifications mentioned: (a) pasteurization is achieved by a treatment involving (i) a high temperature for a short time (at least 72 °C for 15 seconds); (ii) a low temperature for a long time (at least 63 °C for 30 minutes); or (iii) any other combination of time-temperature conditions to obtain an equivalent effect such that the product show, where applicable, a negative reaction to an alkaline phosphatase test immediately after such treatment »
In concern with the time temperature conditions, studies have reported that both batch (63 °C for 30 minutes) and HTST (72°C for 15 seconds) pasteurization of colostrum is effective in destroying viable bacteria for most of the pathogenic species including Mycobacterium bovis E. coli 0157:H7, Salmonella sp., Listeria monocytogenes, Staphylococcus aureus, and Mycoplasma spp.(Green et al., 2002; 2003; Butler et al., 2000; Stabel et al., 2003). However, early research on pasteurizing colostrum, using the conventional methods and temperatures to pasteurize milk, yielded less than acceptable results. Pasteurization resulted in mild to severe thickening or congealing of the colostrum, a reduction of up to 32% of immunoglobulin G (IgG) concentration and lower serum IgG concentrations in calves that were fed pasteurized colostrum (Meylan et al., 1995; Green et al., 2003; Godden et al., 2003; Stabel et al., 2004). It has been determined, however, that this problem can be solved by using a lower-temperature, longer-time approach to heat-treat colostrum. In most situations, heat-treating colostrum at 60°C for 60 minutes in a commercial batch pasteurizer should be sufficient to maintain IgG concentrations and viscosity while eliminating important pathogens including Listeria monocytogenes, E. coli and Salmonella enteritidis, (Godden et al., 2006, McMartin et al., 2006, Elizondo-Salazar et al., 2010).
In concern with the alkaline phosphatase (ALP) test, this enzyme is naturally present in blood and milk of all mammals. As milk ALP is slightly more heat resistant than most pathogenic bacteria, its inactivation has been used for almost 75 years as an indicator of proper pasteurisation. Today, ALP activity is recognized and accepted as the method of choice for the rapid validation of milk product pasteurization (Hammershoj et al, 2010, Rankin et al, 2010). The ALP test is particularly indicated for the control of colostrum pasteurization with on-farm pasteurizers due to frequent failure of adequate pasteurization. In a Wisconsin survey of 31 on-farm systems, the efficacy of waste milk pasteurizers was questionable on 12.9% of operations (Jorgensen et al., 2006) and it was suggested that producers should adopt a routine testing procedure to evaluate on-farm pasteurizer performance such as the alkaline phosphatase test. Reasons for the failure of pasteurization equipment to reach the target time and temperature can include improper equipment settings or calibration, equipment malfunction, lack of enough hot water, or human error, for example, turning off the equipment early in order to finish chores. Without routine monitoring, the producer will never know if the pasteurization program is working or not (Godden 2011).
Unfortunately, positive reactions to the ALP test are routinely observed while colostrum has been properly pasteurized. This could be a consequence of the non-specificity of the currently used ALP tests which all use a phosphate derived substrate which reacts with any kind of phosphatase to produce a coloured or fluorescent derivative. In mammals, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone and placenta. The isoforms cannot be readily differentiated using physico-chemical techniques such as electrofocusing since there was considerable similarity in band pattern among tissues, with only pancreatic and colostral ALP having substantially different isoelectric points from the others (Ellison & Jacobs, 1990). In the bovine, ALPs have been well characterized in the intestine and in the milk (Barman & Gutreund, 1966; Peerebroom, 1966, 1968; Linden 1974, 1976, Chuang & Yang, 1990; Vega-Warner et al, 1999) and they greatly differ, particularly in molecular weight (130 and 185 kDa respectively, Andrews 1965). In the colostrum, ALP characterization is not presently available due to the complexity of this very particular secretion.
Bovine colostrum is elaborated during the last weeks of gestation. Many components are actively concentrated from serum such as immunoglobulins IgG1, or passively transferred such as IgG2, IgM or albumin (1.21 g/l in colostrum instead of 0.13 g/l in mature milk, Levieux et al., 1999). Higher levels of ALP in colostrum than in mature milk have been reported in ewe (Zarelli, 2003, Maden et al, 2004), buffalo (Lombardi et al, 2001) and in the human (ALP level is 4 to 5 times higher than in mature milk, Kocic et al, 2010). In the bovine, the level of ALP is very high in colostrum and falls to a minimum within 1-2 week after parturition (Jenness & Patton, 1959, Linden G & Maraval B, 1979), thus the amount of ALP could help to assess the quality of colostrum (Atyabi et al, 2006). The origin of this increase in colostral ALP is presently unknown, however a participitation of seric ALPs originating from different tissues other than the mammary gland cannot be excluded. Moreover, a high number of white blood cells is usually found in colostrum (3.47-5.57 log10 cells/ml recorded by Conte & Scarantino, 2013). These cells contain a specific leucocyte alkaline phosphatase (LAP). Lastly, bacteria are frequently in higher concentration than in milk. The generally accepted recommendation is that colostrum should have a total bacterial count of less than 100,000 cfu/ml. However a Wisconsin study indicated that more than 50% of fresh colostrum samples obtained from commercial dairy herds had total counts that exceeded this level. It was dertermined that most bacterial contaminations occur during colostrum harvesting and/or storage before and after pasteurization (Stewart et al., 2005, Elizondo-Salazar et al, 2010).
The physicochemical and enzymatic properties of theses different ALPs are not similar. As an example, all mammalian ALP isoenzymes except placental are inhibited by homoarginine and, in the same way, all except the intestinal and placental ones are blocked by levamisole. Their antigenic structure is also different: a sandwich ELISA developed against bovine intestinal ALP was able to detect this ALP down to 3-5 ng/ml but was unable to detect the milk ALP (Levieux D & Geneix 2006, unpublished results). Conversely, a monoclonal antibody produced against the milk bovine ALP allowed detection of 5 ng/ml milk ALP but did not react with the bovine intestinal or Escherichia Coli ALPs. (Geneix et al, 2007). The heat sensitivity of different ALP also greatly differs: most mammalian ALPs are inactivated by heating for ~2 hours at 65°C except the placental isoforms. When adding bovine intestinal ALP to pasteurized milk, 15 % of ALP activity was still present at 60 °C after 120 min of treatment (Fadiloglu et al., 204) while the same residual activity was obtained for the endogenous milk ALP after heating at only 57°C for 120 min. (Levieux et al, 2007). Thus bovine intestinal ALP, which is the only commercially available bovine ALP, cannot be used as a model to study the effect of heat treatments on milk ALP. Lastly, both heat-labile and heat-stable ALP could be produced by microorganisms such as Bacillus anthracis, Bacillus cereus, Bacillus megaterium (Dobozy and Hammer 1969), Micrococcus sadonesis (Glew and Heath, 1971), Saccharomyces cerevisiae (Gorman and Hu, 1969) and the presence of ALP activity in properly pasteurized milk as a consequence of contamination with bacterial ALPs has been reported (Hammer & Olson, 1941).
As a consequence of the differences of heat sensitivity between the many ALPs isoforms potentially present in colostrum, a true control of colostrum pasteurization must be done using a test specific for the milk ALP. An immunological approach based on polyclonal antibodies (PAbs) produced against bovine milk ALP has been proposed by Vega- Warner et al. (1999). The antibodies have been used to develop a competitive indirect ELISA specific for the bovine milk ALP (Vega-Warner et al. 2000). However, the PAbs cross-reacted with heat-denatured ALP and the ELISA was thus not enough specific to verify adequate heat treatment of milk. Chen et al. (2006) used the same approach with PAbs developed in hens. The antibodies cross-reacted with Escherichia coli ALP and their reactivity against bovine ALP in raw milk was not established. Geneix et al (2007) developed a monoclonal antibodies specific of the bovine milk ALP after immunization of mice with a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products but not intestinal or E Coli ALPs. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISAs formats, the capture immunoassay does not require purified ALP. Such a test (PAL Test) is presently commercially available from GTP Immuno.
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